HCV NS5A replication complex inhibitors. Part 4.1 optimization for genotype 1a replicon inhibitory activity

Article

pubs.acs.org/jmc

HCV NS5A Replication Complex Inhibitors. Part 4.¹Optimization 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 (G-

1b) 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.

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

INTRODUCTION

■

Often deemed the quiet epidemic, hepatitis C virus (HCV)

infection aﬀects an estimated 170 million individuals worldwide

including more than 4 million Americans.²Approximately 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.²At

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.^3,4However,

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.⁶Its 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

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

A

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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.¹³BVDV (Luc) CC₅₀> 10 μM for all and BVDV (Luc) EC₅₀> 5 μM for all compounds except 3 (BVDV EC₅₀

b

= 4.7 μM), 7 (BVDV EC₅₀= 3.1 μM), and 14 (BVDV EC₅₀= 0.042 μM).¹³Luc assay value. FRET assay value.

c

d

agents and ribavirin have demonstrated the capacity to cure

chronic HCV infection.^9d−fThe 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).^10,11Herein, 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).

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

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).¹²Although 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.¹³Attention 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.¹⁵Normally 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.¹³In all cases,

b

BVDV (Luc) CC₅₀and BVDV (Luc) EC₅₀are > 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-5-

methoxy-1-isoquinolinoyl (CMIQ) derivative 30,^17cinitiated

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.^10,13cHowever, 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 G-

5a hybrid (EC₅₀< 0.23 nM) replicons.

Table 3. G-1a/1b Replicon Inhibitory Activity for a Series of

Symmetrical N-PhAc- and N-CMIQ-Linked Prolinamide

Analogues

Overlaying the isoquinolinamide fragments of 30 and the 2-

ethylbenzoyl moiety of 3 exhibited single clusters within a

conformational minimum of 0.15 kcal/mol (Figure 4).¹⁸By

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

a

Data represent mean values of at least two experiments with a

maximum of 3-fold variation.¹³In all cases, BVDV (Luc) CC₅₀and

b

BVDV (Luc) EC₅₀are >10 μM except for 37 where BVDV (Luc)

c

CC₅₀= 3.3 μM.¹³EC₅₀(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).¹G-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.

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′-(ethene-

1,2-diyl)dianiline (39) and N-Boc-^L-proline, as shown in

Scheme 1.¹⁹Thus, coupling of 39 and N-Boc-L-proline using

EEDQ²⁰followed by Boc deprotection using either triﬂuoro-

acetic acid (TFA) in CH₂Cl₂or 4 N HCl in dioxane aﬀorded

In order to further validate the G-1a replicon inhibitory

potency achieved with the 3-chloro-5-methoxyisoquinolinoyl

D

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a

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.²⁸Suzuki coupling of triﬂate 52 with phenyl-

boronic acid followed by saponiﬁcation yielded the phenyl

derivative 58.²⁹

Scheme 1. Synthesis of Target Molecules

The 1-isoquinolinecarboxylic acids 89−101 were accessed

through a variety of methods, as illustrated in Scheme 3.^22−24,30

a

Scheme 3. Synthesis of Isoquinoline-1-carboxylic Acids

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.²¹The 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 48.²²⁻²⁴Hence, 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-proline²⁵using

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.

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)

NaBH_4,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 1-

chloro- 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 isoquino-

lines 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.³³In 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 65.³⁴Subsequent sodium

borohydride mediated reduction provided 3-methylisoquinoli-

necarboxylic 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-methylisoquinoline-

carboxylic acid 92 following basic hydrolysis.³⁵A slightly

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

Scheme 2. Synthesis of Picolinic Acids

a

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.

E

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modiﬁed version of a procedure disclosed by Song et al.³⁶was

used to prepare quinolinecarboxylic acids 105 and 106 from m-

anisidine (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

a

b

c

d

e

f

LCMS condition

Col

GT

FR

Sol A

Sol B

method A

method B

method C

method D

method E

method F

method G

method H

method I

method J

method K

method L

3

1

4

5

2

3

5

6

7

8

9

3

2

4

3

2

3

2

3

4

5

A1

A2

A1

A4

A3

A4

A1

B1

B2

B1

B4

B3

B4

B1

a

Scheme 4. Synthesis of Quinoline-2-carboxylic Acids

0.8

4

5

1

4

5

4

a

Reagents and conditions: (a) crotonaldehyde, 2,3-dichloro-1,4-

4

naphthoquinone, conc HCl, n-BuOH, 130 °C, 2 h; (b) SeO₂,

pyridine, 115 °C, 5 h.

4

a

Gradient = 0% B to 100% B; wavelengh (λ) = 220 nm; injection

b

volume = 5 μL unless stated otherwise. Col = 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

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.

c

= XTERRA C18 (4.6 mm × 50 mm, S7). GT = Gradient time (min).

d

e

FR = Flow rate (mL/min). Sol 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;

f

solvent A4 = 10 mM NH₄OAc/5% CH₃CN/90% H₂O. Sol 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.

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 EC₅₀values

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 EC₅₀values 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 Avance^III500 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

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

Journal of Medicinal Chemistry

Article

conservative and would be aﬀected by only 25%, which would be

within the error margin of the assay.

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-(2-phenylacetyl)pyrrolidine-2-carboxamide) (1). 2-Ethoxy-

1-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): t_R= 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). Com-

pound 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): t_R= 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): t_R

= 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). Com-

pound 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): t_R= 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 1-

naphthoic acid in a manner similar to that described for compound 11.

¹H 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): t_R= 2.68

(2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))-

bis(1-(3-ethylpicolinoyl)pyrrolidine-2-carboxamide) (7). Com-

pound 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): t_R= 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). Com-

pound 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): t_R= 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). Com-

pound 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): t_R

=

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

1

that described for compound 11. H 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): t_R= 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 hexa-

ﬂuorophosphate (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

1

11 (52.0 mg, 64%) as an orange solid. H NMR (500 MHz, DMSO-

d₆) 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): t_R= 2.38 min. LCMS (ESI) m/z calcd for

G

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

Journal of Medicinal Chemistry

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): t_R= 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): t_R= 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]⁺.

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): t_R= 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-carboxa-

mide) (18). Compound 18 (40.6 mg, 38%) was prepared as a tan

solid from 41 and 4-chloroisoquinoline-1-carboxylic acid (94) in a

1

manner similar to that described for compound 11. H 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): t_R= 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-carboxa-

mide) (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-carboxa-

mide) (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

(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): t_R= 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

1

a manner similar to that described for compound 11. H 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-carboxa-

mide) (21). Compound 21 (80.4 mg, 76%) was prepared as an orange

solid from 41 and 7-chloroisoquinoline-1-carboxylic acid (97) in a

1

aﬀord 15 (17.1 mg, 34%) as a yellow solid. H 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): t_R= 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): t_R= 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]⁺.

1

manner similar to that described for compound 11. H 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): t_R= 1.81 min. LCMS (ESI) m/z

calcd for C₄₄H₃₇Cl₂N₆O₄783.23; 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-carboxa-

mide) (22). Compound 22 (51.4 mg, 50%) was prepared as an oﬀ-

white solid from 41 and 3-ﬂuoroisoquinoline-1-carboxylic acid (98) in

1

a manner similar to that described for compound 11. H 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): t_R= 2.43 min. LCMS

(ESI) m/z calcd for C₄₄H₃₇N₆O₄S₂, 751.28; found, 751.20 [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-carboxa-

mide) (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. H NMR (500

MHz, DMSO-d₆) δ 10.39/9.70 (s, 2H), 8.31−8.28/8.18−8.15 (m,

1

H

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HRMS (ESI) m/z calcd for C₄₄H₃₇N₆O₄S₂, 751.2845; found, 751.2849

[M + H]⁺.

from 41 and 3-chloro-1-naphthoic acid^30b,cin 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): t_R= 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-2-

carboxamide) (29). Compound 29 (18 mg, 43%) was prepared as a

tan solid from 41 and 3-chloro-5-ﬂuoroisoquinoline-1-carboxylic acid

(2S,2′S)-N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))-

bis(1-(5-ﬂuoroisoquinoline-1-carbonyl)pyrrolidine-2-carboxa-

mide) (23). Compound 23 (57.7 mg, 56%) was prepared as a yellow

solid from 41 and 5-ﬂuoroisoquinoline-1-carboxylic acid (99) in a

1

manner similar to that described for compound 11. H 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): t_R= 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]⁺.

1

(100) in a manner similar to that described for compound 11. H

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): t_R= 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-methylisoquinoline-1-carbonyl)pyrrolidine-2-carbox-

amide) (24). Compound 24 (42.7 mg, 69%) was prepared as a yellow

solid from 41 and 3-methylisoquinoline-1-carboxylic acid (91) in a

1

manner similar to that described for compound 11. H 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): t_R= 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-carbox-

amide) (25). Compound 25 (36.8 mg, 68%) was prepared as an oﬀ-

white 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): t_R=

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-chloro-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-5-

methoxyisoquinoline-1-carboxylic acid (101) in a manner similar to

1

that described for compound 11. H 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): t_R=

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-carboxa-

mide) (31). Compound 31 (49 mg, 46%) was prepared as an orange

solid from 41 and 5-methoxyquinoline-2-carboxylic acid (105) in a

1

manner similar to that described for compound 11. H 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): t_R= 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-(3-methoxyisoquinoline-1-carbonyl)pyrrolidine-2-car-

boxamide) (26). Compound 26 (52.9 mg, 50%) was prepared as an

oﬀ-white solid from 41·2HCl and 3-methoxyisoquinoline-1-carboxylic

1

acid (89) in a manner similar to that described for compound 11. H

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): t_R

= 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-car-

boxamide) (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): t_R= 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-(7-methoxyquinoline-2-carbonyl)pyrrolidine-2-carboxa-

mide) (32). Compound 32 (38.9 mg, 37%) was prepared as a yellow

solid from 41 and 7-methoxyquinoline-2-carboxylic acid (106) in a

1

manner similar to that described for compound 11. H 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):

t_R= 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′-(ethyne-

1,2-diyl)dianiline (39, Bionet Research) and (S)-1-(2-phenylacetyl)-

pyrrolidine-2-carboxylic acid²⁵in 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, DMSO-

d₆) 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): t_R= 1.81 min. LCMS (ESI) m/z

(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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Journal of Medicinal Chemistry

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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-2-

carboxamide) (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(4,1-

phenylene))dipyrrolidine-2-carboxamide dihydrochloride (44·2HCl)

and 3-chloro-5-methoxyisoquinoline-1-carboxylic acid (101) in a

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(4,1-

phenylene))bis(azanediyl)bis(oxomethylene)dipyrrolidine-1-

carboxylate (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 N₂at room temperature. After

1

manner similar to that described for compound 11. H 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): t_R= 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). Com-

pound 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 acid²⁵in a manner similar to that described

15 min, the suspension became homogeneous and was stirred for 16 h

1

before the solvent was removed in vacuo to

/ volume. The

4

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): t_R=

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]⁺.

1

for compound 1. H 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): t_R= 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′-(1H-

pyrazole-3,5-diyl)bis(4,1-phenylene))dipyrrolidine-2-carboxamide tri-

hydrochloride (47·3HCl) and 3-chloro-5-methoxyisoquinoline-1-

carboxylic acid (101) in a manner similar to that described for

Example of Boc Deprotection with TFA in CH₂Cl₂: (2S,2′S)-

N,N′-(4,4′-((E)-Ethene-1,2-diyl)bis(4,1-phenylene))-

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(4,1-

phenylene))bis(azanediyl)bis(oxomethylene)dipyrrolidine-1-carboxy-

late (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 NaHCO₃solution (some 1 N NaOH

solution added), brine, dried over anhydrous Na₂SO₄, and evaporated

to ¹/₄of 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): t_R= 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-1-

carboxylate (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.

1

compound 11. H 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): t_R= 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′-(oxazole-

2,5-diyl)dianiline (48) and (S)-1-(2-phenylacetyl)pyrrolidine-2-car-

boxylic acid²⁵in a manner similar to that described for compound

1

1. H 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): t_R= 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-2-

carboxamide) (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(4,1-

phenylene))dipyrrolidine-2-carboxamide dihydrochloride (50·2HCl)

and 3-chloro-5-methoxyisoquinoline-1-carboxylic acid (101) in a

1

manner similar to that described for compound 11. H 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): t_R= 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′-(Ethene-

1,2-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

4,4′-(Ethyne-1,2-diyl)dianiline (42). Compound 42 was pur-

chased 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(4,1-

phenylene))bis(azanediyl)bis(oxomethylene)dipyrrolidine-1-

carboxylate (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

1

that described for compound 40. H 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

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

Journal of Medicinal Chemistry

Article

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): t_R= 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]⁺.

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-^L-

1

proline in a manner similar to that described for compound 40. H

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): t_R= 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′-(Ethyne-1,2-diyl)bis(4,1-phenylene))-

dipyrrolidine-2-carboxamide Dihydrochloride (44·2HCl). Com-

pound 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(4,1-

phenylene))bis(azanediyl)bis(oxomethylene)dipyrrolidine-1-carboxy-

late (43) in a manner similar to that described for compound

(2S,2′S)-N,N′-(4,4′-(Oxazole-2,5-diyl)bis(4,1-phenylene))-

dipyrrolidine-2-carboxamide Dihydrochloride (50·2HCl). Com-

pound 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(4,1-

phenylene))bis(azanediyl)bis(oxomethylene)dipyrrolidine-1-carboxy-

late (49) in a manner similar to that described for compound

1

41·2HCl. H 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): t_R= 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]⁺.

1

41·2HCl. H 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): t_R= 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]⁺.

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(4-

nitrophenyl)-1H-pyrazole²³in a manner similar to that described for

1

compound 48. H NMR (500 MHz, DMSO-d₆) δ 11.71 (br s, 1H),

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 NaHCO₃solution (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

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): t_R= 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): t_R= 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). Com-

pound 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(4,1-

phenylene))bis(azanediyl)bis(oxomethylene)dipyrrolidine-1-carboxy-

late (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): t_R= 0.77 min. LCMS (ESI) m/z calcd for C₂₅H₂₉N₆O₂, 445.23;

found, 445.12 [M + H]⁺.

1

which was carried forward directly. H 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): t_R= 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]⁺.

4,4′-(Oxazole-2,5-diyl)dianiline (48). A suspension of 2,5-bis(4-

nitrophenyl)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 ¹/₄volume

Methyl 3-Vinylpicolinate (53). Methyl 3-(triﬂuoro-

methylsulfonyloxy)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%

1

EtOAc in hexanes) furnished 53 (130 mg, 40%) as a yellow oil. H

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): t_R

= 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

1

and suction ﬁltration of the precipitate. H NMR (300 MHz, DMSO-

d₆and 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): t_R= 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(4,1-

phenylene))bis(azanediyl)bis(oxomethylene)dipyrrolidine-1-

K

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in the preparation of compound 57. LC (method D): t_R= 1.15 min.

LCMS (ESI) m/z calcd for C₉H₁₂NO₂, 166.09; found, 166.09 [M +

H]⁺.

(s, 3H). LC (method A): t_R= 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

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) di-

chloride−CH₂Cl₂complex (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): t_R= 1.16 min.

LCMS (ESI) m/z calcd for C₁₃H₁₂NO₂, 214.09; found, 214.17 [M +

H]⁺.

1

described for compound 64. H 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): t_R= 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 5-

hydroxisoquinoline (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): t_R= 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.

¹H 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): t_R= 0.92 min. LCMS (ESI) m/z calcd for C₁₀H₁₀NO₂,

176.07; found, 176.03 [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

1

without further puriﬁcation. H 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): t_R= 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): t_R= 0.36 min. LCMS (ESI)

m/z calcd for C₈H₁₀NO₂, 152.07; found, 152.10 [M + H]⁺.

3-Methylisoquinoline (65), 5-methylisoquinoline (66), and the

1-haloisoquinolines 67−75 were purchased from commercial sources

unless noted otherwise.

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 Na₂SO₄and

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): t_R= 0.51 min. LCMS (ESI) m/z

calcd for C₁₂H₁₀NO₂, 200.07, found 200.13 [M + H]⁺. 3-Phenyl-

picolinic acid is now available from A.A. Scientiﬁc (AAS-2846),

Apollo-Inter (OR16992), and Combi-Blocks (YA-5926).

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-chloroisoquinoline^22−24,30(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 CH₂Cl₂on a 25S Thomson single step column using 900

mL of solvent) to yield additional 1-chloro-5-ﬂuoroisoquinoline 2-

oxide (0.16 g, 29%) as a light yellow foam. Both isolates were

3-Methoxyisoquinoline (60). Compound 60 (1.42 g, 32%) was

prepared as a colorless oil from 3-hydroxyisoquinoline (59, Aldrich) in

1

a manner similar to that described for compound 63. H 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

L

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combined and treated with neat POCl₃(3 mL). The mixture was

heated at 80 °C for 3 h before the POCl₃was removed in vacuo. The

residue was taken up in EtOAc, and the solution was washed with

saturated NaHCO₃solution and brine prior to drying over anhydrous

Na₂SO₄and 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): t_R= 2.21 min. LCMS (ESI) m/z calcd for C₉H₅Cl₂FN,

215.98 and 217.98; found, 215.92 and 217.92 [M + H]⁺.

over anhydrous MgSO₄and evaporation. The residue was triturated

with hot MeOH to give intermediate 3-methyl-2-(phenylsulfonyl)-1,2-

dihydroisoquinoline-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-1-

carbonitrile (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 CHCl₃and the organic layer was separated, washed with

saturated NaHCO₃solution, 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): t_R= 1.25 min. LCMS (ESI) m/z calcd

for C₁₁H₉N₂, 169.09; found, 169.10 [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-methoxy-

2,3-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

5-Methylisoquinoline-1-carbonitrile (79). A mixture of 1-

chloro-5-methylisoquinoline^22−24,30(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 CH₂Cl₂as solvent

and ﬂash chromatographed on silica gel (elution with 15% EtOAc in

1

concentrated in vacuo to

/ volume and diluted with Et₂O and

3

hexanes. Suction ﬁltration of the precipitate aﬀorded 2-(hydroxyimi-

no)-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): t_R= 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-1H-

inden-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 CH₂Cl₂and

washed with 1 N NaOH and brine prior to drying over anhydrous

Na₂SO₄and 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): t_R= 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-methoxyisoquino-

line 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): t_R= 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 N₂was 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

NaHCO₃solution and brine prior to being dried over anhydrous

Na₂SO₄and 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): t_R= 1.75 min.

LCMS (ESI) m/z calcd for C₁₁H₉N₂O, 185.07; found, 185.10 [M +

H]⁺.

1

hexanes) to yield 79 (0.27g, 57%) as a yellow solid. H 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):

t_R= 1.25 min. LCMS (ESI) m/z calcd for C₁₁H₉N₂, 169.09; found,

169.15 [M + H]⁺.

3-Chloroisoquinoline-1-carbonitrile (80). 3-Chloroisoquino-

line-1-carbonitrile (80, 400 mg, 42%) was prepared as a light yellow

solid from 1,3-dichloroisoquinoline (67)^22−24,30in a manner similar to

1

that described for compound 88. H NMR (300 MHz, CDCl₃) δ

8.47−8.44 (m, 1H), 8.36 (s, 1H), 8.07−7.97 (m, 3H). LC (method

A): t_R= 1.37 min. LCMS (ESI) m/z calcd for C₁₀H₆ClN₂, 189.02;

found, 188.97 [M + H]⁺.

4-Chloroisoquinoline-1-carbonitrile (81). 4-Chloroisoquino-

line-1-carbonitrile (81, 395 mg, 70%) was prepared as a light yellow

solid from 1,4-dichloroisoquinoline (68)^22−24,30in 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): t_R= 1.45 min. LCMS (ESI) m/z

calcd for C₁₀H₆ClN₂, 189.02; found, 188.98 [M + H]⁺.

5-Chloroisoquinoline-1-carbonitrile (82). 5-Chloroisoquino-

line-1-carbonitrile (82, 490 mg, 52%) was prepared as an oﬀ-white

solid from 1,5-dichloroisoquinoline (69)^22−24,30in 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): t_R= 1.36 min. LCMS (ESI) m/z calcd

for C₁₀H₆ClN₂, 189.02; found, 188.98 [M + H]⁺.

6-Chloroisoquinoline-1-carbonitrile (83). 6-Chloroisoquino-

line-1-carbonitrile (83, 174 mg, 65%) was prepared as yellow solid

from 1,6-dichloroisoquinoline (70)^22−24,30in 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): t_R

= 1.31 min. LCMS (ESI) m/z calcd for C₁₀H₆ClN₂, 189.02; found,

188.98 [M + H]⁺.

7-Chloroisoquinoline-1-carbonitrile (84). 7-Chloroisoquino-

line-1-carbonitrile (84, 238 mg, 25%) was prepared as light yellow

solid from 1,7-dichloroisoquinoline (71)^22−24,30in 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): t_R= 1.35 min. LCMS (ESI) m/z calcd

for C₁₀H₆ClN₂, 189.02; found, 189.01 [M + H]⁺.

3-Fluoroisoquinoline-1-carbonitrile (85). 3-Fluoroisoquinoline-

1-carbonitrile (85, 90.1 mg, 60%) was prepared as an oﬀ-white solid

from 1-bromo-3-ﬂuoroisoquinoline (72)^22−24,30in a manner similar to

that described for compound 88. ¹H NMR (500 MHz, CDCl₃) δ 8.35

3-Methylisoquinoline-1-carbonitrile (78). To a mixture of 3-

methylisoquinoline (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): t_R= 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]⁺.

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): t_R= 0.51

min. LCMS (ESI) m/z calcd for C₁₁H₁₀NO₂, 188.07; found, 188.13

[M + H]⁺.

3-Chloroisoquinoline-1-carboxylic Acid (93). 3-Chloroisoqui-

noline-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): t_R=

1.14 min. LCMS (ESI) m/z calcd for C₁₀H₇ClNO₂, 208.02; found,

208.00 [M + H]⁺.

5-Fluoroisoquinoline-1-carbonitrile (86). 5-Fluoroisoquinoline-

1-carbonitrile (86, 98 mg, 28%) was prepared as an oﬀ-white solid

from 5-ﬂuoro-1-chloroisoquinoline (73)^22−24,30in 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): t_R= 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-Chloro-

5-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): t_R

= 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-chromato-

graphed 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): t_R= 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-Methoxyiso-

quinoline-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

4-Chloroisoquinoline-1-carboxylic Acid (94). 4-Chloroisoqui-

noline-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)

1

using method A. H 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): t_R= 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-Chloroisoqui-

noline-1-carboxylic acid (95, 0.43 g, 41%, two steps) was prepared as a

yellow solid from 1,5-dichloroisoquinoline (69)^22−24,30in 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): t_R= 0.69 min.

LCMS (ESI) m/z calcd for C₁₀H₇ClNO₂, 208.02; found, 208.01 [M +

H]⁺.

6-Chloroisoquinoline-1-carboxylic Acid (96). 6-Chloroisoqui-

noline-1-carboxylic acid (96, 0.10 g, 24.5%, three steps) was prepared

as an oﬀ-white solid from 1,6-dichloroisoquinoline (70)^22−24,30in 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): t_R= 0.60 min.

LCMS (ESI) m/z calcd for C₁₀H₇ClNO₂, 208.02; found, 208.03 [M +

H]⁺.

1

A (see example 98). H 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): t_R= 1.40 min. LCMS (ESI) m/z calcd for C₁₁H₁₀NO₃,

204.07; found, 204.06 [M + H]⁺.

5-Methoxyisoquinoline-1-carboxylic Acid (90). 5-Methoxyiso-

quinoline-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,

1

dried over anhydrous Na₂SO₄, concentrated to

/ volume, and

7-Chloroisoquinoline-1-carboxylic Acid (97). 7-Chloroisoqui-

noline-1-carboxylic acid (97, 240 mg, 25%, two steps) was prepared as

a white solid from 1,7-dichloroisoquinoline (71)^22−24,30in a manner

similar to that described for compound 101 following basic hydrolysis

4

1

suction-ﬁltered to aﬀord 90 (0.44 g, 89%) as a yellow solid. H 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): t_R= 0.70 min.

LCMS (ESI) m/z calcd for C₁₁H₁₀NO₃, 204.07; found, 204.05 [M +

H]⁺.

1

of 7-chloroisoquinoline-1-carbonitrile (84) using method B. H 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): t_R= 0.59 min. LCMS

(ESI) m/z calcd for C₁₀H₇ClNO₂, 208.02; found, 208.00 [M + H]⁺.

Isoquinoline-1-carbonitrile Hydrolysis. Method A: 3-Fluo-

roisoquinoline-1-carboxylic Acid (98). 3-Fluoroisoquinoline-1-

carbonitrile (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

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): t_R= 0.39 min. LCMS (ESI) m/z calcd for C₁₁H₁₀NO₂, 188.07;

found, 188.11 [M + H]⁺.

1

5-Methylisoquinoline-1-carboxylic Acid (92). A suspension of

concentrated to aﬀord additional 98 (29.3 mg, 32%). H NMR (500

5-methylisoquinoline-1-carbonitrile (79, 98.3 mg, 0.55 mmol) in

MHz, DMSO-d₆) δ 14.0 (br s, 1H), 8.59−8.57 (m, 1H), 8.10 (d, J =

N

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

Journal of Medicinal Chemistry

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8.5 Hz, 1H), 7.88−7.85 (m, 2H), 7.74−7.71 (m, 1H). LC (method

A): t_R= 1.33 min. LCMS (ESI) m/z calcd for C₁₀H₇FNO₂, 192.05;

found, 191.97 [M + H]⁺.

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): t_R= 0.66 min. LCMS (ESI) m/z calcd for C₁₁H₁₀NO₃,

204.07; found, 203.95 [M + H]⁺.

Isoquinoline-1-carbonitrile Hydrolysis. Method B: 5-Fluo-

roisoquinoline-1-carboxylic Acid (99). 5-Fluoroisoquinoline-1-

carbonitrile (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%)

7-Methoxyquinoline-2-carboxylic Acid (106).³⁶SeO₂(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,

1

as an oﬀ-white solid. H 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): t_R= 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). 3-

Chloro-5-ﬂuoroisoquinoline-1-carboxylic acid (100, 21 mg, 100%)

was prepared as a white solid following acidic hydrolysis of 3-chloro-5-

ﬂuoroisoquinoline-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): t_R= 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). 3-

Chloro-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

1

85%) as light, reddish-brown needles. H NMR (400 MHz, DMSO-

d₆) 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): t_R= 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.¹³The 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.^13eand

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 ×

10⁶Huh-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.¹³The 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

1

drying at 45 °C for 16 h. H 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): t_R= 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-2-

methylquinoline (104).³⁶Concentrated 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 CH₂Cl₂and 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): t_R= 1.40 min. LCMS (ESI) m/z calcd

for C₁₁H₁₁NO, 174.09; found, did not ionize [M + H]⁺. For

1

compound 104: H 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): t_R= 0.58 min. LCMS (ESI) m/z calcd for

C₁₁H₁₁NO, 174.09; found, 173.96 [M + H]⁺.

5-Methoxyquinoline-2-carboxylic Acid (105).³⁶SeO₂(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

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

Journal of Medicinal Chemistry

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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

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 HRP-

conjugated 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 D-

PBST 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 back-

ground 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 × 10³cells/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 CC₅₀values were calculated using the four-parameter

logistic formula.

̈

period. These numbers were similar to those obtained from naıve

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. EC₅₀values 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 H1920-

27B) 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

5. BVDV Assay Preparation. BVDV analysis was typically

performed following HCV FRET analysis according to a previously

described method and is described brieﬂy here.¹³Following 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.

AUTHOR INFORMATION

Corresponding Author

■

*Phone: 1-203-677-6972. Fax: 1-203-677-7702. E-mail: denis.

stlaurent@bms.com.

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.

ACKNOWLEDGMENTS

■

We thank the Discovery Analytical Services (DAS) staﬀ for

obtaining and analyzing HRMS and H NMR spectra. We also

1

P

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

Journal of Medicinal Chemistry

Article

(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.

thank the reviewers of this manuscript for several insightful

comments and suggestions.

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