PubAnnotation

Id	Subject	Object	Predicate	Lexical cue
T8	0-12	Sentence	denotes	Introduction
T9	13-111	Sentence	denotes	In late 2019, cases of an unknown respiratory tract infection were first reported in Wuhan, China.
T10	112-566	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	567-950	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	951-1299	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	1300-1685	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	1686-2068	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	2069-2482	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	2483-2701	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	2702-2786	Sentence	denotes	Alternative modes of enzyme inhibition are covalently bound irreversible inhibitors.
T18	2787-3429	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	3430-3696	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	3697-3761	Sentence	denotes	Figure 1 Subsite nomenclature for proteolytic enzymes is shown.
T21	3762-3915	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	3916-4053	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	4054-4141	Sentence	denotes	Complimentary regions of the protease active site employ the corresponding S numbering.
T24	4142-4423	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	4424-4645	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	4646-4795	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	4796-5051	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	5052-5201	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	5202-5376	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	5377-5766	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	5767-5916	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	5917-6055	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	6056-6290	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	6291-6568	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	6569-6685	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	6686-6862	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	6863-6964	Sentence	denotes	Figure 3 Cocrystal structure of the covalent adduct of 2 bound to SARS CoV-1 3CLpro (PDB code 6XHO).
T38	6965-7036	Sentence	denotes	The Connolly surface for the inhibitor binding pocket is shown in gray.
T39	7037-7134	Sentence	denotes	The bonds are represented as the dashed lines, with the bond length measured between heavy atoms.
T40	7135-7255	Sentence	denotes	The views are centered on warhead with active-site interactions and of complete inhibitor–protein interactions depicted.
T41	7256-7451	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	7452-7649	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	7650-7841	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	7842-8046	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	8047-8234	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	8235-8442	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	8443-8712	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	8713-9049	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	9051-9073	Sentence	denotes	Results and Discussion
T50	9075-9084	Sentence	denotes	Chemistry
T51	9085-9258	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	9259-9477	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	9478-9529	Sentence	denotes	Scheme 1 Preparation of P1 Synthetic Intermediates
T54	9530-9884	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	9885-9990	Sentence	denotes	Saponification of amino ester 5(33) afforded the corresponding amino acid 6 in nearly quantitative yield.
T56	9991-10159	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	10160-10311	Sentence	denotes	Diazoketone 7 was treated with hydrochloric acid for simultaneous nitrogen deprotection and conversion to the chloromethylketone (CMK) intermediate 10.
T58	10312-10471	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	10472-10656	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	10657-10864	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	10865-10997	Sentence	denotes	A key element for rapid access to substituted methylketone derivatives was the generation of the fully elaborated CMK 12 (Scheme 2).
T62	10998-11106	Sentence	denotes	The utility of the CMK moiety to prepare acyloxy- and hydroxy-substituted methylketones is well established.
T63	11107-11359	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	11360-11485	Sentence	denotes	Reaction of 12 with various carboxylic acids in the presence of cesium fluoride at 60 °C provided acyloxymethylketones 13–28.
T65	11486-11610	Sentence	denotes	Using this synthetic approach with substitution of P2 and amine capping structural elements provided inhibitors 3 and 29–31.
T66	11611-11691	Sentence	denotes	Scheme 2 Preparation of Substituted Acyloxymethylketones and Aminomethylketones
T67	11692-11898	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	11899-12007	Sentence	denotes	Hydroxymethylketone (HMK) derivatives were accessed by two complimentary approaches illustrated in Scheme 3.
T69	12008-12227	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	12228-12317	Sentence	denotes	Hydrogenolytic debenzylation readily afforded the final HMK inhibitors exemplified by 36.
T71	12318-12667	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	12668-12799	Sentence	denotes	Using these two synthetic approaches with substitution of P2 and amine capping structural elements provided inhibitors 4 and 38–50.
T73	12800-12856	Sentence	denotes	Scheme 3 Preparation of Hydroxymethylketone Derivatives
T74	12857-13058	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	13059-13102	Sentence	denotes	K2CO3 (53%), and (vi) CH3I, Ag2O, DCE (9%).
T76	13104-13125	Sentence	denotes	Biological Evaluation
T77	13127-13171	Sentence	denotes	Irreversible Inhibitors of SARS CoV-1 3CLpro
T78	13172-13313	Sentence	denotes	The warhead reactivity and binding affinity to P1′ are both important design considerations to ensure specificity of irreversible inhibitors.
T79	13314-13684	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	13685-13862	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	13863-14100	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	14101-14183	Sentence	denotes	A series of acyloxymethylketone derivatives were prepared, as depicted in Table 1.
T83	14184-14261	Sentence	denotes	Table 1 SARS CoV-1 3CLpro Inhibition Data for Acyloxymethylketone Inhibitors
T84	14262-14291	Sentence	denotes	SARS CoV-1 3CLpro FRETa
T85	14292-14329	Sentence	denotes	entry R kobs/I (M–1 s–1) IC50 (nM)
T86	14330-14356	Sentence	denotes	12 283,039 ± 22,586
T87	14357-14377	Sentence	denotes	13 Me 220 ± 0.5
T88	14378-14404	Sentence	denotes	14 cyc-propyl 182 ± 6
T89	14405-14431	Sentence	denotes	15 tert-butyl 230 ± 5
T90	14432-14449	Sentence	denotes	16 Ph 86 ± 3
T91	14450-14473	Sentence	denotes	17 4-OMe-Ph 79 ± 3
T92	14474-14496	Sentence	denotes	18 4-Me-Ph 87 ± 2
T93	14497-14519	Sentence	denotes	19 4-CN-Ph 53 ± 1
T94	14520-14541	Sentence	denotes	20 4-F-Ph 82 ± 3
T95	14542-14564	Sentence	denotes	21 4-Cl-Ph 97 ± 3
T96	14565-14600	Sentence	denotes	22 2,6-(Cl)2-Ph 62,993 ± 2,501
T97	14601-14633	Sentence	denotes	23 2,6-(F)2-Ph 12,776 ± 594
T98	14634-14666	Sentence	denotes	24 2-OH-4-Cl-Ph 11,525 ± 40
T99	14667-14702	Sentence	denotes	25 2-F, 4-CN-Ph 13,321 ± 2,309
T100	14703-14730	Sentence	denotes	26 2,6-(Me)2-Ph 74 ± 4
T101	14731-14760	Sentence	denotes	27 2,6-(MeO)2-Ph 205 ± 2
T102	14761-14783	Sentence	denotes	28 2-CN-Ph 17 ± 2
T103	14784-14950	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	14951-15042	Sentence	denotes	As expected, chloromethylketone 12 is a potent irreversible inhibitor of SARS CoV-1 3CLpro.
T105	15043-15180	Sentence	denotes	Compounds 22–25 possess the most electron-deficient benzoate leaving groups and display kinetics consistent with irreversible inhibition.
T106	15181-15310	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	15311-15505	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	15506-15709	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	15710-15853	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	15854-16034	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	16035-16145	Sentence	denotes	Specifically, the imidazole hydrogen is directed toward a lone pair electron acceptor from a structural water.
T112	16146-16480	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	16481-16583	Sentence	denotes	Figure 4 Cocrystal structure of the covalent adduct of 28 bound to SARS CoV-1 3CLpro (PDB code 6XHN).
T114	16584-16655	Sentence	denotes	The Connolly surface for the inhibitor binding pocket is shown in gray.
T115	16656-16749	Sentence	denotes	The bonds are represented as dashed lines, with the bond length between heavy atoms depicted.
T116	16750-17191	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	17192-17573	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	17574-17790	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	17791-17878	Sentence	denotes	Replacement of the 4-methoxy indole cap present in 22 with a benzimidazole provided 31.
T120	17879-17990	Sentence	denotes	This inhibitor displayed potent irreversible inhibition kinetics but did not improve solubility compared to 22.
T121	17991-18148	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	18149-18238	Sentence	denotes	Table 2 SARS CoV-1 3CLpro Inhibition Data for P3-Modified Acyloxymethylketone Inhibitors
T123	18239-18405	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	18406-18642	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	18643-18718	Sentence	denotes	Initially, this derivative appeared to display reversible binding kinetics.
T126	18719-18900	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	18901-19052	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	19053-19229	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	19230-19450	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	19451-19677	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	19678-19791	Sentence	denotes	Viral particles themselves are not reliant on 3CLpro activity nor are the remaining coronavirus life cycle steps.
T132	19792-19856	Sentence	denotes	Each event of cell infection initiates newly synthesized 3CLpro.
T133	19857-19984	Sentence	denotes	Because detailed kinetics of this process are not understood, reversible and irreversible inhibitors were investigated equally.
T134	19986-20028	Sentence	denotes	Reversible Inhibitors of SARS CoV-1 3CLpro
T135	20029-20478	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	20479-20822	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	20823-20995	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	20996-21143	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	21144-21211	Sentence	denotes	Table 3 SARS 3CLpro Inhibition Data for P2-Modified HMK Inhibitors
T140	21212-21380	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	21381-21415	Sentence	denotes	c This value is a Ki measurement.
T142	21416-21464	Sentence	denotes	d These values are from a single determination.
T143	21465-21616	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	21617-21760	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	21761-21906	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	21907-22087	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	22088-22284	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	22285-22429	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	22430-22567	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	22568-22737	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	22738-22934	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	22935-23107	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	23108-23377	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	23378-23554	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	23555-23691	Sentence	denotes	Additionally, the terminal α-hydroxyl moiety located at P1′ appeared to provide a potential opportunity to remove a hydrogen bond donor.
T156	23692-23851	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	23852-24084	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	24085-24257	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	24258-24421	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	24422-24612	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	24613-24723	Sentence	denotes	Table 4 summarizes this effort where two of the optimal P2 residues (Leu and β-tert-butyl-Ala) were conserved.
T162	24724-24981	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	24982-25093	Sentence	denotes	The impact of more significant changes to the P3 cap is illustrated by tetrahydrofuranyl derivatives 46 and 47.
T164	25094-25268	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	25269-25432	Sentence	denotes	A noteworthy reduction in biochemical and antiviral potency was observed in comparing each hydroxymethylketone derivative with its corresponding ether counterpart.
T166	25433-25606	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	25607-25914	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	25915-26029	Sentence	denotes	Table 4 SARS CoV-1 3CLpro Inhibition Data for P3- and P2-Modified Hydroxymethyl and Alkoxymethylketone Inhibitors
T169	26030-26198	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	26199-26233	Sentence	denotes	c This value is a Ki measurement.
T171	26234-26282	Sentence	denotes	d These values are from a single determination.
T172	26283-26598	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	26599-26725	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	26726-26902	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	26903-27040	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	27041-27162	Sentence	denotes	Another key active-site interaction is the hydrogen bond between the primary alcohol moiety of 4 and the catalytic His41.
T177	27163-27299	Sentence	denotes	Similar interactions between catalytic His residues and the OH moiety of HMK in other protease–inhibitor complexes have been reported.43
T178	27300-27392	Sentence	denotes	Figure 5 Cocrystal structure of the covalent adduct of 4 bound to SARS CoV-2 3CLpro (6XHM).
T179	27393-27464	Sentence	denotes	The Connolly surface for the inhibitor binding pocket is shown in gray.
T180	27465-27558	Sentence	denotes	The bonds are represented as dashed lines, with the bond length between heavy atoms depicted.
T181	27559-27731	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	27732-27974	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	27975-28050	Sentence	denotes	These three residues partially define the bottom and edge of the S1 pocket.
T184	28051-28208	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	28209-28351	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	28352-28438	Sentence	denotes	The inhibitor P1 NH forms a strong hydrogen bond with the backbone carbonyl of His164.
T187	28439-28599	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	28600-28796	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	28797-29214	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	29215-29312	Sentence	denotes	Hydroxymethylketone 4 was evaluated against a panel of other viral and human proteases (Table 5).
T191	29313-29550	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	29551-29683	Sentence	denotes	High levels of similarity between the catalytic sites of 3CLpro proteases suggest the potential of 4 as a pan-coronavirus inhibitor.
T193	29684-29905	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	29906-29939	Sentence	denotes	Table 5 Protease Inhibition by 4
T195	29940-29991	Sentence	denotes	protease source % inhibition @ 10 μMa IC50 (μM)a
T196	29992-30030	Sentence	denotes	3CLpro SARS CoV-1 0.004 ± 0.0003b
T197	30031-30068	Sentence	denotes	3CLpro SARS CoV-2 0.00027 ± 0.0001b
T198	30069-30102	Sentence	denotes	cathepsin B human 93 1.3 ± 0.1
T199	30103-30129	Sentence	denotes	cathepsin D human 3 >10
T200	30130-30165	Sentence	denotes	leukocyte elastase human –31 >10
T201	30166-30193	Sentence	denotes	chymotrypsin human 8 >10
T202	30194-30219	Sentence	denotes	thrombin human –28 >10
T203	30220-30242	Sentence	denotes	pepsin human –2 >10
T204	30243-30269	Sentence	denotes	caspase 2 human –20 >10
T205	30270-30290	Sentence	denotes	HIV-1 HIV-1 0 >10
T206	30291-30308	Sentence	denotes	HCV HCV 16 >10
T207	30309-30329	Sentence	denotes	HRV HRV 1.7 ± 1
T208	30330-30365	Sentence	denotes	hCoV 229E human 0.004 ± 0.0004
T209	30366-30532	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	30533-30597	Sentence	denotes	b For SARS CoV-1 and CoV-2 3CLpro, Ki values are reported here.
T211	30598-30680	Sentence	denotes	The antiviral specificity of 4 was evaluated against a panel of viruses (Table 6).
T212	30681-30880	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	30881-30934	Sentence	denotes	This compound also does not inhibit the HCV replicon.
T214	30935-31113	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	31114-31144	Sentence	denotes	Table 6 Viral Inhibition by 4
T216	31145-31174	Sentence	denotes	virus EC50 (μM)a CC50 (μM)a
T217	31175-31201	Sentence	denotes	SARS CoV-1 4.8 ± 2.2 452
T218	31202-31229	Sentence	denotes	hCoV 229E 0.09 ± 0.04 >40
T219	31230-31250	Sentence	denotes	HRV-14 >100 >1,260
T220	31251-31271	Sentence	denotes	HRV-16 >100 >1,260
T221	31272-31290	Sentence	denotes	HIV-1 RF >32 >32
T222	31291-31315	Sentence	denotes	HCV replicon >320 >320
T223	31316-31338	Sentence	denotes	HCMV RC256 >100 >100
T224	31339-31505	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	31506-31718	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	31719-31978	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	31979-32254	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	32255-32420	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	32421-32664	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	32665-32935	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	32936-33083	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	33085-33096	Sentence	denotes	Conclusions
T233	33097-33300	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	33301-33417	Sentence	denotes	Two classes, acyloxymethylketones and hydroxymethylketones, were discovered as potent inhibitors of the SARS 3CLpro.
T235	33418-33528	Sentence	denotes	The hydroxymethylketone derivative 4 demonstrated potent SARS CoV-1 inhibition in 3CLpro and antiviral assays.
T236	33529-33875	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	33877-33897	Sentence	denotes	Experimental Section
T238	33899-33917	Sentence	denotes	General Procedures
T239	33918-34030	Sentence	denotes	Unless otherwise noted, materials were obtained from commercial suppliers and used without further purification.
T240	34031-34184	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	34185-34267	Sentence	denotes	Products obtained as solids or high-boiling oils were dried under vacuum (1 mmHg).
T242	34268-34394	Sentence	denotes	Silica gel chromatography was performed on either a CombiFlash (Teledyne ISCO), SP4, or Isolera (Biotage) purification system.
T243	34395-34595	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	34596-34765	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	34766-34936	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	34937-35107	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	35108-35212	Sentence	denotes	The TLC plates were visualized using UV light, p-anisaldehyde, phosphomolybdic acid, or iodine staining.
T248	35213-35274	Sentence	denotes	Microwave-assisted reactions were run in a Biotage Initiator.
T249	35275-35348	Sentence	denotes	1H NMR spectra were recorded on a Bruker XWIN-NMR (400 MHz) spectrometer.
T250	35349-35445	Sentence	denotes	Proton chemical shifts are reported in parts per million downfield from tetramethylsilane (TMS).
T251	35446-35536	Sentence	denotes	1H NMR data are reported as chemical shift (multiplicity, coupling constant, integration).
T252	35537-35697	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	35698-35848	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	35849-35935	Sentence	denotes	The purity of the final products is >95% as determined by HPLC and/or 1H NMR analyses.
T255	35937-36004	Sentence	denotes	N-(tert-Butoxycarbonyl)-3-[(3S)-2-oxopyrrolidin-3-yl]-l-alanine (6)
T256	36005-36293	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	36294-36496	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	36497-36684	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	36685-36870	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	36871-37074	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	37075-37272	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	37274-37362	Sentence	denotes	tert-Butyl ((1S)-3-Diazo-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)carbamate (7)
T263	37363-37535	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	37536-37693	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	37694-37796	Sentence	denotes	The reaction mixture gradually became opaque with a fine white precipitate and after 1 h was filtered.
T266	37797-37969	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	37970-37975	Sentence	denotes	Note:
T268	37976-38109	Sentence	denotes	The diazomethane was generated employing a Diazald kit according to the procedure described in the Aldrich Technical Bulletin AL-180.
T269	38110-38202	Sentence	denotes	The resulting yellow clear solution was gradually warmed to room temperature (RT) over 16 h.
T270	38203-38318	Sentence	denotes	At this time, N2 was bubbled through the reaction to remove excess diazomethane followed by in vacuo concentration.
T271	38319-38403	Sentence	denotes	The resulting residue was diluted with ethyl acetate (100 mL), washed once with sat.
T272	38404-38518	Sentence	denotes	NaHCO3 (50 mL), once with brine (50 mL), dried over MgSO4, filtered, and concentrated to give a crude yellow foam.
T273	38519-38667	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	38668-38888	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	38890-38979	Sentence	denotes	tert-Butyl ((1S)-3-Chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)carbamate (8)
T276	38980-39269	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	39270-39445	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	39446-39664	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	39665-39775	Sentence	denotes	After complete addition, the dark suspension was stirred for 10 min and then warmed to an ambient temperature.
T280	39776-39885	Sentence	denotes	The reaction was diluted with ethyl acetate (500 mL), and the organics were washed with water (250 mL), satd.
T281	39886-40102	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	40103-40221	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	40222-40535	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	40536-40570	Sentence	denotes	Found: C, 51.03; H, 6.93; N, 9.03.
T285	40572-40665	Sentence	denotes	tert-Butyl((1S)-3-(benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)carbamate (9)
T286	40666-40964	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	40965-41248	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	41249-41404	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	41405-41669	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	41670-41912	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	41913-42179	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	42180-42368	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	42369-42654	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	42655-42713	Sentence	denotes	The reaction was quenched by the careful addition of satd.
T295	42714-42844	Sentence	denotes	NH4Cl solution (25 mL), and the mixture was stirred until effervescence ceased and then extracted with ethyl acetate (3 × 120 mL).
T296	42845-43117	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	43118-43338	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	43340-43422	Sentence	denotes	(3S)-3-[(2S)-2-Amino-4-chloro-3-oxobutyl]pyrrolidin-2-one, Hydrochloride Salt (10)
T299	43423-43620	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	43621-43782	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	43783-43888	Sentence	denotes	Upon complete addition, the reaction was warmed to RT over 1 h with the formation of a white precipitate.
T302	43889-44026	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	44027-44253	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	44255-44344	Sentence	denotes	tert-Butyl ((1S)-3-Bromo-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)carbamate (11)
T305	44345-44601	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	44602-44783	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	44784-45043	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	45045-45191	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	45192-45399	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	45400-45524	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	45525-45604	Sentence	denotes	The reaction gradually became opaque with the formation of a gummy precipitate.
T312	45605-45854	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	45855-46146	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	46147-46324	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	46325-46475	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	46476-46601	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	46602-46729	Sentence	denotes	The combined organics were washed once with brine (30 mL), dried over MgSO4, filtered, and concentrated to give a yellow syrup.
T318	46730-46874	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	46875-47366	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	47368-47484	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	47485-47810	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	47811-47984	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	47985-48101	Sentence	denotes	After 3 h, the reaction was cooled to RT, diluted with water (30 mL), and extracted with dichloromethane (4 × 7 mL).
T324	48102-48207	Sentence	denotes	The combined organic layers were washed with water (2 × 20 mL), brine (20 mL), and concentrated in vacuo.
T325	48208-48354	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	48355-48869	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	48870-48935	Sentence	denotes	Anal. calcd for C26H34N4O7·0.65 H2O: C, 59.34; H, 6.76; N, 10.65.
T328	48936-48971	Sentence	denotes	Found: C, 59.41; H, 6.63; N, 10.68.
T329	48973-49105	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	49106-49379	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	49380-49509	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	49510-49972	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	49973-50038	Sentence	denotes	Anal. calcd for C28H36N4O7·0.59 H2O: C, 61.01; H, 6.80; N, 10.16.
T334	50039-50074	Sentence	denotes	Found: C, 61.04; H, 6.72; N, 10.00.
T335	50076-50193	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	50194-50452	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	50453-50583	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	50584-51005	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	51006-51070	Sentence	denotes	Anal. calcd for C29H40N4O7·0.76 H2O: C, 61.07; H, 7.34; N, 9.82.
T340	51071-51105	Sentence	denotes	Found: C, 61.14; H, 7.63; N, 9.59.
T341	51107-51224	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	51225-51483	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	51484-51614	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	51615-52155	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	52156-52220	Sentence	denotes	Anal. calcd for C31H36N4O7·1.04 H2O: C, 62.54; H, 6.45; N, 9.41.
T346	52221-52255	Sentence	denotes	Found: C, 62.49; H, 6.54; N, 9.31.
T347	52257-52383	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	52384-52651	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	52652-52782	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	52783-53303	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	53304-53377	Sentence	denotes	Anal. calcd for C32H38N4O8·0.80 H2O·0.05 DCM: C, 61.56; H, 6.40; N, 8.96.
T352	53378-53412	Sentence	denotes	Found: C, 61.84; H, 6.36; N, 8.58.
T353	53414-53539	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	53540-53806	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	53807-53936	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	53937-54477	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	54478-54551	Sentence	denotes	Anal. calcd for C32H38N4O7·0.10 H2O·0.11 DCM: C, 64.08; H, 6.43; N, 9.31.
T358	54552-54586	Sentence	denotes	Found: C, 64.36; H, 6.41; N, 8.92.
T359	54588-54712	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	54713-54978	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	54979-55109	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	55110-55641	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	55642-55707	Sentence	denotes	Anal. calcd for C32H35N5O7·0.90 H2O: C, 62.21; H, 6.00; N, 11.33.
T364	55708-55743	Sentence	denotes	Found: C, 62.23; H, 6.06; N, 11.58.
T365	55745-55870	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	55871-56137	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	56138-56268	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	56269-56788	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	56789-56863	Sentence	denotes	Anal. calcd for C31H35FN4O7·0.59 H2O·0.06 DCM: C, 61.28; H, 5.98; N, 9.20.
T370	56864-56898	Sentence	denotes	Found: C, 61.27; H, 5.98; N, 9.11.
T371	56900-57025	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	57026-57292	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	57293-57423	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	57424-57971	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	57972-58047	Sentence	denotes	Anal. calcd for C31H35ClN4O7·0.72 H2O·0.05 DCM: C, 59.36; H, 5.86; N, 8.92.
T376	58048-58082	Sentence	denotes	Found: C, 59.31; H, 5.99; N, 9.23.
T377	58084-58213	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	58214-58484	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	58485-58624	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	58625-59061	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	59062-59122	Sentence	denotes	Calcd for C31H34Cl2N4O7·0.2 H2O: C, 57.36; H, 5.34; N, 8.63.
T382	59123-59157	Sentence	denotes	Found: C, 57.23; H, 5.55; N, 8.46.
T383	59158-59222	Sentence	denotes	HRMS (ESI+) calcd for C31H34Cl2N4O7+H1 645.1877, found 645.1871.
T384	59224-59353	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	59354-59633	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	59634-59755	Sentence	denotes	This material was triturated with chloroform/ethyl acetate to afford 180 mg (75%) of the title compound as a white solid.
T387	59756-60278	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	60280-60415	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	60416-60688	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	60689-60829	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	60830-61346	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	61347-61445	Sentence	denotes	Found: C, 58.04; H, 5.78; N, 8.74; HRMS (ESI+) calcd for C31H35ClN4O8+H1 627.2216, found 627.2219.
T393	61447-61580	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	61581-61864	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	61865-62018	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	62019-62561	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	62562-62660	Sentence	denotes	Found: C, 61.47; H, 5.61; N, 10.98; HRMS (ESI+) calcd for C32H34FN5O7+H1 620.2515, found 620.2532.
T398	62662-62791	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	62792-63065	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	63066-63214	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	63215-63694	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	63695-63729	Sentence	denotes	Found: C, 62.95; H, 6.42; N, 8.72.
T403	63730-63791	Sentence	denotes	HRMS (ESI+) calcd for C33H40N4O7+H1 605.2970, found 605.2985.
T404	63793-63923	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	63924-64198	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	64199-64347	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	64348-64899	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	64900-64934	Sentence	denotes	Found: C, 59.49; H, 6.08; N, 8.42.
T409	64936-65060	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	65061-65335	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	65336-65456	Sentence	denotes	This material was triturated with chloroform/ethyl acetate to afford 91 mg (38%) of the title compound as a white solid.
T412	65457-65945	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	65946-66043	Sentence	denotes	Found: C, 63.16; H, 5.96; N, 11.43; HRMS (ESI+) calcd for C32H35N5O7+H1 602.2609, found 602.2610.
T414	66045-66142	Sentence	denotes	(3S)-3-[(tert-Butoxycarbonyl)amino]-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 2,6-Dichlorobenzoate
T415	66143-66384	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	66385-66462	Sentence	denotes	The resulting suspension was placed in a preheated oil bath at 65 °C for 2 h.
T417	66463-66680	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	66681-66830	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	66831-67096	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	67098-67205	Sentence	denotes	N2-(tert-Butoxycarbonyl)-N1-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-l-leucinamide
T421	67206-67415	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	67416-67553	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	67554-67719	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	67720-67847	Sentence	denotes	The combined organics were washed once with brine (30 mL), dried over MgSO4, filtered, and concentrated to give a yellow syrup.
T425	67848-68077	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	68078-68435	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	68436-68501	Sentence	denotes	Anal. calcd for C19H32ClN3O5·0.6 H2O: C, 53.22; H, 7.81; N, 9.80.
T428	68502-68536	Sentence	denotes	Found: C, 53.00; H, 7.65; N, 9.54.
T429	68537-68600	Sentence	denotes	HRMS (ESI+) calcd for C19H32ClN3O5+H1 418.2103, found 418.2091.
T430	68602-68717	Sentence	denotes	(3S)-3-{[N-(tert-Butoxycarbonyl)-l-leucyl]amino}-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 2,6-Dichlorobenzoate (29)
T431	68718-69054	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	69055-69206	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	69207-69565	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	69566-69632	Sentence	denotes	Anal. calcd for C26H35Cl2N3O7·0.5 H2O: C, 53.70; H, 6.24; N, 7.23.
T435	69633-69732	Sentence	denotes	Found: C, 53.74; H, 6.31; N, 7.31; HRMS (ESI+) calcd for C26H35Cl2N3O7+Na 594.1744, found 594.1729.
T436	69734-69834	Sentence	denotes	(3S)-3-[(N-Acetyl-l-leucyl)amino]-2-oxo-4-[(3S)-2-oxopyrrolidin-3-yl]butyl 2,6-Dichlorobenzoate (30)
T437	69835-70171	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	70172-70318	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	70319-70753	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	70754-70788	Sentence	denotes	Found: C, 53.27; H, 5.82; N, 7.84.
T441	70789-70853	Sentence	denotes	HRMS (ESI+) calcd for C23H29Cl2N3O6+H1 514.1506, found 514.1508.
T442	70855-70979	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	70980-71408	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	71409-71555	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	71556-71918	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	71919-71978	Sentence	denotes	Anal. calcd for C29H31Cl2N5O6: C, 56.50; H, 5.07; N, 11.36.
T447	71979-72014	Sentence	denotes	Found: C, 57.28; H, 5.32; N, 11.35.
T448	72015-72079	Sentence	denotes	HRMS (ESI+) calcd for C29H31Cl2N5O6+H1 616.1724, found 616.1729.
T449	72081-72218	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	72219-72630	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	72631-72843	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	72844-73209	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	73210-73276	Sentence	denotes	Anal. calcd for C27H35Cl2N3O7·0.5 H2O: C, 54.64; H, 6.11; N, 7.08.
T454	73277-73311	Sentence	denotes	Found: C, 54.26; H, 6.00; N, 6.87.
T455	73312-73376	Sentence	denotes	HRMS (ESI+) calcd for C27H35Cl2N3O7+H1 584.1925, found 584.1921.
T456	73378-73499	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	73500-73763	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	73764-73911	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	73912-74262	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	74264-74390	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	74391-74684	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	74685-74834	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	74835-75157	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	75159-75314	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	75315-75724	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	75725-75872	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	75873-76327	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	76328-76421	Sentence	denotes	Found: C, 65.65; H, 6.90; N, 9.58; HRMS (ESI+) calcd for C32H40N4O6 577.3021, found 577.3001.
T469	76423-76573	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	76574-76970	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	76971-77122	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	77123-77644	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	77646-77797	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	77798-78224	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	78225-78371	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	78372-78415	Sentence	denotes	MS (ESI+) for C25H34N4O6m/z 487.2 (M + H)+.
T477	78416-78862	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	78863-79008	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	79009-79522	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	79523-79617	Sentence	denotes	Found: C, 61.07; H, 7.09; N, 11.22; HRMS (ESI+) calcd for C25H34N4O6 487.2551, found 487.2541.
T481	79619-79770	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	79771-80195	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	80196-80341	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	80342-80857	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	80859-81005	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	81006-81277	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	81278-81391	Sentence	denotes	This clear pale yellow solution was treated with cesium fluoride (350 mg, 2.3 mmol) followed by heating to 65 °C.
T488	81392-81749	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	81750-81793	Sentence	denotes	MS (ESI+) for C32H36N4O8m/z 605.2 (M + H)+.
T490	81794-82076	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	82077-82168	Sentence	denotes	After 1 h, the volatiles were removed in vacuo (bath < 30 °C) to give a crude yellow glass.
T492	82169-82317	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	82318-82776	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	82778-82900	Sentence	denotes	N1-((1S)-3-(Benzyloxy)-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-N2-(tert-butoxycarbonyl)-N2-methyl-l-leucinamide
T495	82901-83192	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	83193-83350	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	83351-83614	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	83616-83771	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	83772-84185	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	84186-84340	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	84341-84795	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	84797-84953	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	84954-85398	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	85399-85554	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	85555-85941	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	85943-86053	Sentence	denotes	N2-(tert-Butoxycarbonyl)-N1-((1S)-3-chloro-2-oxo-1-{[(3S)-2-oxopyrrolidin-3-yl]methyl}propyl)-l-norleucinamide
T507	86054-86308	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.

T8

0-12

Sentence

denotes

Introduction

T9

13-111

Sentence

denotes

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

T10

112-566

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

567-950

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

951-1299

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

1300-1685

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

1686-2068

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

2069-2482

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

2483-2701

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

2702-2786

Sentence

denotes

Alternative modes of enzyme inhibition are covalently bound irreversible inhibitors.

T18

2787-3429

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

3430-3696

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

3697-3761

Sentence

denotes

Figure 1 Subsite nomenclature for proteolytic enzymes is shown.

T21

3762-3915

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

3916-4053

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

4054-4141

Sentence

denotes

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

T24

4142-4423

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

4424-4645

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

4646-4795

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

4796-5051

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

5052-5201

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

5202-5376

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

5377-5766

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

5767-5916

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

5917-6055

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

6056-6290

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

6291-6568

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

6569-6685

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

6686-6862

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

6863-6964

Sentence

denotes

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

T38

6965-7036

Sentence

denotes

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

T39

7037-7134

Sentence

denotes

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

T40

7135-7255

Sentence

denotes

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

T41

7256-7451

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

7452-7649

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

7650-7841

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

7842-8046

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

8047-8234

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

8235-8442

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

8443-8712

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

8713-9049

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

9051-9073

Sentence

denotes

Results and Discussion

T50

9075-9084

Sentence

denotes

Chemistry

T51

9085-9258

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

9259-9477

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

9478-9529

Sentence

denotes

Scheme 1 Preparation of P1 Synthetic Intermediates

T54

9530-9884

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

9885-9990

Sentence

denotes

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

T56

9991-10159

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

10160-10311

Sentence

denotes

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

T58

10312-10471

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

10472-10656

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

10657-10864

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

10865-10997

Sentence

denotes

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

T62

10998-11106

Sentence

denotes

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

T63

11107-11359

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

11360-11485

Sentence

denotes

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

T65

11486-11610

Sentence

denotes

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

T66

11611-11691

Sentence

denotes

Scheme 2 Preparation of Substituted Acyloxymethylketones and Aminomethylketones

T67

11692-11898

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

11899-12007

Sentence

denotes

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

T69

12008-12227

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

12228-12317

Sentence

denotes

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

T71

12318-12667

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

12668-12799

Sentence

denotes

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

T73

12800-12856

Sentence

denotes

Scheme 3 Preparation of Hydroxymethylketone Derivatives

T74

12857-13058

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

13059-13102

Sentence

denotes

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

T76

13104-13125

Sentence

denotes

Biological Evaluation

T77

13127-13171

Sentence

denotes

Irreversible Inhibitors of SARS CoV-1 3CLpro

T78

13172-13313

Sentence

denotes

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

T79

13314-13684

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

13685-13862

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

13863-14100

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

14101-14183

Sentence

denotes

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

T83

14184-14261

Sentence

denotes

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

T84

14262-14291

Sentence

denotes

SARS CoV-1 3CLpro FRETa

T85

14292-14329

Sentence

denotes

entry R kobs/I (M–1 s–1) IC50 (nM)

T86

14330-14356

Sentence

denotes

12 283,039 ± 22,586

T87

14357-14377

Sentence

denotes

13 Me 220 ± 0.5

T88

14378-14404

Sentence

denotes

14 cyc-propyl 182 ± 6

T89

14405-14431

Sentence

denotes

15 tert-butyl 230 ± 5

T90

14432-14449

Sentence

denotes

16 Ph 86 ± 3

T91

14450-14473

Sentence

denotes

17 4-OMe-Ph 79 ± 3

T92

14474-14496

Sentence

denotes

18 4-Me-Ph 87 ± 2

T93

14497-14519

Sentence

denotes

19 4-CN-Ph 53 ± 1

T94

14520-14541

Sentence

denotes

20 4-F-Ph 82 ± 3

T95

14542-14564

Sentence

denotes

21 4-Cl-Ph 97 ± 3

T96

14565-14600

Sentence

denotes

22 2,6-(Cl)2-Ph 62,993 ± 2,501

T97

14601-14633

Sentence

denotes

23 2,6-(F)2-Ph 12,776 ± 594

T98

14634-14666

Sentence

denotes

24 2-OH-4-Cl-Ph 11,525 ± 40

T99

14667-14702

Sentence

denotes

25 2-F, 4-CN-Ph 13,321 ± 2,309

T100

14703-14730

Sentence

denotes

26 2,6-(Me)2-Ph 74 ± 4

T101

14731-14760

Sentence

denotes

27 2,6-(MeO)2-Ph 205 ± 2

T102

14761-14783

Sentence

denotes

28 2-CN-Ph 17 ± 2

T103

14784-14950

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

14951-15042

Sentence

denotes

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

T105

15043-15180

Sentence

denotes

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

T106

15181-15310

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

15311-15505

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

15506-15709

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

15710-15853

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

15854-16034

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

16035-16145

Sentence

denotes

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

T112

16146-16480

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

16481-16583

Sentence

denotes

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

T114

16584-16655

Sentence

denotes

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

T115

16656-16749

Sentence

denotes

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

T116

16750-17191

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

17192-17573

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

17574-17790

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

17791-17878

Sentence

denotes

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

T120

17879-17990

Sentence

denotes

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

T121

17991-18148

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

18149-18238

Sentence

denotes

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

T123

18239-18405

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

18406-18642

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

18643-18718

Sentence

denotes

Initially, this derivative appeared to display reversible binding kinetics.

T126

18719-18900

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

18901-19052

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

19053-19229

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

19230-19450

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

19451-19677

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

19678-19791

Sentence

denotes

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

T132

19792-19856

Sentence

denotes

Each event of cell infection initiates newly synthesized 3CLpro.

T133

19857-19984

Sentence

denotes

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

T134

19986-20028

Sentence

denotes

Reversible Inhibitors of SARS CoV-1 3CLpro

T135

20029-20478

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

20479-20822

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

20823-20995

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

20996-21143

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

21144-21211

Sentence

denotes

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

T140

21212-21380

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

21381-21415

Sentence

denotes

c This value is a Ki measurement.

T142

21416-21464

Sentence

denotes

d These values are from a single determination.

T143

21465-21616

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

21617-21760

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

21761-21906

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

21907-22087

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

22088-22284

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

22285-22429

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

22430-22567

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

22568-22737

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

22738-22934

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

22935-23107

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

23108-23377

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

23378-23554

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

23555-23691

Sentence

denotes

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

T156

23692-23851

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

23852-24084

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

24085-24257

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

24258-24421

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

24422-24612

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

24613-24723

Sentence

denotes

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

T162

24724-24981

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

24982-25093

Sentence

denotes

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

T164

25094-25268

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

25269-25432

Sentence

denotes

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

T166

25433-25606

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

25607-25914

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

25915-26029

Sentence

denotes

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

T169

26030-26198

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

26199-26233

Sentence

denotes

c This value is a Ki measurement.

T171

26234-26282

Sentence

denotes

d These values are from a single determination.

T172

26283-26598

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

26599-26725

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

26726-26902

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

26903-27040

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

27041-27162

Sentence

denotes

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

T177

27163-27299

Sentence

denotes

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

T178

27300-27392

Sentence

denotes

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

T179

27393-27464

Sentence

denotes

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

T180

27465-27558

Sentence

denotes

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

T181

27559-27731

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

27732-27974

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

27975-28050

Sentence

denotes

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

T184

28051-28208

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

28209-28351

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

28352-28438

Sentence

denotes

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

T187

28439-28599

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

28600-28796

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

28797-29214

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

29215-29312

Sentence

denotes

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

T191

29313-29550

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

29551-29683

Sentence

denotes

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

T193

29684-29905

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

29906-29939

Sentence

denotes

Table 5 Protease Inhibition by 4

T195

29940-29991

Sentence

denotes

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

T196

29992-30030

Sentence

denotes

3CLpro SARS CoV-1 0.004 ± 0.0003b

T197

30031-30068

Sentence

denotes

3CLpro SARS CoV-2 0.00027 ± 0.0001b

T198

30069-30102

Sentence

denotes

cathepsin B human 93 1.3 ± 0.1

T199

30103-30129

Sentence

denotes

cathepsin D human 3 >10

T200

30130-30165

Sentence

denotes

leukocyte elastase human –31 >10

T201

30166-30193

Sentence

denotes

chymotrypsin human 8 >10

T202

30194-30219

Sentence

denotes

thrombin human –28 >10

T203

30220-30242

Sentence

denotes

pepsin human –2 >10

T204

30243-30269

Sentence

denotes

caspase 2 human –20 >10

T205

30270-30290

Sentence

denotes

HIV-1 HIV-1 0 >10

T206

30291-30308

Sentence

denotes

HCV HCV 16 >10

T207

30309-30329

Sentence

denotes

HRV HRV 1.7 ± 1

T208

30330-30365

Sentence

denotes

hCoV 229E human 0.004 ± 0.0004

T209

30366-30532

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

30533-30597

Sentence

denotes

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

T211

30598-30680

Sentence

denotes

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

T212

30681-30880

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

30881-30934

Sentence

denotes

This compound also does not inhibit the HCV replicon.

T214

30935-31113

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

31114-31144

Sentence

denotes

Table 6 Viral Inhibition by 4

T216

31145-31174

Sentence

denotes

virus EC50 (μM)a CC50 (μM)a

T217

31175-31201

Sentence

denotes

SARS CoV-1 4.8 ± 2.2 452

T218

31202-31229

Sentence

denotes

hCoV 229E 0.09 ± 0.04 >40

T219

31230-31250

Sentence

denotes

HRV-14 >100 >1,260

T220

31251-31271

Sentence

denotes

HRV-16 >100 >1,260

T221

31272-31290

Sentence

denotes

HIV-1 RF >32 >32

T222

31291-31315

Sentence

denotes

HCV replicon >320 >320

T223

31316-31338

Sentence

denotes

HCMV RC256 >100 >100

T224

31339-31505

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

31506-31718

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

31719-31978

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

31979-32254

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

32255-32420

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

32421-32664

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

32665-32935

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

32936-33083

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

33085-33096

Sentence

denotes

Conclusions

T233

33097-33300

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

33301-33417

Sentence

denotes

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

T235

33418-33528

Sentence

denotes

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

T236

33529-33875

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

33877-33897

Sentence

denotes

Experimental Section

T238

33899-33917

Sentence

denotes

General Procedures

T239

33918-34030

Sentence

denotes

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

T240

34031-34184

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

34185-34267

Sentence

denotes

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

T242

34268-34394

Sentence

denotes

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

T243

34395-34595

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

34596-34765

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

34766-34936

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

34937-35107

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

35108-35212

Sentence

denotes

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

T248

35213-35274

Sentence

denotes

Microwave-assisted reactions were run in a Biotage Initiator.

T249

35275-35348

Sentence

denotes

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

T250

35349-35445

Sentence

denotes

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

T251

35446-35536

Sentence

denotes

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

T252

35537-35697

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

35698-35848

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

35849-35935

Sentence

denotes

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

T255

35937-36004

Sentence

denotes

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

T256

36005-36293

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

36294-36496

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

36497-36684

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

36685-36870

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

36871-37074

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

37075-37272

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

37274-37362

Sentence

denotes

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

T263

37363-37535

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

37536-37693

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

37694-37796

Sentence

denotes

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

T266

37797-37969

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

37970-37975

Sentence

denotes

Note:

T268

37976-38109

Sentence

denotes

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

T269

38110-38202

Sentence

denotes

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

T270

38203-38318

Sentence

denotes

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

T271

38319-38403

Sentence

denotes

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

T272

38404-38518

Sentence

denotes

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

T273

38519-38667

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

38668-38888

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

38890-38979

Sentence

denotes

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

T276

38980-39269

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

39270-39445

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

39446-39664

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

39665-39775

Sentence

denotes

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

T280

39776-39885

Sentence

denotes

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

T281

39886-40102

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

40103-40221

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

40222-40535

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

40536-40570

Sentence

denotes

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

T285

40572-40665

Sentence

denotes

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

T286

40666-40964

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

40965-41248

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

41249-41404

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

41405-41669

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

41670-41912

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

41913-42179

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

42180-42368

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

42369-42654

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

42655-42713

Sentence

denotes

The reaction was quenched by the careful addition of satd.

T295

42714-42844

Sentence

denotes

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

T296

42845-43117

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

43118-43338

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

43340-43422

Sentence

denotes

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

T299

43423-43620

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

43621-43782

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

43783-43888

Sentence

denotes

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

T302

43889-44026

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

44027-44253

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

44255-44344

Sentence

denotes

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

T305

44345-44601

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

44602-44783

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

44784-45043

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

45045-45191

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

45192-45399

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

45400-45524

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

45525-45604

Sentence

denotes

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

T312

45605-45854

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

45855-46146

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

46147-46324

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

46325-46475

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

46476-46601

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

46602-46729

Sentence

denotes

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

T318

46730-46874

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

46875-47366

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

47368-47484

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

47485-47810

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

47811-47984

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

47985-48101

Sentence

denotes

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

T324

48102-48207

Sentence

denotes

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

T325

48208-48354

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

48355-48869

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

48870-48935

Sentence

denotes

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

T328

48936-48971

Sentence

denotes

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

T329

48973-49105

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

49106-49379

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

49380-49509

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

49510-49972

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

49973-50038

Sentence

denotes

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

T334

50039-50074

Sentence

denotes

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

T335

50076-50193

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

50194-50452

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

50453-50583

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

50584-51005

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

51006-51070

Sentence

denotes

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

T340

51071-51105

Sentence

denotes

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

T341

51107-51224

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

51225-51483

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

51484-51614

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

51615-52155

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

52156-52220

Sentence

denotes

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

T346

52221-52255

Sentence

denotes

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

T347

52257-52383

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

52384-52651

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

52652-52782

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

52783-53303

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

53304-53377

Sentence

denotes

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

T352

53378-53412

Sentence

denotes

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

T353

53414-53539

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

53540-53806

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

53807-53936

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

53937-54477

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

54478-54551

Sentence

denotes

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

T358

54552-54586

Sentence

denotes

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

T359

54588-54712

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

54713-54978

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

54979-55109

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

55110-55641

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

55642-55707

Sentence

denotes

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

T364

55708-55743

Sentence

denotes

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

T365

55745-55870

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

55871-56137

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

56138-56268

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

56269-56788

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

56789-56863

Sentence

denotes

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

T370

56864-56898

Sentence

denotes

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

T371

56900-57025

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

57026-57292

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

57293-57423

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

57424-57971

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

57972-58047

Sentence

denotes

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

T376

58048-58082

Sentence

denotes

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

T377

58084-58213

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

58214-58484

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

58485-58624

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

58625-59061

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

59062-59122

Sentence

denotes

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

T382

59123-59157

Sentence

denotes

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

T383

59158-59222

Sentence

denotes

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

T384

59224-59353

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

59354-59633

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

59634-59755

Sentence

denotes

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

T387

59756-60278

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

60280-60415

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

60416-60688

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

60689-60829

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

60830-61346

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

61347-61445

Sentence

denotes

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

T393

61447-61580

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

61581-61864

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

61865-62018

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

62019-62561

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

62562-62660

Sentence

denotes

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

T398

62662-62791

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

62792-63065

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

63066-63214

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

63215-63694

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

63695-63729

Sentence

denotes

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

T403

63730-63791

Sentence

denotes

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

T404

63793-63923

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

63924-64198

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

64199-64347

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

64348-64899

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

64900-64934

Sentence

denotes

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

T409

64936-65060

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

65061-65335

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

65336-65456

Sentence

denotes

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

T412

65457-65945

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

65946-66043

Sentence

denotes

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

T414

66045-66142

Sentence

denotes

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

T415

66143-66384

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

66385-66462

Sentence

denotes

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

T417

66463-66680

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

66681-66830

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

66831-67096

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

67098-67205

Sentence

denotes

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

T421

67206-67415

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

67416-67553

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

67554-67719

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

67720-67847

Sentence

denotes

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

T425

67848-68077

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

68078-68435

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

68436-68501

Sentence

denotes

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

T428

68502-68536

Sentence

denotes

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

T429

68537-68600

Sentence

denotes

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

T430

68602-68717

Sentence

denotes

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

T431

68718-69054

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

69055-69206

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

69207-69565

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

69566-69632

Sentence

denotes

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

T435

69633-69732

Sentence

denotes

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

T436

69734-69834

Sentence

denotes

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

T437

69835-70171

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

70172-70318

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

70319-70753

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

70754-70788

Sentence

denotes

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

T441

70789-70853

Sentence

denotes

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

T442

70855-70979

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

70980-71408

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

71409-71555

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

71556-71918

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

71919-71978

Sentence

denotes

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

T447

71979-72014

Sentence

denotes

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

T448

72015-72079

Sentence

denotes

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

T449

72081-72218

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

72219-72630

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

72631-72843

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

72844-73209

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

73210-73276

Sentence

denotes

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

T454

73277-73311

Sentence

denotes

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

T455

73312-73376

Sentence

denotes

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

T456

73378-73499

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

73500-73763

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

73764-73911

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

73912-74262

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

74264-74390

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

74391-74684

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

74685-74834

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

74835-75157

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

75159-75314

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

75315-75724

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

75725-75872

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

75873-76327

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

76328-76421

Sentence

denotes

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

T469

76423-76573

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

76574-76970

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

76971-77122

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

77123-77644

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

77646-77797

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

77798-78224

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

78225-78371

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

78372-78415

Sentence

denotes

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

T477

78416-78862

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

78863-79008

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

79009-79522

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

79523-79617

Sentence

denotes

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

T481

79619-79770

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

79771-80195

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

80196-80341

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

80342-80857

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

80859-81005

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

81006-81277

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

81278-81391

Sentence

denotes

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

T488

81392-81749

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

81750-81793

Sentence

denotes

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

T490

81794-82076

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

82077-82168

Sentence

denotes

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

T492

82169-82317

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

82318-82776

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

82778-82900

Sentence

denotes

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

T495

82901-83192

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

83193-83350

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

83351-83614

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

83616-83771

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

83772-84185

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

84186-84340

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

84341-84795

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

84797-84953

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

84954-85398

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

85399-85554

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

85555-85941

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

85943-86053

Sentence

denotes

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

T507

86054-86308

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

86309-86461

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

86462-86613

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

86614-86840

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

86842-86975

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

86976-87372

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

87373-87521

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

87522-88014

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

88015-88050

Sentence

denotes

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

T516

88052-88191

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

88192-88609

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

88610-88753

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

88754-89285

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

89286-89383

Sentence

denotes

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

T521

89385-89452

Sentence

denotes

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

T522

89453-89681

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

89682-89797

Sentence

denotes

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

T524

89798-89904

Sentence

denotes

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

T525

89905-90056

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

90057-90448

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

90450-90504

Sentence

denotes

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

T528

90505-90804

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

90805-90974

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

90975-91057

Sentence

denotes

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

T531

91058-91390

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

91391-91641

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

91643-91688

Sentence

denotes

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

T534

91689-91986

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

91987-92094

Sentence

denotes

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

T536

92095-92271

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

92272-92527

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

92529-92648

Sentence

denotes

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

T539

92649-92939

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

92940-93102

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

93103-93519

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

93521-93663

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

93664-94069

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

94070-94219

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

94220-94704

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

94706-94854

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

94855-95278

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

95279-95425

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

95426-95962

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

95964-96102

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

96103-96477

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

96478-96759

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

96760-96881

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

96882-97153

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

97154-97220

Sentence

denotes

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

T556

97221-97527

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

97529-97683

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

97684-98106

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

98107-98395

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

98396-98517

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

98518-98841

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

98842-98928

Sentence

denotes

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

T563

98929-99473

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

99475-99630

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

99631-99960

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

99961-100225

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

100226-100648

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

100649-100684

Sentence

denotes

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

T569

100686-100797

Sentence

denotes

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

T570

100798-101052

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

101053-101210

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

101211-101539

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

101541-101666

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

101667-102058

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

102059-102216

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

102217-102697

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

102699-102830

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

102831-103238

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

103239-103401

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

103402-103966

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

103967-104002

Sentence

denotes

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

T582

104004-104139

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

104140-104571

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

104572-104727

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

104728-105185

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

105186-105282

Sentence

denotes

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

T587

105284-105425

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

105426-105842

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

105843-105992

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

105993-106534

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

106535-106629

Sentence

denotes

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

T592

106631-106772

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

106773-107188

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

107189-107337

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

107338-107833

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

107834-107928

Sentence

denotes

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

T597

107930-108070

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

108071-108353

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

108354-108502

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

108503-109016

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

109018-109148

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

109149-109584

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

109585-109834

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

109835-110265

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

110267-110403

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

110404-110811

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

110812-111133

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

111134-111555

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

111556-111618

Sentence

denotes

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

T610

111619-111653

Sentence

denotes

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

T611

111654-111714

Sentence

denotes

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

T612

111716-111852

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

111853-112262

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

112263-112515

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

112516-112913

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

112914-112975

Sentence

denotes

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

T617

112977-113124

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

113125-113440

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

113441-113621

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

113622-113819

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

113820-113973

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

113974-114490

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

114492-114623

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

114624-114928

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

114929-115091

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

115092-115530

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

115532-115669

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

115670-116080

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

116081-116243

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

116244-116704

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

116705-116739

Sentence

denotes

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

T632

116741-116878

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

116879-117296

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

117297-117451

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

117452-117908

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

117910-117953

Sentence

denotes

SARS CoV-1 Protease FRET Assay and Analysis

T637

117954-118079

Sentence

denotes

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

T638

118080-118226

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

118227-118269

Sentence

denotes

The fluorescence of the cleaved TAMRA (ex.

T640

118270-118280

Sentence

denotes

558 nm/em.

T641

118281-118383

Sentence

denotes

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

T642

118384-118503

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

118504-118701

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

118702-118782

Sentence

denotes

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

T645

118783-119032

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

119033-119173

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

119174-119411

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

119412-119515

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

119516-119640

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

119641-119866

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

119868-119911

Sentence

denotes

SARS CoV-2 Protease FRET Assay and Analysis

T652

119912-120066

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

120067-120477

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

120478-120584

Sentence

denotes

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

T655

120585-120709

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

120710-120836

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

120837-121012

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

121013-121101

Sentence

denotes

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

T659

121102-121328

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

121330-121357

Sentence

denotes

SARS CoV-1 Antiviral Assays

T661

121358-121404

Sentence

denotes

All assays were performed in BSL3 containment.

T662

121405-121499

Sentence

denotes

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

T663

121500-121799

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

121800-121955

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

121956-122118

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

122119-122305

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

122306-122479

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

122480-122733

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

122734-122997

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

122999-123025

Sentence

denotes

hCOV 229E Antiviral assays

T671

123026-123109

Sentence

denotes

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

T672

123110-123335

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

123336-123591

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

123592-123845

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

123846-124104

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

124106-124128

Sentence

denotes

Other Antiviral Assays

T677

124129-124290

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

124292-124341

Sentence

denotes

Expression and Purification of CoV-2 3CL Protease

T679

124342-124593

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

124594-124720

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

124721-124866

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

124867-124921

Sentence

denotes

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

T683

124922-125029

Sentence

denotes

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

T684

125030-125083

Sentence

denotes

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

T685

125084-125219

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

125220-125279

Sentence

denotes

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

T687

125280-125384

Sentence

denotes

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

T688

125385-125670

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

125671-125824

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

125826-125873

Sentence

denotes

Crystallization/Soak Protocols for SARS1 with 2

T691

125874-125967

Sentence

denotes

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

T692

125968-126080

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

126081-126238

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

126239-126460

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

126461-126583

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

126584-126718

Sentence

denotes

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

T697

126720-126768

Sentence

denotes

Crystallization/Soak Protocols for SARS1 with 28

T698

126769-126863

Sentence

denotes

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

T699

126864-126977

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

126978-127135

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

127136-127404

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

127405-127527

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

127528-127662

Sentence

denotes

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

T704

127664-127735

Sentence

denotes

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

T705

127737-127759

Sentence

denotes

LJEC2520 + PF-00835231

T706

127760-127840

Sentence

denotes

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

T707

127841-127971

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

127972-128129

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

128130-128356

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

128357-128477

Sentence

denotes

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

T711

128478-128610

Sentence

denotes

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

T712

128612-128634

Sentence

denotes

LJEC2521 + PF-00835231

T713

128635-128718

Sentence

denotes

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

T714

128719-128851

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

128852-129009

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

129010-129246

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

129247-129382

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

129383-129515

Sentence

denotes

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

T719

129517-129539

Sentence

denotes

LJEC2522 + PF-00835231

T720

129540-129610

Sentence

denotes

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

T721

129611-129807

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

129808-130050

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

130051-130192

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

130193-130338

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

130339-130478

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

130479-130503

Sentence

denotes

Structure determination.

T727

130504-130653

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

130654-130927

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

130928-131143

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.

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