PubAnnotation

Id	Subject	Object	Predicate	Lexical cue
T1	0-126	Sentence	denotes	Discovery of Ketone-Based Covalent Inhibitors of Coronavirus 3CL Proteases for the Potential Therapeutic Treatment of COVID-19
T2	128-136	Sentence	denotes	Abstract
T3	137-349	Sentence	denotes	The novel coronavirus disease COVID-19 that emerged in 2019 is caused by the virus SARS CoV-2 and named for its close genetic similarity to SARS CoV-1 that caused severe acute respiratory syndrome (SARS) in 2002.
T4	350-584	Sentence	denotes	Both SARS coronavirus genomes encode two overlapping large polyproteins, which are cleaved at specific sites by a 3C-like cysteine protease (3CLpro) in a post-translational processing step that is critical for coronavirus replication.
T5	585-711	Sentence	denotes	The 3CLpro sequences for CoV-1 and CoV-2 viruses are 100% identical in the catalytic domain that carries out protein cleavage.
T6	712-942	Sentence	denotes	A research effort that focused on the discovery of reversible and irreversible ketone-based inhibitors of SARS CoV-1 3CLpro employing ligand-protease structures solved by X-ray crystallography led to the identification of 3 and 4.
T7	943-1140	Sentence	denotes	Preclinical experiments reveal 4 (PF-00835231) as a potent inhibitor of CoV-2 3CLpro with suitable pharmaceutical properties to warrant further development as an intravenous treatment for COVID-19.
T8	1142-1154	Sentence	denotes	Introduction
T9	1155-1253	Sentence	denotes	In late 2019, cases of an unknown respiratory tract infection were first reported in Wuhan, China.
T10	1254-1708	Sentence	denotes	By February 2020, the novel coronavirus SARS CoV-2 was identified as the causative agent for COVID-19.1,2 Genome analysis of this virus revealed a high similarity to SARS CoV-1, the coronavirus that caused severe acute respiratory syndrome (SARS) in 2002–2003.2−4 Like SARS CoV-1, which resulted in 799 deaths among the 8464 probable cases,5 SARS CoV-2 can induce fever, coughing, and difficulty breathing that rapidly becomes more serious in some cases.
T11	1709-2092	Sentence	denotes	The spread of SARS CoV-2 has been more extensive than that of SARS CoV-1, causing a global pandemic with the current number of worldwide infections surpassing eight million and deaths surpassing 400,000.6 The SARS CoV-1 genome encodes for two large polyproteins pp1a (∼450 kDa) and pp1ab (∼750 kDa) that contain overlapping sequences and include a 3C-like cysteine protease (3CLpro).
T12	2093-2441	Sentence	denotes	The function of this internally encoded 3CLpro is integral to the processing of these proteins and critical for viral replication.7 The SARS CoV-1 3CLpro shares a high degree of structural homology and similar substrate specificity with the coronavirus 3C-like cysteine proteases of hCoV 229E and TGEV8 but is most similar to the SARS CoV-2 3CLpro.
T13	2442-2827	Sentence	denotes	Specifically, the SARS CoV-1 and SARS CoV-2 share 96% identity between their respective 3CLpro sequences and 100% identity in the active site.8 A recent report by Dai et al. demonstrates that crystallographic information and structure–activity relationships obtained with the SARS CoV-1 3CLpro could facilitate the design of potent SARS CoV-2 3CLpro inhibitors with antiviral potency.9
T14	2828-3210	Sentence	denotes	There are numerous reports of reversible cysteine protease inhibitors, which include aldehydes,10−13 thio- or oxymethylketones,14 cyclic ketones,15 amidomethylketones,16 nitriles,17,18 or various 1,2-dicarbonyl motifs.19,20 The electrophilic carbon of these chemotypes reacts reversibly with the sulfur atom of an active-site cysteine forming a covalently bound tetrahedral complex.
T15	3211-3624	Sentence	denotes	Stabilization of this charged protein–ligand transition state by an “oxyanion hole” present in the active site has been observed with certain inhibitor classes by X-ray crystallography and NMR.21,22 A recent report describes the optimization of a series of peptidomimetics designed with an α-ketoamide warhead, which achieves broad-spectrum inhibition against the Mpro for several coronaviruses and enteroviruses.
T16	3625-3843	Sentence	denotes	Importantly, the reported protease potencies correlate to antiviral potencies, and the authors suggest that the reported SARS CoV-1 3CLpro activity of their lead could be predictive of the SARS CoV-2 3CLpro activity.23
T17	3844-3928	Sentence	denotes	Alternative modes of enzyme inhibition are covalently bound irreversible inhibitors.
T18	3929-4571	Sentence	denotes	While chemically reactive affinity labels such as chloromethylketones (CMKs) have demonstrated potent protease inhibition and in certain instances in vivo activity, their inherent chemical reactivity precludes development as clinical agents due to safety concerns.24 However, acyloxymethylketones first reported by Krantz as “quiescent affinity labels” for cathepsin B have demonstrated potent levels of enzyme inhibition with relatively low chemical reactivity.25,26 Mechanistic studies of this class of inhibitors with the cysteine protease caspase-1 support the intermediacy of a thiohemiketal arising from the initial 1,2-carbonyl attack.
T19	4572-4838	Sentence	denotes	Orientation and stabilization of the acyloxy leaving group within the S1′ domain (Figure 1) of the protease can facilitate SN2 displacement via sulfur migration, resulting in irreversible or bimodal (reversible inhibition followed by slow inactivation) inhibition.27
T20	4839-4903	Sentence	denotes	Figure 1 Subsite nomenclature for proteolytic enzymes is shown.
T21	4904-5057	Sentence	denotes	Amino acid residues to the left of the polypeptide scissile amide bond are numbered sequentially, beginning with P1 and increasing toward the N-terminus.
T22	5058-5195	Sentence	denotes	Amino acid residues to the right of the scissile bond are numbered sequentially, beginning with P1′ and increasing toward the C-terminus.
T23	5196-5283	Sentence	denotes	Complimentary regions of the protease active site employ the corresponding S numbering.
T24	5284-5565	Sentence	denotes	Following the SARS outbreak, a CoV-1 3CLpro homology model was published in 2003 with comparisons made to the human rhinovirus (HRV) 3Cpro.28 Although these two proteases share very little sequence conservation, the substrate consensus sequences have a Gln residue in common at P1.
T25	5566-5787	Sentence	denotes	Analysis of the HRV 3Cpro crystal structure with 1 (rupintrivir; Figure 2) revealed binding interactions that are like those observed for 1 bound to TGEV 3CLpro, which was used in the construction of their homology model.
T26	5788-5937	Sentence	denotes	The authors concluded that Michael acceptor 1 would serve as a starting point to expedite the identification of a potent SARS CoV-1 3CLpro inhibitor.
T27	5938-6193	Sentence	denotes	Figure 2 HRV clinical candidate rupintrivir and SARS CoV-1 3CLpro inhibitor 2 employing Michael acceptor-based warheads along with optimized SARS CoV-1 3CLpro inhibitors 3 and 4 containing a carbonyl designed for attack by the catalytic cysteine residue.
T28	6194-6343	Sentence	denotes	Performing a similar analysis, we tested 1 and additional HRV 3Cpro inhibitors that displayed very weak to unmeasurable SARS CoV-1 3CLpro inhibition.
T29	6344-6518	Sentence	denotes	Concurrently, new Michael acceptor derivatives were designed that tested P1/P2 alterations and truncation of the P3/P4 binding motif to optimize SARS CoV-1 3CLpro inhibition.
T30	6519-6908	Sentence	denotes	Compound 2, a derivative that contains an indole capping group at P2, displayed modest levels of irreversible inhibition (kobs/I = 586 ± 11 M–1 s–1) that enabled cocrystallization in complex with SARS CoV-1 3CLpro (PDB code 6XHO).29 Comparison of this structure with the complex of 1 in HRV 3Cpro helped to rationalize the poor performance of the warhead, which had been successful in HRV.
T31	6909-7058	Sentence	denotes	The structure of the SARS CoV-1 3CLpro complex revealed an eclipsed torsion about the resulting sp3 α,β-carbons from the Michael acceptor (Figure 3).
T32	7059-7197	Sentence	denotes	Furthermore, the carbonyl oxygen sp2 lone pair electrons are not aligned well with either 3CLpro hydrogen bond donor of the oxyanion hole.
T33	7198-7432	Sentence	denotes	Specifically, the hydrogen bond between the carbonyl oxygen and the Gly143 NH in the SARS CoV-1 3CLpro complex is 3.4 Å, while the corresponding hydrogen bond distance is a much more favorable 2.8 Å in the complex of 1 with HRV 3Cpro.
T34	7433-7710	Sentence	denotes	In contrast to these unfavorable interactions, the constrained lactam30 at the P1 site, which was designed to be isosteric with the highly conserved P1 glutamine present in all SARS CoV substrates, is well positioned in the S1 pocket making a favorable hydrogen bond to His163.
T35	7711-7827	Sentence	denotes	The NH of the lactam is within the hydrogen bond distance to Glu166 and the backbone oxygen of Phe140 in the 3CLpro.
T36	7828-8004	Sentence	denotes	Additionally, the NH of the indole P2 capping moiety makes a hydrogen bond with the protein backbone while it extends across an otherwise lipophilic surface over the P3 pocket.
T37	8005-8106	Sentence	denotes	Figure 3 Cocrystal structure of the covalent adduct of 2 bound to SARS CoV-1 3CLpro (PDB code 6XHO).
T38	8107-8178	Sentence	denotes	The Connolly surface for the inhibitor binding pocket is shown in gray.
T39	8179-8276	Sentence	denotes	The bonds are represented as the dashed lines, with the bond length measured between heavy atoms.
T40	8277-8397	Sentence	denotes	The views are centered on warhead with active-site interactions and of complete inhibitor–protein interactions depicted.
T41	8398-8593	Sentence	denotes	Although the structure of 2 in complex with SARS CoV-1 3CLpro is the final product, the above analysis is expected to apply similarly to the more relevant and structurally close transition state.
T42	8594-8791	Sentence	denotes	Accordingly, a focused design strategy aimed at replacing the warhead while retaining the P1 lactam and utilizing the methoxy indole capping group in the early rounds of optimization was initiated.
T43	8792-8983	Sentence	denotes	Specifically, an ideal electrophilic warhead would be bioisosteric, with the scissile amide carbonyl of peptidyl substrates ensuring proper alignment within the oxyanion hole of the protease.
T44	8984-9188	Sentence	denotes	To better mimic the tetrahedral intermediate generated in the amide bond cleavage, we designed ketone-based covalent reversible and irreversible inhibitors of SARS CoV-1 3CLpro, as illustrated by 3 and 4.
T45	9189-9376	Sentence	denotes	Compound 4 was selected as a development candidate for SARS CoV-1 but with the successful public health response that ended the 2003 pandemic, the clinical advancement of 4 was suspended.
T46	9377-9584	Sentence	denotes	Following the COVID-19 outbreak, testing has demonstrated that 4 is a potent inhibitor of the SARS CoV-2 3CLpro (Ki = 0.27 ± 0.1 nM) and a cocrystal structure with 4 bound in the active site has been solved.
T47	9585-9854	Sentence	denotes	In addition to the potent inhibition of the 3CLpro and viral replication of several coronaviruses, 4 possesses solubility as well as metabolic and chemical stability characteristics, which are consistent with a continuous infusion IV therapeutic treatment for COVID-19.
T48	9855-10191	Sentence	denotes	We report the research focused on the discovery of reversible and irreversible ketone-based inhibitors of SARS CoV-1 3CLpro employing ligand-protease structures solved by X-ray crystallography, which led to the identification of 4 as a molecule warranting further evaluation for its potential to treat coronaviruses, including COVID-19.
T49	10193-10215	Sentence	denotes	Results and Discussion
T50	10217-10226	Sentence	denotes	Chemistry
T51	10227-10400	Sentence	denotes	Central to our strategy of examining ketone-based thiophiles was the preparation of an array of intermediates containing diverse methylketone moieties and protecting groups.
T52	10401-10619	Sentence	denotes	The preparation of halomethylketone intermediates was accomplished in a two-step procedure by the generation of a diazoketone intermediate31 or directly by a modified Kowalski–Haque reaction,32 as depicted in Scheme 1.
T53	10620-10671	Sentence	denotes	Scheme 1 Preparation of P1 Synthetic Intermediates
T54	10672-11026	Sentence	denotes	(i) 3 M NaOH, MeOH (95%), (ii) isobutylchloroformate (IBCF), triethylamine (TEA), tetrahydrofuran (THF), (iii) CH2N2/ether (92%), (iv) 4 M HCl/dioxane (83%), (v) 1.0 equiv 48% HBr (83%), (vi) HNCH3(OCH3), EDC, HOBt, NMM, dichloromethane (DCM) (79%), (vii) Mg, HgCl2, BOM-Cl, THF, −78°C (50%), and (viii) lithium diisopropylamide (LDA), ClCH2I, THF (54%).
T55	11027-11132	Sentence	denotes	Saponification of amino ester 5(33) afforded the corresponding amino acid 6 in nearly quantitative yield.
T56	11133-11301	Sentence	denotes	Acid 6 was treated with isobutylchloroformate and triethylamine, and the resulting mixed anhydride was reacted with diazomethane34 to provide high isolated yields of 7.
T57	11302-11453	Sentence	denotes	Diazoketone 7 was treated with hydrochloric acid for simultaneous nitrogen deprotection and conversion to the chloromethylketone (CMK) intermediate 10.
T58	11454-11613	Sentence	denotes	Alternatively, the N-Boc-protected bromomethylketone was prepared by treatment of 7 with stoichiometric quantities of 48% HBr in dichloromethane to provide 11.
T59	11614-11798	Sentence	denotes	Another preparation of CMK intermediates that avoids the use of diazomethane was accomplished by the reaction of 5 with excess LDA and chloroiodomethane to afford 8 in moderate yields.
T60	11799-12006	Sentence	denotes	A direct approach to oxymethylketone intermediate 9 was achieved by conversion of acid 6 to the corresponding Weinreb amide followed by treatment with the Grignard generated from benzyl chloromethyl ether.35
T61	12007-12139	Sentence	denotes	A key element for rapid access to substituted methylketone derivatives was the generation of the fully elaborated CMK 12 (Scheme 2).
T62	12140-12248	Sentence	denotes	The utility of the CMK moiety to prepare acyloxy- and hydroxy-substituted methylketones is well established.
T63	12249-12501	Sentence	denotes	Elaboration of 10 in a sequence of deprotection/1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU)-mediated peptide couplings provided CMK derivative 12 in moderate yield with no epimerization observed.
T64	12502-12627	Sentence	denotes	Reaction of 12 with various carboxylic acids in the presence of cesium fluoride at 60 °C provided acyloxymethylketones 13–28.
T65	12628-12752	Sentence	denotes	Using this synthetic approach with substitution of P2 and amine capping structural elements provided inhibitors 3 and 29–31.
T66	12753-12833	Sentence	denotes	Scheme 2 Preparation of Substituted Acyloxymethylketones and Aminomethylketones
T67	12834-13040	Sentence	denotes	(i) Boc-Leu-OH, HATU, NMM, N,N-dimethylformamide (DMF), 0 °C (64%), (ii) 4 M HCl/dioxane, (iii) HATU, 4-MeO-2-indolecarboxylic acid, NMM, DMF, 0 °C (61%), and (iv) CsF, DMF, carboxylic acid, 60 °C (43–91%).
T68	13041-13149	Sentence	denotes	Hydroxymethylketone (HMK) derivatives were accessed by two complimentary approaches illustrated in Scheme 3.
T69	13150-13369	Sentence	denotes	Elaboration of the P1′-protected oxygen intermediate 9 by sequential amide bond coupling reactions provided benzylether derivatives such as 34 in moderate yields with a low propensity for epimerization at the P1 center.
T70	13370-13459	Sentence	denotes	Hydrogenolytic debenzylation readily afforded the final HMK inhibitors exemplified by 36.
T71	13460-13809	Sentence	denotes	Alternatively, fully elaborated CMK derivatives such as 35 were reacted with benzoylformic acid followed by methanolysis to generate HMK final products such as 36.36 Alkylation of the terminal hydroxyl was achieved under microwave-assisted conditions in the presence of silver (I) oxide to afford alkoxymethylketone inhibitors, as illustrated by 37.
T72	13810-13941	Sentence	denotes	Using these two synthetic approaches with substitution of P2 and amine capping structural elements provided inhibitors 4 and 38–50.
T73	13942-13998	Sentence	denotes	Scheme 3 Preparation of Hydroxymethylketone Derivatives
T74	13999-14200	Sentence	denotes	(i) 4 M HCl/dioxane, (ii) Boc-Leu-OH, HATU, NMM, DMF, 0 °C, (iii) HATU, 4-MeO-2-indolecarboxylic acid, NMM, 0 °C, (iv) Pd/C, H2, EtOH (77%), (v) CsF, DMF, benzoylformic acid, 60 °C, and then MeOH, cat.
T75	14201-14244	Sentence	denotes	K2CO3 (53%), and (vi) CH3I, Ag2O, DCE (9%).
T76	14246-14267	Sentence	denotes	Biological Evaluation
T77	14269-14313	Sentence	denotes	Irreversible Inhibitors of SARS CoV-1 3CLpro
T78	14314-14455	Sentence	denotes	The warhead reactivity and binding affinity to P1′ are both important design considerations to ensure specificity of irreversible inhibitors.
T79	14456-14826	Sentence	denotes	Krantz has demonstrated that the inherent chemical reactivity of acyloxymethylketones can be tuned to inhibit cysteine proteases through the modulation of substituent effects on the carboxylate leaving group.37 The importance of leaving group “strength” was confirmed by the strong dependence between the pKa of the leaving carboxylate and cathepsin B enzyme inhibition.
T80	14827-15004	Sentence	denotes	These otherwise weak electrophiles are elegant “quiescent” inhibitors that harness the very same interactions with catalytic residues that lead to proteolysis rate acceleration.
T81	15005-15242	Sentence	denotes	Molecular modeling of a 2,6-dichlorobenzoate design with SARS CoV-1 3CLpro indicated that a low strain conformation of the ketone carbonyl was aligned in the oxyanion hole and the substituted benzoate is accommodated within the P1′ site.
T82	15243-15325	Sentence	denotes	A series of acyloxymethylketone derivatives were prepared, as depicted in Table 1.
T83	15326-15403	Sentence	denotes	Table 1 SARS CoV-1 3CLpro Inhibition Data for Acyloxymethylketone Inhibitors
T84	15404-15433	Sentence	denotes	SARS CoV-1 3CLpro FRETa
T85	15434-15471	Sentence	denotes	entry R kobs/I (M–1 s–1) IC50 (nM)
T86	15472-15498	Sentence	denotes	12 283,039 ± 22,586
T87	15499-15519	Sentence	denotes	13 Me 220 ± 0.5
T88	15520-15546	Sentence	denotes	14 cyc-propyl 182 ± 6
T89	15547-15573	Sentence	denotes	15 tert-butyl 230 ± 5
T90	15574-15591	Sentence	denotes	16 Ph 86 ± 3
T91	15592-15615	Sentence	denotes	17 4-OMe-Ph 79 ± 3
T92	15616-15638	Sentence	denotes	18 4-Me-Ph 87 ± 2
T93	15639-15661	Sentence	denotes	19 4-CN-Ph 53 ± 1
T94	15662-15683	Sentence	denotes	20 4-F-Ph 82 ± 3
T95	15684-15706	Sentence	denotes	21 4-Cl-Ph 97 ± 3
T96	15707-15742	Sentence	denotes	22 2,6-(Cl)2-Ph 62,993 ± 2,501
T97	15743-15775	Sentence	denotes	23 2,6-(F)2-Ph 12,776 ± 594
T98	15776-15808	Sentence	denotes	24 2-OH-4-Cl-Ph 11,525 ± 40
T99	15809-15844	Sentence	denotes	25 2-F, 4-CN-Ph 13,321 ± 2,309
T100	15845-15872	Sentence	denotes	26 2,6-(Me)2-Ph 74 ± 4
T101	15873-15902	Sentence	denotes	27 2,6-(MeO)2-Ph 205 ± 2
T102	15903-15925	Sentence	denotes	28 2-CN-Ph 17 ± 2
T103	15926-16092	Sentence	denotes	a See the Experimental Section for details on assay methods; the values were calculated from at least eight data points with at least two independent determinations.
T104	16093-16184	Sentence	denotes	As expected, chloromethylketone 12 is a potent irreversible inhibitor of SARS CoV-1 3CLpro.
T105	16185-16322	Sentence	denotes	Compounds 22–25 possess the most electron-deficient benzoate leaving groups and display kinetics consistent with irreversible inhibition.
T106	16323-16452	Sentence	denotes	The remaining entries appear to be reversible inhibitors in the time scale of the assay, with 28 possessing the most potent IC50.
T107	16453-16647	Sentence	denotes	A crystal structure (PDB code 6XHN)38 of 28 in complex with SARS CoV-1 3CLpro at 2.25 Å shows a covalent adduct, which demonstrates the bimodal activity of certain acyloxy inhibitors (Figure 4).
T108	16648-16851	Sentence	denotes	In this structure, the electron density for the 2-cyanobenzoate moiety is absent and the 3CLpro active-site cysteine (Cys145) sulfur forms a covalent bond to the methylene carbon (1.8 Å C–S bond length).
T109	16852-16995	Sentence	denotes	The ketone carbonyl is positioned within the oxyanion hole and as such engaging in hydrogen bonds with backbone NH groups of Gly143 and Cys145.
T110	16996-17176	Sentence	denotes	A detailed analysis of an extended hydrogen bond network from catalytic His41 reveals that the side-chain imidazole serves as a hydrogen bond donor to the interior of the protease.
T111	17177-17287	Sentence	denotes	Specifically, the imidazole hydrogen is directed toward a lone pair electron acceptor from a structural water.
T112	17288-17622	Sentence	denotes	We can conclude that this water must be an acceptor to His41 as one of the water hydrogens is engaged with acceptor electrons on the side chain of Asp187, and the second hydrogen engages a terminating acceptor backbone carbonyl on Asp176 through a short network that includes the side chains of an internal, neutral His164 and Thr175.
T113	17623-17725	Sentence	denotes	Figure 4 Cocrystal structure of the covalent adduct of 28 bound to SARS CoV-1 3CLpro (PDB code 6XHN).
T114	17726-17797	Sentence	denotes	The Connolly surface for the inhibitor binding pocket is shown in gray.
T115	17798-17891	Sentence	denotes	The bonds are represented as dashed lines, with the bond length between heavy atoms depicted.
T116	17892-18333	Sentence	denotes	The 2,6-dichlorobenzoate derivative 22 displays the highest levels of potency in the SARS CoV-1 3CLpro and in antiviral cytopathic effect assays in Vero 76 cells (EC50 = 0.29 ± 0.19 μM).39 We note here that the assay in Vero 76 cells provided data establishing antiviral activity, but the translation of potency measured in Vero cells, which have high efflux potential, to the potency achievable in human lung cells was unknown (vide infra).
T117	18334-18715	Sentence	denotes	As expected, 22 possesses low levels of reactivity toward endogenous nucleophiles such as glutathione (t1/2 > 60 min)40 and exhibits high levels of stability in human plasma (t1/2 > 240 min).41 Although 22 displays a promising activity profile, it possesses very poor solubility in clinically relevant IV vehicle formulations, thus limiting its development as an IV clinical agent.
T118	18716-18932	Sentence	denotes	To improve the solubility characteristics of inhibitors incorporating the lipophilic 2,6-dichlorobenzoate, a limited set of inhibitors containing smaller or more polar amine capping fragments were prepared (Table 2).
T119	18933-19020	Sentence	denotes	Replacement of the 4-methoxy indole cap present in 22 with a benzimidazole provided 31.
T120	19021-19132	Sentence	denotes	This inhibitor displayed potent irreversible inhibition kinetics but did not improve solubility compared to 22.
T121	19133-19290	Sentence	denotes	Reduction in the size of the P2 capping element as represented by 29 and 30 provided derivatives that possessed weak reversible SARS CoV-1 3CLpro inhibition.
T122	19291-19380	Sentence	denotes	Table 2 SARS CoV-1 3CLpro Inhibition Data for P3-Modified Acyloxymethylketone Inhibitors
T123	19381-19547	Sentence	denotes	a See the Experimental Section for details on assay methods; the values were calculated from at least eight data points with at least two independent determinations.
T124	19548-19784	Sentence	denotes	Another design to replace the 4-methoxy indole was the (2R)-tetrahydrofuran-2-carboxylate, which molecular modeling suggested could make additional favorable protein interactions with a Gln189 side chain and led to the preparation of 3.
T125	19785-19860	Sentence	denotes	Initially, this derivative appeared to display reversible binding kinetics.
T126	19861-20042	Sentence	denotes	However, further kinetic studies of compound 3 with the SARS CoV-1 3CLpro protein demonstrated irreversible time-dependent inactivation of this enzyme (kobs/I = 5834 ± 151 M–1 s–1).
T127	20043-20194	Sentence	denotes	Additionally, 3 possessed potent antiviral activity in Vero cells (EC50 = 2.4 μM) and solubility in vehicle formulations almost 20-fold higher than 22.
T128	20195-20371	Sentence	denotes	Evaluation of the pharmacokinetic profile of 3 in rat suggested that this inhibitor possessed properties potentially sufficient for an IV continuous infusion dosing paradigm.42
T129	20372-20592	Sentence	denotes	Although irreversible inhibition can provide an extended pharmacodynamic effect when protein resynthesis rate is slow compared to drug clearance, such extended effects for 3CLpro inhibition were not necessarily expected.
T130	20593-20819	Sentence	denotes	The 3CLpro-mediated proteolysis of newly expressed viral polyproteins is essential to virus replication; however, this activity only occurs during a single step in the virus life cycle that closely follows each cell infection.
T131	20820-20933	Sentence	denotes	Viral particles themselves are not reliant on 3CLpro activity nor are the remaining coronavirus life cycle steps.
T132	20934-20998	Sentence	denotes	Each event of cell infection initiates newly synthesized 3CLpro.
T133	20999-21126	Sentence	denotes	Because detailed kinetics of this process are not understood, reversible and irreversible inhibitors were investigated equally.
T134	21128-21170	Sentence	denotes	Reversible Inhibitors of SARS CoV-1 3CLpro
T135	21171-21620	Sentence	denotes	Peptidyl HMK inhibitors and their corresponding ethers have been designed as potent reversible cysteine protease inhibitors of cathepsin K34 and cruzain.43 High-resolution crystal structures of two such inhibitors complexed with cruzain highlighted the presence of a strong hydrogen bond between the catalytic His and the α-hydroxyl moiety of the inhibitor but interestingly no covalent bond between the active-site Cys and the ketone pharmacophore.
T136	21621-21964	Sentence	denotes	Utilizing the crystal structure of 28 in complex with SARS CoV-1 3CLpro, molecular modeling of an HMK-based design indicated the potential for establishing a covalent bond between the sulfur of the active-site cysteine (Cys145) and the HMK carbonyl as well as hydrogen bond interactions between catalytic His41 and the terminal hydroxy moiety.
T137	21965-22137	Sentence	denotes	Additionally, the structure of 28 suggested the possibility to increase the lipophilic filling of the deep hydrophobic S2 pocket as a strategy to increase binding affinity.
T138	22138-22285	Sentence	denotes	To evaluate these structure-based observations, a series of reversible HMK inhibitors containing P2 diversity were prepared as depicted in Table 3.
T139	22286-22353	Sentence	denotes	Table 3 SARS 3CLpro Inhibition Data for P2-Modified HMK Inhibitors
T140	22354-22522	Sentence	denotes	a&b See the Experimental Section for details on assay methods; the values were calculated from at least eight data points with at least two independent determinations.
T141	22523-22557	Sentence	denotes	c This value is a Ki measurement.
T142	22558-22606	Sentence	denotes	d These values are from a single determination.
T143	22607-22758	Sentence	denotes	As suggested by molecular modeling, the S2 site appears to accommodate a variety of linear, branched, and cyclic alkyl moieties (entries 36 and 39–41).
T144	22759-22902	Sentence	denotes	Interestingly, the P2 Phe derivative 42 displayed less potent inhibition of the SARS CoV-1 3CLpro than the corresponding saturated analogue 41.
T145	22903-23048	Sentence	denotes	Also noteworthy is the large attenuation in enzyme inhibition seen with P2-N-methyl-Leu inhibitor 38 (IC50 = 83 nM) as compared to 4 (Ki = 4 nM).
T146	23049-23229	Sentence	denotes	This loss of potency is consistent with the inhibitor ligand interactions observed with 2 and 4 that show the P2 NH involved in a hydrogen bond with the side-chain amide of Gln189.
T147	23230-23426	Sentence	denotes	Additionally, the methyl substitution in 38 would be expected to alter the conformation of the 4-methoxy indole cap and perturb the observed ligand–enzyme hydrogen bond network present in 2 and 4.
T148	23427-23571	Sentence	denotes	The results for 4 and the other examples in Table 3 reveal a high antiviral EC50/enzyme IC50 ratio, which may arise from poor cell permeability.
T149	23572-23709	Sentence	denotes	Indeed, these HMK inhibitors exhibit very low permeability and high levels of efflux beyond the sensitivity of the Caco-2 in vitro assay.
T150	23710-23879	Sentence	denotes	However, the impact of high efflux on antiviral potencies from Vero 76 cells (derived from monkey kidney) versus disease-relevant human lung epithelial cells is unknown.
T151	23880-24076	Sentence	denotes	To better understand the observed high antiviral EC50/enzyme IC50 ratio for 4, we evaluated the role of efflux in the Vero 76 cell line by the in vitro experimental design discussed further below.
T152	24077-24249	Sentence	denotes	Concurrently, a strategy to design molecules reducing efflux by active transporters, such as P-glycoprotein, was pursued to decrease high antiviral EC50/enzyme IC50 ratios.
T153	24250-24519	Sentence	denotes	An analysis of the physicochemical properties of 4 suggested that increasing logP, reducing polar surface area (PSA), and reducing the number of hydrogen bond donors/acceptors were design strategies with the potential to improve cellular permeability and reduce efflux.
T154	24520-24696	Sentence	denotes	An examination of the crystal structure of 2 in complex with SARS CoV-1 3CLpro suggested a limited functional role for the 4-methoxy substituent contained in the P3 indole cap.
T155	24697-24833	Sentence	denotes	Additionally, the terminal α-hydroxyl moiety located at P1′ appeared to provide a potential opportunity to remove a hydrogen bond donor.
T156	24834-24993	Sentence	denotes	Although it was anticipated that the α-hydroxyl moiety would form a hydrogen bond to the catalytic His41, the directionality of this interaction was uncertain.
T157	24994-25226	Sentence	denotes	As noted earlier, His41 presents a hydrogen bond donor to the interior of the protease, leaving an acceptor to participate in the proton transfer cycling from neutral to cationic in the putative catalytic mechanism44 of proteolysis.
T158	25227-25399	Sentence	denotes	If His41 is protonated in the inhibitor-bound complex, a lone pair from the α-hydroxyl moiety would function as a hydrogen bond acceptor from the proton on the His residue.
T159	25400-25563	Sentence	denotes	On the other hand, if His41 is deprotonated, the hydrogen of the α-hydroxyl moiety would act as a hydrogen bond donor to the nitrogen lone pair of the His residue.
T160	25564-25754	Sentence	denotes	Consistent with the strategy to decrease PSA and hydrogen bond donors, a series of inhibitors were prepared that contained alterations to the P3 cap and substitution of the α-hydroxyl group.
T161	25755-25865	Sentence	denotes	Table 4 summarizes this effort where two of the optimal P2 residues (Leu and β-tert-butyl-Ala) were conserved.
T162	25866-26123	Sentence	denotes	Removal of the methoxy group from the indole generally led to slightly weaker potency in both the enzymatic and antiviral assays across pairs (43 vs 36, 44 vs 37, 50 vs 48 and 49 vs 4) and therefore no improvement in the antiviral/protease inhibition ratio.
T163	26124-26235	Sentence	denotes	The impact of more significant changes to the P3 cap is illustrated by tetrahydrofuranyl derivatives 46 and 47.
T164	26236-26410	Sentence	denotes	These derivatives lack the lipophilic aryl ring and NH hydrogen bond donor present in the indole P3 cap and exhibit a pronounced reduction in enzymatic and antiviral potency.
T165	26411-26574	Sentence	denotes	A noteworthy reduction in biochemical and antiviral potency was observed in comparing each hydroxymethylketone derivative with its corresponding ether counterpart.
T166	26575-26748	Sentence	denotes	These results suggest that modest structural and physicochemical alterations of 4 fail to significantly decrease the high EC50/IC50 ratios for this class of peptidomimetics.
T167	26749-27056	Sentence	denotes	However, in vitro analysis of 4 revealed this derivative to possess high levels of metabolic stability in human liver microsomes (t1/2 = 107 min).45 Further evaluation of this breakthrough derivative in several aqueous-based formulations suitable for IV administration indicated high levels of solubility.46
T168	27057-27171	Sentence	denotes	Table 4 SARS CoV-1 3CLpro Inhibition Data for P3- and P2-Modified Hydroxymethyl and Alkoxymethylketone Inhibitors
T169	27172-27340	Sentence	denotes	a&b See the Experimental Section for details on assay methods; the values were calculated from at least eight data points with at least two independent determinations.
T170	27341-27375	Sentence	denotes	c This value is a Ki measurement.
T171	27376-27424	Sentence	denotes	d These values are from a single determination.
T172	27425-27740	Sentence	denotes	Cocrystal structures of 4 bound to the 3CLpro of both SARS CoV-1 and CoV-2 were solved at 1.47 and 1.26 Å resolutions, respectively (PDB codes 6XHL and 6XHM).47 As expected, the ligand binding sites of the main protease from SARS CoV-1 and SARS CoV-2 are conserved in sequence and are nearly identical structurally.
T173	27741-27867	Sentence	denotes	The schematic diagram in Figure 5 is representative of the covalent adduct between 4 and the 3CLpro from both CoV-1 and CoV-2.
T174	27868-28044	Sentence	denotes	The warhead HMK carbonyl carbon of 4 forms a covalent bond to the 3CLpro active-site cysteine (Cys145) sulfur generating a tetrahedral carbinol complex (1.8 Å C–S bond length).
T175	28045-28182	Sentence	denotes	This carbinol hydroxyl forms hydrogen bonds with the backbone NH of Cys145 and with the amide NH of Gly143 via a bridging water molecule.
T176	28183-28304	Sentence	denotes	Another key active-site interaction is the hydrogen bond between the primary alcohol moiety of 4 and the catalytic His41.
T177	28305-28441	Sentence	denotes	Similar interactions between catalytic His residues and the OH moiety of HMK in other protease–inhibitor complexes have been reported.43
T178	28442-28534	Sentence	denotes	Figure 5 Cocrystal structure of the covalent adduct of 4 bound to SARS CoV-2 3CLpro (6XHM).
T179	28535-28606	Sentence	denotes	The Connolly surface for the inhibitor binding pocket is shown in gray.
T180	28607-28700	Sentence	denotes	The bonds are represented as dashed lines, with the bond length between heavy atoms depicted.
T181	28701-28873	Sentence	denotes	The schematic rendering of the active site with dashed lines represented as hydrogen bonds with key residues and curved lines to show S1 and S2 binding pockets is depicted.
T182	28874-29116	Sentence	denotes	In S1, the lactam carbonyl of 4 forms a strong hydrogen bond with the side chain of His163, while the lactam NH is within the hydrogen-bonding distance but poorly aligned with the side-chain oxygen of Glu166 and the backbone oxygen of Phe140.
T183	29117-29192	Sentence	denotes	These three residues partially define the bottom and edge of the S1 pocket.
T184	29193-29350	Sentence	denotes	Additionally, the backbone carbonyl and NH of Glu166 form β-sheet-like hydrogen-bonding interactions with the NH and C2-carbonyl of the indole fragment of 4.
T185	29351-29493	Sentence	denotes	The NH of the P2 Leu of inhibitor 4 and the side chain of Gln189 form a hydrogen bond, while the carbonyl of P2 Leu is exposed to the solvent.
T186	29494-29580	Sentence	denotes	The inhibitor P1 NH forms a strong hydrogen bond with the backbone carbonyl of His164.
T187	29581-29741	Sentence	denotes	The inhibitor’s lipophilic leucine residue is bound to the SARS 3CLpro in the hydrophobic S2 pocket formed by residues Asp187, Arg188, Gln189, Met49, and His41.
T188	29742-29938	Sentence	denotes	The indole portion of 4 does not protrude into the S3 pocket but rather rests across a partially collapsed S3, making extended van der Waals interactions across the 189–191 residue backbone atoms.
T189	29939-30356	Sentence	denotes	The closed S3 is the only significant conformational difference compared to a recently reported structure of SARS CoV-2 3CLpro inhibited by α-ketoamides.8 The energetics of this extended interaction, the associated protein conformation stabilities, or warhead difference may contribute to the 3-order-of-magnitude increased SARS CoV-2 3CLpro potency of HMK 4 (see below) compared to that of the referenced ketoamides.
T190	30357-30454	Sentence	denotes	Hydroxymethylketone 4 was evaluated against a panel of other viral and human proteases (Table 5).
T191	30455-30692	Sentence	denotes	In general, 4 is highly selective for 3CLpro inhibition, displaying IC50 values of >10 μM against many of the other proteases and possessing modest levels of inhibition of cathepsin B (IC50 = 1.3 μM) and rhinovirus 3Cpro (IC50 = 1.7 μM).
T192	30693-30825	Sentence	denotes	High levels of similarity between the catalytic sites of 3CLpro proteases suggest the potential of 4 as a pan-coronavirus inhibitor.
T193	30826-31047	Sentence	denotes	Testing revealed that 4 demonstrates potent inhibition of the related hCoV 229E protease (IC50 = 0.004 μM) but more importantly, given the current global pandemic, potently inhibits SARS CoV-2 3CLpro with a Ki of 0.27 nM.
T194	31048-31081	Sentence	denotes	Table 5 Protease Inhibition by 4
T195	31082-31133	Sentence	denotes	protease source % inhibition @ 10 μMa IC50 (μM)a
T196	31134-31172	Sentence	denotes	3CLpro SARS CoV-1 0.004 ± 0.0003b
T197	31173-31210	Sentence	denotes	3CLpro SARS CoV-2 0.00027 ± 0.0001b
T198	31211-31244	Sentence	denotes	cathepsin B human 93 1.3 ± 0.1
T199	31245-31271	Sentence	denotes	cathepsin D human 3 >10
T200	31272-31307	Sentence	denotes	leukocyte elastase human –31 >10
T201	31308-31335	Sentence	denotes	chymotrypsin human 8 >10
T202	31336-31361	Sentence	denotes	thrombin human –28 >10
T203	31362-31384	Sentence	denotes	pepsin human –2 >10
T204	31385-31411	Sentence	denotes	caspase 2 human –20 >10
T205	31412-31432	Sentence	denotes	HIV-1 HIV-1 0 >10
T206	31433-31450	Sentence	denotes	HCV HCV 16 >10
T207	31451-31471	Sentence	denotes	HRV HRV 1.7 ± 1
T208	31472-31507	Sentence	denotes	hCoV 229E human 0.004 ± 0.0004
T209	31508-31674	Sentence	denotes	a See the Experimental Section for details on assay methods; the values were calculated from at least eight data points with at least two independent determinations.
T210	31675-31739	Sentence	denotes	b For SARS CoV-1 and CoV-2 3CLpro, Ki values are reported here.
T211	31740-31822	Sentence	denotes	The antiviral specificity of 4 was evaluated against a panel of viruses (Table 6).
T212	31823-32022	Sentence	denotes	No antiviral activity was observed when 4 was tested against two human rhinovirus strains (HRV-14 and HRV-16), human immunodeficiency virus-1 (HIV-1), or human cytomegalovirus (HCMV) in cell culture.
T213	32023-32076	Sentence	denotes	This compound also does not inhibit the HCV replicon.
T214	32077-32255	Sentence	denotes	As anticipated by the potent hCoV 229E protease activity, 4 is a potent inhibitor of the human coronavirus hCoV 229E in MRC-5 cells (human lung-derived) with an EC50 of 0.090 μM.
T215	32256-32286	Sentence	denotes	Table 6 Viral Inhibition by 4
T216	32287-32316	Sentence	denotes	virus EC50 (μM)a CC50 (μM)a
T217	32317-32343	Sentence	denotes	SARS CoV-1 4.8 ± 2.2 452
T218	32344-32371	Sentence	denotes	hCoV 229E 0.09 ± 0.04 >40
T219	32372-32392	Sentence	denotes	HRV-14 >100 >1,260
T220	32393-32413	Sentence	denotes	HRV-16 >100 >1,260
T221	32414-32432	Sentence	denotes	HIV-1 RF >32 >32
T222	32433-32457	Sentence	denotes	HCV replicon >320 >320
T223	32458-32480	Sentence	denotes	HCMV RC256 >100 >100
T224	32481-32647	Sentence	denotes	a See the Experimental Section for details on assay methods; the values were calculated from at least eight data points with at least two independent determinations.
T225	32648-32860	Sentence	denotes	The hCoV 229E antiviral potency is more than 50-fold greater than that for SARS CoV-1 in the Vero 76 line, even though 4 possesses nearly equivalent 3CLpro biochemical potency for the hCoV 229E and CoV-1 viruses.
T226	32861-33120	Sentence	denotes	While undefined differences between hCoV 229E and SARS CoV-1 could account for these large differences in antiviral EC50/enzyme IC50 ratios, different active transporter expression levels between MRC-5 and Vero 76 cell lines may also be a contributing factor.
T227	33121-33396	Sentence	denotes	Consistent with the previously stated goal to characterize the impact of efflux on potency measured in the SARS CoV-1 antiviral assay, we co-dosed a known P-glycoprotein transport inhibitor at a fixed concentration (0.5 μM CP-100356)48 in a full dose–response of inhibitor 4.
T228	33397-33562	Sentence	denotes	This resulted in a pronounced potency shift of SARS CoV-1 antiviral activity (EC50 = 0.11 μM) that now more closely matched the antiviral potency seen for hCoV 229E.
T229	33563-33806	Sentence	denotes	As a control, the same co-dosing of efflux inhibitor led to no such shift in the hCoV 229E antiviral potency of 4, demonstrating that the human lung-derived MRC-5 cells show less P-glycoprotein-based transporter under similar assay conditions.
T230	33807-34077	Sentence	denotes	These results suggest that the high antiviral EC50/enzyme IC50 ratio observed in Vero 76 cells is an artifact of the high efflux potential of that assay cell line and may underestimate the antiviral potency in human lung cells, the relevant tissue for SARS and COVID-19.
T231	34078-34225	Sentence	denotes	Assays to directly measure the antiviral potency of 4 against SARS CoV-2 in clinically relevant human lung cells are currently under investigation.
T232	34227-34238	Sentence	denotes	Conclusions
T233	34239-34442	Sentence	denotes	This report details the efforts to identify irreversible and reversible peptidomimetic inhibitors of the SARS CoV-1 3CLpro as potential IV agents for the treatment of SARS and other coronavirus diseases.
T234	34443-34559	Sentence	denotes	Two classes, acyloxymethylketones and hydroxymethylketones, were discovered as potent inhibitors of the SARS 3CLpro.
T235	34560-34670	Sentence	denotes	The hydroxymethylketone derivative 4 demonstrated potent SARS CoV-1 inhibition in 3CLpro and antiviral assays.
T236	34671-35017	Sentence	denotes	Additionally, 4 possesses acceptable solubility, stability in plasma, and low in vitro and in vivo clearances suitable for further development as a coronavirus treatment.49 Importantly, the potent inhibition of SARS CoV-2 3CLpro has prompted further preclinical investigations of 4 as a potential therapeutic treatment for patients with COVID-19.
T237	35019-35039	Sentence	denotes	Experimental Section
T238	35041-35059	Sentence	denotes	General Procedures
T239	35060-35172	Sentence	denotes	Unless otherwise noted, materials were obtained from commercial suppliers and used without further purification.
T240	35173-35326	Sentence	denotes	The removal of solvent under reduced pressure or concentration refers to distillation using a Büchi rotary evaporator attached to a vacuum pump (3 mmHg).
T241	35327-35409	Sentence	denotes	Products obtained as solids or high-boiling oils were dried under vacuum (1 mmHg).
T242	35410-35536	Sentence	denotes	Silica gel chromatography was performed on either a CombiFlash (Teledyne ISCO), SP4, or Isolera (Biotage) purification system.
T243	35537-35737	Sentence	denotes	All reactions were performed under a positive pressure of nitrogen or argon or with a drying tube at an ambient temperature (unless otherwise stated) in anhydrous solvents, unless otherwise indicated.
T244	35738-35907	Sentence	denotes	Analytical thin-layer chromatography (TLC) was performed on glass-backed silica gel 60 F254 plates (Analtech, 0.25 mm), eluting with the appropriate solvent ratio (v/v).
T245	35908-36078	Sentence	denotes	The reactions were assayed by high-performance liquid chromatography–mass spectrometry (LCMS) or TLC and terminated as judged by the consumption of the starting material.
T246	36079-36249	Sentence	denotes	LCMS utilized wavelengths of 254 and 220 nm and either electrospray ionization (ESI) in positive mode or atmospheric-pressure chemical ionization (APCI) in positive mode.
T247	36250-36354	Sentence	denotes	The TLC plates were visualized using UV light, p-anisaldehyde, phosphomolybdic acid, or iodine staining.
T248	36355-36416	Sentence	denotes	Microwave-assisted reactions were run in a Biotage Initiator.
T249	36417-36490	Sentence	denotes	1H NMR spectra were recorded on a Bruker XWIN-NMR (400 MHz) spectrometer.
T250	36491-36587	Sentence	denotes	Proton chemical shifts are reported in parts per million downfield from tetramethylsilane (TMS).
T251	36588-36678	Sentence	denotes	1H NMR data are reported as chemical shift (multiplicity, coupling constant, integration).
T252	36679-36839	Sentence	denotes	Multiplicities are denoted as follows: s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublets; dt, doublet of triplets; and brs, broad singlet.
T253	36840-36990	Sentence	denotes	For spectra obtained in CDCl3, DMSO-d6, and CD3OD, the residual protons (7.27, 2.50, and 3.31 ppm, respectively) were used as the internal references.
T254	36991-37077	Sentence	denotes	The purity of the final products is >95% as determined by HPLC and/or 1H NMR analyses.
T255	37079-37146	Sentence	denotes	N-(tert-Butoxycarbonyl)-3-[(3S)-2-oxopyrrolidin-3-yl]-l-alanine (6)
T256	37147-37435	Sentence	denotes	A 3 L multineck flask equipped with an overhead stirrer and internal thermometer was charged with methyl N-(tert-butoxycarbonyl)-3-[(3S)-2-oxopyrrolidin-3-yl]-l-alaninate (compound 5)33 (205 g, 716 mmol) followed by methanol (1 L), and the solution was cooled to 0 °C using ice/NaCl bath.
T257	37436-37638	Sentence	denotes	NaOH (115 g in 950 mL water, 2.9 mol) solution was precooled to 0 °C and then added to the flask via a pressure-equalizing dropping funnel at such a rate to maintain the internal temperature below 5 °C.
T258	37639-37826	Sentence	denotes	The resulting solution was stirred at 0 °C for 1 h before neutralizing with conc. hydrochloric acid (keeping the internal temperature below 10 °C) and then removing the methanol in vacuo.
T259	37827-38012	Sentence	denotes	The residue was diluted with ethyl acetate (400 mL), acidified to pH 3 with conc. hydrochloric acid, and then the mixture was transferred to a sep funnel, and the organics were removed.
T260	38013-38216	Sentence	denotes	The aqueous was extracted with ethyl acetate (2 × 400 mL), and the combined organics were washed with brine, dried over MgSO4, filtered, and concentrated to yield the title compound as a white foam, 95%.
T261	38217-38414	Sentence	denotes	1H NMR (400 MHz, MeOD) δ 3.98–4.28 (m, 1H), 3.25–3.41 (m, 2H), 2.44–2.57 (m, 1H), 2.29–2.41 (m, 1H), 2.03–2.14 (m, 1H), 1.73–1.90 (m, 2H), 1.44 (s, 9H); MS (APCI−) for C12H20N2O5m/z 272.3 (M – H)−.
T262	38416-38504	Sentence	denotes	tert-Butyl ((1S)-3-Diazo-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)carbamate (7)
T263	38505-38677	Sentence	denotes	A solution of N-(tert-butoxycarbonyl)-3-[(3S)-2-oxopyrrolidin-3-yl]-l-alanine (2.72 g, 10.0 mmol) in THF (100 mL) was placed under an atmosphere of N2 and cooled to −23 °C.
T264	38678-38835	Sentence	denotes	The resulting clear colorless solution was successively treated with triethylamine (2.1 mL, 15.0 mmol) followed by isobutylchloroformate (1.6 mL, 12.0 mmol).
T265	38836-38938	Sentence	denotes	The reaction mixture gradually became opaque with a fine white precipitate and after 1 h was filtered.
T266	38939-39111	Sentence	denotes	The colorless filtrate was transferred to a nonground joint flask, cooled to 0 °C, and slowly treated with a solution of diazomethane (∼35 mL, ∼16.6 mmol) in diethyl ether.
T267	39112-39117	Sentence	denotes	Note:
T268	39118-39251	Sentence	denotes	The diazomethane was generated employing a Diazald kit according to the procedure described in the Aldrich Technical Bulletin AL-180.
T269	39252-39344	Sentence	denotes	The resulting yellow clear solution was gradually warmed to room temperature (RT) over 16 h.
T270	39345-39460	Sentence	denotes	At this time, N2 was bubbled through the reaction to remove excess diazomethane followed by in vacuo concentration.
T271	39461-39545	Sentence	denotes	The resulting residue was diluted with ethyl acetate (100 mL), washed once with sat.
T272	39546-39660	Sentence	denotes	NaHCO3 (50 mL), once with brine (50 mL), dried over MgSO4, filtered, and concentrated to give a crude yellow foam.
T273	39661-39809	Sentence	denotes	This material was purified by LC (150 g 230–400 SiO2, 3–4% methanol/chloroform) to afford 2.72 g (92%) of the title compound as a light yellow foam.
T274	39810-40030	Sentence	denotes	1H NMR (DMSO-d6) δ 7.63 (bs, 1H), 7.42 (d, J = 8 Hz, 1H), 6.06 (bs, 1H), 3.96 (m, 1H), 3.13 (m, 2H), 2.21 (m, 1H), 2.01 (m, 1H), 1.86 (m, 1H), 1.63–1.52 (m, 2H), 1.38 (s, 9H); MS (ESI+) for C13H20N4O4m/z 319.0 (M + Na)+.
T275	40032-40121	Sentence	denotes	tert-Butyl ((1S)-3-Chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)carbamate (8)
T276	40122-40411	Sentence	denotes	A three-neck flame-dried flask equipped with a nitrogen inlet and internal thermometer was charged with methyl N-(tert-butoxycarbonyl)-3-[(3S)-2-oxopyrrolidin-3-yl]-l-alaninate (10 g, 35 mmol), THF (200 mL), and chloroiodomethane (10.2 mL, 140 mmol), and the solution was cooled to −77 °C.
T277	40412-40587	Sentence	denotes	LDA (140 mL, 210 mmol, 1.5 M mono-THF complex in cyclohexane) was added via a pressure-equalizing dropping funnel at such a rate to keep the internal temperature below −70 °C.
T278	40588-40806	Sentence	denotes	After complete addition, the reaction was stirred for an additional hour and quenched with a mixture of AcOH (33 mL) and THF (200 mL) with a rate of addition regulated to maintain the internal temperature below −65 °C.
T279	40807-40917	Sentence	denotes	After complete addition, the dark suspension was stirred for 10 min and then warmed to an ambient temperature.
T280	40918-41027	Sentence	denotes	The reaction was diluted with ethyl acetate (500 mL), and the organics were washed with water (250 mL), satd.
T281	41028-41244	Sentence	denotes	NaHCO3 (250 mL), and brine (250 mL), dried over MgSO4, filtered, and the solvents were removed in vacuo to yield the crude product as a dark oil, which was purified by flash chromatography eluting with ethyl acetate.
T282	41245-41363	Sentence	denotes	The resulting solid was triturated with diethyl ether to afford the title compound as a pale yellow solid, 5.7 g, 54%.
T283	41364-41677	Sentence	denotes	1H NMR (400 MHz, DMSO-d6) δ 7.88 (s, 1H), 7.75 (d, J = 7.6 Hz, 1H), 4.73–4.94 (m, 2H), 4.37 (m, 1H), 3.28–3.43 (m, 2H), 2.46 (m, 1H), 2.30–2.40 (m, 1H), 2.02–2.14 (m, 1H), 1.77–1.95 (m, 2H), 1.60 (s, 9H); MS (API-ES+) for C13H21N2O4Cl m/z 327.1 (M + Na)+; anal. calcd for C13H21ClN2O4: C, 51.23; H, 6.95; N, 9.19.
T284	41678-41712	Sentence	denotes	Found: C, 51.03; H, 6.93; N, 9.03.
T285	41714-41807	Sentence	denotes	tert-Butyl((1S)-3-(benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)carbamate (9)
T286	41808-42106	Sentence	denotes	To a 3 L multinecked flask equipped with an overhead stirrer, nitrogen inlet, and internal thermometer was charged N-(tert-butoxycarbonyl)-3-[(3S)-2-oxopyrrolidin-3-yl]-l-alanine (190 g, 698 mmol) followed by dichloromethane (1100 mL) and the solution was cooled to 0 °C employing an ice/NaCl bath.
T287	42107-42390	Sentence	denotes	N,O-Dimethylhydroxylamine hydrochloric acid salt (68 g, 698 mmol) was added followed by N-methylmorpholine (230 mL, 2.09 mol), HOBt.hydrate (106 g, 698 mmol), and EDCI (147 g, 768 mmol), and the mixture was stirred at 0 °C under nitrogen for 6 h before quenching with water (500 mL).
T288	42391-42546	Sentence	denotes	The biphasic mixture was transferred to a sep funnel, and the organics were isolated, washed with 1 M hydrochloric acid (2 × 500 mL), water (400 mL), satd.
T289	42547-42811	Sentence	denotes	NaHCO3 (2 × 700 mL), and brine (300 mL) and then dried over MgSO4, filtered, and the solvents were evaporated in vacuo to yield tert-butyl ((1S)-2-[methoxy(methyl)amino]-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}ethyl)carbamate as a pale yellow solid, 173 g, 79%.
T290	42812-43054	Sentence	denotes	1H NMR (400 MHz, CDCl3) δ 5.92 (s, 1H), 5.39 (d, J = 8.8 Hz, 1H), 4.66 (td, J = 9.6, 2.8 Hz, 1H), 3.77 (s, 3H), 3.33 (dd, J = 9.3, 4.0 Hz, 2H), 3.19 (s, 2H), 2.41–2.62 (m, 2H), 2.05–2.16 (m, 1H), 1.75–1.95 (m, 1H), 1.67 (m, 1H), 1.41 (s, 9H).
T291	43055-43321	Sentence	denotes	To a 100 mL multinecked flask equipped with a stirrer bar, nitrogen inlet, and internal thermometer were charged magnesium turnings (dried in oven at 100 °C overnight, 1.04 g, 43 mmol) and HgCl2 (774 mg, 2.85 mmol), and the flask was purged with nitrogen for 10 min.
T292	43322-43510	Sentence	denotes	THF (50 mL) was added, and the suspension was cooled to −45 °C before adding BOM-Cl (5.94 mL, 43 mmol), and the resulting suspension was stirred for 5 h, the temperature returning to 5 °C.
T293	43511-43796	Sentence	denotes	The suspension was recooled to −50 °C, and tert-butyl ((1S)-2-[methoxy(methyl)amino]-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}ethyl)carbamate (1.5 g, 4.76 mmol) was added, and the thick suspension was stirred for 48 h under nitrogen and was allowed to come to an ambient temperature.
T294	43797-43855	Sentence	denotes	The reaction was quenched by the careful addition of satd.
T295	43856-43986	Sentence	denotes	NH4Cl solution (25 mL), and the mixture was stirred until effervescence ceased and then extracted with ethyl acetate (3 × 120 mL).
T296	43987-44259	Sentence	denotes	The combined organics were dried over MgSO4, filtered, and the solvents were evaporated in vacuo to yield a crude orange gum, which was purified by flash chromatography, eluting with 1–3% methanol/dichloromethane to afford the title compound as a clear glass, 900 mg, 50%.
T297	44260-44480	Sentence	denotes	1H NMR (400 MHz, CDCl3) δ 7.21–7.33 (m, 5H), 5.63–5.73 (m, 2H), 4.52 (m, 3H), 4.19 (q, J = 17.4 Hz, 2H), 3.19–3.25 (m, 2H), 2.29–2.42 (m, 2H), 1.68–1.93 (m, 3H), 1.35 (s, 9H); MS (APCI+) for C20H28N2O5m/z 378.1 (M + H)+.
T298	44482-44564	Sentence	denotes	(3S)-3-[(2S)-2-Amino-4-chloro-3-oxobutyl]pyrrolidin-2-one, Hydrochloride Salt (10)
T299	44565-44762	Sentence	denotes	A solution of tert-butyl ((1S)-3-diazo-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl) carbamate (592 mg, 2.0 mmol) in 1,4-dioxane (7 mL) was placed under an atmosphere of N2 and cooled to 0 °C.
T300	44763-44924	Sentence	denotes	This clear pale yellow solution was dropwise treated with a solution of 4 M hydrochloric acid in 1,4-dioxane (5 mL, 20 mmol) with copious gas evolution observed.
T301	44925-45030	Sentence	denotes	Upon complete addition, the reaction was warmed to RT over 1 h with the formation of a white precipitate.
T302	45031-45168	Sentence	denotes	The solid was collected by filtration, washed with diethyl ether, and dried to give 401 mg (83%) of the title compound as a white powder.
T303	45169-45395	Sentence	denotes	1H NMR (DMSO-d6) δ 8.76 (bs, 3H), 7.96 (s, 1H), 4.93 (d, J = 16 Hz, 1H), 4.80 (d, J = 16 Hz, 1H), 4.28 (m, 1H), 3.18 (m, 2H), 2.61 (m, 1H), 2.30 (m, 1H), 1.93 (m, 2H), 1.70 (m, 1H); MS (ESI+) for C8H13ClN2O2m/z 205.0 (M + H)+.
T304	45397-45486	Sentence	denotes	tert-Butyl ((1S)-3-Bromo-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)carbamate (11)
T305	45487-45743	Sentence	denotes	A solution of tert-butyl ((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)carbamate (1.26 g, 4.3 mmol) in dichloromethane (107 mL) at 0 °C under nitrogen was treated with 48% hydrobromic acid (0.48 mL, 4.3 mmol) with effervescence observed.
T306	45744-45925	Sentence	denotes	The reaction was stirred at 0 °C for 1 h, washed once with water (30 mL), dried over MgSO4, filtered, and concentrated to afford 1.23 g (83%) of the title compound as a white solid.
T307	45926-46185	Sentence	denotes	1H NMR (400 MHz, DMSO-d6) δ 7.64 (s, 1H), 7.51 (d, J = 8 Hz, 1H), 4.46 (d, J = 16 Hz, 1H), 4.41 (d, J = 16 Hz, 1H), 4.19 (m, 1H), 3.13 (m, 2H), 2.26 (m, 1H), 2.13 (m, 1H), 1.87 (m, 1H), 1.63 (m, 2H), 1.38 (s, 9H); MS (ESI+) for C13H21BrN2O4m/z 371.0 (M + H)+.
T308	46187-46333	Sentence	denotes	N-((1S)-1-{[((1S)-3-Chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-1H-indole-2-carboxamide (12)
T309	46334-46541	Sentence	denotes	A solution of 29, N2-(tert-butoxycarbonyl)-N1-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-l-leucinamide (244 mg, 0.59 mmol) in 1,4-dioxane (1.0 mL) was placed under an atmosphere of N2.
T310	46542-46666	Sentence	denotes	This clear colorless solution was treated with a solution of 4 M hydrochloric acid in 1,4-dioxane with no observable change.
T311	46667-46746	Sentence	denotes	The reaction gradually became opaque with the formation of a gummy precipitate.
T312	46747-46996	Sentence	denotes	After 2 h, the volatiles were removed in vacuo, diluted with 1:1 ethanol/1,4-dioxane, concentrated, and high-vacuum-dried to give N1-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-l-leucinamide hydrochloride as an off-white solid.
T313	46997-47288	Sentence	denotes	1H NMR (DMSO-d6) δ 9.18 (d, J = 8 Hz, 1H), 8.40 (bs, 3H), 7.69 (s, 1H), 4.68 (s, 2H), 4.49 (m, 1H), 3.81 (m, 1H), 3.15 (m, 2H), 2.37 (m, 1H), 2.17 (m, 1H), 1.92 (m, 1H), 1.67 (m, 3H), 1.55 (m, 2H), 0.92 (d, J = 4 Hz, 3H), 0.88 (d, J = 4 Hz, 3H); MS (ESI+) for C14H24ClN3O3m/z 318.1 (M + H)+.
T314	47289-47466	Sentence	denotes	A solution of the crude hydrochloride salt and 4-methoxy-1H-indole-2-carboxylic acid (123 mg, 0.64 mmol) in DMF (2.5 mL) was placed under an atmosphere of N2 and cooled to 0 °C.
T315	47467-47617	Sentence	denotes	This pale yellow solution was successively treated with HATU (245 mg, 0.64 mmol) and N-methylmorpholine (0.14 mL, 1.29 mmol) turning a brighter color.
T316	47618-47743	Sentence	denotes	After 30 min, the reaction was quenched with 1:1 ice/sat NaHCO3 (25 mL) and extracted three times with ethyl acetate (20 mL).
T317	47744-47871	Sentence	denotes	The combined organics were washed once with brine (30 mL), dried over MgSO4, filtered, and concentrated to give a yellow syrup.
T318	47872-48016	Sentence	denotes	This material was purified by LC (30 g 230–400 SiO2, 4% methanol/chloroform) to afford 167 mg (58%) of the title compound as an off-white solid.
T319	48017-48508	Sentence	denotes	1H NMR (DMSO-d6) δ 11.59 (s, 1H), 8.62 (d, J = 8 Hz, 1H), 8.44 (d, J = 4 Hz, 1H), 7.65 (s, 1H), 7.38 (s, 1H), 7.10 (t, J = 8 Hz, 1H), 7.02 (d, J = 8 Hz, 1H), 6.51 (d, J = 8 Hz, 1H), 4.60 (d, J = 16 Hz, 1H), 4.58 (d, J = 16 Hz, 1H), 4.46 (m, 2H), 3.89 (s, 3H), 3.11 (m, 2H), 2.29 (m, 1H), 2.11 (m, 1H), 1.99 (m, 1H), 1.76–1.54 (m, 5H), 0.95 (d, J = 8 Hz, 3H), 0.9 (d, J = 8 Hz, 3H); MS (ESI+) for C24H31ClN4O5m/z 491.1 (M + H)+; HRMS (ESI+) calcd for C24H31ClN4O5+H1 491.2056, found 491.2058.
T320	48510-48626	Sentence	denotes	(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl Acetate (13)
T321	48627-48952	Sentence	denotes	An oven-dried 40 mL scintillation vial with a spinbar was charged with acetic acid (27 mg, 0.46 mmol) followed by a solution of N-((1S)-1-{[((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-1H-indole-2-carboxamide (172 mg, 0.35 mmol) in DMF (3.5 mL) and purged with N2.
T322	48953-49126	Sentence	denotes	This pale yellow solution was then treated with CsF (122 mg, 0.81 mmol), sealed with a Teflon-lined screwcap, and heated at 65 °C on a reaction block with vigorous stirring.
T323	49127-49243	Sentence	denotes	After 3 h, the reaction was cooled to RT, diluted with water (30 mL), and extracted with dichloromethane (4 × 7 mL).
T324	49244-49349	Sentence	denotes	The combined organic layers were washed with water (2 × 20 mL), brine (20 mL), and concentrated in vacuo.
T325	49350-49496	Sentence	denotes	This material was purified by Biotage MPLC (25 M, 2.5–4.5% methanol/dichloromethane) to afford 78 mg (45%) of the title compound as a white solid.
T326	49497-50011	Sentence	denotes	1H NMR (400 MHz, DMSO-d6) δ 11.57 (s, 1H), 8.57 (d, J = 7.8 Hz, 1H), 8.43 (d, J = 7.6 Hz, 1H), 7.64 (s, 1H), 7.36 (d, J = 1.8 Hz, 1H), 7.08 (t, J = 8.0 Hz, 1H), 6.99 (d, J = 8.1 Hz, 1H), 6.49 (d, J = 7.6 Hz, 1H), 4.83 (d, J = 3.0 Hz, 1H), 4.76–4.95 (m, 1H), 4.35–4.50 (m, 2H), 3.87 (s, 3H), 3.03–3.17 (m, 2H), 2.22–2.35 (m, 1H), 2.09–2.22 (m, 1H), 2.07 (s, 3H), 1.90–2.04 (m, 1H), 1.65–1.77 (m, 2H), 1.48–1.65 (m, 3H), 0.94 (d, J = 6.3 Hz, 3H), 0.89 (d, J = 6.3 Hz, 3H); MS (ESI+) for C26H34N4O7m/z 515.2 (M + H)+.
T327	50012-50077	Sentence	denotes	Anal. calcd for C26H34N4O7·0.65 H2O: C, 59.34; H, 6.76; N, 10.65.
T328	50078-50113	Sentence	denotes	Found: C, 59.41; H, 6.63; N, 10.68.
T329	50115-50247	Sentence	denotes	(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl Cyclopropanecarboxylate (14)
T330	50248-50521	Sentence	denotes	Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting cyclopropanecarboxylic acid and making noncritical variations provided a crude product.
T331	50522-50651	Sentence	denotes	This material was purified by Biotage MPLC (25 M, 2.5–4.5% methanol/dichloromethane) to afford 82 mg (43%) of the title compound.
T332	50652-51114	Sentence	denotes	1H NMR (400 MHz, DMSO-d6) δ 11.57 (d, J = 2.0 Hz, 1H), 8.56 (d, J = 7.8 Hz, 1H), 8.43 (d, J = 7.6 Hz, 1H), 7.63 (s, 1H), 7.36 (d, J = 1.5 Hz, 1H), 7.08 (t, J = 8.0 Hz, 1H), 6.97–7.02 (m, 1H), 6.49 (d, J = 7.6 Hz, 1H), 4.85 (d, 1H), 4.78–4.96 (m, 1H), 4.33–4.51 (m, 2H), 3.87 (s, 3H), 3.02–3.16 (m, 2H), 2.22–2.35 (m, 1H), 2.01–2.11 (m, 1H), 1.89–2.00 (m, 1H), 1.65–1.77 (m, 3H), 1.46–1.65 (m, 3H), 0.81–0.98 (m, 10 H); MS (ESI+) for C28H36N4O7m/z 541.2 (M + H)+.
T333	51115-51180	Sentence	denotes	Anal. calcd for C28H36N4O7·0.59 H2O: C, 61.01; H, 6.80; N, 10.16.
T334	51181-51216	Sentence	denotes	Found: C, 61.04; H, 6.72; N, 10.00.
T335	51218-51335	Sentence	denotes	(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl Pivalate (15)
T336	51336-51594	Sentence	denotes	Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting pivalic acid and making noncritical variations provided a crude product.
T337	51595-51725	Sentence	denotes	This material was purified by Biotage MPLC (25 M, 2.5–4.5% methanol/dichloromethane) to afford 152 mg (78%) of the title compound.
T338	51726-52147	Sentence	denotes	1H NMR (400 MHz, DMSO-d6) δ 11.58 (s, 1H), 8.56 (d, J = 7.8 Hz, 1H), 8.44 (d, J = 7.8 Hz, 1H), 7.64 (s, 1H), 7.36 (s, 1H), 7.08 (t, J = 8.0 Hz, 1H), 6.99 (d, 1H), 6.49 (d, J = 7.6 Hz, 1H), 4.84 (s, 1H), 4.77–4.94 (m, 1H), 4.34–4.51 (m, 2H), 3.87 (s, 3H), 3.02–3.16 (m, 2H), 1.91–2.36 (m, 3H), 1.48–1.78 (m, 5H), 1.16 (s, 9H), 0.94 (d, J = 6.3 Hz, 3H), 0.89 (d, J = 6.3 Hz, 3H); MS (ESI+) for C29H40N4O7m/z 557.2 (M + H)+.
T339	52148-52212	Sentence	denotes	Anal. calcd for C29H40N4O7·0.76 H2O: C, 61.07; H, 7.34; N, 9.82.
T340	52213-52247	Sentence	denotes	Found: C, 61.14; H, 7.63; N, 9.59.
T341	52249-52366	Sentence	denotes	(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl Benzoate (16)
T342	52367-52625	Sentence	denotes	Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting benzoic acid and making noncritical variations provided a crude product.
T343	52626-52756	Sentence	denotes	This material was purified by Biotage MPLC (25 M, 2.5–4.5% methanol/dichloromethane) to afford 183 mg (91%) of the title compound.
T344	52757-53297	Sentence	denotes	1H NMR (400 MHz, DMSO-d6) δ 11.59 (s, 1H), 8.64 (d, J = 8.1 Hz, 1H), 8.46 (d, J = 7.6 Hz, 1H), 7.98 (d, J = 8.1 Hz, 2H), 7.91–7.95 (m, 1H), 7.66–7.71 (m, 1H), 7.66 (s, 1H), 7.52–7.57 (m, 1H), 7.37 (d, J = 1.8 Hz, 1H), 7.08 (t, J = 8.0 Hz, 1H), 6.96–7.02 (m, 1H), 6.49 (d, J = 7.6 Hz, 1H), 5.13 (s, 1H), 5.06–5.24 (m, 1H), 4.44–4.53 (m, 2H), 3.87 (s, 3H), 3.04–3.15 (m, 2H), 2.34 (m, 1H), 2.07–2.27 (m, 1H), 1.98–2.07 (m, 1H), 1.52–1.79 (m, 5H), 0.94 (d, J = 6.3 Hz, 3H), 0.89 (d, J = 6.3 Hz, 3H); MS (ESI+) for C31H36N4O7m/z 577.2 (M + H)+.
T345	53298-53362	Sentence	denotes	Anal. calcd for C31H36N4O7·1.04 H2O: C, 62.54; H, 6.45; N, 9.41.
T346	53363-53397	Sentence	denotes	Found: C, 62.49; H, 6.54; N, 9.31.
T347	53399-53525	Sentence	denotes	(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 4-Methoxybenzoate (17)
T348	53526-53793	Sentence	denotes	Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting 4-methoxybenzoic acid and making noncritical variations provided a crude product.
T349	53794-53924	Sentence	denotes	This material was purified by Biotage MPLC (25 M, 2.5–4.5% methanol/dichloromethane) to afford 145 mg (68%) of the title compound.
T350	53925-54445	Sentence	denotes	1H NMR (400 MHz, DMSO-d6) δ 11.59 (d, J = 1.8 Hz, 1H), 8.62 (d, J = 7.8 Hz, 1H), 8.46 (d, J = 7.6 Hz, 1H), 7.93 (d, J = 9.1 Hz, 2H), 7.65 (s, 1H), 7.37 (d, J = 2.0 Hz, 1H), 7.04–7.11 (m, 2H), 7.00 (dd, J = 8.6, 3.0 Hz, 2H), 6.50 (d, J = 7.6 Hz, 1H), 5.07 (s, 1H), 5.01–5.18 (m, 1H), 4.44–4.51 (m, 2H), 3.87 (s, 3H), 3.83 (s, 3H), 3.04–3.17 (m, 2H), 2.28–2.38 (m, 1H), 2.07–2.25 (m, 1H), 1.97–2.06 (m, 1H), 1.50–1.79 (m, 5H), 0.94 (d, J = 6.3 Hz, 3H), 0.89 (d, J = 6.3 Hz, 3H); MS (ESI+) for C32H38N4O8m/z 607.2 (M + H)+.
T351	54446-54519	Sentence	denotes	Anal. calcd for C32H38N4O8·0.80 H2O·0.05 DCM: C, 61.56; H, 6.40; N, 8.96.
T352	54520-54554	Sentence	denotes	Found: C, 61.84; H, 6.36; N, 8.58.
T353	54556-54681	Sentence	denotes	(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 4-Methylbenzoate (18)
T354	54682-54948	Sentence	denotes	Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting 4-methylbenzoic acid and making noncritical variations provided a crude product.
T355	54949-55078	Sentence	denotes	This material was purified by Biotage MPLC (25 M, 2.5–4.5% methanol/dichloromethane) to afford 92 mg (44%) of the title compound.
T356	55079-55619	Sentence	denotes	1H NMR (400 MHz, DMSO-d6) δ 11.59 (d, J = 1.8 Hz, 1H), 8.63 (d, J = 8.1 Hz, 1H), 8.46 (d, J = 7.6 Hz, 1H), 7.87 (d, J = 8.1 Hz, 2H), 7.65 (s, 1H), 7.37 (d, J = 1.8 Hz, 1H), 7.34 (d, J = 8.3 Hz, 2H), 7.08 (t, J = 8.0 Hz, 1H), 6.97–7.01 (m, 1H), 6.50 (d, J = 7.8 Hz, 1H), 5.09 (s, 1H), 5.03–5.20 (m, 1H), 4.43–4.52 (m, 2H), 3.87 (s, 3H), 3.04–3.17 (m, 2H), 2.38 (s, 3H), 2.28–2.34 (m, 1H), 2.06–2.14 (m, 1H), 1.96–2.06 (m, 1H), 1.51–1.78 (m, 5H), 0.94 (d, J = 6.3 Hz, 3H), 0.89 (d, J = 6.3 Hz, 3H); MS (ESI+) for C32H38N4O7m/z 591.2 (M + H)+.
T357	55620-55693	Sentence	denotes	Anal. calcd for C32H38N4O7·0.10 H2O·0.11 DCM: C, 64.08; H, 6.43; N, 9.31.
T358	55694-55728	Sentence	denotes	Found: C, 64.36; H, 6.41; N, 8.92.
T359	55730-55854	Sentence	denotes	(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 4-Cyanobenzoate (19)
T360	55855-56120	Sentence	denotes	Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting 4-cyanobenzoic acid and making noncritical variations provided a crude product.
T361	56121-56251	Sentence	denotes	This material was purified by Biotage MPLC (25 M, 2.5–4.5% methanol/dichloromethane) to afford 159 mg (75%) of the title compound.
T362	56252-56783	Sentence	denotes	1H NMR (400 MHz, DMSO-d6) δ 11.58 (d, J = 2.0 Hz, 1H), 8.64 (d, J = 8.1 Hz, 1H), 8.46 (d, J = 7.6 Hz, 1H), 8.09–8.14 (m, 2H), 7.99–8.05 (m, 2H), 7.66 (s, 1H), 7.37 (d, J = 1.5 Hz, 1H), 7.08 (t, J = 8.0 Hz, 1H), 6.99 (d, J = 8.3 Hz, 1H), 6.50 (d, J = 7.6 Hz, 1H), 5.17 (d, J = 2.8 Hz, 1H), 5.11–5.27 (m, 1H), 4.44–4.53 (m, 2H), 3.87 (s, 3H), 3.04–3.17 (m, 2H), 2.28–2.38 (m, 1H), 2.07–2.26 (m, 1H), 1.97–2.06 (m, 1H), 1.51–1.78 (m, 5H), 0.94 (d, J = 6.3 Hz, 3H), 0.89 (d, J = 6.3 Hz, 3H); MS (ESI+) for C32H35N5O7m/z 602.2 (M + H)+.
T363	56784-56849	Sentence	denotes	Anal. calcd for C32H35N5O7·0.90 H2O: C, 62.21; H, 6.00; N, 11.33.
T364	56850-56885	Sentence	denotes	Found: C, 62.23; H, 6.06; N, 11.58.
T365	56887-57012	Sentence	denotes	(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 4-Fluorobenzoate (20)
T366	57013-57279	Sentence	denotes	Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting 4-fluorobenzoic acid and making noncritical variations provided a crude product.
T367	57280-57410	Sentence	denotes	This material was purified by Biotage MPLC (25 M, 2.5–4.5% methanol/dichloromethane) to afford 136 mg (65%) of the title compound.
T368	57411-57930	Sentence	denotes	1H NMR (400 MHz, DMSO-d6) δ 11.58 (d, J = 2.0 Hz, 1H), 8.63 (d, J = 7.8 Hz, 1H), 8.46 (d, J = 7.6 Hz, 1H), 8.04 (dd, J = 8.6, 5.6 Hz, 2H), 7.65 (s, 1H), 7.35–7.41 (m, 3H), 7.08 (t, J = 8.0 Hz, 1H), 6.99 (d, J = 8.1 Hz, 1H), 6.50 (d, J = 7.6 Hz, 1H), 5.12 (d, J = 1.3 Hz, 1H), 5.06–5.23 (m, 1H), 4.44–4.52 (m, 2H), 3.87 (s, 3H), 3.04–3.17 (m, 2H), 2.28–2.38 (m, 1H), 2.07–2.26 (m, 1H), 1.98–2.06 (m, 1H), 1.52–1.79 (m, 5H), 0.94 (d, J = 6.3 Hz, 3H), 0.89 (d, J = 6.3 Hz, 3H); MS (ESI+) for C31H35FN4O7m/z 595.2 (M + H)+.
T369	57931-58005	Sentence	denotes	Anal. calcd for C31H35FN4O7·0.59 H2O·0.06 DCM: C, 61.28; H, 5.98; N, 9.20.
T370	58006-58040	Sentence	denotes	Found: C, 61.27; H, 5.98; N, 9.11.
T371	58042-58167	Sentence	denotes	(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 4-Chlorobenzoate (21)
T372	58168-58434	Sentence	denotes	Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting 4-chlorobenzoic acid and making noncritical variations provided a crude product.
T373	58435-58565	Sentence	denotes	This material was purified by Biotage MPLC (25 M, 2.5–4.5% methanol/dichloromethane) to afford 172 mg (80%) of the title compound.
T374	58566-59113	Sentence	denotes	1H NMR (400 MHz, DMSO-d6) δ 11.58 (d, J = 1.8 Hz, 1H), 8.63 (d, J = 7.8 Hz, 1H), 8.46 (d, J = 7.6 Hz, 1H), 7.98 (d, J = 8.6 Hz, 2H), 7.65 (s, 1H), 7.62 (d, J = 8.6 Hz, 2H), 7.37 (d, J = 1.8 Hz, 1H), 7.08 (t, J = 8.0 Hz, 1H), 6.99 (d, J = 8.3 Hz, 1H), 6.50 (d, J = 7.6 Hz, 1H), 5.13 (d, J = 1.5 Hz, 1H), 5.07–5.23 (m, 1H), 4.44–4.52 (m, 2H), 3.87 (s, 3H), 3.04–3.14 (m, 2H), 2.28–2.38 (m, 1H), 2.06–2.26 (m, 1H), 1.97–2.06 (m, 1H), 1.51–1.78 (m, 5H), 0.94 (d, J = 6.3 Hz, 3H), 0.89 (d, J = 6.3 Hz, 3H); MS (ESI+) for C31H35ClN4O7m/z 611.1 (M + H)+.
T375	59114-59189	Sentence	denotes	Anal. calcd for C31H35ClN4O7·0.72 H2O·0.05 DCM: C, 59.36; H, 5.86; N, 8.92.
T376	59190-59224	Sentence	denotes	Found: C, 59.31; H, 5.99; N, 9.23.
T377	59226-59355	Sentence	denotes	(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 2,6-Dichlorobenzoate (22)
T378	59356-59626	Sentence	denotes	Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting 2,6-dichlorobenzoic acid and making noncritical variations provided a crude product.
T379	59627-59766	Sentence	denotes	This material was purified by LC (20 g 230–400 SiO2, 3% methanol/chloroform) to afford 114 mg (65%) of the title compound as a white solid.
T380	59767-60203	Sentence	denotes	1H NMR (DMSO-d6) δ 11.57 (d, J = 2 Hz, 1H), 8.62 (d, J = 8 Hz, 1H), 8.46 (d, J = 4 Hz, 1H), 7.65–7.53 (m, 4H), 7.36 (s, 1H), 7.08 (t, J = 8 Hz, 1H), 6.99 (d, J = 8 Hz, 1H), 6.49 (d, J = 8 Hz, 1H), 5.19 (d, J = 20 Hz, 1H), 5.15 (d, J = 20 Hz, 1H), 4.51 (m, 2H), 3.87 (s, 3H), 3.11 (m, 2H), 2.30 (m, 1H), 2.06 (m, 2H), 1.76–1.51 (m, 5H), 0.94 (d, J = 8 Hz, 3H), 0.89 (d, J = 8 Hz, 3H); MS (ESI+) for C31H34Cl2N4O7m/z 645.1 (M + H)+; Anal.
T381	60204-60264	Sentence	denotes	Calcd for C31H34Cl2N4O7·0.2 H2O: C, 57.36; H, 5.34; N, 8.63.
T382	60265-60299	Sentence	denotes	Found: C, 57.23; H, 5.55; N, 8.46.
T383	60300-60364	Sentence	denotes	HRMS (ESI+) calcd for C31H34Cl2N4O7+H1 645.1877, found 645.1871.
T384	60366-60495	Sentence	denotes	(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 2,6-Difluorobenzoate (23)
T385	60496-60775	Sentence	denotes	Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting 2,6-difluorobenzoic acid and making noncritical variations provided a crude brown oily solid.
T386	60776-60897	Sentence	denotes	This material was triturated with chloroform/ethyl acetate to afford 180 mg (75%) of the title compound as a white solid.
T387	60898-61420	Sentence	denotes	1H NMR (DMSO-d6) δ 11.65 (d, J = 2 Hz, 1H), 8.68 (d, J = 8 Hz, 1H), 8.54 (d, J = 8 Hz, 1H), 7.73–7.65 (m, 2H), 7.36 (s, 1H), 7.27 (t, J = 8 Hz, 1H), 7.08 (t, J = 8 Hz, 1H), 6.99 (d, J = 8 Hz, 1H), 6.49 (d, J = 8 Hz, 1H), 5.19 (d, J = 16 Hz, 1H), 5.14 (d, J = 16 Hz, 1H), 4.48 (m, 2H), 3.87 (s, 3H), 3.11 (m, 2H), 2.32 (m, 1H), 2.11–1.91 (m, 2H), 1.81–1.56 (m, 5H), 0.94 (d, J = 4 Hz, 3H), 0.89 (d, J = 4 Hz, 3H); MS (ESI+) for C31H34F2N4O7m/z 613.2 (M + H)+; HRMS (ESI+) calcd for C31H34F2N4O7+H1 613.2469, found 613.2476.
T388	61422-61557	Sentence	denotes	(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 4-Chloro-2-hydroxybenzoate (24)
T389	61558-61830	Sentence	denotes	Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting 4-chlorosalicylic acid and making noncritical variations provided a crude brown syrup.
T390	61831-61971	Sentence	denotes	This material was purified by Biotage MPLC (25 M, 2–3% methanol/dichloromethane) to afford 64 mg (25%) of the title compound as a tan solid.
T391	61972-62488	Sentence	denotes	1H NMR (DMSO-d6) δ 11.58 (s, 1H), 10.50 (bs, 1H), 8.65 (d, J = 8 Hz, 1H), 8.46 (d, J = 8 Hz, 1H), 7.81 (d, J = 8 Hz, 1H), 7.63 (m, 1H), 7.37 (s, 1H), 7.08 (m, 2H), 7.00 (m, 2H), 6.50 (d, J = 8 Hz, 1H), 5.16 (d, J = 16 Hz, 2H), 5.11 (d, J = 16 Hz, 2H), 4.49 (m, 2H), 3.87 (s, 3H), 3.13 (m, 2H), 2.32 (m, 1H), 2.11–1.98 (m, 2H), 1.76–1.55 (m, 5H), 0.94 (d, J = 6 Hz, 3H), 0.89 (d, J = 6 Hz, 3H); MS (ESI-) for C31H35ClN4O8m/z 625.1 (M – H)−; anal. calcd for C31H35ClN4O8·0.1 H2O·0.14 CHCl3: C, 57.93; H, 5.52; N, 8.68.
T392	62489-62587	Sentence	denotes	Found: C, 58.04; H, 5.78; N, 8.74; HRMS (ESI+) calcd for C31H35ClN4O8+H1 627.2216, found 627.2219.
T393	62589-62722	Sentence	denotes	(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 4-Cyano-2-fluorobenzoate (25)
T394	62723-63006	Sentence	denotes	Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting 4-cyano-2-fluorobenzoic acid and making noncritical variations provided a crude oily brown solid.
T395	63007-63160	Sentence	denotes	This material was purified by Biotage MPLC (25 M, 2.5–3.5% methanol/dichloromethane) to afford 119 mg (48%) of the title compound as an off-white powder.
T396	63161-63703	Sentence	denotes	1H NMR (DMSO-d6) δ 11.58 (d, J = 2 Hz, 1H), 8.63 (d, J = 8 Hz, 1H), 8.46 (d, J = 8 Hz, 1H), 8.06 (m, 2H), 7.83 (d, J = 8 Hz, 1H), 7.65 (s, 1H), 7.36 (d, J = 2 Hz, 1H), 7.08 (t, J = 8 Hz, 1H), 6.99 (d, J = 8 Hz, 1H), 6.50 (d, J = 8 Hz, 1H), 5.16 (d, J = 16 Hz, 1H), 5.11 (d, J = 16 Hz, 1H), 4.49 (m, 2H), 3.87 (s, 3H), 3.11 (m, 2H), 2.32 (m, 1H), 2.10–1.98 (m, 2H), 1.77–1.53 (m, 5H), 0.94 (d, J = 6 Hz, 3H), 0.89 (d, J = 6 Hz, 3H); MS (ESI+) for C32H34FN5O7m/z 620.1 (M + H)+; anal. calcd for C32H34FN5O7·0.3 H2O: C, 61.49; H, 5.58; N, 11.20.
T397	63704-63802	Sentence	denotes	Found: C, 61.47; H, 5.61; N, 10.98; HRMS (ESI+) calcd for C32H34FN5O7+H1 620.2515, found 620.2532.
T398	63804-63933	Sentence	denotes	(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 2,6-Dimethylbenzoate (26)
T399	63934-64207	Sentence	denotes	Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting 2,6-dimethylbenzoic acid and making noncritical variations provided a crude brown foam.
T400	64208-64356	Sentence	denotes	This material was purified by Biotage MPLC (25 M, 3–4% methanol/dichloromethane) to afford 177 mg (73%) of the title compound as an off-white glass.
T401	64357-64836	Sentence	denotes	1H NMR (DMSO-d6) δ 11.58 (d, J = 2 Hz, 1H), 8.63 (d, J = 8 Hz, 1H), 8.46 (d, J = 8 Hz, 1H), 7.65 (s, 1H), 7.36 (s, 1H), 7.25 (t, J = 8 Hz, 1H), 7.08 (m, 3H), 7.00 (d, J = 8 Hz, 1H), 6.49 (d, J = 8 Hz, 1H), 5.11 (s, 2H), 4.51 (m, 2H), 3.87 (s, 3H), 3.10 (m, 2H), 2.32–2.21 (m, 7H), 2.07 (m, 2H), 1.78–1.53 (m, 5H), 0.94 (d, J = 8 Hz, 3H), 0.89 (d, J = 8 Hz, 3H); MS (ESI+) for C33H40N4O7m/z 605.2 (M + H)+; anal. calcd for C33H40N4O7·0.3 H2O·0.2 CHCl3: C, 62.90; H, 6.49; N, 8.84.
T402	64837-64871	Sentence	denotes	Found: C, 62.95; H, 6.42; N, 8.72.
T403	64872-64933	Sentence	denotes	HRMS (ESI+) calcd for C33H40N4O7+H1 605.2970, found 605.2985.
T404	64935-65065	Sentence	denotes	(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 2,6-Dimethoxybenzoate (27)
T405	65066-65340	Sentence	denotes	Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting 2,6-dimethoxybenzoic acid and making noncritical variations provided a crude brown foam.
T406	65341-65489	Sentence	denotes	This material was purified by Biotage MPLC (25 M, 3–4% methanol/dichloromethane) to afford 169 mg (66%) of the title compound as an off-white solid.
T407	65490-66041	Sentence	denotes	1H NMR (DMSO-d6) δ 11.58 (d, J = 2 Hz, 1H), 8.55 (d, J = 8 Hz, 1H), 8.45 (d, J = 8 Hz, 1H), 7.64 (s, 1H), 7.40–7.35 (m, 2H), 7.08 (t, J = 8 Hz, 1H), 7.00 (d, J = 8 Hz, 1H), 6.71 (d, J = 8 Hz, 1H), 6.50 (d, J = 8 Hz, 1H), 5.01 (d, J = 16 Hz, 1H), 4.95 (d, J = 16 Hz, 1H), 4.53–4.48 (m, 2H), 3.87 (s, 3H), 3.75 (s, 6H), 3.08 (m, 2H), 2.31 (m, 1H), 2.11–1.90 (m, 2H), 1.75–1.55 (m, 5H), 0.94 (d, J = 8 Hz, 3H), 0.89 (d, J = 8 Hz, 3H); MS (ESI+) for C33H40N4O9m/z 637.2 (M + H)+; anal. calcd for C33H40N4O9·0.25 H2O·0.25 CHCl3: C, 59.51; H, 6.12; N, 8.35.
T408	66042-66076	Sentence	denotes	Found: C, 59.49; H, 6.08; N, 8.42.
T409	66078-66202	Sentence	denotes	(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 2-Cyanobenzoate (28)
T410	66203-66477	Sentence	denotes	Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting 2-cyanobenzoic acid and making noncritical variations provided a crude brown oily solid.
T411	66478-66598	Sentence	denotes	This material was triturated with chloroform/ethyl acetate to afford 91 mg (38%) of the title compound as a white solid.
T412	66599-67087	Sentence	denotes	1H NMR (DMSO-d6) δ 11.58 (s, 1H), 8.65 (d, J = 8 Hz, 1H), 8.46 (d, J = 8 Hz, 1H), 8.17 (m, 1H), 8.04 (m, 1H), 7.88 (m, 1H), 7.65 (s, 1H), 7.37 (s, 1H), 7.08 (t, J = 8 Hz, 1H), 6.99 (d, J = 8 Hz, 1H), 6.49 (d, J = 8 Hz, 1H), 5.19 (m, 2H), 4.50 (m, 2H), 3.87 (s, 3H), 3.11 (m, 2H), 2.32 (m, 1H), 2.07–1.97 (m, 2H), 1.76–1.56 (m, 5H), 0.94 (d, J = 6 Hz, 3H), 0.89 (d, J = 6 Hz, 3H); MS (ESI+) for C32H35N5O7m/z 602.2 (M + H)+; anal. calcd for C32H35N5O7·0.4 H2O: C, 63.12, H, 5.93; N, 11.50.
T413	67088-67185	Sentence	denotes	Found: C, 63.16; H, 5.96; N, 11.43; HRMS (ESI+) calcd for C32H35N5O7+H1 602.2609, found 602.2610.
T414	67187-67284	Sentence	denotes	(3S)-3-[(tert-Butoxycarbonyl)amino]-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 2,6-Dichlorobenzoate
T415	67285-67526	Sentence	denotes	A solution of tert-butyl ((1S)-3-bromo-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)carbamate (1.17 g, 3.2 mmol) in DMF (16 mL) was treated with 2,6-dichlorobenzoic acid (794 mg, 4.2 mmol) followed by cesium fluoride (1.18 g, 7.4 mmol).
T416	67527-67604	Sentence	denotes	The resulting suspension was placed in a preheated oil bath at 65 °C for 2 h.
T417	67605-67822	Sentence	denotes	The reaction was cooled to an ambient temperature, diluted with ethyl acetate (100 mL), washed once with water (40 mL) and with brine (40 mL), dried over MgSO4, filtered, and concentrated to give a crude yellow syrup.
T418	67823-67972	Sentence	denotes	This material was purified by Biotage MPLC (25 M, 4.5% methanol/dichloromethane) to afford 1.19 mg (80%) of the title compound as an off-white glass.
T419	67973-68238	Sentence	denotes	1H NMR (400 MHz, DMSO-d6) δ 7.60 (m, 5H), 5.35 (s, 2H), 4.32 (m, 1H), 3.23 (m, 2H), 2.24 (m, 1H), 2.11 (m, 1H), 1.86 (m, 1H), 1.67 (m, 2H), 1.35 (s, 9H); MS (ESI+) for C20H24Cl2N2O6m/z 481.0 (M + Na)+; HRMS (ESI+) calcd for C20H24Cl2N2O6+H 481.0903, found 481.0890.
T420	68240-68347	Sentence	denotes	N2-(tert-Butoxycarbonyl)-N1-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-l-leucinamide
T421	68348-68557	Sentence	denotes	A solution of (3S)-3-[(2S)-2-amino-4-chloro-3-oxobutyl]pyrrolidin-2-one hydrochloride (391 mg, 1.6 mmol) and Boc-Leu-OH (412 mg, 1.8 mmol) in DMF (9 mL) was placed under an atmosphere of N2 and cooled to 0 °C.
T422	68558-68695	Sentence	denotes	This clear pale yellow solution was successively treated with HATU (678 mg, 1.8 mmol) followed by N-methylmorpholine (0.41 mL, 3.7 mmol).
T423	68696-68861	Sentence	denotes	The reaction mixture gradually became opaque and after 1 h was quenched with 1:1 ice/sat aqueous NaHCO3 (40 mL) and extracted three times with ethyl acetate (40 mL).
T424	68862-68989	Sentence	denotes	The combined organics were washed once with brine (30 mL), dried over MgSO4, filtered, and concentrated to give a yellow syrup.
T425	68990-69219	Sentence	denotes	This material was purified by LC (50 g 230–400 SiO2, 3–5% methanol/chloroform) to afford 636 mg (40%) of N2-(tert-butoxycarbonyl)-N1-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-l-leucinamide as a white foam.
T426	69220-69577	Sentence	denotes	1H NMR (DMSO-d6) δ 8.47 (d, J = 8 Hz, 1H), 7.64 (s, 1H), 7.04 (d, J = 8 Hz, 1H), 4.60 (d, J = 16 Hz, 1H), 4.53 (d, J = 16 Hz, 1H), 4.40 (m, 1H), 3.90 (m, 1H), 3.16 (m, 1H), 3.08 (m, 1H), 2.24 (m, 1H), 2.10 (m, 1H), 1.98 (m, 1H), 1.63 (m, 2H), 1.45–1.37 (m, 11 H), 0.89 (d, J = 4 Hz, 3H), 0.85 (d, J = 4 Hz, 3H); MS (ESI+) for C19H32ClN3O5m/z 418.1 (M + H)+.
T427	69578-69643	Sentence	denotes	Anal. calcd for C19H32ClN3O5·0.6 H2O: C, 53.22; H, 7.81; N, 9.80.
T428	69644-69678	Sentence	denotes	Found: C, 53.00; H, 7.65; N, 9.54.
T429	69679-69742	Sentence	denotes	HRMS (ESI+) calcd for C19H32ClN3O5+H1 418.2103, found 418.2091.
T430	69744-69859	Sentence	denotes	(3S)-3-{[N-(tert-Butoxycarbonyl)-l-leucyl]amino}-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 2,6-Dichlorobenzoate (29)
T431	69860-70196	Sentence	denotes	Following the procedure described for the preparation of N2-(tert-butoxycarbonyl)-N1-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-l-leucinamide but substituting (3S)-3-[(tert-butoxycarbonyl)amino]-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 2,6-dichlorobenzoate and making noncritical variations provided a brown foam.
T432	70197-70348	Sentence	denotes	This material was purified by LC (50 g 230–400 SiO2, 2.5–3.5% methanol/chloroform) to afford 413 mg (61%) of the title compound as a light yellow foam.
T433	70349-70707	Sentence	denotes	1H NMR (DMSO-d6) δ 8.47 (d, J = 8 Hz, 1H), 7.60 (m, 4H), 7.05 (d, J = 8 Hz, 1H), 5.19 (d, J = 16 Hz, 1H), 5.13 (d, J = 16 Hz, 1H), 4.47 (m, 1H), 3.95 (m, 1H), 3.16 (m, 1H), 3.08 (m, 1H), 2.27 (m, 1H), 2.10 (m, 1H), 2.03 (m, 1H), 1.65 (m, 2H), 1.47–1.37 (m, 11 H), 0.89 (d, J = 4 Hz, 3H), 0.85 (d, J = 4 Hz, 3H); MS (ESI+) for C26H35Cl2N3O7m/z 572.1 (M + H)+.
T434	70708-70774	Sentence	denotes	Anal. calcd for C26H35Cl2N3O7·0.5 H2O: C, 53.70; H, 6.24; N, 7.23.
T435	70775-70874	Sentence	denotes	Found: C, 53.74; H, 6.31; N, 7.31; HRMS (ESI+) calcd for C26H35Cl2N3O7+Na 594.1744, found 594.1729.
T436	70876-70976	Sentence	denotes	(3S)-3-[(N-Acetyl-l-leucyl)amino]-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 2,6-Dichlorobenzoate (30)
T437	70977-71313	Sentence	denotes	Following the procedure described for the preparation of N2-acetyl-N1-[(1S)-1-(chloroacetyl)-4-(dimethylamino)-4-oxobutyl]-N2-methyl-l-leucinamide but substituting (3S)-3-{[N-(tert-butoxycarbonyl)-l-leucyl]amino}-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 2,6-dichlorobenzoate and making noncritical variations provided a pale yellow oil.
T438	71314-71460	Sentence	denotes	This material was purified by Biotage MPLC (25 M, 3.5–4.5% methanol/dichloromethane) to afford 161 mg (78%) of the title compound as a white foam.
T439	71461-71895	Sentence	denotes	1H NMR (DMSO-d6) δ 8.56 (d, J = 8 Hz, 1H), 8.06 (d, J = 8 Hz, 1H), 7.65–7.53 (m, 4H), 5.17 (d, J = 16 Hz, 1H), 5.10 (d, J = 16 Hz, 1H), 4.44 (m, 1H), 4.25 (m, 1H), 3.12 (m, 2H), 2.25 (m, 1H), 2.06 (m, 1H), 1.97 (m, 1H), 1.84 (s, 3H), 1.63 (m, 3H), 1.45 (m, 2H), 0.90 (d, J = 8 Hz, 3H), 0.85 (d, J = 8 Hz, 3H); MS (ESI+) for C23H29Cl2N3O6m/z 514.0 (M + H)+; anal. calcd for C23H29Cl2N3O6·0.25 H2O·0.1 EtOAc: C, 53.26; H, 5.79; N, 7.96.
T440	71896-71930	Sentence	denotes	Found: C, 53.27; H, 5.82; N, 7.84.
T441	71931-71995	Sentence	denotes	HRMS (ESI+) calcd for C23H29Cl2N3O6+H1 514.1506, found 514.1508.
T442	71997-72121	Sentence	denotes	(3S)-3-{[N-(1H-Benzimidazol-2-ylcarbonyl)-l-leucyl]amino}-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 2,6-Dichlorobenzoate (31)
T443	72122-72550	Sentence	denotes	Following the procedure described for the preparation of N-((1S)-1-{[((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-1H-indole-2-carboxamide but substituting (3S)-3-{[N-(tert-butoxycarbonyl)-l-leucyl]amino}-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 2,6-dichlorobenzoate and 1H-benzimidazole-2-carboxylic acid and making noncritical variations provided a crude yellow oil.
T444	72551-72697	Sentence	denotes	This material was purified by Biotage MPLC (25 M, 3–4% methanol/dichloromethane) to afford 95 mg (72%) of the title compound as an off-white foam.
T445	72698-73060	Sentence	denotes	1H NMR (DMSO-d6) δ 13.32 (s, 1H), 8.89 (d, J = 8 Hz, 1H), 8.60 (d, J = 8 Hz, 1H), 7.74 (d, J = 8 Hz, 1H), 7.59 (m, 5H), 7.29 (m, 2H), 5.20 (d, J = 20 Hz, 1H), 5.15 (d, J = 20 Hz, 1H), 4.56 (m, 2H), 3.12 (m, 2H), 2.30 (m, 1H), 2.01 (m, 2H), 1.83 (m, 1H), 1.65 (m, 4H), 0.92 (d, J = 8 Hz, 3H), 0.89 (d, J = 8 Hz, 3H); MS (ESI+) for C29H31Cl2N5O6m/z 616.0 (M + H)+.
T446	73061-73120	Sentence	denotes	Anal. calcd for C29H31Cl2N5O6: C, 56.50; H, 5.07; N, 11.36.
T447	73121-73156	Sentence	denotes	Found: C, 57.28; H, 5.32; N, 11.35.
T448	73157-73221	Sentence	denotes	HRMS (ESI+) calcd for C29H31Cl2N5O6+H1 616.1724, found 616.1729.
T449	73223-73360	Sentence	denotes	(3S)-3-({4-Methyl-N-[(2R)-tetrahydrofuran-2-ylcarbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 2,6-Dichlorobenzoate (3)
T450	73361-73772	Sentence	denotes	Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting N1-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-4-methyl-N2-[(2R)-tetrahydrofuran-2-ylcarbonyl]-l-leucinamide and 2,6-dichlorobenzoic acid and making noncritical variations provided a light amber residue.
T451	73773-73985	Sentence	denotes	The residue was purified by preparative HPLC (Luna 10μ C18) eluting with a gradient of MeCN containing 0.1% AcOH in water containing 0.1% AcOH to give 0.155 g (54%) of the title compound as a cream colored solid.
T452	73986-74351	Sentence	denotes	1H NMR (300 MHz, DMSO-d6) δ 8.52 (d, J = 8 Hz, 1H), 7.79 (d, J = 8 Hz, 1H), 7.71 (s, 1H), 7.66–7.55 (m, 3H), 5.18 (s, 2H), 4.54–4.40 (m, 1H), 4.37–4.35 (m, 1H), 4.25 (m, 1H), 3.98–3.91 (m, 1H), 3.84–3.72 (m, 1H), 3.23–3.07 (m, 2H), 2.33–2.23 (m, 1H), 2.16–2.05 (m, 2H), 1.86–1.74 (m, 3H), 1.72–1.62 (m, 5H), 0.89 (s, 9H); MS (ESI+) for C27H35Cl2N3O7m/z 584 (M + H).
T453	74352-74418	Sentence	denotes	Anal. calcd for C27H35Cl2N3O7·0.5 H2O: C, 54.64; H, 6.11; N, 7.08.
T454	74419-74453	Sentence	denotes	Found: C, 54.26; H, 6.00; N, 6.87.
T455	74454-74518	Sentence	denotes	HRMS (ESI+) calcd for C27H35Cl2N3O7+H1 584.1925, found 584.1921.
T456	74520-74641	Sentence	denotes	N2-(tert-Butoxycarbonyl)-N1-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-4-methyl-l-leucinamide (32)
T457	74642-74905	Sentence	denotes	Following the procedure described for the preparation of N2-(tert-butoxycarbonyl)-N1-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-l-leucinamide but substituting Boc-(β t-butyl)-Ala-OH and making noncritical variations provided a golden syrup.
T458	74906-75053	Sentence	denotes	This material was purified by Biotage MPLC (65i column, 2.5–3.5% methanol/chloroform) to afford 3.41 g (40%) of the title compound as a white foam.
T459	75054-75404	Sentence	denotes	1H NMR (DMSO-d6) δ 8.39 (d, J = 8 Hz, 1H), 7.64 (s, 1H), 7.00 (d, J = 8 Hz, 1H), 4.58 (d, J = 16 Hz, 1H), 4.52 (d, J = 16 Hz, 1H), 4.38 (m, 1H), 3.92 (m, 1H), 3.15 (t, J = 8 Hz, 1H), 3.07 (q, J = 8 Hz, 1H), 2.22 (m, 1H), 2.09 (m, 1H), 1.97 (m, 1H), 1.62 (m, 2H), 1.50 (m, 2H), 1.36 (s, 9H), 0.88 (s, 9H); MS (ESI+) for C20H34ClN3O6m/z 432.1 (M + H)+.
T460	75406-75532	Sentence	denotes	N1-((1S)-3-(Benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-N2-(tert-butoxycarbonyl)-4-methyl-l-leucinamide (33)
T461	75533-75826	Sentence	denotes	Following the procedure described for the preparation of tert-butyl [(1S)-2-[((1S)-3-(benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]-1-(cyclohexylmethyl)-2-oxoethyl]carbamate but substituting Boc-(β t-butyl)-Ala-OH and making noncritical variations provided a brown syrup.
T462	75827-75976	Sentence	denotes	This material was purified by LC (33 g 230–400 SiO2, 1–2.5% methanol/chloroform) to afford 258 mg (55%) of the title compound as a light yellow foam.
T463	75977-76299	Sentence	denotes	1H NMR (DMSO-d6) δ 8.23 (d, J = 8 Hz, 1H), 7.62 (s, 1H), 7.32 (m, 5H), 6.96 (d, J = 8 Hz, 1H), 4.46 (s, 2H), 4.35–4.33 (m, 2H), 4.21 (d, J = 16 Hz, 1H), 3.95 (m, 1H), 3.09 (m, 2H), 2.24 (m, 1H), 2.07 (m, 1H), 1.92 (m, 1H), 1.58 (m, 2H), 1.46 (m, 2H), 1.35 (s, 9H), 0.87 (m, 9H); MS (ESI+) for C27H41N3O6m/z 504.2 (M + H)+.
T464	76301-76456	Sentence	denotes	N-((1S)-1-{[((1S)-3-(Benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3,3-dimethylbutyl)-4-methoxy-1H-indole-2-carboxamide (34)
T465	76457-76866	Sentence	denotes	Following the procedure described for the preparation of N-[(1S)-2-[((1S)-3-(benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]-1-(cyclohexylmethyl)-2-oxoethyl]-4-methoxy-1H-indole-2-carboxamide but substituting N1-((1S)-3-(benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-N2-(tert-butoxycarbonyl)-4-methyl-l-leucinamide and making noncritical variations provided a golden syrup.
T466	76867-77014	Sentence	denotes	This material was purified by Biotage MPLC (25 M column, 3% methanol/chloroform) to afford 173 mg (60%) of the title compound as an off-white foam.
T467	77015-77469	Sentence	denotes	1H NMR (400 MHz, DMSO-d6) δ 11.58 (s, 1H), 8.43 (app t, J = 8 Hz, 2H), 7.62 (s, 1H), 7.32 (m, 6H), 7.08 (t, J = 8 Hz, 1H), 7.00 (d, J = 8 Hz, 1H), 6.49 (d, J = 8 Hz, 1H), 4.52 (m, 1H), 4.47–4.35 (m, 4H), 4.22 (d, J = 16 Hz, 1H), 3.87 (s, 3H), 3.05 (m, 2H), 2.28 (m, 1H), 2.06 (m, 1H), 1.93 (m, 1H), 1.64–1.57 (m, 3H), 0.92 (s, 9H); MS (ESI+) for C32H40N4O6m/z 577.2 (M + H)+; anal. calcd for C32H40N4O6·0.33 methanol·0.25 H2O: C, 65.62; H, 7.13; N, 9.47.
T468	77470-77563	Sentence	denotes	Found: C, 65.65; H, 6.90; N, 9.58; HRMS (ESI+) calcd for C32H40N4O6 577.3021, found 577.3001.
T469	77565-77715	Sentence	denotes	N-((1S)-1-{[((1S)-3-Chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3,3-dimethylbutyl)-4-methoxy-1H-indole-2-carboxamide (35)
T470	77716-78112	Sentence	denotes	Following the procedure described for the preparation of N-((1S)-1-{[((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-1H-indole-2-carboxamide but substituting N2-(tert-butoxycarbonyl)-N1-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-4-methyl-l-leucinamide and making noncritical variations provided a pale yellow solid.
T471	78113-78264	Sentence	denotes	This material was purified by Biotage MPLC (40 M column, 2–4.5% methanol/chloroform) to afford 920 g (60%) of the title compound as an off-white solid.
T472	78265-78786	Sentence	denotes	1H NMR (400 MHz, DMSO-d6) δ 11.59 (d, J = 1.8 Hz, 1H), 8.54 (d, J = 7.8 Hz, 1H), 8.45 (d, J = 8.1 Hz, 1H), 7.64 (s, 1H), 7.32 (d, J = 1.8 Hz, 1H), 7.08 (t, J = 8.0 Hz, 1H), 7.00 (d, J = 8.3 Hz, 1H), 6.49 (d, J = 7.6 Hz, 1H), 4.52–4.62 (m, 1H), 4.40–4.51 (m, 2H), 3.87 (s, 3H), 3.01–3.16 (m, 2H), 2.19–2.31 (m, 1H), 2.03–2.12 (m, 1H), 1.91–2.01 (m, 1H), 1.81 (dd, J = 14.1, 9.9 Hz, 1H), 1.54–1.72 (m, 3H), 0.93 (s, 9H); MS (ESI+) for C25H33ClN4O5m/z 505.1 (M + H)+; HRMS (ESI+) calcd for (M + H)+ 505.2212, found 505.2204.
T473	78788-78939	Sentence	denotes	N-((1S)-1-{[((1S)-3-Hydroxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3,3-dimethylbutyl)-4-methoxy-1H-indole-2-carboxamide (36)
T474	78940-79366	Sentence	denotes	Following the procedure described for the preparation of N-((1S)-1-{[((1S)-3-hydroxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-1H-indole-2-carboxamide but substituting N-((1S)-1-{[((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3,3-dimethylbutyl)-4-methoxy-1H-indole-2-carboxamide and making noncritical variations provided a crude brown glass.
T475	79367-79513	Sentence	denotes	This material was purified by Biotage MPLC (40 M column, 2–10% methanol/chloroform) to afford 415 mg (53%) of the title compound as a white solid.
T476	79514-79557	Sentence	denotes	MS (ESI+) for C25H34N4O6m/z 487.2 (M + H)+.
T477	79558-80004	Sentence	denotes	Alternatively, following the procedure described for the preparation of N-{(1S)-1-(cyclohexylmethyl)-2-[((1S)-3-hydroxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]-2-oxoethyl}-4-methoxy-1H-indole-2-carboxamide but substituting N-((1S)-1-{[((1S)-3-(benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3,3-dimethylbutyl)-4-methoxy-1H-indole-2-carboxamide and making noncritical variations provided a tan solid.
T478	80005-80150	Sentence	denotes	This material was purified by Biotage MPLC (25 M column, 6–7% methanol/chloroform) to afford 100 mg (77%) of the title compound as a white solid.
T479	80151-80664	Sentence	denotes	1H NMR (DMSO-d6) δ 11.58 (s, 1H), 8.42 (d, J = 8 Hz, 1H), 8.37 (d, J = 8 Hz, 1H), 7.62 (s, 1H), 7.32 (s, 1H), 7.08 (t, J = 8 Hz, 1H), 6.99 (d, J = 8 Hz, 1H), 6.49 (d, J = 8 Hz, 1H), 5.04 (t, J = 8 Hz, 1H), 4.52 (m, 1H), 4.42 (m, 1H), 4.24 (dd, J = 8, 20 Hz, 1H), 4.12 (dd, J = 8, 20 Hz, 1H), 3.87 (s, 3H), 3.09 (m, 2H), 2.25 (m, 1H), 2.06 (m, 1H), 1.93 (m, 1H), 1.80 (dd, J = 8, 16 Hz, 1H), 0.93 (s, 9H); MS (ESI+) for C25H34N4O6m/z 487.2 (M + H)+; anal. calcd for C25H34N4O6·0.3 H2O: C, 61.03; H, 7.09; N, 11.39.
T480	80665-80759	Sentence	denotes	Found: C, 61.07; H, 7.09; N, 11.22; HRMS (ESI+) calcd for C25H34N4O6 487.2551, found 487.2541.
T481	80761-80912	Sentence	denotes	4-Methoxy-N-((1S)-1-{[((1S)-3-methoxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3,3-dimethylbutyl)-1H-indole-2-carboxamide (37)
T482	80913-81337	Sentence	denotes	Following the procedure described for the preparation of 4-methoxy-N-((1S)-1-{[((1S)-3-methoxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-4-methylpentyl)-1H-indole-2-carboxamide but substituting N-((1S)-1-{[((1S)-3-hydroxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3,3-dimethylbutyl)-4-methoxy-1H-indole-2-carboxamide and making noncritical variations provided a crude product.
T483	81338-81483	Sentence	denotes	This material was purified by Biotage MPLC (40S column, 2–5% methanol/chloroform) to afford 21 mg (9%) of the title compound as a pale tan solid.
T484	81484-81999	Sentence	denotes	1H NMR (400 MHz, DMSO-d6) δ 11.58 (s, 1H), 8.42 (d, J = 6.8 Hz, 1H), 7.62 (s, 1H), 7.32 (s, 1H), 7.08 (t, J = 7.7 Hz, 1H), 7.00 (d, J = 7.8 Hz, 1H), 6.49 (d, J = 7.3 Hz, 1H), 4.49–4.55 (m, 1H), 4.32–4.41 (m, 1H), 4.21–4.30 (m, 1H), 4.12 (t, J = 16.9 Hz, 1H), 3.87 (s, 3H), 3.23 (s, 3H), 3.01–3.16 (m, 2H), 2.20–2.32 (m, 1H), 2.03–2.15 (m, 1H), 1.87–1.98 (m, 1H), 1.73–1.85 (m, 1H), 1.54–1.70 (m, 3H), 0.93 (s, 9H); MS (ESI+) for C26H36N4O6m/z 501.2 (M + H)+; HRMS (ESI+) calcd for (M + H)+ 501.2708, found 501.2701.
T485	82001-82147	Sentence	denotes	N-((1S)-1-{[((1S)-3-Hydroxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-1H-indole-2-carboxamide (4)
T486	82148-82419	Sentence	denotes	A solution of N-((1S)-1-{[((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-1H-indole-2-carboxamide (488 mg, 0.99 mmol) and benzoylformic acid (195 mg, 1.3 mmol) in DMF (6.5 mL) was placed under an atmosphere of N2.
T487	82420-82533	Sentence	denotes	This clear pale yellow solution was treated with cesium fluoride (350 mg, 2.3 mmol) followed by heating to 65 °C.
T488	82534-82891	Sentence	denotes	After 4 h, the now yellow suspension was cooled to RT, diluted with ethyl acetate (60 mL), washed three times with water (30 mL) and once with brine (30 mL), dried over MgSO4, filtered, and concentrated to give (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl oxo(phenyl)acetate as a crude yellow foam.
T489	82892-82935	Sentence	denotes	MS (ESI+) for C32H36N4O8m/z 605.2 (M + H)+.
T490	82936-83218	Sentence	denotes	A solution of the crude (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl oxo(phenyl)acetate in methanol (40 mL) was placed under an atmosphere of N2 and treated with potassium carbonate (7 mg, 0.05 mmol) with vigorous stirring.
T491	83219-83310	Sentence	denotes	After 1 h, the volatiles were removed in vacuo (bath < 30 °C) to give a crude yellow glass.
T492	83311-83459	Sentence	denotes	This material was purified by Biotage MPLC (25 M column, 6% methanol/chloroform) to afford 346 mg (73%) of the title compound as an off-white solid.
T493	83460-83918	Sentence	denotes	1H NMR (DMSO-d6) δ 11.56 (s, 1H), 8.44 (d, J = 8 Hz, 1H), 8.39 (d, J = 8 Hz, 1H), 7.61 (s, 1H), 7.35 (s, 1H), 7.08 (t, J = 8 Hz, 1H), 6.99 (d, J = 8 Hz, 1H), 6.49 (d, J = 8 Hz, 1H), 5.04 (t, J = 8 Hz, 1H), 4.46 (m, 2H), 4.25 (dd, J = 8, 20 Hz, 1H), 4.13 (dd, J = 8, 20 Hz, 1H), 3.87 (s, 3H), 3.10 (m, 2H), 2.28 (m, 1H), 2.08 (m, 1H), 1.92 (m, 1H), 1.70–1.53 (m, 5H), 0.93 (d, J = 8 Hz, 3H), 0.89 (d, J = 8 Hz, 3H); MS (ESI+) for C24H32N4O6m/z 473.2 (M + H)+.
T494	83920-84042	Sentence	denotes	N1-((1S)-3-(Benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-N2-(tert-butoxycarbonyl)-N2-methyl-l-leucinamide
T495	84043-84334	Sentence	denotes	Following the procedure described for the preparation of tert-butyl [(1S)-2-[((1S)-3-(benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]-1-(cyclohexylmethyl)-2-oxoethyl]carbamate but substituting Boc-N-methyl-Leu and making noncritical variations provided a crude brown oil.
T496	84335-84492	Sentence	denotes	This material was purified by Biotage flash chromatography, eluting with methanol/dichloromethane to afford the title compound (123 mg, 47%) as a yellow gum.
T497	84493-84756	Sentence	denotes	1H NMR (400 MHz, CDCl3) δ 7.22–7.33 (m, 5H), 5.76 (bd, 1H), 4.73 (s, 1H), 4.46–4.59 (m, 2H), 4.08–4.27 (m, 2H), 3.14–3.28 (m, 2H), 2.68 (s, 3H), 2.16–2.38 (m, 2H), 1.88–2.00 (m, 1H), 1.56–1.86 (m, 5H), 1.41 (s, 9H), 0.94 (d, J = 8 Hz, 3H), 0.89 (d, J = 8 Hz, 3H).
T498	84758-84913	Sentence	denotes	N-((1S)-1-{[((1S)-3-(Benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-N-methyl-1H-indole-2-carboxamide
T499	84914-85327	Sentence	denotes	Following the procedure described for the preparation of N-[(1S)-2-[((1S)-3-(benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]-1-(cyclohexylmethyl)-2-oxoethyl]-4-methoxy-1H-indole-2-carboxamide but substituting N1-((1S)-3-(benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-N2-(tert-butoxycarbonyl)-N2-methyl-l-leucinamide and making noncritical variations provided a crude brown oil.
T500	85328-85482	Sentence	denotes	This material was purified by Biotage flash chromatography, eluting with methanol/dichloromethane to afford the title compound as a clear oil, 67 mg, 48%.

T1

Sentence

denotes

Discovery of Ketone-Based Covalent Inhibitors of Coronavirus 3CL Proteases for the Potential Therapeutic Treatment of COVID-19

T2

128-136

Sentence

denotes

Abstract

T3

137-349

Sentence

denotes

The novel coronavirus disease COVID-19 that emerged in 2019 is caused by the virus SARS CoV-2 and named for its close genetic similarity to SARS CoV-1 that caused severe acute respiratory syndrome (SARS) in 2002.

T4

350-584

Sentence

denotes

Both SARS coronavirus genomes encode two overlapping large polyproteins, which are cleaved at specific sites by a 3C-like cysteine protease (3CLpro) in a post-translational processing step that is critical for coronavirus replication.

T5

585-711

Sentence

denotes

The 3CLpro sequences for CoV-1 and CoV-2 viruses are 100% identical in the catalytic domain that carries out protein cleavage.

T6

712-942

Sentence

denotes

A research effort that focused on the discovery of reversible and irreversible ketone-based inhibitors of SARS CoV-1 3CLpro employing ligand-protease structures solved by X-ray crystallography led to the identification of 3 and 4.

T7

943-1140

Sentence

denotes

Preclinical experiments reveal 4 (PF-00835231) as a potent inhibitor of CoV-2 3CLpro with suitable pharmaceutical properties to warrant further development as an intravenous treatment for COVID-19.

T8

1142-1154

Sentence

denotes

Introduction

T9

1155-1253

Sentence

denotes

In late 2019, cases of an unknown respiratory tract infection were first reported in Wuhan, China.

T10

1254-1708

Sentence

denotes

By February 2020, the novel coronavirus SARS CoV-2 was identified as the causative agent for COVID-19.1,2 Genome analysis of this virus revealed a high similarity to SARS CoV-1, the coronavirus that caused severe acute respiratory syndrome (SARS) in 2002–2003.2−4 Like SARS CoV-1, which resulted in 799 deaths among the 8464 probable cases,5 SARS CoV-2 can induce fever, coughing, and difficulty breathing that rapidly becomes more serious in some cases.

T11

1709-2092

Sentence

denotes

The spread of SARS CoV-2 has been more extensive than that of SARS CoV-1, causing a global pandemic with the current number of worldwide infections surpassing eight million and deaths surpassing 400,000.6 The SARS CoV-1 genome encodes for two large polyproteins pp1a (∼450 kDa) and pp1ab (∼750 kDa) that contain overlapping sequences and include a 3C-like cysteine protease (3CLpro).

T12

2093-2441

Sentence

denotes

The function of this internally encoded 3CLpro is integral to the processing of these proteins and critical for viral replication.7 The SARS CoV-1 3CLpro shares a high degree of structural homology and similar substrate specificity with the coronavirus 3C-like cysteine proteases of hCoV 229E and TGEV8 but is most similar to the SARS CoV-2 3CLpro.

T13

2442-2827

Sentence

denotes

Specifically, the SARS CoV-1 and SARS CoV-2 share 96% identity between their respective 3CLpro sequences and 100% identity in the active site.8 A recent report by Dai et al. demonstrates that crystallographic information and structure–activity relationships obtained with the SARS CoV-1 3CLpro could facilitate the design of potent SARS CoV-2 3CLpro inhibitors with antiviral potency.9

T14

2828-3210

Sentence

denotes

There are numerous reports of reversible cysteine protease inhibitors, which include aldehydes,10−13 thio- or oxymethylketones,14 cyclic ketones,15 amidomethylketones,16 nitriles,17,18 or various 1,2-dicarbonyl motifs.19,20 The electrophilic carbon of these chemotypes reacts reversibly with the sulfur atom of an active-site cysteine forming a covalently bound tetrahedral complex.

T15

3211-3624

Sentence

denotes

Stabilization of this charged protein–ligand transition state by an “oxyanion hole” present in the active site has been observed with certain inhibitor classes by X-ray crystallography and NMR.21,22 A recent report describes the optimization of a series of peptidomimetics designed with an α-ketoamide warhead, which achieves broad-spectrum inhibition against the Mpro for several coronaviruses and enteroviruses.

T16

3625-3843

Sentence

denotes

Importantly, the reported protease potencies correlate to antiviral potencies, and the authors suggest that the reported SARS CoV-1 3CLpro activity of their lead could be predictive of the SARS CoV-2 3CLpro activity.23

T17

3844-3928

Sentence

denotes

Alternative modes of enzyme inhibition are covalently bound irreversible inhibitors.

T18

3929-4571

Sentence

denotes

While chemically reactive affinity labels such as chloromethylketones (CMKs) have demonstrated potent protease inhibition and in certain instances in vivo activity, their inherent chemical reactivity precludes development as clinical agents due to safety concerns.24 However, acyloxymethylketones first reported by Krantz as “quiescent affinity labels” for cathepsin B have demonstrated potent levels of enzyme inhibition with relatively low chemical reactivity.25,26 Mechanistic studies of this class of inhibitors with the cysteine protease caspase-1 support the intermediacy of a thiohemiketal arising from the initial 1,2-carbonyl attack.

T19

4572-4838

Sentence

denotes

Orientation and stabilization of the acyloxy leaving group within the S1′ domain (Figure 1) of the protease can facilitate SN2 displacement via sulfur migration, resulting in irreversible or bimodal (reversible inhibition followed by slow inactivation) inhibition.27

T20

4839-4903

Sentence

denotes

Figure 1 Subsite nomenclature for proteolytic enzymes is shown.

T21

4904-5057

Sentence

denotes

Amino acid residues to the left of the polypeptide scissile amide bond are numbered sequentially, beginning with P1 and increasing toward the N-terminus.

T22

5058-5195

Sentence

denotes

Amino acid residues to the right of the scissile bond are numbered sequentially, beginning with P1′ and increasing toward the C-terminus.

T23

5196-5283

Sentence

denotes

Complimentary regions of the protease active site employ the corresponding S numbering.

T24

5284-5565

Sentence

denotes

Following the SARS outbreak, a CoV-1 3CLpro homology model was published in 2003 with comparisons made to the human rhinovirus (HRV) 3Cpro.28 Although these two proteases share very little sequence conservation, the substrate consensus sequences have a Gln residue in common at P1.

T25

5566-5787

Sentence

denotes

Analysis of the HRV 3Cpro crystal structure with 1 (rupintrivir; Figure 2) revealed binding interactions that are like those observed for 1 bound to TGEV 3CLpro, which was used in the construction of their homology model.

T26

5788-5937

Sentence

denotes

The authors concluded that Michael acceptor 1 would serve as a starting point to expedite the identification of a potent SARS CoV-1 3CLpro inhibitor.

T27

5938-6193

Sentence

denotes

Figure 2 HRV clinical candidate rupintrivir and SARS CoV-1 3CLpro inhibitor 2 employing Michael acceptor-based warheads along with optimized SARS CoV-1 3CLpro inhibitors 3 and 4 containing a carbonyl designed for attack by the catalytic cysteine residue.

T28

6194-6343

Sentence

denotes

Performing a similar analysis, we tested 1 and additional HRV 3Cpro inhibitors that displayed very weak to unmeasurable SARS CoV-1 3CLpro inhibition.

T29

6344-6518

Sentence

denotes

Concurrently, new Michael acceptor derivatives were designed that tested P1/P2 alterations and truncation of the P3/P4 binding motif to optimize SARS CoV-1 3CLpro inhibition.

T30

6519-6908

Sentence

denotes

Compound 2, a derivative that contains an indole capping group at P2, displayed modest levels of irreversible inhibition (kobs/I = 586 ± 11 M–1 s–1) that enabled cocrystallization in complex with SARS CoV-1 3CLpro (PDB code 6XHO).29 Comparison of this structure with the complex of 1 in HRV 3Cpro helped to rationalize the poor performance of the warhead, which had been successful in HRV.

T31

6909-7058

Sentence

denotes

The structure of the SARS CoV-1 3CLpro complex revealed an eclipsed torsion about the resulting sp3 α,β-carbons from the Michael acceptor (Figure 3).

T32

7059-7197

Sentence

denotes

Furthermore, the carbonyl oxygen sp2 lone pair electrons are not aligned well with either 3CLpro hydrogen bond donor of the oxyanion hole.

T33

7198-7432

Sentence

denotes

Specifically, the hydrogen bond between the carbonyl oxygen and the Gly143 NH in the SARS CoV-1 3CLpro complex is 3.4 Å, while the corresponding hydrogen bond distance is a much more favorable 2.8 Å in the complex of 1 with HRV 3Cpro.

T34

7433-7710

Sentence

denotes

In contrast to these unfavorable interactions, the constrained lactam30 at the P1 site, which was designed to be isosteric with the highly conserved P1 glutamine present in all SARS CoV substrates, is well positioned in the S1 pocket making a favorable hydrogen bond to His163.

T35

7711-7827

Sentence

denotes

The NH of the lactam is within the hydrogen bond distance to Glu166 and the backbone oxygen of Phe140 in the 3CLpro.

T36

7828-8004

Sentence

denotes

Additionally, the NH of the indole P2 capping moiety makes a hydrogen bond with the protein backbone while it extends across an otherwise lipophilic surface over the P3 pocket.

T37

8005-8106

Sentence

denotes

Figure 3 Cocrystal structure of the covalent adduct of 2 bound to SARS CoV-1 3CLpro (PDB code 6XHO).

T38

8107-8178

Sentence

denotes

The Connolly surface for the inhibitor binding pocket is shown in gray.

T39

8179-8276

Sentence

denotes

The bonds are represented as the dashed lines, with the bond length measured between heavy atoms.

T40

8277-8397

Sentence

denotes

The views are centered on warhead with active-site interactions and of complete inhibitor–protein interactions depicted.

T41

8398-8593

Sentence

denotes

Although the structure of 2 in complex with SARS CoV-1 3CLpro is the final product, the above analysis is expected to apply similarly to the more relevant and structurally close transition state.

T42

8594-8791

Sentence

denotes

Accordingly, a focused design strategy aimed at replacing the warhead while retaining the P1 lactam and utilizing the methoxy indole capping group in the early rounds of optimization was initiated.

T43

8792-8983

Sentence

denotes

Specifically, an ideal electrophilic warhead would be bioisosteric, with the scissile amide carbonyl of peptidyl substrates ensuring proper alignment within the oxyanion hole of the protease.

T44

8984-9188

Sentence

denotes

To better mimic the tetrahedral intermediate generated in the amide bond cleavage, we designed ketone-based covalent reversible and irreversible inhibitors of SARS CoV-1 3CLpro, as illustrated by 3 and 4.

T45

9189-9376

Sentence

denotes

Compound 4 was selected as a development candidate for SARS CoV-1 but with the successful public health response that ended the 2003 pandemic, the clinical advancement of 4 was suspended.

T46

9377-9584

Sentence

denotes

Following the COVID-19 outbreak, testing has demonstrated that 4 is a potent inhibitor of the SARS CoV-2 3CLpro (Ki = 0.27 ± 0.1 nM) and a cocrystal structure with 4 bound in the active site has been solved.

T47

9585-9854

Sentence

denotes

In addition to the potent inhibition of the 3CLpro and viral replication of several coronaviruses, 4 possesses solubility as well as metabolic and chemical stability characteristics, which are consistent with a continuous infusion IV therapeutic treatment for COVID-19.

T48

9855-10191

Sentence

denotes

We report the research focused on the discovery of reversible and irreversible ketone-based inhibitors of SARS CoV-1 3CLpro employing ligand-protease structures solved by X-ray crystallography, which led to the identification of 4 as a molecule warranting further evaluation for its potential to treat coronaviruses, including COVID-19.

T49

10193-10215

Sentence

denotes

Results and Discussion

T50

10217-10226

Sentence

denotes

Chemistry

T51

10227-10400

Sentence

denotes

Central to our strategy of examining ketone-based thiophiles was the preparation of an array of intermediates containing diverse methylketone moieties and protecting groups.

T52

10401-10619

Sentence

denotes

The preparation of halomethylketone intermediates was accomplished in a two-step procedure by the generation of a diazoketone intermediate31 or directly by a modified Kowalski–Haque reaction,32 as depicted in Scheme 1.

T53

10620-10671

Sentence

denotes

Scheme 1 Preparation of P1 Synthetic Intermediates

T54

10672-11026

Sentence

denotes

(i) 3 M NaOH, MeOH (95%), (ii) isobutylchloroformate (IBCF), triethylamine (TEA), tetrahydrofuran (THF), (iii) CH2N2/ether (92%), (iv) 4 M HCl/dioxane (83%), (v) 1.0 equiv 48% HBr (83%), (vi) HNCH3(OCH3), EDC, HOBt, NMM, dichloromethane (DCM) (79%), (vii) Mg, HgCl2, BOM-Cl, THF, −78°C (50%), and (viii) lithium diisopropylamide (LDA), ClCH2I, THF (54%).

T55

11027-11132

Sentence

denotes

Saponification of amino ester 5(33) afforded the corresponding amino acid 6 in nearly quantitative yield.

T56

11133-11301

Sentence

denotes

Acid 6 was treated with isobutylchloroformate and triethylamine, and the resulting mixed anhydride was reacted with diazomethane34 to provide high isolated yields of 7.

T57

11302-11453

Sentence

denotes

Diazoketone 7 was treated with hydrochloric acid for simultaneous nitrogen deprotection and conversion to the chloromethylketone (CMK) intermediate 10.

T58

11454-11613

Sentence

denotes

Alternatively, the N-Boc-protected bromomethylketone was prepared by treatment of 7 with stoichiometric quantities of 48% HBr in dichloromethane to provide 11.

T59

11614-11798

Sentence

denotes

Another preparation of CMK intermediates that avoids the use of diazomethane was accomplished by the reaction of 5 with excess LDA and chloroiodomethane to afford 8 in moderate yields.

T60

11799-12006

Sentence

denotes

A direct approach to oxymethylketone intermediate 9 was achieved by conversion of acid 6 to the corresponding Weinreb amide followed by treatment with the Grignard generated from benzyl chloromethyl ether.35

T61

12007-12139

Sentence

denotes

A key element for rapid access to substituted methylketone derivatives was the generation of the fully elaborated CMK 12 (Scheme 2).

T62

12140-12248

Sentence

denotes

The utility of the CMK moiety to prepare acyloxy- and hydroxy-substituted methylketones is well established.

T63

12249-12501

Sentence

denotes

Elaboration of 10 in a sequence of deprotection/1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU)-mediated peptide couplings provided CMK derivative 12 in moderate yield with no epimerization observed.

T64

12502-12627

Sentence

denotes

Reaction of 12 with various carboxylic acids in the presence of cesium fluoride at 60 °C provided acyloxymethylketones 13–28.

T65

12628-12752

Sentence

denotes

Using this synthetic approach with substitution of P2 and amine capping structural elements provided inhibitors 3 and 29–31.

T66

12753-12833

Sentence

denotes

Scheme 2 Preparation of Substituted Acyloxymethylketones and Aminomethylketones

T67

12834-13040

Sentence

denotes

(i) Boc-Leu-OH, HATU, NMM, N,N-dimethylformamide (DMF), 0 °C (64%), (ii) 4 M HCl/dioxane, (iii) HATU, 4-MeO-2-indolecarboxylic acid, NMM, DMF, 0 °C (61%), and (iv) CsF, DMF, carboxylic acid, 60 °C (43–91%).

T68

13041-13149

Sentence

denotes

Hydroxymethylketone (HMK) derivatives were accessed by two complimentary approaches illustrated in Scheme 3.

T69

13150-13369

Sentence

denotes

Elaboration of the P1′-protected oxygen intermediate 9 by sequential amide bond coupling reactions provided benzylether derivatives such as 34 in moderate yields with a low propensity for epimerization at the P1 center.

T70

13370-13459

Sentence

denotes

Hydrogenolytic debenzylation readily afforded the final HMK inhibitors exemplified by 36.

T71

13460-13809

Sentence

denotes

Alternatively, fully elaborated CMK derivatives such as 35 were reacted with benzoylformic acid followed by methanolysis to generate HMK final products such as 36.36 Alkylation of the terminal hydroxyl was achieved under microwave-assisted conditions in the presence of silver (I) oxide to afford alkoxymethylketone inhibitors, as illustrated by 37.

T72

13810-13941

Sentence

denotes

Using these two synthetic approaches with substitution of P2 and amine capping structural elements provided inhibitors 4 and 38–50.

T73

13942-13998

Sentence

denotes

Scheme 3 Preparation of Hydroxymethylketone Derivatives

T74

13999-14200

Sentence

denotes

(i) 4 M HCl/dioxane, (ii) Boc-Leu-OH, HATU, NMM, DMF, 0 °C, (iii) HATU, 4-MeO-2-indolecarboxylic acid, NMM, 0 °C, (iv) Pd/C, H2, EtOH (77%), (v) CsF, DMF, benzoylformic acid, 60 °C, and then MeOH, cat.

T75

14201-14244

Sentence

denotes

K2CO3 (53%), and (vi) CH3I, Ag2O, DCE (9%).

T76

14246-14267

Sentence

denotes

Biological Evaluation

T77

14269-14313

Sentence

denotes

Irreversible Inhibitors of SARS CoV-1 3CLpro

T78

14314-14455

Sentence

denotes

The warhead reactivity and binding affinity to P1′ are both important design considerations to ensure specificity of irreversible inhibitors.

T79

14456-14826

Sentence

denotes

Krantz has demonstrated that the inherent chemical reactivity of acyloxymethylketones can be tuned to inhibit cysteine proteases through the modulation of substituent effects on the carboxylate leaving group.37 The importance of leaving group “strength” was confirmed by the strong dependence between the pKa of the leaving carboxylate and cathepsin B enzyme inhibition.

T80

14827-15004

Sentence

denotes

These otherwise weak electrophiles are elegant “quiescent” inhibitors that harness the very same interactions with catalytic residues that lead to proteolysis rate acceleration.

T81

15005-15242

Sentence

denotes

Molecular modeling of a 2,6-dichlorobenzoate design with SARS CoV-1 3CLpro indicated that a low strain conformation of the ketone carbonyl was aligned in the oxyanion hole and the substituted benzoate is accommodated within the P1′ site.

T82

15243-15325

Sentence

denotes

A series of acyloxymethylketone derivatives were prepared, as depicted in Table 1.

T83

15326-15403

Sentence

denotes

Table 1 SARS CoV-1 3CLpro Inhibition Data for Acyloxymethylketone Inhibitors

T84

15404-15433

Sentence

denotes

SARS CoV-1 3CLpro FRETa

T85

15434-15471

Sentence

denotes

entry R kobs/I (M–1 s–1) IC50 (nM)

T86

15472-15498

Sentence

denotes

12 283,039 ± 22,586

T87

15499-15519

Sentence

denotes

13 Me 220 ± 0.5

T88

15520-15546

Sentence

denotes

14 cyc-propyl 182 ± 6

T89

15547-15573

Sentence

denotes

15 tert-butyl 230 ± 5

T90

15574-15591

Sentence

denotes

16 Ph 86 ± 3

T91

15592-15615

Sentence

denotes

17 4-OMe-Ph 79 ± 3

T92

15616-15638

Sentence

denotes

18 4-Me-Ph 87 ± 2

T93

15639-15661

Sentence

denotes

19 4-CN-Ph 53 ± 1

T94

15662-15683

Sentence

denotes

20 4-F-Ph 82 ± 3

T95

15684-15706

Sentence

denotes

21 4-Cl-Ph 97 ± 3

T96

15707-15742

Sentence

denotes

22 2,6-(Cl)2-Ph 62,993 ± 2,501

T97

15743-15775

Sentence

denotes

23 2,6-(F)2-Ph 12,776 ± 594

T98

15776-15808

Sentence

denotes

24 2-OH-4-Cl-Ph 11,525 ± 40

T99

15809-15844

Sentence

denotes

25 2-F, 4-CN-Ph 13,321 ± 2,309

T100

15845-15872

Sentence

denotes

26 2,6-(Me)2-Ph 74 ± 4

T101

15873-15902

Sentence

denotes

27 2,6-(MeO)2-Ph 205 ± 2

T102

15903-15925

Sentence

denotes

28 2-CN-Ph 17 ± 2

T103

15926-16092

Sentence

denotes

a See the Experimental Section for details on assay methods; the values were calculated from at least eight data points with at least two independent determinations.

T104

16093-16184

Sentence

denotes

As expected, chloromethylketone 12 is a potent irreversible inhibitor of SARS CoV-1 3CLpro.

T105

16185-16322

Sentence

denotes

Compounds 22–25 possess the most electron-deficient benzoate leaving groups and display kinetics consistent with irreversible inhibition.

T106

16323-16452

Sentence

denotes

The remaining entries appear to be reversible inhibitors in the time scale of the assay, with 28 possessing the most potent IC50.

T107

16453-16647

Sentence

denotes

A crystal structure (PDB code 6XHN)38 of 28 in complex with SARS CoV-1 3CLpro at 2.25 Å shows a covalent adduct, which demonstrates the bimodal activity of certain acyloxy inhibitors (Figure 4).

T108

16648-16851

Sentence

denotes

In this structure, the electron density for the 2-cyanobenzoate moiety is absent and the 3CLpro active-site cysteine (Cys145) sulfur forms a covalent bond to the methylene carbon (1.8 Å C–S bond length).

T109

16852-16995

Sentence

denotes

The ketone carbonyl is positioned within the oxyanion hole and as such engaging in hydrogen bonds with backbone NH groups of Gly143 and Cys145.

T110

16996-17176

Sentence

denotes

A detailed analysis of an extended hydrogen bond network from catalytic His41 reveals that the side-chain imidazole serves as a hydrogen bond donor to the interior of the protease.

T111

17177-17287

Sentence

denotes

Specifically, the imidazole hydrogen is directed toward a lone pair electron acceptor from a structural water.

T112

17288-17622

Sentence

denotes

We can conclude that this water must be an acceptor to His41 as one of the water hydrogens is engaged with acceptor electrons on the side chain of Asp187, and the second hydrogen engages a terminating acceptor backbone carbonyl on Asp176 through a short network that includes the side chains of an internal, neutral His164 and Thr175.

T113

17623-17725

Sentence

denotes

Figure 4 Cocrystal structure of the covalent adduct of 28 bound to SARS CoV-1 3CLpro (PDB code 6XHN).

T114

17726-17797

Sentence

denotes

The Connolly surface for the inhibitor binding pocket is shown in gray.

T115

17798-17891

Sentence

denotes

The bonds are represented as dashed lines, with the bond length between heavy atoms depicted.

T116

17892-18333

Sentence

denotes

The 2,6-dichlorobenzoate derivative 22 displays the highest levels of potency in the SARS CoV-1 3CLpro and in antiviral cytopathic effect assays in Vero 76 cells (EC50 = 0.29 ± 0.19 μM).39 We note here that the assay in Vero 76 cells provided data establishing antiviral activity, but the translation of potency measured in Vero cells, which have high efflux potential, to the potency achievable in human lung cells was unknown (vide infra).

T117

18334-18715

Sentence

denotes

As expected, 22 possesses low levels of reactivity toward endogenous nucleophiles such as glutathione (t1/2 > 60 min)40 and exhibits high levels of stability in human plasma (t1/2 > 240 min).41 Although 22 displays a promising activity profile, it possesses very poor solubility in clinically relevant IV vehicle formulations, thus limiting its development as an IV clinical agent.

T118

18716-18932

Sentence

denotes

To improve the solubility characteristics of inhibitors incorporating the lipophilic 2,6-dichlorobenzoate, a limited set of inhibitors containing smaller or more polar amine capping fragments were prepared (Table 2).

T119

18933-19020

Sentence

denotes

Replacement of the 4-methoxy indole cap present in 22 with a benzimidazole provided 31.

T120

19021-19132

Sentence

denotes

This inhibitor displayed potent irreversible inhibition kinetics but did not improve solubility compared to 22.

T121

19133-19290

Sentence

denotes

Reduction in the size of the P2 capping element as represented by 29 and 30 provided derivatives that possessed weak reversible SARS CoV-1 3CLpro inhibition.

T122

19291-19380

Sentence

denotes

Table 2 SARS CoV-1 3CLpro Inhibition Data for P3-Modified Acyloxymethylketone Inhibitors

T123

19381-19547

Sentence

denotes

a See the Experimental Section for details on assay methods; the values were calculated from at least eight data points with at least two independent determinations.

T124

19548-19784

Sentence

denotes

Another design to replace the 4-methoxy indole was the (2R)-tetrahydrofuran-2-carboxylate, which molecular modeling suggested could make additional favorable protein interactions with a Gln189 side chain and led to the preparation of 3.

T125

19785-19860

Sentence

denotes

Initially, this derivative appeared to display reversible binding kinetics.

T126

19861-20042

Sentence

denotes

However, further kinetic studies of compound 3 with the SARS CoV-1 3CLpro protein demonstrated irreversible time-dependent inactivation of this enzyme (kobs/I = 5834 ± 151 M–1 s–1).

T127

20043-20194

Sentence

denotes

Additionally, 3 possessed potent antiviral activity in Vero cells (EC50 = 2.4 μM) and solubility in vehicle formulations almost 20-fold higher than 22.

T128

20195-20371

Sentence

denotes

Evaluation of the pharmacokinetic profile of 3 in rat suggested that this inhibitor possessed properties potentially sufficient for an IV continuous infusion dosing paradigm.42

T129

20372-20592

Sentence

denotes

Although irreversible inhibition can provide an extended pharmacodynamic effect when protein resynthesis rate is slow compared to drug clearance, such extended effects for 3CLpro inhibition were not necessarily expected.

T130

20593-20819

Sentence

denotes

The 3CLpro-mediated proteolysis of newly expressed viral polyproteins is essential to virus replication; however, this activity only occurs during a single step in the virus life cycle that closely follows each cell infection.

T131

20820-20933

Sentence

denotes

Viral particles themselves are not reliant on 3CLpro activity nor are the remaining coronavirus life cycle steps.

T132

20934-20998

Sentence

denotes

Each event of cell infection initiates newly synthesized 3CLpro.

T133

20999-21126

Sentence

denotes

Because detailed kinetics of this process are not understood, reversible and irreversible inhibitors were investigated equally.

T134

21128-21170

Sentence

denotes

Reversible Inhibitors of SARS CoV-1 3CLpro

T135

21171-21620

Sentence

denotes

Peptidyl HMK inhibitors and their corresponding ethers have been designed as potent reversible cysteine protease inhibitors of cathepsin K34 and cruzain.43 High-resolution crystal structures of two such inhibitors complexed with cruzain highlighted the presence of a strong hydrogen bond between the catalytic His and the α-hydroxyl moiety of the inhibitor but interestingly no covalent bond between the active-site Cys and the ketone pharmacophore.

T136

21621-21964

Sentence

denotes

Utilizing the crystal structure of 28 in complex with SARS CoV-1 3CLpro, molecular modeling of an HMK-based design indicated the potential for establishing a covalent bond between the sulfur of the active-site cysteine (Cys145) and the HMK carbonyl as well as hydrogen bond interactions between catalytic His41 and the terminal hydroxy moiety.

T137

21965-22137

Sentence

denotes

Additionally, the structure of 28 suggested the possibility to increase the lipophilic filling of the deep hydrophobic S2 pocket as a strategy to increase binding affinity.

T138

22138-22285

Sentence

denotes

To evaluate these structure-based observations, a series of reversible HMK inhibitors containing P2 diversity were prepared as depicted in Table 3.

T139

22286-22353

Sentence

denotes

Table 3 SARS 3CLpro Inhibition Data for P2-Modified HMK Inhibitors

T140

22354-22522

Sentence

denotes

a&b See the Experimental Section for details on assay methods; the values were calculated from at least eight data points with at least two independent determinations.

T141

22523-22557

Sentence

denotes

c This value is a Ki measurement.

T142

22558-22606

Sentence

denotes

d These values are from a single determination.

T143

22607-22758

Sentence

denotes

As suggested by molecular modeling, the S2 site appears to accommodate a variety of linear, branched, and cyclic alkyl moieties (entries 36 and 39–41).

T144

22759-22902

Sentence

denotes

Interestingly, the P2 Phe derivative 42 displayed less potent inhibition of the SARS CoV-1 3CLpro than the corresponding saturated analogue 41.

T145

22903-23048

Sentence

denotes

Also noteworthy is the large attenuation in enzyme inhibition seen with P2-N-methyl-Leu inhibitor 38 (IC50 = 83 nM) as compared to 4 (Ki = 4 nM).

T146

23049-23229

Sentence

denotes

This loss of potency is consistent with the inhibitor ligand interactions observed with 2 and 4 that show the P2 NH involved in a hydrogen bond with the side-chain amide of Gln189.

T147

23230-23426

Sentence

denotes

Additionally, the methyl substitution in 38 would be expected to alter the conformation of the 4-methoxy indole cap and perturb the observed ligand–enzyme hydrogen bond network present in 2 and 4.

T148

23427-23571

Sentence

denotes

The results for 4 and the other examples in Table 3 reveal a high antiviral EC50/enzyme IC50 ratio, which may arise from poor cell permeability.

T149

23572-23709

Sentence

denotes

Indeed, these HMK inhibitors exhibit very low permeability and high levels of efflux beyond the sensitivity of the Caco-2 in vitro assay.

T150

23710-23879

Sentence

denotes

However, the impact of high efflux on antiviral potencies from Vero 76 cells (derived from monkey kidney) versus disease-relevant human lung epithelial cells is unknown.

T151

23880-24076

Sentence

denotes

To better understand the observed high antiviral EC50/enzyme IC50 ratio for 4, we evaluated the role of efflux in the Vero 76 cell line by the in vitro experimental design discussed further below.

T152

24077-24249

Sentence

denotes

Concurrently, a strategy to design molecules reducing efflux by active transporters, such as P-glycoprotein, was pursued to decrease high antiviral EC50/enzyme IC50 ratios.

T153

24250-24519

Sentence

denotes

An analysis of the physicochemical properties of 4 suggested that increasing logP, reducing polar surface area (PSA), and reducing the number of hydrogen bond donors/acceptors were design strategies with the potential to improve cellular permeability and reduce efflux.

T154

24520-24696

Sentence

denotes

An examination of the crystal structure of 2 in complex with SARS CoV-1 3CLpro suggested a limited functional role for the 4-methoxy substituent contained in the P3 indole cap.

T155

24697-24833

Sentence

denotes

Additionally, the terminal α-hydroxyl moiety located at P1′ appeared to provide a potential opportunity to remove a hydrogen bond donor.

T156

24834-24993

Sentence

denotes

Although it was anticipated that the α-hydroxyl moiety would form a hydrogen bond to the catalytic His41, the directionality of this interaction was uncertain.

T157

24994-25226

Sentence

denotes

As noted earlier, His41 presents a hydrogen bond donor to the interior of the protease, leaving an acceptor to participate in the proton transfer cycling from neutral to cationic in the putative catalytic mechanism44 of proteolysis.

T158

25227-25399

Sentence

denotes

If His41 is protonated in the inhibitor-bound complex, a lone pair from the α-hydroxyl moiety would function as a hydrogen bond acceptor from the proton on the His residue.

T159

25400-25563

Sentence

denotes

On the other hand, if His41 is deprotonated, the hydrogen of the α-hydroxyl moiety would act as a hydrogen bond donor to the nitrogen lone pair of the His residue.

T160

25564-25754

Sentence

denotes

Consistent with the strategy to decrease PSA and hydrogen bond donors, a series of inhibitors were prepared that contained alterations to the P3 cap and substitution of the α-hydroxyl group.

T161

25755-25865

Sentence

denotes

Table 4 summarizes this effort where two of the optimal P2 residues (Leu and β-tert-butyl-Ala) were conserved.

T162

25866-26123

Sentence

denotes

Removal of the methoxy group from the indole generally led to slightly weaker potency in both the enzymatic and antiviral assays across pairs (43 vs 36, 44 vs 37, 50 vs 48 and 49 vs 4) and therefore no improvement in the antiviral/protease inhibition ratio.

T163

26124-26235

Sentence

denotes

The impact of more significant changes to the P3 cap is illustrated by tetrahydrofuranyl derivatives 46 and 47.

T164

26236-26410

Sentence

denotes

These derivatives lack the lipophilic aryl ring and NH hydrogen bond donor present in the indole P3 cap and exhibit a pronounced reduction in enzymatic and antiviral potency.

T165

26411-26574

Sentence

denotes

A noteworthy reduction in biochemical and antiviral potency was observed in comparing each hydroxymethylketone derivative with its corresponding ether counterpart.

T166

26575-26748

Sentence

denotes

These results suggest that modest structural and physicochemical alterations of 4 fail to significantly decrease the high EC50/IC50 ratios for this class of peptidomimetics.

T167

26749-27056

Sentence

denotes

However, in vitro analysis of 4 revealed this derivative to possess high levels of metabolic stability in human liver microsomes (t1/2 = 107 min).45 Further evaluation of this breakthrough derivative in several aqueous-based formulations suitable for IV administration indicated high levels of solubility.46

T168

27057-27171

Sentence

denotes

Table 4 SARS CoV-1 3CLpro Inhibition Data for P3- and P2-Modified Hydroxymethyl and Alkoxymethylketone Inhibitors

T169

27172-27340

Sentence

denotes

a&b See the Experimental Section for details on assay methods; the values were calculated from at least eight data points with at least two independent determinations.

T170

27341-27375

Sentence

denotes

c This value is a Ki measurement.

T171

27376-27424

Sentence

denotes

d These values are from a single determination.

T172

27425-27740

Sentence

denotes

Cocrystal structures of 4 bound to the 3CLpro of both SARS CoV-1 and CoV-2 were solved at 1.47 and 1.26 Å resolutions, respectively (PDB codes 6XHL and 6XHM).47 As expected, the ligand binding sites of the main protease from SARS CoV-1 and SARS CoV-2 are conserved in sequence and are nearly identical structurally.

T173

27741-27867

Sentence

denotes

The schematic diagram in Figure 5 is representative of the covalent adduct between 4 and the 3CLpro from both CoV-1 and CoV-2.

T174

27868-28044

Sentence

denotes

The warhead HMK carbonyl carbon of 4 forms a covalent bond to the 3CLpro active-site cysteine (Cys145) sulfur generating a tetrahedral carbinol complex (1.8 Å C–S bond length).

T175

28045-28182

Sentence

denotes

This carbinol hydroxyl forms hydrogen bonds with the backbone NH of Cys145 and with the amide NH of Gly143 via a bridging water molecule.

T176

28183-28304

Sentence

denotes

Another key active-site interaction is the hydrogen bond between the primary alcohol moiety of 4 and the catalytic His41.

T177

28305-28441

Sentence

denotes

Similar interactions between catalytic His residues and the OH moiety of HMK in other protease–inhibitor complexes have been reported.43

T178

28442-28534

Sentence

denotes

Figure 5 Cocrystal structure of the covalent adduct of 4 bound to SARS CoV-2 3CLpro (6XHM).

T179

28535-28606

Sentence

denotes

The Connolly surface for the inhibitor binding pocket is shown in gray.

T180

28607-28700

Sentence

denotes

The bonds are represented as dashed lines, with the bond length between heavy atoms depicted.

T181

28701-28873

Sentence

denotes

The schematic rendering of the active site with dashed lines represented as hydrogen bonds with key residues and curved lines to show S1 and S2 binding pockets is depicted.

T182

28874-29116

Sentence

denotes

In S1, the lactam carbonyl of 4 forms a strong hydrogen bond with the side chain of His163, while the lactam NH is within the hydrogen-bonding distance but poorly aligned with the side-chain oxygen of Glu166 and the backbone oxygen of Phe140.

T183

29117-29192

Sentence

denotes

These three residues partially define the bottom and edge of the S1 pocket.

T184

29193-29350

Sentence

denotes

Additionally, the backbone carbonyl and NH of Glu166 form β-sheet-like hydrogen-bonding interactions with the NH and C2-carbonyl of the indole fragment of 4.

T185

29351-29493

Sentence

denotes

The NH of the P2 Leu of inhibitor 4 and the side chain of Gln189 form a hydrogen bond, while the carbonyl of P2 Leu is exposed to the solvent.

T186

29494-29580

Sentence

denotes

The inhibitor P1 NH forms a strong hydrogen bond with the backbone carbonyl of His164.

T187

29581-29741

Sentence

denotes

The inhibitor’s lipophilic leucine residue is bound to the SARS 3CLpro in the hydrophobic S2 pocket formed by residues Asp187, Arg188, Gln189, Met49, and His41.

T188

29742-29938

Sentence

denotes

The indole portion of 4 does not protrude into the S3 pocket but rather rests across a partially collapsed S3, making extended van der Waals interactions across the 189–191 residue backbone atoms.

T189

29939-30356

Sentence

denotes

The closed S3 is the only significant conformational difference compared to a recently reported structure of SARS CoV-2 3CLpro inhibited by α-ketoamides.8 The energetics of this extended interaction, the associated protein conformation stabilities, or warhead difference may contribute to the 3-order-of-magnitude increased SARS CoV-2 3CLpro potency of HMK 4 (see below) compared to that of the referenced ketoamides.

T190

30357-30454

Sentence

denotes

Hydroxymethylketone 4 was evaluated against a panel of other viral and human proteases (Table 5).

T191

30455-30692

Sentence

denotes

In general, 4 is highly selective for 3CLpro inhibition, displaying IC50 values of >10 μM against many of the other proteases and possessing modest levels of inhibition of cathepsin B (IC50 = 1.3 μM) and rhinovirus 3Cpro (IC50 = 1.7 μM).

T192

30693-30825

Sentence

denotes

High levels of similarity between the catalytic sites of 3CLpro proteases suggest the potential of 4 as a pan-coronavirus inhibitor.

T193

30826-31047

Sentence

denotes

Testing revealed that 4 demonstrates potent inhibition of the related hCoV 229E protease (IC50 = 0.004 μM) but more importantly, given the current global pandemic, potently inhibits SARS CoV-2 3CLpro with a Ki of 0.27 nM.

T194

31048-31081

Sentence

denotes

Table 5 Protease Inhibition by 4

T195

31082-31133

Sentence

denotes

protease source % inhibition @ 10 μMa IC50 (μM)a

T196

31134-31172

Sentence

denotes

3CLpro SARS CoV-1 0.004 ± 0.0003b

T197

31173-31210

Sentence

denotes

3CLpro SARS CoV-2 0.00027 ± 0.0001b

T198

31211-31244

Sentence

denotes

cathepsin B human 93 1.3 ± 0.1

T199

31245-31271

Sentence

denotes

cathepsin D human 3 >10

T200

31272-31307

Sentence

denotes

leukocyte elastase human –31 >10

T201

31308-31335

Sentence

denotes

chymotrypsin human 8 >10

T202

31336-31361

Sentence

denotes

thrombin human –28 >10

T203

31362-31384

Sentence

denotes

pepsin human –2 >10

T204

31385-31411

Sentence

denotes

caspase 2 human –20 >10

T205

31412-31432

Sentence

denotes

HIV-1 HIV-1 0 >10

T206

31433-31450

Sentence

denotes

HCV HCV 16 >10

T207

31451-31471

Sentence

denotes

HRV HRV 1.7 ± 1

T208

31472-31507

Sentence

denotes

hCoV 229E human 0.004 ± 0.0004

T209

31508-31674

Sentence

denotes

a See the Experimental Section for details on assay methods; the values were calculated from at least eight data points with at least two independent determinations.

T210

31675-31739

Sentence

denotes

b For SARS CoV-1 and CoV-2 3CLpro, Ki values are reported here.

T211

31740-31822

Sentence

denotes

The antiviral specificity of 4 was evaluated against a panel of viruses (Table 6).

T212

31823-32022

Sentence

denotes

No antiviral activity was observed when 4 was tested against two human rhinovirus strains (HRV-14 and HRV-16), human immunodeficiency virus-1 (HIV-1), or human cytomegalovirus (HCMV) in cell culture.

T213

32023-32076

Sentence

denotes

This compound also does not inhibit the HCV replicon.

T214

32077-32255

Sentence

denotes

As anticipated by the potent hCoV 229E protease activity, 4 is a potent inhibitor of the human coronavirus hCoV 229E in MRC-5 cells (human lung-derived) with an EC50 of 0.090 μM.

T215

32256-32286

Sentence

denotes

Table 6 Viral Inhibition by 4

T216

32287-32316

Sentence

denotes

virus EC50 (μM)a CC50 (μM)a

T217

32317-32343

Sentence

denotes

SARS CoV-1 4.8 ± 2.2 452

T218

32344-32371

Sentence

denotes

hCoV 229E 0.09 ± 0.04 >40

T219

32372-32392

Sentence

denotes

HRV-14 >100 >1,260

T220

32393-32413

Sentence

denotes

HRV-16 >100 >1,260

T221

32414-32432

Sentence

denotes

HIV-1 RF >32 >32

T222

32433-32457

Sentence

denotes

HCV replicon >320 >320

T223

32458-32480

Sentence

denotes

HCMV RC256 >100 >100

T224

32481-32647

Sentence

denotes

a See the Experimental Section for details on assay methods; the values were calculated from at least eight data points with at least two independent determinations.

T225

32648-32860

Sentence

denotes

The hCoV 229E antiviral potency is more than 50-fold greater than that for SARS CoV-1 in the Vero 76 line, even though 4 possesses nearly equivalent 3CLpro biochemical potency for the hCoV 229E and CoV-1 viruses.

T226

32861-33120

Sentence

denotes

While undefined differences between hCoV 229E and SARS CoV-1 could account for these large differences in antiviral EC50/enzyme IC50 ratios, different active transporter expression levels between MRC-5 and Vero 76 cell lines may also be a contributing factor.

T227

33121-33396

Sentence

denotes

Consistent with the previously stated goal to characterize the impact of efflux on potency measured in the SARS CoV-1 antiviral assay, we co-dosed a known P-glycoprotein transport inhibitor at a fixed concentration (0.5 μM CP-100356)48 in a full dose–response of inhibitor 4.

T228

33397-33562

Sentence

denotes

This resulted in a pronounced potency shift of SARS CoV-1 antiviral activity (EC50 = 0.11 μM) that now more closely matched the antiviral potency seen for hCoV 229E.

T229

33563-33806

Sentence

denotes

As a control, the same co-dosing of efflux inhibitor led to no such shift in the hCoV 229E antiviral potency of 4, demonstrating that the human lung-derived MRC-5 cells show less P-glycoprotein-based transporter under similar assay conditions.

T230

33807-34077

Sentence

denotes

These results suggest that the high antiviral EC50/enzyme IC50 ratio observed in Vero 76 cells is an artifact of the high efflux potential of that assay cell line and may underestimate the antiviral potency in human lung cells, the relevant tissue for SARS and COVID-19.

T231

34078-34225

Sentence

denotes

Assays to directly measure the antiviral potency of 4 against SARS CoV-2 in clinically relevant human lung cells are currently under investigation.

T232

34227-34238

Sentence

denotes

Conclusions

T233

34239-34442

Sentence

denotes

This report details the efforts to identify irreversible and reversible peptidomimetic inhibitors of the SARS CoV-1 3CLpro as potential IV agents for the treatment of SARS and other coronavirus diseases.

T234

34443-34559

Sentence

denotes

Two classes, acyloxymethylketones and hydroxymethylketones, were discovered as potent inhibitors of the SARS 3CLpro.

T235

34560-34670

Sentence

denotes

The hydroxymethylketone derivative 4 demonstrated potent SARS CoV-1 inhibition in 3CLpro and antiviral assays.

T236

34671-35017

Sentence

denotes

Additionally, 4 possesses acceptable solubility, stability in plasma, and low in vitro and in vivo clearances suitable for further development as a coronavirus treatment.49 Importantly, the potent inhibition of SARS CoV-2 3CLpro has prompted further preclinical investigations of 4 as a potential therapeutic treatment for patients with COVID-19.

T237

35019-35039

Sentence

denotes

Experimental Section

T238

35041-35059

Sentence

denotes

General Procedures

T239

35060-35172

Sentence

denotes

Unless otherwise noted, materials were obtained from commercial suppliers and used without further purification.

T240

35173-35326

Sentence

denotes

The removal of solvent under reduced pressure or concentration refers to distillation using a Büchi rotary evaporator attached to a vacuum pump (3 mmHg).

T241

35327-35409

Sentence

denotes

Products obtained as solids or high-boiling oils were dried under vacuum (1 mmHg).

T242

35410-35536

Sentence

denotes

Silica gel chromatography was performed on either a CombiFlash (Teledyne ISCO), SP4, or Isolera (Biotage) purification system.

T243

35537-35737

Sentence

denotes

All reactions were performed under a positive pressure of nitrogen or argon or with a drying tube at an ambient temperature (unless otherwise stated) in anhydrous solvents, unless otherwise indicated.

T244

35738-35907

Sentence

denotes

Analytical thin-layer chromatography (TLC) was performed on glass-backed silica gel 60 F254 plates (Analtech, 0.25 mm), eluting with the appropriate solvent ratio (v/v).

T245

35908-36078

Sentence

denotes

The reactions were assayed by high-performance liquid chromatography–mass spectrometry (LCMS) or TLC and terminated as judged by the consumption of the starting material.

T246

36079-36249

Sentence

denotes

LCMS utilized wavelengths of 254 and 220 nm and either electrospray ionization (ESI) in positive mode or atmospheric-pressure chemical ionization (APCI) in positive mode.

T247

36250-36354

Sentence

denotes

The TLC plates were visualized using UV light, p-anisaldehyde, phosphomolybdic acid, or iodine staining.

T248

36355-36416

Sentence

denotes

Microwave-assisted reactions were run in a Biotage Initiator.

T249

36417-36490

Sentence

denotes

1H NMR spectra were recorded on a Bruker XWIN-NMR (400 MHz) spectrometer.

T250

36491-36587

Sentence

denotes

Proton chemical shifts are reported in parts per million downfield from tetramethylsilane (TMS).

T251

36588-36678

Sentence

denotes

1H NMR data are reported as chemical shift (multiplicity, coupling constant, integration).

T252

36679-36839

Sentence

denotes

Multiplicities are denoted as follows: s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublets; dt, doublet of triplets; and brs, broad singlet.

T253

36840-36990

Sentence

denotes

For spectra obtained in CDCl3, DMSO-d6, and CD3OD, the residual protons (7.27, 2.50, and 3.31 ppm, respectively) were used as the internal references.

T254

36991-37077

Sentence

denotes

The purity of the final products is >95% as determined by HPLC and/or 1H NMR analyses.

T255

37079-37146

Sentence

denotes

N-(tert-Butoxycarbonyl)-3-[(3S)-2-oxopyrrolidin-3-yl]-l-alanine (6)

T256

37147-37435

Sentence

denotes

A 3 L multineck flask equipped with an overhead stirrer and internal thermometer was charged with methyl N-(tert-butoxycarbonyl)-3-[(3S)-2-oxopyrrolidin-3-yl]-l-alaninate (compound 5)33 (205 g, 716 mmol) followed by methanol (1 L), and the solution was cooled to 0 °C using ice/NaCl bath.

T257

37436-37638

Sentence

denotes

NaOH (115 g in 950 mL water, 2.9 mol) solution was precooled to 0 °C and then added to the flask via a pressure-equalizing dropping funnel at such a rate to maintain the internal temperature below 5 °C.

T258

37639-37826

Sentence

denotes

The resulting solution was stirred at 0 °C for 1 h before neutralizing with conc. hydrochloric acid (keeping the internal temperature below 10 °C) and then removing the methanol in vacuo.

T259

37827-38012

Sentence

denotes

The residue was diluted with ethyl acetate (400 mL), acidified to pH 3 with conc. hydrochloric acid, and then the mixture was transferred to a sep funnel, and the organics were removed.

T260

38013-38216

Sentence

denotes

The aqueous was extracted with ethyl acetate (2 × 400 mL), and the combined organics were washed with brine, dried over MgSO4, filtered, and concentrated to yield the title compound as a white foam, 95%.

T261

38217-38414

Sentence

denotes

1H NMR (400 MHz, MeOD) δ 3.98–4.28 (m, 1H), 3.25–3.41 (m, 2H), 2.44–2.57 (m, 1H), 2.29–2.41 (m, 1H), 2.03–2.14 (m, 1H), 1.73–1.90 (m, 2H), 1.44 (s, 9H); MS (APCI−) for C12H20N2O5m/z 272.3 (M – H)−.

T262

38416-38504

Sentence

denotes

tert-Butyl ((1S)-3-Diazo-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)carbamate (7)

T263

38505-38677

Sentence

denotes

A solution of N-(tert-butoxycarbonyl)-3-[(3S)-2-oxopyrrolidin-3-yl]-l-alanine (2.72 g, 10.0 mmol) in THF (100 mL) was placed under an atmosphere of N2 and cooled to −23 °C.

T264

38678-38835

Sentence

denotes

The resulting clear colorless solution was successively treated with triethylamine (2.1 mL, 15.0 mmol) followed by isobutylchloroformate (1.6 mL, 12.0 mmol).

T265

38836-38938

Sentence

denotes

The reaction mixture gradually became opaque with a fine white precipitate and after 1 h was filtered.

T266

38939-39111

Sentence

denotes

The colorless filtrate was transferred to a nonground joint flask, cooled to 0 °C, and slowly treated with a solution of diazomethane (∼35 mL, ∼16.6 mmol) in diethyl ether.

T267

39112-39117

Sentence

denotes

Note:

T268

39118-39251

Sentence

denotes

The diazomethane was generated employing a Diazald kit according to the procedure described in the Aldrich Technical Bulletin AL-180.

T269

39252-39344

Sentence

denotes

The resulting yellow clear solution was gradually warmed to room temperature (RT) over 16 h.

T270

39345-39460

Sentence

denotes

At this time, N2 was bubbled through the reaction to remove excess diazomethane followed by in vacuo concentration.

T271

39461-39545

Sentence

denotes

The resulting residue was diluted with ethyl acetate (100 mL), washed once with sat.

T272

39546-39660

Sentence

denotes

NaHCO3 (50 mL), once with brine (50 mL), dried over MgSO4, filtered, and concentrated to give a crude yellow foam.

T273

39661-39809

Sentence

denotes

This material was purified by LC (150 g 230–400 SiO2, 3–4% methanol/chloroform) to afford 2.72 g (92%) of the title compound as a light yellow foam.

T274

39810-40030

Sentence

denotes

1H NMR (DMSO-d6) δ 7.63 (bs, 1H), 7.42 (d, J = 8 Hz, 1H), 6.06 (bs, 1H), 3.96 (m, 1H), 3.13 (m, 2H), 2.21 (m, 1H), 2.01 (m, 1H), 1.86 (m, 1H), 1.63–1.52 (m, 2H), 1.38 (s, 9H); MS (ESI+) for C13H20N4O4m/z 319.0 (M + Na)+.

T275

40032-40121

Sentence

denotes

tert-Butyl ((1S)-3-Chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)carbamate (8)

T276

40122-40411

Sentence

denotes

A three-neck flame-dried flask equipped with a nitrogen inlet and internal thermometer was charged with methyl N-(tert-butoxycarbonyl)-3-[(3S)-2-oxopyrrolidin-3-yl]-l-alaninate (10 g, 35 mmol), THF (200 mL), and chloroiodomethane (10.2 mL, 140 mmol), and the solution was cooled to −77 °C.

T277

40412-40587

Sentence

denotes

LDA (140 mL, 210 mmol, 1.5 M mono-THF complex in cyclohexane) was added via a pressure-equalizing dropping funnel at such a rate to keep the internal temperature below −70 °C.

T278

40588-40806

Sentence

denotes

After complete addition, the reaction was stirred for an additional hour and quenched with a mixture of AcOH (33 mL) and THF (200 mL) with a rate of addition regulated to maintain the internal temperature below −65 °C.

T279

40807-40917

Sentence

denotes

After complete addition, the dark suspension was stirred for 10 min and then warmed to an ambient temperature.

T280

40918-41027

Sentence

denotes

The reaction was diluted with ethyl acetate (500 mL), and the organics were washed with water (250 mL), satd.

T281

41028-41244

Sentence

denotes

NaHCO3 (250 mL), and brine (250 mL), dried over MgSO4, filtered, and the solvents were removed in vacuo to yield the crude product as a dark oil, which was purified by flash chromatography eluting with ethyl acetate.

T282

41245-41363

Sentence

denotes

The resulting solid was triturated with diethyl ether to afford the title compound as a pale yellow solid, 5.7 g, 54%.

T283

41364-41677

Sentence

denotes

1H NMR (400 MHz, DMSO-d6) δ 7.88 (s, 1H), 7.75 (d, J = 7.6 Hz, 1H), 4.73–4.94 (m, 2H), 4.37 (m, 1H), 3.28–3.43 (m, 2H), 2.46 (m, 1H), 2.30–2.40 (m, 1H), 2.02–2.14 (m, 1H), 1.77–1.95 (m, 2H), 1.60 (s, 9H); MS (API-ES+) for C13H21N2O4Cl m/z 327.1 (M + Na)+; anal. calcd for C13H21ClN2O4: C, 51.23; H, 6.95; N, 9.19.

T284

41678-41712

Sentence

denotes

Found: C, 51.03; H, 6.93; N, 9.03.

T285

41714-41807

Sentence

denotes

tert-Butyl((1S)-3-(benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)carbamate (9)

T286

41808-42106

Sentence

denotes

To a 3 L multinecked flask equipped with an overhead stirrer, nitrogen inlet, and internal thermometer was charged N-(tert-butoxycarbonyl)-3-[(3S)-2-oxopyrrolidin-3-yl]-l-alanine (190 g, 698 mmol) followed by dichloromethane (1100 mL) and the solution was cooled to 0 °C employing an ice/NaCl bath.

T287

42107-42390

Sentence

denotes

N,O-Dimethylhydroxylamine hydrochloric acid salt (68 g, 698 mmol) was added followed by N-methylmorpholine (230 mL, 2.09 mol), HOBt.hydrate (106 g, 698 mmol), and EDCI (147 g, 768 mmol), and the mixture was stirred at 0 °C under nitrogen for 6 h before quenching with water (500 mL).

T288

42391-42546

Sentence

denotes

The biphasic mixture was transferred to a sep funnel, and the organics were isolated, washed with 1 M hydrochloric acid (2 × 500 mL), water (400 mL), satd.

T289

42547-42811

Sentence

denotes

NaHCO3 (2 × 700 mL), and brine (300 mL) and then dried over MgSO4, filtered, and the solvents were evaporated in vacuo to yield tert-butyl ((1S)-2-[methoxy(methyl)amino]-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}ethyl)carbamate as a pale yellow solid, 173 g, 79%.

T290

42812-43054

Sentence

denotes

1H NMR (400 MHz, CDCl3) δ 5.92 (s, 1H), 5.39 (d, J = 8.8 Hz, 1H), 4.66 (td, J = 9.6, 2.8 Hz, 1H), 3.77 (s, 3H), 3.33 (dd, J = 9.3, 4.0 Hz, 2H), 3.19 (s, 2H), 2.41–2.62 (m, 2H), 2.05–2.16 (m, 1H), 1.75–1.95 (m, 1H), 1.67 (m, 1H), 1.41 (s, 9H).

T291

43055-43321

Sentence

denotes

To a 100 mL multinecked flask equipped with a stirrer bar, nitrogen inlet, and internal thermometer were charged magnesium turnings (dried in oven at 100 °C overnight, 1.04 g, 43 mmol) and HgCl2 (774 mg, 2.85 mmol), and the flask was purged with nitrogen for 10 min.

T292

43322-43510

Sentence

denotes

THF (50 mL) was added, and the suspension was cooled to −45 °C before adding BOM-Cl (5.94 mL, 43 mmol), and the resulting suspension was stirred for 5 h, the temperature returning to 5 °C.

T293

43511-43796

Sentence

denotes

The suspension was recooled to −50 °C, and tert-butyl ((1S)-2-[methoxy(methyl)amino]-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}ethyl)carbamate (1.5 g, 4.76 mmol) was added, and the thick suspension was stirred for 48 h under nitrogen and was allowed to come to an ambient temperature.

T294

43797-43855

Sentence

denotes

The reaction was quenched by the careful addition of satd.

T295

43856-43986

Sentence

denotes

NH4Cl solution (25 mL), and the mixture was stirred until effervescence ceased and then extracted with ethyl acetate (3 × 120 mL).

T296

43987-44259

Sentence

denotes

The combined organics were dried over MgSO4, filtered, and the solvents were evaporated in vacuo to yield a crude orange gum, which was purified by flash chromatography, eluting with 1–3% methanol/dichloromethane to afford the title compound as a clear glass, 900 mg, 50%.

T297

44260-44480

Sentence

denotes

1H NMR (400 MHz, CDCl3) δ 7.21–7.33 (m, 5H), 5.63–5.73 (m, 2H), 4.52 (m, 3H), 4.19 (q, J = 17.4 Hz, 2H), 3.19–3.25 (m, 2H), 2.29–2.42 (m, 2H), 1.68–1.93 (m, 3H), 1.35 (s, 9H); MS (APCI+) for C20H28N2O5m/z 378.1 (M + H)+.

T298

44482-44564

Sentence

denotes

(3S)-3-[(2S)-2-Amino-4-chloro-3-oxobutyl]pyrrolidin-2-one, Hydrochloride Salt (10)

T299

44565-44762

Sentence

denotes

A solution of tert-butyl ((1S)-3-diazo-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl) carbamate (592 mg, 2.0 mmol) in 1,4-dioxane (7 mL) was placed under an atmosphere of N2 and cooled to 0 °C.

T300

44763-44924

Sentence

denotes

This clear pale yellow solution was dropwise treated with a solution of 4 M hydrochloric acid in 1,4-dioxane (5 mL, 20 mmol) with copious gas evolution observed.

T301

44925-45030

Sentence

denotes

Upon complete addition, the reaction was warmed to RT over 1 h with the formation of a white precipitate.

T302

45031-45168

Sentence

denotes

The solid was collected by filtration, washed with diethyl ether, and dried to give 401 mg (83%) of the title compound as a white powder.

T303

45169-45395

Sentence

denotes

1H NMR (DMSO-d6) δ 8.76 (bs, 3H), 7.96 (s, 1H), 4.93 (d, J = 16 Hz, 1H), 4.80 (d, J = 16 Hz, 1H), 4.28 (m, 1H), 3.18 (m, 2H), 2.61 (m, 1H), 2.30 (m, 1H), 1.93 (m, 2H), 1.70 (m, 1H); MS (ESI+) for C8H13ClN2O2m/z 205.0 (M + H)+.

T304

45397-45486

Sentence

denotes

tert-Butyl ((1S)-3-Bromo-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)carbamate (11)

T305

45487-45743

Sentence

denotes

A solution of tert-butyl ((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)carbamate (1.26 g, 4.3 mmol) in dichloromethane (107 mL) at 0 °C under nitrogen was treated with 48% hydrobromic acid (0.48 mL, 4.3 mmol) with effervescence observed.

T306

45744-45925

Sentence

denotes

The reaction was stirred at 0 °C for 1 h, washed once with water (30 mL), dried over MgSO4, filtered, and concentrated to afford 1.23 g (83%) of the title compound as a white solid.

T307

45926-46185

Sentence

denotes

1H NMR (400 MHz, DMSO-d6) δ 7.64 (s, 1H), 7.51 (d, J = 8 Hz, 1H), 4.46 (d, J = 16 Hz, 1H), 4.41 (d, J = 16 Hz, 1H), 4.19 (m, 1H), 3.13 (m, 2H), 2.26 (m, 1H), 2.13 (m, 1H), 1.87 (m, 1H), 1.63 (m, 2H), 1.38 (s, 9H); MS (ESI+) for C13H21BrN2O4m/z 371.0 (M + H)+.

T308

46187-46333

Sentence

denotes

N-((1S)-1-{[((1S)-3-Chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-1H-indole-2-carboxamide (12)

T309

46334-46541

Sentence

denotes

A solution of 29, N2-(tert-butoxycarbonyl)-N1-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-l-leucinamide (244 mg, 0.59 mmol) in 1,4-dioxane (1.0 mL) was placed under an atmosphere of N2.

T310

46542-46666

Sentence

denotes

This clear colorless solution was treated with a solution of 4 M hydrochloric acid in 1,4-dioxane with no observable change.

T311

46667-46746

Sentence

denotes

The reaction gradually became opaque with the formation of a gummy precipitate.

T312

46747-46996

Sentence

denotes

After 2 h, the volatiles were removed in vacuo, diluted with 1:1 ethanol/1,4-dioxane, concentrated, and high-vacuum-dried to give N1-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-l-leucinamide hydrochloride as an off-white solid.

T313

46997-47288

Sentence

denotes

1H NMR (DMSO-d6) δ 9.18 (d, J = 8 Hz, 1H), 8.40 (bs, 3H), 7.69 (s, 1H), 4.68 (s, 2H), 4.49 (m, 1H), 3.81 (m, 1H), 3.15 (m, 2H), 2.37 (m, 1H), 2.17 (m, 1H), 1.92 (m, 1H), 1.67 (m, 3H), 1.55 (m, 2H), 0.92 (d, J = 4 Hz, 3H), 0.88 (d, J = 4 Hz, 3H); MS (ESI+) for C14H24ClN3O3m/z 318.1 (M + H)+.

T314

47289-47466

Sentence

denotes

A solution of the crude hydrochloride salt and 4-methoxy-1H-indole-2-carboxylic acid (123 mg, 0.64 mmol) in DMF (2.5 mL) was placed under an atmosphere of N2 and cooled to 0 °C.

T315

47467-47617

Sentence

denotes

This pale yellow solution was successively treated with HATU (245 mg, 0.64 mmol) and N-methylmorpholine (0.14 mL, 1.29 mmol) turning a brighter color.

T316

47618-47743

Sentence

denotes

After 30 min, the reaction was quenched with 1:1 ice/sat NaHCO3 (25 mL) and extracted three times with ethyl acetate (20 mL).

T317

47744-47871

Sentence

denotes

The combined organics were washed once with brine (30 mL), dried over MgSO4, filtered, and concentrated to give a yellow syrup.

T318

47872-48016

Sentence

denotes

This material was purified by LC (30 g 230–400 SiO2, 4% methanol/chloroform) to afford 167 mg (58%) of the title compound as an off-white solid.

T319

48017-48508

Sentence

denotes

1H NMR (DMSO-d6) δ 11.59 (s, 1H), 8.62 (d, J = 8 Hz, 1H), 8.44 (d, J = 4 Hz, 1H), 7.65 (s, 1H), 7.38 (s, 1H), 7.10 (t, J = 8 Hz, 1H), 7.02 (d, J = 8 Hz, 1H), 6.51 (d, J = 8 Hz, 1H), 4.60 (d, J = 16 Hz, 1H), 4.58 (d, J = 16 Hz, 1H), 4.46 (m, 2H), 3.89 (s, 3H), 3.11 (m, 2H), 2.29 (m, 1H), 2.11 (m, 1H), 1.99 (m, 1H), 1.76–1.54 (m, 5H), 0.95 (d, J = 8 Hz, 3H), 0.9 (d, J = 8 Hz, 3H); MS (ESI+) for C24H31ClN4O5m/z 491.1 (M + H)+; HRMS (ESI+) calcd for C24H31ClN4O5+H1 491.2056, found 491.2058.

T320

48510-48626

Sentence

denotes

(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl Acetate (13)

T321

48627-48952

Sentence

denotes

An oven-dried 40 mL scintillation vial with a spinbar was charged with acetic acid (27 mg, 0.46 mmol) followed by a solution of N-((1S)-1-{[((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-1H-indole-2-carboxamide (172 mg, 0.35 mmol) in DMF (3.5 mL) and purged with N2.

T322

48953-49126

Sentence

denotes

This pale yellow solution was then treated with CsF (122 mg, 0.81 mmol), sealed with a Teflon-lined screwcap, and heated at 65 °C on a reaction block with vigorous stirring.

T323

49127-49243

Sentence

denotes

After 3 h, the reaction was cooled to RT, diluted with water (30 mL), and extracted with dichloromethane (4 × 7 mL).

T324

49244-49349

Sentence

denotes

The combined organic layers were washed with water (2 × 20 mL), brine (20 mL), and concentrated in vacuo.

T325

49350-49496

Sentence

denotes

This material was purified by Biotage MPLC (25 M, 2.5–4.5% methanol/dichloromethane) to afford 78 mg (45%) of the title compound as a white solid.

T326

49497-50011

Sentence

denotes

1H NMR (400 MHz, DMSO-d6) δ 11.57 (s, 1H), 8.57 (d, J = 7.8 Hz, 1H), 8.43 (d, J = 7.6 Hz, 1H), 7.64 (s, 1H), 7.36 (d, J = 1.8 Hz, 1H), 7.08 (t, J = 8.0 Hz, 1H), 6.99 (d, J = 8.1 Hz, 1H), 6.49 (d, J = 7.6 Hz, 1H), 4.83 (d, J = 3.0 Hz, 1H), 4.76–4.95 (m, 1H), 4.35–4.50 (m, 2H), 3.87 (s, 3H), 3.03–3.17 (m, 2H), 2.22–2.35 (m, 1H), 2.09–2.22 (m, 1H), 2.07 (s, 3H), 1.90–2.04 (m, 1H), 1.65–1.77 (m, 2H), 1.48–1.65 (m, 3H), 0.94 (d, J = 6.3 Hz, 3H), 0.89 (d, J = 6.3 Hz, 3H); MS (ESI+) for C26H34N4O7m/z 515.2 (M + H)+.

T327

50012-50077

Sentence

denotes

Anal. calcd for C26H34N4O7·0.65 H2O: C, 59.34; H, 6.76; N, 10.65.

T328

50078-50113

Sentence

denotes

Found: C, 59.41; H, 6.63; N, 10.68.

T329

50115-50247

Sentence

denotes

(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl Cyclopropanecarboxylate (14)

T330

50248-50521

Sentence

denotes

Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting cyclopropanecarboxylic acid and making noncritical variations provided a crude product.

T331

50522-50651

Sentence

denotes

This material was purified by Biotage MPLC (25 M, 2.5–4.5% methanol/dichloromethane) to afford 82 mg (43%) of the title compound.

T332

50652-51114

Sentence

denotes

1H NMR (400 MHz, DMSO-d6) δ 11.57 (d, J = 2.0 Hz, 1H), 8.56 (d, J = 7.8 Hz, 1H), 8.43 (d, J = 7.6 Hz, 1H), 7.63 (s, 1H), 7.36 (d, J = 1.5 Hz, 1H), 7.08 (t, J = 8.0 Hz, 1H), 6.97–7.02 (m, 1H), 6.49 (d, J = 7.6 Hz, 1H), 4.85 (d, 1H), 4.78–4.96 (m, 1H), 4.33–4.51 (m, 2H), 3.87 (s, 3H), 3.02–3.16 (m, 2H), 2.22–2.35 (m, 1H), 2.01–2.11 (m, 1H), 1.89–2.00 (m, 1H), 1.65–1.77 (m, 3H), 1.46–1.65 (m, 3H), 0.81–0.98 (m, 10 H); MS (ESI+) for C28H36N4O7m/z 541.2 (M + H)+.

T333

51115-51180

Sentence

denotes

Anal. calcd for C28H36N4O7·0.59 H2O: C, 61.01; H, 6.80; N, 10.16.

T334

51181-51216

Sentence

denotes

Found: C, 61.04; H, 6.72; N, 10.00.

T335

51218-51335

Sentence

denotes

(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl Pivalate (15)

T336

51336-51594

Sentence

denotes

Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting pivalic acid and making noncritical variations provided a crude product.

T337

51595-51725

Sentence

denotes

This material was purified by Biotage MPLC (25 M, 2.5–4.5% methanol/dichloromethane) to afford 152 mg (78%) of the title compound.

T338

51726-52147

Sentence

denotes

1H NMR (400 MHz, DMSO-d6) δ 11.58 (s, 1H), 8.56 (d, J = 7.8 Hz, 1H), 8.44 (d, J = 7.8 Hz, 1H), 7.64 (s, 1H), 7.36 (s, 1H), 7.08 (t, J = 8.0 Hz, 1H), 6.99 (d, 1H), 6.49 (d, J = 7.6 Hz, 1H), 4.84 (s, 1H), 4.77–4.94 (m, 1H), 4.34–4.51 (m, 2H), 3.87 (s, 3H), 3.02–3.16 (m, 2H), 1.91–2.36 (m, 3H), 1.48–1.78 (m, 5H), 1.16 (s, 9H), 0.94 (d, J = 6.3 Hz, 3H), 0.89 (d, J = 6.3 Hz, 3H); MS (ESI+) for C29H40N4O7m/z 557.2 (M + H)+.

T339

52148-52212

Sentence

denotes

Anal. calcd for C29H40N4O7·0.76 H2O: C, 61.07; H, 7.34; N, 9.82.

T340

52213-52247

Sentence

denotes

Found: C, 61.14; H, 7.63; N, 9.59.

T341

52249-52366

Sentence

denotes

(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl Benzoate (16)

T342

52367-52625

Sentence

denotes

Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting benzoic acid and making noncritical variations provided a crude product.

T343

52626-52756

Sentence

denotes

This material was purified by Biotage MPLC (25 M, 2.5–4.5% methanol/dichloromethane) to afford 183 mg (91%) of the title compound.

T344

52757-53297

Sentence

denotes

1H NMR (400 MHz, DMSO-d6) δ 11.59 (s, 1H), 8.64 (d, J = 8.1 Hz, 1H), 8.46 (d, J = 7.6 Hz, 1H), 7.98 (d, J = 8.1 Hz, 2H), 7.91–7.95 (m, 1H), 7.66–7.71 (m, 1H), 7.66 (s, 1H), 7.52–7.57 (m, 1H), 7.37 (d, J = 1.8 Hz, 1H), 7.08 (t, J = 8.0 Hz, 1H), 6.96–7.02 (m, 1H), 6.49 (d, J = 7.6 Hz, 1H), 5.13 (s, 1H), 5.06–5.24 (m, 1H), 4.44–4.53 (m, 2H), 3.87 (s, 3H), 3.04–3.15 (m, 2H), 2.34 (m, 1H), 2.07–2.27 (m, 1H), 1.98–2.07 (m, 1H), 1.52–1.79 (m, 5H), 0.94 (d, J = 6.3 Hz, 3H), 0.89 (d, J = 6.3 Hz, 3H); MS (ESI+) for C31H36N4O7m/z 577.2 (M + H)+.

T345

53298-53362

Sentence

denotes

Anal. calcd for C31H36N4O7·1.04 H2O: C, 62.54; H, 6.45; N, 9.41.

T346

53363-53397

Sentence

denotes

Found: C, 62.49; H, 6.54; N, 9.31.

T347

53399-53525

Sentence

denotes

(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 4-Methoxybenzoate (17)

T348

53526-53793

Sentence

denotes

Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting 4-methoxybenzoic acid and making noncritical variations provided a crude product.

T349

53794-53924

Sentence

denotes

This material was purified by Biotage MPLC (25 M, 2.5–4.5% methanol/dichloromethane) to afford 145 mg (68%) of the title compound.

T350

53925-54445

Sentence

denotes

1H NMR (400 MHz, DMSO-d6) δ 11.59 (d, J = 1.8 Hz, 1H), 8.62 (d, J = 7.8 Hz, 1H), 8.46 (d, J = 7.6 Hz, 1H), 7.93 (d, J = 9.1 Hz, 2H), 7.65 (s, 1H), 7.37 (d, J = 2.0 Hz, 1H), 7.04–7.11 (m, 2H), 7.00 (dd, J = 8.6, 3.0 Hz, 2H), 6.50 (d, J = 7.6 Hz, 1H), 5.07 (s, 1H), 5.01–5.18 (m, 1H), 4.44–4.51 (m, 2H), 3.87 (s, 3H), 3.83 (s, 3H), 3.04–3.17 (m, 2H), 2.28–2.38 (m, 1H), 2.07–2.25 (m, 1H), 1.97–2.06 (m, 1H), 1.50–1.79 (m, 5H), 0.94 (d, J = 6.3 Hz, 3H), 0.89 (d, J = 6.3 Hz, 3H); MS (ESI+) for C32H38N4O8m/z 607.2 (M + H)+.

T351

54446-54519

Sentence

denotes

Anal. calcd for C32H38N4O8·0.80 H2O·0.05 DCM: C, 61.56; H, 6.40; N, 8.96.

T352

54520-54554

Sentence

denotes

Found: C, 61.84; H, 6.36; N, 8.58.

T353

54556-54681

Sentence

denotes

(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 4-Methylbenzoate (18)

T354

54682-54948

Sentence

denotes

Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting 4-methylbenzoic acid and making noncritical variations provided a crude product.

T355

54949-55078

Sentence

denotes

This material was purified by Biotage MPLC (25 M, 2.5–4.5% methanol/dichloromethane) to afford 92 mg (44%) of the title compound.

T356

55079-55619

Sentence

denotes

1H NMR (400 MHz, DMSO-d6) δ 11.59 (d, J = 1.8 Hz, 1H), 8.63 (d, J = 8.1 Hz, 1H), 8.46 (d, J = 7.6 Hz, 1H), 7.87 (d, J = 8.1 Hz, 2H), 7.65 (s, 1H), 7.37 (d, J = 1.8 Hz, 1H), 7.34 (d, J = 8.3 Hz, 2H), 7.08 (t, J = 8.0 Hz, 1H), 6.97–7.01 (m, 1H), 6.50 (d, J = 7.8 Hz, 1H), 5.09 (s, 1H), 5.03–5.20 (m, 1H), 4.43–4.52 (m, 2H), 3.87 (s, 3H), 3.04–3.17 (m, 2H), 2.38 (s, 3H), 2.28–2.34 (m, 1H), 2.06–2.14 (m, 1H), 1.96–2.06 (m, 1H), 1.51–1.78 (m, 5H), 0.94 (d, J = 6.3 Hz, 3H), 0.89 (d, J = 6.3 Hz, 3H); MS (ESI+) for C32H38N4O7m/z 591.2 (M + H)+.

T357

55620-55693

Sentence

denotes

Anal. calcd for C32H38N4O7·0.10 H2O·0.11 DCM: C, 64.08; H, 6.43; N, 9.31.

T358

55694-55728

Sentence

denotes

Found: C, 64.36; H, 6.41; N, 8.92.

T359

55730-55854

Sentence

denotes

(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 4-Cyanobenzoate (19)

T360

55855-56120

Sentence

denotes

Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting 4-cyanobenzoic acid and making noncritical variations provided a crude product.

T361

56121-56251

Sentence

denotes

This material was purified by Biotage MPLC (25 M, 2.5–4.5% methanol/dichloromethane) to afford 159 mg (75%) of the title compound.

T362

56252-56783

Sentence

denotes

1H NMR (400 MHz, DMSO-d6) δ 11.58 (d, J = 2.0 Hz, 1H), 8.64 (d, J = 8.1 Hz, 1H), 8.46 (d, J = 7.6 Hz, 1H), 8.09–8.14 (m, 2H), 7.99–8.05 (m, 2H), 7.66 (s, 1H), 7.37 (d, J = 1.5 Hz, 1H), 7.08 (t, J = 8.0 Hz, 1H), 6.99 (d, J = 8.3 Hz, 1H), 6.50 (d, J = 7.6 Hz, 1H), 5.17 (d, J = 2.8 Hz, 1H), 5.11–5.27 (m, 1H), 4.44–4.53 (m, 2H), 3.87 (s, 3H), 3.04–3.17 (m, 2H), 2.28–2.38 (m, 1H), 2.07–2.26 (m, 1H), 1.97–2.06 (m, 1H), 1.51–1.78 (m, 5H), 0.94 (d, J = 6.3 Hz, 3H), 0.89 (d, J = 6.3 Hz, 3H); MS (ESI+) for C32H35N5O7m/z 602.2 (M + H)+.

T363

56784-56849

Sentence

denotes

Anal. calcd for C32H35N5O7·0.90 H2O: C, 62.21; H, 6.00; N, 11.33.

T364

56850-56885

Sentence

denotes

Found: C, 62.23; H, 6.06; N, 11.58.

T365

56887-57012

Sentence

denotes

(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 4-Fluorobenzoate (20)

T366

57013-57279

Sentence

denotes

Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting 4-fluorobenzoic acid and making noncritical variations provided a crude product.

T367

57280-57410

Sentence

denotes

This material was purified by Biotage MPLC (25 M, 2.5–4.5% methanol/dichloromethane) to afford 136 mg (65%) of the title compound.

T368

57411-57930

Sentence

denotes

1H NMR (400 MHz, DMSO-d6) δ 11.58 (d, J = 2.0 Hz, 1H), 8.63 (d, J = 7.8 Hz, 1H), 8.46 (d, J = 7.6 Hz, 1H), 8.04 (dd, J = 8.6, 5.6 Hz, 2H), 7.65 (s, 1H), 7.35–7.41 (m, 3H), 7.08 (t, J = 8.0 Hz, 1H), 6.99 (d, J = 8.1 Hz, 1H), 6.50 (d, J = 7.6 Hz, 1H), 5.12 (d, J = 1.3 Hz, 1H), 5.06–5.23 (m, 1H), 4.44–4.52 (m, 2H), 3.87 (s, 3H), 3.04–3.17 (m, 2H), 2.28–2.38 (m, 1H), 2.07–2.26 (m, 1H), 1.98–2.06 (m, 1H), 1.52–1.79 (m, 5H), 0.94 (d, J = 6.3 Hz, 3H), 0.89 (d, J = 6.3 Hz, 3H); MS (ESI+) for C31H35FN4O7m/z 595.2 (M + H)+.

T369

57931-58005

Sentence

denotes

Anal. calcd for C31H35FN4O7·0.59 H2O·0.06 DCM: C, 61.28; H, 5.98; N, 9.20.

T370

58006-58040

Sentence

denotes

Found: C, 61.27; H, 5.98; N, 9.11.

T371

58042-58167

Sentence

denotes

(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 4-Chlorobenzoate (21)

T372

58168-58434

Sentence

denotes

Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting 4-chlorobenzoic acid and making noncritical variations provided a crude product.

T373

58435-58565

Sentence

denotes

This material was purified by Biotage MPLC (25 M, 2.5–4.5% methanol/dichloromethane) to afford 172 mg (80%) of the title compound.

T374

58566-59113

Sentence

denotes

1H NMR (400 MHz, DMSO-d6) δ 11.58 (d, J = 1.8 Hz, 1H), 8.63 (d, J = 7.8 Hz, 1H), 8.46 (d, J = 7.6 Hz, 1H), 7.98 (d, J = 8.6 Hz, 2H), 7.65 (s, 1H), 7.62 (d, J = 8.6 Hz, 2H), 7.37 (d, J = 1.8 Hz, 1H), 7.08 (t, J = 8.0 Hz, 1H), 6.99 (d, J = 8.3 Hz, 1H), 6.50 (d, J = 7.6 Hz, 1H), 5.13 (d, J = 1.5 Hz, 1H), 5.07–5.23 (m, 1H), 4.44–4.52 (m, 2H), 3.87 (s, 3H), 3.04–3.14 (m, 2H), 2.28–2.38 (m, 1H), 2.06–2.26 (m, 1H), 1.97–2.06 (m, 1H), 1.51–1.78 (m, 5H), 0.94 (d, J = 6.3 Hz, 3H), 0.89 (d, J = 6.3 Hz, 3H); MS (ESI+) for C31H35ClN4O7m/z 611.1 (M + H)+.

T375

59114-59189

Sentence

denotes

Anal. calcd for C31H35ClN4O7·0.72 H2O·0.05 DCM: C, 59.36; H, 5.86; N, 8.92.

T376

59190-59224

Sentence

denotes

Found: C, 59.31; H, 5.99; N, 9.23.

T377

59226-59355

Sentence

denotes

(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 2,6-Dichlorobenzoate (22)

T378

59356-59626

Sentence

denotes

Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting 2,6-dichlorobenzoic acid and making noncritical variations provided a crude product.

T379

59627-59766

Sentence

denotes

This material was purified by LC (20 g 230–400 SiO2, 3% methanol/chloroform) to afford 114 mg (65%) of the title compound as a white solid.

T380

59767-60203

Sentence

denotes

1H NMR (DMSO-d6) δ 11.57 (d, J = 2 Hz, 1H), 8.62 (d, J = 8 Hz, 1H), 8.46 (d, J = 4 Hz, 1H), 7.65–7.53 (m, 4H), 7.36 (s, 1H), 7.08 (t, J = 8 Hz, 1H), 6.99 (d, J = 8 Hz, 1H), 6.49 (d, J = 8 Hz, 1H), 5.19 (d, J = 20 Hz, 1H), 5.15 (d, J = 20 Hz, 1H), 4.51 (m, 2H), 3.87 (s, 3H), 3.11 (m, 2H), 2.30 (m, 1H), 2.06 (m, 2H), 1.76–1.51 (m, 5H), 0.94 (d, J = 8 Hz, 3H), 0.89 (d, J = 8 Hz, 3H); MS (ESI+) for C31H34Cl2N4O7m/z 645.1 (M + H)+; Anal.

T381

60204-60264

Sentence

denotes

Calcd for C31H34Cl2N4O7·0.2 H2O: C, 57.36; H, 5.34; N, 8.63.

T382

60265-60299

Sentence

denotes

Found: C, 57.23; H, 5.55; N, 8.46.

T383

60300-60364

Sentence

denotes

HRMS (ESI+) calcd for C31H34Cl2N4O7+H1 645.1877, found 645.1871.

T384

60366-60495

Sentence

denotes

(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 2,6-Difluorobenzoate (23)

T385

60496-60775

Sentence

denotes

Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting 2,6-difluorobenzoic acid and making noncritical variations provided a crude brown oily solid.

T386

60776-60897

Sentence

denotes

This material was triturated with chloroform/ethyl acetate to afford 180 mg (75%) of the title compound as a white solid.

T387

60898-61420

Sentence

denotes

1H NMR (DMSO-d6) δ 11.65 (d, J = 2 Hz, 1H), 8.68 (d, J = 8 Hz, 1H), 8.54 (d, J = 8 Hz, 1H), 7.73–7.65 (m, 2H), 7.36 (s, 1H), 7.27 (t, J = 8 Hz, 1H), 7.08 (t, J = 8 Hz, 1H), 6.99 (d, J = 8 Hz, 1H), 6.49 (d, J = 8 Hz, 1H), 5.19 (d, J = 16 Hz, 1H), 5.14 (d, J = 16 Hz, 1H), 4.48 (m, 2H), 3.87 (s, 3H), 3.11 (m, 2H), 2.32 (m, 1H), 2.11–1.91 (m, 2H), 1.81–1.56 (m, 5H), 0.94 (d, J = 4 Hz, 3H), 0.89 (d, J = 4 Hz, 3H); MS (ESI+) for C31H34F2N4O7m/z 613.2 (M + H)+; HRMS (ESI+) calcd for C31H34F2N4O7+H1 613.2469, found 613.2476.

T388

61422-61557

Sentence

denotes

(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 4-Chloro-2-hydroxybenzoate (24)

T389

61558-61830

Sentence

denotes

Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting 4-chlorosalicylic acid and making noncritical variations provided a crude brown syrup.

T390

61831-61971

Sentence

denotes

This material was purified by Biotage MPLC (25 M, 2–3% methanol/dichloromethane) to afford 64 mg (25%) of the title compound as a tan solid.

T391

61972-62488

Sentence

denotes

1H NMR (DMSO-d6) δ 11.58 (s, 1H), 10.50 (bs, 1H), 8.65 (d, J = 8 Hz, 1H), 8.46 (d, J = 8 Hz, 1H), 7.81 (d, J = 8 Hz, 1H), 7.63 (m, 1H), 7.37 (s, 1H), 7.08 (m, 2H), 7.00 (m, 2H), 6.50 (d, J = 8 Hz, 1H), 5.16 (d, J = 16 Hz, 2H), 5.11 (d, J = 16 Hz, 2H), 4.49 (m, 2H), 3.87 (s, 3H), 3.13 (m, 2H), 2.32 (m, 1H), 2.11–1.98 (m, 2H), 1.76–1.55 (m, 5H), 0.94 (d, J = 6 Hz, 3H), 0.89 (d, J = 6 Hz, 3H); MS (ESI-) for C31H35ClN4O8m/z 625.1 (M – H)−; anal. calcd for C31H35ClN4O8·0.1 H2O·0.14 CHCl3: C, 57.93; H, 5.52; N, 8.68.

T392

62489-62587

Sentence

denotes

Found: C, 58.04; H, 5.78; N, 8.74; HRMS (ESI+) calcd for C31H35ClN4O8+H1 627.2216, found 627.2219.

T393

62589-62722

Sentence

denotes

(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 4-Cyano-2-fluorobenzoate (25)

T394

62723-63006

Sentence

denotes

Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting 4-cyano-2-fluorobenzoic acid and making noncritical variations provided a crude oily brown solid.

T395

63007-63160

Sentence

denotes

This material was purified by Biotage MPLC (25 M, 2.5–3.5% methanol/dichloromethane) to afford 119 mg (48%) of the title compound as an off-white powder.

T396

63161-63703

Sentence

denotes

1H NMR (DMSO-d6) δ 11.58 (d, J = 2 Hz, 1H), 8.63 (d, J = 8 Hz, 1H), 8.46 (d, J = 8 Hz, 1H), 8.06 (m, 2H), 7.83 (d, J = 8 Hz, 1H), 7.65 (s, 1H), 7.36 (d, J = 2 Hz, 1H), 7.08 (t, J = 8 Hz, 1H), 6.99 (d, J = 8 Hz, 1H), 6.50 (d, J = 8 Hz, 1H), 5.16 (d, J = 16 Hz, 1H), 5.11 (d, J = 16 Hz, 1H), 4.49 (m, 2H), 3.87 (s, 3H), 3.11 (m, 2H), 2.32 (m, 1H), 2.10–1.98 (m, 2H), 1.77–1.53 (m, 5H), 0.94 (d, J = 6 Hz, 3H), 0.89 (d, J = 6 Hz, 3H); MS (ESI+) for C32H34FN5O7m/z 620.1 (M + H)+; anal. calcd for C32H34FN5O7·0.3 H2O: C, 61.49; H, 5.58; N, 11.20.

T397

63704-63802

Sentence

denotes

Found: C, 61.47; H, 5.61; N, 10.98; HRMS (ESI+) calcd for C32H34FN5O7+H1 620.2515, found 620.2532.

T398

63804-63933

Sentence

denotes

(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 2,6-Dimethylbenzoate (26)

T399

63934-64207

Sentence

denotes

Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting 2,6-dimethylbenzoic acid and making noncritical variations provided a crude brown foam.

T400

64208-64356

Sentence

denotes

This material was purified by Biotage MPLC (25 M, 3–4% methanol/dichloromethane) to afford 177 mg (73%) of the title compound as an off-white glass.

T401

64357-64836

Sentence

denotes

1H NMR (DMSO-d6) δ 11.58 (d, J = 2 Hz, 1H), 8.63 (d, J = 8 Hz, 1H), 8.46 (d, J = 8 Hz, 1H), 7.65 (s, 1H), 7.36 (s, 1H), 7.25 (t, J = 8 Hz, 1H), 7.08 (m, 3H), 7.00 (d, J = 8 Hz, 1H), 6.49 (d, J = 8 Hz, 1H), 5.11 (s, 2H), 4.51 (m, 2H), 3.87 (s, 3H), 3.10 (m, 2H), 2.32–2.21 (m, 7H), 2.07 (m, 2H), 1.78–1.53 (m, 5H), 0.94 (d, J = 8 Hz, 3H), 0.89 (d, J = 8 Hz, 3H); MS (ESI+) for C33H40N4O7m/z 605.2 (M + H)+; anal. calcd for C33H40N4O7·0.3 H2O·0.2 CHCl3: C, 62.90; H, 6.49; N, 8.84.

T402

64837-64871

Sentence

denotes

Found: C, 62.95; H, 6.42; N, 8.72.

T403

64872-64933

Sentence

denotes

HRMS (ESI+) calcd for C33H40N4O7+H1 605.2970, found 605.2985.

T404

64935-65065

Sentence

denotes

(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 2,6-Dimethoxybenzoate (27)

T405

65066-65340

Sentence

denotes

Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting 2,6-dimethoxybenzoic acid and making noncritical variations provided a crude brown foam.

T406

65341-65489

Sentence

denotes

This material was purified by Biotage MPLC (25 M, 3–4% methanol/dichloromethane) to afford 169 mg (66%) of the title compound as an off-white solid.

T407

65490-66041

Sentence

denotes

1H NMR (DMSO-d6) δ 11.58 (d, J = 2 Hz, 1H), 8.55 (d, J = 8 Hz, 1H), 8.45 (d, J = 8 Hz, 1H), 7.64 (s, 1H), 7.40–7.35 (m, 2H), 7.08 (t, J = 8 Hz, 1H), 7.00 (d, J = 8 Hz, 1H), 6.71 (d, J = 8 Hz, 1H), 6.50 (d, J = 8 Hz, 1H), 5.01 (d, J = 16 Hz, 1H), 4.95 (d, J = 16 Hz, 1H), 4.53–4.48 (m, 2H), 3.87 (s, 3H), 3.75 (s, 6H), 3.08 (m, 2H), 2.31 (m, 1H), 2.11–1.90 (m, 2H), 1.75–1.55 (m, 5H), 0.94 (d, J = 8 Hz, 3H), 0.89 (d, J = 8 Hz, 3H); MS (ESI+) for C33H40N4O9m/z 637.2 (M + H)+; anal. calcd for C33H40N4O9·0.25 H2O·0.25 CHCl3: C, 59.51; H, 6.12; N, 8.35.

T408

66042-66076

Sentence

denotes

Found: C, 59.49; H, 6.08; N, 8.42.

T409

66078-66202

Sentence

denotes

(3S)-3-({N-[(4-Methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 2-Cyanobenzoate (28)

T410

66203-66477

Sentence

denotes

Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting 2-cyanobenzoic acid and making noncritical variations provided a crude brown oily solid.

T411

66478-66598

Sentence

denotes

This material was triturated with chloroform/ethyl acetate to afford 91 mg (38%) of the title compound as a white solid.

T412

66599-67087

Sentence

denotes

1H NMR (DMSO-d6) δ 11.58 (s, 1H), 8.65 (d, J = 8 Hz, 1H), 8.46 (d, J = 8 Hz, 1H), 8.17 (m, 1H), 8.04 (m, 1H), 7.88 (m, 1H), 7.65 (s, 1H), 7.37 (s, 1H), 7.08 (t, J = 8 Hz, 1H), 6.99 (d, J = 8 Hz, 1H), 6.49 (d, J = 8 Hz, 1H), 5.19 (m, 2H), 4.50 (m, 2H), 3.87 (s, 3H), 3.11 (m, 2H), 2.32 (m, 1H), 2.07–1.97 (m, 2H), 1.76–1.56 (m, 5H), 0.94 (d, J = 6 Hz, 3H), 0.89 (d, J = 6 Hz, 3H); MS (ESI+) for C32H35N5O7m/z 602.2 (M + H)+; anal. calcd for C32H35N5O7·0.4 H2O: C, 63.12, H, 5.93; N, 11.50.

T413

67088-67185

Sentence

denotes

Found: C, 63.16; H, 5.96; N, 11.43; HRMS (ESI+) calcd for C32H35N5O7+H1 602.2609, found 602.2610.

T414

67187-67284

Sentence

denotes

(3S)-3-[(tert-Butoxycarbonyl)amino]-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 2,6-Dichlorobenzoate

T415

67285-67526

Sentence

denotes

A solution of tert-butyl ((1S)-3-bromo-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)carbamate (1.17 g, 3.2 mmol) in DMF (16 mL) was treated with 2,6-dichlorobenzoic acid (794 mg, 4.2 mmol) followed by cesium fluoride (1.18 g, 7.4 mmol).

T416

67527-67604

Sentence

denotes

The resulting suspension was placed in a preheated oil bath at 65 °C for 2 h.

T417

67605-67822

Sentence

denotes

The reaction was cooled to an ambient temperature, diluted with ethyl acetate (100 mL), washed once with water (40 mL) and with brine (40 mL), dried over MgSO4, filtered, and concentrated to give a crude yellow syrup.

T418

67823-67972

Sentence

denotes

This material was purified by Biotage MPLC (25 M, 4.5% methanol/dichloromethane) to afford 1.19 mg (80%) of the title compound as an off-white glass.

T419

67973-68238

Sentence

denotes

1H NMR (400 MHz, DMSO-d6) δ 7.60 (m, 5H), 5.35 (s, 2H), 4.32 (m, 1H), 3.23 (m, 2H), 2.24 (m, 1H), 2.11 (m, 1H), 1.86 (m, 1H), 1.67 (m, 2H), 1.35 (s, 9H); MS (ESI+) for C20H24Cl2N2O6m/z 481.0 (M + Na)+; HRMS (ESI+) calcd for C20H24Cl2N2O6+H 481.0903, found 481.0890.

T420

68240-68347

Sentence

denotes

N2-(tert-Butoxycarbonyl)-N1-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-l-leucinamide

T421

68348-68557

Sentence

denotes

A solution of (3S)-3-[(2S)-2-amino-4-chloro-3-oxobutyl]pyrrolidin-2-one hydrochloride (391 mg, 1.6 mmol) and Boc-Leu-OH (412 mg, 1.8 mmol) in DMF (9 mL) was placed under an atmosphere of N2 and cooled to 0 °C.

T422

68558-68695

Sentence

denotes

This clear pale yellow solution was successively treated with HATU (678 mg, 1.8 mmol) followed by N-methylmorpholine (0.41 mL, 3.7 mmol).

T423

68696-68861

Sentence

denotes

The reaction mixture gradually became opaque and after 1 h was quenched with 1:1 ice/sat aqueous NaHCO3 (40 mL) and extracted three times with ethyl acetate (40 mL).

T424

68862-68989

Sentence

denotes

The combined organics were washed once with brine (30 mL), dried over MgSO4, filtered, and concentrated to give a yellow syrup.

T425

68990-69219

Sentence

denotes

This material was purified by LC (50 g 230–400 SiO2, 3–5% methanol/chloroform) to afford 636 mg (40%) of N2-(tert-butoxycarbonyl)-N1-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-l-leucinamide as a white foam.

T426

69220-69577

Sentence

denotes

1H NMR (DMSO-d6) δ 8.47 (d, J = 8 Hz, 1H), 7.64 (s, 1H), 7.04 (d, J = 8 Hz, 1H), 4.60 (d, J = 16 Hz, 1H), 4.53 (d, J = 16 Hz, 1H), 4.40 (m, 1H), 3.90 (m, 1H), 3.16 (m, 1H), 3.08 (m, 1H), 2.24 (m, 1H), 2.10 (m, 1H), 1.98 (m, 1H), 1.63 (m, 2H), 1.45–1.37 (m, 11 H), 0.89 (d, J = 4 Hz, 3H), 0.85 (d, J = 4 Hz, 3H); MS (ESI+) for C19H32ClN3O5m/z 418.1 (M + H)+.

T427

69578-69643

Sentence

denotes

Anal. calcd for C19H32ClN3O5·0.6 H2O: C, 53.22; H, 7.81; N, 9.80.

T428

69644-69678

Sentence

denotes

Found: C, 53.00; H, 7.65; N, 9.54.

T429

69679-69742

Sentence

denotes

HRMS (ESI+) calcd for C19H32ClN3O5+H1 418.2103, found 418.2091.

T430

69744-69859

Sentence

denotes

(3S)-3-{[N-(tert-Butoxycarbonyl)-l-leucyl]amino}-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 2,6-Dichlorobenzoate (29)

T431

69860-70196

Sentence

denotes

Following the procedure described for the preparation of N2-(tert-butoxycarbonyl)-N1-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-l-leucinamide but substituting (3S)-3-[(tert-butoxycarbonyl)amino]-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 2,6-dichlorobenzoate and making noncritical variations provided a brown foam.

T432

70197-70348

Sentence

denotes

This material was purified by LC (50 g 230–400 SiO2, 2.5–3.5% methanol/chloroform) to afford 413 mg (61%) of the title compound as a light yellow foam.

T433

70349-70707

Sentence

denotes

1H NMR (DMSO-d6) δ 8.47 (d, J = 8 Hz, 1H), 7.60 (m, 4H), 7.05 (d, J = 8 Hz, 1H), 5.19 (d, J = 16 Hz, 1H), 5.13 (d, J = 16 Hz, 1H), 4.47 (m, 1H), 3.95 (m, 1H), 3.16 (m, 1H), 3.08 (m, 1H), 2.27 (m, 1H), 2.10 (m, 1H), 2.03 (m, 1H), 1.65 (m, 2H), 1.47–1.37 (m, 11 H), 0.89 (d, J = 4 Hz, 3H), 0.85 (d, J = 4 Hz, 3H); MS (ESI+) for C26H35Cl2N3O7m/z 572.1 (M + H)+.

T434

70708-70774

Sentence

denotes

Anal. calcd for C26H35Cl2N3O7·0.5 H2O: C, 53.70; H, 6.24; N, 7.23.

T435

70775-70874

Sentence

denotes

Found: C, 53.74; H, 6.31; N, 7.31; HRMS (ESI+) calcd for C26H35Cl2N3O7+Na 594.1744, found 594.1729.

T436

70876-70976

Sentence

denotes

(3S)-3-[(N-Acetyl-l-leucyl)amino]-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 2,6-Dichlorobenzoate (30)

T437

70977-71313

Sentence

denotes

Following the procedure described for the preparation of N2-acetyl-N1-[(1S)-1-(chloroacetyl)-4-(dimethylamino)-4-oxobutyl]-N2-methyl-l-leucinamide but substituting (3S)-3-{[N-(tert-butoxycarbonyl)-l-leucyl]amino}-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 2,6-dichlorobenzoate and making noncritical variations provided a pale yellow oil.

T438

71314-71460

Sentence

denotes

This material was purified by Biotage MPLC (25 M, 3.5–4.5% methanol/dichloromethane) to afford 161 mg (78%) of the title compound as a white foam.

T439

71461-71895

Sentence

denotes

1H NMR (DMSO-d6) δ 8.56 (d, J = 8 Hz, 1H), 8.06 (d, J = 8 Hz, 1H), 7.65–7.53 (m, 4H), 5.17 (d, J = 16 Hz, 1H), 5.10 (d, J = 16 Hz, 1H), 4.44 (m, 1H), 4.25 (m, 1H), 3.12 (m, 2H), 2.25 (m, 1H), 2.06 (m, 1H), 1.97 (m, 1H), 1.84 (s, 3H), 1.63 (m, 3H), 1.45 (m, 2H), 0.90 (d, J = 8 Hz, 3H), 0.85 (d, J = 8 Hz, 3H); MS (ESI+) for C23H29Cl2N3O6m/z 514.0 (M + H)+; anal. calcd for C23H29Cl2N3O6·0.25 H2O·0.1 EtOAc: C, 53.26; H, 5.79; N, 7.96.

T440

71896-71930

Sentence

denotes

Found: C, 53.27; H, 5.82; N, 7.84.

T441

71931-71995

Sentence

denotes

HRMS (ESI+) calcd for C23H29Cl2N3O6+H1 514.1506, found 514.1508.

T442

71997-72121

Sentence

denotes

(3S)-3-{[N-(1H-Benzimidazol-2-ylcarbonyl)-l-leucyl]amino}-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 2,6-Dichlorobenzoate (31)

T443

72122-72550

Sentence

denotes

Following the procedure described for the preparation of N-((1S)-1-{[((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-1H-indole-2-carboxamide but substituting (3S)-3-{[N-(tert-butoxycarbonyl)-l-leucyl]amino}-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 2,6-dichlorobenzoate and 1H-benzimidazole-2-carboxylic acid and making noncritical variations provided a crude yellow oil.

T444

72551-72697

Sentence

denotes

This material was purified by Biotage MPLC (25 M, 3–4% methanol/dichloromethane) to afford 95 mg (72%) of the title compound as an off-white foam.

T445

72698-73060

Sentence

denotes

1H NMR (DMSO-d6) δ 13.32 (s, 1H), 8.89 (d, J = 8 Hz, 1H), 8.60 (d, J = 8 Hz, 1H), 7.74 (d, J = 8 Hz, 1H), 7.59 (m, 5H), 7.29 (m, 2H), 5.20 (d, J = 20 Hz, 1H), 5.15 (d, J = 20 Hz, 1H), 4.56 (m, 2H), 3.12 (m, 2H), 2.30 (m, 1H), 2.01 (m, 2H), 1.83 (m, 1H), 1.65 (m, 4H), 0.92 (d, J = 8 Hz, 3H), 0.89 (d, J = 8 Hz, 3H); MS (ESI+) for C29H31Cl2N5O6m/z 616.0 (M + H)+.

T446

73061-73120

Sentence

denotes

Anal. calcd for C29H31Cl2N5O6: C, 56.50; H, 5.07; N, 11.36.

T447

73121-73156

Sentence

denotes

Found: C, 57.28; H, 5.32; N, 11.35.

T448

73157-73221

Sentence

denotes

HRMS (ESI+) calcd for C29H31Cl2N5O6+H1 616.1724, found 616.1729.

T449

73223-73360

Sentence

denotes

(3S)-3-({4-Methyl-N-[(2R)-tetrahydrofuran-2-ylcarbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 2,6-Dichlorobenzoate (3)

T450

73361-73772

Sentence

denotes

Following the procedure described for the preparation of (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl acetate but substituting N1-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-4-methyl-N2-[(2R)-tetrahydrofuran-2-ylcarbonyl]-l-leucinamide and 2,6-dichlorobenzoic acid and making noncritical variations provided a light amber residue.

T451

73773-73985

Sentence

denotes

The residue was purified by preparative HPLC (Luna 10μ C18) eluting with a gradient of MeCN containing 0.1% AcOH in water containing 0.1% AcOH to give 0.155 g (54%) of the title compound as a cream colored solid.

T452

73986-74351

Sentence

denotes

1H NMR (300 MHz, DMSO-d6) δ 8.52 (d, J = 8 Hz, 1H), 7.79 (d, J = 8 Hz, 1H), 7.71 (s, 1H), 7.66–7.55 (m, 3H), 5.18 (s, 2H), 4.54–4.40 (m, 1H), 4.37–4.35 (m, 1H), 4.25 (m, 1H), 3.98–3.91 (m, 1H), 3.84–3.72 (m, 1H), 3.23–3.07 (m, 2H), 2.33–2.23 (m, 1H), 2.16–2.05 (m, 2H), 1.86–1.74 (m, 3H), 1.72–1.62 (m, 5H), 0.89 (s, 9H); MS (ESI+) for C27H35Cl2N3O7m/z 584 (M + H).

T453

74352-74418

Sentence

denotes

Anal. calcd for C27H35Cl2N3O7·0.5 H2O: C, 54.64; H, 6.11; N, 7.08.

T454

74419-74453

Sentence

denotes

Found: C, 54.26; H, 6.00; N, 6.87.

T455

74454-74518

Sentence

denotes

HRMS (ESI+) calcd for C27H35Cl2N3O7+H1 584.1925, found 584.1921.

T456

74520-74641

Sentence

denotes

N2-(tert-Butoxycarbonyl)-N1-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-4-methyl-l-leucinamide (32)

T457

74642-74905

Sentence

denotes

Following the procedure described for the preparation of N2-(tert-butoxycarbonyl)-N1-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-l-leucinamide but substituting Boc-(β t-butyl)-Ala-OH and making noncritical variations provided a golden syrup.

T458

74906-75053

Sentence

denotes

This material was purified by Biotage MPLC (65i column, 2.5–3.5% methanol/chloroform) to afford 3.41 g (40%) of the title compound as a white foam.

T459

75054-75404

Sentence

denotes

1H NMR (DMSO-d6) δ 8.39 (d, J = 8 Hz, 1H), 7.64 (s, 1H), 7.00 (d, J = 8 Hz, 1H), 4.58 (d, J = 16 Hz, 1H), 4.52 (d, J = 16 Hz, 1H), 4.38 (m, 1H), 3.92 (m, 1H), 3.15 (t, J = 8 Hz, 1H), 3.07 (q, J = 8 Hz, 1H), 2.22 (m, 1H), 2.09 (m, 1H), 1.97 (m, 1H), 1.62 (m, 2H), 1.50 (m, 2H), 1.36 (s, 9H), 0.88 (s, 9H); MS (ESI+) for C20H34ClN3O6m/z 432.1 (M + H)+.

T460

75406-75532

Sentence

denotes

N1-((1S)-3-(Benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-N2-(tert-butoxycarbonyl)-4-methyl-l-leucinamide (33)

T461

75533-75826

Sentence

denotes

Following the procedure described for the preparation of tert-butyl [(1S)-2-[((1S)-3-(benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]-1-(cyclohexylmethyl)-2-oxoethyl]carbamate but substituting Boc-(β t-butyl)-Ala-OH and making noncritical variations provided a brown syrup.

T462

75827-75976

Sentence

denotes

This material was purified by LC (33 g 230–400 SiO2, 1–2.5% methanol/chloroform) to afford 258 mg (55%) of the title compound as a light yellow foam.

T463

75977-76299

Sentence

denotes

1H NMR (DMSO-d6) δ 8.23 (d, J = 8 Hz, 1H), 7.62 (s, 1H), 7.32 (m, 5H), 6.96 (d, J = 8 Hz, 1H), 4.46 (s, 2H), 4.35–4.33 (m, 2H), 4.21 (d, J = 16 Hz, 1H), 3.95 (m, 1H), 3.09 (m, 2H), 2.24 (m, 1H), 2.07 (m, 1H), 1.92 (m, 1H), 1.58 (m, 2H), 1.46 (m, 2H), 1.35 (s, 9H), 0.87 (m, 9H); MS (ESI+) for C27H41N3O6m/z 504.2 (M + H)+.

T464

76301-76456

Sentence

denotes

N-((1S)-1-{[((1S)-3-(Benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3,3-dimethylbutyl)-4-methoxy-1H-indole-2-carboxamide (34)

T465

76457-76866

Sentence

denotes

Following the procedure described for the preparation of N-[(1S)-2-[((1S)-3-(benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]-1-(cyclohexylmethyl)-2-oxoethyl]-4-methoxy-1H-indole-2-carboxamide but substituting N1-((1S)-3-(benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-N2-(tert-butoxycarbonyl)-4-methyl-l-leucinamide and making noncritical variations provided a golden syrup.

T466

76867-77014

Sentence

denotes

This material was purified by Biotage MPLC (25 M column, 3% methanol/chloroform) to afford 173 mg (60%) of the title compound as an off-white foam.

T467

77015-77469

Sentence

denotes

1H NMR (400 MHz, DMSO-d6) δ 11.58 (s, 1H), 8.43 (app t, J = 8 Hz, 2H), 7.62 (s, 1H), 7.32 (m, 6H), 7.08 (t, J = 8 Hz, 1H), 7.00 (d, J = 8 Hz, 1H), 6.49 (d, J = 8 Hz, 1H), 4.52 (m, 1H), 4.47–4.35 (m, 4H), 4.22 (d, J = 16 Hz, 1H), 3.87 (s, 3H), 3.05 (m, 2H), 2.28 (m, 1H), 2.06 (m, 1H), 1.93 (m, 1H), 1.64–1.57 (m, 3H), 0.92 (s, 9H); MS (ESI+) for C32H40N4O6m/z 577.2 (M + H)+; anal. calcd for C32H40N4O6·0.33 methanol·0.25 H2O: C, 65.62; H, 7.13; N, 9.47.

T468

77470-77563

Sentence

denotes

Found: C, 65.65; H, 6.90; N, 9.58; HRMS (ESI+) calcd for C32H40N4O6 577.3021, found 577.3001.

T469

77565-77715

Sentence

denotes

N-((1S)-1-{[((1S)-3-Chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3,3-dimethylbutyl)-4-methoxy-1H-indole-2-carboxamide (35)

T470

77716-78112

Sentence

denotes

Following the procedure described for the preparation of N-((1S)-1-{[((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-1H-indole-2-carboxamide but substituting N2-(tert-butoxycarbonyl)-N1-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-4-methyl-l-leucinamide and making noncritical variations provided a pale yellow solid.

T471

78113-78264

Sentence

denotes

This material was purified by Biotage MPLC (40 M column, 2–4.5% methanol/chloroform) to afford 920 g (60%) of the title compound as an off-white solid.

T472

78265-78786

Sentence

denotes

1H NMR (400 MHz, DMSO-d6) δ 11.59 (d, J = 1.8 Hz, 1H), 8.54 (d, J = 7.8 Hz, 1H), 8.45 (d, J = 8.1 Hz, 1H), 7.64 (s, 1H), 7.32 (d, J = 1.8 Hz, 1H), 7.08 (t, J = 8.0 Hz, 1H), 7.00 (d, J = 8.3 Hz, 1H), 6.49 (d, J = 7.6 Hz, 1H), 4.52–4.62 (m, 1H), 4.40–4.51 (m, 2H), 3.87 (s, 3H), 3.01–3.16 (m, 2H), 2.19–2.31 (m, 1H), 2.03–2.12 (m, 1H), 1.91–2.01 (m, 1H), 1.81 (dd, J = 14.1, 9.9 Hz, 1H), 1.54–1.72 (m, 3H), 0.93 (s, 9H); MS (ESI+) for C25H33ClN4O5m/z 505.1 (M + H)+; HRMS (ESI+) calcd for (M + H)+ 505.2212, found 505.2204.

T473

78788-78939

Sentence

denotes

N-((1S)-1-{[((1S)-3-Hydroxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3,3-dimethylbutyl)-4-methoxy-1H-indole-2-carboxamide (36)

T474

78940-79366

Sentence

denotes

Following the procedure described for the preparation of N-((1S)-1-{[((1S)-3-hydroxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-1H-indole-2-carboxamide but substituting N-((1S)-1-{[((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3,3-dimethylbutyl)-4-methoxy-1H-indole-2-carboxamide and making noncritical variations provided a crude brown glass.

T475

79367-79513

Sentence

denotes

This material was purified by Biotage MPLC (40 M column, 2–10% methanol/chloroform) to afford 415 mg (53%) of the title compound as a white solid.

T476

79514-79557

Sentence

denotes

MS (ESI+) for C25H34N4O6m/z 487.2 (M + H)+.

T477

79558-80004

Sentence

denotes

Alternatively, following the procedure described for the preparation of N-{(1S)-1-(cyclohexylmethyl)-2-[((1S)-3-hydroxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]-2-oxoethyl}-4-methoxy-1H-indole-2-carboxamide but substituting N-((1S)-1-{[((1S)-3-(benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3,3-dimethylbutyl)-4-methoxy-1H-indole-2-carboxamide and making noncritical variations provided a tan solid.

T478

80005-80150

Sentence

denotes

This material was purified by Biotage MPLC (25 M column, 6–7% methanol/chloroform) to afford 100 mg (77%) of the title compound as a white solid.

T479

80151-80664

Sentence

denotes

1H NMR (DMSO-d6) δ 11.58 (s, 1H), 8.42 (d, J = 8 Hz, 1H), 8.37 (d, J = 8 Hz, 1H), 7.62 (s, 1H), 7.32 (s, 1H), 7.08 (t, J = 8 Hz, 1H), 6.99 (d, J = 8 Hz, 1H), 6.49 (d, J = 8 Hz, 1H), 5.04 (t, J = 8 Hz, 1H), 4.52 (m, 1H), 4.42 (m, 1H), 4.24 (dd, J = 8, 20 Hz, 1H), 4.12 (dd, J = 8, 20 Hz, 1H), 3.87 (s, 3H), 3.09 (m, 2H), 2.25 (m, 1H), 2.06 (m, 1H), 1.93 (m, 1H), 1.80 (dd, J = 8, 16 Hz, 1H), 0.93 (s, 9H); MS (ESI+) for C25H34N4O6m/z 487.2 (M + H)+; anal. calcd for C25H34N4O6·0.3 H2O: C, 61.03; H, 7.09; N, 11.39.

T480

80665-80759

Sentence

denotes

Found: C, 61.07; H, 7.09; N, 11.22; HRMS (ESI+) calcd for C25H34N4O6 487.2551, found 487.2541.

T481

80761-80912

Sentence

denotes

4-Methoxy-N-((1S)-1-{[((1S)-3-methoxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3,3-dimethylbutyl)-1H-indole-2-carboxamide (37)

T482

80913-81337

Sentence

denotes

Following the procedure described for the preparation of 4-methoxy-N-((1S)-1-{[((1S)-3-methoxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-4-methylpentyl)-1H-indole-2-carboxamide but substituting N-((1S)-1-{[((1S)-3-hydroxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3,3-dimethylbutyl)-4-methoxy-1H-indole-2-carboxamide and making noncritical variations provided a crude product.

T483

81338-81483

Sentence

denotes

This material was purified by Biotage MPLC (40S column, 2–5% methanol/chloroform) to afford 21 mg (9%) of the title compound as a pale tan solid.

T484

81484-81999

Sentence

denotes

1H NMR (400 MHz, DMSO-d6) δ 11.58 (s, 1H), 8.42 (d, J = 6.8 Hz, 1H), 7.62 (s, 1H), 7.32 (s, 1H), 7.08 (t, J = 7.7 Hz, 1H), 7.00 (d, J = 7.8 Hz, 1H), 6.49 (d, J = 7.3 Hz, 1H), 4.49–4.55 (m, 1H), 4.32–4.41 (m, 1H), 4.21–4.30 (m, 1H), 4.12 (t, J = 16.9 Hz, 1H), 3.87 (s, 3H), 3.23 (s, 3H), 3.01–3.16 (m, 2H), 2.20–2.32 (m, 1H), 2.03–2.15 (m, 1H), 1.87–1.98 (m, 1H), 1.73–1.85 (m, 1H), 1.54–1.70 (m, 3H), 0.93 (s, 9H); MS (ESI+) for C26H36N4O6m/z 501.2 (M + H)+; HRMS (ESI+) calcd for (M + H)+ 501.2708, found 501.2701.

T485

82001-82147

Sentence

denotes

N-((1S)-1-{[((1S)-3-Hydroxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-1H-indole-2-carboxamide (4)

T486

82148-82419

Sentence

denotes

A solution of N-((1S)-1-{[((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-1H-indole-2-carboxamide (488 mg, 0.99 mmol) and benzoylformic acid (195 mg, 1.3 mmol) in DMF (6.5 mL) was placed under an atmosphere of N2.

T487

82420-82533

Sentence

denotes

This clear pale yellow solution was treated with cesium fluoride (350 mg, 2.3 mmol) followed by heating to 65 °C.

T488

82534-82891

Sentence

denotes

After 4 h, the now yellow suspension was cooled to RT, diluted with ethyl acetate (60 mL), washed three times with water (30 mL) and once with brine (30 mL), dried over MgSO4, filtered, and concentrated to give (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl oxo(phenyl)acetate as a crude yellow foam.

T489

82892-82935

Sentence

denotes

MS (ESI+) for C32H36N4O8m/z 605.2 (M + H)+.

T490

82936-83218

Sentence

denotes

A solution of the crude (3S)-3-({N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-leucyl}amino)-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl oxo(phenyl)acetate in methanol (40 mL) was placed under an atmosphere of N2 and treated with potassium carbonate (7 mg, 0.05 mmol) with vigorous stirring.

T491

83219-83310

Sentence

denotes

After 1 h, the volatiles were removed in vacuo (bath < 30 °C) to give a crude yellow glass.

T492

83311-83459

Sentence

denotes

This material was purified by Biotage MPLC (25 M column, 6% methanol/chloroform) to afford 346 mg (73%) of the title compound as an off-white solid.

T493

83460-83918

Sentence

denotes

1H NMR (DMSO-d6) δ 11.56 (s, 1H), 8.44 (d, J = 8 Hz, 1H), 8.39 (d, J = 8 Hz, 1H), 7.61 (s, 1H), 7.35 (s, 1H), 7.08 (t, J = 8 Hz, 1H), 6.99 (d, J = 8 Hz, 1H), 6.49 (d, J = 8 Hz, 1H), 5.04 (t, J = 8 Hz, 1H), 4.46 (m, 2H), 4.25 (dd, J = 8, 20 Hz, 1H), 4.13 (dd, J = 8, 20 Hz, 1H), 3.87 (s, 3H), 3.10 (m, 2H), 2.28 (m, 1H), 2.08 (m, 1H), 1.92 (m, 1H), 1.70–1.53 (m, 5H), 0.93 (d, J = 8 Hz, 3H), 0.89 (d, J = 8 Hz, 3H); MS (ESI+) for C24H32N4O6m/z 473.2 (M + H)+.

T494

83920-84042

Sentence

denotes

N1-((1S)-3-(Benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-N2-(tert-butoxycarbonyl)-N2-methyl-l-leucinamide

T495

84043-84334

Sentence

denotes

Following the procedure described for the preparation of tert-butyl [(1S)-2-[((1S)-3-(benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]-1-(cyclohexylmethyl)-2-oxoethyl]carbamate but substituting Boc-N-methyl-Leu and making noncritical variations provided a crude brown oil.

T496

84335-84492

Sentence

denotes

This material was purified by Biotage flash chromatography, eluting with methanol/dichloromethane to afford the title compound (123 mg, 47%) as a yellow gum.

T497

84493-84756

Sentence

denotes

1H NMR (400 MHz, CDCl3) δ 7.22–7.33 (m, 5H), 5.76 (bd, 1H), 4.73 (s, 1H), 4.46–4.59 (m, 2H), 4.08–4.27 (m, 2H), 3.14–3.28 (m, 2H), 2.68 (s, 3H), 2.16–2.38 (m, 2H), 1.88–2.00 (m, 1H), 1.56–1.86 (m, 5H), 1.41 (s, 9H), 0.94 (d, J = 8 Hz, 3H), 0.89 (d, J = 8 Hz, 3H).

T498

84758-84913

Sentence

denotes

N-((1S)-1-{[((1S)-3-(Benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-N-methyl-1H-indole-2-carboxamide

T499

84914-85327

Sentence

denotes

Following the procedure described for the preparation of N-[(1S)-2-[((1S)-3-(benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]-1-(cyclohexylmethyl)-2-oxoethyl]-4-methoxy-1H-indole-2-carboxamide but substituting N1-((1S)-3-(benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-N2-(tert-butoxycarbonyl)-N2-methyl-l-leucinamide and making noncritical variations provided a crude brown oil.

T500

85328-85482

Sentence

denotes

This material was purified by Biotage flash chromatography, eluting with methanol/dichloromethane to afford the title compound as a clear oil, 67 mg, 48%.

T501

85483-85937

Sentence

denotes

1H NMR (400 MHz, CDCl3) δ 9.83 (s, 1H), 7.95 (s, 1H), 7.26–7.36 (m, 5H), 7.18 (t, J = 8.0 Hz, 1H), 7.04 (d, J = 8.3 Hz, 1H), 7.00 (s, 1H), 6.48 (d, J = 7.8 Hz, 1H), 5.87 (s, 1H), 5.18–5.27 (m, 1H), 4.70 (s, 1H), 4.50–4.63 (m, 2H), 4.16–4.36 (m, 2H), 3.93 (s, 3H), 3.32 (s, 3H), 3.03–3.18 (m, 2H), 2.22 (s, 2H), 1.91–2.02 (m, 1H), 1.81 (t, J = 7.3 Hz, 3H), 1.66–1.72 (m, 1H), 1.55 (s, 1H), 0.87–1.01 (m, 6H); MS (API-ES−) for C32H40N4O6m/z 576.7 (M – H)−.

T502

85939-86095

Sentence

denotes

N-((1S)-1-{[((1S)-3-Hydroxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-N-methyl-1H-indole-2-carboxamide (38)

T503

86096-86540

Sentence

denotes

Following the procedure described for the preparation of N-{(1S)-1-(cyclohexylmethyl)-2-[((1S)-3-hydroxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]-2-oxoethyl}-4-methoxy-1H-indole-2-carboxamide but substituting N-((1S)-1-{[((1S)-3-(benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-N-methyl-1H-indole-2-carboxamide and making noncritical variations provided a crude clear glass.

T504

86541-86696

Sentence

denotes

This material was purified by Biotage flash chromatography, eluting with methanol/dichloromethane to afford the title compound as a white foam, 42 mg, 82%.

T505

86697-87083

Sentence

denotes

1H NMR (400 MHz, CDCl3) δ 10.16 (s, 1H), 8.26 (s, 1H), 7.18 (t, J = 8.0 Hz, 1H), 7.05 (d, J = 8.1 Hz, 1H), 6.99 (d, J = 2.3 Hz, 1H), 6.48 (d, J = 7.6 Hz, 1H), 6.09 (s, 1H), 5.14–5.27 (m, 1H), 4.51–4.65 (m, 1H), 4.25–4.50 (m, 2H), 3.94 (s, 3H), 3.26–3.46 (m, 3H), 3.11–3.24 (m, 1H), 3.01–3.11 (m, 1H), 1.48–2.39 (m, 9H), 0.88–1.02 (m, 6H); MS (API-ES–-) for C25H34N4O6m/z 485.3 (M – H)−.

T506

87085-87195

Sentence

denotes

N2-(tert-Butoxycarbonyl)-N1-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-l-norleucinamide

T507

87196-87450

Sentence

denotes

Following the procedure described for the preparation of N2-(tert-butoxycarbonyl)-N1-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-l-leucinamide but substituting Boc-NorLeu-OH and making noncritical variations provided a golden syrup.

T508

87451-87603

Sentence

denotes

This material was purified by LC (100 g 230–400 SiO2, 2.5–3.5% methanol/chloroform) to afford 727 mg (42%) of the title compound as a light yellow foam.

T509

87604-87755

Sentence

denotes

1H NMR (DMSO-d6) δ 8.45 (d, J = 8 Hz, 1H), 7.62 (s, 1H), 7.00 (d, J = 8 Hz, 1H), 4.59 (d, J = 16 Hz, 1H), 4.53 (d, J = 16 foam in a 42% isolated yield.

T510

87756-87982

Sentence

denotes

1H Hz, 1H), 4.38 (m, 1H), 3.80 (m, 1H), 3.15 (m, 1H), 3.06 (m, 1H), 2.22 (m, 1H), 2.07 (m, 1H), 1.98 (m, 1H), 1.63–1.51 (m, 4H), 1.36 (m, 9H), 1.24 (m, 4H), 0.83 (t, J = 8 Hz, 3H); MS (ESI+) for C19H32ClN3O5m/z 418.1 (M + H)+.

T511

87984-88117

Sentence

denotes

N-((1S)-1-{[((1S)-3-Chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}pentyl)-4-methoxy-1H-indole-2-carboxamide

T512

88118-88514

Sentence

denotes

Following the procedure described for the preparation of N-((1S)-1-{[((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-1H-indole-2-carboxamide but substituting N2-(tert-butoxycarbonyl)-N1-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-l-norleucinamide and making no other critical variations provided a crude yellow foam.

T513

88515-88663

Sentence

denotes

This material was purified by Biotage MPLC (25 M column, 3% methanol/chloroform) to afford 254 mg (63%) of the title compound as an off-white solid.

T514

88664-89156

Sentence

denotes

1H NMR (DMSO-d6) δ 11.56 (s, 1H), 8.57 (d, J = 8 Hz, 1H), 8.39 (d, J = 4 Hz, 1H), 7.62 (s, 1H), 7.36 (s, 1H), 7.08 (t, J = 8 Hz, 1H), 6.99 (d, J = 8 Hz, 1H), 6.49 (d, J = 8 Hz, 1H), 4.58 (d, J = 16 Hz, 1H), 4.56 (d, J = 16 Hz, 1H), 4.43 (m, 1H), 4.35 (m, 1H), 3.87 (s, 3H), 3.09 (m, 2H), 2.27 (m, 1H), 2.08 (m, 1H), 1.97 (m, 1H), 1.74–1.54 (m, 4H), 1.30 (m, 4H), 0.86 (t, J = 8 Hz, 3H); MS (ESI+) for C24H31N4O5Cl m/z 491.1 (M + H)+; anal. calcd for C24H31ClN4O5: C, 58.71; H, 6.36; N, 11.41.

T515

89157-89192

Sentence

denotes

Found: C, 58.66; H, 6.45; N, 11.22.

T516

89194-89333

Sentence

denotes

N-((1S)-1-{[((1S)-3-Hydroxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}pentyl)-4-methoxy-1H-indole-2-carboxamide (39)

T517

89334-89751

Sentence

denotes

Following the procedure described for the preparation of N-((1S)-1-{[((1S)-3-hydroxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-1H-indole-2-carboxamide but substituting of N-((1S)-1-{[((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}pentyl)-4-methoxy-1H-indole-2-carboxamide and making noncritical variations provided a crude yellow foam.

T518

89752-89895

Sentence

denotes

This material was purified by Biotage MPLC (25 M column, 5–6% methanol/chloroform) to afford 82 mg (35%) of the title compound as a white foam.

T519

89896-90427

Sentence

denotes

1H NMR (DMSO-d6) δ 11.57 (s, 1H), 8.42 (d, J = 8 Hz, 1H), 8.38 (d, J = 8 Hz, 1H), 7.62 (s, 1H), 7.35 (s, 1H), 7.09 (t, J = 8 Hz, 1H), 6.99 (d, J = 8 Hz, 1H), 6.49 (d, J = 8 Hz, 1H), 5.06 (t, J = 8 Hz, 1H), 4.44 (m, 2H), 4.25 (dd, J = 8, 20 Hz, 1H), 4.14 (dd, J = 8, 20 Hz, 1H), 3.87 (s, 3H), 3.08 (m, 2H), 2.28 (m, 1H), 2.10 (m, 1H), 1.91 (m, 1H), 1.74–1.54 (m, 4H), 1.32 (m, 4H), 0.87 (t, J = 8 Hz, 3H); MS (ESI+) for C24H32N4O6m/z 473.2 (M + H)+; anal. calcd for C24H32N4O6·0.6 H2O·0.2 ethyl acetate: C, 59.46; H, 7.01; N, 11.18.

T520

90428-90525

Sentence

denotes

Found: C, 53.37; H, 6.94; N, 11.23; HRMS (ESI+) calcd for C24H32N4O6+H1 473.2395, found 473.2382.

T521

90527-90594

Sentence

denotes

Methyl N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-5-methyl-l-norleucinate

T522

90595-90823

Sentence

denotes

To a solution of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-5-methyl-l-norleucine (2.14 g, 5.8 mmol) in methanol (15 mL) is added toluene (30 mL) followed by the dropwise addition of TMS-diazomethane (2.9 mL, 2 M in hexane, 5.8 mmol).

T523

90824-90939

Sentence

denotes

TLC analysis indicated incomplete reaction, and TMS-diazomethane was added dropwise until a yellow color persisted.

T524

90940-91046

Sentence

denotes

At this time, the reaction was quenched by the addition of AcOH (1 mL) followed by concentration in vacuo.

T525

91047-91198

Sentence

denotes

The residue was purified by Biotage flash chromatography, eluting with ethyl acetate/hexane to afford the title compound as a white solid, 2.18 g, 98%.

T526

91199-91590

Sentence

denotes

1H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 7.6 Hz, 2H), 7.60 (dd, J = 7.2, 3.9 Hz, 2H), 7.40 (t, J = 7.2 Hz, 2H), 7.31 (t, J = 7.5 Hz, 2H), 5.26 (d, J = 8.6 Hz, 1H), 4.31–4.51 (m, 3H), 4.23 (t, J = 7.1 Hz, 1H), 3.75 (s, 3H), 1.78–1.93 (m, 1H), 1.60–1.76 (m, 1H), 1.45–1.60 (m, 1H), 1.05–1.34 (m, 2H), 0.88 (d, J = 4 Hz, 3H), 0.86 (d, J = 4 Hz, 3H); MS (APCI+) for C23H27NO4m/z 160.1 (M-Fmoc+H)+.

T527

91592-91646

Sentence

denotes

Methyl N-(tert-Butoxycarbonyl)-5-methyl-l-norleucinate

T528

91647-91946

Sentence

denotes

To a solution of methyl N-[(9H-fluoren-9-ylmethoxy)carbonyl]-5-methyl-l-norleucinate (2.18 g, 5.72 mmol) in DMF (50 mL) was added KF (2.33 g, 40.04 mmol) followed by triethylamine (1.70 mL, 12.24 mmol) and di-tert-butyl dicarbonate (7.39 mmol), and the mixture was stirred at an ambient temperature.

T529

91947-92116

Sentence

denotes

After 4 h, TLC analysis indicated incomplete reaction and the reaction mixture was treated with a second portion of KF (2.7 g, 46.55 mmol) and BOC2O (800 mg, 3.67 mmol).

T530

92117-92199

Sentence

denotes

After 16 h, the mixture was diluted with diethyl ether (300 mL), washed with satd.

T531

92200-92532

Sentence

denotes

NaHCO3 (2 × 50 mL), 1 M hydrochloric acid (2 × 50 mL), NaHCO3 (50 mL), and brine (50 mL), dried over MgSO4, filtered, and the solvents were evaporated in vacuo to yield the crude product, which was purified by Biotage flash chromatography eluting with dichloromethane/hexane to afford the title compound as a clear oil, 980 mg, 66%.

T532

92533-92783

Sentence

denotes

1H NMR (400 MHz, CDCl3) δ 4.96 (d, J = 6.8 Hz, 1H), 4.21–4.32 (m, 1H), 3.72 (s, 3H), 1.72–1.85 (m, 1H), 1.46–1.66 (m, 2H), 1.43 (s, 9H), 1.11–1.29 (m, 2H), 0.88 (d, J = 4 Hz, 3H), 0.86 (d, J = 4 Hz, 3H); MS (API-ES+) for C13H25NO4m/z 282.2 (M + Na)+.

T533

92785-92830

Sentence

denotes

N-(tert-Butoxycarbonyl)-5-methyl-l-norleucine

T534

92831-93128

Sentence

denotes

To a solution of methyl N-(tert-butoxycarbonyl)-5-methyl-l-norleucinate (980 mg, 3.78 mmol) in THF (30 mL) at 0 °C was added a solution (precooled to 5 °C) of LiOH (1 M, 11.3 mL, 11.33 mmol), and the resulting mixture was stirred at 0 °C for 1 h and then allowed to warm to an ambient temperature.

T535

93129-93236

Sentence

denotes

The reaction was acidified to pH 2 with 1 M hydrochloric acid and extracted with ethyl acetate (3 × 60 mL).

T536

93237-93413

Sentence

denotes

The combined organics were washed with brine (100 mL), dried over MgSO4, filtered, and the solvent was removed in vacuo to yield the title compound as a clear oil, 990 mg, 99%.

T537

93414-93669

Sentence

denotes

1H NMR (400 MHz, CDCl3) δ 4.96 (d, J = 7.8 Hz, 1H), 4.23–4.34 (m, 1H), 1.75–1.93 (m, 2H), 1.60–1.72 (m, 1H), 1.50–1.59 (m, 1H), 1.44 (s, 9H), 1.19–1.30 (m, 1H), 0.88 (d, J = 4 Hz, 3H), 0.86 (d, J = 4 Hz, 3H); MS (API-ES+) for C12H23NO4m/z 268.1 (M + Na)+.

T538

93671-93790

Sentence

denotes

N2-(tert-Butoxycarbonyl)-N1-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-5-methyl-l-norleucinamide

T539

93791-94081

Sentence

denotes

Following the procedure described for the preparation of N2-(tert-butoxycarbonyl)-N1-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-l-leucinamide but substituting N-(tert-butoxycarbonyl)-5-methyl-l-norleucine and making noncritical variations provided a crude golden oil.

T540

94082-94244

Sentence

denotes

This material was purified by Biotage flash chromatography, eluting with methanol/dichloromethane to afford the title compound as an off-white solid, 360 mg, 41%.

T541

94245-94661

Sentence

denotes

1H NMR (400 MHz, DMSO-d6) δ 8.45 (d, J = 8.1 Hz, 1H), 7.62 (s, 1H), 7.02 (d, J = 7.1 Hz, 1H), 4.49–4.62 (m, 2H), 4.33–4.44 (m, 1H), 3.78 (m, 1H), 3.15 (t, J = 8.7 Hz, 1H), 3.00–3.10 (m, 1H), 2.18–2.30 (m, 1H), 2.04–2.14 (m, 1H), 1.92–2.02 (m, 1H), 1.40–1.68 (m, 5H), 1.36 (s, 9H), 1.05–1.25 (m, J = 7.3 Hz, 2H), 0.83 (d, J = 1.52 Hz, 3H), 0.82 (d, J = 1.52 Hz, 3H); MS (API-ES+) for C20H34N3O5Cl m/z 454.2 (M + Na)+.

T542

94663-94805

Sentence

denotes

N-((1S)-1-{[((1S)-3-Chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-4-methylpentyl)-4-methoxy-1H-indole-2-carboxamide

T543

94806-95211

Sentence

denotes

Following the procedure described for the preparation of N-((1S)-1-{[((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-1H-indole-2-carboxamide but substituting N2-(tert-butoxycarbonyl)-N1-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-5-methyl-l-norleucinamide and making no other critical variations provided a crude yellow foam.

T544

95212-95361

Sentence

denotes

This material was purified by Biotage MPLC (25 M column, 2.5–3.5% methanol/chloroform) to afford 307 mg (73%) of the title compound as a white solid.

T545

95362-95846

Sentence

denotes

1H NMR (DMSO-d6) δ 11.57 (s, 1H), 8.59 (d, J = 8 Hz, 1H), 8.41 (d, J = 4 Hz, 1H), 7.64 (s, 1H), 7.37 (s, 1H), 7.09 (t, J = 8 Hz, 1H), 7.00 (d, J = 8 Hz, 1H), 6.49 (d, J = 8 Hz, 1H), 4.59 (s, 2H), 4.44 (m, 1H), 4.35 (m, 1H), 3.87 (s, 3H), 3.08 (m, 2H), 2.26 (m, 1H), 2.07 (m, 1H), 1.98 (m, 1H), 1.70–1.51 (m, 5H), 1.25 (m, 2H), 0.88 (d, J = 4 Hz, 3H), 0.86 (d, J = 4 Hz, 3H); MS (ESI+) for C25H33ClN4O5m/z 505.2 (M + H)+; HRMS (ESI+) calcd for C25H33ClN4O5+H1 505.2212, found 505.2204.

T546

95848-95996

Sentence

denotes

N-((1S)-1-{[((1S)-3-Hydroxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-4-methylpentyl)-4-methoxy-1H-indole-2-carboxamide (40)

T547

95997-96420

Sentence

denotes

Following the procedure described for the preparation of N-((1S)-1-{[((1S)-3-hydroxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-1H-indole-2-carboxamide but substituting N-((1S)-1-{[((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-4-methylpentyl)-4-methoxy-1H-indole-2-carboxamide and making noncritical variations provided a crude yellow foam.

T548

96421-96567

Sentence

denotes

This material was purified by Biotage MPLC (25 M column, 5–5.5% methanol/chloroform) to afford 135 mg (50%) of the title compound as a white foam.

T549

96568-97104

Sentence

denotes

1H NMR (DMSO-d6) δ 11.57 (s, 1H), 8.42 (d, J = 8 Hz, 1H), 8.38 (d, J = 8 Hz, 1H), 7.62 (s, 1H), 7.35 (s, 1H), 7.09 (t, J = 8 Hz, 1H), 6.99 (d, J = 8 Hz, 1H), 6.49 (d, J = 8 Hz, 1H), 5.06 (t, J = 8 Hz, 1H), 4.47–4.30 (m, 2H), 4.25 (dd, J = 8, 20 Hz, 1H), 4.14 (dd, J = 8, 20 Hz, 1H), 3.87 (s, 3H), 3.09 (m, 2H), 2.30 (m, 1H), 2.08 (m, 1H), 1.92 (m, 1H), 1.72–1.51 (m, 5H), 1.25 (m, 2H), 0.87 (d, J = 4 Hz, 3H), 0.86 (d, J = 4 Hz, 3H); MS (ESI+) for C25H34N4O6m/z 487.1 (M + H)+; HRMS (ESI+) calcd for C25H34N4O6 487.2551, found 487.2541.

T550

97106-97244

Sentence

denotes

tert-Butyl [(1S)-2-[((1S)-3-(Benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]-1-(cyclohexylmethyl)-2-oxoethyl]carbamate

T551

97245-97619

Sentence

denotes

To a solution of tert-butyl ((1S)-3-(benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)carbamate (200 mg, 0.53 mmol) in dioxane (5 mL) was added 4 M hydrochloric acid/dioxane (5 mL), and the solution was stirred at an ambient temperature for 4 h before removing the solvents in vacuo, azeotroping the residue with toluene (2 × 10 mL), and drying in vacuo for 1 h.

T552

97620-97901

Sentence

denotes

The crude hydrochloride salt was taken into DMF (3 mL), and the solution was cooled to 0 °C before adding N-Boc-cyclohexylalanine-OH (139 mg, 0.53 mmol), collidine (156 μL, 1.22 mmol), and HATU (194 mg, 0.53 mmol) in order, and the resulting suspension was stirred at 0 °C for 5 h.

T553

97902-98023

Sentence

denotes

The reaction was quenched by the addition of water (30 mL), and the mixture was extracted with diethyl ether (3 × 75 mL).

T554

98024-98295

Sentence

denotes

The combined organics were dried over MgSO4, filtered, and the solvents were removed in vacuo to yield the crude product, which was purified by flash chromatography, eluting with 1–3% methanol/dichloromethane to afford the title compound as a pale brown gum, 205 mg, 76%.

T555

98296-98362

Sentence

denotes

The product was contaminated with ∼20% of another diastereoisomer.

T556

98363-98669

Sentence

denotes

1H NMR (400 MHz, CDCl3) δ 7.69 (d, J = 5.6 Hz, 1H), 7.26–7.42 (m, 5H), 5.93 (s, 1H), 4.94 (d, J = 7.6 Hz, 1H), 4.72 (m, 1H), 4.60 (d, J = 11.6 Hz, 1H), 4.55 (d, J = 11.6 Hz, 1H), 4.14–4.34 (m, 3H), 3.27 (m, 2H), 2.23–2.51 (m, 3H), 1.56–2.05 (m, 9H), 1.33–1.48 (m, 9H), 1.05–1.29 (m, 4H), 0.80–1.03 (m, 2H).

T557

98671-98825

Sentence

denotes

N-[(1S)-2-[((1S)-3-(Benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]-1-(cyclohexylmethyl)-2-oxoethyl]-4-methoxy-1H-indole-2-carboxamide

T558

98826-99248

Sentence

denotes

To a solution of tert-butyl [(1S)-2-[((1S)-3-(benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]-1-(cyclohexylmethyl)-2-oxoethyl]carbamate (200 mg, 0.38 mmol) in dioxane (4 mL) was added 4 M hydrochloric acid/dioxane (4 mL), and the solution was stirred at an ambient temperature for 2 h before removing the solvents in vacuo, azeotroping the residue with toluene (2 × 10 mL), and drying in vacuo for 1 h.

T559

99249-99537

Sentence

denotes

The crude hydrochloride salt was taken into DMF (3 mL), and the solution was cooled to 0 °C before adding 4-methoxy-indole-2-carboxylic acid (73 mg, 0.38 mmol), collidine (125 μL, 0.95 mmol), and HATU (144 mg, 0.38 mmol) in order, and the resulting suspension was stirred at 0 °C for 6 h.

T560

99538-99659

Sentence

denotes

The reaction was quenched by the addition of water (20 mL), and the mixture was extracted with diethyl ether (3 × 50 mL).

T561

99660-99983

Sentence

denotes

The combined organics were washed with water (20 mL) and brine (20 mL), dried over MgSO4, filtered, and the solvents were removed in vacuo to yield the crude product, which was purified by Biotage flash chromatography, eluting with 1–3% methanol/dichloromethane to afford the title compound as a pale brown gum, 90 mg, 39%.

T562

99984-100070

Sentence

denotes

The product was contaminated with ∼20% of another diastereomer from the previous step.

T563

100071-100615

Sentence

denotes

1H NMR (400 MHz, CDCl3) δ 9.59 (s, 1H), 8.13 (d, J = 6.6 Hz, 1H), 7.21–7.32 (m, 5H), 7.11 (t, J = 8.0 Hz, 1H), 7.02 (d, J = 1.5 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 6.72–6.78 (m, 1H), 6.42 (d, J = 7.8 Hz, 1H), 5.97 (s, 1H), 4.73 (m, 1H), 4.64 (m, 1H), 4.53 (d, J = 11.6 Hz, 1H), 4.46 (d, J = 11.6 Hz, 1H), 4.26 (d, J = 17.2 Hz, 1H), 4.15 (d, J = 17.4 Hz, 1H), 3.87 (s, 3H), 3.08–3.16 (m, 2H), 2.27–2.38 (m, 1H), 1.92 (m, 1H), 1.49–1.84 (m, 9H), 1.26–1.42 (m, 1H), 0.98–1.23 (m, 4H), 0.78–0.98 (m, 2H); MS (APCI+) for C34H42N4O6m/z 603.2 (M + H)+.

T564

100617-100772

Sentence

denotes

N-{(1S)-1-(Cyclohexylmethyl)-2-[((1S)-3-hydroxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]-2-oxoethyl}-4-methoxy-1H-indole-2-carboxamide (41)

T565

100773-101102

Sentence

denotes

To a solution of N-[(1S)-2-[((1S)-3-(benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]-1-(cyclohexylmethyl)-2-oxoethyl]-4-methoxy-1H-indole-2-carboxamide (80 mg, 0.13 mmol) in EtOH (3 mL) was added 10% Pd/C (50 mg), and the suspension was hydrogenated at an ambient temperature under H2 (1 atm balloon) for 5 h.

T566

101103-101367

Sentence

denotes

The catalyst was removed by filtration, and the solvents were evaporated in vacuo to yield the crude product, which was purified by Biotage flash chromatography, eluting with 2–10% methanol/dichloromethane to afford the title compound, 37 mg, 55% as a white solid.

T567

101368-101790

Sentence

denotes

1H NMR (400 MHz, MeOD) δ 7.19 (s, 1H), 7.05 (t, J = 8.0 Hz, 1H), 6.93 (d, J = 8.1 Hz, 1H), 6.41 (d, J = 7.8 Hz, 1H), 4.52–4.59 (m, 2H), 4.27 (m, 2H), 3.83 (s, 3H), 3.08–3.21 (m, 2H), 2.47 (m, 1H), 2.13–2.24 (m, 1H), 1.92–2.03 (m, 1H), 1.53–1.79 (m, 9H), 1.31–1.45 (m, 1H), 1.04–1.29 (m, 3H), 0.81–1.02 (m, 2H); MS (APCI+) for C27H36N4O6m/z 513.2. (M + H)+; anal. calcd for C27H36N4O6· 0.8 H2O: C, 61.53; H, 7.19; N, 10.63.

T568

101791-101826

Sentence

denotes

Found: C, 61.83; H, 7.12; N, 10.27.

T569

101828-101939

Sentence

denotes

N-(tert-Butoxycarbonyl)-N-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-l-phenylalaninamide

T570

101940-102194

Sentence

denotes

Following the procedure described for the preparation of N2-(tert-butoxycarbonyl)-N1-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-l-leucinamide but substituting Boc-Phe-OH and making noncritical variations provided a crude brown oil.

T571

102195-102352

Sentence

denotes

This material was purified by Biotage flash chromatography, eluting with methanol/dichloromethane to afford the title compound as a white solid, 351 mg, 36%.

T572

102353-102681

Sentence

denotes

1H NMR (400 MHz, CDCl3) δ 8.02 (s, 1H), 7.19–7.31 (m, 5H), 5.77 (s, 1H), 5.10 (d, J = 6.1 Hz, 1H), 4.52–4.57 (m, 1H), 4.44 (m, 1H), 4.01–4.12 (m, 2H), 3.26–3.37 (m, 2H), 3.01–3.08 (m, 2H), 2.29–2.37 (m, 1H), 2.15–2.25 (m, 1H), 1.98–2.06 (m, 1H), 1.75–1.91 (m, 2H), 1.40 (s, 9H); MS (API-ES−) for C22H30N3O5Cl m/z 450.2 (M – H)−.

T573

102683-102808

Sentence

denotes

N-((1S)-3-Chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-phenylalaninamide

T574

102809-103200

Sentence

denotes

Following the procedure described for the preparation of N-((1S)-1-{[((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-1H-indole-2-carboxamide but substituting N-(tert-butoxycarbonyl)-N-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-l-phenylalaninamide and making noncritical variations provided a crude green solid.

T575

103201-103358

Sentence

denotes

This material was purified by Biotage flash chromatography, eluting with methanol/dichloromethane to afford the title compound as a white solid, 300 mg, 91%.

T576

103359-103839

Sentence

denotes

1H NMR (400 MHz, DMSO-d6) δ 11.52 (d, J = 2.0 Hz, 1H), 8.68 (d, J = 7.8 Hz, 1H), 8.61 (d, J = 7.8 Hz, 1H), 7.61 (s, 1H), 7.37 (d, J = 7.1 Hz, 2H), 7.31 (d, J = 1.8 Hz, 1H), 7.26 (t, J = 7.6 Hz, 2H), 7.16 (t, J = 7.2 Hz, 1H), 7.07 (t, 1H), 6.97 (d, J = 8.1 Hz, 1H), 6.49 (d, J = 7.6 Hz, 1H), 4.68 (m, 1H), 4.38–4.49 (m, 3H), 3.88 (s, 3H), 2.97–3.17 (m, 4H), 2.20–2.32 (m, 1H), 2.02–2.14 (m, 1H), 1.93–2.02 (m, 1H), 1.52–1.69 (m, 2H); MS (APCI±) for C27H29N4O5Cl m/z 526.0 (M + H)−.

T577

103841-103972

Sentence

denotes

N-((1S)-3-Hydroxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-phenylalaninamide (42)

T578

103973-104380

Sentence

denotes

Following the procedure described for the preparation of N-((1S)-1-{[((1S)-3-hydroxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-1H-indole-2-carboxamide but substituting N-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-N-[(4-methoxy-1H-indol-2-yl)carbonyl]-l-phenylalaninamide and making noncritical variations provided a crude greenish gum.

T579

104381-104543

Sentence

denotes

This material was purified by Biotage flash chromatography, eluting with methanol/dichloromethane to afford the title compound as an off-white solid, 123 mg, 44%.

T580

104544-105108

Sentence

denotes

1H NMR (400 MHz, DMSO-d6) δ 11.50 (d, J = 2.0 Hz, 1H), 8.58 (dd, J = 8.2, 3.9 Hz, 2H), 7.63 (s, 1H), 7.35–7.43 (m, 2H), 7.31 (d, J = 1.8 Hz, 1H), 7.27 (t, J = 7.6 Hz, 2H), 7.17 (t, J = 7.3 Hz, 1H), 7.08 (t, J = 8.0 Hz, 1H), 6.98 (d, J = 8.1 Hz, 1H), 6.48 (d, J = 7.8 Hz, 1H), 5.06 (t, J = 6.1 Hz, 1H), 4.72 (m, 1H), 4.48 (m, 1H), 4.16 (m, 2H), 3.89 (s, 3H), 2.97–3.18 (m, 4H), 2.24–2.36 (m, 1H), 2.04–2.18 (m, 1H), 1.88–2.01 (m, 1H), 1.55–1.76 (m, 2H); MS (APCI+) for C27H30N4O6m/z 507.1 (M + H)+; anal. calcd for C27H30N4O6· 1.25 H2O: C, 61.29; H, 6.19; N, 10.59.

T581

105109-105144

Sentence

denotes

Found: C, 61.37; H, 6.06; N, 10.49.

T582

105146-105281

Sentence

denotes

N-((1S)-1-{[((1S)-3-Chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3,3-dimethylbutyl)-1H-indole-2-carboxamide

T583

105282-105713

Sentence

denotes

Following the procedure described for the preparation of N-((1S)-1-{[((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-1H-indole-2-carboxamide but substituting N2-(tert-butoxycarbonyl)-N1-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-4-methyl-l-leucinamide and indole-2-carboxylic acid and making no other critical variations provided a crude yellow foam.

T584

105714-105869

Sentence

denotes

This material was purified by Biotage MPLC (40 M column, 2.5–3.5% methanol/chloroform) to afford 1.06 g (62%) of the title compound as a light yellow foam.

T585

105870-106327

Sentence

denotes

1H NMR (DMSO-d6) δ 11.59 (s, 1H), 8.58 (d, J = 8 Hz, 1H), 8.50 (d, J = 8 Hz, 1H), 7.64 (s, 1H), 7.61 (d, J = 8 Hz, 1H), 7.41 (d, J = 8 Hz, 1H), 7.23 (s, 1H), 7.17 (t, J = 8 Hz, 1H), 7.02 (t, J = 8 Hz, 1H), 4.62–4.50 (m, 3H), 4.45 (m, 1H), 3.10 (m, 2H), 2.25 (m, 1H), 2.08 (m, 1H), 1.96 (m, 1H), 1.80 (m, 1H), 1.72–1.58 (m, 3H), 0.94 (s, 9H); MS (ESI+) for C24H31ClN4O4m/z 475.1 (M + H)+; anal. calcd for C24H31ClN4O4·0.35 CHCl3: C, 56.59; H, 6.12; N, 10.84.

T586

106328-106424

Sentence

denotes

Found: C, 56.38; H, 6.18; N, 10.75; HRMS (ESI+) calcd for C24H31ClN4O4 475.2107, found 475.2122.

T587

106426-106567

Sentence

denotes

N-((1S)-1-{[((1S)-3-Hydroxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3,3-dimethylbutyl)-1H-indole-2-carboxamide (43)

T588

106568-106984

Sentence

denotes

Following the procedure described for the preparation of N-((1S)-1-{[((1S)-3-hydroxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-1H-indole-2-carboxamide but substituting N-((1S)-1-{[((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3,3-dimethylbutyl)-1H-indole-2-carboxamide and making noncritical variations provided a crude yellow foam.

T589

106985-107134

Sentence

denotes

This material was purified by Biotage MPLC (40 M column, 4.5–5.5% methanol/chloroform) to afford 730 mg (72%) of the title compound as a white solid.

T590

107135-107676

Sentence

denotes

1H NMR (DMSO-d6) δ 11.59 (s, 1H), 8.49 (d, J = 8 Hz, 1H), 8.43 (d, J = 8 Hz, 1H), 7.62 (s, 1H), 7.60 (s, 1H), 7.41 (d, J = 8 Hz, 1H), 7.23 (s, 1H), 7.17 (t, J = 8 Hz, 1H), 7.02 (t, J = 8 Hz, 1H), 5.05 (t, J = 8 Hz, 1H), 4.56 (m, 1H), 4.43 (m, 1H), 4.25 (dd, J = 8, 20 Hz, 1H), 4.13 (dd, J = 8, 20 Hz, 1H), 3.10 (m, 2H), 2.25 (m, 1H), 2.07 (m, 1H), 1.93 (m, 1H), 1.80 (m, 1H), 1.64 (m, 3H), 0.94 (s, 9H); MS (ESI+) for C24H32N4O5m/z 457.1 (M + H)+; anal. calcd for C24H32N4O5·0.2 CHCl3·0.2 ethyl acetate·0.25 H2O: C, 59.75; H, 6.88; N, 11.15.

T591

107677-107771

Sentence

denotes

Found: C, 59.67; H, 6.72; N, 11.03; HRMS (ESI+) calcd for C24H32N4O5 457.2446, found 457.2439.

T592

107773-107914

Sentence

denotes

N-((1S)-1-{[((1S)-3-Methoxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3,3-dimethylbutyl)-1H-indole-2-carboxamide (44)

T593

107915-108330

Sentence

denotes

Following the procedure described for the preparation of 4-methoxy-N-((1S)-1-{[((1S)-3-methoxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-4-methylpentyl)-1H-indole-2-carboxamide but substituting N-((1S)-1-{[((1S)-3-hydroxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3,3-dimethylbutyl)-1H-indole-2-carboxamide and making noncritical variations provided a crude tan foam.

T594

108331-108479

Sentence

denotes

This material was purified by Biotage MPLC (25 M column, 3.5–4.5% methanol/chloroform) to afford 43 mg (15%) of the title compound as a white solid.

T595

108480-108975

Sentence

denotes

1H NMR (DMSO-d6) δ 11.58 (s, 1H), 8.48 (apar t, J = 8 Hz, 2H), 7.61 (apar d, J = 8 Hz, 2H), 7.41 (d, J = 8 Hz, 1H), 7.23 (s, 1H), 7.17 (t, J = 8 Hz, 1H), 7.02 (t, J = 8 Hz, 1H), 4.55 (m, 1H), 4.37 (m, 1H), 4.25 (d, J = 20 Hz, 1H), 4.10 (d, J = 20 Hz, 1H), 3.23 (s, 3H), 3.06 (m, 2H), 2.27 (m, 1H), 2.07 (m, 1H), 1.91 (m, 1H), 1.79 (m, 1H), 1.69 (m, 3H), 0.94 (s, 9H); MS (ESI+) for C25H34N4O5m/z 471.2 (M + H)+; anal. calcd for C25H34N4O5·0.2 ethyl acetate·0.75 H2O: C, 61.76; H, 7.46; N, 11.17.

T596

108976-109070

Sentence

denotes

Found: C, 61.85; H, 7.15; N, 11.02; HRMS (ESI+) calcd for C25H34N4O5 471.2602, found 471.2595.

T597

109072-109212

Sentence

denotes

N-((1S)-1-{[((1S)-3-Ethoxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3,3-dimethylbutyl)-1H-indole-2-carboxamide (45)

T598

109213-109495

Sentence

denotes

Following the procedure described for the preparation of N-((1S)-1-{[((1S)-3-methoxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3,3-dimethylbutyl)-1H-indole-2-carboxamide but substituting iodoethane and making noncritical variations provided a crude tan foam.

T599

109496-109644

Sentence

denotes

This material was purified by Biotage MPLC (25 M column, 3–5% methanol/chloroform) to afford 19 mg (6%) of the title compound as an off-white solid.

T600

109645-110158

Sentence

denotes

1H NMR (DMSO-d6) δ 11.58 (s, 1H), 8.48 (d, J = 8 Hz, 1H), 8.45 (d, J = 8 Hz, 1H), 7.61 (apar d, J = 8 Hz, 2H), 7.41 (d, J = 8 Hz, 1H), 7.23 (s, 1H), 7.17 (t, J = 8 Hz, 1H), 7.02 (t, J = 8 Hz, 1H), 4.56 (m, 1H), 4.40 (m, 1H), 4.28 (d, J = 16 Hz, 1H), 4.13 (d, J = 16 Hz, 1H), 3.41 (m, 2H), 3.06 (m, 2H), 2.26 (m, 1H), 2.09 (m, 1H), 1.91 (m, 1H), 1.82 (m, 1H), 1.63 (m, 3H), 1.08 (t, J = 8 Hz, 3H), 0.94 (s, 9H); MS (ESI+) for C26H36N4O5m/z 485.2 (M + H)+; HRMS (ESI+) calcd for C26H36N4O5 485.2759, found 485.2756.

T601

110160-110290

Sentence

denotes

N1-((1S)-3-Chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-4-methyl-N2-[(2R)-tetrahydrofuran-2-ylcarbonyl]-l-leucinamide

T602

110291-110726

Sentence

denotes

Following the procedure described for the preparation of N-((1S)-1-{[((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-1H-indole-2-carboxamide but substituting N2-(tert-butoxycarbonyl)-N1-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-4-methyl-l-leucinamide and (2R)-tetrahydrofuran-2-carboxylic acid and making noncritical variations provided a crude product.

T603

110727-110976

Sentence

denotes

This crude material was purified by Biotage MPLC (40 M cartridge, chloroform mobile phase with 2% methanol followed by 3% methanol, sample loaded in chloroform) resulting in the isolation of 1.13 g (61%) of the title compound as a light yellow foam.

T604

110977-111407

Sentence

denotes

Rf = 0.27 (95:5 dichloromethane/methanol); 1H NMR (400 MHz, DMSO-d6) δ 8.47 (d, J = 8 Hz, 1H), 7.72 (d, J = 8 Hz, 1H), 7.68 (s, 1H), 4.55 (s, 2H), 4.44–4.36 (m, 1H), 4.35–4.27 (m, 1H), 4.21 (dd, J = 8, 5 Hz, 1H), 3.95–3.86 (m, 1H), 3.79–3.71 (m, 1H), 3.18–3.07 (m, 2H), 2.21 (td, J = 9, 4 Hz, 1H), 2.13–2.03 (m, 2H), 1.97–1.88 (m, 1H), 1.85–1.74 (m, 3H), 1.68–1.56 (m, 4H), 0.88 (s, 9H); MS (ESI+) for C20H32ClN3O5m/z 430 (M + H).

T605

111409-111545

Sentence

denotes

N1-((1S)-3-Hydroxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-4-methyl-N2-[(2R)-tetrahydrofuran-2-ylcarbonyl]-l-leucinamide (46)

T606

111546-111953

Sentence

denotes

Following the procedure described for the preparation of N-((1S)-1-{[((1S)-3-hydroxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-1H-indole-2-carboxamide but substituting N1-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-4-methyl-N2-[(2R)-tetrahydrofuran-2-ylcarbonyl]-l-leucinamide and making noncritical variations provided a crude product.

T607

111954-112275

Sentence

denotes

This material was purified by a series of two radial chromatographies (first −2 mm plate, 90:10 dichloromethane/methanol to 90:20, sample loaded in 90:10) (second −1 mm plates, 90:10 dichloromethane/methanol to 95:5, sample loaded in dichloromethane) to provide 0.402 g (46%) of the title compound as a light yellow foam.

T608

112276-112697

Sentence

denotes

Rf = 0.44 (90:10 dichloromethane/methanol); 1H NMR (400 MHz, DMSO-d6) δ 8.27 (d, J = 8 Hz, 1H), 7.71 (d, J = 9 Hz, 1H), 7.65 (s, 1H), 5.11 (t, J = 6 Hz, 1H), 4.46–4.38 (m, 1H), 4.37–4.30 (m, 1H), 4.24–4.15 (m, 2H), 4.14–4.08 (m, 1H), 3.96–3.87 (m, 1H), 3.79–3.70 (m, 1H), 3.18–3.06 (m, 2H), 2.25–2.16 (m, 1H), 2.13–2.02 (m, 2H), 1.87–1.75 (m, 4H), 1.66–1.54 (m, 4H), 0.88 (s, 9H); MS (ESI+) for C20H33N3O6m/z 412 (M + H).

T609

112698-112760

Sentence

denotes

Anal. calcd for C20H33N3O6·0.5H2O: C, 57.12; H, 8.16; N, 9.99.

T610

112761-112795

Sentence

denotes

Found: C, 57.25; H, 7.93; N, 9.68.

T611

112796-112856

Sentence

denotes

HRMS (ESI+) calcd for C20H33N3O6+H 412.2442, found 412.2447.

T612

112858-112994

Sentence

denotes

N1-((1S)-3-Methoxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-4-methyl-N2-[(2R)-tetrahydrofuran-2-ylcarbonyl]-l-leucinamide (47)

T613

112995-113404

Sentence

denotes

Following the procedure described for the preparation of 4-methoxy-N-((1S)-1-{[((1S)-3-methoxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-4-methylpentyl)-1H-indole-2-carboxamide but substituting N1-((1S)-3-hydroxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-4-methyl-N2-[(2R)-tetrahydrofuran-2-ylcarbonyl]-l-leucinamide and making noncritical variations provided a crude product.

T614

113405-113657

Sentence

denotes

This material was purified by radial chromatography (1 mm plate, 95:5 dichloromethane/methanol, sample loaded in dichloromethane) resulting in the isolation of 45.6 mg (22%) of the title compound as a light orange gum and as a mixture of diastereomers.

T615

113658-114055

Sentence

denotes

Rf = 0.30 (95:5 dichloromethane/methanol); 1H NMR (400 MHz, DMSO-d6, major diastereomer) δ 8.33 (d, J = 8 Hz, 1H), 7.70 (d, J = 9 Hz, 1H), 7.66 (s, 1H), 4.39–4.28 (m, 2H), 4.24–4.07 (m, 3H), 3.94–3.86 (m, 1H), 3.75 (q, J = 7 Hz, 1H), 3.24 (s, 3H), 3.18–3.06 (m, 2H), 2.27–2.15 (m, 1H), 2.14–2.03 (m, 2H), 1.92–1.73 (m, 4H), 1.67–1.53 (m, 4H), 0.88 (s, 9H); MS (ESI+) for C21H35N3O6m/z 426 (M + H).

T616

114056-114117

Sentence

denotes

HRMS (ESI+) calcd for C21H35N3O6+H1 426.2599, found 426.2604.

T617

114119-114266

Sentence

denotes

4-Methoxy-N-((1S)-1-{[((1S)-3-methoxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-1H-indole-2-carboxamide (48)

T618

114267-114582

Sentence

denotes

A solution of N-((1S)-1-{[((1S)-3-hydroxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-1H-indole-2-carboxamide (185 mg, 0.39 mmol), iodomethane (0.12 mL, 2.0 mmol), and silver(I) oxide (182 mg, 0.79 mmol) in dichloromethane (12 mL) was placed under an atmosphere of N2.

T619

114583-114763

Sentence

denotes

The resulting black thick suspension was heated to reflux for 18 h, treated with a second portion of iodomethane (0.12 mL, 2.0 mmol), and returned to reflux for an additional 24 h.

T620

114764-114961

Sentence

denotes

The reaction was cooled to RT, diluted with dichloromethane (20 mL), washed once with water (20 mL), once with brine (20 mL), dried over MgSO4, filtered, and concentrated to give a crude tan solid.

T621

114962-115115

Sentence

denotes

This material was purified by Biotage MPLC (25 M column, 3.5–4.5% methanol/chloroform) to afford 19 mg (10%) of the title compound as an off-white solid.

T622

115116-115632

Sentence

denotes

1H NMR (DMSO-d6) δ 11.57 (s, 1H), 8.48 (d, J = 8 Hz, 1H), 8.40 (d, J = 8 Hz, 1H), 7.63 (s, 1H), 7.35 (s, 1H), 7.08 (t, J = 8 Hz, 1H), 6.99 (d, J = 8 Hz, 1H), 6.49 (d, J = 8 Hz, 1H), 4.47 (m, 1H), 4.38 (m, 1H), 4.25 (d, J = 16 Hz, 1H), 4.11 (d, J = 16 Hz, 1H), 3.87 (s, 3H), 3.23 (s, 3H), 3.08 (m, 2H), 2.31 (m, 1H), 2.08 (m, 1H), 1.92 (m, 1H), 1.72–1.51 (m, 5H), 0.93 (d, J = 8 Hz, 3H), 0.88 (d, J = 8 Hz, 3H); MS (ESI+) for C25H34N4O6m/z 487.2 (M + H)+; HRMS (ESI+) calcd for C25H34N4O6+H1 487.2551, found 487.2563.

T623

115634-115765

Sentence

denotes

N-((1S)-1-{[((1S)-3-Chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-1H-indole-2-carboxamide

T624

115766-116070

Sentence

denotes

Following the procedure described for the preparation of N-((1S)-1-{[((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-1H-indole-2-carboxamide but substituting indole-2-carboxylic acid and making noncritical variations provided a crude orange foam.

T625

116071-116233

Sentence

denotes

This material was purified by Biotage flash chromatography, eluting with methanol/dichloromethane to afford the title compound as an off-white solid, 1.75 g, 82%.

T626

116234-116672

Sentence

denotes

1H NMR (400 MHz, DMSO-d6) δ 11.58 (s, 1H), 8.64 (d, J = 7.8 Hz, 1H), 8.50 (d, J = 7.8 Hz, 1H), 7.55–7.69 (m, 2H), 7.42 (d, J = 8.3 Hz, 1H), 7.26 (d, J = 1.5 Hz, 1H), 7.16 (m, 1H), 7.02 (t, J = 7.5 Hz, 1H), 4.53–4.66 (m, 2H), 4.39–4.52 (m, 2H), 2.94–3.18 (m, 2H), 2.22–2.35 (m, 1H), 2.03–2.16 (m, 1H), 1.89–2.02 (m, 1H), 1.45–1.82 (m, 5H), 0.93 (d, J = 6.3 Hz, 3H), 0.88 (d, J = 6.3 Hz, 3H); MS (APCI−) for C23H29N4O4Cl m/z 459.1 (M – H)−.

T627

116674-116811

Sentence

denotes

N-((1S)-1-{[((1S)-3-Hydroxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-1H-indole-2-carboxamide (49)

T628

116812-117222

Sentence

denotes

Following the procedure described for the preparation of N-((1S)-1-{[((1S)-3-hydroxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-4-methoxy-1H-indole-2-carboxamide but substituting N-((1S)-1-{[((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-1H-indole-2-carboxamide and making noncritical variations provided a crude black gum.

T629

117223-117385

Sentence

denotes

This material was purified by Biotage flash chromatography, eluting with methanol/dichloromethane to afford the title compound as an off-white solid, 218 mg, 18%.

T630

117386-117846

Sentence

denotes

1H NMR (400 MHz, DMSO-d6) δ 11.58 (s, 1H), 8.42–8.61 (m, 2H), 7.61 (d, J = 8.6 Hz, 2H), 7.41 (d, J = 8.1 Hz, 1H), 7.26 (s, 1H), 7.17 (t, J = 7.6 Hz, 1H), 7.02 (t, J = 7.5 Hz, 1H), 5.02 (t, 1H), 4.38–4.58 (m, 2H), 4.09–4.32 (m, 2H), 3.01–3.17 (m, 2H), 2.24–2.38 (m, 1H), 2.03–2.22 (m, 2H), 1.92 (m, 1H), 1.46–1.77 (m, 4H), 0.85–0.99 (m, 6H); MS (APCI+) for C23H30N4O5m/z 443.1 (M + H)+; anal. calcd for C23H30N4O5·0.55 H2O·0.05 DCM: C, 60.62; H, 6.89; N, 12.27.

T631

117847-117881

Sentence

denotes

Found: C, 60.83; H, 6.90; N,11.93.

T632

117883-118020

Sentence

denotes

N-((1S)-1-{[((1S)-3-Methoxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-1H-indole-2-carboxamide (50)

T633

118021-118438

Sentence

denotes

Following the procedure described for the preparation of 4-methoxy-N-((1S)-1-{[((1S)-3-methoxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-4-methylpentyl)-1H-indole-2-carboxamide but substituting N-((1S)-1-{[((1S)-3-hydroxy-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)amino]carbonyl}-3-methylbutyl)-1H-indole-2-carboxamide and making noncritical variations provided a crude pale brown oil.

T634

118439-118593

Sentence

denotes

This material was purified by Biotage flash chromatography, eluting with methanol/dichloromethane to afford the title compound as a white foam, 15 mg, 7%.

T635

118594-119050

Sentence

denotes

1H NMR (CDCl3) δ 9.52 (s, 1H), 8.38 (d, J = 6.1 Hz, 1H), 7.62 (d, J = 8.1 Hz, 1H), 7.40 (dd, J = 8.1, 3.8 Hz, 1H), 7.20–7.29 (m, 1H), 7.11 (t, J = 7.5 Hz, 1H), 6.98 (dd, J = 11.4, 1.5 Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H), 5.98 (d, J = 13.1 Hz, 1H), 4.71–4.88 (m, 1H), 4.63 (m, 1H), 4.08–4.33 (m, 2H), 3.38–3.44 (s, 3H), 3.08–3.33 (m, 2H), 2.28–2.63 (m, 2H), 2.13–2.25 (m, 1H), 1.55–2.10 (m, 4H), 0.85–1.03 (m, 6H); MS (APCI−) for C24H32N4O5m/z 455.2 (M – H)−.

T636

119052-119095

Sentence

denotes

SARS CoV-1 Protease FRET Assay and Analysis

T637

119096-119221

Sentence

denotes

Proteolytic activity of coronavirus 3CL protease is measured using a continuous fluorescence resonance energy-transfer assay.

T638

119222-119368

Sentence

denotes

The SARS 3CLpro FRET assay measures the protease-catalyzed cleavage of TAMRA-SITSAVLQSGFRKMK-(DABCYL)-OH to TAMRA-SITSAVLQ and SGFRKMK(DABCYL)-OH.

T639

119369-119411

Sentence

denotes

The fluorescence of the cleaved TAMRA (ex.

T640

119412-119422

Sentence

denotes

558 nm/em.

T641

119423-119525

Sentence

denotes

581 nm) peptide was measured using a TECAN SAFIRE fluorescence plate reader over the course of 10 min.

T642

119526-119645

Sentence

denotes

Typical reaction solutions contained 20 mM HEPES (pH 7.0), 1 mM EDTA, 4.0 FRET substrate, 4% DMSO, and 0.005% Tween-20.

T643

119646-119843

Sentence

denotes

Assays were initiated with the addition of 25 nM SARS CoV-1 3CLpro nucleotide sequence 9985–10902 of the Urbani strain of SARS coronavirus complete genome sequence (NCBI accession number AY278741).

T644

119844-119924

Sentence

denotes

Percent inhibition was determined in duplicate at a 0.001 mM level of inhibitor.

T645

119925-120174

Sentence

denotes

Data was analyzed with the nonlinear regression analysis program KaleidaGraph using the equationwhere offset equals the fluorescence signal of the uncleaved peptide substrate, and limit equals the fluorescence of the fully cleaved peptide substrate.

T646

120175-120315

Sentence

denotes

The kobs is the first-order rate constant for this reaction and, in the absence of any inhibitor, represents the utilization of a substrate.

T647

120316-120553

Sentence

denotes

In an enzyme start reaction, which contains an irreversible inhibitor, and where the calculated limit is less than 20% of the theoretical maximum limit, the calculated kobs represents the rate of inactivation of coronavirus 3CL protease.

T648

120554-120657

Sentence

denotes

The slope (kobs/I) of a plot of kobs vs [I] is a measure of the avidity of the inhibitor for an enzyme.

T649

120658-120782

Sentence

denotes

For very fast irreversible inhibitors, kobs/I is calculated from observations at only one or two [I] rather than as a slope.

T650

120783-121008

Sentence

denotes

For the determination of IC50 values for reversible inhibitors, the data was analyzed with the nonlinear regression analysis programs Xlfit3.05 (IDBS, Guildford, U.K.) or GraphPad Prism 8.02 (GraphPad Software San Diego, CA).

T651

121010-121053

Sentence

denotes

SARS CoV-2 Protease FRET Assay and Analysis

T652

121054-121208

Sentence

denotes

The proteolytic activity of the main protease, 3CLpro, of SARS CoV-2 was monitored using a continuous fluorescence resonance energy-transfer (FRET) assay.

T653

121209-121619

Sentence

denotes

The SARS CoV-2 3CLpro assay measures the activity of full-length SARS CoV-2 3CL protease to cleave a synthetic fluorogenic substrate peptide with the following sequence DABCYL-KTSAVLQ-SGFRKME-EDANS modeled on a consensus peptide.50 The fluorescence of the cleaved EDANS peptide (excitation 340 nm/emission 490 nm) is measured using a fluorescence intensity protocol on a Flexstation reader (Molecular Devices).

T654

121620-121726

Sentence

denotes

The fluorescent signal is reduced in the presence of PF-00835231, a potent inhibitor of SARS CoV-2 3CLpro.

T655

121727-121851

Sentence

denotes

The assay reaction buffer contained 20 mM Tris–HCl (pH 7.3), 100 nM NaCl, 1 mM EDTA, 5 mM TCEP, and 25 μM peptide substrate.

T656

121852-121978

Sentence

denotes

Enzyme reactions were initiated with the addition of 15 nM SARS CoV-2 3CL protease and allowed to proceed for 60 min at 23 °C.

T657

121979-122154

Sentence

denotes

Percent inhibition or activity was calculated based on control wells containing no compound (0% inhibition/100% activity) and a control compound (100% inhibition/0% activity).

T658

122155-122243

Sentence

denotes

IC50 values were generated using a four-parameter fit model using ABASE software (IDBS).

T659

122244-122470

Sentence

denotes

Ki values were fit to the Morrison equation with the enzyme concentration parameter fixed to 15 nM, the Km parameter fixed to 14 μM, and the substrate concentration parameter fixed to 25 μM using Activity Base software (IDBS).

T660

122472-122499

Sentence

denotes

SARS CoV-1 Antiviral Assays

T661

122500-122546

Sentence

denotes

All assays were performed in BSL3 containment.

T662

122547-122641

Sentence

denotes

Vero 76 cells were plated at 10 000 cells per well using phenol red-free DMEM or IMEM (Gibco).

T663

122642-122941

Sentence

denotes

After adhering for 2 h at 37 °C, various concentrations of compound (320, 100, 33, 10, 3.3, 1.0, 0.3, or 0.1 μM) were added and the cells were infected with 2.6 × 103 PFU/well SARS CoV Toronto-2 (provided as a gift from Dr. Heinz Feldman (NIAID, Hamilton, MT)) or mock-infected with the medium only.

T664

122942-123097

Sentence

denotes

After 66 h, cell viability was determined using either the neutral red method or the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI).

T665

123098-123260

Sentence

denotes

For the neutral red method, the cells were washed twice with PBS, and 100 μL of DMEM pH 4.5 containing 0.066% neutral red was added to the cells for 2 h at 37 °C.

T666

123261-123447

Sentence

denotes

The cells were again washed twice with PBS, 100 μL of buffer solution (50% EtOH, 1% acetic acid) was added, and the OD was read at 540 nM after a 10 min incubation at 37 °C with shaking.

T667

123448-123621

Sentence

denotes

Data are expressed as the percent of neutral red or luminescent signal in wells of compound-treated cells compared to the signal in wells of uninfected, compound-free cells.

T668

123622-123875

Sentence

denotes

The 50% effective concentration (EC50) is calculated as the concentration of the compound that increases the percent of the neutral red or luminescent signal in infected, compound-treated cells to 50% of that produced by uninfected, compound-free cells.

T669

123876-124139

Sentence

denotes

The 50% cytotoxicity concentration (CC50) is calculated as the concentration of the compound that decreases the percent of the neutral red or luminescent signal in uninfected, compound-treated cells to 50% of that produced in uninfected, compound-free cells.51,52

T670

124141-124167

Sentence

denotes

hCOV 229E Antiviral assays

T671

124168-124251

Sentence

denotes

MRC-5 cells were plated at 10 000 cells per well using phenol red-free MEM (Gibco).

T672

124252-124477

Sentence

denotes

Various concentrations of compound (320, 100, 33, 10, 3.3, 1.0, 0.3, or 0.1 μM) were added, and the cells were infected with an amount of hCoV 229E, which caused 80% cell death in 4 days or mock-infected with the medium only.

T673

124478-124733

Sentence

denotes

After 4 days, cell viability was determined using the XTT dye reduction method.53 Data are expressed as the percent of neutral red or luminescent signal in wells of compound-treated cells compared to the signal in wells of uninfected, compound-free cells.

T674

124734-124987

Sentence

denotes

The 50% effective concentration (EC50) is calculated as the concentration of the compound that increases the percent of the neutral red or luminescent signal in infected, compound-treated cells to 50% of that produced by uninfected, compound-free cells.

T675

124988-125246

Sentence

denotes

The 50% cytotoxicity concentration (CC50) is calculated as the concentration of the compound that decreases the percent of the neutral red or luminescent signal in uninfected, compound-treated cells to 50% of that produced in uninfected, compound-free cells.

T676

125248-125270

Sentence

denotes

Other Antiviral Assays

T677

125271-125432

Sentence

denotes

The following antiviral assays for HCMV,54 HIV-RF,55 HRV-14, and HRV-16,54 as well as the HCV replicon,56,57 were performed as described in the above references.

T678

125434-125483

Sentence

denotes

Expression and Purification of CoV-2 3CL Protease

T679

125484-125735

Sentence

denotes

The expression and purification strategy followed previous studies on SARS CoV-1 3CLpro.58 Genes encoding SARS CoV-1 3CLpro and SARS CoV-2 Mpro MN908947.3 were synthesized with codon usage optimized for Escherichia coli expression (Genscript and IDT).

T680

125736-125862

Sentence

denotes

Genes were cloned into a modified pET24a vector to produce a TEV-cleavable N-terminal 6xHis tag under the T7 promoter control.

T681

125863-126008

Sentence

denotes

BL21(DE3) cells (LifeTech) were grown in Terrific Broth (Teknova) at 37 °C until an OD600 = 0.6–0.8 and induced with 0.4 M IPTG for 5 h at 30 °C.

T682

126009-126063

Sentence

denotes

Cell pellets were stored at −80 °C until purification.

T683

126064-126171

Sentence

denotes

Cell pellets were resuspended and lysed by microfluidization in Buffer A (20 mM Tris pH 8.0 + 150 mM NaCl).

T684

126172-126225

Sentence

denotes

Lysates were clarified at 25,000 × g for 1 h at 4 °C.

T685

126226-126361

Sentence

denotes

The soluble lysate was loaded onto a nickel affinity column (Probond) and washed sequentially with Buffer A + 5 mM and 20 mM imidazole.

T686

126362-126421

Sentence

denotes

The proteases were eluted with Buffer A + 300 mM imidazole.

T687

126422-126526

Sentence

denotes

The His-tag was removed by TEV protease (1:40) during an overnight dialysis step in Buffer A + 1 mM DTT.

T688

126527-126812

Sentence

denotes

The CoV Mpro proteins were further purified on a HiTrap Q hp (GE Healthcare), and the Q-flow-through material was concentrated and loaded onto a 26/60 Superdex-75 (GE Healthcare) gel filtration column equilibrated with Buffer B (20 mM Tris pH 7.8 + 150 mM NaCl + 1 mM EDTA + 1 mM DTT).

T689

126813-126966

Sentence

denotes

Final proteins were concentrated from 5 to 25 mg/mL and were directly moved to crystallization experiments or snap-frozen in liquid nitrogen for storage.

T690

126968-127015

Sentence

denotes

Crystallization/Soak Protocols for SARS1 with 2

T691

127016-127109

Sentence

denotes

Crystals of compound 2 bound to SARS1/CoV-1 DC2 protease were produced via cocrystallization.

T692

127110-127222

Sentence

denotes

SARS1/CoV-1 DC2 protease at 10.00 mg/mL was incubated with a 3-fold molar excess of compound 2 for 18 h at 4 °C.

T693

127223-127380

Sentence

denotes

The complex was then passed through a 0.45 μM cellulose acetate spin filter and set up for crystallization using an NT-8 crystallization robot (Formulatrix).

T694

127381-127602

Sentence

denotes

Using MRC-2 crystallization plates, wells containing 40 μL of 4% w/v PEG 6000, 0.1 M MES pH 6.0, and 2.5 mM DTT were dispensed, and then sitting drops consisting of 0.3 μL protein were set up against a 0.3 μL well buffer.

T695

127603-127725

Sentence

denotes

Crystallization plates were incubated at 21 °C, and rectangular crystals measuring 0.1 × 0.25 × 0.25 mm3 grew within 24 h.

T696

127726-127860

Sentence

denotes

Cocrystals were flash-frozen in liquid nitrogen after being passed through a cryo consisting of a well buffer containing 20% glycerol.

T697

127862-127910

Sentence

denotes

Crystallization/Soak Protocols for SARS1 with 28

T698

127911-128005

Sentence

denotes

Crystals of compound 28 bound to SARS1/CoV-1 DC2 protease were produced via cocrystallization.

T699

128006-128119

Sentence

denotes

SARS1/CoV-1 DC2 protease at 10.00 mg/mL was incubated with a 3-fold molar excess of compound 28 for 18 h at 4 °C.

T700

128120-128277

Sentence

denotes

The complex was then passed through a 0.45 μM cellulose acetate spin filter and set up for crystallization using an NT-8 crystallization robot (Formulatrix).

T701

128278-128546

Sentence

denotes

Using MRC-2 crystallization plates, wells containing 40 μL of 10% w/v PEG 8000, 0.2 M sodium chloride, 0.1 M sodium potassium phosphate pH 6.2, and 10 mM TCEP were dispensed, and then sitting drops consisting of 0.3 μL protein were set up against a 0.3 μL well buffer.

T702

128547-128669

Sentence

denotes

Crystallization plates were incubated at 21 °C, and rectangular crystals measuring 0.1 × 0.25 × 0.25 mm3 grew within 24 h.

T703

128670-128804

Sentence

denotes

Cocrystals were flash-frozen in liquid nitrogen after being passed through a cryo consisting of a well buffer containing 20% glycerol.

T704

128806-128877

Sentence

denotes

Crystallization/Soak Protocols for SARS1 and SARS2 with 4 (PF-00835231)

T705

128879-128901

Sentence

denotes

LJEC2520 + PF-00835231

T706

128902-128982

Sentence

denotes

Crystals of PF-00835231 bound to SARS1/CoV-1 DC2 protease via cocrystallization:

T707

128983-129113

Sentence

denotes

Freshly prepared SARS1/CoV-1 DC2 protease at 10.00 mg/mL was incubated with a 3-fold molar excess of PF-00835231 for 18 h at 4 °C.

T708

129114-129271

Sentence

denotes

The complex was then passed through a 0.45 μM cellulose acetate spin filter and set up for crystallization using an NT-8 crystallization robot (Formulatrix).

T709

129272-129498

Sentence

denotes

Using MRC-2 crystallization plates, wells containing 40 μL of 10% w/v PEG 6000, 0.1 M MES pH 6.0, and 10% glycerol were dispensed, and then sitting drops consisting of 0.15 μL protein were set up against a 0.15 μL well buffer.

T710

129499-129619

Sentence

denotes

Crystallization plates were incubated at 21 °C, and rectangular crystals measuring 0.2 × 0.25 × 0.15 mm3 grew overnight.

T711

129620-129752

Sentence

denotes

Crystals were flash-frozen in liquid nitrogen after being passed through a cryo consisting of a well buffer containing 20% glycerol.

T712

129754-129776

Sentence

denotes

LJEC2521 + PF-00835231

T713

129777-129860

Sentence

denotes

Crystals of PF-00835231 bound to SARS2/CoV-2 mature protease via cocrystallization:

T714

129861-129993

Sentence

denotes

Freshly prepared SARS2/CoV-2 mature protease at 11.3 mg/mL was incubated with a 3-fold molar excess of PF-00835231 for 18 h at 4 °C.

T715

129994-130151

Sentence

denotes

The complex was then passed through a 0.45 μM cellulose acetate spin filter and set up for crystallization using an NT-8 crystallization robot (Formulatrix).

T716

130152-130388

Sentence

denotes

Using MRC-2 crystallization plates, wells containing 40 μL of 15% v/v 2-propanol, 0.1 M citric acid pH 5.0, and 10% w/v PEG 10 000 were dispensed, and then sitting drops consisting of 0.3 μL protein and a 0.3 μL well buffer were set up.

T717

130389-130524

Sentence

denotes

Crystallization plates were incubated at 13 °C, and crystals shaped as rectangular blocks measuring 0.1 × 0.2 × 0.2 mm3 grew overnight.

T718

130525-130657

Sentence

denotes

Crystals were flash-frozen in liquid nitrogen after being passed through a cryo consisting of a well buffer containing 20% glycerol.

T719

130659-130681

Sentence

denotes

LJEC2522 + PF-00835231

T720

130682-130752

Sentence

denotes

Crystals of PF-00835231 bound to SARS2/CoV-2 DC2 protease via soaking:

T721

130753-130949

Sentence

denotes

Freshly prepared SARS2/CoV-2 DC2 protease at 25.8 mg/mL was passed through a 0.45 mM cellulose acetate spin filter and set up for crystallization using an NT-8 crystallization robot (Formulatrix).

T722

130950-131192

Sentence

denotes

Using MRC-2 crystallization plates, wells containing 40 mL of 0.2 M potassium sodium tartrate tetrahydrate and 20% w/v PEG 3350 were dispensed, and then sitting drops consisting of 0.3 μL protein were set up in a 1:1 ratio with a well buffer.

T723

131193-131334

Sentence

denotes

Crystallization plates were incubated at 13 °C, and large crystals shaped as beveled plates measuring 0.35 × 0.2 × 0.025 mm3 grew after 48 h.

T724

131335-131480

Sentence

denotes

PF-00835231 (in 100% DMSO solution) was introduced to the drop (in situ), at a final concentration of 1 mM, and then incubated at 13 °C for 24 h.

T725

131481-131620

Sentence

denotes

Soaked crystals were flash-frozen in liquid nitrogen after being passed through a cryo consisting of a well buffer containing 20% glycerol.

T726

131621-131645

Sentence

denotes

Structure determination.

T727

131646-131795

Sentence

denotes

X-ray diffraction data were collected at the IMCA-CAT 17-ID beamline of Advanced Photon Source at Argonne National Labs and processed using autoPROC.

T728

131796-132069

Sentence

denotes

Structure of the SARS CoV main protease in complex with PF-835231 was determined by molecular replacement using the published protein structure (PDBID 1Q2W) as the starting model in program Phaser and refined iteratively using autoBUSTER followed by model building in Coot.

T729

132070-132285

Sentence

denotes

Structure of the SARS CoV-2 main protease in complex with PF-835231 was determined similarly by rigid body refinement using the SARS CoV main protease structure in complex with the same ligand as the starting model.

T730

132287-132319

Sentence

denotes

Supporting Information Available

T731

132320-132502

Sentence

denotes

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01063.Molecular formula strings for all compounds with biological data (CSV)

T732

132503-132535

Sentence

denotes

HPLC purity for compound 4 (PDF)

T733

132537-132559

Sentence

denotes

Supplementary Material

T734

132560-132580

Sentence

denotes

jm0c01063_si_001.csv

T735

132581-132601

Sentence

denotes

jm0c01063_si_002.pdf

T736

132603-132623

Sentence

denotes

Author Contributions

T737

132624-132692

Sentence

denotes

The manuscript was written through the contributions of all authors.

T738

132693-132764

Sentence

denotes

All authors have given approval to the final version of the manuscript.

T739

132766-132877

Sentence

denotes

Funding for the antiviral testing was provided by NIAID under a cooperative agreement between NIAID and Pfizer.

T740

132879-132931

Sentence

denotes

The authors declare no competing financial interest.

T741

132933-132948

Sentence

denotes

Acknowledgments

T742

132949-133101

Sentence

denotes

The authors would like to thank the Pfizer La Jolla Discovery Technology group who were responsible for all HRMS, preparative HPLC, and SFC separations.

T743

133102-133186

Sentence

denotes

Antiviral testing was performed at the Southern Research Institute (Birmingham, AL).

T744

133187-133329

Sentence

denotes

The authors wish to thank Dr. Heinz Feldman (NIAID, Hamilton, MT) for the kind gift of the SARS CoV Toronto-2 cell line enabling this testing.

T745

133330-133446

Sentence

denotes

The authors wish to thank Cathy Laughlin, Christopher Tseng, and Jack Secrist for their roles in this collaboration.

T746

133447-133535

Sentence

denotes

The authors would also like to thank Kevin Freeman-Cook, Charlotte Allerton, and Gina M.

T747

133536-133631

Sentence

denotes

Yanochko-Hoffman for critically reading the manuscript and offering valued editing suggestions.

PMC:7571312 JSON TXT 18 Projects

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