HCV NS5A Replication Complex Inhibitors. Part 4.1 Optimization for

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HCV NS5A Replication Complex Inhibitors. Part 4.1 Optimization for Genotype 1a Replicon Inhibitory Activity Denis R. St. Laurent,*,† Michael H. Serrano-Wu,†,∥ Makonen Belema,† Min Ding,† Hua Fang,‡ Min Gao,‡ Jason T. Goodrich,† Rudolph G. Krause,‡ Julie A. Lemm,‡ Mengping Liu,‡ Omar D. Lopez,† Van N. Nguyen,† Peter T. Nower,‡ Donald R. O’Boyle II,‡ Bradley C. Pearce,§ Jeffrey L. Romine,† Lourdes Valera,‡ Jin-Hua Sun,‡ Ying-Kai Wang,‡ Fukang Yang,† Xuejie Yang,†,⊥ Nicholas A. Meanwell,† and Lawrence B. Snyder† Departments of †Medicinal Chemistry, ‡Virology, and §Computer-Aided Drug Design, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, Connecticut 06492, United States ABSTRACT: A series of symmetrical E-stilbene prolinamides that originated from the library-synthesized lead 3 was studied with respect to HCV genotype 1a (G-1a) and genotype 1b (G1b) replicon inhibition and selectivity against BVDV and cytotoxicity. SAR emerging from an examination of the prolinamide cap region revealed 11 to be a selective HCV NS5A inhibitor exhibiting submicromolar potency against both G-1a and G-1b replicons. Additional structural refinements resulted in the identification of 30 as a potent, dual G-1a/1b HCV NS5A inhibitor.

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INTRODUCTION

to the lack of an effective protective immune response against infection.8 Daclatasvir (2, Figure 1) is a potent HCV inhibitor that provided the first clinical proof-of-concept for the relevance of

Often deemed the quiet epidemic, hepatitis C virus (HCV) infection affects an estimated 170 million individuals worldwide including more than 4 million Americans.2 Approximately 85% of infected patients progress to a chronic condition that is often asymptomatic but that, without treatment, may lead to cirrhosis, hepatocellular carcinoma, and ultimately death.2 At present, chronic HCV infection is the leading cause of liver failure and consequently liver transplantation in the U.S. Optimal therapy has evolved to rely upon a combination of pegylated interferon-α (PEG-IFN-α) and ribavirin with a protease inhibitor administered for 24−48 weeks.3,4 However, the significant incidence of adverse side effects associated with each of these therapeutic agents as well as the suboptimal response rate observed in G-1-infected patients, which is attributed to the selection of resistant variants particularly in those that have an underlying poor response to PEG-IFN-α, provides an impetus to develop effective therapies with improved efficacy and tolerability.5 HCV is a positive strand RNA virus that is classified into six major genotypes with over 100 subtypes and is a member of the Flaviviridae family.6 Its genome encodes for a polyprotein of approximately 3200 amino acids that is processed by host signal peptidases and by HCV-encoded proteases into three structural proteins (core, E1, E2), an ion channel (p7), and six nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B), which are collectively responsible for viral replication.6,7 HCV exhibits a high replication rate and a poor mechanistic control of genome fidelity that, taken together, may contribute © XXXX American Chemical Society

Figure 1. Prototype stilbene prolinamide 1 and daclatasvir (2).

targeting the NS5A protein and has demonstrated the potential to cure G-1b HCV infection when administered as part of a combination with the NS3 protease inhibitor asunaprevir, G-1a infection in combination with asunaprevir, PEG-IFN-α and ribavirin, and both G-1a and G-1b when dosed in conjunction with the nucleoside-based NS5B inhibitor sofosbuvir.9a−c,10 More recently, other regimens comprising direct-acting antiviral Special Issue: HCV Therapies Received: December 6, 2012

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dx.doi.org/10.1021/jm301796k | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Table 1. Structure and G-1a/1b Replicon Inhibitory Activity of Symmetrical E-Stilbene Prolinamides

a

Data represent mean values of at least two experiments with a maximum of 3-fold variation. G-1a values are from ELISA and G-1b values are from FRET assays unless noted otherwise.13 bBVDV (Luc) CC50 > 10 μM for all and BVDV (Luc) EC50 > 5 μM for all compounds except 3 (BVDV EC50 = 4.7 μM), 7 (BVDV EC50 = 3.1 μM), and 14 (BVDV EC50 = 0.042 μM).13 cLuc assay value. dFRET assay value.

partly because the desethyl analogue 4 exhibited a G-1a inhibitory potency that was 7-fold weaker than that of 3, albeit accompanied with a 10-fold relative enhancement in G-1b potency. However, an overlay of conformationally minimized fragments of 3 and 4 (truncated to reduce complexity) revealed similar dihedral angles (−114.6° vs −116.6°) between the benzamide carbonyl and the aryl rings (Figure 2).14 The dihedral angle defined between one fork of the aryl ring and the benzamide carbonyl differ by just 2°. This observation indicated that the enhanced G-1a activity of analogue 3 was not likely due to a sterically driven ortho effect, leading to

agents and ribavirin have demonstrated the capacity to cure chronic HCV infection.9d−f The campaign that discovered daclatasvir (2) had its origins in the stilbene prolinamide lead 1, which interestingly exhibited potent inhibitory activity toward a G-1b replicon (EC50 = 86 pM) but essentially no activity toward G-1a (EC50 > 10 μM).10,11 Herein, we report an expansion of the SAR survey associated with 1 that led to the capture of G-1a replicon inhibition in the stilbene prolinamide series, a critical step in the program that ultimately led to the discovery of daclatasvir (2).

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RESULTS AND DISCUSSION Shortly after the discovery of compound 1, a lead optimization effort was initiated to generate small libraries of stilbene prolinamides that resulted in the identification of stilbene 3, a pivotal compound first reported by Snyder et al. that exhibited modest G-1a replicon inhibitory activity with EC50 = 0.95 μM (Table 1).12 Although the G-1b replicon inhibitory activity of 3 was substantially weaker than prototype 1, its modest selectivity for HCV versus BVDV (EC50 = 5 μM) and minimal cytotoxicity (CC50 > 30 μM) suggested an opportunity for further exploration.13 Attention was initially focused on the ortho-positioned ethyl group and its potential role in modulating planarity between the cap and pyrrolidine ring

Figure 2. Proline ring overlay of the lowest energy conformation of truncated 3 (yellow) with 4 (purple). The dihedral angle referred to in the discussion is indicated in green. B



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consideration of a second model.15 Normally for an ortho effect, the ethyl group would change the out-of-plane bend significantly; however, in this case the proline ring and observed hydrophobic collapse/π-stacking interactions favor similar nonplanar conformations. Figure 3 depicts a topological

Table 2. Structure and G-1a/1b Replicon Inhibitory Activity of Symmetrical E-Stilbene N-Prolinamides

Figure 3. Topological overlay of the G-1b active PhAc cap of 1 with the G-1a active 2-ethylpicolinoyl cap of 7 which predicted the design of compounds 6 and 11.

hybridization of the cap elements of prototypes 1 and 3 that led to conjecture that the inhibitory activity toward G-1a replication may be the result of a specific interaction accessible to 3 but not to 1. In probing the ortho region of 3, the topologically similar vinyl analogue 5 (EC50 = 1.1 μM) was found to be equivalent to 3 with respect to G-1a inhibitory potency, further suggesting that the G-1a activity is related to the benzoyl aromatic ring or an effect of the ring rather than the presence of an ortho appendage. However, the simple hybridization of 1 and 3 produced the fused naphthyl analogue 6, which exhibited poor inhibitory activity (EC50 > 10 μM) toward the G-1a replicon. As part of the study, a nitrogen atom was introduced into the benzoyl ring of 3 which afforded pyridine analogue 7, a compound exhibiting similar G-1a inhibitory activity to 3, along with enhanced G-1b inhibitory potency. This observation initiated the synthesis of a series of analogues, compounds 8− 13 in Table 1, from which the isoquinolinamide 11 emerged as a compound of interest that exhibited reasonable inhibitory activity toward both G-1a and G-1b replicons. Evaluation of the topological isomer variants 12−14 revealed modest potency variation toward both genotypes but the two bis-aza analogues of 11, compounds 15 and 16, demonstrated similar inhibitory potency toward the G-1a replicon but differed significantly (100-fold) in potency toward G-1b.16 Comparison of 11 with the analogous naphthyl derivative 6 revealed an enhancement in both G-1a (>25-fold) and G-1b (∼75-fold) replicon inhibitory activity, highlighting the important contribution of the nitrogen atom. This observation led to the preparation and evaluation of additional derivatives of isoquinoline 11, and the compounds comprising this phase of the study are compiled in Table 2. In order to quickly gauge where substitution may enhance inhibitory activity, a chlorine atom scan was conducted across positions 3−7 of the isoquinoline ring by taking advantage of the availability of relevant synthetic precursors from an accompanying HCV NS3 inhibitor program. From this effort it became apparent that regioisomer 17 was more potent than the alternative isomers, with G-1a EC50 = 62 nM and G-1b EC50 = 23 nM. The sensitivity of the G-1a SAR is most effectively illustrated by regioisomer 21 which suffered a significant potency loss in the G-1a replicon but with minimal impact in the G-1b replicon. Structure−activity studies at the C-3 position of 11 revealed the fluoro analogue 22 to be equipotent and the Me derivative 24 to be several-fold weaker toward G-1a, whereas the 3-MeO compound 26 suffered a

a

Data represent mean values of at least two experiments with a maximum of 3-fold variation. G-1a values are from ELISA and G-1b values are from FRET assays unless noted otherwise.13 bIn all cases, BVDV (Luc) CC50 and BVDV (Luc) EC50 are > 10 μM except for 11 (BVDV EC50 = 6.4 μM), 20 (BVDV EC50 = 2.1 μM), and 23 (BVDV EC50 = 7.9 μM).13

substantial loss of inhibitory activity toward the G-1a replicon with only minimal effect on G-1b inhibition. The notable reduction in potency of the 3-Cl naphthyl analogue 28 when compared with the 3-Cl isoquinoline 17 lends further support to the observation that the presence of the nitrogen atom provides a productive impact on G-1a inhibitory potency.17a,b On the basis of the SAR pattern gleaned from the monosubstitution studies, dual substitution patterns were next examined while retaining the chlorine at the 3-position. Similar potency gains were achieved upon introduction of substituents into the 5-position, as observed with the analogues 29 and 30. The excellent G-1a and G-1b data obtained for the 3-chloro-5methoxy-1-isoquinolinoyl (CMIQ) derivative 30,17c initiated additional profiling, and this molecule was found to exhibit inhibitory activity toward a panel of HCV genotype variants, including G-2a JFH (EC50 = 2.2 nM), G-2a NIH (EC50 = 14 C



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Figure 4. Stereoview of proline ring and N-substituent overlay of truncated 3 (yellow) with truncated 30 (blue).

nM), G-3a (EC50 = 3.2 nM), and G-5a hybrid (EC50 = 3.0 nM), with an excellent selectivity index based on the measured cytotoxicity, CC50 > 10.0 μM.10,13c However, 30 exhibited weaker activity (EC50 > 1000 nM) toward a G-4a hybrid replicon. By way of comparison, stilbene prolinamide 1 exhibited an inferior profile toward G-1a (EC50 > 10000 nM), G-2a JFH (EC50 = 79 nM) and G-2a NIH (EC50 = 260 nM) replicons. In contrast, 1 exhibited stronger activity toward G-3a (EC50 = 0.30 nM), G-4a hybrid (EC50 = 157 nM), and G5a hybrid (EC50 < 0.23 nM) replicons. Overlaying the isoquinolinamide fragments of 30 and the 2ethylbenzoyl moiety of 3 exhibited single clusters within a conformational minimum of 0.15 kcal/mol (Figure 4).18 By comparison, the more flexible phenacetyl (PhAc) cap found in 1 exhibits visible edge-to-face π-stacking with the anilide ring and, when overlaid with 30, shows poorer conformational alignment (Figure 5). This difference may suggest a bioactive conformation in the G-1b NS5A pocket that more closely mimics that of compound 1, while that of the G-1a variant pocket more closely aligns with compound 30.18

Table 3. G-1a/1b Replicon Inhibitory Activity for a Series of Symmetrical N-PhAc- and N-CMIQ-Linked Prolinamide Analogues

a

Data represent mean values of at least two experiments with a maximum of 3-fold variation.13 bIn all cases, BVDV (Luc) CC50 and BVDV (Luc) EC50 are >10 μM except for 37 where BVDV (Luc) CC50 = 3.3 μM.13 cEC50 (ELISA) = 19 nM.

(CMIQ) motif, this cap was introduced into an alkyne-linked core and evaluated side-by-side with its PhAc analogue. The alkyne-based core proved to be a suitable replacement for the stilbene, and the profile of compound 33 mimicked that of 1 with good G-1b replicon inhibitory potency and poor G-1a replicon inhibition (Table 3).1 G-1a inhibitory activity was confirmed with compound 34 bearing the CMIQ cap, demonstrating good inhibitory activity against the G-1a replicon relative to the PhAc-capped 33 while simultaneously maintaining the G-1b replicon inhibitory activity. Moreover, a similar trend was seen for heterocyclic cores, as reflected in 35−38 where the CMIQ analogues 36 and 38 were consistently superior in both G-1a and G-1b replicons to the less potent PhAc counterparts 35 and 37.

Figure 5. Proline ring overlay of the phenacetyl cap of 1 (light blue) with the cap element of 30 (blue) showing poor conformational alignment.

In a concurrent effort, the effect of monosubstitution on potency was also briefly examined within the context of the quinolinamide 13, which differs from 11 by its topological point of attachment to the heterocycle. Even though the G-1b potency of 31, which was within 4- to 5-fold of isoquinolinamide 27, was encouraging (Table 3), follow-up effort failed to further enhance the G-1a activity in general, and most of the analogues prepared in this quinoline cap series suffered from a poor selectivity index with respect to cytotoxicity (data not shown). The position of the substituents about the quinolinamide nucleus had a large impact on potency, as reflected in the difference between analogues 31 and 32, which was >79-fold and >15-fold for G-1a and G-1b potency, respectively. Although 32 from this series showed noteworthy potency, data for the quinolinamide nucleus seemed less predictable overall; therefore, the more potent isoquinolinamide series was pursued. In order to further validate the G-1a replicon inhibitory potency achieved with the 3-chloro-5-methoxyisoquinolinoyl

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CHEMISTRY Synthetic access to the acyl-capped E-stilbene prolinamides compiled in Table 1 was generally secured from commercially available carboxylic acids and E-stilbene prolinamide 41 which, in turn, was prepared in a two-step sequence from 4,4′-(ethene1,2-diyl)dianiline (39) and N-Boc-L-proline, as shown in Scheme 1.19 Thus, coupling of 39 and N-Boc-L-proline using EEDQ20 followed by Boc deprotection using either trifluoroacetic acid (TFA) in CH2Cl2 or 4 N HCl in dioxane afforded D



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Scheme 1. Synthesis of Target Moleculesa

of triflate 52 with tri(n-butyl)vinylstannane in the presence of bis(triphenylphosphine)palladium(II) dichloride followed by saponification gave 3-vinylpicolinic acid (56) in good yield.27 An extended reaction time of 16 h at 100 °C caused concominant hydrolysis of the methyl ester, and subsequent hydrogenation of the vinyl precursor gave 3-ethylpicolinic acid (57) directly.28 Suzuki coupling of triflate 52 with phenylboronic acid followed by saponification yielded the phenyl derivative 58.29 The 1-isoquinolinecarboxylic acids 89−101 were accessed through a variety of methods, as illustrated in Scheme 3.22−24,30 Scheme 3. Synthesis of Isoquinoline-1-carboxylic Acidsa

a

Reagents and conditions: (a) N-Boc-L-proline, EEDQ, CH2Cl2, rt; (b) TFA, CH2Cl2, 0 °C to rt or 4 N HCl in dioxane, MeOH, 0 °C to rt; (c) Cap-OH, EDCI, DIEA, THF, rt or Cap-OH, HATU, DIEA, DMF, rt.

41 in excellent overall yield.21 The latter method was often preferred in order to avoid contamination of final targets with trifluoroacetamide side products. The general coupling condition outlined in Scheme 1 was also applied to the stilbene bioisostere 4,4′-(ethyne-1,2-diyl)dianiline (42) as well as heterocycles 45 and 48.22−24 Hence, direct coupling of 42, 45, and 48 with N-Boc-L-proline using HATU gave the respective precursors 43, 46, and 49 which were elaborated to the corresponding parent molecules 44, 47, and 50 under the noted conditions. Similarly, as with all phenylacetyl (PhAc)capped prolinamides shown in Tables 1 and 3, direct coupling of cores such as 44, 47, and 50 with N-PhAc-L-proline25 using EEDQ furnished the corresponding PhAc-capped target molecules 33, 35, and 37 in excellent yield. EDCI or HATU was employed to mediate coupling of the arylcarboxylic acids with E-stilbene prolinamide 41 or heterocyclic prolinamides 44, 47, and 50 in the presence of N,N′-diisopropylethylamine.26 Purification of final targets 1−38 was performed by reverse phase preparative HPLC using MeOH or CH3CN buffered with 0.1% TFA as the mobile phase. The 3-substituted picolinic acids 56−58 were prepared from methyl 3-hydroxypicolinic acid (51) through its triflate intermediate (52), as summarized in Scheme 2. Stille reaction

a

See Table 2 for a full accounting of R = halogen. Reagents and conditions: (a) PPh3, MeOH, DEAD, rt; (b) mCPBA, CH2Cl2, rt; (c) TMSCN, CH3CN, 75 °C; (d) KCN, PhSO2Cl, H2O, CH2Cl2; (e) NaBH4, EtOH, rt; (f) KCN, MeSO2Na, 18-crown-6, DMF; (g) Pd(OAc)2, KCN, TMEDA, dpppe, toluene, 150 °C; (h) 12 N HCl, 80 °C; (i) 5 N NaOH, 85 °C or 6 N NaOH, dioxane, reflux.

Many of the acids were prepared from the corresponding 1chloro- or 1-bromoisoquinolines 66−75 and were subject to Beller’s procedure involving palladium-assisted insertion of cyanide.31 The resultant 1-isoquinolinecarbonitriles 76−88 were subjected to basic or acidic hydrolysis at elevated temperature to provide the desired 1-isoquinolinecarboxylic acids. Methoxy analogues 89 and 90 were prepared through a two-step sequence involving first oxidation of the respective isoquinolines 60 and 63 with m-CPBA followed by rearrangement and concurrent cyanide incorporation via trimethylsilyl cyanide.32 Again, basic or acidic hydrolysis at elevated temperature readily afforded the 1-isoquinolinecarboxylic acids 89 and 90. Notably, the 3- and 5-methoxyisoquinolines 60 and 63 were secured from their respective hydroxy analogues 59 and 62 through a Mitsunobu protocol.33 In contrast to the palladium-assisted insertion of cyanide, 3-methylisoquinolinecarbonitrile (78) was prepared by the phenylsulfonyl-assisted insertion of cyanide into the parent isoquinoline ring 65.34 Subsequent sodium borohydride mediated reduction provided 3-methylisoquinolinecarboxylic acid 91 after basic hydrolysis. Direct displacement of 1-bromo-5-methylisoquinoline (66) with potassium cyanide assisted by sodium methylsulfinate and 18-crown-6 in DMF was the method of choice for preparing 5-methylisoquinolinecarboxylic acid 92 following basic hydrolysis.35 A slightly

Scheme 2. Synthesis of Picolinic Acidsa

Reagents and conditions: (a) Tf2O, TEA, CH2Cl2, 0 °C; (b) nBu3SnCHCH2, PdCl2(PPh3)2, LiCl, DMF, 100 °C, 4 or 16 h;28 (c) PhB(OH)2, Pd(dppf)Cl2·DCM, K3PO4, THF, reflux; (d) 1 N NaOH, MeOH, rt; (e) LiOH, H2O, THF, rt or NaOH, H2O, MeOH, rt. a

E



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modified version of a procedure disclosed by Song et al.36 was used to prepare quinolinecarboxylic acids 105 and 106 from manisidine (102) via the intermediacy of methylquinolines 103 and 104, respectively (see Scheme 4).

Table 4. LCMS Conditions Used in the Characterization of All Compounds Found in the Experimental Section LCMS conditiona method method method method method method method method method method method method

Scheme 4. Synthesis of Quinoline-2-carboxylic Acidsa

a

Reagents and conditions: (a) crotonaldehyde, 2,3-dichloro-1,4naphthoquinone, conc HCl, n-BuOH, 130 °C, 2 h; (b) SeO2, pyridine, 115 °C, 5 h.

GTc

FRd

Sol Ae

Sol Bf

3 3 1 4 5 2 3 5 6 7 8 9

3 2 4 3 2 2 3 2 3 3 3 3

4 5 0.8 4 5 1 4 5 4 4 4 4

A1 A1 A2 A1 A4 A3 A4 A1 A1 A1 A1 A1

B1 B1 B2 B1 B4 B3 B4 B1 B1 B1 B1 B1

a Gradient = 0% B to 100% B; wavelengh (λ) = 220 nm; injection volume = 5 μL unless stated otherwise. bCol = column where column 1 = Phenomenex-Luna C18 (2.0 mm × 50 mm, 3 μm); column 2 = Phenomenex-Luna C18 (2.0 mm × 30 mm, 3 μm); column 3 = XTERRA C18 (3.0 mm × 50 mm, S7); column 4 = XTERRA C18 (4.6 mm × 50 mm, S5); column 5 = XTERRA C18 (4.6 mm × 30 mm, S5); column 6 = Phenomenex-Luna C18 (3.0 mm × 50 mm, 5 μm); column 7 = Phenomenex-Luna C18 (4.6 mm × 50 mm, S10); column 8 = Phenomenex-Luna C18 (3.0 mm × 50 mm, S7); column 9 = XTERRA C18 (4.6 mm × 50 mm, S7). cGT = Gradient time (min). d FR = Flow rate (mL/min). eSol A = solvent A where solvent A1 = 0.1% TFA/10% MeOH/90% H2O; solvent A2 = 0.1% TFA/5% MeOH/95% H2O; solvent A3 = 0.1% TFA/10% CH3CN/90% H2O; solvent A4 = 10 mM NH4OAc/5% CH3CN/90% H2O. fSol B = solvent B where solvent B1 = 0.1% TFA/90% MeOH/10% H2O; solvent B2 = 0.1% TFA/95% MeOH/5% H2O; solvent B3 = 10 mM NH4OAc/90% CH3CN/10% H2O; solvent B4 = 10 mM NH4OAc/ 95% CH3CN/5% H2O.

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CONCLUSION In summary, a series of symmetrical E-stilbene N-capped prolinamides was examined as inhibitors of HCV NS5A with the objective of introducing significant G-1a replicon inhibitory activity into the chemotype. It was discovered that isoquinolinoyl caps imparted G-1a replicon inhibition to stilbene prolinamide lead 1 and an optimization effort uncovered analogue 30 with excellent inhibitory potency toward both G-1a and G-1b replicons (EC50 = 37 nM and EC50 = 7 nM, respectively) and a panel of HCV genotype variants that included G-2a JFH (EC50 = 2.2 nM), G-2a NIH (EC50 = 14 nM), G-3a (EC50 = 3.2 nM), and G-5a (EC50 = 3.0 nM), with good selectivity against BVDV (EC50 > 10 μM) and minimal cytotoxicity (CC50 > 10 μM). Demonstrating that dual G-1a/1b HCV replicon inhibition could be achieved with this NS5A inhibitor chemotype was an essential step for the campaign that ultimately led to the discovery of daclatasvir (2), and this study describes the key underlying observations.

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Colb

height maximum, fwhm). The instrument was calibrated daily according to the manufacturer’s specifications, resulting in mass accuracy of ≤5 ppm. The operating software Xcalibur was used to calculate theoretical mass-to-charge values and to process the obtained data. A Horizon flash column and collector station (Biotage Inc.) was used to conduct silica gel flash chromatography (solvent systems indicated in parentheses). Preparative HPLC was performed on a Shimadzu instrument using a Phenomenex Gemini or Luna column (30 mm × 100 mm, S10) operating at 40 mL/min at 220 nm with an injection volume of 500−2000 μL using one of the four solvent pairs described in the LCMS table. The gradients varied between 0% B or 20% B to 100% B over 14−30 min unless mentioned otherwise in the experimental procedure. Representative procedures and physical properties for synthesized compounds are described below. Purification of final targets was performed by reverse phase preparative HPLC using methanol or acetonitrile buffered with 0.1% TFA as mobile phase. The final products were isolated as TFA salts upon evaporation of the mobile phase with the assumption that each basic functionality in the final product (e.g., pyridine, isoquinoline, quinoline, etc.) formed full integral TFA salts, which is reflected in the reaction yield and EC50 calculation. In the cases examined, it was found that the final targets had TFA contents as high as 2 mol equiv when dried in the manner described above. For example, the percent TFA content of 31 and 32 was analyzed by 19F NMR and found to be between 0.63 and 0.70 mol equiv when using 2-fluoroisonicotinic acid as an internal standard. It is believed that this reflects residual TFA content and not salt formation. To be on the conservative side, we have calculated the EC50 values with the assumption that each analogue contains 2 mol equiv of TFA. The addition of up to 2 mol equiv of TFA reduces the percent API by approximately 25% on average. This means that the EC50 values are

EXPERIMENTAL SECTION

All reagents were purchased from commercial suppliers and used without purification unless noted otherwise. All anhydrous reactions were performed under a nitrogen atmosphere using Sure/Seal solvents from Aldrich. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on a Bruker AM FT 300 MHz spectrometer or on a Bruker AvanceIII 500 MHz spectrometer or on a Bruker Ultrashield 400 MHz spectrometer, each equipped with a 5 mm TXI cryoprobe. All spectra were determined in the solvents indicated, and chemical shifts are reported in δ units downfield from the internal standard tetramethylsilane (TMS) with interproton coupling constants reported in hertz (Hz). Multiplicity patterns are designated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad; dd, doublet of doublet; dt, doublet of triplet; dq, doublet of quartet. Most spectra were analyzed using the ACDLABS SpecManager 12.0 program software. At ambient temperature, most of the final products exist predominantly as a mixture of rotamers in DMSO-d6. Liquid chromatography (LC)/mass spectrometry (MS) analyses were performed on a Shimadzu LC instrument coupled to a Water Micromass ZQ instrument, and the LCMS conditions are shown in Table 4. All final compounds had purity of ≥95% except for compound 8 (see experimental procedure). High resolution mass spectrometry (HRMS) analyses were performed on a Thermo Scientific Finnegan MAT900 (magnetic sector MS, polypropylene glycol reference) mass spectrometer or on a Fourier Transform Orbitrap mass spectrometer (Exactive, Thermo Fisher Scientific, San Jose, CA) in positive or negative ionization electrospray mode operating at 25,000 resolution (full width at half F



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min. LCMS (ESI) m/z calcd for C46H41N4O4, 713.31; found, 713.50 [M + H]+. HRMS (ESI) m/z calcd for C46H41N4O4, 713.3128; found, 713.3142 [M + H]+. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis(1-(3-ethylpicolinoyl)pyrrolidine-2-carboxamide) (7). Compound 7 (4.0 mg, 11%) was prepared as an off-white solid from 41 and 3-ethylpicolinic acid (57) in a manner similar to that described for compound 11. 1H NMR (300 MHz, DMSO-d6) δ 10.25/9.61 (s, 2H), 8.43/8.32 (dd, J = 4.4, 1.1 Hz, 2H), 7.80−7.26 (series of m, 12H), 7.13−7.01 (m, 2H), 4.65/4.52 (dd, J = 8.8, 4.4 Hz, 2H), 3.69 (br s, 1H), 3.30−3.32 (m, 3H), 2.65 (q, J = 7.7 Hz, 4H), 2.32−2.24 (m, 2H), 2.00−1.82 (m, 6H), 1.00/0.86 (t, J = 7.3 Hz, 6H). LC (method D): tR = 2.43 min. LCMS (ESI) m/z calcd for C40H43N6O4, 671.33; found, 671.36 [M + H]+. HRMS (ESI) m/z calcd for C40H43N6O4, 671.3346; found, 671.3368 [M + H]+. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis(1-(4-ethylpicolinoyl)pyrrolidine-2-carboxamide) (8). Compound 8 (49.9 mg, 53%) was prepared as an orange solid from 41 and 4-ethylpicolinic acid (Toronto Research Chemical) in a manner similar to that described for compound 11. Repeated purification attempts on compound 8 yielded a purity of 82% which was sufficiently pure to carry forward and use as-is. 1H NMR (500 MHz, DMSO-d6) δ 10.18/ 9.90 (s, 2H), 8.54 (d, J = 5.2 Hz, 1H), 8.33 (t, J = 4.6 Hz, 1H), 7.66− 7.04 (series of m, 14H), 5.07 (br s, 1H), 4.65 (br s, 1H), 3.87−3.84 (m, 1H), 3.80−3.75 (m, 1H) 3.72−3.67 (m, 2H), 2.71 (q, J = 7.6 Hz, 2H), 2.61 (q, J = 7.6 Hz, 2H), 2.35−2.24 (m, 2H), 2.03−1.88 (m, 6H), 1.22 (t, J = 7.6 Hz, 3H), 1.12 (t, J = 7.6 Hz, 3H). LC (method A): tR = 2.16 min. LCMS (ESI) m/z calcd for C40H43N6O4, 671.33; found, 671.35 [M + H]+. HRMS (ESI) m/z calcd for C40H43N6O4, 671.3346; found, 671.3327 [M + H]+. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis(1-(3-vinylpicolinoyl)pyrrolidine-2-carboxamide) (9). Compound 9 (6.1 mg, 22%) was prepared as an off-white solid from 41 and 3-vinylpicolinic acid (56) in a manner similar to that described for compound 11. 1H NMR (300 MHz, DMSO-d6) δ 10.29/9.64 (s, 2H), 8.50/8.38 (dd, J = 4.3, 1.1 Hz, 2H), 8.22/8.01 (d, J = 7.3 Hz, 2H) 7.68−7.27 (series of m, 10H), 7.14−7.07 (m, 2H), 7.01−6.76 (m, 2H), 6.04/5.83 (d, J = 17.6 Hz, 2H), 5.48/5.38 (d, J = 11.3 Hz, 2H), 4.67−4.63/4.54−4.50 (m, 2H), 3.72−3.68 (m, 1H), 3.27−3.16 (m, 3H), 2.33−2.27 (m, 2H), 2.0−1.83 (m, 6H). LC (method D): tR = 2.42 min. LCMS (ESI) m/z calcd for C40H39N6O4, 667.30; found, 667.37 [M + H]+. HRMS (ESI) m/z calcd for C40H39N6O4, 667.3033; found, 667.3002 [M + H]+. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis(1-(3-phenylpicolinoyl)pyrrolidine-2-carboxamide) (10). Compound 10 (48.6 mg, 46.5%) was prepared as a straw-colored solid from 41 and 3-phenylpicolinic acid (58) in a manner similar to that described for compound 11. 1H NMR (500 MHz, DMSO-d6) δ 10.15/10.97 (s, 2H), 8.62/8.46 (dd, J = 4.6, 1.5 Hz, 2H), 7.96/7.82 (dd, J = 7.9, 1.5 Hz, 2H), 7.63−7.01(series of m, 22H), 4.56/4.50 (dd, J = 8.4, 4.1 Hz, 2H), 3.55/3.33 (br s, 4H), 2.12−2.08 (m, 2H), 1.96− 1.93/1.79/1.68 (m, 6H). LC (method A): tR = 2.47 min. LCMS (ESI) m/z calcd for C48H43N6O4, 767.33; found, 767.61 [M + H]+. HRMS (ESI) m/z calcd for C48H43N6O4, 767.3346; found, 767.3336 [M + H]+. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis(1-(isoquinoline-1-carbonyl)pyrrolidine-2-carboxamide) (11). O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU, 72.4 mg, 0.19 mmol, 2.2 equiv) was added in one portion to a solution of 41 (35 mg, 0.087 mmol, 1.0 equiv), isoquinoline-1-carboxylic acid (33 mg, 0.19 mmol, 2.2 equiv), and N,N′-diisopropylethylamine (60 μL, 0.35 mmol, 4 equiv) in anhydrous DMF (1.5 mL). The mixture was stirred at room temperature for 3 h before it was purified by reverse phase preparative HPLC to provide 11 (52.0 mg, 64%) as an orange solid. 1H NMR (500 MHz, DMSOd6) δ 10.35/9.55 (s, 2H), 8.54−8.53/8.40−8.39 (s, 2H), 8.28−8.27/ 8.16−8.14 (s, 2H), 8.07−7.53 (series of m, 14H), 7.32−6.97 (series of m, 4H), 4.78−4.77/4.46 (m, 2H), 3.82/3.33/3.20 (br s, 4H), 2.36− 2.35 (m, 2H), 2.04−2.01 (m, 2H), 1.96−1.93 (m, 2H), 1.88−1.86 (m, 2H). LC (method A): tR = 2.38 min. LCMS (ESI) m/z calcd for

conservative and would be affected by only 25%, which would be within the error margin of the assay. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis(1-(2-phenylacetyl)pyrrolidine-2-carboxamide) (1). 2-Ethoxy1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ, 568 mg, 2.3 mmol) was added in one portion to a suspension of 4,4′-diaminostilbene (39, 210 mg, 1.0 mmol) and (S)-1-(2-phenylacetyl)pyrrolidine-2-carboxylic acid25 (513 mg, 2.2 mmol) in dry CH2Cl2 (10 mL) under nitrogen at ambient temperature. The suspension was stirred for 1.5 h, diluted with CH2Cl2, and washed with 1 N HCl solution. Some insoluble product was isolated from the aqueous phase by extraction with CH2Cl2. The organic layers were combined, dried over anhydrous Na2SO4, and evaporated. The residue was subjected to flash chromatography on silica gel (gradient elution with 0% MeOH to 30% MeOH in CH2Cl2) to afford 1 (568 mg, 87%) as a white solid. 1H NMR (500 MHz, DMSO-d6) δ 10.27/10.05 (s, 2H), 7.62−7.58 (m, 4H), 7.55−7.50 (m, 4H), 7.32−7.17 (m, 10H), 7.11−7.09 (m, 2H), 4.66−4.63/4.45−4.53 (m, 2H), 3.70 (s, 4H), 3.68−3.63 (m, 2H), 3.61−3.57 (m, 2H), 2.17−2.13 (m, 2H), 2.03−1.98 (m, 2H), 1.93− 1.88 (m, 4H). LC (method B): tR = 1.73 min. LCMS (ESI) m/z calcd for C40H41N4O4, 641.31; found, 641.51 [M + H]+. HRMS (ESI) m/z calcd for C40H41N4O4, 641.30 96; found, 641.3143 [M + H]+. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis(1-(2-ethylbenzoyl)pyrrolidine-2-carboxamide) (3). Compound 3 (43.6 mg, 62%) was prepared as a white solid from (2S,2′S)-N,N′-(4,4′-((E)-ethene-1,2-diyl)bis(4,1-phenylene))dipyrrolidine-2-carboxamide dihydrochloride (41) and 2-ethylbenzoic acid in a manner similar to that described for compound 11. 1H NMR (500 MHz, CDCl3) δ 9.83 (s, 2H), 7.45 (d, J = 7.9 Hz, 4H), 7.37− 7.33 (m, 2H), 7.29−7.27 (m, 6H), 7.25−7.24 (m, 4H), 6.85 (br s, 2H), 5.00 (dd, J = 7.8, 3.5 Hz, 2H), 3.37−3.32 (m, 2H), 3.29−3.24 (m, 2H), 2.75−2.60 (m, 4H), 2.17−2.02 (m, 8H), 1.21 (t, J = 7.6 Hz, 6H). LC (method C): tR = 4.18 min. LCMS (ESI) m/z calcd for C42H45N4O4, 669.42; found, 669.68 [M + H]+. HRMS (ESI) m/z calcd for C42H45N4O4, 669.3435; found, 669.3407 [M + H]+. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis(1-(benzoyl)pyrrolidine-2-carboxamide) (4). Compound 4 (69.0 mg, 65%) was prepared as a white solid from 41 and benzoic acid in a manner similar to that described for compound 11. 1H NMR (500 MHz, DMSO-d6) δ 10.15/9.78 (s, 2H), 7.65 (d, J = 8.5 Hz, 3H), 7.57−7.53 (m, 6H), 7.49−7.44 (m, 6H), 7.38−7.34 (m, 3H), 7.12− 7.09 (m, 2H), 4.61/4.39 (dd, J = 7.9, 5.3 Hz, 2H), 3.67−3.49 (series of m, 4H), 2.31−2.27 (m, 2H), 2.03−1.80 (m, 6H). LC (method C): tR = 3.68 min. LCMS (ESI) m/z calcd for C38H37N4O4, 613.28; found, 613.53 [M + H]+. HRMS (ESI) m/z calcd for C38H37N4O4, 613.2809; found, 613.2781 [M + H]+. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis(1-(2-vinylbenzoyl)pyrrolidine-2-carboxamide) (5). Compound 5 (30.4 mg, 43%) was prepared as an off-white solid from 41 and 2-vinylbenzoic acid (Alfa Aesar) in a manner similar to that described for compound 11. 1H NMR (500 MHz, MeOH-d4) δ 10.24/ 9.61 (s, 2H), 7.77−7.74 (m, 1.5H), 7.67−7.62 (m, 3.5H), 7.57−7.53 (m, 3H), 7.47−7.42 (m, 2.5H), 7.39−7.32 (m, 2.5H), 7.30−7.27 (m, 2H), 7.23−7.17 (m, 1H), 7.13/7.09 (s, 2H), 6.96/6.69 (dd, J = 18.0, 11.6 Hz, 2H), 5.93/5.81 (d, J = 17.6 Hz, 2H), 5.38−5.32 (m, 2H), 4.64/4.10 (dd, J = 8.1, 4.1 Hz, 2H), 3.71−3.63 (m, 1H), 3.25−3.20 (m, 1.5H), 3.18−3.14 (m, 1.5H), 2.33−2.26 (m, 2H), 2.01−1.91 (m, 4H), 1.85−1.81 (m, 2H). LC (method C): tR = 4.00 min. LCMS (ESI) m/z calcd for C42H41N4O4, 665.31; found, 665.60 [M + H]+. HRMS (ESI) m/z calcd for C42H41N4O4, 665.3122; found, 665.3105 [M + H]+. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis(1-(1-naphthoyl)pyrrolidine-2-carboxamide) (6). Compound 6 (37.2 mg, 60%) was prepared as a white solid from 41 and 1naphthoic acid in a manner similar to that described for compound 11. 1 H NMR (400 MHz, DMSO-d6) δ 10.30/9.46 (s, 2H), 8.17 (d, J = 8.2 Hz, 2H), 8.00 (d, J = 7.9 Hz, 3H), 7.92−7.81 (m, 1H), 7.72−7.69 (m, 3H), 7.64−7.51 (m, 11H), 7.41−7.36 (m, 2H), 7.15/7.07 (s, 2H), 4.77 (dd, J = 8.5, 4.9 Hz, 2H), 3.88−3.71 (m, 1H), 3.24−3.16 (m, 3H), 2.39−2.21 (m, 2H), 2.05−1.80 (m, 6H). LC (method A): tR = 2.68 G



Article

C44H39N6O4, 715.30; found, 715.51 [M + H]+. HRMS (ESI) m/z calcd for C44H39N6O4, 715.3033; found, 715.3038 [M + H]+. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis(1-(isoquinoline-3-carbonyl)pyrrolidine-2-carboxamide) (12). Compound 12 (14.4 mg, 13%) was prepared as an orange solid from 41 and isoquinoline-3-carboxylic acid in a manner similar to that described for compound 11. 1H NMR (500 MHz, DMSO-d6) δ 10.21/9.95 (s, 2H), 9.39/9.19 (s, 2H), 8.35−8.08 (series of m, 6H), 7.88−7.64 (series of m, 6H), 7.55−7.38 (series of m, 6H), 7.12−6.99 (m, 2H), 5.23/4.71 (br s, 2H), 3.88/3.37/3.17 (br s, 4H), 2.36−2.37 (m, 2H), 2.04−1.88 (m, 6H). LC (method A): tR = 2.24 min. LCMS (ESI) m/z calcd for C44H39N6O4, 715.30; found, 715.64 [M + H]+. HRMS (ESI) m/z calcd for C44H39N6O4, 715.3033; found, 715.3023 [M + H]+. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis(1-(quinoline-2-carbonyl)pyrrolidine-2-carboxamide) (13). Compound 13 (29.8 mg, 27%) was prepared as an orange solid from 41 and quinoline-2-carboxylic acid in a manner similar to that described for compound 11. 1H NMR (500 MHz, DMSO-d6) δ 10.23/10.03 (s, 2H), 8.51/8.43 (d, J = 8.5 Hz, 2H), 8.12/8.07 (d, J = 8.3 Hz, 2H), 7.97−7.93 (m, 3H), 7.88−7.82 (m, 2H), 7.73−7.51 (series of m, 6H), 7.43−7.40 (m, 5H), 7.13−7.00 (m, 2H), 5.31/4.71 (dd, J = 8.2, 3.7 Hz, 2H), 3.97/3.79/3.47 (br s, 4H), 2.40−2.28 (m, 2H), 2.10−1.92 (m, 6H). LC (method A): tR = 2.37 min. LCMS (ESI) m/z calcd for C44H39N6O4, 715.30; found, 715.59 [M + H]+. HRMS (ESI) m/z calcd for C44H39N6O4, 715.3033; found, 715.3038 [M + H]+. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis(1-(isoquinoline-4-carbonyl)-pyrrolidine-2-carboxamide) (14). Compound 14 (49.4 mg, 66%) was prepared as a yellow solid from 41 and isoquinoline-4-carboxylic acid in a manner similar to that described for compound 11. 1H NMR (500 MHz, DMSO-d6) δ 10.41 (s, 1H), 9.61/9.47 (s, 2H), 8.65/8.54 (s, 2H), 8.38/8.31/8.21 (d, J = 8.2 Hz, 3H), 8.10−7.78 (series of m, 5H), 7.80−7.34 (series of m, 8H), 7.16−7.06 (m, 3H), 4.80 (dd, J = 8.5, 4.9 Hz, 2H), 3.84−3.81 (m, 1H), 3.33−3.22 (m, 3H), 2.42−2.29 (m, 2H), 2.06−1.83 (m, 6H). LC (method F): tR = 1.14 min. LCMS (ESI) m/z calcd for C44H39N6O4, 715.15; found, 715.31 [M + H]+. HRMS (ESI) m/z calcd for C44H39N6O4, 715.3027; found, 715.3005 [M + H]+. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis(1-(quinazoline-4-carbonyl)pyrrolidine-2-carboxamide) (15). 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 31 mg, 0.165 mmol) was added in one portion to a stirred solution of 41 (25 mg, 0.053 mmol), 1-hydroxybenztriazole (HOBT, 22 mg, 0.165 mmol), quinazoline-4-carboxylic acid (available from Anichem Inc.) (39 mg, 0.165 mmol), and triethylamine (0.11 mL, 0.825 mmol) in anhydrous DMF (2.0 mL) at room temperature. The mixture was stirred for 16 h before the solvent was removed in vacuo, and the residue was purified by reverse phase preparative HPLC to afford 15 (17.1 mg, 34%) as a yellow solid. 1H NMR (500 MHz, DMSO-d6) δ 10.38 (s, 2H), 9.77 (br s, 2H), 7.62−7.65 (m, 2H), 7.61 (d, J = 8.9 Hz, 4H), 7.60−7.58 (m, 2H), 7.54 (d, J = 8.6 Hz, 4H), 7.11 (s, 2H), 4.75−4.73 (m, 2H), 3.65−3.61 (m, 2H), 3.53−3.50 (m, 2H), 2.03−2.00 (m, 2H), 1.92−1.85 (m, 6H). LC (method E): tR = 1.94 min. LCMS (ESI) m/z calcd for C42H37N8O4, 717.29; found, 717.45 [M + H]+. HRMS (ESI) m/z calcd for C42H37N8O4, 717.2932; found, 717.2911 [M + H]+. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis(1-(phthalazine-1-carbonyl)pyrrolidine-2-carboxamide) (16). Compound 16 (8.0 mg, 15%) was prepared as a yellow solid from 41 and phthalazine-1-carboxylic acid (available from Ark Pharm, Inc.) in a manner similar to that described for compound 15. LC (method E): tR = 1.87 min. LCMS (ESI) m/z calcd for C42H37N8O4, 717.29; found, 717.37 [M + H]+. HRMS (ESI) m/z calcd for C42H37N8O4, 717.2932; found, 717.2911 [M + H]+. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis(1-(3-chloroisoquinoline-1-carbonyl)pyrrolidine-2-carboxamide) (17). Compound 17 (74 mg, 38%, free base) was prepared as a white solid from 41 and 3-chloroisoquinoline-1-carboxylic acid (93) in a manner similar to that described for compound 11. 1H NMR (500 MHz, DMSO-d6) δ 10.39/9.70 (s, 2H), 8.31−8.28/8.18−8.15 (m,

3.5H), 8.08−8.03 (m, 2H), 7.92−7.89 (m, 2H), 7.83−7.77 (m, 2H), 7.72−7.67 (m, 3.5H), 7.62−7.53 (m, 3H), 7.34−7.27 (m, 1H), 7.16 (s, 1H), 7.05−6.94 (m, 2H), 4.77/4.54 (dd, J = 8.6, 4.9 Hz, 2H), 3.82 (br s, 1H), 3.37−3.33 (m, 1.5H) 3.25−3.20 (m, 1.5H), 2.40−2.35 (m, 2H), 2.05−1.87 (m, 6H). LC (method C): tR = 4.07 min. LCMS (ESI) m/z calcd for C44H37Cl2N6O4, 783.23; found, 783.34 [M + H]+. HRMS (ESI) m/z calcd for C44H37Cl2N6O4, 783.2253; found, 783.2250 [M + H]+. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis(1-(4-chloroisoquinoline-1-carbonyl)pyrrolidine-2-carboxamide) (18). Compound 18 (40.6 mg, 38%) was prepared as a tan solid from 41 and 4-chloroisoquinoline-1-carboxylic acid (94) in a manner similar to that described for compound 11. 1H NMR (500 MHz, DMSO-d6) δ 10.39/9.57 (s, 2H), 8.70/8.53−8.82 (m, 2H), 8.37−8.18 (series of m, 3.5H), 8.07−8.03 (m, 2H), 7.94−7.89/7.82− 7.78 (m, 2.5H), 7.72−7.68 (m, 3H), 7.59/7.54 (d, J = 8.8 Hz, 3H), 7.30/7.25 (d, J = 8.8 Hz, 1), 7.16 (s, 1H), 7.05−6.91 (m, 2H), 4.77/ 4.55 (dd, J = 8.9, 4.9 Hz, 2H), 3.84−3.80 (m, 1H), 3.37−3.33 (m, 1.5H) 3.25−3.19 (m, 1.5H), 2.39−2.35 (m, 2H), 2.06−1.85 (m, 6H). LC (method A): tR = 2.71 min. LCMS (ESI) m/z calcd for C44H37Cl2N6O4, 783.23; found, 783.23 [M + H]+. HRMS (ESI) m/z calcd for C44H35Cl2N6O4, 781.2097; found, 781.2109 [M − H]−. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis(1-(5-chloroisoquinoline-1-carbonyl)pyrrolidine-2-carboxamide) (19). Compound 19 (24.7 mg, 30%, free base) was prepared as a light green solid from 41 and 5-chloroisoquinoline-1-carboxylic acid (95) in a manner similar to that described for compound 11. 1H NMR (300 MHz, DMSO-d6) δ 10.39/9.55 (s, 2H), 8.72−8.70/8.57−8.54 (m, 2H), 8.30−7.90 (series of m, 6H), 7.82−7.53 (m, 8H), 7.31/7.26 (d, J = 8.8 Hz, 1H), 7.16/7.05 (s, 2H), 6.95−6.90 (m, 1H), 4.78/4.55 (dd, J = 8.4, 4.4 Hz, 2H), 3.84−3.80 (m, 1H), 3.37−3.29 (m, 1.5H) 3.22−3.15 (m, 1.5H), 2.41−2.28 (m, 2H), 2.06−1.85 (m, 6H). LC (method B): t R = 1.91 min. LCMS (ESI) m/z calcd for C44H3737Cl2N6O4, 785.22; found, 785.22 [M + H]+. HRMS (ESI) m/z calcd for C44H37Cl2N6O4, 783.2253; found, 783.2228 [M + H]+. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis(1-(6-chloroisoquinoline-1-carbonyl)pyrrolidine-2-carboxamide) (20). Compound 20 (33.8 mg, 43%, free base) was prepared as a tan solid from 41 and 6-chloroisoquinoline-1-carboxylic acid (96) in a manner similar to that described for compound 11. 1H NMR (500 MHz, CDCl3) δ 10.04 (s, 2H), 8.54 (d, J = 5.9 Hz, 2H), 8.37 (d, J = 8.8 Hz, 2H), 7.85 (d, J = 1.8 Hz, 2H), 7.66−7.60 (m, 4H), 7.38 (d, J = 8.8 Hz, 4H), 7.03 (d, J = 8.0 Hz, 4H), 6.69 (s, 2H), 5.06−5.04 (m, 2H), 3.58−3.52 (m, 2H), 3.38−3.34 (m, 2H), 2.09−1.92 (m, 8H). LC (method B): t R = 1.88 min. LCMS (ESI) m/z calcd for C44H37Cl2N6O4, 783.23; found, 783.08 [M + H]+. HRMS (ESI) m/ z calcd for C44H37Cl2N6O4, 783.2253; found, 783.2236 [M + H]+. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis(1-(7-chloroisoquinoline-1-carbonyl)pyrrolidine-2-carboxamide) (21). Compound 21 (80.4 mg, 76%) was prepared as an orange solid from 41 and 7-chloroisoquinoline-1-carboxylic acid (97) in a manner similar to that described for compound 11. 1H NMR (300 MHz, CDCl3) δ 9.82 (s, 2H), 8.64 (d, J = 5.9 Hz, 2H), 8.57 (d, J = 1.1 Hz, 2H), 7.99 (t, J = 2.9 Hz, 2H), 7.96 (s, 2H), 7.89−7.86 (m, 2H), 7.37 (d, J = 8.8 Hz, 4H), 7.08 (d, J = 8.8 Hz, 4H), 6.70 (s, 2H), 5.23 (dd, J = 8.1, 5.1 Hz, 2H), 3.60−3.52 (m, 4H), 2.49−2.44 (m, 2H), 2.28−2.03 (m, 6H). LC (method B): tR = 1.81 min. LCMS (ESI) m/z calcd for C44H37Cl2N6O4 783.23; found, 783.15 [M + H]+. HRMS (ESI) m/z calcd for C44H37Cl2N6O4, 783.2253; found, 783.2231 [M + H]+. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis(1-(3-fluoroisoquinoline-1-carbonyl)pyrrolidine-2-carboxamide) (22). Compound 22 (51.4 mg, 50%) was prepared as an offwhite solid from 41 and 3-fluoroisoquinoline-1-carboxylic acid (98) in a manner similar to that described for compound 11. 1H NMR (500 MHz, DMSO-d6) δ 10.39/9.66 (s, 2H), 8.31/8.15 (d, J = 8.6 Hz, 2H), 8.09/7.91 (d, J = 8.6 Hz, 2H), 7.89−7.53 (series of m, 12H), 7.32/7.27 (d, J = 8.8 Hz, 1H), 7.16−6.93 (series of m, 3H), 4.77/4.56 (dd, J = 8.2, 4.9 Hz, 2H), 3.37−3.33 (m, 2H), 3.25−3.21 (m, 2H), 2.40−2.35 (m, 2H), 2.05−1.88 (m, 6H). LC (method A): tR = 2.43 min. LCMS (ESI) m/z calcd for C44H37N6O4S2, 751.28; found, 751.20 [M + H]+. H



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from 41 and 3-chloro-1-naphthoic acid30b,c in a manner similar to that described for compound 11. 1H NMR (500 MHz, DMSO-d6) δ 10.34/9.57 (s, 2H), 8.16 (d, J = 1.8 Hz, 2H), 8.03−8.00 (m, 2H), 7.71−7.55 (series of m, 13H), 7.16/7.08 (s, 2H), 4.75 (dd, J = 8.4, 5.0 Hz, 2H), 3.82−3.79/3.23−3.20 (m, 4H), 2.38−2.32 (m, 2H), 2.03− 1.81 (m, 6H). LC (method H): tR = 1.90 min. LCMS (ESI) m/z calcd for C46H39Cl2N4O4, 781.23; found, 781.55 [M + H]+. HRMS (ESI) m/z calcd for C46H39Cl2N4O4, 781.2348; found, 781.2352 [M + H]+. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis(1-(3-chloro-5-fluoroisoquinoline-1-carbonyl)pyrrolidine-2carboxamide) (29). Compound 29 (18 mg, 43%) was prepared as a tan solid from 41 and 3-chloro-5-fluoroisoquinoline-1-carboxylic acid (100) in a manner similar to that described for compound 11. 1H NMR (400 MHz, DMSO-d6) δ 10.40/9.71 (s, 2H), 8.10/8.01 (s, 2H), 8.17−8.14/8.01−7.98 (m, 2H), 7.86−7.76 (m, 3H), 7.71−7.62 (m, 4H), 7.59/7.55 (d, J = 8.8 Hz, 3H), 7.34/7.29 (d, J = 8.8 Hz, 1H), 7.16 (s, 1H), 7.06−6.96 (m, 2H), 4.78/4.52 (dd, J = 8.2, 4.5 Hz, 2H), 3.83−380 (t, J = 6.8 Hz, 1H), 3.39−3.33/3.27−3.21 (m, 3H), 2.43− 2.33 (m, 2H), 2.07−1.87 (m, 6H). LC (method A): tR = 2.85 min. LCMS (ESI) m/z calcd for C44H35Cl2F2N6O4, 819.21; found, 819.37 [M + H]+. HRMS (ESI) m/z calcd for C44H35Cl2F2N6O4, 819.2065; found, 819.2029 [M + H]+. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis (1-(3-c hloro-5-methoxyisoquinoline-1-carbonyl)pyrrolidine-2-carboxamide) (30). Compound 30 (15.0 mg, 37%) was prepared as a peach-colored solid from 41 and 3-chloro-5methoxyisoquinoline-1-carboxylic acid (101) in a manner similar to that described for compound 11. 1H NMR (500 MHz, DMSO-d6) δ 10.39/9.64 (s, 2H), 8.09/7.93 (s, 2H), 7.87−7.53 (series of m, 10H), 7.36−7.02 (series of m, 6H), 4.76/4.47 (dd, J = 8.3, 4.4 Hz, 2H), 4.03/ 3.92 (s, 6H), 3.80−3.79 (m, 1H), 3.34−3.28 (m, 1.5H), 3.23−3.18 (m, 1.5H), 2.40−2.33 (m, 2H), 2.05−1.86 (m, 6H). LC (method A): tR = 2.78 min. LCMS (ESI) m/z calcd for C46H41Cl2N6O6, 843.25; found, 843.40 [M + H]+. HRMS (ESI) m/z calcd for C46H41Cl2N6O6, 843.2465; found, 843.2443 [M + H]+. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis(1-(5-methoxyquinoline-2-carbonyl)pyrrolidine-2-carboxamide) (31). Compound 31 (49 mg, 46%) was prepared as an orange solid from 41 and 5-methoxyquinoline-2-carboxylic acid (105) in a manner similar to that described for compound 11. 1H NMR (400 MHz, DMSO-d6) 10.26 (d, J = 3.0 Hz, 0.8H), 10.07 (d, J = 2.7, 1.2H), 8.65 (d, J = 8.6 Hz, 0.8H), 8.57 (d, J = 8.8 Hz, 1.2H), 7.94 (d, J = 8.8 Hz, 1.2H), 7.83−7.41 (series of m, 13.0H), 7.17−7.01 (series of m, 3.9H), 5.30 (dd, J = 8.2, 3.6 Hz, 1.2H), 4.70 (m, 0.8H), 4.02 (s, 2.5H), 3.95−3.90 (m, 5.2H), 3.82−3.71 (m, 2.4H), 2.42−1.90 (m, 8.0H). LC (method B): tR = 1.69 min. LCMS (ESI) m/z calcd for C46H43N6O6, 775.23; found, 775.29 [M + H]+. HRMS (ESI) m/z calcd for C46H43N6O6, 775.3239; found, 775.3220 [M + H]+. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis(1-(7-methoxyquinoline-2-carbonyl)pyrrolidine-2-carboxamide) (32). Compound 32 (38.9 mg, 37%) was prepared as a yellow solid from 41 and 7-methoxyquinoline-2-carboxylic acid (106) in a manner similar to that described for compound 11. 1H NMR (400 MHz, DMSO-d6) 10.26 (s, 0.6H), 10.17 (s, 1.4H), 8.42 (d, J = 8.3 Hz, 0.6H), 8.34 (d, J = 8.6 Hz, 1.4H), 7.96 (d, J = 9.1 Hz, 0.6H), 7.87− 7.83 (m, 2.8H), 7.69−7.03 (series of m, 16.6H), 5.24 (dd, J = 8.5, 3.1 Hz, 1.4H), 4.7 (dd, J = 8.1, 4.2 Hz, 0.6H), 3.98−3.93 (m, 3H), 3.78− 3.72 (m, 2.8H), 3.52 (s, 4.2H), 2.40−1.90 (m, 8.0H). LC (method B): tR = 1.70 min. LCMS (ESI) m/z calcd for C46H43N6O6, 775.32; found, 775.24 [M + H]+. HRMS (ESI) m/z calcd for C46H43N6O6, 775.3239; found, 775.3213 [M + H]+. (2S,2′S)-N,N′-(4,4′-(Ethyne-1,2-diyl)bis(4,1-phenylene))bis(1(2-phenylacetyl)pyrrolidine-2-carboxamide) (33). Compound 33 (153 mg, 50%) was prepared as a white solid from 4,4′-(ethyne1,2-diyl)dianiline (39, Bionet Research) and (S)-1-(2-phenylacetyl)pyrrolidine-2-carboxylic acid25 in a manner similar to that described for compound 1 but employing N,N′-diisopropylcarbodiimide (DCI) as the coupling agent instead of EEDQ. 1H NMR (500 MHz, DMSOd6) δ 10.19 (s, 2H), 7.56−7.70 (m, 4H), 7.39−7.53 (m, 4H), 7.10− 7.36 (m, 10H), 4.44 (dd, J = 8.5, 3.7 Hz, 2H), 3.35−3.76 (m, 8H), 1.78−2.22 (m, 8H). LC (method B): tR = 1.81 min. LCMS (ESI) m/z

HRMS (ESI) m/z calcd for C44H37N6O4S2, 751.2845; found, 751.2849 [M + H]+. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis(1-(5-fluoroisoquinoline-1-carbonyl)pyrrolidine-2-carboxamide) (23). Compound 23 (57.7 mg, 56%) was prepared as a yellow solid from 41 and 5-fluoroisoquinoline-1-carboxylic acid (99) in a manner similar to that described for compound 11. 1H NMR (500 MHz, CDCl3) δ 9.79 (s, 2H), 8.71 (d, J = 6.1 Hz, 2H), 8.40 (d, J = 8.6 Hz, 2H), 8.28 (d, J = 6.1 Hz, 2H), 7.90−7.01 (series of m, 12H), 6.65 (s, 2H), 5.21−5.17 (m, 2H), 3.48−3.45 (m, 4H), 2.47−2.43 (m, 2H), 2.23−2.14 (m, 4H), 2.01−2.01 (m, 2H). LC (method B): tR = 1.74 min. LCMS (ESI) m/z calcd for C44H37FN6O4, 751.28; found, 751.27 [M + H]+. HRMS (ESI) m/z calcd for C44H37FN6O4, 751.2844; found, 751.2856 [M + H]+. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis(1-(3-methylisoquinoline-1-carbonyl)pyrrolidine-2-carboxamide) (24). Compound 24 (42.7 mg, 69%) was prepared as a yellow solid from 41 and 3-methylisoquinoline-1-carboxylic acid (91) in a manner similar to that described for compound 11. 1H NMR (500 MHz, DMSO-d6) δ 10.38/9.60 (s, 2H), 8.21/8.12 (d, J = 8.2 Hz, 2H), 7.95 (d, J = 8.4 Hz, 1.5H), 7.80−7.77 (m, 3.5H), 7.72−7.68 (m, 5H), 7.59/7.54 (d, J = 8.2 Hz, 4H), 7.16 (s, 1H), 7.11−6.95 (m, 2H), 4.77/ 4.53 (dd, J = 8.2, 4.6 Hz, 2H), 3.83 (br s, 1H), 3.35−3.30 (m, 1.5H), 3.21−3.17 (m, 1.5H), 2.64/2.47 (s, 6H), 2.40−2.26 (m, 2H), 2.04− 1.85 (m, 6H). LC (method C): tR = 3.87 min. LCMS (ESI) m/z calcd for C46H43N6O4, 743.34; found, 743.41 [M + H]+. HRMS (ESI) m/z calcd for C46H43N6O4, 743.3340; found, 743.3308 [M + H]+. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis(1-(5-methylisoquinoline-1-carbonyl)pyrrolidine-2-carboxamide) (25). Compound 25 (36.8 mg, 68%) was prepared as an offwhite solid from 41 and 5-methylisoquinoline-1-carboxylic acid (92) in a manner similar to that described for compound 11. 1H NMR (500 MHz, DMSO-d6) δ 10.24/9.96 (s, 2H), 9.36/9.17 (s, 2H), 8.21/8.12 (d, J = 22.9 Hz, 2H), 8.07−7.93 (m, 2H), 7.70−7.64 (m, 6H), 7.56− 7.51 (m, 2H), 7.42−7.40 (m, 4H), 7.13−7.0 (m, 2H), 5.15/4.70 (br s, 2H), 3.95 (br s, 1H), 3.86 (br s, 1H), 3.77 (br s, 2H), 2.69/2.63 (s, 6H), 2.36−2.25 (m, 2H), 2.09−1.90 (m, 6H). LC (method C): tR = 3.94 min. LCMS (ESI) m/z calcd for C46H43N6O4, 743.34; found, 743.41 [M + H]+. HRMS (ESI) m/z calcd for C46H43N6O4, 743.3340; found, 743.3320 [M + H]+. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis(1-(3-methoxyisoquinoline-1-carbonyl)pyrrolidine-2-carboxamide) (26). Compound 26 (52.9 mg, 50%) was prepared as an off-white solid from 41·2HCl and 3-methoxyisoquinoline-1-carboxylic acid (89) in a manner similar to that described for compound 11. 1H NMR (300 MHz, DMSO-d6) δ 10.36/9.60 (s, 2H), 8.19−8.17/8.01− 8.00 (m, 2H), 7.90/7.76 (d, J = 8.6 Hz, 2H), 7.72−7.68 (m, 4H), 7.62−7.58 (m, 3H), 7.55−7.49/7.43−7.40 (m, 3H), 7.35 (d, J = 8.6 Hz, 1H), 7.31/7.16 (s, 3H), 7.07−6.95 (m, 2H), 4.77/4.51 (dd, J = 8.5, 4.6 Hz, 2H), 3.97/3.83 (s, 6H), 3.41−3.36 (m, 2H), 3.25−3.20 (m, 2H), 2.40−2.28 (m, 2H), 2.06−1.85 (m, 6H). LC (method A): tR = 2.59 min. LCMS (ESI) m/z calcd for C46H43N6O6, 775.32; found, 775.32 [M + H]+. HRMS (ESI) m/z calcd for C46H43N6O6, 775.3244; found, 775.3236 [M + H]+. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis(1-(5-methoxyisoquinoline-1-carbonyl)pyrrolidine-2-carboxamide) (27). Compound 27 (70.9 mg, 67%) was prepared as an orange solid from 41 and 5-methoxyisoquinoline-1-carboxylic acid (90) in a manner similar to that described for compound 11. 1H NMR (500 MHz, DMSO-d6) δ 10.37/9.52 (s, 2H), 8.53−8.51/8.39−8.37 (m, 2H), 8.05/7.90 (d, J = 5.8 Hz, 2H), 7.83−7.80 (m, 1H), 7.72− 7.64 (m, 5H), 7.60−7.53 (m, 3H), 7.33−7.29 (m, 2H), 7.16/7.05 (s, 2H), 6.99/6.95 (d, J = 8.9 Hz, 1H), 4.76/4.53 (dd, J = 8.5, 4.1 Hz, 2H), 4.03/3.91 (s, 6H), 3.82−3.79 (m, 1H), 3.31−3.26 (m, 1.5H), 3.20−3.15 (m, 1.5H), 2.39−2.26 (m, 2H), 2.05−1.83 (m, 6H). LC (method A): tR = 2.49 min. LCMS (ESI) m/z calcd for C46H43N6O6, 775.32; found, 775.35 [M + H]+. HRMS (ESI) m/z calcd for C46H43N6O6, 775.3244; found, 775.3259 [M + H]+. (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))bis(1-(3-chloro-1-naphthoyl)pyrrolidine-2-carboxamide) (28). Compound 28 (51 mg, 54%) was prepared as an off-white solid I



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calcd for C40H39N4O4, 639.30; found, 639.49 [M + H]+. HRMS (ESI) m/z calcd for C40H37N4O4, 637.2815; found, 637.2820 [M − H]−. (2S,2′S)-N,N′-(4,4′-(Ethyne-1,2-diyl)bis(4,1-phenylene))bis(1(3-chloro-5-methoxyisoquinoline-1-carbonyl)pyrrolidine-2carboxamide) (34). Compound 34 (32.6 mg, 49%) was prepared as a tan solid from (2S,2′S)-N,N′-(4,4′-(ethyne-1,2-diyl)bis(4,1phenylene))dipyrrolidine-2-carboxamide dihydrochloride (44·2HCl) and 3-chloro-5-methoxyisoquinoline-1-carboxylic acid (101) in a manner similar to that described for compound 11. 1H NMR (400 MHz, DMSO-d6) δ 10.52/9.76 (s, 2H), 8.09/7.93 (s, 2H), 7.86−7.49 (series of m, 10H), 7.36−7.09 (series of m, 4H), 4.76/4.45 (dd, J = 8.3, 4.6 Hz, 2H), 4.03/3.94 (s, 6H), 3.82−3.79 (m, 1H), 3.34−3.28 (m, 1.5H), 3.23−3.17 (m, 1.5H), 2.41−2.30 (m, 2H), 2.05−1.86 (m, 6H). LC (method A): tR = 2.79 min. LCMS (ESI) m/z calcd for C46H39Cl2N6O6, 841.23; found, 841.24 [M + H]+. HRMS (ESI) m/z calcd for C46H39Cl2N6O6, 841.2308; found, 841.2325 [M + H]+. (2S,2′S)-N,N′-(4,4′-(1H-Pyrazole-3,5-diyl)bis(4,1-phenylene))bis(1-(2-phenylacetyl)pyrrolidine-2-carboxamide) (35). Compound 35 (61 mg, 64%) was prepared as a white solid from 4,4′(1H-pyrazole-3,5-diyl)dianiline (45) and (S)-1-(2-phenylacetyl)pyrrolidine-2-carboxylic acid25 in a manner similar to that described for compound 1. 1H NMR (500 MHz, DMSO-d6) δ 10.09/10.32 (s, 2H), 7.75 (d, J = 8.8 Hz, 4H), 7.65 (d, J = 8.8 Hz, 4H), 7.26 (m, 12H), 4.45 (dd, J = 8.2, 3.5 Hz, 2H), 3.72 (s, 4H), 3.61 (m, 4H), 2.15 (m, 2H), 1.92 (m, 6H). LC (method H): tR = 1.71 min. LCMS (ESI) m/z calcd for C41H41N6O4, 681.31; found, 681.21 [M + H]+. HRMS (ESI) m/z calcd for C41H41N6O4, 681.3184; found, 681.3168 [M + H]+. (2S,2′S)-N,N′-(4,4′-(1H-Pyrazole-3,5-diyl)bis(4,1-phenylene))bis(1-(3-chloro-5-methoxyisoquinoline-1-carbonyl)pyrrolidine-2-carboxamide) (36). Compound 36 (30 mg, 35%) was prepared as an off-white solid from 2S,2′S)-N,N′-(4,4′-(1Hpyrazole-3,5-diyl)bis(4,1-phenylene))dipyrrolidine-2-carboxamide trihydrochloride (47·3HCl) and 3-chloro-5-methoxyisoquinoline-1carboxylic acid (101) in a manner similar to that described for compound 11. 1H NMR (500 MHz, DMSO-d6, missing pyrrole NH) δ 10.43/9.67 (s, 2H), 8.09−7.55 (series of m, 13H), 7.36−7.08 (series of m, 4H), 4.78/4.47 (dd, J = 8.2, 4.6 Hz, 2H), 4.03/3.90 (s, 6H), 3.81−3.80 (m, 1H), 3.34−3.31 (m, 1.5H), 3.22−3.19 (m, 1.5H), 2.41−2.37 (m, 2H), 2.03−1.86 (m, 6H). LC (method H): tR = 2.17 min. LCMS (ESI) m/z calcd for C47H41Cl2N8O6, 883.25; found, 883.43 [M + H]+. HRMS (ESI) m/z calcd for C47H39Cl2N8O6, 881.2370; found, 881.2401 [M − H]−. (2S,2′S)-N,N′-(4,4′-(Oxazole-2,5-diyl)bis(4,1-phenylene))bis(1-(2-phenylacetyl)pyrrolidine-2-carboxamide) (37). Compound 37 (49 mg, 59%) was prepared as a yellow solid from 4,4′-(oxazole2,5-diyl)dianiline (48) and (S)-1-(2-phenylacetyl)pyrrolidine-2-carboxylic acid25 in a manner similar to that described for compound 1. 1H NMR (500 MHz, DMSO-d6) δ 7.82−7.68 (m, 8H), 7.34−7.21 (m, 11H), 4.46 (br s, 2H), 3.72 (s, 4H), 3.70−3.64 (m, 2H), 3.64− 3.58 (m, 2H), 2.22−2.13 (m, 2H), 2.07−1.99 (m, 2H), 1.93 (br s, 4H). LC (method I): tR = 2.66 min. LCMS (ESI) m/z calcd for C41H39N5O5, 681.80; found, 682.26 [M + H]+. HRMS (ESI) m/z calcd for C41H40N5O5, 682.3024; found, 682.3002. (2S,2′S)-N,N′-(4,4′-(Oxazole-2,5-diyl)bis(4,1-phenylene))bis(1-(3-chloro-5-methoxyisoquinoline-1-carbonyl)pyrrolidine-2carboxamide) (38). Compound 38 (41 mg, 40%) was prepared as an off-white solid from (2S,2′S)-N,N′-(4,4′-(oxazole-2,5-diyl)bis(4,1phenylene))dipyrrolidine-2-carboxamide dihydrochloride (50·2HCl) and 3-chloro-5-methoxyisoquinoline-1-carboxylic acid (101) in a manner similar to that described for compound 11. 1H NMR (500 MHz, DMSO-d6) δ 10.56/9.81 (m, 2H), 8.07 (m, 3H), 7.94−7.53 (series of m, 11H), 7.36 (m, 2H), 7.20 (m, 1H), 4.78/4.46 (m, 2H), 4.03/3.90 (s, 6H), 3.83−3.79 (m, 1H), 3.36−3.28 (m, 1.5H), 3.25− 3.19 (m, 1.5H), 2.41−2.37 (m, 2H), 2.10−1.85 (m, 6H). LC (method H): tR = 2.26 min. LCMS (ESI) m/z calcd for C47H40Cl2N7O7, 884.24; found, 884.42 [M + H]+. HRMS (ESI) m/z calcd for C47H38Cl2N7O7, 882.2210; found, 882.2207 [M − H]−. 4,4′-(Ethene-1,2-diyl)dianiline Free Base (39). 4,4′-(Ethene1,2-diyl)dianiline dihydrochloride (39, Aldrich, 15.0 g, 53.0 mmol) was taken up in EtOAc (400 mL), and concentrated NH4OH solution (28%, ∼200 mL) was added. The suspension was vigorously stirred for

1 h at room temperature before 1 N NaOH (∼50 mL) was added. The suspension which had a pH of ∼11 was stirred further at room temperature for 1 h before it was suction-filtered to yield a tan precipitate. The filtrate was washed with brine and dried over anhydrous Na2SO4. The original precipitate was taken up in CH2Cl2 and stirred at room temperature for 1 h with concentrated NH4OH solution (∼50 mL) before it was suction-filtered a second time to yield a tan precipitate. The filtrate was washed with brine and dried over anhydrous Na2SO4. Evaporation of the combined, dried filtrate afforded 4,4′-(ethene-1,2-diyl)dianiline (39, 10.56 g, 94.9% recovery) as a tan-colored free base when combined with the original precipitate which was used directly without further purification. (2S,2′S)-tert-Butyl 2,2′-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1phenylene))bis(azanediyl)bis(oxomethylene)dipyrrolidine-1carboxylate (40). EEDQ (22.58 g, 91.3 mmol) was added in one portion to a stirred suspension of 4,4′-(ethene-1,2-diyl)dianiline (39, free base, 8.00 g, 38.1 mmol) and N-Boc-L-proline (18.83 g, 87.5 mmol) in dry CH2Cl2 (250 mL) under N2 at room temperature. After 15 min, the suspension became homogeneous and was stirred for 16 h before the solvent was removed in vacuo to 1/4 volume. The suspension was diluted with Et2O and suction-filtered to provide 40 (21.62 g, 94%) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ 10.04 (s, 2H), 7.61 (d, J = 8.8 Hz, 4H), 7.51 (d, J = 8.4 Hz, 4H), 7.09 (s, 2H), 4.30−4.17 (2m, 2H), 3.45−3.34 (2m, 4H), 2.25−2.12 (m, 2H), 1.95−1.72 (m, 6H), 1.40/1.27 (s, 18H). LC (method A): tR = 2.56 min. LCMS (ESI) m/z calcd for C34H45N4O6, 605.33; found, 605.35 [M + H]+. HRMS (ESI) m/z calcd for C34H45N4O6, 605.3339; found, 605.3365 [M + H]+. Example of Boc Deprotection with TFA in CH2Cl2: (2S,2′S)N , N ′ - ( 4 , 4 ′ - ( (E ) - E t h e n e - 1 , 2 - d i y l ) b i s (4 , 1 - p h e n y l e n e ) ) dipyrrolidine-2-carboxamide (41, Free Base). A suspension of (2S,2′S)-tert-butyl 2,2′-N,N′-(4,4′-((E)-ethene-1,2-diyl)bis(4,1phenylene))bis(azanediyl)bis(oxomethylene)dipyrrolidine-1-carboxylate (40, 21.6 g, 35.72 mmol) in dry CH2Cl2 (500 mL) was treated with CF3CO2H (50 mL) at room temperature under N2. The mixture was stirred for 3 h before additional CF3CO2H (20 mL) was added. After being stirred for an additional 4 h at room temperature, the mixture was concentrated in vacuo. The residue was dissolved in EtOAc, washed with saturated NaHCO3 solution (some 1 N NaOH solution added), brine, dried over anhydrous Na2SO4, and evaporated to 1/4 of its original volume to provide 41 (11.21 g, 78%, free base) as a white solid after suction filtration. 1H NMR (500 MHz, DMSO-d6) δ 9.99 (s, 2H), 7.66 (d, J = 8.5 Hz, 4H), 7.51 (d, J = 8.5 Hz, 4H), 7.10 (s, 2H), 3.68 (dd, J = 8.8, 6.0 Hz, 2H), 3.05 (br s, 2H), 2.89 (t, J = 7.0 Hz, 4H), 2.07−2.01 (m, 2H), 1.80−1.75 (m, 2H), 1.68−1.64 (m, 4H). LC (method A): tR = 1.20 min. LCMS (ESI) m/z calcd for C24H29N4O2, 405.23; found, 405.43 [M + H]+. HRMS (ESI) m/z calcd for C24H29N4O2, 405.2291; found, 405.2293 [M + H]+. Example of Boc Deprotection with 4 N HCl in Dioxane: (2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))dipyrrolidine-2-carboxamide Dihydrochloride (41·2HCl). A suspension of (2S,2′S)-tert-butyl 2,2′-N,N′-(4,4′-((E)-ethene-1,2-diyl)bis(4,1-phenylene))bis(azanediyl)bis(oxomethylene)dipyrrolidine-1carboxylate (40, 8.29 g, 14.28 mmol) in dry dioxane (30 mL) was treated with 4 N HCl in dioxane (30 mL) at 0 °C under N2. The mixture was stirred at 0 °C for 1 h before MeOH (5 mL) was added. The mixture was allowed to warm to room temperature where it was stirred for an additional 3 h before the suspension was diluted with Et2O (30 mL) and suction-filtered to yield 41·2HCl (6.2 g, 91%) as an off-white solid. See characterization above. 4,4′-(Ethyne-1,2-diyl)dianiline (42). Compound 42 was purchased from Bionet Research (A Trading Division of Key Organics Ltd.), U.K., as a free base with a minimal purity of 90% and was used as is. (2S,2′S)-tert-Butyl 2,2′-N,N′-(4,4′-(Ethyne-1,2-diyl)bis(4,1phenylene))bis(azanediyl)bis(oxomethylene)dipyrrolidine-1carboxylate (43). Compound 43 (6.45 g, 97%) was prepared as an off-white solid from 42 and N-Boc-L-proline in a manner similar to that described for compound 40. 1H NMR (500 MHz, DMSO-d6) δ 10.16 (br s, 2 H), 7.66 (d, J = 8.3 Hz, 4H), 7.47 (d, J = 8.3 Hz, 4H), 4.26 (dd, J = 8.1, 2.7 Hz, 0.75H), 4.20 (dd, J = 8.0, 4.1 Hz, 1.25H), J



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carboxylate (49). Compound 49 (6.2 g, 80.4%) was prepared as an orange solid from 4,4′-(oxazole-2,5-diyl)dianiline (48) and N-Boc-Lproline in a manner similar to that described for compound 40. 1H NMR (400 MHz, DMSO-d6) δ 10.28 (s, 1H), 10.17 (s, 1H), 8.04− 8.02 (m, 2H), 7.79−7.68 (m, 7H), 4.28−4.19 (m, 2H), 3.46−3.32 (m, 4H), 2.25−2.17 (m, 2H), 1.95−1.80 (2m, 6H), 1.41/1.27 (s, 18H). LC (method J): tR = 2.95 min. LCMS (ESI) m/z calcd for C35H44N5O7, 646.63; found, 646.51 [M + H]+. HRMS (ESI) m/z calcd for C35H44N5O7, 646.3241; found, 646.3254 [M + H]+. (2S,2′S)-N,N′-(4,4′-(Oxazole-2,5-diyl)bis(4,1-phenylene))dipyrrolidine-2-carboxamide Dihydrochloride (50·2HCl). Compound 50·2HCl (4.91 g, 98%) was prepared as an orange solid from (2S,2′S)-tert-butyl 2,2′-N,N′-(4,4′-(oxazole-2,5-diyl)bis(4,1phenylene))bis(azanediyl)bis(oxomethylene)dipyrrolidine-1-carboxylate (49) in a manner similar to that described for compound 41·2HCl. 1H NMR (400 MHz, DMSO-d6) δ 10.39−10.38 and 9.64− 9.62 (m, 2H), 8.09 (s, 1H), 7.93−7.02 (series of m, 8H), 4.78−4.75 and 4.48−4.45 (m, 2H), 3.34−3.28 (m, 2H), 3.23−3.18 (m, 2H), 2.40−2.30 (m, 2H), 2.10−1.80 (m, 6H). LC (method J): tR = 1.64 min. LCMS calcd for C25H28N5O3, 446.22; found, 446.50 [M + H]+. HRMS (ESI) m/z calcd for C25H28N5O3, 446.2192; found, 446.2207 [M + H]+. Methyl 3-(Trifluoromethylsulfonyloxy)picolinate (52). To a cold (0 °C) mixture of methyl 3-hydroxypicolinic acid (51, Aldrich, 2.5 g, 16.3 mmol) and Et3N (2.5 mL, 18.0 mmol) in dry CH2Cl2 (180 mL) was added triflic anhydride (5.0 g, 18.0 mmol) under an atmosphere of N2. The mixture was stirred for 1 h at 0 °C, warmed to room temperature, and stirred for 1 h. The mixture was quenched with saturated NaHCO3 solution (10 mL), and the organic layer was separated, washed with brine, and dried over anhydrous MgSO4. Evaporation of the solvent gave 52 (3.38 g, 73%) as a dark brown oil which was carried forward directly. 1H NMR (300 MHz, CDCl3) δ 8.76 (dd, J = 4.4, 1.5 Hz, 1H), 7.72 (dd, J = 8.4, 1.5 Hz, 1H), 7.62 (dd, J = 8.4, 4.4 Hz, 1H), 4.04 (s, 3H). LC (method D): tR = 1.93 min. LCMS (ESI) m/z calcd for C8H7F3NO5, 286.00; found, 286.08 [M + H]+. Alternative Route: Methyl 3-(Trifluoromethylsulfonyloxy)picolinate (52). 4-Nitrophenyl trifluoromethanesulfonate (271 mg, 1.0 mmol) was added in one portion to a stirred suspension of methyl 3-hydroxypicolinate (51, Aldrich, 153 mg, 1.0 mmol) and powdered K2CO3 (276 mg, 2.0 mmol) in anhydrous DMF (10 mL) at room temperature. The mixture was stirred for 2 h before it was diluted with EtOAc, washed with 1 N NaOH and brine, dried over anhydrous MgSO4, filtered, and concentrated to furnish 52 as a light yellow oil which was used directly as-is. 1H NMR (300 MHz, CDCl3) δ 8.76 (dd, J = 4.4, 1.5 Hz, 1H), 7.72 (dd, J = 8.4, 1.5 Hz, 1H), 7.62 (dd, J = 8.4, 4.4 Hz, 1H), 4.04 (s, 3H); LRMS (ESI) m/z calcd for C8H7F3NO5S, 286.00; found, 286.14 [M + H]+. Methyl 3-Vinylpicolinate (53). Methyl 3-(trifluoromethylsulfonyloxy)picolinate (52, 570 mg, 2.0 mmol) was taken up in anhydrous DMF (3 mL) and treated with tributyl(vinyl)stannane (761 mg, 2.4 mmol), LiCl (254 mg, 6.0 mmol), and (Ph3P)2Pd(II)Cl2 (42 mg, 0.06 mmol). The mixture was heated for 4 h at 100 °C, cooled to room temperature, and the solvent was removed in vacuo. The residue was partitioned between hexanes and CH3CN, and the CH3CN layer was washed twice with hexanes before the combined CH3CN extract was dried over anhydrous MgSO4, filtered through Celite, and concentrated. Purification of the residue by flash chromatography on silica gel (gradient elution from 25% to 65% EtOAc in hexanes) furnished 53 (130 mg, 40%) as a yellow oil. 1H NMR (300 MHz, CDCl3) δ 8.60 (dd, J = 4.6, 1.7 Hz, 1 H), 7.94 (dd, J = 7.9, 1.3 Hz, 1H), 7.29−7.53 (m, 2H), 5.72 (dd, J = 17.4, 0.9 Hz, 1H), 5.47 (dd, J = 11.0, 0.7 Hz, 1H), 3.99 (s, 3H). LC (method D): tR = 1.29 min. LCMS (ESI) m/z calcd for C9H10NO2, 164.07; found, 164.06 [M + H]+. Methyl 3-Ethylpicolinate (54). A solution of 53 (120 mg, 0.74 mmol) in EtOH (5 mL) was subjected to balloon hydrogenation for 1 h at room temperature using 10% Pd/C (25 mg) as catalyst. The suspension was filtered through Celite and the pad of Celite was rinsed with MeOH to afford a yellow solution of 54 which was used directly

3.45−3.39 (m, 2H), 3.39−3.30 (m, 2H), 2.26−2.12 (m, 2H), 1.97− 1.74 (m, 6H), 1.40/1.27 (s, 18). LC (method A): tR = 2.53 min. LCMS (ESI) m/z calcd for C34H43N4O6, 603.32; found, 603.39 [M + H]+. HRMS (ESI) m/z calcd for C34H43N4O6, 603.3177; found, 603.3154 [M + H]+. (2S,2′S)-N,N′-(4,4′-(Ethyne-1,2-diyl)bis(4,1-phenylene))dipyrrolidine-2-carboxamide Dihydrochloride (44·2HCl). Compound 44·2HCl (4.00 g, 78%) was prepared as light tan solid from (2S,2′S)-tert-butyl 2,2′-N,N′-(4,4′-(ethyne-1,2-diyl)bis(4,1phenylene))bis(azanediyl)bis(oxomethylene)dipyrrolidine-1-carboxylate (43) in a manner similar to that described for compound 41·2HCl. 1H NMR (500 MHz, DMSO-d6) δ 11.60 (s, 2 H), 10.06− 9.88 (br s, 2H), 8.77−8.57 (br s, 2H), 7.72 (d, J = 8.8 Hz, 4H), 7.53 (d, J = 8.5 Hz, 4H), 4.48−4.37 (m, 2H), 3.73−3.53 (m, 2H), 3.34− 3.20 (m, 2H), 2.47−2.35 (m, 2H), 2.02−1.89 (m, 6H). LC (method A): tR = 1.30 min. LCMS (ESI) m/z calcd for C24H27N4O2, 403.21; found, 403.29 [M + H]+. HRMS (ESI) m/z calcd for C24H27N4O2, 403.2129; found, 403.2113 [M + H]+. 4,4′-(1H-Pyrazole-3,5-diyl)dianiline (45). Compound 45 (0.78 g, 97%) was prepared as an off-white solid from 3,5-bis(4nitrophenyl)-1H-pyrazole23 in a manner similar to that described for compound 48. 1H NMR (500 MHz, DMSO-d6) δ 11.71 (br s, 1H), 7.52 (s, 1H), 7.11 (m, 4H), 6.48 (m, 4H), 5.17 (br s, 2H), 4.94 (br s, 2H). LC (method L): tR = 0.87 min. LCMS (ESI) m/z calcd for C15H15N4, 251.12; found, 251.29 [M + H]+. (2S,2′S)-tert-Butyl 2,2′-N,N′-(4,4′-(1H-Pyrazole-3,5-diyl)bis(4,1-phenylene))bis(azanediyl)bis(oxomethylene)dipyrrolidine-1-carboxylate (46). Compound 46 (2.20 g, 85%) was prepared as an off-white solid from 4,4′-(1H-pyrazole-3,5-diyl)dianiline (45) and N-Boc-L-proline in a manner similar to that described for compound 40. 1H NMR (500 MHz, DMSO-d6) δ 10.16 (d, J = 2.9 Hz, 2H), 8.08 (m, 4H), 7.67 (m, 4H), 7.34 (m, 1H), 4.39 (m, 2H), 3.49 (m, 4H), 2.27 (m, 2H), 1.93 (m, 6H), 1.41 /1.27 (s, 18H). LC (method H): tR = 1.70 min. LCMS (ESI) m/z calcd for C35H45N6O6, 645.33; found, 645.38 [M + H]+. (2S,2′S)-N,N′-(4,4′-(1H-Pyrazole-3,5-diyl)bis(4,1-phenylene))dipyrrolidine-2-carboxamide Trihydrochloride (47·3HCl). Compound 47·3HCl (0.26 g, 87%) was prepared as a brown solid from (2S,2′S)-tert-butyl 2,2′-N,N′-(4,4′-(1H-pyrazole-3,5-diyl)bis(4,1phenylene))bis(azanediyl)bis(oxomethylene)dipyrrolidine-1-carboxylate (46) in a manner similar to that described for compound 41·2HCl. A solution of compound 47·3HCl in MeOH (2 mL) was free-based using a UCT CHQAX12M6 (2.0 g) cartridge and MeOH elution. 1H NMR (500 MHz, DMSO-d6, missing pyrrole NH) δ 10.16 (s, 2H), 8.08 (m, 4H), 7.67 (m, 4H), 7.34 (m, 1H), 4.39 (m, 2H), 3.49 (m, 4H), 2.50 (br s, 2H), 2.24 (m, 2H), 1.95 (m, 6H). LC (method H): tR = 0.77 min. LCMS (ESI) m/z calcd for C25H29N6O2, 445.23; found, 445.12 [M + H]+. 4,4′-(Oxazole-2,5-diyl)dianiline (48). A suspension of 2,5-bis(4nitrophenyl)oxazole23 (9.83 g, 31.6 mmol) in MeOH (100 mL) and EtOAc (100 mL) was subjected to balloon hydrogenation over 20% Pd(OH)2/C (1.0 g) for 6 h at ambient temperature before it was suction-filtered through Celite and concentrated in vacuo to yield the crude product as a reddish solid. The solid was dissolved in hot MeOH and the solution cooled to ambient temperature to afford a precipitate which was suctioned-filtered to furnish crop 1 of 48 (1.70 g, 21%) as a brick-colored solid. The filtrate was concentrated in vacuo, and this residue was dissolved in a minimal amount of hot MeOH and cooled to afford a second crop of 48 (2.90 g, 37%) as a reddish-brown solid. The filtrate was concentrated in vacuo and the residue was subjected to flash chromatography on silica gel (gradient elution from 50% to 100% EtOAc in hexanes) to yield a third and final crop of 48 (2.30 g, 29%) as an orange solid after concentration of the eluant to 1/4 volume and suction filtration of the precipitate. 1H NMR (300 MHz, DMSOd6 and D2O) δ 7.72 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H), 7.40 (s, 1H), 6.83 (d, J = 8.05 Hz, 2H), 6.69 (d, J = 8.05 Hz, 2H). LC (method K): tR = 1.17 min. LCMS (ESI) m/z calcd for C15H13N3O, 252.11; found, 252.05 [M + H]+. (2S,2′S)-tert-Butyl 2,2′-N,N′-(4,4′-(Oxazole-2,5-diyl)bis(4,1phenylene))bis(azanediyl)bis(oxomethylene)dipyrrolidine-1K



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in the preparation of compound 57. LC (method D): tR = 1.15 min. LCMS (ESI) m/z calcd for C9H12NO2, 166.09; found, 166.09 [M + H]+. Methyl 3-Phenylpicolinate (55). Compound 52 (285 mg, 1.0 mmol) was taken up in anydrous THF (10 mL) and treated with powdered K2PO4 (318 mg, 1.50 mmol), phenylboronic acid (146 mg, 1.2 mmol), and bis(diphenylphosphinylferrocene)palladium(II) dichloride−CH2Cl2 complex (16 mg, 0.02 mmol). The mixture was heated at reflux for 16 h before it was cooled to room temperature and partitioned between EtOAc and H2O. The organic phase was separated, washed with brine, dried over anhydrous Na2SO4, and concentrated. Purification of the residue by flash chromatography on silica gel (gradient elution with 10−70% EtOAc in hexanes on a 25S Thomson single step column) furnished 55 (159 mg, 75%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 9.30 (s, 1H), 8.36 (dd, J = 2.2, 8.3 Hz, 1H), 8.07 (dd, J = 2.2, 8.4 Hz, 2H), 7.83 (dd, J = 0.8, 8.0 Hz, 1H), 7.53−7.45 (m, 3H), 3.98 (s, 3H). LC (method B): tR = 1.16 min. LCMS (ESI) m/z calcd for C13H12NO2, 214.09; found, 214.17 [M + H]+. 3-Vinylpicolinic Acid (56). Compound 52 (570 mg, 2.0 mmol) was taken up in anhydrous DMF (3 mL) and treated with tributyl(vinyl)stannane (761 mg, 2.4 mmol), LiCl (254 mg, 6.0 mmol), and (Ph3P)2Pd(II)Cl2 (42 mg, 0.06 mmol). The mixture was heated for 16 h at 100 °C before it was cooled to room temperature and treated with saturated KF solution (3 mL). After being stirred for 4 h, the mixture was filtered through Celite and the pad of Celite rinsed with EtOAc. The aqueous phase of the filtrate was separated, concentrated, and acidified with 4 N HCl in dioxane. The residue was taken up in MeOH and the suspension sonicated for 2 min and suction-filtered to remove the inorganic salts. Evaporation of the filtrate afforded 56 (260 mg, 87%) as a green solid which was slightly contaminated with inorganic salts but was taken forward directly without further purification. 1H NMR (500 MHz, DMSO-d6) δ 8.21 (d, J = 3.7 Hz, 1H), 7.85 (dd, J = 7.7, 1.5 Hz, 1H), 7.09 (dd, J = 7.7, 4.8 Hz, 1H), 6.98 (dd, J = 17.9, 11.3 Hz, 1H), 5.74 (dd, J = 17.9, 1.5 Hz, 1H), 5.20 (dd, J = 11.0, 1.1 Hz, 1H). LC (method D): tR = 0.39 min. LCMS (ESI) m/z calcd for C8H8NO2, 150.05; found, 150.07 [M + H]+. 3-Ethylpicolinic Acid (57). The solution of 54 was diluted with THF (5 mL), treated with LiOH (115 mg, 4.80 mmol), and stirred at ambient temperature for 2 days before the solvent was removed in vacuo. The residue was treated with 4 N HCl in dioxane (∼5 mL) and the mixture was concentrated in vacuo to yield 57 (35 mg, 40%, two steps) as a yellow solid which was slightly contaminated with inorganic salts but used directly without further purification. 1H NMR (300 MHz, DMSO-d6) δ 8.47 (dd, J = 4.8, 1.5 Hz, 1H), 7.86 (dd, J = 7.9, 1.3 Hz, 1H), 7.53 (dd, J = 7.7, 4.8 Hz, 1H), 2.82 (q, J = 7.3 Hz, 2H), 1.17 (t, J = 7.5 Hz, 3H). LC (method D): tR = 0.36 min. LCMS (ESI) m/z calcd for C8H10NO2, 152.07; found, 152.10 [M + H]+. 3-Phenylpicolinic Acid (58). Methyl 3-phenylpicolinate (55, 159 mg, 0.75 mmol) was taken up in MeOH (3 mL) and treated with 1 N NaOH (1 mL). The mixture was stirred at ambient temperature for 1 h before additional 1 N NaOH (1.5 mL) was added. After being stirred for an additional 2 h, the mixture was heated to 50 °C for 16 h before it was cooled to room temperature and acidified with 1 N HCl to pH ∼2. The mixture was extracted with CH2Cl2 (3 × 10 mL) and the combined organic extract was dried over anhydrous Na2SO4 and evaporated to yield 58 (45.9 mg, 31%) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 13.3 (br s, 1H), 8.79−8.69/8.61−8.57/8.46−8.40 (m, 1H), 8.12−8.08/8.02/7.93−7.89/7.81−7.79 (m, 1H), 7.62−7.28 (series of m, 6H). LC (method B): tR = 0.51 min. LCMS (ESI) m/z calcd for C12H10NO2, 200.07, found 200.13 [M + H]+. 3-Phenylpicolinic acid is now available from A.A. Scientific (AAS-2846), Apollo-Inter (OR16992), and Combi-Blocks (YA-5926). 3-Methoxyisoquinoline (60). Compound 60 (1.42 g, 32%) was prepared as a colorless oil from 3-hydroxyisoquinoline (59, Aldrich) in a manner similar to that described for compound 63. 1H NMR (500 MHz, CDCl3) δ 8.93 (s, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.65 (d, J = 8.5 Hz, 1H), 7.55−7.51 (m, 1H), 7.35−7.32 (m, 1H), 6.97 (s, 1H), 4.02

(s, 3H). LC (method A): tR = 0.98 min. LCMS (ESI) m/z calcd for C10H10NO, 160.08; found, 160.07 [M + H]+. 3-Methoxyisoquinoline N-Oxide (61). 3-Methoxyisoquinoline N-oxide (61) (1.36 g, 28%, two steps) was prepared as a white solid from 3-methoxyisoquinoline (60) in a manner similar to that described for compound 64. 1H NMR (500 MHz, CDCl3) δ 8.86 (s, 1H), 7.68 (dd, J = 13.7, 8.2 Hz, 2H), 7.54 (t, J = 7.5 Hz, 1H), 7.50− 7.43 (m, 1H), 7.06 (s, 1H), 4.14 (s, 3H). LC (method A): tR = 0.70 min. LCMS (ESI) m/z calcd for C10H10NO2, 176.07; found, 176.14 [M + H]+. 5-Methoxyisoquinoline (63). To a stirred suspension of 5hydroxisoquinoline (62, Aldrich, 2.0 g, 13.8 mmol) and Ph3P (4.3 g, 16.5 mmol) in dry THF (20 mL) was added dry MeOH (0.8 mL) and diethyl azodicarboxylate (DEAD, 3.0 mL, 16.5 mmol) portionwise. The mixture was stirred at room temperature for 20 h before it was diluted with EtOAc, washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated. The residue was preabsorbed onto silica gel and purified by flash chromatography on silica gel (elution with 40% EtOAc in hexanes) to afford 63 (2.59 g, 45%) as a light yellow solid. The sample was contaminated with reduced DEAD but was taken forward directly. 1H NMR (500 MHz, CDCl3) δ 9.19 (s, 1H), 8.51 (d, J = 6.0 Hz, 1H), 7.99 (d, J = 6.0 Hz, 1H), 7.52−7.50 (m, 2H), 7.00− 6.99 (m, 1H), 4.01 (s, 3H). LC (method A): tR = 0.66 min. LCMS (ESI) m/z calcd for C10H10NO, 160.08; found, 160.10 [M + H]+. 5-Methoxyisoquinoline N-Oxide (64). m-Chloroperbenzoic acid (77%, 3.42 g, 19.8 mmol) was added in one portion to a stirred solution of 63 (2.34 g, 14.7 mmol) in anhydrous CH2Cl2 (50 mL) at room temperature. After the mixture was stirred for 20 h, powdered K2CO3 (2.0 g) was added, and the mixture was stirred further for 1 h before it was filtered and concentrated to furnish 64 (2.15 g, 83%) as a pale, yellow solid which was sufficiently pure to carry forward directly. 1 H NMR (400 MHz, CDCl3) δ 8.73 (d, J = 1.5 Hz, 1H), 8.11 (dd, J = 7.3, 1.7 Hz, 1H), 8.04 (d, J = 7.1 Hz, 1H), 7.52 (t, J = 8.1 Hz, 1H), 7.28 (d, J = 8.3 Hz, 1H), 6.91 (d, J = 7.8 Hz, 1H), 4.00 (s, 3H). LC (method A): tR = 0.92 min. LCMS (ESI) m/z calcd for C10H10NO2, 176.07; found, 176.03 [M + H]+. 3-Methylisoquinoline (65), 5-methylisoquinoline (66), and the 1-haloisoquinolines 67−75 were purchased from commercial sources unless noted otherwise.

1,3-Dichloro-5-fluoroisoquinoline (74). m-Chloroperbenzoic acid (77%, 0.62 g, 3.58 mmol) was added in one portion to a stirred solution of 5-fluoro-1-chloroisoquinoline22−24,30 (73, 0.50 g, 2.75 mmol) in anhydrous CH2Cl2 (20 mL) at room temperature. After the mixture was stirred for 2 h, additional mCPBA (0.62 g) was added and the mixture was stirred further at room temperature for 16 h before additional CH2Cl2 (20 mL) was added as well as powdered K2CO3 (2.5 g). The mixture was stirred for 1 h before it was filtered and concentrated to furnish 1-chloro-5-fluoroisoquinoline 2-oxide (0.13 g, 24%) as a white solid. The filtrate was purified by flash chromatography on silica gel (gradient elution from 0% to 5% MeOH in CH2Cl2 on a 25S Thomson single step column using 900 mL of solvent) to yield additional 1-chloro-5-fluoroisoquinoline 2oxide (0.16 g, 29%) as a light yellow foam. Both isolates were L



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over anhydrous MgSO4 and evaporation. The residue was triturated with hot MeOH to give intermediate 3-methyl-2-(phenylsulfonyl)-1,2dihydroisoquinoline-1-carbonitrile as a tan solid (94.2 mg, 27%) after suction filtration. NaBH4 (87.2 mg, 2.31 mmol) was added to a stirred suspension of 3-methyl-2-(phenylsulfonyl)-1,2-dihydroisoquinoline-1carbonitrile (65.9 mg, 0.21 mmol) in absolute EtOH (5 mL) at ambient temperature. The mixture was stirred for 2 h before the solvent was removed in vacuo. The residue was partitioned between H2O and CHCl3 and the organic layer was separated, washed with saturated NaHCO3 solution, dried over anhydrous MgSO4, and concentrated in vacuo to afford 78 (47.1 mg, 27%, two steps) as a tan solid. 1H NMR (500 MHz, CDCl3) δ 8.28 (d, J = 8.2 Hz, 1H), 7.83 (d, J = 8.2 Hz, 1H), 7.78−7.73 (m, 1H), 7.72 (s, 1H), 7.71−7.68 (m, 1H), 2.74 (s, 3H). LC (method B): tR = 1.25 min. LCMS (ESI) m/z calcd for C11H9N2, 169.09; found, 169.10 [M + H]+. 5-Methylisoquinoline-1-carbonitrile (79). A mixture of 1chloro-5-methylisoquinoline22−24,30 (66, 0.50 g, 2.81 mmol), sodium methanesulfinate (0.58, 5.63 mmol), KCN (0.55 g, 8.44 mmol), and 18-crown-6 (2.97 g, 11.26 mmol) in dry DMF (30 mL) was heated at 140 °C for 6 h before the solvent was removed in vacuo. The residue was diluted with H2O (60 mL) and the mixture was extracted with EtOAc (3 × 20 mL) and the combined organic extract was washed with brine, dried over anhydrous MgSO4, and evaporated. The dark brown residue was preabsorbed onto silica gel using CH2Cl2 as solvent and flash chromatographed on silica gel (elution with 15% EtOAc in hexanes) to yield 79 (0.27g, 57%) as a yellow solid. 1H NMR (500 MHz, DMSO-d6) δ 9.42 (d, J = 0.9 Hz, 1H), 8.68 (s, 1H), 8.11 (dd, J = 6.7, 2.4 Hz, 1H), 7.87−7.72 (m, 2H), 2.71 (s, 3H). LC (method B): tR = 1.25 min. LCMS (ESI) m/z calcd for C11H9N2, 169.09; found, 169.15 [M + H]+. 3-Chloroisoquinoline-1-carbonitrile (80). 3-Chloroisoquinoline-1-carbonitrile (80, 400 mg, 42%) was prepared as a light yellow solid from 1,3-dichloroisoquinoline (67)22−24,30 in a manner similar to that described for compound 88. 1H NMR (300 MHz, CDCl3) δ 8.47−8.44 (m, 1H), 8.36 (s, 1H), 8.07−7.97 (m, 3H). LC (method A): tR = 1.37 min. LCMS (ESI) m/z calcd for C10H6ClN2, 189.02; found, 188.97 [M + H]+. 4-Chloroisoquinoline-1-carbonitrile (81). 4-Chloroisoquinoline-1-carbonitrile (81, 395 mg, 70%) was prepared as a light yellow solid from 1,4-dichloroisoquinoline (68)22−24,30 in a manner similar to that described for compound 88. 1H NMR (300 MHz, CDCl3) δ 8.98 (s, 1H), 8.50−8.47 (m, 1H), 8.33−8.31 (m, 1H), 8.09−8.06 (m, 1H), 8.02−7.98 (m, 1H). LC (method A): tR = 1.45 min. LCMS (ESI) m/z calcd for C10H6ClN2, 189.02; found, 188.98 [M + H]+. 5-Chloroisoquinoline-1-carbonitrile (82). 5-Chloroisoquinoline-1-carbonitrile (82, 490 mg, 52%) was prepared as an off-white solid from 1,5-dichloroisoquinoline (69)22−24,30 in a manner similar to that described for compound 88. 1H NMR (300 MHz, CDCl3) δ 8.76 (d, J = 5.7 Hz, 1H), 8.31−8.28 (m, 2H), 7.92−7.89 (m, 1H), 7.75− 7.70 (m, 1H). LC (method A): tR = 1.36 min. LCMS (ESI) m/z calcd for C10H6ClN2, 189.02; found, 188.98 [M + H]+. 6-Chloroisoquinoline-1-carbonitrile (83). 6-Chloroisoquinoline-1-carbonitrile (83, 174 mg, 65%) was prepared as yellow solid from 1,6-dichloroisoquinoline (70)22−24,30 in a manner similar to that described for compound 77. 1H NMR (300 MHz, CDCl3) δ 8.67 (d, J = 5.7 Hz, 1H), 8.30 (d, J = 9.0 Hz, 1H), 7.94 (d, J = 1.8 Hz, 1H), 7.82 (d, J = 5.7 Hz, 1H), 7.74 (dd, J = 9.0, 1.8 Hz, 1H). LC (method B): tR = 1.31 min. LCMS (ESI) m/z calcd for C10H6ClN2, 189.02; found, 188.98 [M + H]+. 7-Chloroisoquinoline-1-carbonitrile (84). 7-Chloroisoquinoline-1-carbonitrile (84, 238 mg, 25%) was prepared as light yellow solid from 1,7-dichloroisoquinoline (71)22−24,30 in a manner similar to that described for compound 88. 1H NMR (300 MHz, CDCl3) δ 8.76 (d, J = 5.7 Hz, 1H), 8.35−8.34 (m, 1H), 7.92−7.88 (m, 2H), 7.78− 7.75 (m, 1H). LC (method B): tR = 1.35 min. LCMS (ESI) m/z calcd for C10H6ClN2, 189.02; found, 189.01 [M + H]+. 3-Fluoroisoquinoline-1-carbonitrile (85). 3-Fluoroisoquinoline1-carbonitrile (85, 90.1 mg, 60%) was prepared as an off-white solid from 1-bromo-3-fluoroisoquinoline (72)22−24,30 in a manner similar to that described for compound 88. 1H NMR (500 MHz, CDCl3) δ 8.35

combined and treated with neat POCl3 (3 mL). The mixture was heated at 80 °C for 3 h before the POCl3 was removed in vacuo. The residue was taken up in EtOAc, and the solution was washed with saturated NaHCO3 solution and brine prior to drying over anhydrous Na2SO4 and concentration. Purification of the residue by flash chromatography on silica gel (gradient elution from 0% to 25% EtOAc in hexanes on a 25S Thomson single step column using 900 mL of solvent) afforded 74 (70 mg, 12%, two steps) as a white solid. LC (method A): tR = 2.21 min. LCMS (ESI) m/z calcd for C9H5Cl2FN, 215.98 and 217.98; found, 215.92 and 217.92 [M + H]+. 1,3-Dichloro-5-methoxyisoquinoline (75). n-Butyl nitrite (9.7 mL, 83.25 mmol) was added to a cold (0 °C) solution of 4-methoxy2,3-dihydro-1H-inden-1-one (Aldrich, 9.0 g, 55.62 mmol) in anhydrous THF (160 mL) under N2. A saturated solution of HCl in EtOH (3.6 mL) was added in ∼0.5 mL portions to the cold (0 °C) reaction mixture. The mixture was stirred at 0 °C for 2 h before it was concentrated in vacuo to 1/3 volume and diluted with Et2O and hexanes. Suction filtration of the precipitate afforded 2-(hydroxyimino)-4-methoxy-2,3-dihydro-1H-inden-1-one (10.50 g, 99%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 12.66 (s, 1H), 7.51−7.44 (m, 1H), 7.33 (dd, J = 7.6, 2.9 Hz, 2H), 3.98−3.81 (m, 3H), 3.60 (s, 2H). LC (method A): tR = 1.37 min. LCMS (ESI) m/z calcd for C10H10NO3, 192.07; found, 192.08 [M + H]+. POCl3 (325 mL) was added to 2-(hydroxyimino)-4-methoxy-2,3-dihydro-1Hinden-1-one in a 1 L round-bottom flask equipped with a drying tube. The suspension was cooled to 0 °C before it was treated with PCl5 (7.9 mL, 12.6 g, 60.56 mmol) portionwise. After 30 min at 0 °C, HCl was bubbled into the reaction mixture for 20 min before it was warmed to 60 °C for 4.5 h. Afterward, additional PCl5 (7.9 mL) was added before the mixture was heated to 100 °C for 2 h and allowed to stir at room temperature for 14 h. The solvent was removed in vacuo and cold H2O was added cautiously to the reaction mixture with vigorous stirring for 30 min before the precipitate was suction-filtered to yield an off-white solid. The solid was taken up in CH2Cl2 and washed with 1 N NaOH and brine prior to drying over anhydrous Na2SO4 and evaporation of the solvent to afford 75 (12.2 g, 98%) as an off-white solid. 1H NMR (400 MHz, CDCl3) δ 8.04 (d, J = 1.0 Hz, 1H), 7.86−7.81 (m, 1H), 7.56 (dd, J = 8.6, 7.8 Hz, 1H), 7.05 (d, J = 7.8 Hz, 1H), 4.01 (s, 3H). LC (method A): tR = 2.35 min. LCMS (ESI) m/z calcd for C10H8Cl2NO, 228.00; found, 227.95 [M + H]+. 3-Methoxyisoquinoline-1-carbonitrile (76). Compound 76 (0.71 g, 52%) was prepared as a white solid from 3-methoxyisoquinoline N-oxide (61) in a manner similar to that described for compound 77. 1H NMR (500 MHz, CDCl3) δ 8.19 (d, J = 8.5 Hz, 1H), 7.76 (d, J = 8.5 Hz, 1H), 7.64 (t, J = 7.5 Hz, 1H), 7.55−7.52 (m, 1H), 7.29 (s, 1H), 4.06 (s, 3H). LC (method A): tR = 1.96 min. LCMS (ESI) m/z calcd for C11H9N2O, 185.07; found, 185.05 [M + H]+. 5-Methoxyisoquinoline-1-carbonitrile (77). To a stirred solution of 5-methoxyisoquinoline N-oxide (64, 0.70 g, 4.0 mmol) and Et3N (1.1 mL, 8.0 mmol) in dry CH3CN (20 mL) at room temperature under N2 was added trimethylsilyl cyanide (1.60 mL, 12.0 mmol). The mixture was heated at 75 °C for 20 h before it was cooled to room temperature, diluted with EtOAc, and washed with saturated NaHCO3 solution and brine prior to being dried over anhydrous Na2SO4 and evaporated. Purification of the residue by flash chromatography on silica gel (gradient elution from 5% to 25% EtOAc in hexanes) yielded 77 (0.50 g, 47.5%) as a white, crystalline solid along with additional material (0.22 g, 22%) recovered from the filtrate. 1H NMR (CDCl3, 500 MHz) δ 8.63 (d, J = 5.5 Hz, 1H), 8.26 (d, J = 5.5 Hz, 1H), 7.88 (d, J = 8.5 Hz, 1H), 7.69 (t, J = 8.0 Hz, 1H), 7.08 (d, J = 7.5 Hz, 1H), 4.04 (s, 3H). LC (method A): tR = 1.75 min. LCMS (ESI) m/z calcd for C11H9N2O, 185.07; found, 185.10 [M + H]+. 3-Methylisoquinoline-1-carbonitrile (78). To a mixture of 3methylisoquinoline (65, 0.30 g, 2.10 mmol) and KCN (0.41 g, 6.29 mmol, 3 equiv) in H2O (1 mL) and CH2Cl2 (3 mL) was added PhSO2Cl (0.35 μL, 2.72 mmol, 1.3 equiv) dropwise over 0.5 h at room temperature. The mixture was stirred for 16 h before it was extracted with CH2Cl2 (3 × 5 mL), and the combined organic extract was separated and washed with 5% HCl and 5% NaOH prior to drying M



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(d, J = 8.5 Hz, 1H), 7.93 (d, J = 8.5 Hz, 1H), 7.83 (t, J = 7.6 Hz, 1H), 7.77−7.73 (m, 1H), 7.55 (s, 1H). LC (method A): tR = 1.60 min. LCMS (ESI) m/z calcd for C10H6FN2, 173.05, found 172.99 [M + H]+. HRMS (ESI) m/z calcd for C10H6FN2, 173.0515; found, 173.0520 [M + H]+. 5-Fluoroisoquinoline-1-carbonitrile (86). 5-Fluoroisoquinoline1-carbonitrile (86, 98 mg, 28%) was prepared as an off-white solid from 5-fluoro-1-chloroisoquinoline (73)22−24,30 in a manner similar to that described for compound 88. 1H NMR (300 MHz, CDCl3) δ 8.69 (d, J = 5.9 Hz, 1H), 8.14−8.04 (m, 2H), 7.73 (td, J = 8.1, 5.3 Hz, 1H), 7.48 (dd, J = 9.1, 8.4 Hz, 1H). LC (method B): tR = 1.17 min. LCMS (ESI) m/z calcd for C10H6FN2, 173.05; found, 173.02 [M + H]+. 3-Chloro-5-fluoroisoquinoline-1-carbonitrile (87). 3-Chloro5-fluoroisoquinoline-1-carbonitrile (87, 18 mg, 27%) was prepared as a white solid from 5-fluoro-1,3-dichloroisoquinoline (74) in a manner similar to that described for compound 88. 1H NMR (500 MHz, CDCl3) δ 8.18 (d, J = 0.6 Hz, 1H), 8.13 (d, J = 8.5 Hz, 1H), 7.75 (td, J = 8.1, 5.2 Hz, 1H), 7.52 (dd, J = 8.9, 7.9 Hz, 1H). LC (method A): tR = 2.01 min. LCMS (ESI) m/z calcd for C10H5ClFN2, 207.01; found, 206.91 [M + H]+. 3-Chloro-5-methoxyisoquinoline-1-carbonitrile (88). To a thick-walled, screw-top vial containing an argon-degassed suspension of 1,3-dichloro-5-methoxyisoquinoline (75, 1.80 g, 7.89 mmol), KCN (0.51 g, 7.89 mmol), 1,5-bis(diphenylphosphino)pentane (0.35 g, 7.79 mmol), and Pd(II)OAc2 (88.6 mg, 0.39 mmol) in anhydrous toluene (36 mL) was added N,N,N′,N′-tetramethylethylenediamine (0.47 mL, 3.11 mmol). The vial was sealed, heated at 150 °C for 14 h, and then allowed to cool to room temperature. The reaction mixture was preabsorbed onto silica gel directly with THF and flash-chromatographed on silica gel (elution with 10% EtOAc in hexanes) to afford 88 (0.95 g, 55%) as a yellow, fluffy solid. 1H NMR (400 MHz, CDCl3) δ 8.32 (d, J = 1.0 Hz, 1H), 7.85 (dt, J = 8.4, 0.9 Hz, 1H), 7.67 (dd, J = 8.6, 7.8 Hz, 1H), 7.25 (s, 1H), 7.09 (d, J = 7.8 Hz, 1H), 4.04 (s, 3H). LC (method A): tR = 2.16 min. LCMS (ESI) m/z calcd for C11H7ClN2O, 219.03; found, 218.96 [M + H]+. HRMS (ESI) m/z calcd for C11H8ClN2O, 219.0325; found, 219.0323 [M − H]−. 3-Methoxyisoquinoline-1-carboxylic Acid (89). 3-Methoxyisoquinoline-1-carboxylic acid (89, 0.53 g, 17%, four steps) was prepared from commercially available 3-hydroxyisoquinoline (59, Aldrich) in a manner similar to that described for compound 90 following acidic hydrolysis of 3-methoxyisoquinoline-1-carbonitrile (76) using method A (see example 98). 1H NMR (400 MHz, DMSO-d6) δ 13.66 (br s, 1H), 8.42 (d, J = 8.8 Hz, 1H), 7.92 (d, J = 8.3 Hz, 1H), 7.70 (t, J = 7.6 Hz, 1H), 7.51 (t, J = 7.7 Hz, 1H), 7.43 (s, 1H), 3.98 (s, 3H). LC (method A): tR = 1.40 min. LCMS (ESI) m/z calcd for C11H10NO3, 204.07; found, 204.06 [M + H]+. 5-Methoxyisoquinoline-1-carboxylic Acid (90). 5-Methoxyisoquinoline-1-carbonitrile (77, 0.45 g, 2.44 mmol) was treated with 5 N NaOH (10 mL), and the resulting suspension was heated at 85 °C for 4 h, cooled to ambient temperature, diluted with CH2Cl2, and acidified with 1 N HCl. The organic phase was separated, washed with brine, dried over anhydrous Na2SO4, concentrated to 1/4 volume, and suction-filtered to afford 90 (0.44 g, 89%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 13.60 (br s, 1H), 8.56 (d, J = 6.0 Hz, 1H), 8.16 (d, J = 6.0 Hz, 1H), 8.06 (d, J = 8.8 Hz, 1H), 7.71−7.67 (m, 1H), 7.30 (d, J = 8.0 Hz, 1H), 4.02 (s, 3H). LC (method A): tR = 0.70 min. LCMS (ESI) m/z calcd for C11H10NO3, 204.07; found, 204.05 [M + H]+. 3-Methylisoquinoline-1-carboxylic Acid (91). A suspension of 3-methylisoquinoline-1-carbonitrile (78, 129.9 mg, 0.77 mmol) in dioxane (2 mL) was treated with 6 N NaOH (4 mL), and the resulting slurry was heated to reflux for 16 h before it was cooled to room temperature and concentrated in vacuo to remove most of the dioxane. The residue was acidified with 1 N HCl until the pH was ∼5, extracted with CHCl3 (4 × 10 mL), dried over anhydrous MgSO4, and evaporated to yield 91 (64.0 mg, 44%) as a yellow solid. LC (method B): tR = 0.39 min. LCMS (ESI) m/z calcd for C11H10NO2, 188.07; found, 188.11 [M + H]+. 5-Methylisoquinoline-1-carboxylic Acid (92). A suspension of 5-methylisoquinoline-1-carbonitrile (79, 98.3 mg, 0.55 mmol) in

dioxane (2 mL) was treated with 6 N NaOH (3 mL), and the resulting slurry was refluxed for 20 h before it was cooled to room temperature and concentrated in vacuo to remove most of the dioxane. The residue was acidified with 6 N HCl until the pH was ∼3, extracted with EtOAc (2 × 20 mL), dried over anhydrous MgSO4, and evaporated to yield 92 (90.2 mg, 97%) as a yellow solid. 1H NMR (500 MHz, DMSO-d6) δ 13.27−13.02 (br s, 1H), 9.38 (s, 1H), 8.60 (s, 1H), 8.08 (t, J = 4.6 Hz, 1H), 7.77−7.66 (m, 2H), 2.72 (s, 3H). LC (method B): tR = 0.51 min. LCMS (ESI) m/z calcd for C11H10NO2, 188.07; found, 188.13 [M + H]+. 3-Chloroisoquinoline-1-carboxylic Acid (93). 3-Chloroisoquinoline-1-carboxylic acid (93, 0.37 g, 36%, two steps) was prepared as an off-white solid from commercially available 1,3-dichloroisoquinoline (67) in a manner similar to that described for compound 101 following acidic hydrolysis of 3-chloroisoquinoline-1-carbonitrile (80) using method A. 1H NMR (300 MHz, DMSO-d6) δ 14.07 (br s, 1H), 8.51 (d, J = 8.8 Hz, 1H), 8.27 (s, 1H), 8.07 (d, J = 8.1 Hz, 1H), 7.90 (dd, J = 8.1, 7.0 Hz, 1H), 7.85−7.74 (m, 1H). LC (method B): tR = 1.14 min. LCMS (ESI) m/z calcd for C10H7ClNO2, 208.02; found, 208.00 [M + H]+. 4-Chloroisoquinoline-1-carboxylic Acid (94). 4-Chloroisoquinoline-1-carboxylic acid (94, 0.15 g, 24%, two steps) was prepared as an off-white solid from commercially available 1,4-dichloroisoquinoline (68) in a manner similar to that described for compound 101 following acidic hydrolysis of 4-chloroisoquinoline-1-carbonitrile (81) using method A. 1H NMR (500 MHz, DMSO-d6, missing CO2H) δ 8.73 (s, 1H), 8.62 (d, J = 8.5 Hz, 1H), 8.28 (d, J = 8.5 Hz, 1H), 8.04 (ddd, J = 8.4, 7.0, 1.1 Hz, 1H), 7.91 (ddd, J = 8.4, 7.0, 1.1 Hz, 1H). LC (method A): tR = 1.49 min. LCMS (ESI) m/z calcd for C10H7ClNO2, 208.02; found, 208.00 [M + H]+. HRMS (ESI) m/z calcd for C10H7ClNO2, 208.0165; found, 208.0160 [M + H]+. 5-Chloroisoquinoline-1-carboxylic Acid (95). 5-Chloroisoquinoline-1-carboxylic acid (95, 0.43 g, 41%, two steps) was prepared as a yellow solid from 1,5-dichloroisoquinoline (69)22−24,30 in a manner similar to that described for compound 101 following basic hydrolysis of 5-chloroisoquinoline-1-carbonitrile (82) using method B (see example 99). 1H NMR (300 MHz, DMSO-d6, missing CO2H) δ 8.74−8.70 (m, 1H), 8.52−8.46 (m, 1H), 8.25−8.19 (m, 1H), 8.08− 8.02 (m, 1H), 7.80−7.73 (m, 1H). LC (method B): tR = 0.69 min. LCMS (ESI) m/z calcd for C10H7ClNO2, 208.02; found, 208.01 [M + H]+. 6-Chloroisoquinoline-1-carboxylic Acid (96). 6-Chloroisoquinoline-1-carboxylic acid (96, 0.10 g, 24.5%, three steps) was prepared as an off-white solid from 1,6-dichloroisoquinoline (70)22−24,30 in a manner similar to that described for compound 101 following basic hydrolysis of 6-chloroisoquinoline-1-carbonitrile acid (83) using method B. 1H NMR (300 MHz, DMSO-d6, missing CO2H) δ 8.67−8.56 (m, 1H), 8.28−8.21 (m, 1H), 8.08−8.01 (m, 1H), 7.84− 7.76 (m, 1H), 5.75−5.72 (m, 1H). LC (method B): tR = 0.60 min. LCMS (ESI) m/z calcd for C10H7ClNO2, 208.02; found, 208.03 [M + H]+. 7-Chloroisoquinoline-1-carboxylic Acid (97). 7-Chloroisoquinoline-1-carboxylic acid (97, 240 mg, 25%, two steps) was prepared as a white solid from 1,7-dichloroisoquinoline (71)22−24,30 in a manner similar to that described for compound 101 following basic hydrolysis of 7-chloroisoquinoline-1-carbonitrile (84) using method B. 1H NMR (300 MHz, DMSO-d6, missing CO2H) δ 8.72 (d, J = 2.2 Hz, 1H), 8.63 (d, J = 5.5 Hz, 1H), 8.16 (d, J = 8.8 Hz, 1H), 8.13 (d, J = 5.9 Hz, 1H), 7.89 (dd, J = 9.0, 2.0 Hz, 1H). LC (method B): tR = 0.59 min. LCMS (ESI) m/z calcd for C10H7ClNO2, 208.02; found, 208.00 [M + H]+. Isoquinoline-1-carbonitrile Hydrolysis. Method A: 3-Fluoroisoquinoline-1-carboxylic Acid (98). 3-Fluoroisoquinoline-1carbonitrile (85, 83 mg, 0.48 mmol) was treated with 12 N HCl (3 mL), and the resulting slurry was heated at 80 °C for 16 h before it was cooled to room temperature and diluted with H2O (3 mL). The suspension was stirred for 10 min and then filtered to afford 98 (44.1 mg, 48%) as an off-white solid. The filtrate was diluted with CH2Cl2 and washed with brine, dried over anhydrous Na2SO4, and concentrated to afford additional 98 (29.3 mg, 32%). 1H NMR (500 MHz, DMSO-d6) δ 14.0 (br s, 1H), 8.59−8.57 (m, 1H), 8.10 (d, J = N



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residue was treated with 10 mL of H2O and the mixture heated to reflux. The solution was decanted away from a black residue and the volatile component was removed in vacuo to afford crude product as an orange solid. Recrystallization from MeOH at room temperature afforded 105 (92 mg, 19%) as an off-white solid. 1H NMR (500 MHz, DMSO-d6) 8.56 (d, J = 8.5 Hz, 1H), 8.05 (d, J = 8.8 Hz, 1H), 7.74− 7.68 (m, 2H), 7.10 (dd, J = 7.4, 1.3 Hz, 1H), 4.00 (s, 3H). LC (method B): tR = 0.66 min. LCMS (ESI) m/z calcd for C11H10NO3, 204.07; found, 203.95 [M + H]+. 7-Methoxyquinoline-2-carboxylic Acid (106).36 SeO2 (1.05 g, 9.46 mmol) was added to a solution of 104 (0.49 g, 2.85 mmol) in pyridine (10 mL), and the reaction mixture was heated with an oil bath at 115 °C for 5.3 h. H2O (12 mL) and 1 N NaOH (2 mL) were added, and the mixture was brought to reflux and then allowed to stand at ambient temperature. The precipitate was filtered, washed with copious amounts of H2O, and dried in vacuo to afford 106 (0.49 g, 85%) as light, reddish-brown needles. 1H NMR (400 MHz, DMSOd6) 13.28 (br s, 1H), 8.46 (d, J = 8.3 Hz, 1H), 7.98 (two overlapping d, 2H), 7.52 (d, J = 2.2 Hz, 1H), 7.39 (dd, J = 9.1, 2.4 Hz, 1H), 3.95 (s, 3H). LC (method B): tR = 0.58 min. LCMS (ESI) m/z calcd for C11H10NO3, 204.07; found, 203.94 [M + H]+. Biology Studies. The following HCV replicon assays were utilized for compound evaluations, and their preparation and use have been described in detail.13 The assays used to determine compound potency included FRET-based, ELISA-based, and Luciferase-based methods that yielded equivalent value ranges. 1. HCV Replicon Cell Line Preparation. The HCV replicon cell line was isolated from colonies as described by Lohman et al.13e and used for all experiments. The HCV replicon has the nucleic acid sequence set forth in EMBL accession no. AJ242652, the coding sequence of which is from nucleotide 1801 to nucleotide 7758. The coding sequence of the published HCV replicon was synthesized by Operon Technologies, Inc. (Alameda, CA), and the full-length replicon was then assembled in plasmid pGem9zf (+) (Promega, Madison, WI) using standard molecular biology techniques. The replicon consists of (i) the HCV 5′-UTR fused to the first 12 amino acids of the capsid protein, (ii) the neomycin phosphotransferase gene (neo), (iii) the IRES from encephalomyocarditis virus (EMCV), and (iv) HCV NS3 to NS5B genes and the HCV 3′-UTR. Plasmid DNAs were linearized with ScaI, and RNA transcripts were synthesized in vitro using the T7 MegaScript transcription kit (Ambion, Austin, TX) according to the manufacturer’s directions. To generate cell lines, 4 × 106 Huh-7 cells (kindly provided by R. Bartenschlager and available from Health Science Research Resources Bank, Japan Health Sciences Foundation) were electroporated with 10 μg of RNA transcript and plated into 100 mm dishes. After 24 h, selective medium containing 1.0 mg/mL of G418 was added, and medium was changed every 3−5 days. Approximately 4 weeks after electroporation, small colonies were visible that were isolated and expanded for further analysis. These cell lines were maintained at 37 °C, 5% CO2, 100% relative humidity in DMEM (Gibco-BRL, Rockville, MD) with 10% heat inactivated calf serum (Sigma), penicillin/streptomycin (Gibco-BRL, Rockville, MD), and Geneticin (Gibco-BRL, Rockville, MD) at 1 mg/mL. One of the cell lines (deposited as ATCC accession no. PTA-4583 in the American Type Culture Collection) which had approximately 3000 copies of HCV replicon RNA/cell was used for development of the assay (HCV 1b-377-neo replicon cells). 2. FRET Assay Preparation. The HCV FRET screening assay was performed as previously described.13 The FRET peptide (Anaspec, Inc., San Jose, CA) contains a fluorescence donor, EDANS, near one end of the peptide and an acceptor, DABCYL, near the other end.13d The fluorescence of the peptide is quenched by intermolecular resonance energy transfer (RET) between the donor and the acceptor, but as the NS3 protease cleaves the peptide, the products are released from RET quenching and the fluorescence of the donor becomes apparent. The assay reagent was made as follows: 5× luciferase cell culture lysis buffer (Promega, Madison, WI) diluted to 1× with H2O, NaCl added to 150 mM final concentration, the FRET peptide diluted to 20 μM final concentration from a 2 mM stock. Cells were trypsinized, plated in a 96-well plate (10K per well), and allowed to

8.5 Hz, 1H), 7.88−7.85 (m, 2H), 7.74−7.71 (m, 1H). LC (method A): tR = 1.33 min. LCMS (ESI) m/z calcd for C10H7FNO2, 192.05; found, 191.97 [M + H]+. Isoquinoline-1-carbonitrile Hydrolysis. Method B: 5-Fluoroisoquinoline-1-carboxylic Acid (99). 5-Fluoroisoquinoline-1carbonitrile (86, 98 mg, 0.57 mmol) was treated with 5 N NaOH (3 mL), and the resulting slurry was heated at 80 °C for 16 h before it was cooled to room temperature and acidified with 1 N HCl (15 mL). The mixture was diluted with CH2Cl2, washed with brine, dried over anhydrous Na2SO4, and concentrated to afford 99 (108.8 mg, 100%) as an off-white solid. 1H NMR (300 MHz, DMSO-d6) δ 13.85 (br s, 1H), 8.68 (d, J = 5.9 Hz, 1H), 8.39 (d, J = 8.1 Hz, 1H), 8.14 (dd, J = 5.9, 0.7 Hz, 1H), 7.83−7.68 (m, 2H). LC (method B): tR = 0.41 min. LCMS (ESI) m/z calcd for C10H7FNO2, 192.05; found, 192.03 [M + H]+. 3-Chloro-5-fluoroisoquinoline-1-carboxylic Acid (100). 3Chloro-5-fluoroisoquinoline-1-carboxylic acid (100, 21 mg, 100%) was prepared as a white solid following acidic hydrolysis of 3-chloro-5fluoroisoquinoline-1-carbonitrile (87) using method A. 1H NMR (500 MHz, CDCl3) δ 11.54−10.90 (br m, 1H), 9.34 (d, J = 8.9 Hz, 1H), 8.27 (s, 1H), 7.71 (ddd, J = 8.8, 7.9, 5.6 Hz, 1H), 7.54−7.46 (m, 1H). LC (method A): tR = 1.70 min. LCMS (ESI) m/z calcd for C10H6ClFNO2, 226.01; found, 226.03 [M + H]+. HRMS (ESI) m/z calcd for C10H4ClFNO2, 223.9915; found, 223.9905 [M − H]−. 3-Chloro-5-methoxyisoquinoline-1-carboxylic Acid (101). 3Chloro-5-methoxyisoquinoline-1-carbonitrile (88, 0.95 g, 4.43 mmol) was treated with 12 N HCl (40 mL), and the resulting slurry was heated at 90 °C for 48 h in a thick-walled, sealed, screw-top tube before it was cooled to room temperature and poured into cold H2O (40 mL). The suspension was stirred for 10 min and suction-filtered to afford 101 (0.975 g, 87%) as a yellow solid following vacuum oven drying at 45 °C for 16 h. 1H NMR (400 MHz, DMSO-d6, CO2H missing) δ 8.17 (d, J = 0.7 Hz, 1H), 8.01 (d, J = 8.6 Hz, 1H), 7.70 (dd, J = 8.6, 7.8 Hz, 1H), 7.35 (d, J = 7.6 Hz, 1H), 4.02 (s, 3H). LC (method A): tR = 1.78 min. LCMS (ESI) m/z calcd for C11H9ClNO3, 238.03; found, 238.09 [M + H]+. HRMS (ESI) m/z calcd for C11H7ClNO3, 236.0114; found, 236.0122 [M − H]−. 5-Methoxy-2-methylquinoline (103) and 7-Methoxy-2methylquinoline (104).36 Concentrated HCl (5.0 mL) was carefully added to m-anisidine (102, 2.46 g, 20 mmol), followed by the addition of 2,3-dichloro-1,4-naphthoquinone (4.53 g, 20 mmol) and n-butanol (10 mL). The resultant heterogeneous mixture was lowered into a preheated 130 °C oil bath, and a solution of crotonaldehyde (2.0 mL, 24 mmol) in n-butanol (2 mL) was added dropwise over 30 min using an addition funnel. The reaction mixture was heated for an additional 2 h, allowed to cool to ambient temperature, poured over ice, and neutralized by the addition of a sufficient amount of solid NaOH. The mixture was extracted with EtOAc and the organic phase was washed with brine, dried over anhydrous MgSO4, filtered, and evaporated in vacuo to afford a brown residue that was preabsorbed onto silica gel with CH2Cl2 and subjected to flash column chromatography on silica gel (gradient elution with 5−40% EtOAc in hexane) to afford 103 (0.42 g, 12%) as a brown oil and impure 104, which was repurified by flash column chromatography on silica gel (gradient elution with 0− 25% EtOAc in hexane) to afford a cleaner sample of 104 (1.52 g, 44%) as a brown oil. For compound 103: 1H NMR (400 MHz, DMSO-d6) δ 8.39 (d, J = 8.6 Hz, 1H), 7.61 t, J = 8.2 Hz, 1H), 7.48 (d, J = 8.3 Hz, 1H), 7.38 (d, J = 8.6 Hz, 1H), 6.98 (d, J = 7.5 Hz, 1H), 3.97 (s, 3H), 2.63 (s, 3H). LC (method B): tR = 1.40 min. LCMS (ESI) m/z calcd for C11H11NO, 174.09; found, did not ionize [M + H]+. For compound 104: 1H NMR (400 MHz, DMSO-d6) δ 8.14 (d, J = 8.3 Hz, 1H), 7.81 (d, J = 9.0 Hz, 1H), 7.31 (d, J = 2.4 Hz, 1H), 7.25 (d, J = 8.3 Hz, 1H), 7.17 (dd, J = 8.9, 2.6 Hz, 1H), 3.90 (s, 3H), 2.61 (s, 3H). LC (method B): tR = 0.58 min. LCMS (ESI) m/z calcd for C11H11NO, 174.09; found, 173.96 [M + H]+. 5-Methoxyquinoline-2-carboxylic Acid (105).36 SeO2 (1.04 g, 9.37 mmol) was added to a solution of 103 (0.42 g, 2.42 mmol) in pyridine (10 mL), and the reaction mixture was heated with an oil bath at 115 °C until the starting material was consumed. H2O (2 mL) was added, and the volatile components were removed in vacuo. The O



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attach overnight in 200 μL of medium. The next day, the test compounds were added to columns 1−10. Column 11 was medium plus DMSO only (0.5% v/v final concentration), and column 12 contained a titration of interferon or another inhibitor as a control. At various times later (typically 72 h), 10% final volume Alamar blue (Trek Diagnostics, Cleveland, OH) was added per well. The plates were returned to the incubator for 2−5 h and then read in the Cytofluor 4000 instrument (PE Biosystems) to determine Alamar blue conversion in each well as a measure of cellular toxicity. After reading the Alamar blue fluorescence, plates were rinsed 2× with PBS and then used for FRET assay by the addition of 30 μL per well of the FRET peptide assay reagent (described above). The plate was then placed into the Cytofluor instrument which had been set to 340 nm excitation and 490 nm emission, automatic mode for 20 cycles, and the plate was read in a kinetic mode. Typically, the signal-to-noise ratio using an end point analysis after the reads was at least 3-fold. Compound analysis depended upon the quantification of the relative HCV replicon inhibition and the relative cytotoxicity values. To calculate cytotoxicity values, the average Alamar blue fluorescence signal from the control wells in row 11 was set as 100% nontoxic. The individual signals in each of the compound test wells were then divided by the average control signal and multiplied by 100% to determine percent cytotoxicity. To calculate the HCV replicon inhibition values, an average background value FRET signal was obtained from the two wells containing the highest amount of inhibitor at the end of the assay period. These numbers were similar to those obtained from naı̈ve Huh-7 cells (results not shown). The background numbers were then subtracted from the average FRET signal obtained from the control wells in row 11, and this number was used as 100% activity. The individual signals in each of the compound test wells were then divided by the averaged control value after background subtraction and multiplied by 100% to determine percent activity. EC50 values for an interferon titration or a compound titration were calculated as the concentration that caused a 50% reduction in FRET activity. The two numbers generated from the compound plates, percent cytotoxicity and percent activity, were then tabulated for comparisons. 3. ELISA Assay Preparation. The ELISA assay consisted of the following steps. HCV genotype 1a (H77c) replicon cells (1.5 × 104) in 80 μL of DMEM (Invitrogen, catalog no. 11965-084) plus 10% FBS (Sigma, catalog no. F4135) were seeded in a 96-well assay plate (Costar 3904, VWR, catalog no. 29443-152) and allowed to attach overnight at 37 °C in a tissue culture incubator (5% CO2, 100% humidity). Compounds of interest were serially diluted in DMEM without FBS. The diluted compounds (120 μL of the solution) were then transferred to the assay-plated cells and maintained at 37 °C for 5 days in a tissue culture incubator. The medium was then discarded. The plates were washed 3× with 200 μL of D-PBS (Invitrogen, catalog no. 70011-644), and the plates were dried overnight at 37 °C (0% humidity incubator) to fix the cells to the plate. The dried assay plate was either used immediately or stored at −80 °C. For ELISA, the fixed replicon cells were soaked in 100 μL of D-PBST (0.02% v/v Tween 20 in D-PBS) and shaken for 5 min at room temperature. The solution was removed, and the cells were incubated in 100 μL of a 0.2% Triton X-100 solution in D-PBS for 60 min. The cells were then rinsed with 100 μL of D-PBST, drained, and washed 2× with 200 μL of D-PBST (with shaking for 5 min). The wells were blocked by addition of 150 μL of blocker casein in PBS (Pierce, catalog no. 37528) with shaking for 30 min before addition of antisera. Two antibodies were used: the first was antiserum 203W-αNS5-GK2.4, and the second was prepared in-house (in rabbit against G-1b (Con 1) NS5A recombinant purified from E. coli). Another antiserum was a monoclonal derived from a peptide covering the NS5A-NS5B cleavage site of G-1a purchased from US Biological (catalog no. H1920-27B). Antiserum was used at a 1:25000 dilution (for 203W-αNS5-GK2.4) or 1:10000 (for H192027B) in blocker casein buffer. Antiserum in blocking buffer was added to wells (45 μL) and allowed to incubate at room temperature for 60 min with gentle shaking. Following incubation the antiserum was removed and the plates were rinsed once with 200 μL of D-PBST, followed by 2× washes using 200 μL of H-TBST (1 M NaCl, 10 mM Tris-HCl, pH 7.4; 0.5% v/v Tween 20) shaking for 4 min, and then

rinsed 1× with 200 μL of D-PBST. The secondary antibody was added to the wells (45 μL of diluted HRP-conjugated goat anti-rabbit IgG (Bio-Rad catalog no. 170-6515) for 203W-αNS5-GK2.4 or HRPconjugated goat anti-mouse IgG (Bio-Rad, catalog no. 170-6516) for H1920-27B. The secondary antibodies were diluted in blocker casein (1:20000 dilution) and incubated for 45 min at room temperature, and the assay plate was then rinsed 1× with 200 μL of D-PBST and 2× with 200 μL of H-TBST with 4 min of shaking. To reduce the background more, the assay plate was rinsed again with 200 μL of DPBST and washed once using 200 μL of D-PBST with 5 min of shaking. To generate signal, equal amounts of SuperSignal ELISA Femto Lumino/enhancer solution and SuperSignal ELISA Femto stable peroxide solution (Pierce, catalog no. 37075) were mixed and 50 μL of mixed solution was added to each well. The relative light units were determined on a TopCount NXT (Packard) luminescence reader, and the data were converted into an Excel file. The percentage of inhibition by the test compounds was calculated by comparison to wells without any compound treatment (100% signal). The background was calibrated by adding 500 nM NS3 inhibitor (known to result in 100% inhibition, data not shown). When these conditions were used, the ELISA assay screen window coefficient (Z′) ranged from 0.5 to 0.75, indicating acceptable variation. 4. Luciferase Assay Preparation. To evaluate compound efficacy, titrated compounds were transferred to sterile 384-well tissue culture treated plates, and the plates were seeded with HCV replicon cells (50 μL at a density of 2.4 × 103 cells/well) in DMEM containing 4% FCS (final DMSO concentration at 0.5% v/v). After 3 days of incubation at 37 °C, cells were analyzed for Renilla luciferase activity using the EnduRen substrate (Promega catalog no. E6485) according to the manufacturer’s directions. Briefly, the EnduRen substrate was diluted in DMEM and then added to the plates to a final concentration of 7.5 mM. The plates were incubated for at least 1 h at 37 °C and then read on a Viewlux Imager (Perkin-Elmer) using a luminescence program. The 50% effective concentration (EC50) was calculated using the four-parameter logistic formula noted above. To assess the cytotoxicity of compounds, Cell Titer-Blue (Promega) was added to the EnduRen-containing plates and incubated for at least 4 h at 37 °C. The fluorescence signal from each well was read using a Viewlux imager. All CC50 values were calculated using the four-parameter logistic formula. 5. BVDV Assay Preparation. BVDV analysis was typically performed following HCV FRET analysis according to a previously described method and is described briefly here.13 Following FRET assay for HCV activity, 40 μL of luciferase substrate (Promega kit for firefly luciferase E4550) was added to each well and the 96-well plate was placed in a Packard Top Count instrument programmed for luciferase measurements. The percent BVDV inhibition was quantified relative to a specific BVDV test compound in the wells, while wells containing HCV inhibitor only were used as 100% BVDV luciferase activity.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 1-203-677-6972. Fax: 1-203-677-7702. E-mail: denis. [email protected]. Present Addresses ∥

For M.H.S-W.: Broad Institute, Therapeutics Development and Translational Sciences, 320 Charles Street, Room 2169, Cambridge, MA 02141. ⊥ For X.Y.: Department of Clinical Information Science, AstraZeneca, Wilmington, DE 19803. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Discovery Analytical Services (DAS) staff for obtaining and analyzing HRMS and 1H NMR spectra. We also P



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(b) Ribeiro, R. M.; Li, H.; Wang, S.; Stoddard, M. B.; Learn, G. H.; Korber, B. T.; Bhattacharya, T.; Guedj, J.; Parrish, E. H.; Hahn, B. H.; Shaw, G. M.; Perelson, A. S. Quantifying the diversification of hepatitis C virus (HCV) during primary infection: estimates of the in vivo mutation rate. Pathogens 2012, 8, 1−13. (9) (a) Reviriego, C. Daclatasvir dihydrochloride. Drugs Future 2011, 36, 735−739. (b) Lok, A. S.; Gardiner, D. F.; Lawitz, E.; Martorell, C.; Everson, G. T.; Ghalib, R.; Reindollar, R.; Rustgi, V.; McPhee, F.; Wind-Rotolo, M.; Persson, A.; Zhu, K.; Dimitrova, D. I.; Eley, T.; Guo, T.; Grasela, D. M.; Pasquinelli, C. Preliminary study of two antiviral agents for hepatitis C genotype 1. N. Engl. J. Med. 2012, 366, 216−224. (c) Chayama, K.; Takahashi, S.; Toyota, J.; Karino, Y.; Ikeda, K.; Ishikawa, H.; Watanabe, H.; McPhee, F.; Hughes, E.; Kumada, H. Dual therapy with the nonstructural protein 5A inhibitor, daclatasvir, and the nonstructural protein 3 protease inhibitor, asunaprevir, in hepatitis C virus genotype 1b-infected null responders. Hepatology 2012, 55, 742−748. (d) Poordad, F.; Lawitz, E.; Kowdley, K. V.; Cohen, D. E.; Podsadecki, T.; Siggelkow, S.; Heckaman, M.; Larsen, L.; Menon, R.; Koev, G.; Tripathi, R.; Pilot-Matias, T.; Bernstein, B. Exploratory study of oral combination antiviral therapy for hepatitis C. N. Engl. J. Med. 2013, 368, 45−53. (e) Gane, E. J.; Stedman, C. A.; Hyland, R. H.; Ding, X.; Svarovskaia, E.; Symonds, W. T.; Hindes, R. G.; Berrey, M. M. Nucleotide polymerase inhibitor sofosbuvir plus ribavirin for hepatitis C. N. Engl. J. Med. 2013, 368, 34−44. (f) Poordad, F.; Lawitz, E.; Kowdley, K. V.; Cohen, D. E.; Podsadecki, T.; Siggelkow, S.; Heckaman, M.; Larsen, L.; Menon, R.; Koev, G.; Tripathi, R.; PilotMatias, T.; Bernstein, B. Exploratory study of oral combination antiviral therapy for hepatitis C. N. Engl. J. Med. 2013, 368, 45−53. (10) Gao, M.; Nettles, R. E.; Belema, M.; Snyder, L. B.; Nguyen, V. N.; Fridell, R. A.; Serrano-Wu, M. H.; Langley, D. R.; Sun, J.-H.; O’Boyle, D. R., II; Lemm, J. A.; Wang, C.; Knipe, J. O.; Chien, C.; Colonno, R. J.; Grasela, D. M.; Meanwell, N. A.; Hamann, L. G. Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. Nature 2010, 465, 96−100. (11) Romine, J. L.; St. Laurent, D. R.; Leet, J. E.; Martin, S. W.; Serrano-Wu, M. H.; Yang, F.; Gao, M.; O’Boyle, D. R., II; Lemm, J. A.; Sun, J.-H.; Nower, P. T.; Huang, X.; Deshpande, M. S.; Meanwell, N. A.; Snyder, L. B. Inhibitors of HCV NS5A: from iminothiazolidinones to symmetrical stilbenes. ACS Med. Chem. Lett. 2011, 2, 224−229. (12) St. Laurent, D. R.; Belema, M.; Gao, M.; Goodrich, J.; Kakarla, R.; Knipe, J. O.; Lemm, J. A.; Liu, M.; Lopez, O. D.; Nguyen, V. N.; Nower, P. T.; O’Boyle, D. R., II; Qiu, Y.; Romine, J. L.; Serrano-Wu, M. H.; Sun, J.-H.; Valera, L.; Yang, F.; Yang, X.; Meanwell, N. A.; Snyder, L. B. HCV NS5A replication complex inhibitors. Part 2: Investigation of stilbene prolinamides. Bioorg. Med. Chem. Lett. 2012, 22, 6063−66. (13) (a) O’Boyle, D. R., II; Nower, P. T.; Lemm, J. A.; Valera, L.; Sun, J.-H.; Rigat, K.; Colonno, R.; Gao, M. Development of a cellbased high-throughput specificity screen using a hepatitis C virusbovine viral diarrhea virus dual replicon assay. Antimicrob. Agents Chemother. 2005, 49, 1346−1353. (b) Sun, J. H.; Lemm, J. A.; O’Boyle, D. R., II; Racela, J.; Colonno, R.; Gao, M. Specific inhibition of bovine viral diarrhea virus replicase. J. Virol. 2003, 77, 6753−6760. (c) Scheel, T. K. H.; Gottwein, J. M.; Mikkelsen, L. S.; Jensen, T. B.; Bukh, J. Recombinant HCV variants with NS5A from genotypes 1−7 have different sensitivities to an NS5A inhibitor but not interferon-α. Gastroenterolgy 2011, 140, 1032−1042. (d) Taliani, M.; Bianchi, E.; Narjes, F.; Fossatelli, M.; Urbani, A.; Steinkühler, C.; De Francesco, R.; Pessi, A. A continuous assay of hepatitis C virus protease based on resonance energy transfer depsipeptide substrates. Anal. Biochem. 1996, 240, 60−67. (e) Lohman, V.; Korner, F.; Koch, J.-O..; Herian, U.; Theilmann, L.; Bartenschlager, R. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 1999, 285, 110−113. (14) (a) Potential conformational differences between caps were investigated using a mixed torsional/low mode sampling protocol within Macromodel [Schrodinger, Maestro, version 9.2, OPLS2005 FF, water solvent]. An overlay [rmsd minimized proline ring] of full

thank the reviewers of this manuscript for several insightful comments and suggestions.

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REFERENCES

(1) Paper 3 in this series: Lopez, O. D.; Nguyen, V. N.; St. Laurent, D. R.; Belema, M.; Serrano-Wu, M. H.; Goodrich, J. T.; Yang, F.; Qiu, Y.; Ripka, A. S.; Nower, P. T.; Valera, L.; Liu, M.; O’Boyle, D. R., II; Sun, J.-H.; Fridell, R. A.; Lemm, J. A.; Gao, M.; Good, A. C.; Meanwell, N. A.; Snyder, L. B. HCV NS5A replication complex inhibitors. Part 3: Discovery of potent analogs with distinct core topologies. Bioorg. Med. Chem. Lett. 2013, 23, 779−784. (2) (a) Lavanchy, D. Evolving epidemiology of hepatitis C virus. Clin. Microbiol. Inf. 2011, 17, 107−115. (b) Pawlotsky, J.-M. The science of direct-acting antiviral and host-targeted agent therapy. A review. Antiviral Ther. 2012, 17, 1109−1117. (c) Thompson, A. J.; Locarnini, S. Direct-acting antiviral agents for the treatment of HCV. An editorial. Antiviral Ther. 2012, 17, 1105−1107. (3) Ghany, M. G.; Nelson, D. R.; Strader, D. B.; Thomas, D. L.; Seeff, L. B. An update on treatment of genotype 1 chronic hepatitis C virus infection: 2011 practice guideline by the American Association for the Study of Liver Diseases. Hepatology 2011, 54, 1433−1444. (4) (a) Buehler, S.; Bartenschlager, R. New targets for the antiviral therapy of chronic hepatitis C. Liver Int. 2012, 32 (Suppl. 1), 9−16. (b) Sheridan, C. New Merck and Vertex drugs raise standard of care in hepatitis C. Nat. Biotechnol. 2011, 29, 553−554. (c) Cheng, K.-C.; Gupta, S.; Wang, H.; Uss, A. S.; Njoroge, G. F.; Hughes, E. Current drug discovery strategies for treatment of hepatitis C virus infection. J. Pharm. Pharmacol. 2011, 63, 883−892. (d) Yoshida, T.; Kondoh, M.; Yagi, K. Promising targets for anti-hepatitis C virus agents. Curr. Med. Chem. 2011, 18, 1239−1244. (e) Vermehren, J.; Sarrazin, C. New HCV therapies on the horizon. Clin. Microbiol. Infect. 2011, 17, 122− 134. (5) (a) Sulkowski, M. S.; Cooper, C.; Hunyady, B.; Jia, J.; Ogurtsov, P.; Peck-Radosavljevic, M.; Shiffman, M. L.; Yurdaydin, C.; Dalgard, O. Management of adverse effects of Peg-IFN and ribavirin therapy for hepatitis C. Nat. Rev. Gastroenterol. Hepatol. 2011, 8, 212−223. (b) Brillanti, S.; Mazzella, G.; Roda, E. Ribavirin for chronic hepatitis C: and the mystery goes on. Dig. Liver Dis. 2011, 43, 425−430. (c) Kemp, W.; Roberts, S. Pegylated interferon and ribavirin for the treatment of chronic hepatitis C. Clin. Med. Insights: Ther. 2011, 3, 137−150. (d) Vezali, E.; Aghemo, A.; Colombo, M. A review of the treatment of chronic hepatitis C virus infection in cirrhosis. Clin. Ther. 2010, 32, 2117−2138. (6) (a) Bartenschlager, R.; Penin, F.; Lohman, V.; Andre, P. Assembly of infectious hepatitis C virus particles. Trends Microbiol. 2011, 19, 95−103. (b) Buehler, S.; Bartenschlager, R. Molecular mechanisms of hepatitis C virus (HCV) replicationimplications for the development of antiviral drugs. Z. Gastroenterol. 2011, 49, 836− 844. (c) Popescu, C.-I.; Rouille, Y.; Dubuisson, J. HCV replication and assembly: a play in one act. Future Virol. 2011, 6, 985−995. (d) Jones, D. M.; McLauchlan, J. Hepatitis C virus: assembly and release of virus particles. J. Biol. Chem. 2010, 285, 22733−22739. (e) Lo-Ten-Foe, J.; Richter, M.; Niesters, H. G. M. Hepatitis C Virus. In Molecular Detection of Human Viral Pathogens; Liu, D., Ed.; CRC Press: Boca Raton, FL, 2011; pp 189−200. (7) (a) Targett-Adams, P.; Belda, O. Small molecule inhibitors of the hepatitis C virus-encoded NS5A protein. Virus Res. 2012, 170, 1−14. (b) Cordek, D. G.; Bechtel, J. T.; Maynard, A. T.; Kazmierski, W. M.; Cameron, C. E. Targeting the NS5A protein of HCV: an emerging option. Drugs Future 2011, 691−711. (c) Tan, S.-L.; Katze, M. G. How hepatitis C virus counteracts the interferon response: the jury is still out on NS5A. Virology 2001, 284, 1−12. (d) Lee, C. Discovery of hepatitis C virus NS5A inhibitors as a new class of anti-HCV therapy. Arch. Pharmacol. Res. 2011, 34, 1403−1407. (e) Najarro, P.; Mathews, N.; Cockerill, S. Hepatitis C; Tan, S.-L., He, Y., Eds; Caister Academic Press; Norfolk, U.K., 2011; Chapter 14, pp 271−292. (8) (a) Rong, L.; Perelson, A. S. Treatment of hepatitis C virus infection with interferon and small molecule direct antivirals: viral kinetics and modeling. Crit. Rev. Immunol. 2010, 30, 131−148. Q



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length compound 4 with a truncated version showed maintenance of symmetry and convergence on the conformational minimum. (15) Brameld, K. A.; Kuhn, B.; Reuter, D. C.; Stahl, M. Small molecule conformational preferences derived from crystal structure data. A medicinal chemistry focused analysis. J. Chem. Inf. Model. 2008, 48, 1−24. (16) All of the isomers were prepared except for position 6 (i.e., isoquinoline-5-carboxyl), and only a representative set of isomers is shown in Table 1. All of these isomers resulted in reduced G-1a inhibitory activity. (17) (a) Wilcken, R.; Zimmermann, M. O.; Lange, A.; Joerger, A. C.; Boeckler, F. M. Principles and applications of halogen bonding in medicinal chemistry and chemical biology. J. Med. Chem. 2013, 56, 1363−1388. (b) The difference in potency between 19 and 25 is surprising, especially in light of the potency of methoxy analogue 27. The G-1b inhibitory data for this triad (19, 25, 27) appear to be consistent with what might be expected, and although G-1a inhibition is more sensitive to SAR variation, this result appears as an outlier. Interestingly, this trend is reversed for the same triad at the 3-position in compounds 17, 24, and 26, where this time it is the methoxy analogue that is inactive and the methyl analogue that is active; however, G-1b data again appear to be consistent with SAR trends. It is noteworthy that since compound 25 exhibited an EC50 of

HCV NS5A Replication Complex Inhibitors. Part 4.1 Optimization for

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