Optimization of novel indole-2-carboxamide inhibitors of neurotropic alphavirus replication

Article

pubs.acs.org/jmc

Optimization of Novel Indole-2-carboxamide Inhibitors of

Neurotropic Alphavirus Replication

Janice A. Sindac,^‡Scott J. Barraza,^‡Craig J. Dobry,^§Jianming Xiang,^⊥Pennelope K. Blakely,^∥

David N. Irani,^∥Richard F. Keep,^⊥David J. Miller,*^,§,#and Scott D. Larsen*^,‡,†,#

‡

§

^†Vahlteich Medicinal Chemistry Core and Department of Medicinal Chemistry, College of Pharmacy, Departments of Internal

∥

⊥

Medicine and Microbiology and Immunology, Department of Neurology, Department of Neurosurgery, University of Michigan,

Ann Arbor, Michigan 48109, United States

ABSTRACT: Neurotropic alphaviruses, which include western equine

encephalitis virus (WEEV) and Fort Morgan virus, are mosquito-borne

pathogens that infect the central nervous system causing acute and

potentially fatal encephalitis. We previously reported a novel series of

indole-2-carboxamides as alphavirus replication inhibitors, one of

which conferred protection against neuroadapted Sindbis virus

infection in mice. We describe here further development of this

series, resulting in 10-fold improvement in potency in a WEEV

replicon assay and up to 40-fold increases in half-lives in mouse liver

microsomes. Using a rhodamine123 uptake assay in MDR1-MDCKII

cells, we were able to identify structural modiﬁcations that markedly reduce recognition by P-glycoprotein, the key eﬄux

transporter at the blood−brain barrier. In a preliminary mouse PK study, we were able to demonstrate that two new analogues

could achieve higher and/or longer plasma drug exposures than our previous lead and that one compound achieved measurable

drug levels in the brain.

novel series of thienopyrrole derivatives (represented by 1 in

Figure 1) active against WEEV and related alphaviruses.⁶

INTRODUCTION

■

Alphaviruses are mosquito-borne pathogens that cause disease

outbreaks in humans and animals worldwide.¹The neurotropic

alphaviruses, which include western equine encephalitis virus

(WEEV), infect the central nervous system (CNS), causing

acute and potentially fatal encephalitis. In addition to natural

insect-borne disease transmission,²these pathogens could be

aerosolized and released into a population center as potential

bioterrorism agents.^3,4As a result, the neurotropic alphaviruses

are considered Category B Priority Pathogens by the National

Institute of Allergy and Infectious Diseases (NIAID).⁵There

are no FDA-approved vaccines or antiviral drugs active against

neurotropic alphaviruses, and thus there remains a pressing

need for novel therapies to combat either naturally occurring or

intentional outbreaks from these highly virulent pathogens.

Alphaviruses such as WEEV contain a single-stranded,

positive polarity RNA genome that serves as a direct template

for translation and replication.¹The genome encodes both

nonstructural proteins having RNA polymerase, protease,

helicase, and methyltransferase activities and structural capsid

and envelope proteins, which are translated from a subgenomic

RNA that is produced during viral RNA replication. However,

alphavirus structural proteins are dispensable for RNA

replication and can be replaced with easily measured reporter

genes to generate noninfectious replicons that facilitate drug

discovery and development under reduced biosafety conditions.

We previously generated WEEV replicons containing a ﬁreﬂy

luciferase (fLUC) reporter gene, developed a cell-based assay

amenable to high-throughput screening (HTS), and identiﬁed a

Figure 1. Original HTS hit 1 and initial key replicon SAR.

We subsequently undertook the synthesis of indole

analogues of 1, designed to improve potency and metabolic

stability.⁷Initial structure−activity relationship (SAR) develop-

ment of the terminal amide led to the discovery of enantiomer

3 (CCG-203926, Figure 1). Although 3 achieved only a modest

improvement in potency compared to 2, it proved to be

signiﬁcantly more stable to oxidative metabolism by liver

micorosmes. Compound 3 was subsequently advanced to

Received: August 28, 2013

Published: October 23, 2013

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preclinical eﬃcacy studies in mice, where it conferred

protection against infection caused by a related alphavirus,

neuroadapted Sindbis virus.⁷During the course of our

investigation, we noted that 4-pyridylmethyl amide 4 possessed

increased potency relative to simple benzyl amide 2 (Figure 1)

and that simply moving the nitrogen to the 3-position (5)

resulted in a 2-fold loss in activity. These results and the

enantiospeciﬁc activity of 3 strongly suggested that the amide

moiety was making close contact with the unknown molecular

target.

In the present work, we therefore expanded our investigation

of the benzylamide moiety in an eﬀort to further enhance

antiviral potency and also initiated an investigation of the N-

benzyl position. To improve the potential for achieving in vivo

activity, we also explored modiﬁcations of the indole template

that reduced molecular weight and/or lipophilicity to improve

aqueous solubility. Finally, we introduced an assay to estimate

aﬃnity of new compounds for P-glycoprotein (Pgp), the major

eﬄux transporter at the blood−brain barrier (BBB),⁸in order

to identify compounds with the greatest potential for CNS

penetration in future in vivo studies.

Pyrrole analogue 29a was prepared as shown in Scheme 3.

Methyl 1H-pyrrole-2-carboxylate 11 was alkylated with 4-

chlorobenzyl chloride in the presence of potassium carbonate.

Saponiﬁcation resulted in acid 12. Amide bond formation with

ethyl isonipecotate and subsequent hydrolysis generated

carboxylic acid 13. A ﬁnal peptide coupling resulted in 29a.

A convenient synthon for the synthesis of several indole

replacement analogues is amine 16 (Scheme 4). Its synthesis

began with the Boc protection of ethyl isonipecotate 14 using

di-tert-butyl dicarbonate. The Boc-protected ester was hydro-

lyzed to acid 15 and subsequently coupled with 2-(4-

pyridyl)ethylamine. Deprotection with 4M HCl aﬀorded

piperidine amide 16.

The synthesis of imidazole analogue 29b (Scheme 5)

commenced with the alkylation of ethyl 1H-imidazole-2-

carboxylate (17b) with 1-chloro-4-(chloromethyl)benzene.

Ester hydrolysis then gave 18b. The ﬁnal step proceeded

through a peptide coupling with amine 16. The synthesis of

ﬂuoropyrrole 29k proceeded through similar steps; however, it

utilized methyl 4-ﬂuoro-1H-pyrrole-2-carboxylate 17k, which

was synthesized according to a previously described proce-

dure.^11,12

Urea analogue 29d was prepared as shown in Scheme 6.

Addition of ethyl isonipecotate to 1-chloro-4-(2-

isocyanatoethyl)benzene resulted in urea 20. The ester was

saponiﬁed followed by peptide bond formation with 2-(4-

pyridyl)ethylamine to generate 29d.

The synthesis of indole-modiﬁed analogues 29c and 29h

proceeded through similar synthetic steps (Scheme 7). It began

with a coupling reaction between benzimidazole or 6-

ﬂuoroindole carboxylates (21c or 21h, respectively) and ethyl

isonipecotate. Alkylation with 1-chloro-4-(chloromethyl)-

benzene resulted in 22c or 22h. Subsequent hydrolysis and

peptide coupling with 2-(4-pyridyl)ethylamine resulted in the

ﬁnal analogues.

RESULTS

■

Chemistry. Indole carboxamide analogues 27 were

generated as shown in Scheme 1 via standard peptide coupling

Scheme 1. Preparation of Terminal Carboxamide Analogues

a

27

Scheme 8 summarizes the preparation of three more indole-

modiﬁed analogues. Compounds 29f and 29g utilized ethyl

indole-2-carboxylate 8 for their indole starting material, while

29j utilized azaindole ester 23j synthesized according to

procedures described in the literature.^13,14Each were N-

alkylated under basic conditions and saponiﬁed to give

intermediate acids 24f, 24g, and 24j. Final coupling reactions

with piperidine 16 provided the ﬁnal compounds.

Finally, the preparation of indoles 29e and 29i started with 8

and 25i, respectively (Scheme 9). Each was N-alkylated,

saponiﬁed, and coupled with ethyl isonipecotate to give amides

26e and 26i. After ester hydrolysis, the ethyl pyridine motif was

appended using an amide bond coupling to generate the

desired compounds.

WEEV Replicon SAR. All new analogues were tested in the

WEEV replicon assay as previously described.⁷Speciﬁc

modiﬁcations to the terminal amide are summarized in Table

1. Combining the two favorable modiﬁcations from our

previous SAR noted in Figure 1 (4-pyridyl amide and α-methyl

group) improved activity by about 3-fold in the racemate 27a.

As anticipated, the (R)-enantiomer 27b proved to be more

active than the racemate, consistent with what we observed

previously with 3.⁷Replacement of the pyridine ring of 4 with

basic amines (27c and 27d) or isosteric heterocycles (27e and

27f) both greatly reduced potency.

a

Reagents and conditions: (a) R¹R²NH, EDCI, HOBt, DCM, DIEA,

RT, overnight.

conditions using our previously synthesized carboxylic acid

intermediate 7 and commercially available amines, except for 3-

(pyridin-4-yl)propan-1-amine used to prepare 27j, which was

synthesized as previously reported.^9,10The various indole

carboxamide analogues synthesized are speciﬁed in Table 1.

Analogues 28 varying the indole N-substituent were prepared

as in Scheme 2. All contain a terminal N-methylbenzyl

carboxamide (Table 2). We found that attempts to alkylate

intermediate 10 lacking the amide N-methyl group under basic

conditions resulted in decomposition, double alkylation, or no

reaction. Our initial SAR established that methylation of the

amide did not signiﬁcantly compromise activity (compare 28a

in Table 2 with 2 in Figure 1), so we maintained the N-Me

benzylamide through the course of this small SAR series.

Hydrolysis of ethyl indole-2-carboxylate 8 followed by an

EDCI-mediated coupling with ethyl isonipecotate generated

intermediate 9 (Scheme 2). Subsequent hydrolysis followed by

amide formation with N-methylbenzylamine aﬀorded the

desired key advanced common intermediate 10. Final

compounds were then obtained through base-mediated N-

alkylations of the indole.

The optimal distance between the pyridine and the

carboxamide was established by generating homologues 27g

and 27j. Relative to analogue 4, the 10-fold increase in potency

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Table 1. WEEV Replicon and In Vitro ADME Data for Carboxamide Analogues

a

Inhibition of luciferase expression in WEEV replicon assay. Ribavirin as positive control has an IC₅₀in the assay of 16 μM. Values are mean of at

b

least n = 3 independent experiments SE. Cell viability determined by inhibition of cellular reduction of MTT. Values are mean of at least n = 3

independent experiments. Log of eﬀective permeability (cm/s) determined using PAMPA Explorer (pION) with BBB lipid mixture measured at pH

= 7.4. Half-life in mouse liver microsome incubations. Values are mean of ≥2 independent incubations. Rhodamine 123 uptake was measured in

MDR1-MDCKII cells utilizing Glomax multidetection system (Promega). “MDR1 recognition” was assessed by measuring uptake in the presence of

c

d

e

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Table 1. continued

MDR inhibitor, tariquidar (5 μM), and either 30 μM of antiviral or vehicle, and calculating: (C_av− C_veh) × 100/(C_tar− C_veh), where C_av

=

concentration of rhodamine 123 in the presence of antiviral, C_veh= concentration in the presence of vehicle, C_tar= concentration of rhodamine 123

f

in the presence of tariquidar. In the presence of tariquidar, rhodamine 123 uptake was 1123 54% of vehicle controls (n = 44). Kinetic solubility

measured using the same assay media as WEEV replicon assay, except with 10% fetal bovine serum. See Experimental Section for methods and

detailed synthetic procedures.

a

Scheme 2. Preparation of N-Alkyl Indole Analogues

a

Reagents and conditions: (a) LiOH, THF, H₂O; (b) ethyl piperidine-4-carboxylate, EDCI, HOBt, DIEA, THF, RT, overnight; (c) N-

methylbenzylamine, EDCI, HOBt, DCM, DIEA, DCM, RT, overnight; (d) X-R³, base, DMF, overnight, RT, X = I, Br, Cl, OMs.

Table 2. WEEV Replicon and PAMPA Data for N-Alkyl

Indole Analogues

introduced some cytotoxicity, contrary to what we had earlier

observed with 28a. Additional conformationally biased

analogues (27k, 27l, and 27n) decreased potency compared

to 27g. Replacement of the pyridine of 27g with an imidazole,

in an attempt to introduce greater hydrogen-bonding potential

(27o), was not productive. Various substituted phenethyl

amides were also explored, ranging from hydrogen bonding

(27p, 27r) to lipophilic (27q, 27s, 27t), but none matched the

potency of pyridine 27g. Finally, amides 27u−x were prepared

to improve solubility or reduce molecular weight, but all caused

unacceptable potency reductions in the WEEV replicon assay.

In addition to the variations in the amide group, substitution

at the N1 position of the indole was explored (Table 2).

Replacing the 4-chloro group of the benzyl motif in 28a with

other aromatic substituents or hydrogen did not improve

activity (28b−d, 28h, 28j). Overall, the activity seemed to be

more dependent on size than electronegativity, with H and

OMe having the best activity among the new analogues.

Aliphatic substitution (28f, 28g) or acetylation (28e) resulted

in less active or inactive analogues. Replacement of the phenyl

with 4-pyridine slightly diminished potency (28i).

On the basis of the results outlined in Tables 1 and 2, the

optimal 4-pyridylethyl amide and N-4-chlorobenzyl moieties

were retained for an investigation of the indole template SAR

(Table 3). Replacement with a pyrrole (29a) to reduce

molecular weight maintained potency and actually diminished

cytotoxicity compared to 27g, indicating a pyrrole may be a

viable substitute for the indole. Decreasing lipophilicity with an

imidazole (29b), a benzoimidazole (29c), or an azaindole (29j)

scaﬀold decreased potency. Removal of the aromatic ring

altogether (29d) resulted in nearly complete loss of activity,

demonstrating the importance of an aromatic ring or a rigid

scaﬀold for antiviral activity. Compounds 29h and 29i were

synthesized to attenuate the potential for CYP450-mediated

metabolism of the indole scaﬀold by decreasing the electron

density of the indole. These analogues possessed activity and

cytotoxicity similar to 27g. However, a similar attempt to

a

CC₅₀

(μM)

BBB-PAMPA (log

R¹

IC₅₀(μM)

P_eff)

28a

4-Cl-PhCH₂

16.9 4.4

87.0

>100

99.2

>50

28b 4-NO₂−PhCH₂61.3 35.5

28c

4-CN-PhCH₂

95.9 7.0

16.5 0.8

>50

−4.27 0.03

28d 4-MeO-PhCH₂

28e

28f

28g

Ac

−5.01 0.09

−4.39 0.05

MeOCH₂CH₂

i-Bu

>50

27.9 3.6

17.0 1.1

24.2 3.6

35.1 43.5

58.2

64.8

80.3

90.3

28h PhCH₂

28i

28j

4-Pyr-CH₂

4-CF₃-PhCH₂

a

Inhibition of luciferase expression in WEEV replicon assay. Ribavirin

as positive control has an IC₅₀in the assay of 16 μM. Values are mean

of at least n = 3 independent experiments SE. Cell viability

determined by inhibition of cellular reduction of MTT. Values are

mean of at least n = 3 independent experiments. Log of eﬀective

permeability (cm/s) determined using PAMPA Explorer (pION) with

BBB lipid mixture measured at pH = 7.4. See Experimental Section for

methods and detailed synthetic procedures.

with addition of one methylene (27g) and 5-fold decrease with

addition of a second methylene (27j) clearly indicates that the

ethylene linker of 27g is optimal. Very interestingly, there is a

nearly 40-fold decrease in potency as the nitrogen in the

pyridine ring is migrated from para to ortho (27g−i),

demonstrating the key importance of the nitrogen being in

the 4-position of the pyridylethyl amide. N-Methylation of the

amide of 27g (27m) diminished potency by 3-fold and

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a

Scheme 3. Preparation of Pyrrole Analogue 29a

a

Reagents and conditions: (a) 1-chloro-4-(chloromethyl)benzene, cat. NaI, K₂CO₃, DMF, 90 °C; (b) 10% NaOH (aq), EtOH, RT to 50 °C,

overnight; (c) ethyl isonipecotate, EDCI, HOBT, DIPEA, DCM, RT, 24 h; (d) LiOH, H₂O, EtOH, RT, 24 h; (e) 2-(4-pyridyl)ethylamine, EDCI,

HOBT, TEA, DCM.

a

Scheme 4. Preparation of Isonipecotamide Intermediate 16

a

Reagents and conditions: (a) Boc₂O, TEA, DCM, overnight; (b) 10% NaOH (aq), EtOH, 4 h; (c) 2-(4-pyridyl)ethylamine, EDCI, HOBt, DIEA,

DCM; (d) 4N HCl in 1,4-dioxane, Et₂O.

a

Scheme 5. Preparation of Monocyclic Template Analogues

a

Reagents and conditions: (a) 1-chloro-4-(chloromethyl)benzene, Na₂CO₃, DMF, RT, 24 h; (b) 10% NaOH (aq), EtOH, RT, 15 h; (c) 16, EDCI,

HOBt, DIEA, DCM, RT, 24 h.

increase metabolic stability of pyrrole 29a with a ﬂuoro

analogue (29k) resulted in a signiﬁcant increase in toxicity.

Finally, a few modiﬁcations of the N1-indole position of 27g

were investigated to improve solubility and/or metabolic

stability. Replacing the benzyl motif with a methyl group

(29e) eliminated activity, but removing the 4-chloro group was

tolerated with only a small reduction in activity (29f). Insertion

of ortho-ﬂuoro groups (29g) also did not overly diminish

activity but did increase cytotoxicity as evidenced by a decline

in the CC₅₀/IC₅₀ratio below our target of 50.

Solubility. Aqueous solubility was an important physico-

chemical property targeted during our synthetic eﬀorts. Poor

solubility can result in low bioavailability after oral or

intraperitoneal (IP) dosing.¹⁵The kinetic solubility of selected

compounds in our assay media was measured using a simple

precipitation assay.^15,16As expected, pyridyl amides (e.g., 4 and

27g) were about 4-fold more soluble than previous lead

compound 3. Extending the pyridyl alkyl amide chain (27j)

reduced solubility by half. Decreasing the molecular weight

resulted in an increase in solubility (e.g., 27v), which was

particularly dramatic when transitioning from a bicyclic

aromatic core to a monocyclic core. This is evident when

comparing 27g vs 29a, 29b vs 29c, and 29i vs 29k. Finally,

increasing polarity (e.g., benzimidazole 29c and azaindole 29j

vs indole 27g) was eﬀective at improving solubility.

BBB Permeability. Because our candidate inhibitors need

to exert antiviral activity within the brain, compounds with

good blood−brain barrier (BBB) permeability are a priority. An

important physical property for BBB permeability is high

passive permeability. Selected compounds were tested using the

PAMPA Explorer program from pION with the PAMPA−BBB

lipid mixture.^17,18Because of the low solubility of our

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a

same assay. Higher values indicate greater recognition by Pgp

and thus higher likelihood for eﬄux at the BBB. Our data

indicates a small direct correlation between lipophilicity and

Pgp recognition (r = 0.5). A similar trend can be seen for the

relationship between molecular weight and Pgp interaction (r =

0.6). Interestingly, topological polar surface area (TPSA)

actually has a small inverse relationship to Pgp recognition in

our series (r = −0.3), contrary to the prevailing concept that

TPSA contributes to recognition by Pgp.²²Some direct SAR

comparisons are worth noting. N-Methylation of the amide of

27g (27m) actually increased Pgp recognition, suggesting that

conformational eﬀects are more important than lowering the

TPSA, which normally reduces recognition. Similarly, con-

formational restriction through cyclization (27n vs 27g)

increased Pgp recognition. The most eﬀective modiﬁcations

to attenuate Pgp recognition were replacing the indole template

with a monocyclic template (e.g., 29a and 29b) and decreasing

the lipophilicity of the indole (29c and 29j).

Scheme 6. Preparation of Urea Analogue 29d

a

Reagents and conditions: (a) ethyl isonipecotate, DCM, RT, 1 h; (b)

10% NaOH (aq), EtOH, 50 °C, 2 h; (c) 2-(4-pyridyl)ethylamine,

EDCI, HOBt, TEA, DCM, RT, overnight.

compounds, we utilized a cosolvent system containing 20%

acetonitrile in donor wells.¹⁹Data are shown in Tables 1−3. In

general, compounds with log P_eff> −4.7 are categorized as

having high permeability while those exhibiting log P_eff≤ −6

are considered to have low permeability.¹⁷Most of our

compounds fell between these standards, indicating that our

compounds would be predicted to exhibit moderate passive

permeability through the BBB. Somewhat surprisingly, our data

indicate no direct correlation between PAMPA−BBB perme-

ability and either molecular weight or lipophilicity (r = −0.03

and r = 0.03, respectively) within this class of compounds.

The BBB expresses high levels of the eﬄux transporter, P-

glycoprotein (Pgp/MDR1).⁸Pgp facilitates the eﬄux of

xenobiotics from the CNS. For our inhibitors to have the

greatest potential for antiviral activity within the CNS,

interactions with Pgp should be minimized. The degree to

which Pgp interacts with our compounds was measured using a

rhodamine 123 uptake assay in MDCK cells transfected with

human Pgp (MDR1-MDCKII). Rhodamine 123 is a known

Pgp substrate that is actively eﬄuxed from MDR1-MDCKII

cells.²⁰In this assay, cellular uptake of rhodamine 123 is

measured in the absence or presence of a test compound. If the

test compound interacts with Pgp, it will impede eﬄux of

rhodamine 123, thereby increasing its intracellular concen-

tration. This ﬂuorescent assay is similar to the [³H]vinblastine

assay we have used previously in these cells.²¹Tariquidar, a

known inhibitor of Pgp, is included as a control. Results are

included in Tables 1 and 3 as “MDR1 Recognition”, where the

increase in rhodamine 123 uptake in the presence of a test

compound is reported as a percent of the increased rhodamine

123 uptake in the presence of control inhibitor tariquidar in the

Antiviral Activity. Selected compounds were advanced for

testing of their capacity to inhibit the replication of infectious

virus directly in cells. We used two parallel assays to measure

activity against infectious virus: reduction in cytopathic eﬀect

(CPE) and extracellular virus titers. For initial experiments, we

used infectious WEEV, which requires Biosafety Level-3

containment, and focused studies on initial analogues 27g,

29a, and 27a. For subsequent experiments we used Fort

Morgan virus (FMV), a WEEV-serogroup alphavirus that can

be safely handled under Biosafety Level-2 conditions.²³There

was excellent correlation between results with WEEV and FMV

using both CPE reduction (R = 0.96, p < 0.005) and virus titer

(R = 0.92, p < 0.01) assays. Of the eight novel compounds

examined, all but 29j had activity in viral titer assays equivalent

to or superior than our previous lead 3 and all analogues had

superior activity in CPE reduction assays (Table 4). Analogues

27g, 27a, and 29h were particularly eﬀective, reducing viral

titers by approximately 10-fold more than 3.

Pharmacokinetics. Selected compounds were assessed for

stability to oxidative metabolism by mouse liver microsomes

(MLMs), and results are included in Tables 1 and 3. Our

previous lead compound 3 was included for comparison and to

control for a change in the lot of microsomes from that used in

our previous studies. In Table 1, it can be seen that

transitioning to a heterocyclic carboxamide aryl group (27g

and 27o) signiﬁcantly improved metabolic stability, perhaps

due to reduced lipophilicity. Branching of the alkyl chain (27b)

improved stability further, consistent with what we observed in

our earlier work⁷and suggesting that oxidation of the alkyl

chain is one metabolic pathway. In Table 3, it can be seen that

a

Scheme 7. Preparation of Indole-Modiﬁed Analogues

a

Reagents and conditions: (a) ethyl isonipecotate, EDCI, HOBt, DIEA, DCM; (b) 1-chloro-4-(chloromethyl)benzene, Cs₂CO₃, DMF, 80 °C; (c)

LiOH, THF/H₂O; (d) EDCI, HOBt or DMAP, DIEA, 2-(4-pyridyl)ethylamine, DCM.

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a

Scheme 8. Preparation of Indole-Modiﬁed Analogues

a

Reagents and conditions: (a) base, R⁴-X (see Experimental Section), DMF; (b) 10% NaOH (aq), THF or EtOH, RT, 24 h; (c) 16, EDCI, HOBt or

DMAP, DIEA or TEA, THF or DCM, RT, overnight.

a

Scheme 9. Preparation of Indole-Modiﬁed Analogues

a

Reagents and conditions: (a) R⁴-X, K₂CO₃or Cs₂CO₃, DMF, 80 °C; (b) LiOH, THF/H₂O; (c) ethyl isonipecotate, EDCI, HOBt or DMAP,

DIEA, DCM; (d) EDC, HOBt or DMAP, DIEA, 2-(4-pyridyl)ethylamine, DCM.

replacement of the indole ring of 27g with pyrrole (29a)

eroded metabolic stability despite lowering lipophilicity,

suggesting that the central aromatic ring is a major site of

metabolism. This is consistent with the improved stability

realized by replacement of the indole with the more electron-

deﬁcient benzimidazole ring of 29c. Remarkably, simply

removing the chlorine of 27g dramatically improved metabolic

stability (29f), probably due to reduced overall lipophilicity and

strongly suggesting that oxidation of the N-benzyl aromatic ring

is not a major metabolic pathway.

DISCUSSION AND CONCLUSION

■

In this work, our objectives were to develop analogues of our

novel antialphaviral indole carboxamide 3 possessing greater

potency and enhanced potential for in vivo eﬃcacy against

alphavirus infection. Although protection against infection by

neuroadapted Sindbis virus in mice was achieved with 3 in our

previous work, the in vitro ADME properties of this early lead

compound were clearly not optimal, especially in light of its

modest potency.

To improve potency, we focused on expanding our

investigation of the terminal carboxamide because the

enantiospeciﬁc activity of 3 suggested that the amide was

making intimate contact with the unknown molecular target.

Six compounds were identiﬁed with submicromolar activity

against our WEEV replicon, four of which achieved 10-fold

improvements over 3. The SAR leading to the optimal terminal

4-pyridylethyl amide (e.g., 27g−i) is consistent with our

hypothesis that this substituent is making intimate contact with

the binding site, probably via hydrogen bonding through the

pyridyl nitrogen. We also initiated an exploration of the SAR of

the indole N-substituent. This was less fruitful; neither

alteration of the aromatic substitution nor replacement with

alkyl groups improved activity. Antiviral activity of replicon

actives was conﬁrmed with studies of WEEV- or FMV-infected

neuronal cells. In all cases, cell viability was improved relative to

lead 3 at equal concentrations, and in all but one case, viral

titers were reduced below that achieved by 3. Two compounds

reduced titers by over a log relative to 3 (27a and 29h).

With regard to improving potential for in vivo activity, we

focused on both physical properties (aqueous solubility, passive

lipid bilayer permeability) and in vitro predictors of

pharmacokinetics (stability to phase I metabolism by mouse

liver mirosomes, recognition by the BBB eﬄux transporter

Pgp). Increases in solubility were realized, as expected, by

reducing lipophilicity or molecular weight (e.g., 29a−d,29j).

Signiﬁcant improvements in stability to metabolism by mouse

liver microsomes were achieved through reductions in overall

lipophilicity and/or electron density of the central indole ring.

Two of the most stable compounds (29c and 29f) achieved

20−35-fold increases in half-lives relative to lead compound 3.

Two new compounds (27g and 29a) were selected for

preliminary in vivo PK studies in comparison to compound 3

that was previously shown to have some in vivo eﬃcacy.⁷

Compound 27g was one of our most potent analogues and

displayed signiﬁcantly improved in vitro metabolic stability

compared to compound 3. Compound 29a also possessed

excellent potency, combined with a very low potential for Pgp

recognition as determined by the rhodamine 123 uptake assay.

C57BL/6 mice were injected IP with compounds 3, 27g, or

29a. Brain and plasma samples were collected from duplicate

mice at multiple time points and drug levels quantiﬁed via LC/

MS/MS (Table 5). Compound 29a achieved the highest

plasma concentration and demonstrated measurable brain

exposure at 30 min but was cleared rapidly from both

compartments. Analogue 27g had the highest plasma

concentration at 2 h and was the only compound with

measurable plasma levels at 12 h; however, no drug was

consistently detected in brain. Nonetheless, it was reassuring to

learn that the analogue with the greatest in vivo stability (27g)

was also the one with the best in vitro stability, and the only

analogue that achieved measurable levels in the brain (29a) was

the one with the lowest apparent Pgp recognition in the

rhodamine 123 uptake assay. These ﬁndings provide signiﬁcant

validation of our in vitro approach to selecting compounds for

progression to in vivo studies. Although neither 27g nor 29a

have ideal PK properties, both appear superior to our previous

in vivo active 3 in important ways. 29a achieved measurable

levels in the brain, while 27g exhibited higher drug levels at all

time points.

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a

Table 3. WEEV Replicon and In Vitro ADME Data for Template Analogues

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Table 3. continued

a

Inhibition of luciferase expression in WEEV replicon assay. Ribavirin as positive control has an IC₅₀in the assay of 16 μM. Values are mean of at

least n = 3 independent experiments SE. Cell viability determined by inhibition of cellular reduction of MTT. Values are mean of at least n = 3

independent experiments. Log of eﬀective permeability (cm/s) determined using PAMPA Explorer (pION) with BBB lipid mixture measured at pH

= 7.4. Half-life in mouse liver microsome incubations. Values are mean of ≥2 independent incubations. Rhodamine 123 uptake was measured in

MDR1-MDCKII cells utilizing a Glomax multidetection system (Promega). “MDR1 recognition” was assessed by measuring uptake in the presence

of MDR inhibitor, tariquidar (5 μM), and either 30 μM of antiviral or vehicle, and calculating: (C_av− C_veh) × 100/(C_tar− C_veh), where C_av

=

concentration of rhodamine 123 in the presence of antiviral, C_veh= concentration in the presence of vehicle, C_tar= concentration of rhodamine 123

in the presence of tariquidar. In the presence of tariquidar, rhodamine 123 uptake was 1123 54% of vehicle controls (n = 44). Kinetic solubility

measured using the same assay media as WEEV replicon assay, except with 10% fetal bovine serum. See Experimental Section for methods and

detailed synthetic procedures.

a

Table 4. Antiviral Data for Selected Analogues

WEEV

viability (% uninfected control)

FMV

viability (% uninfected control)

compound (25 μM)

titer (× 10⁶pfu/mL)

DMSO

25.7 5.2

9.3 3.4

8.8 1.0*

0.5 0.2**

4.9 2.3*

0.7 0.4**

ND

22.7 3.1

35.3 1.0

36.7 1.4**

60.7 4.7**

59.1 6.1**

63.2 2.6**

ND

47.1 10.5

53.5 6.6

39.3 13.4

4.9 1.9*

23.8 2.9

3.7 1.1**

6.4 3.2*

13.3 4.2*

15.8 9.8

16.4 4.6*

6.8 1.5**

1.6 0.3**

69.4 43.7

39.2 1.9

Ribavirin

32.6 1.7

b

3

47.4 8.0

27g

29a

27a

27b

27k

27o

29c

29f

71.9 1.9**

63.0 5.9*

68.2 2.4**

65.5 11.9*

74.0 7.7**

70.3 9.2*

69.7 13.7

79.4 6.6**

69.2 12.1*

66.1 4.9**

ND

29h

29j

ND

a

Assays utilized the alphaviruses, western equine encephalitis virus (WEEV), and Fort Morgan virus (FMV). Infections were done in cultured human

BE(2)-C neuronal cells. Viability was measured using MTT assay, and viral titers were measured using plaque reduction assays at 25 μM. Values are

b

mean SEM of n = 4 independent experiments. p value <0.05* or 0.005** compared to DMSO control. Results for 3 antiviral activity against

WEEV have been previously published⁷and are reproduced here for convenience to compare results with FMV and between 3 and newly generated

analogues. ND = not determined.

undertaken to “validate” our in vitro approach to selecting

compounds for future in vivo infection studies. The results

correlated well with our predictive in vitro assays for Pgp eﬄux

and metabolism, with measurable brain levels being observed

only with the compound with the lowest value in our MDR1

recognition assay (29a, CCG-206381²⁴) and the highest plasma

levels being achieved by the compound with the longest half-life

in mouse liver microsome incubations (27g, CCG-205432²⁴).

Both of these compounds achieved higher plasma and/or brain

levels than our previous lead 3, which has already successfully

protected mice from alphaviral infection. We therefore intend

to advance both compounds into in vivo eﬃcacy studies in mice

with WEEV encephalitis, the results of which will be reported

in due course.

Table 5. In Vivo Exposure Following IP Administration to

Mice

a

plasma (ng/mL)

30 min 2 h 12 h

brain (ng/g)

2 h

compd (dose)

30 min

12 h

3 (30 mg/kg)

10

59

30

79

5

BLQ

23

BLQ

19

BLQ

27g (30 mg/kg)

29a (20 mg/kg)

124

BLQ

a

Six-week old female C57BL/6 mice were injected IP with a single

indicated dose. The data shown are mean values from two mice at each

time point. BLQ: below the level of quantiﬁcation.

Collectively, our structure−metabolism relationship data

suggest that two sites for metabolism of this class of

compounds are the alkyl chain of the terminal amide (27b vs

27g) and the pyrrole ring of the indole (27g vs 29c). Oxidation

of the indole N-benzyl group, on the other hand, is unlikely

based on the greater stability of 29f vs 27g. Finally, we

demonstrated that apparent recognition by MDR1 (Pgp), a

potential signiﬁcant barrier to penetration of the BBB, could be

attenuated by speciﬁc modiﬁcations to the indole ring. In

particular, replacement of the bicyclic indole with monocyclic

templates (e.g., 29a and 29b) or incorporation of a nitrogen

into the indole (29c and 29j) strongly diminished or virtually

completely eliminated recognition by Pgp as determined by our

assay.

EXPERIMENTAL SECTION

■

General Synthetic Procedures. All reagents were used as

received from commercial sources unless otherwise noted. ¹H and

¹³C spectra were obtained in DMSO-d₆or CDCl₃at room

temperature, unless otherwise noted, on a Varian Inova 400 MHz,

Varian Inova 500 MHz, or Bruker Avance DRX 500 instruments.

Chemical shifts for the ¹H NMR and ¹³C NMR spectra were recorded

in parts per million (ppm) on the δ scale from an internal standard of

residual tetramethylsilane (0 ppm). Rotamers are described as a ratio

of rotamer A and B if possible. Otherwise, if the rotamers cannot be

distinguished, the NMR peaks are described as multiplets. Mass

spectroscopy data were obtained on a Waters Corporation LCT.

HPLC retention times were recorded in minutes (min) using an

Agilent 1100 series with an Agilent Zorbax Eclipse Plus−C18 column

A preliminary PK study comparing the plasma and brain

levels of three diverse compounds after a single IP dose was

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with the gradient 10% ACN/water (1 min), 10−90% ACN/water (6

min), and 90% ACN/water (2 min). Solvent abbreviations used:

MeOH (methanol), DCM (dichloromethane), EtOAc (ethyl acetate),

Hex (hexanes), DMSO (dimethylsulfoxide), DMF (dimethylforma-

mide), H₂O (water), THF (tetrahydrofuran), ACN (acetonitrile).

Reagent abbreviations used: HOBt (1-hydroxy-1,2,3-benzotriazole),

EDCI (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochlor-

ide), DMAP (4-dimethylaminopyridine), DIEA (diisopropylethyl-

amine), TEA (triethylamine), PPh₃(triphenylphosphine), MgSO₄

(magnesium sulfate), Na₂SO₄(sodium sulfate), NaHCO₃(sodium

bicarbonate), Na₂CO₃(sodium carbonate), Cs₂CO₃(sodium carbo-

nate), NH₄Cl (ammonium chloride), K₂CO₃(potassium carbonate),

KOH (potassium hydroxide), HCl (hydrogen chloride). Assay

abbreviations: LUC (luciferase), MTT ((3-(4,5-dimethylthiazol-2-yl)-

2,5-diphenyltetrazolium bromide. All anhydrous reactions were run

under an atmosphere of dry nitrogen.

MHz, DMSO-d₆) 1.7:1 mixture of rotamers δ 11.59−11.53 (m, 1H),

7.64−7.56 (m, 1H), 7.45−7.13 (m, 7H), 7.09−6.99 (m, 1H), 6.78

(rotamer A, s), 6.75 (rotamer B, s, 1H), 4.69 (rotamer A, s, 1H),

4.54−4.37 (m, 3H), 3.28−2.92 (m, 5H), 2.79 (rotamer B, s, 1H),

1.86−1.77 (m, 1H), 1.75−1.55 (m, 3H). TOF ES+ MS: 376.2 (M +

H), 398.1 (M + Na). HPLC retention time = 6.10 min; purity = 93%.

1-(4-Chlorobenzyl)-1H-pyrrole-2-carboxylic Acid (12). Methyl 1H-

pyrrole-2-carboxylate 11 (2.77 g, 22.14 mmol) was dissolved in

anhydrous DMF (15 mL). Granular potassium carbonate (1.94 g, 26.6

mmol) was added, followed by the addition of 4-chlorobenzylchloride

(1.80 mL, 26.6 mmol). The reaction was permitted to stir at 60 °C for

24 h, then again 90 °C for 48 h. Cesium carbonate (21.6 g, 66.4

mmol) was then added, and the reaction was allowed to stir at 90 °C

for 24 h, then sodium iodide (332 mg, 2.214 mmol) was added and

stirring continued at 90 °C for 24 h. At this time, the reaction was

allowed to cool to RT, diluted with ethyl acetate, and washed with

brine (3×). The organic phase was then dried (magnesium sulfate),

concentrated in vacuo, and puriﬁed by ﬂash chromatography (150 g

silica, 5% ethyl acetate/hexanes) to provide methyl 1-(4-chloroben-

zyl)-1H-pyrrole-2-carboxylate as colorless crystals. Yield: 4.91 g (89%).

¹H NMR (400 MHz, CDCl₃) δ 7.32 (d, J = 4.7 Hz, 1H), 7.26 (d, J =

3.5 Hz, 1H), 7.05−6.98 (m, 3H), 6.92−6.86 (m, 1H), 6.20 (dd, J =

3.9, 2.6 Hz, 1H), 5.52 (s, 2H), 3.76 (s, 3H).

Ethyl 1-(1H-Indole-2-carbonyl)piperidine-4-carboxylate (9). Ethyl

1H-indole-2-carboxylate (8, 3.0 g, 16 mmol) and lithium hydroxide

and H₂O (3.3 g, 79 mmol) were dissolved in 2/1 water/THF (30

mL). The reaction was stirred overnight. The reaction was diluted with

water and diethyl ether. The water layer was washed with diethyl ether

twice. The aqueous layer was acidiﬁed to pH 2 using 2N HCl. The

suspension was extracted using ethyl acetate. The organic layer was

washed with saturated sodium chloride solution, dried over

magnesium sulfate, ﬁltered, and concentrated to obtain 1H-Indole-2-

To methyl 1-(4-chlorobenzyl)-1H-pyrrole-2-carboxylate (4.5 g,

18.02 mmol) was added to EtOH (50 mL) and 10% aq NaOH (20

mL) and stirred overnight at RT, then again overnight at 50 °C to give

completion. The ethanol was stripped by rotary evaporation until

material began to precipitate, at which point the aqueous mixture was

cooled in an ice bath and acidiﬁed with concentrated HCl, which

elicited further precipitation. The precipitate was collected over a ﬁlter

and washed with a small amount of cold 1 M HCl and hexanes. The

material was then dried under high vacuum to provide the title

1

carboxylic acid as a white solid. Yield: 2.39 g, 94%. H NMR (400

MHz, DMSO-d₆) δ 12.95 (s, 1H), 11.74 (s, 1H), 7.67−7.60 (m, 1H),

7.47−7.39 (m, 1H), 7.28−7.19 (m, 1H), 7.11−7.01 (m, 2H). 1H-

indole-2-carboxylic acid (2.49 g, 15.45 mmol), EDCI (4.44 g, 23.18

mmol), and HOBt (3.55 g, 23.18 mmol) were dissolved in THF (30

mL). The reaction was allowed to stir for 15 min before DIEA (4.05

mL, 23.18 mmol) and ethyl piperidine-4-carboxylate (3.57 mL, 23.18

mmol) were added to the reaction. The reaction was diluted with

water and ethyl acetate. The organic layer was washed with saturated

sodium bicarbonate, 1 M HCl, and saturated sodium chloride. The

organic layer was then dried over magnesium sulfate, ﬁltered, and

concentrated. The resulting solid was triturated with ethyl acetate to

obtain the product as a white solid. Yield: 3.27 g, 71%. ¹H NMR (400

MHz, DMSO-d₆) δ 11.55 (s, 1H), 7.60 (d, J = 8.1 Hz, 1H), 7.41 (d, J

= 8.2 Hz, 1H), 7.22−7.13 (m, 1H), 7.08−7.00 (m, 1H), 6.80−6.74 (m,

1H), 4.34 (dt, J = 13.4, 3.9 Hz, 2H), 4.09 (q, J = 7.1 Hz, 2H), 3.18 (bs,

2H), 2.76−2.64 (m, 1H), 1.99−1.88 (m, 2H), 1.65−1.50 (m, 2H),

1.20 (t, J = 7.1 Hz, 3H).

1

compound as a white powder. Yield: 4.20 g, (99%). H NMR (400

MHz, DMSO-d₆) δ 7.34 (d, J = 8.2 Hz, 2H), 7.19 (s, 1H), 7.06 (d, J =

8.2 Hz, 2H), 6.82 (s, 1H), 6.16−6.08 (m, 1H), 5.53 (s, 2H).

1-(1-(4-Chlorobenzyl)-1H-pyrrole-2-carbonyl)piperidine-4-car-

boxylic Acid (13). The following were added sequentially to anhydrous

DMF (20 mL), 12 (2.0 g mg, 8.5 mmol), DIEA (4.45 mL, 25.5

mmol), EDCI (1.79 g, 9.34 mmol), and HOBt (1.43 g, 9.34 mmol),

and this solution was allowed to stir at RT for 30 min. Finally, ethyl

isonipecotate (1.44 mL, 9.34 mmol) was added and the reaction was

permitted to stir at RT for 24 h. At this time, DCM was removed in

vacuo and the residue was dissolved in ethyl acetate, which was

subsequently washed with 1 M HCl (3×), water (2×), 10% aq sodium

carbonate (2×), and at last brine (1×). The organic layer was then

dried (magnesium sulfate) and concentrated in vacuo. The residue was

crystallized from ethyl acetate/hexanes via the slow evaporation of

solvent at RT to give ethyl 1-(1-(4-chlorobenzyl)-1H-pyrrole-2-

carbonyl)piperidine-4-carboxylate as large translucent crystals. Yield:

2.16 g (68%). ¹H NMR (400 MHz, CDCl₃) δ 7.24 (d, J = 8.4 Hz, 2H),

7.03 (d, J = 8.4 Hz, 2H), 6.84−6.74 (m, 1H), 6.31 (dd, J = 3.7, 1.5 Hz,

1H), 6.18−6.06 (m, 1H), 5.27 (s, 2H), 4.21 (d, J = 11.5 Hz, 2H), 4.14

(q, J = 7.1 Hz, 2H), 2.94 (t, J = 11.5 Hz, 2H), 2.46 (tt, J = 10.7, 4.0 Hz,

1H), 1.79 (d, J = 12.7 Hz, 2H), 1.56−1.30 (m, 2H), 1.25 (t, J = 7.1 Hz,

3H).

N-Benzyl-1-(1H-indole-2-carbonyl)-N-methylpiperidine-4-carbox-

amide (10). Ester 9 (3.0 g, 10 mmol) and lithium hydroxide, H₂O

(4.19 g, 100 mmol)) were dissolved in THF (ratio, 1; volume, 20 mL)

and water (ratio, 2; volume, 40.0 mL) and allowed to stir overnight at

room temperature. The reaction was diluted with water and extracted

with diethyl ether. The aqueous layer was acidiﬁed using 2N HCl to

pH ∼2. A ﬁne, white suspension resulted. The aqueous layer was

extracted with ethyl acetate twice. The combined ethyl acetate layers

were dried over magnesium sulfate, ﬁltered, and concentrated to aﬀord

the 1-(1H-indole-2-carbonyl)piperidine-4-carboxylic acid as a white

solid. Yield: 678 mg, 25%. ¹H NMR (400 MHz, DMSO-d₆) δ 12.33 (s,

1H), 11.54 (s, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.41 (d, J = 7.5 Hz, 1H),

7.22−7.13 (m, 1H), 7.08−6.99 (m, 1H), 6.79−6.74 (m, 1H), 4.32 (d, J

= 13.3 Hz, 2H), 3.17 (bs, 1H), 2.65−2.54 (m, 1H), 1.96−1.87 (m,

2H), 1.63−1.49 (m, 2H). HPLC retention time = 5.31 min; purity

>95%. 1-(1H-Indole-2-carbonyl)piperidine-4-carboxylic acid (1.00g,

3.67 mmol), EDCI (1.408 g, 7.34 mmol), and HOBt hydrate (1.125 g,

7.34 mmol) were dissolved in DCM (10 mL). The reaction was

allowed to stir for 10 min before adding DIEA (1.283 mL, 7.34 mmol)

and N-methyl-1-phenylmethanamine (0.836 mL, 7.34 mmol). The

reaction was allowed to stir overnight at room temperature. The

reaction was diluted with water and ethyl acetate. The organic phase

was washed with saturated sodium bicarbonate, 1N HCl, and saturated

sodium chloride solution. The organic layer was dried over magnesium

sulfate, ﬁltered, and concentrated. The crude material was puriﬁed by

silica gel chromatography using 30−60% ethyl acetate:hexanes to

Ethyl 1-(1-(4-chlorobenzyl)-1H-pyrrole-2-carbonyl)piperidine-4-

carboxylate (700 mg, 1.867 mmol) was dissolved in EtOH (60 mL)

and water (20 mL). Granular LiOH (134 mg, 5.60 mmol) was added

to this, and the reaction was allowed to stir at RT for 24 h. After this

time, most of the solvent was removed in vacuo, and the remaining

aqueous solution was chilled to 0 °C and acidiﬁed by concentrated

HCl, which elicited a thick, sticky precipitate that adhered to the ﬂask.

The aqueous solution was decanted oﬀ, a small amount of 1 N HCl

was added, and the material sonicated. The aqueous solution was again

decanted, and a small amount of ice-cold water was added, mixed, and

decanted. Remaining water and HCl was then removed by rotary

evaporation, followed by high-vacuum drying to aﬀord the title

1

compound as a white powder. Yield: 620 mg (96%). H NMR (400

MHz, DMSO-d₆) δ 7.33 (dd, J = 9.0, 2.7 Hz, 2H), 7.14−6.97 (m, 2H),

6.27 (dd, J = 3.6, 1.5 Hz, 1H), 6.10−5.94 (m, 1H), 5.25 (s, 2H), 3.98

1

obtain product as a white solid. Yield: 1.03 g, 75%. H NMR (400

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(d, J = 11.0 Hz, 2H), 3.03−2.80 (m, 2H), 2.35−2.19 (m, 1H), 1.64 (d,

J = 11.1 Hz, 2H), 1.24 (d, J = 11.5 Hz, 2H).

cool to room temperature. The reaction was diluted with a 1:1

solution of ethyl acetate/diethyl ether, washed with water (3×) and

brine (1×), dried with magnesium sulfate, and concentrated in vacuo.

The resulting brown residue was puriﬁed via ﬂash chromatography (60

g silica, 10% ethyl acetate/hexanes) to aﬀord methyl 1-(4-

chlorobenzyl)-4-ﬂuoro-1H-pyrrole-2-carboxylate a light-yellow oil.

Yield: 203 mg (83%). H NMR (400 MHz, CDCl₃) δ 7.27 (d, J =

8.3 Hz, 2H), 7.03 (d, J = 8.3 Hz, 2H), 6.69−6.59 (m, 2H), 5.44 (s,

2H), 3.75 (s, 3H).

1-(tert-Butoxycarbonyl)piperidine-4-carboxylic Acid (15). Ethyl

piperidine-4-carboxylate 14 was converted to 1-tert-butyl 4-ethyl

piperidine-1,4-dicarboxylate as previously described.²⁵The ester (3.00

g, 11.66 mmol) was dissolved in ethanol (20 mL), and 10% aqueous

sodium hydroxide (10 mL) and stirred at room temperature for 4 h. At

this time, as much ethanol as possible was stripped oﬀ in vacuo, and

the remaining mostly aqueous solution was cooled in an ice bath and

acidiﬁed with concentrated HCl. The resulting precipitate was

collected over a ﬁlter and washed with cold water, dried over the

ﬁlter, then ﬁnally dried further under high vacuum to aﬀord the desired

1

Methyl 1-(4-chlorobenzyl)-4-ﬂuoro-1H-pyrrole-2-carboxylate (150

mg, 0.56 mmol) was dissolved in ethanol (10 mL) at room

temperature. Then 10% aqueous sodium hydroxide (2 mL) was

added, and the reaction was stirred for 18 h at room temperature. At

this time, solvent was stripped oﬀ in vacuo until material began to

precipitate. Additional water was added (2 mL), and the solution was

cooled in an ice bath, then acidiﬁed with concentrated HCl. The

resulting precipitate was collected over a ﬁlter and washed with cold 1

M HCl, then dried under high vacuum to aﬀord the desired carboxylic

1

carboxylic acid as a white powder. Yield: 2.43 g, 91%. H NMR (400

MHz, DMSO-d₆) δ 12.25 (s, 1H), 3.81 (d, J = 13.3 Hz, 2H), 2.78 (s,

2H), 2.38 (tt, J = 11.1, 3.9 Hz, 1H), 1.76 (dd, J = 13.3, 3.1 Hz, 2H),

1.47−1.24 (m, 10H).

N-(2-(Pyridin-4-yl)ethyl)piperidine-4-carboxamide Dihydrochlor-

ide (16). The following was added to DCM: 15 (600 mg, 2.62 mmol),

DIEA (1.37 mL, 7.85 mmol), EDCI (552 mg, 2.88 mL), HOBt (441

mg, 2.88 mmol), and 2-(4-pyridylethyl)amine (0.34 mL, 2.88 mL).

The solution was stirred at rt for 17 h, at which time the DCM was

stripped oﬀ and 10% aq sodium carbonate was added. Material was

extracted out with EtOAc (3×). The organic extractions were pooled,

dried over magnesium sulfate, and concentrated in vacuo. The residue

was taken up in a small amount of EtOAc and diethyl ether was added.

The precipitate was collected over a ﬁlter and washed with diethyl

ether to give tert-butyl 4-((2-(pyridin-4-yl)ethyl)carbamoyl)piperidine-

1

acid as a white powder. Yield: 111 mg (78%). H NMR (400 MHz,

CDCl₃) δ 7.29 (d, J = 8.3 Hz, 2H), 7.04 (d, J = 8.3 Hz, 2H), 6.79 (d, J

= 2.0 Hz, 1H), 6.71−6.65 (m, 1H), 5.43 (s, 2H).

Ethyl 1-((4-Chlorophenethyl)carbamoyl)piperidine-4-carboxylate

(20). 1-Chloro-4-(2-isocyanatoethyl)benzene 19 (0.45 mL, 2.94

mmol) was dissolved in DCM (10 mL) at RT. Ethyl isonipecotate

was then added dropwise, which elicited precipitation. The slurry was

allowed to stir at RT for 1 h, at which time the precipitate was

collected and dried over a ﬁlter to aﬀord ethyl 1-((4-chlorophenethyl)-

carbamoyl)piperidine-4-carboxylate as an oﬀ-white granular solid. This

material was taken into the next reaction in this crude form. Yield: 916

mg (92%).

1

1-carboxylate as an oﬀ-white solid. Yield: 634 mg (73%). H NMR

(400 MHz, CDCl₃) δ 8.49 (d, J = 5.9 Hz, 2H), 7.09 (d, J = 5.9 Hz,

2H), 5.59 (t, J = 5.3 Hz, 1H), 4.22−3.95 (m, 2H), 3.52 (q, J = 6.8 Hz,

2H), 2.81 (t, J = 6.9 Hz, 2H), 2.68 (t, J = 12.5 Hz, 2H), 2.14 (tt, J =

11.6, 3.8 Hz, 1H), 1.77−1.65 (m, 2H), 1.56 (qd, J = 12.1, 4.3 Hz, 2H),

1.43 (s, 9H).

Ethyl 1-(1-(4-Chlorobenzyl)-1H-benzo[d]imidazole-2-carbonyl)-

piperidine-4-carboxylate (22c). 1H-Benzo[d]imidazole-2-carboxylic

acid 21c, H₂O (500 mg, 2.78 mmol), EDCI (1170 mg, 6.11 mmol),

and HOBt (825 mg, 6.11 mmol) were dissolved in DCM. The

reaction was allowed to stir for 10 min before DIEA (1.066 mL, 6.11

mmol) and ethyl piperidine-4-carboxylate (0.941 mL, 6.11 mmol)

were added. The reaction was allowed to stir overnight at room

temperature. The reaction was diluted with water and ethyl acetate.

The organic phase was washed with 1N HCl, saturated sodium

bicarbonate, and saturated sodium chloride solution. The ethyl acetate

layer was dried over magnesium sulfate, ﬁltered, and concentrated. The

resulting crude material was triturated with ethyl acetate to obtain

ethyl 1-(1H-benzo[d]imidazole-2-carbonyl)piperidine-4-carboxylate as

a white solid. Yield: 113 mg (14%). ¹H NMR (400 MHz, DMSO-d₆) δ

13.09 (s, 1H), 7.63 (dd, J = 84.6, 8.0 Hz, 2H), 7.28 (dt, 2H), 5.30 (d, J

= 13.5 Hz, 1H), 4.42 (d, J = 13.0 Hz, 1H), 4.09 (q, J = 14.2, 7.0 Hz,

2H), 3.48 (t, J = 11.7 Hz, 1H), 3.05 (t, J = 11.3 Hz, 1H), 2.77−2.65

(m, 1H), 1.97 (d, J = 13.7 Hz, 2H), 1.69−1.52 (m, 2H), 1.19 (t, J = 7.1

Hz, 3H). TOF ES+ MS: 302.1 (M + H), 324.1 (M + Na). HPLC

retention time = 5.44 min; purity >95%.

tert-Butyl 4-((2-(pyridin-4-yl)ethyl)carbamoyl)piperidine-1-carbox-

ylate (575 mg, 1.72 mmol) was suspended in diethyl ether at rt, and 4

M HCl in dioxane (6 mL, 24 mmol) was added. The mixture was

stirred at rt for 30 min, at which time the organic solution was

decanted oﬀ and the solid material collected and dried in vacuo. The

title compound was thus obtained as a tan powder. Yield: 448 mg

1

(97%). H NMR (400 MHz, DMSO-d₆) δ 9.22 (bs, 1H), 8.91 (bs,

1H), 8.79 (d, J = 6.4 Hz, 2H), 8.18 (t, J = 5.6 Hz, 1H), 7.89 (d, J = 6.3

Hz, 2H), 3.40 (q, J = 6.2 Hz, 2H), 3.15 (d, J = 12.6 Hz, 2H), 3.00 (t, J

= 6.4 Hz, 2H), 2.83−2.69 (m, 2H), 2.39−2.26 (m, 1H), 1.80−1.59 (m,

4H). TOF ES+ MS: (M + H) 234.2, (M + Na) 256.1.

1-(4-Chlorobenzyl)-1H-imidazole-2-carboxylic Acid (18b). Ethyl

1H-imidazole-2-carboxylate 17b (4.00 g, 28.5 mmol), p-chlorobenzyl-

chloride (4.38 mL, 34.3 mmol), and sodium carbonate (3.63 g, 34.3

mmol) was dissolved in DMF (8 mL). The solution was stirred at rt

for 24 h, at which time water was added and material was extracted

with EtOAc. The organic phase was collected, dried over magnesium

sulfate, and decanted. Puriﬁcation accomplished via silica gel ﬂash

chromatography (150 g silica, 10% EtOAc/hexanes to 80% EtOAc/

hexanes). Ethyl 1-(4-chlorobenzyl)-1H-imidazole-2-carboxylate was

Ethyl 1-(1H-benzo[d]imidazole-2-carbonyl)piperidine-4-carboxy-

late (80 mg, 0.265 mmol) and cesium carbonate (130 mg, 0.398

mmol) were dissolved in DMF (Volume: 2.0 mL). 1-Chloro-4-

(chloromethyl)benzene (0.051 mL, 0.40 mmol) was added, and the

reaction was heated at 80 °C overnight. After 18 h, the reaction was

cooled to room temperature and diluted with water and ethyl acetate.

The organic phase was washed with saturated sodium chloride four

times before it was dried over magnesium sulfate, ﬁltered, and

concentrated. The isolated beige solid was taken directly to the next

1

obtained as a clear, yellow-tinted oil. Yield: 7.28 g (96%). H NMR

(400 MHz, CDCl₃) δ 7.29 (d, J = 8.4 Hz, 2H), 7.18 (d, J = 0.9 Hz,

1H), 7.09 (d, J = 8.4 Hz, 2H), 7.06 (d, J = 0.9 Hz, 1H), 5.59 (s, 2H),

4.37 (q, J = 7.2 Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H).

Ethyl 1-(4-chlorobenzyl)-1H-imidazole-2-carboxylate (7.28 g, 27.5

mmol) was dissolved in EtOH (10 mL) and 10% aq NaOH (20 mL)

and stirred at rt for 15 h. The solvent was then stripped oﬀ, water was

added, and the solution acidiﬁed with HCl. The resulting precipitate

was collected over a ﬁlter and washed with 1 M HCl and dried to

aﬀord the title compound as a white powder. This material was taken

directly into the next step. Yield: 6.506 g (100%).

1-(4-Chlorobenzyl)-4-ﬂuoro-1H-pyrrole-2-carboxylic Acid (18k).

Methyl 4-ﬂuoro-1H-pyrrole-2-carboxylate 17k (130 mg, 0.91 mmol)

was dissolved in anhydrous DMF at room temperature, followed by

the addition of potassium carbonate (151 mg, 1.09 mmol) and 1-

chloro-4-(chloromethyl)benzene (0.14 mL, 1.09 mmol). The reaction

was then stirred for 30 h at 60 °C, after which time it was allowed to

1

step without puriﬁcation. Yield: 85 mg (75%). H NMR (400 MHz,

DMSO-d₆) δ 7.74 (d, J = 7.7 Hz, 1H), 7.66 (d, J = 7.7 Hz, 1H), 7.49−

7.27 (m, 6H), 5.63−5.48 (m, 1H), 5.15 (s, 1H), 4.42−4.32 (m, 1H),

4.08 (q, J = 8 Hz, 8 Hz, 2H), 3.97−3.86 (m, 1H), 3.22−3.10 (m, 1H),

3.05−2.93 (m, 1H), 2.70−2.55 (m, 1H), 1.97−1.88 (m, 1H), 1.76−

1.67 (m, 1H), 1.53−1.38 (m, 1H), 1.21 (t, J = 8 Hz, 3H). TOF ES+

MS: 426.0 (M + H), 448.0 (M + Na).

Ethyl 1-(1-(4-Chlorobenzyl)-6-ﬂuoro-1H-indole-2-carbonyl)-

piperidine-4-carboxylate (22h). 6-Fluoro-1H-indole-2-carboxylic

acid 21h (500 mg, 2.79 mmol), EDCI (1070 mg, 5.58 mmol), and

HOBt (754 mg, 5.58 mmol) were dissolved in DCM (14 mL). The

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reaction was allowed to stir for 20 min before adding ethyl piperidine-

4-carboxylate (0.860 mL, 5.58 mmol) and DIEA (0.975 mL, 5.58

mmol). The reaction was stirred at room temperature overnight. After

18 h, it was diluted with water and ethyl acetate. The organic phase

was washed with 1N HCl, saturated sodium carbonate, and saturated

sodium chloride. It was dried over sodium sulfate, ﬁltered, and

concentrated. No further puriﬁcation was done on the material. Yield:

140 mg (16%). ¹H NMR (500 MHz, DMSO-d₆) δ 11.64 (s, 1H), 7.62

(dd, J = 8.7, 5.5 Hz, 1H), 7.13 (dd, J = 10.0, 2.5 Hz, 1H), 6.96−6.88

(m, 1H), 6.81 (dd, J = 2.3, 0.9 Hz, 1H), 4.33 (dt, J = 13.3, 3.2 Hz, 2H),

4.09 (q, J = 7.1 Hz, 2H), 3.19−3.08 (m, 2H), 2.75−2.65 (m, 1H),

1.97−1.89 (m, 2H), 1.57 (q, J = 11.1 Hz, 2H), 1.20 (t, J = 7.1 Hz, 3H).

TOF ES+ MS: 319.0 (M + H), 341.0 (M + Na). HPLC retention time

= 6.76 min; purity >95%

dropwise as a solution in DMF (3 mL). It was allowed to stir at room

temperature overnight. The reaction was diluted with saturated

ammonium chloride and a mixture of ethyl acetate/diethyl ether. The

aqueous phase was washed with another aliquot of ethyl acetate/

diethyl ether. The organic phases were combined and washed with

saturate sodium chloride, dried over sodium sulfate, ﬁltered, and

concentrated. The resulting crude material was puriﬁed via SP1

Biotage (25 g silica gel column) using a gradient of 100% hexanes to

30% ethyl acetate/hexanes. The product ethyl 1-(2,6-diﬂuorobenzyl)-

1H-indole-2-carboxylate was isolated as a white solid. Yield: 586 mg

1

(70%). H NMR (500 MHz, DMSO-d₆) δ 7.68 (dt, J = 8.0, 1.0 Hz,

1H), 7.50 (dd, J = 8.4, 1.0 Hz, 1H), 7.39−7.27 (m, 3H), 7.16−7.09

(m, 1H), 7.08−7.01 (m, 2H), 5.99 (s, 2H), 4.32 (q, J = 7.1 Hz, 2H),

1.31 (t, J = 7.1 Hz, 3H). TOF ES+ MS: 316.1(M + H), 338.0 (M +

Na). HPLC retention time = 8.53 min; purity >95%.

Ethyl 1-(6-ﬂuoro-1H-indole-2-carbonyl)piperidine-4-carboxylate

(140 mg, 0.440 mmol) and Cs₂CO₃(287 mg, 0.880 mmol) were

dissolved in DMF (5.0 mL). 1-Chloro-4-(chloromethyl)benzene

(0.617 mL, 4.83 mmol) was added as a liquid. The reaction was

heated at 60 °C overnight. The reaction was cooled and diluted with

water and ethyl acetate. The aqueous layer was washed with another

aliquot of ethyl acetate. The combined organic phases were washed

with saturated sodium chloride solution four times. It was dried over

sodium sulfate, ﬁltered, and concentrated. The crude oil was puriﬁed

using ﬂash chromatography (0−10% ethyl acetate/hexanes). The

The ester from above (500 mg, 1.59 mmol) was dissolved in THF

(3 mL) and aqueous sodium hydroxide (3.96 mL, 7.93 mmol). The

reaction was stirred for 3 h before it was concentrated under vacuum

until white precipitate formed. The resulting suspension was cooled

using an ice bath and diluted with water until a stir bar was spinning

freely. Then 2N HCl was added dropwise until the aqueous solution

reached pH 2. It was diluted with ethyl acetate (2×). The organic

phases were combined and washed with saturated sodium chloride,

dried over sodium sulfate, ﬁltered, and concentrated. The resulting

solid 24g was taken to the next step without puriﬁcation. Yield: 396

mg, 87%. ¹H NMR (500 MHz, DMSO-d₆) δ 12.99 (s, 1H), 7.67 (d, J

= 7.9 Hz, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.40−7.23 (m, 3H), 7.14−

6.99 (m, 3H), 6.02 (s, 2H). TOF ES+ MS: 288.1 (M + H), 310.1 (M

+ Na). HPLC retention time = 7.10 min; purity >95%.

1

product was isolated as a white solid. Yield: 170 mg (87%). H NMR

(500 MHz, DMSO-d₆) δ 7.64 (dd, J = 8.7, 5.5 Hz, 1H), 7.54 (dd, J =

10.5, 2.3 Hz, 1H), 7.36−7.29 (m, 2H), 7.10−7.04 (m, 2H), 7.02−6.94

(m, 1H), 6.75 (d, J = 0.8 Hz, 1H), 5.50−5.46 (m, 2H), 4.30 (bs, 1H),

4.07 (q, J = 7.1 Hz, 2H), 3.88 (bs, 1H), 2.99 (m, 2H), 2.62−2.52 (m,

2H), 2.01−1.54 (m, 4H), 1.18 (t, J = 7.1 Hz, 3H). TOF ES+ MS:

443.1 (M + H), 465.0 (M + Na). HPLC retention time = 8.39 min;

purity >95%.

1-(4-Chlorobenzyl)-1H-pyrrolo[2,3-c]pyridine-2-carboxylic Acid

(24j). Ethyl 6-azaindole-2-carboxylate 23j (440 mg, 2.31 mmol) was

dissolved in anhydrous DMF (15 mL) at RT under nitrogen in oven-

dried glassware. Granular potassium tert-butoxide (390 mg, 3.47

mmol) was added, which elicited a red color. This solution was

allowed to stir for 30 min at RT under nitrogen. After this time, 4-

chlorobenzylchloride (325 μL, 2.54 mmol) was added and the reaction

stirred for 14 h. The reaction was then taken up in ethyl acetate/

diethyl ether (1:1) and washed with water (4×) and brine (1×), then

dried (magnesium sulfate) and concentrated in vacuo. The resulting

residue was puriﬁed by ﬂash chromatography (150 g silica, 20% ethyl

acetate/hexanes eluent) to give ethyl 1-(4-chlorobenzyl)-1H-pyrrolo-

[2,3-c]pyridine-2-carboxylate as oﬀ-white needles. Yield: 475 mg

1-Benzyl-1H-indole-2-carboxylic Acid (24f). Ester 8 (1.0 g, 5.3

mmol) and potassium carbonate (1.461 g, 10.57 mmol) were dissolved

in DMF (15 mL) and heated to 60 °C. The reaction was stirred for 20

min before the addition of (bromomethyl)benzene (0.942 mL, 7.93

mmol). The reaction was stirred overnight at 60 °C. After cooling to

room temperature, it was diluted with water and an ethyl acetate/

diethyl ether mixture. The aqueous phase was washed with another

aliquot of the same organic mixture. The organic phases were

combined and washed with saturated sodium chloride (2×), dried over

sodium sulfate, ﬁltered, and concentrated. The resulting crude material

was puriﬁed via SP1 biotage (25 g silica cartridge) with 0−40% ethyl

acetate/hexanes gradient. Ethyl 1-benzyl-1H-indole-2-carboxylate was

1

(65%). H NMR (400 MHz, CDCl₃) δ 8.83 (s, 1H), 8.31 (d, J = 5.5

Hz, 1H), 7.58 (d, J = 5.5 Hz, 1H), 7.32 (s, 1H), 7.22 (d, J = 8.2 Hz,

2H), 7.00 (d, J = 8.1 Hz, 2H), 5.85 (s, 2H), 4.35 (q, J = 7.1 Hz, 2H),

1.37 (t, J = 7.1 Hz, 3H).

1

obtained as an oﬀ-white solid. Yield: 396 mg, 27%. H NMR (500

MHz, DMSO-d₆) δ 7.72 (dt, J = 8.0, 1.0 Hz, 1H), 7.57 (dd, J = 8.5, 0.9

Hz, 1H), 7.38 (d, J = 0.9 Hz, 1H), 7.35−7.11 (m, 5H), 7.05−6.99 (m,

2H), 5.86 (s, 2H), 4.28 (q, J = 7.1 Hz, 2H), 1.28 (t, J = 7.1 Hz, 3H).

HPLC retention time = 8.58 min; purity >95%.

Ethyl 1-(4-chlorobenzyl)-1H-pyrrolo[2,3-c]pyridine-2-carboxylate

(300 mg, 0.953 mmol) was dissolved into ethanol (8 mL) at RT,

followed by the addition of 10% aqueous NaOH (8 mL), which

initially caused precipitation, but homogeneity was achieved over time.

The reaction was allowed to stir at RT for 12 h, at which time the

solvent was removed in vacuo. The residue was taken up in a small

amount of water (5 mL), the pH was adjusted to ∼4, and material was

extracted with ethyl acetate (8×). The extracts were combined and the

solvent removed again in vacuo to provide the title compound as a ﬁne

white powder. Yield: 226 mg (83%). ¹H NMR (400 MHz, DMSO-d₆)

δ 9.64 (s, 1H), 8.40 (d, J = 6.3 Hz, 1H), 8.26 (d, J = 6.3 Hz, 1H), 7.61

(s, 1H), 7.35 (d, J = 8.5 Hz, 2H), 7.10 (d, J = 8.5 Hz, 2H), 6.06 (s,

2H).

The ester from above (390 mg, 1.396 mmol) was dissolved in THF

(1 mL) and 2N aqueous sodium hydroxide (3.49 mL, 6.98 mmol).

The reaction was stirred for 3 h before it was concentrated under

vacuum until a white precipitate formed. The resulting suspension was

cooled by an ice bath and diluted with water until a stir bar was able to

stir. Then 2N HCl was added dropwise until pH 2 was reached. The

acidic aqueous suspension was extracted with ethyl acetate. The

aqueous layer was washed with another aliquot of ethyl acetate. The

organic phases were combined and washed with saturated sodium

chloride, ﬁltered, and concentrated. No further puriﬁcation of 24f was

1

necessary. Yield: 339 mg, 27%. H NMR (500 MHz, DMSO-d₆) δ

Ethyl 1-(1-Methyl-1H-indole-2-carbonyl)piperidine-4-carboxylate

(26e). Ethyl 1H-indole-2-carboxylate 8 (1.00 g, 5.29 mmol) and

K₂CO₃(1.46 g, 10.57 mmol) were dissolved in DMF (10 mL).

Iodomethane (0.99 mL, 16 mmol) was added, and the reaction was

stirred at 60 °C overnight. The reaction was dissolved in water and

diethyl ether. The water layer was washed with diethyl ether twice.

The organic layers were combined and washed with brine twice. It was

then dried over magnesium sulfate, ﬁltered, and concentrated. The

crude product ethyl 1-methyl-1H-indole-2-carboxylate was taken

12.97 (s, 1H), 7.70 (dt, J = 8.1, 0.9 Hz, 1H), 7.54 (dd, J = 8.5, 1.0 Hz,

1H), 7.35−7.17 (m, 5H), 7.16−7.09 (m, 1H), 7.05−7.00 (m, 2H),

5.88 (s, 2H). TOF ES+ MS: 252.1(M + H), 274.1(M + Na). HPLC

retention time = 7.13 min; purity >95%.

1-(2,6-Diﬂuorobenzyl)-1H-indole-2-carboxylic Acid (24g). Sodium

hydride (60 wt %, 127 mg, 3.17 mmol) was suspended in DMF (8

mL) and cooled with an ice bath. The reaction was stirred at for 20

min before a DMF (2 mL) solution of 8 (500 mg, 2.64 mmol) was

added dropwise. The reaction was allowed to stir for 20 min before 2-

(bromomethyl)-1,3-diﬂuorobenzene (821 mg, 3.96 mmol) was added

1

directly into the next step. H NMR (400 MHz, DMSO-d₆) δ 7.69

(d, J = 8.0 Hz, 1H), 7.58 (d, J = 8.6 Hz, 1H), 7.35 (t, J = 7.8 Hz, 1H),

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7.27 (s, 1H), 7.14 (t, J = 7.5 Hz, 1H), 4.32 (q, J = 7.1 Hz, 2H), 4.03 (s,

3H), 1.34 (t, J = 7.1 Hz, 3H). TOF ES+ MS: 204.1 (M + H). HPLC

retention time = 7.85 min; purity >95%.

mmol). The reaction was stirred at room temperature overnight. After

18 h, it was diluted with water and ethyl acetate. The organic phase

was washed with 1N HCl, saturated sodium carbonate, and saturated

sodium chloride. It was dried over sodium sulfate, ﬁltered, and

concentrated. The crude mixture was triturated with diethyl ether and

ethyl acetate. The product 26i was isolated as a pale-yellow oil solid.

Yield: 90 mg (100%). ¹H NMR (500 MHz, DMSO-d₆) δ 7.62 (dd, J =

9.1, 4.4 Hz, 1H), 7.40 (dd, J = 9.5, 2.6 Hz, 1H), 7.35−7.30 (m, 6H),

7.11−7.05 (m, 9H), 6.71 (s, 1H), 5.49 (s, 2H), 4.31 (bs, 1H), 4.07 (q,

J = 7.1, 7.1 Hz, 2H), 3.83 (bs, 1H), 3.08 (bs, 1H), 2.92 (bs, 1H),

2.62−2.53 (m, 1H), 1.86 (bs, 1H), 1.66 (bs, 1H), 1.40 (bs, 1H), 1.18

(t, J = 7.1 Hz, 3H), 1.05 (bs, 1H). TOF ES+ MS: 443.0 (M + H),

465.0 (M + Na). HPLC retention time = 8.28 min; purity >95%.

Representative Procedure for Generating Analogues 27

from 7. 1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-(1-(pyridin-

4-yl)ethyl)piperidine-4-carboxamide (27a). Acid 7⁷(100 mg, 0.25

mmol), EDCI (121 mg, 0.63 mmol), and HOBt (85 mg, 0.63 mmol)

were dissolved in 3.0 mL of DCM. The reaction was stirred at room

temperature for 10 min before DIEA (0.110 mL, 0.63 mmol) and 1-

(pyridin-4-yl)ethanamine (77 mg, 0.63 mmol) as a 1.0 mL DCM

solution were added. The reaction was allowed to stir at room

temperature overnight. The reaction was diluted with water and

extracted 3× with ethyl acetate. The organic phase was washed with

saturated sodium bicarbonate (twice) and saturated sodium chloride.

The organic phase was dried over magnesium sulfate, ﬁltered, and

concentrated. The crude material was triturated with ether and ethyl

acetate and ﬁltered to obtain 27a as a white solid. Yield: 37.9 mg, 30%.

¹H NMR (400 MHz, DMSO-d₆) δ 8.52−8.45 (m, 2H), 8.35 (d, J = 7.7

Ethyl 1-methyl-1H-indole-2-carboxylate (1.0 g, 4.92 mmol) and

lithium hydroxide (1.178 g, 49.2 mmol) were dissolved in 2/1 water/

THF (6 mL). The reaction was allowed to stir overnight. The reaction

was diluted with water and washed with diethyl ether. The water layer

was then acidiﬁed with 1N HCl to pH 2. The resulting suspension was

extracted with ethyl acetate. The organic layer was washed with

saturated sodium chloride, dried over magnesium sulfate, ﬁltered, and

concentrated to obtain pure 1-methyl-1H-indole-2-carboxylic acid as a

1

white solid. Yield: 560 mg (65%). H NMR (400 MHz, DMSO-d₆) δ

12.91 (s, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.57 (d, J = 8.5 Hz, 1H), 7.37−

7.29 (m, 1H), 7.22 (s, 1H), 7.16−7.08 (m, 1H), 4.02 (s, 3H). TOF ES

+ MS: 176.1 (M + H). HPLC retention time = 6.06 min; purity >95%.

1-Methyl-1H-indole-2-carboxylic acid (200 mg, 1.14 mmol), HOBt

(231 mg, 1.712 mmol), and EDCI (328 mg, 1.712 mmol) were

dissolved in DCM (Volume: 4 mL). The reaction was allowed to stir

for 10 min before adding DIEA (0.299 mL, 1.71 mmol) and ethyl

piperidine-4-carboxylate (0.264 mL, 1.71 mmol). The reaction was

stirred overnight. The reaction was diluted with water and extracted

with ethyl acetate. The organic layer was washed with saturated

sodium bicarbonate and 1N HCl. This was followed by a wash with

saturated sodium chloride. The organic phase was dried over

magnesium sulfate, ﬁltered, and concentrated to obtain 26e as a

white solid. No further puriﬁcation was required. Yield: 214 mg, 60%.

¹H NMR (400 MHz, DMSO-d₆) δ 7.57 (d, J = 8.0 Hz, 1H), 7.48 (d, J

= 9.0 Hz, 1H), 7.21 (d, J = 15.3 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 6.61

(s, 1H), 4.37−3.83 (m, 4H), 3.71 (s, 3H), 3.10 (bs, 2H), 2.70−2.59

(m, 1H), 1.87 (bs, 2H), 1.61−1.46 (m, 2H), 1.16 (t, J = 7.1 Hz, 3H).

TOF ES+ MS: 315.1 (M + H), 337.1 (M + Na). HPLC retention time

= 6.99 min; purity >95%.

Hz, 1H), 7.62 (d, J = 7.8 Hz, 1H), 7.53 (d, J = 8.8 Hz, 1H), 7.37−7.17

(m, 5H), 7.14−7.05 (m, 3H), 6.72 (s, 1H), 5.48 (s, 2H), 4.87 (p, J =

7.1 Hz, 1H), 4.03 (m, 2H), 2.95 (bs, 2H), 2.49−2.42 (m, 1H), 1.79−

1.28 (m, 7H). TOF ES+ MS: 501.2 (M + H). HPLC retention time =

5.73 min; purity >95%.

Ethyl 1-(1-(4-Chlorobenzyl)-5-ﬂuoro-1H-indole-2-carbonyl)-

piperidine-4-carboxylate (26i). Ethyl 5-ﬂuoro-1H-indole-2-carboxy-

late 25i (0.500 g, 2.413 mmol) and Cs₂CO₃(1.179 g, 3.62 mmol)

were dissolved in DMF (15 mL). 1-Chloro-4-(chloromethyl)benzene

(0.617 mL, 4.83 mmol) was added as a liquid. The reaction was heated

at 60 °C overnight. The reaction was cooled and diluted with water

and ethyl acetate. The aqueous layer was washed with another aliquot

of ethyl acetate. The combined organic phases were washed with

saturated sodium chloride solution four times. It was dried over

sodium sulfate, ﬁltered, and concentrated. The crude oil was puriﬁed

by ﬂash chromatography using 0−10% ethyl acetate/hexanes. The

product ethyl 1-(4-chlorobenzyl)-5-ﬂuoro-1H-indole-2-carboxylate

was isolated as a clear oil. Yield: 600 mg (75%). ¹H NMR (500

MHz, DMSO-d₆) δ 7.63 (dd, J = 9.0, 5.3 Hz, 1H), 7.51 (dd, J = 9.4,

2.4 Hz, 1H), 7.47−7.41 (m, 1H), 7.37−7.33 (m, 2H), 7.21 (td, J = 9.3,

2.5 Hz, 1H), 7.03 (d, J = 8.5 Hz, 2H), 5.85 (s, 2H), 4.28 (q, J = 7.1 Hz,

2H), 1.28 (t, J = 7.1 Hz, 3H). HPLC retention time = 9.06 min; purity

>95%.

(R)-1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-(1-(pyridin-4-

yl)ethyl)piperidine-4-carboxamide (27b). Synthesized from 7 and

(R)-1-(pyridin-4-yl)ethanamine as described for 27a. The crude solid

was recrystallized using diethyl ether/ethyl acetate. The product was

1

obtained as a white solid after ﬁltration. Yield: 55%. H NMR (500

MHz, DMSO-d₆) δ 8.51−8.46 (m, 2H), 8.37 (d, J = 7.7 Hz, 1H), 7.62

(d, J = 7.9 Hz, 1H), 7.54 (d, J = 8.3 Hz, 1H), 7.36−7.25 (m, 4H),

7.25−7.18 (m, 1H), 7.13−7.06 (m, 3H), 6.73 (s, 1H), 5.48 (s, 2H),

4.88 (p, J = 7.2 Hz, 1H), 4.44 (bs, 1H), 4.02 (bs, 1H), 3.13−2.70 (m,

2H), 2.50−2.44 (m, 1H), 1.85−1.30 (m, 7H). TOF ES+ MS: 501.2

(M + H). HPLC retention time = 5.62 min; purity >95%.

(1-(4-Chlorobenzyl)-1H-indol-2-yl)(4-(4-methylpiperazine-1-

carbonyl)piperidin-1-yl)methanone (27c). Synthesized from 7 and 4-

methylpiperazine as described for 27a. The crude material was puriﬁed

by medium pressure chromatography using 100% DCM to 5% 7 M

ammonia in MeOH/95% DCM. The product was obtained as a white

1

solid. Yield: 57%. H NMR (400 MHz, DMSO-d₆) δ 7.62 (d, J = 7.9

Ethyl 1-(4-chlorobenzyl)-5-ﬂuoro-1H-indole-2-carboxylate (465

mg, 1.402 mmol) and lithium hydroxide, H₂O (588 mg, 14.0 mmol)

were dissolved in 2/1 water/THF (12 mL). The reaction was allowed

to stir overnight at room temperature. The reaction was diluted with

water and diethyl ether. The organic phase was removed, and the

aqueous phase was acidiﬁed using 2N HCl to pH ∼2. The resulting

suspension was extracted with ethyl acetate. The aqueous phase was

washed with another aliquot of ethyl acetate. The organic phases were

combined, washed with saturated sodium chloride, ﬁltered, and

concentrated to obtain the product 1-(4-chlorobenzyl)-5-ﬂuoro-1H-

indole-2-carboxylic acid. No further puriﬁcation was performed. Yield:

130 mg (31%). ¹H NMR (500 MHz, DMSO-d₆) δ 13.14 (s, 1H), 7.58

(dd, J = 9.2, 4.4 Hz, 1H), 7.49 (dd, J = 9.4, 2.6 Hz, 1H), 7.37−7.28 (m,

3H), 7.18 (td, J = 9.2, 2.6 Hz, 1H), 7.06−7.00 (m, 2H), 5.86 (s, 2H).

HPLC retention time = 7.55 min; purity >95%.

Hz, 1H), 7.54 (d, J = 8.7 Hz, 1H), 7.37−7.29 (m, 2H), 7.26−7.17 (m,

1H), 7.14−7.05 (m, 3H), 6.73 (s, 1H), 5.49 (s, 2H), 4.52−3.90 (m,

2H), 3.57−3.39 (m, 4H), 3.19−2.78 (m, 3H), 2.39−2.11 (m, 7H),

1.77−1.20 (m, 4H). TOF ES+ MS: 479.1 (M + H). HPLC retention

time = 5.37 min; purity = 92%.

1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-((1-methylpiperi-

din-4-yl)methyl)piperidine-4-carboxamide (27d). Synthesized from

7 and (1-methylpiperidin-4-yl)methanamine as described for 27a. The

crude product was triturated with diethyl ether/ethyl acetate to aﬀord

the product as a white solid. Yield: 65%. ¹H NMR (400 MHz, DMSO-

d₆) δ 7.79 (t, J = 5.8 Hz, 1H), 7.62 (d, J = 7.9 Hz, 1H), 7.54 (d, J = 8.0

Hz, 1H), 7.38−7.29 (m, 2H), 7.26−7.17 (m, 1H), 7.15−7.05 (m, 3H),

6.72 (s, 1H), 5.49 (s, 2H), 4.61−3.82 (m, 2H), 2.99−2.73 (m, 4H),

2.44−2.30 (m, 1H), 2.19 (s, 3H), 1.96−1.05 (m, 13H). TOF ES+ MS:

507.1 (M + H). HPLC retention time = 5.41 min; purity >95%.

1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-((1-methyl-1H-

pyrazol-3-yl)methyl)piperidine-4-carboxamide (27e). Synthesized

from 7 and (1-methyl-1H-pyrazol-3-yl)methanamine as described for

27a. The crude product was triturated with ethyl acetate to obtain

1-(4-Chlorobenzyl)-5-ﬂuoro-1H-indole-2-carboxylic acid (130 mg,

0.428 mmol), EDCI (164 mg, 0.856 mmol), and DMAP (105 mg,

0.856 mmol) were dissolved in DCM (Volume: 5 mL). The reaction

was allowed to stir for 20 min before adding ethyl piperidine-4-

carboxylate (0.132 mL, 0.856 mmol) and DIEA (0.150 mL, 0.856

1

white solid as the product. Yield: 18%. H NMR (400 MHz, DMSO-

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d₆) δ 8.17 (t, J = 5.7 Hz, 1H), 7.66−7.50 (m, 3H), 7.38−7.29 (m, 2H),

7.25−7.16 (m, 1H), 7.15−7.05 (m, 3H), 6.72 (s, 1H), 6.04 (d, J = 2.2

Hz, 1H), 5.49 (s, 2H), 4.17 (m, 4H), 3.76 (s, 3H), 2.92 (bs, 2H),

2.47−2.37 (m, 1H), 1.81−1.23 (m, 4H). TOF ES+ MS: 490.1 (M +

H). HPLC retention time = 6.62 min; purity = 94%.

(pyridin-4-yl)propan-1-amine as described for 27a. The crude material

was triturated in ethyl acetate to obtain product. Yield: 40%. ¹H NMR

(500 MHz, CDCl₃) δ 8.58−8.52 (m, 2H), 7.68−7.61 (m, 1H), 7.37−

7.31 (m, 1H), 7.31−7.10 (m, 5H), 7.06−6.99 (m, 2H), 6.62 (s, 1H),

5.46 (s, 2H), 5.31 (t, J = 5.1 Hz, 1H), 4.68−3.98 (m, 3H), 3.64−3.54

(m, 1H), 3.37−3.26 (m, 1H), 3.04−2.96 (m, 1H), 2.88−2.81 (m, 1H),

2.22−2.13 (m, 1H), 1.85−1.20 (m, 6H). TOF ES+ MS: 515.1 (M +

H). HPLC retention time = 5.80 min; purity = 85%.

1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-((1-methyl-1H-

imidazol-4-yl)methyl)piperidine-4-carboxamide (27f). Synthesized

from 7 and (1-methyl-1H-imidazol-4-yl)methanamine as described for

27a. The crude material was triturated with ether and ethyl acetate and

1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-(2-methyl-2-(pyri-

din-4-yl)propyl)piperidine-4-carboxamide (27l). Synthesized from 7

and 2-methyl-2-(pyridin-4-yl)propan-1-amine as described for 27a.

The crude material was triturated in ethyl acetate. The white solid was

1

ﬁltered to obtain white solid as a product. Yield: 23%. H NMR (400

MHz, DMSO-d₆) δ 8.07 (t, J = 5.5 Hz, 1H), 7.62 (d, J = 7.9 Hz, 1H),

7.53 (d, J = 8.3 Hz, 1H), 7.46 (s, 1H), 7.38−7.30 (m, 2H), 7.26−7.16

(m, 1H), 7.15−7.05 (m, 3H), 6.87 (s, 1H), 6.73 (s, 1H), 5.49 (s, 2H),

4.49−3.92 (m, 4H), 3.32 (s, 3H), 2.90 (bs, 2H), 2.48−2.36 (m, 1H),

1.78−1.33 (m, 4H). TOF ES+ MS: 490.1 (M + H). HPLC retention

time = 5.48 min; purity >95%.

1

ﬁltered to obtain product. Yield: 23%. H NMR (500 MHz, DMSO-

d₆) δ 8.49−8.45 (m, 2H), 7.69−7.59 (m, 2H), 7.53 (d, J = 8.5 Hz,

1H), 7.36−7.31 (m, 4H), 7.25−7.17 (m, 1H), 7.14−7.06 (m, 3H),

6.71 (s, 1H), 5.48 (s, 2H), 4.55−3.79 (m, 2H), 3.28 (d, J = 6.2 Hz,

2H), 2.88 (bs, 2H), 2.42−2.34 (m, 1H), 1.67−1.19 (m, 10H). TOF ES

+ MS: 529.3 (M + H). HPLC retention time = 6.01 min; purity >95%.

1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-methyl-N-(2-(pyr-

idin-4-yl)ethyl)piperidine-4-carboxamide (27m). Synthesized from 7

and N-methyl-2-(pyridin-4-yl)ethanamine as described for 27a. The

resulting crude material was puriﬁed using SP1 Biotage chromatog-

raphy with a 25 g silica cartridge and a gradient of 20% ethyl acetate/

hexanes to 100% ethyl acetate. The product was obtained as a white

1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-(2-(pyridin-4-yl)-

ethyl)piperidine-4-carboxamide (27g). Synthesized from 7 and 2-

(pyridin-4-yl)ethanamine as described for 27a. The crude material was

triturated with ethyl acetate to aﬀord the product as a white solid.

Yield: 328 mg, 80%. ¹H NMR (500 MHz, DMSO-d₆) δ 8.48−8.42 (m,

2H), 7.89 (t, J = 5.5 Hz, 1H), 7.63 (d, J = 7.8 Hz, 1H), 7.54 (d, J = 8.5

Hz, 1H), 7.37−7.30 (m, 2H), 7.25−7.18 (m, 3H), 7.13−7.07 (m, 3H),

6.71 (s, 1H), 5.48 (s, 2H), 4.53−3.82 (m, 2H), 3.32−3.29 (m, 2H),

3.08−2.67 (m, 4H), 2.36−2.27 (m, 1H), 1.75−1.18 (m, 4H). ¹³C

NMR (500 MHz, DMSO-d₆) 226.99, 183.03, 173.53, 161.90, 149.39,

148.39, 137.21, 136.81, 131.91, 131.80, 128.80, 128.49, 126.16, 124.27,

123.15, 121.41, 120.27, 110.67, 103.40, 46.13, 41.42, 38.79, 34.20,

14.12, 14.09. Anal. Calcd for C₂₉H₂₉ClN₄O₂: C, 69.52%; H, 5.83%; N,

11.18%. Found: C, 69.28%; H, 5.88%; N, 11.23%. TOF ES+ MS:

501.0 (M + H). HPLC retention time = 5.50 min; purity >95%.

1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-(2-(pyridin-3-yl)-

ethyl)piperidine-4-carboxamide (27h). Synthesized from 7 and 2-

(pyridin-3-yl)ethanamine-HCl as described for 27a. The reaction was

diluted with water and ethyl acetate. The organic layer was washed

with saturated sodium bicarbonate/10% aqeuous citric acid solution,

followed by saturated sodium chloride solution. The organic layer was

dried over magnesium sulfate, ﬁltered, and concentrated. The resulting

solid was triturated with ethyl acetate to obtain the product as a white

1

solid. Yield: 32%. H NMR (400 MHz, DMSO-d₆) 1.3:1 mixture of

rotamers δ 8.49−8.45 (m, 2H), 7.64−7.61 (m, 1H), 7.54 (t, J = 10 Hz,

1H), 7.35−7.30 (m, 3H), 7.24−7.19 (m, 2H), 7.12−7.08 (m, 3H),

6.72 (rotamer A, s), 6.70 (rotamer B, s, 1H), 4.50−4.75 (m, 2H), 4.41

(bs, 1H), 3.97 (bs, 1H), 3.63 (t, J = 6.8 Hz, 1H), 3.53 (t, J = 7.3 Hz,

1H), 3.09−2.69 (m, 8H), 2.84−2.50 (m, 1H), 1.17−1.10 (m, 4H).

TOF ES+ MS: 515.3 (M + H). HPLC retention time = 5.84 min;

purity >95%.

1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-((2,3-

dihydrofuro[2,3-c]pyridin-3-yl)methyl)piperidine-4-carboxamide

(27n). Synthesized from 7 and (2,3-dihydrofuro[2,3-c]pyridin-3-

yl)methanamine as described for 27a. The crude material was

triturated in ethyl acetate, ﬁltered, and concentrated to obtain a

white solid. Yield: 50%. ¹H NMR (500 MHz, DMSO-d₆) δ 8.14−8.06

(m, 3H), 7.63 (d, J = 7.9 Hz, 1H), 7.55 (d, J = 8.3 Hz, 1H), 7.37−7.28

(m, 4H), 7.25−7.18 (m, 1H), 7.14−7.06 (m, 2H), 6.72 (s, 1H), 5.49

(s, 2H), 4.63−4.55 (m, 1H), 4.39−4.32 (m, 2H), 4.14−3.87 (m, 2H),

3.73−3.63 (m, 1H), 3.17 (d, J = 5.3 Hz, 1H), 2.86−2.82 (m, 2H),

2.40−2.34 (m, 1H), 1.76−1.15 (m, 4H). TOF ES+ MS: 529.1 (M +

H). HPLC retention time = 5.78 min; purity >95%.

1

solid. Yield: 20%. H NMR (400 MHz, DMSO-d₆) δ 8.44−8.37 (m,

2H), 7.88 (t, J = 5.7 Hz, 1H), 7.66−7.50 (m, 3H), 7.38−7.26 (m, 3H),

7.26−7.17 (m, 1H), 7.14−7.06 (m, 3H), 6.71 (s, 1H), 5.48 (s, 2H),

4.63−3.80 (m, 2H), 3.34−3.25 (m, 2H), 2.89 (bs, 2H), 2.73 (t, J = 7.0

Hz, 2H), 2.38−2.27 (m, 1H), 1.74−1.21 (m, 4H). TOF ES+ MS:

501.0 (M + H), 523.1 (M + Na). HPLC retention time = 5.46 min;

purity >95%.

N-(2-(1H-Imidazol-1-yl)ethyl)-1-(1-(4-chlorobenzyl)-1H-indole-2-

carbonyl)piperidine-4-carboxamide (27o). Synthesized from 7 and

2-(1H-imidazol-1-yl)ethanamine-HCl as described for 27a. The

resulting crude solid was triturated with ethyl acetate to give a white

1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-(2-(pyridin-2-yl)-

ethyl)piperidine-4-carboxamide (27i). Synthesized from 7 and 2-

(pyridin-2-yl)ethanamine as described for 27a. The crude material was

triturated with ether and ethyl acetate and ﬁltered to obtain the

product as a white solid. Yield: 10%. ¹H NMR (400 MHz, DMSO-d₆)

δ 8.52−8.45 (m, 1H), 7.87 (t, J = 5.6 Hz, 1H), 7.72−7.66 (m, 1H),

7.63 (d, J = 7.9 Hz, 1H), 7.54 (d, J = 8.3 Hz, 1H), 7.38−7.30 (m, 2H),

7.25−7.17 (m, 3H), 7.14−7.05 (m, 3H), 6.72 (s, 1H), 5.49 (s, 2H),

4.52−3.78 (m, 2H), 3.40 (q, J = 6.9 Hz, 2H), 3.08−2.76 (m, 4H),

2.38−2.29 (m, 1H), 1.74−1.23 (m, 4H). TOF ES+ MS: 501.1 (M +

H), 523.1 (M + Na). HPLC retention time = 5.51 min; purity >95%.

1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-(3-(pyridin-4-yl)-

propyl)piperidine-4-carboxamide (27j). Synthesized from 7 and 3-

(pyridin-4-yl)propan-1-amine as described for 27a. The crude solid

was triturated with ethyl acetate, giving a white solid. Yield: 40 mg,

31%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.48−8.41 (m, 2H), 7.85 (t, J

= 5.6 Hz, 1H), 7.62 (d, J = 7.9 Hz, 1H), 7.54 (d, J = 8.4 Hz, 1H),

7.37−7.30 (m, 2H), 7.26−7.17 (m, 3H), 7.14−7.05 (m, 3H), 6.72 (s,

1H), 5.49 (s, 2H), 4.41 (bs, 2H), 4.14−3.99 (m, 4H), 3.10−2.75 (m,

4H), 2.62−2.50 (m, 2H), 2.38−2.30 (m, 1H), 1.77−1.64 (m, 4H),

1.49−1.25 (m, 2H). TOF ES+ MS: 514.9 (M + H). HPLC retention

time = 5.58 min; purity >95%.

1

solid. Yield: 18%. H NMR (500 MHz, DMSO-d₆) δ 7.94 (t, J = 5.6

Hz, 1H), 7.63 (d, J = 7.9 Hz, 1H), 7.57−7.51 (m, 2H), 7.37−7.31 (m,

2H), 7.25−7.18 (m, 1H), 7.13−7.06 (m, 4H), 6.87 (s, 1H), 6.71 (s,

1H), 5.49 (s, 2H), 4.39 (s, 1H), 4.07−3.99 (m, 3H), 3.39−3.31 (m,

2H), 2.93 (bs, 2H), 2.39−2.29 (m, 1H), 1.79−1.20 (m, 4H). TOF ES

+ MS: 490.1 (M + H). HPLC retention time = 5.69 min; purity =

90%.

1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-(3-

methoxyphenethyl)piperidine-4-carboxamide (27p). Synthesized

from 7 and 2-(3-methoxyphenyl)ethanamine as described for 27a.

The crude material was triturated with ethyl acetate to obtain product

as a white solid. Yield: 67%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.85 (t,

J = 5.6 Hz, 1H), 7.63 (d, J = 7.8 Hz, 1H), 7.54 (d, J = 8.4 Hz, 1H),

7.37−7.30 (m, 2H), 7.26−7.14 (m, 2H), 7.14−7.06 (m, 3H), 6.78−

6.73 (m, 3H), 6.71 (s, 1H), 5.49 (s, 2H), 4.51−3.86 (m, 2H), 3.72 (s,

3H), 3.26 (q, J = 6.7 Hz, 2H), 2.94 (s, 2H), 2.67 (t, J = 7.2 Hz, 2H),

2.37−2.30 (m, 1H), 1.62 (s, 2H), 1.37 (s, 2H). TOF ES+ MS: 530.1

(M + H), 552.1 (M + Na). HPLC retention time = 7.86 min; purity

>95%.

1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-(4-

ﬂuorophenethyl)piperidine-4-carboxamide (27q). Synthesized from

7 and 2-(4-ﬂuorophenyl)ethanamine as described for 27a. The crude

1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-(2-(pyridin-4-yl)-

propyl)piperidine-4-carboxamide (27k). Synthesized from 7 and 2-

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material was triturated with ethyl acetate to obtain product as a white

solid. Yield: 85%. H NMR (400 MHz, DMSO-d₆) δ 7.84 (t, J = 5.7

NMR (400 MHz, DMSO-d₆) δ 7.63 (d, J = 7.8 Hz, 1H), 7.54 (d, J =

8.1 Hz, 1H), 7.38−7.30 (m, 2H), 7.26−7.17 (m, 1H), 7.16−7.05 (m,

3H), 6.73 (s, 1H), 5.50 (s, 2H), 4.63−3.86 (m, 2H), 3.47 (t, J = 6.7

Hz, 2H), 3.27 (t, J = 6.9 Hz, 2H), 3.13−2.80 (m, 2H), 2.77−2.64 (m,

1H), 1.87 (p, J = 6.7 Hz, 2H), 1.81−1.27 (m, 6H). TOF ES+ MS:

450.0 (M + H). HPLC retention time = 7.35 min; purity >95%.

(1-(4-Chlorobenzyl)-1H-indol-2-yl)(4-(morpholine-4-carbonyl)-

piperidin-1-yl)methanone (27x). Synthesized from 7 and morpholine

as described for 27a. Puriﬁed by ﬂash chromatography with a gradient

of 100% DCM to 5% 7 M ammonia in methanol diluted with 95%

1

Hz, 1H), 7.62 (d, J = 7.9 Hz, 1H), 7.54 (d, J = 8.4 Hz, 1H), 7.37−7.30

(m, 2H), 7.26−7.17 (m, 3H), 7.14−7.05 (m, 5H), 6.71 (s, 1H), 5.49

(s, 2H), 4.52−3.78 (m, 2H), 3.25 (q, J = 6.9 Hz, 2H), 2.91 (bs, 2H),

2.69 (t, J = 7.2 Hz, 2H), 2.38−2.28 (m, 1H), 1.62 (bs, 2H), 1.36 (bs,

2H). TOF ES+ MS: 518.1 (M + H), 540.1 (M + Na). HPLC retention

time = 7.91 min; purity >95%.

1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-(4-

methoxyphenethyl)piperidine-4-carboxamide (27r). Synthesized

from 7 and 2-(4-methoxyphenyl)ethanamine as described for 27a.

The crude material was triturated with ethyl acetate to obtain product

as a white solid. Yield: 63%. ¹H NMR (400 MHz, DMSO-d₆) δ 7.83 (t,

J = 5.6 Hz, 1H), 7.63 (d, J = 7.8 Hz, 1H), 7.54 (d, J = 8.4 Hz, 1H),

7.37−7.30 (m, 2H), 7.26−7.17 (m, 1H), 7.14−7.05 (m, 6H), 6.88−

6.81 (m, 2H), 6.71 (s, 1H), 5.49 (s, 2H), 4.49−3.84 (m, 2H), 3.71 (s,

3H), 3.22 (q, J = 6.8 Hz, 2H), 2.91 (bs, 2H), 2.63 (t, J = 7.3 Hz, 2H),

2.39−2.28 (m, 1H), 1.62 (bs, 2H), 1.37 (bs, 2H). TOF ES+ MS: 530.1

(M + H), 552.1 (M + Na). HPLC retention time = 7.81 min; purity

>95%.

1

DCM. The product was obtained as a white solid. Yield: 73%. H

NMR (400 MHz, DMSO-d₆) δ 7.63 (d, J = 7.8 Hz, 1H), 7.54 (d, J =

8.4 Hz, 1H), 7.37−7.29 (m, 2H), 7.26−7.17 (m, 1H), 7.14−7.05 (m,

3H), 6.73 (s, 1H), 5.49 (s, 2H), 4.43 (bs, 1H), 4.01 (bs, 1H), 3.58−

3.38 (m, 8H), 3.11−2.85 (m, 3H), 1.77−1.20 (m, 4H). TOF ES+ MS:

466.1 (M + H). HPLC retention time = 7.00 min; purity >95%.

N-Benzyl-1-(1-(4-chlorobenzyl)-1H-indole-2-carbonyl)-N-methyl-

piperidine-4-carboxamide (28a). Compound 7⁷(150 mg, 0.378

mmol), HOBt (102 mg, 0.756 mmol), and EDCI (145 mg, 0.756

mmol) were dissolved in 1.5 mL of DCM. The reaction was allowed to

stir for 10 min before N-methyl-1-phenylmethanamine (45.8 mg,

0.378 mmol) and DIEA (0.132 mL, 0.756 mmol) were added. The

reaction was stirred for two days before it was diluted with water and

ethyl acetate. The layers were separated, and the organic layer was

washed with the following saturated aqueous solutions: citric acid,

sodium bicarbonate, and sodium chloride. The organic layer was dried

over magnesium sulfate, ﬁltered, and concentrated. The crude mixture

was puriﬁed using 50% ethyl acetate/hexanes. The product was

1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-(4-

chlorophenethyl)piperidine-4-carboxamide (27s). Synthesized from

7 and 2-(4-chlorophenyl)ethanamine as described for 27a. The crude

material was triturated in ethyl acetate to give the product as a white

1

solid. Yield: 45%. H NMR (400 MHz, DMSO-d₆) δ 7.85 (t, J = 5.7

Hz, 1H), 7.63 (d, J = 7.8 Hz, 1H), 7.54 (d, J = 8.4 Hz, 1H), 7.37−7.30

(m, 4H), 7.26−7.17 (m, 3H), 7.14−7.06 (m, 3H), 6.71 (s, 1H), 5.49

(s, 2H), 4.54−3.86 (m, 2H), 3.26 (q, J = 6.7 Hz, 2H), 2.90 (bs, 2H),

2.69 (t, J = 7.1 Hz, 2H), 2.36−2.30 (m, 1H), 1.76−1.22 (m, 4H). TOF

ES+ MS: 534.1 (M + H), 556.1 (M + Na). HPLC retention time =

8.25 min; purity >95%.

1

obtained as a white solid. Yield: 64 mg (34%). H NMR (400 MHz,

DMSO-d₆) 1.5:1 mixture of rotamers δ 7.67−7.59 (m, 1H), 7.58−7.50

(m, 1H), 7.41−7.17 (m, 8H), 7.14−7.07 (m, 3H), 6.75 (rotamer A, s),

6.71 (rotamer B, s, 1H), 5.53−5.47 (m, 2H), 4.65 (rotamer A, s, 1H),

4.53−4.33 (m, 2H), 4.01 (bs, 1H), 3.20−2.89 (m, 5H), 2.78 (rotamer

B, s, 1H), 1.52 (bm, 4H). TOF ES+ MS: 500 (M + H). HPLC

retention time = 8.23 min; purity = 94%.

1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N-(4-

isopropylphenethyl)piperidine-4-carboxamide (27t). Synthesized

from 7 and 2-(4-isopropylphenyl)ethanamine as described for 27a.

The crude material puriﬁed using medium pressure silica gel

chromatography with a gradient of 0−60% ethyl acetate/hexanes to

N-Benzyl-N-methyl-1-(1-(4-nitrobenzyl)-1H-indole-2-carbonyl)-

piperidine-4-carboxamide (28b). Compound 10 (50 mg, 0.133

mmol) was added to a suspension of sodium hydride (60 wt % in oil,

8.0 mg, 0.20 mmol) in DMF (2 mL). The reaction was allowed to stir

for 10 min at 60 °C. 1-(Chloromethyl)-4-nitrobenzene (45.7 mg,

0.266 mmol) and potassium iodide (44.2 mg, 0.266 mmol) were

added, and the reaction was stirred overnight at 60 °C. The reaction

was diluted with water and ethyl acetate. The organic layer was washed

with water and saturated sodium chloride solution. It was dried over

magnesium sulfate, ﬁltered, and concentrated. The crude material was

puriﬁed using 40−100% ethyl acetate/ hexanes to aﬀord the product

1

obtain product as a white solid. Yield: 37%. H NMR (500 MHz,

DMSO-d₆) δ 7.89 (t, J = 5.7 Hz, 1H), 7.62 (d, J = 7.9 Hz, 1H), 7.55

(d, J = 8.3 Hz, 1H), 7.37−7.31 (m, 2H), 7.26−7.18 (m, 1H), 7.17−

7.07 (m, 7H), 6.71 (s, 1H), 5.49 (s, 2H), 4.52−3.83 (m, 2H), 3.24 (q,

J = 6.9 Hz, 2H), 3.10−2.75 (m, 3H), 2.66 (t, J = 7.4 Hz, 2H), 2.39−

2.31 (m, 1H), 1.85−1.10 (m, 10H). TOF ES+ MS: 542.2 (M + H),

564.1 (M + Na). HPLC retention time = 8.71 min; purity >95%.

(S)-(1-(4-Chlorobenzyl)-1H-indol-2-yl)(4-(2-(hydroxymethyl)-

pyrrolidine-1-carbonyl)piperidin-1-yl)methanone (27u). Synthesized

from 7 and ^L-prolinol as described for 27a. The crude material was

puriﬁed using ﬂash chromatography with a gradient of 100% DCM to

5% 7 M ammonia in methanol diluted with 95% DCM. The product

was obtained as a white solid. Yield: 98%. ¹H NMR (400 MHz,

DMSO-d₆) δ 7.62 (d, J = 8.1 Hz, 1H), 7.58−7.50 (m, 1H), 7.39−7.30

(m, 2H), 7.26−7.17 (m, 1H), 7.17−7.05 (m, 3H), 6.73 (s, 1H), 5.50

(s, 2H), 4.60−3.87 (m, 3H), 3.53−3.43 (m, 2H), 3.29−3.16 (m, 1H),

3.14−2.77 (m, 2H), 2.72−2.64 (m, 1H), 1.98−1.19 (m, 8H). TOF ES

+ MS: 480.1 (M + H). HPLC retention time = 6.82 min; purity =

94%.

1-(1-(4-Chlorobenzyl)-1H-indole-2-carbonyl)-N,N-dimethylpiperi-

dine-4-carboxamide (27v). Synthesized from 7 and dimethylamine as

described for 27a. Puriﬁed by ﬂash chromatography with a gradient of

100% DCM to 5% 7 M ammonia in methanol diluted with 95% DCM.

The product was obtained as a white solid. Yield: 68%. 1H NMR (400

MHz, DMSO-d₆) δ 7.63 (d, J = 7.8 Hz, 1H), 7.54 (d, J = 8.4 Hz, 1H),

7.38−7.27 (m, 2H), 7.26−7.17 (m, 1H), 7.13−7.08 (m, 3H), 6.73 (s,

1H), 5.50 (s, 2H), 4.44 (bs, 1H), 3.99 (bs, 1H), 3.19−2.84 (m, 6H),

2.81 (s, 3H), 1.62 (bs, 2H), 1.36 (bs, 2H). TOF ES+ MS: 424.0 (M +

H). HPLC retention time = 7.09 min; purity >95%.

1

as a white solid. Yield: 11 mg, 16%. H NMR (400 MHz, DMSO-d₆)

1.7:1 mixture of rotamers δ 8.20−8.11 (m, 2H), 7.70−7.62 (m, 1H),

7.55 (d, J = 8.4 Hz, 1H), 7.40−7.10 (m, 9H), 6.80 (rotamer A, s), 6.77

(rotamer B, s, 1H), 5.70−5.63 (m, 2H), 4.62 (rotamer A, s, 1H),

4.49−4.25 (m, 2H), 4.08 (bs, 1H), 3.18−2.87 (m, 5H), 2.74 (rotamer

B, s, 1H), 1.39 (bm, 4H). TOF ES+ MS: 511.1 (M + H). HPLC

retention time = 7.56 min; purity >95%.

N-Benzyl-1-(1-(4-cyanobenzyl)-1H-indole-2-carbonyl)-N-methyl-

piperidine-4-carboxamide (28c). Sodium iodide (99 mg, 0.660

mmol) and 4-(chloromethyl)benzonitrile (100 mg, 0.660 mmol)

were dissolved in acetone (2.2 mL) and allowed to stir at room

temperature for 4 h. The resulting solid was ﬁltered and the ﬁltrate

concentrated to obtain crude 4-(iodomethyl)benzonitrile (130 mg,

0.535 mmol, 81% yield) as an orange oil. It was taken directly to the

next step without characterization and puriﬁcation. Compound 10

(100 mg, 0.266 mmol) was dissolved in 1.0 mL of DMF and added to

a suspension of sodium hydride (12.78 mg, 0.320 mmol) in 2.0 of

DMF. The reaction was stirred at 80 °C for 10 min before adding 4-

(iodomethyl)benzonitrile (129 mg, 0.533 mmol) dissolved in 2.0 mL

of DMF. The reaction was allowed to stir overnight. After 18 h, the

reaction was cooled to room temperature before diluting with water

and ethyl acetate. The organic phase was washed with water followed

by saturated sodium chloride, dried over magnesium sulfate, ﬁltered,

and concentrated. The crude material was puriﬁed via ﬂash column

(1-(4-Chlorobenzyl)-1H-indol-2-yl)(4-(pyrrolidine-1-carbonyl)-

piperidin-1-yl)methanone (27w). Synthesized from 7 and pyrrolidine

as described for 27a. Puriﬁed by ﬂash chromatography with a gradient

of 100% DCM to 5% 7 M ammonia in methanol diluted with 95%

1

DCM. The product was obtained as a white solid. Yield: 73%. H

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chromatography using a gradient of 50% ethyl acetate/hexanes to

stirred at 80 °C for 10 min before adding 1-iodo-2-methylpropane

(0.061 mL, 0.53 mmol). The reaction was allowed to stir overnight.

After 18 h, the reaction was cooled to room temperature before

diluting with water and ethyl acetate. The organic phase was washed

with water, followed by saturated sodium chloride, dried over

magnesium sulfate, ﬁltered, and concentrated. The crude material

was puriﬁed via column chromatography using 50% ethyl acetate/

hexanes to 100% ethyl acetate. The puriﬁcation provided the product

as a white solid. Yield: 28 mg (25%). ¹H NMR (400 MHz, DMSO-d₆)

δ 7.64−7.50 (m, 2H), 7.42−7.15 (m, 6H), 7.12−7.03 (m, 1H), 6.67

(rotamer A, s), 6.64 (rotamer B, s, 1H), 4.67 (rotamer A, s, 1H),

4.60−4.26 (s, 3H), 4.24−3.98 (m, 2H), 3.00 (s, 5H), 2.79 (s, 1H),

2.06−1.92 (m, 1H), 1.78 (bs, 1H), 1.72−1.49 (m, 3H), 0.84−0.71 (m,

6H). TOF ES+ MS: 432.2 (M + H), 454.2 (M + Na). HPLC retention

time = 7.80 min; purity = 94%.

100% ethyl acetate. The puriﬁcation provided product as a white solid.

1

Yield: 50 mg, 38%. H NMR (400 MHz, DMSO-d₆) 1.7:1 mixture of

rotamers δ 7.78−7.70 (m, 2H), 7.69−7.61 (m, 1H), 7.52 (d, J = 8.4

Hz, 1H), 7.43−7.07 (m, 9H), 6.78 (rotamer A, s,), 6.75 (rotamer B, s,

1H), 5.64−5.57 (m, 2H), 4.64 (rotamer A, s, 1H), 4.52−4.25 (m, 2H),

4.04 (bs, 1H), 3.18−2.84 (m, 5H), 2.78 (rotamer B, s, 1H), 1.78−1.17

(m, 4H). TOF ES+ MS: 491.1 (M + H). HPLC retention time = 7.54

min; purity = 94%.

N-Benzyl-1-(1-(4-methoxybenzyl)-1H-indole-2-carbonyl)-N-

methylpiperidine-4-carboxamide (28d). Compound 10 (100 mg,

0.266 mmol) was added to a suspension of sodium hydride (12.8 mg,

0.32 mmol) in THF (1.5 mL). The reaction was stirred for 10 min

before 1-(bromomethyl)-4-methoxybenzene (0.039 mL, 0.27 mmol)

was added. The reaction was allowed to stir at room temperature

overnight. The reaction was diluted with water and extracted with

ethyl acetate. The organic layer was washed with water and saturated

sodium chloride solution. It was dried over magnesium sulfate, ﬁltered,

and concentrated. The crude material was puriﬁed by ﬂash

chromatography using a gradient of 50−100% ethyl acetate in hexanes

N-Benzyl-1-(1-benzyl-1H-indole-2-carbonyl)-N-methylpiperidine-

4-carboxamide (28h). Compound 10 (50 mg, 0.133 mmol) was

dissolved in 0.5 mL of DMF and cooled to 0 °C. Lithium

bis(trimethylsilyl)amide (0.146 mL, 0.146 mmol) was added dropwise,

and the reaction was allowed to stir for 10 min. Benzyl bromide (0.024

mL, 0.20 mmol) was added to the reaction, and it was allowed to stir

overnight at room temperature. The reaction was quenched with

water. It was then extracted with ethyl acetate. The organic extracts

were washed with saturated sodium chloride solution three times and

dried over magnesium sulfate, ﬁltered, and concentrated. The crude

material was puriﬁed through ﬂash silica gel chromatography using

25% EtOAc/DCM to give the product as a white solid. Yield: 41 mg

1

to obtain the product as a white solid. Yield: 42 mg (32%). H NMR

(400 MHz, DMSO-d₆) 1.7:1 mixture of rotamers δ 7.66−7.57 (m,

2H), 7.40−7.04 (m, 8H), 6.85−6.81 (m, 3H), 6.69 (rotamer A, s),

6.65 (rotamer B, s, 1H), 5.46−5.39 (m, 2H), 4.64 (rotamer A, s, 1H),

4.53−4.20 (m, 2H), 3.97 (bs, 1H), 3.69−3.66 (m, 3H), 3.19−2.83 (m,

5H), 2.78 (rotamer B, s, 1H), 1.82−1.24 (m, 4H). TOF ES+ MS:

496.1 (M + H), 518.1 (M + Na). HPLC retention time = 7.83 min;

purity = 92%.

1

(66%). H NMR (400 MHz, DMSO-d₆) 1.6:1 mixture of rotamers δ

7.68−7.52 (m, 2H), 7.45−6.98 (m, 12H), 6.72 (rotamer A, s), 6.69

(rotamer B, s, 1H), 5.54−5.41 (m, 2H), 4.64 (s, 1H), 4.52−4.23 (m,

2H), 4.01 (bs, 1H), 3.18−2.85 (m, 5H), 2.78 (rotamer B, s, 1H),

1.73−1.23 (m, 4H). TOF ES+ MS: 466.2 (M + H), 488.2 (M + Na).

HPLC retention time = 7.86 min; purity >95%.

1-(1-Acetyl-1H-indole-2-carbonyl)-N-benzyl-N-methylpiperidine-

4-carboxamide (28e). A solution of compound 10 (100 mg, 0.27

mmol) in 1 mL of DMF was added to a suspension of sodium hydride

(11.7 mg, 0.29 mmol) in 2.0 mL of DMF. The reaction was stirred at

80 °C for 10 min before adding acetic anhydride (0.050 mL, 0.53

mmol). The reaction was allowed to stir for 5 h before it was cooled to

room temperature and diluted with ammonium chloride and ethyl

acetate. The organic phase was washed with saturated ammonium

chloride twice, followed by saturated sodium chloride, dried over

magnesium sulfate, ﬁltered, and concentrated. The crude material was

puriﬁed via ﬂash chromatography using a gradient of 50% ethyl

acetate/hexanes to 100% ethyl acetate. Puriﬁcation aﬀorded the

product as a white. Yield: 13 mg (12%). ¹H NMR (400 MHz, DMSO-

d₆) 1.6:1 mixture of rotamers δ 8.15−8.07 (m, 1H), 7.69−7.61 (m,

1H), 7.44−7.15 (m, 6H), 6.91 (rotamer A, s,), 6.88 (rotamer B, s, 1H),

4.67 (rotamer A, s, 1H), 4.56−4.33 (m, 2H), 4.00−3.84 (m, 1H),

3.21−2.85 (m, 5H), 2.79 (s, 1H), 2.61 (rotamer A, s, 2H), 2.59

(rotamer B, s, 1H), 1.91−1.48 (m, 4H). TOF ES+ MS: 418.1 (M +

H), 440.1 (M + Na). HPLC retention time = 6.52 min; purity >95%.

N-Benzyl-1-(1-(2-methoxyethyl)-1H-indole-2-carbonyl)-N-meth-

ylpiperidine-4-carboxamide (28f). Compound 10 (50 mg, 0.13

mmol) dissolved in 0.5 mL of DMF was added to a suspension of

sodium hydride (7.99 mg, 0.200 mmol) in 1.0 mL of DMF. The

reaction was heated at 70 °C for 10 min before adding 2-methoxyethyl

4-methylbenzenesulfonate (0.144 mL, 0.266 mmol). The reaction was

allowed to stir overnight. After 18 h, the reaction was cooled to room

temperature before diluting with water and ethyl acetate. The organic

phase was washed with water followed by saturated sodium chloride,

dried over magnesium sulfate, ﬁltered, and concentrated. The crude

material was puriﬁed via ﬂash column chromatography using a gradient

of 50−80% ethyl acetate/hexanes. The puriﬁcation provided the

N-Benzyl-N-methyl-1-(1-(pyridin-4-ylmethyl)-1H-indole-2-

carbonyl)piperidine-4-carboxamide (28i). Pyridin-4-ylmethanol (180

mg, 1.65 mmol) and TEA (0.22 mL, 1.6 mmol) were dissolved in

THF (7 mL) and cooled to 0 °C. Methanesulfonyl chloride (0.125

mL, 1.6 mmol) was added dropwise, and the reaction was allowed to

warm to room temperature and stirred overnight. To a suspension of

60 wt % sodium hydride (21.5 mg, 0.538 mmol) in 3.5 mL of DMF,

compound 10 (200 mg, 0.533 mmol) was added as a 1.0 mL DMF

solution. The reaction was allowed to stir for 10 min before a 0.5 mL

DMF solution of mesylated alcohol was added. The reaction was

stirred at room temperature overnight. After 15 h, the reaction was

diluted with water and ethyl acetate. The aqueous phase was washed

with another aliquot of ethyl acetate. The combined organic phases

were washed with saturated sodium chloride, dried over magnesium

sulfate, ﬁltered, and concentrated. The crude black oil was puriﬁed via

SP1 Biotage using a gradient of 100% ethyl acetate to 10% methanol/

ethyl acetate. The product isolated as a white solid. Yield: 25 mg

1

(10%). H NMR (500 MHz, DMSO-d₆) 1.8:1 mixture of rotamers δ

8.48−8.42 (m, 2H), 7.69−7.62 (m, 1H), 7.51−7.45 (m, 1H), 7.41−

7.08 (m, 7H), 7.02−6.96 (m, 2H), 6.80 (rotamer A, s), 6.77 (rotamer

B, s, 1H), 5.59−5.53 (m, 2H), 4.65 (rotamer A, s, 1H), 4.52−4.48 (m,

2H), 4.11 (bs, 1H), 3.17 (d, J = 5.3 Hz, 2H), 3.10−2.87 (m, H), 2.77

(rotamer B, s, 1H), 1.86−1.33 (bm, 4H). TOF ES+ MS: 467.0 (M +

H). HPLC retention time = 5.52 min; purity >95%.

N-Benzyl-N-methyl-1-(1-(4-(triﬂuoromethyl)benzyl)-1H-indole-2-

carbonyl)piperidine-4-carboxamide (28j). Compound 10 (200 mg,

0.533 mmol), Cs₂CO₃(347 mg, 1.065 mmol), and 1-(bromomethyl)-

4-(triﬂuoromethyl)benzene (0.124 mL, 0.799 mmol) were dissolved in

DMF (5.0 mL) and heated to 70 °C overnight. The reaction was

diluted with water and ethyl acetate and ether (1:1). The aqueous

layer was back extracted with ethyl acetate. The organic phase was

washed with saturated sodium chloride (3×), dried over sodium

sulfate, ﬁltered, and concentrated. The resulting crude material was

puriﬁed via SP1 Biotage with a gradient of 30% ethyl acetate/hexanes

to 100% ethyl acetate. The product was isolated as a white solid. Yield:

90 mg (32%). ¹H NMR (500 MHz, DMSO-d₆) 2:1 mixture of

rotamers δ 7.68−7.61 (m, 3H), 7.55−7.49 (m, 1H), 7.41−7.07 (m,

1

product as a white solid. Yield: 20 mg (35%). H NMR (400 MHz,

DMSO-d₆) 1.7:1 mixture of rotamers δ 7.63−7.50 (m, 2H), 7.42−7.15

(m, 6H), 7.13−7.03 (m, 1H), 6.64 (rotamer A, s), 6.60 (rotamer B, s,

1H), 4.68 (s, 1H), 4.61−4.37 (m, 4H), 4.18 (bs, 1H), 3.62−3.44 (m,

2H), 3.19−3.14 (m, 3H), 3.11−2.92 (m, 5H), 2.79 (s, 1H), 1.89−1.48

(m, 4H). TOF ES+ MS: 434.2 (M + H), 456.1 (M + Na). HPLC

retention time = 7.03 min; purity >95%.

N-Benzyl-1-(1-isobutyl-1H-indole-2-carbonyl)-N-methylpiperi-

dine-4-carboxamide (28g). Compound 10 (100 mg, 0.27 mmol) was

dissolved in 1.0 mL of DMF and added to a suspension of sodium

hydride (12.8 mg, 0.32 mmol) in 2.0 mL of DMF. The reaction was

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9H), 6.79 (rotamer A, s), 6.75 (rotamer B, s, 1H), 5.64−5.59 (m, 2H),

4.64 (s, 1H), 4.51−4.25 (m, 1H), 4.09 (bs, 1H), 2.96 (s, 5H), 2.77 (s,

1H), 1.86−1.22 (m, 4H). TOF ES+ MS: 534.0 (M + H), 556.0 (M +

Na). HPLC retention time = 8.25 min; purity = 92%.

19 mg (30%). ¹H NMR (500 MHz, DMSO-d₆) δ 8.48−8.43 (m, 2H),

7.92 (t, J = 5.7 Hz, 1H), 7.74 (d, J = 8.8 Hz, 1H), 7.61 (d, J = 8.1 Hz,

1H), 7.42−7.19 (m, 8H), 5.61−5.49 (m, 2H), 4.45 (d, J = 13.3 Hz,

1H), 3.98 (d, J = 13.6 Hz, 1H), 3.35−3.28 (m, 2H), 3.10−3.00 (m,

1H), 2.92−2.83 (m, 1H), 2.73 (t, J = 7.0 Hz, 2H), 2.41−2.31 (m, 1H),

1.79−1.31 (m, 4H). TOF ES+ MS: 502.1 (M + H), 524.1 (M + Na).

HPLC retention time = 5.20 min; purity >95%.

1-(1-(4-Chlorobenzyl)-1H-pyrrole-2-carbonyl)-N-(2-(pyridin-4-yl)-

ethyl)piperidine-4-carboxamide (29a). The following were added

sequentially to DCM: 13 (600 mg, 1.73 mmol), TEA (0.725 mL, 5.19

mmol), EDCI (365 mg, 1.903 mmol), and HOBt (291 mg, 1.903

mmol). This was allowed to stir at rt for 30 min, at which time

pyridylethylamine (0.227 mL, 1.90 mmol) was added. Stirring

continued for 18 h. At this time, the DCM and TEA were stripped

oﬀ and the residue was taken up in EtOAc and washed with 10%

aqueous sodium carbonate (3×). The organic phase was collected,

dried over magnesium sulfate, and concentrated in vacuo. The

resulting solid/oil mixture was recrystallized from EtOAc to aﬀord the

title compound as small, white crystals. Yield: 586 mg (75%). ¹H

NMR (400 MHz, CDCl₃) δ 8.50 (d, J = 5.7 Hz, 2H), 7.21 (d, J = 8.3

Hz, 2H), 7.09 (d, J = 5.6 Hz, 2H), 7.02 (d, J = 8.2 Hz, 2H), 6.78 (s,

1H), 6.30 (dd, J = 3.6, 1.2 Hz, 1H), 6.13−6.09 (m, 1H), 5.48 (t, J = 5.4

Hz, 1H), 5.26 (s, 2H), 4.32 (d, J = 12.7 Hz, 2H), 3.53 (q, J = 6.8 Hz,

2H), 2.82 (t, J = 7.0 Hz, 4H), 2.19 (tt, J = 11.2, 3.6 Hz, 1H), 1.68 (d, J

= 14.4 Hz, 2H), 1.35 (q, J = 11.0, 9.8 Hz, 2H). Anal. Calcd for

C₂₅H₂₇ClN₄O₂: C, 66.58%; H, 6.04%; N, 12.42%. Found: C, 66.68%;

H, 6.32%; N, 12.38%. TOF ES+ MS: (M + H) 451.2, (M + Na) 473.2.

HPLC retention time = 5.03 min, >95% purity.

N1-(4-Chlorophenethyl)-N4-(2-(pyridin-4-yl)ethyl)piperidine-1,4-

dicarboxamide (29d). Ethyl 1-((4-chlorophenethyl)carbamoyl)-

piperidine-4-carboxylate 20 (807 mg, 2.382 mmol) was dissolved in

EtOH (20 mL), and 10% aq NaOH (15 mL) was added. The resulting

mixture was warmed to 50 °C and stirred for 2 h. The now

homogeneous solution was then concentrated in vacuo, a small

amount of water was added back, and the solution chilled in an ice

bath. The solution was then acidiﬁed with concentrated HCl, and the

resulting precipitate was collected over a ﬁlter, washed with 1 M HCl

and ice-cold water, ﬁlter-dried, then further dried under high vacuum

to aﬀord 1-((4-chlorophenethyl)carbamoyl)piperidine-4-carboxylic

1

acid as a white powder. Yield: 631 mg (85%). H NMR (400 MHz,

DMSO-d₆) δ 12.21 (s, 1H), 7.30 (d, J = 8.4 Hz, 2H), 7.18 (d, J = 8.4

Hz, 2H), 6.54 (t, J = 5.4 Hz, 1H), 3.80 (d, J = 13.3 Hz, 2H), 3.18 (q, J

= 7.0 Hz, 2H), 2.76−2.62 (m, 4H), 2.36 (ddt, J = 11.0, 7.6, 3.9 Hz,

1H), 1.75−1.64 (m, 2H), 1.40−1.25 (m, 2H).

1-((4-Chlorophenethyl)carbamoyl)piperidine-4-carboxylic acid (60

mg, 0.193 mmol), TEA (81 μL, 0.579 mmol), EDCI (41 mg, 0.212

mmol), HOBt (33 mg, 0.212 mmol), and 4-(2-aminoethyl)pyridine

(25 μL, 0.212 mmol) were all added sequentially to DCM (5 mL) at

RT. This solution was allowed to stir for 16 h at RT, after which time

the reaction was diluted with ethyl acetate and washed with water

(3×), 10% aq sodium carbonate (3×), and brine (1×), then dried

(magnesium sulfate) and concentrated in vacuo. The resulting residue

was then crystallized from ethyl acetate and washed with diethyl ether

1-(1-(4-Chlorobenzyl)-1H-imidazole-2-carbonyl)-N-(2-(pyridin-4-

yl)ethyl)piperidine-4-carboxamide (29b). The following were added

sequentially to DCM (5 mL): 18b (73 mg, 0.309 mmol), 16 (100 mg,

0.371 mmol), DIEA (162 μL, 0.927 mmol), EDCI (65 mg, 0.340

mmol), and HOBt (52 mg, 0.340 mmol). The solution was allowed to

stir at RT for 24 h, after which time the DCM was stripped oﬀ in

vacuo and the resulting residue was taken up on 10% aq sodium

carbonate. Material was then extracted with ethyl acetate. The extract

was dried (magnesium sulfate) and concentrated in vacuo. The residue

was then triturated with ethyl acetate/hexanes and collected over a

ﬁlter. The impure yellow powder was then recrystallized from ethyl

acetate to provide the title compound as oﬀ-white crystals. Yield: 89

1

to provide the title compound as a tan solid. Yield: 46 mg (57%). H

NMR (400 MHz, CDCl₃) δ 8.61−8.26 (m, 2H), 7.30−7.19 (m, 2H),

7.17−7.01 (m, 4H), 5.91−5.66 (m, 1H), 4.59 (dd, J = 13.4, 5.6 Hz,

1H), 3.86 (d, J = 13.2 Hz, 2H), 3.53 (q, J = 6.7 Hz, 2H), 3.42 (q, J =

6.8 Hz, 2H), 2.82 (t, J = 6.8 Hz, 2H), 2.79−2.61 (m, 4H), 2.17 (t, J =

9.6 Hz, 1H), 1.73 (d, J = 12.9 Hz, 2H), 1.57 (qd, J = 12.9, 12.5, 3.8 Hz,

2H). TOF ES+ MS: (M + H) 415.2, (M + Na) 437.2. HPLC retention

time = 4.70 min, >95% purity.

1-(1-Methyl-1H-indole-2-carbonyl)-N-(2-(pyridin-4-yl)ethyl)-

piperidine-4-carboxamide (29e). 26e (210 mg, 0.668 mmol) and

lithium hydroxide/H₂O (280 mg, 6.68 mmol) were dissolved in 2/1

water/THF (2.3 mL). The reaction was stirred for 6 h before it was

diluted with water and diethyl ether. The aqueous layer was acidiﬁed

using 2N HCl to pH ∼ 2. The suspension was extracted with ethyl

acetate. The organic phase was washed with saturated sodium chloride,

dried over magnesium sulfate, ﬁltered, and concentrated to obtain

product as a white solid. No puriﬁcation was performed on the

material. The crude acid was taken directly to the next step. TOF ES+

MS: 287.1 (M + H), 309.1 (M + Na). HPLC retention time = 5.60

min; purity = 94%.

1

mg (64%). H NMR (400 MHz, CDCl₃) δ 8.50 (d, J = 5.2 Hz, 2H),

7.28 (d, J = 8.3 Hz, 2H), 7.15−7.07 (m, 4H), 7.05 (s, 1H), 6.95 (s,

1H), 5.59 (t, J = 5.7 Hz, 1H), 5.35 (d, J = 8.9 Hz, 2H), 4.58 (d, J =

13.4 Hz, 2H), 3.53 (q, J = 6.7 Hz, 2H), 3.10 (t, J = 13.7 Hz, 1H),

2.87−2.71 (m, 3H), 2.29 (tt, J = 11.0, 3.4 Hz, 1H), 2.00−1.41 (m,

4H). TOF ES+ MS: (M + H) 452.2, (M + Na) 474.2. HPLC retention

time = 4.11 min, >90% purity.

1-(1-(4-Chlorobenzyl)-1H-benzo[d]imidazole-2-carbonyl)-N-(2-

(pyridin-4-yl)ethyl)piperidine-4-carboxamide (29c). 22c (60 mg,

0.141 mmol) and lithium hydroxide/H₂O (23.6 mg, 0.564 mmol)

were dissolved in 2/1 water/THF (0.75 mL). The reaction was stirred

for 2 h before it was diluted with water and diethyl ether. The aqueous

phase was washed with diethyl ether twice. It was then acidiﬁed to pH

∼ 2 and extracted with ethyl acetate. The organic phase was washed

with saturated sodium chloride, dried over magnesium sulfate, ﬁltered,

and concentrated to obtain crude acid as a white solid. No further

1

1-(1-Methyl-1H-indole-2-carbonyl)piperidine-4-carboxylic acid

(100 mg, 0.349 mmol), EDCI (100 mg, 0.524 mmol), and HOBt

(70.8 mg, 0.524 mmol) were dissolved in DCM (volume, 3.0 mL).

The reaction was stirred for 10 min before 2-(pyridin-4-yl)ethanamine

(0.059 mL, 0.524 mmol) and DIEA (0.091 mL, 0.524 mmol). The

reaction was stirred overnight at room temperature. The reaction was

diluted with water and ethyl acetate. The organic layer was washed

with saturated sodium bicarbonate solution and ﬁnally saturated

sodium chloride. the organic layer was then dried over magnesium

sulfate, ﬁltered, and concentrated. The resulting solid was triturated in

puriﬁcation was performed. Yield: 54 mg, 96%. H NMR (400 MHz,

DMSO-d₆) δ 12.31 (s, 1H), 7.74 (d, J = 7.8 Hz, 1H), 7.63 (d, J = 7.7

Hz, 2H), 7.41−7.20 (m, 6H), 5.55 (s, 2H), 4.39−4.30 (m, 1H), 3.95−

3.86 (m, 1H), 3.21−3.09 (m, 1H), 3.06−2.95 (m, 1H), 1.98−1.85 (m,

1H), 1.76−1.68 (m, 1H), 1.50−1.42 (m, 1H), 1.30−1.22 (m, 1H).

TOF ES+ MS: 398.1 (M + H), 420.1 (M + Na). HPLC retention time

= 6.01 min; purity >95%.

The crude acid from above (50 mg, 0.13 mmol), EDCI (48.2 mg,

0.251 mmol), and HOBt (34.0 mg, 0.251 mmol) were dissolved in

DCM (2.0 mL). The reaction was allowed to stir for 10 min before the

addition of DIEA (0.044 mL, 0.251 mmol) and 2-(pyridin-4-

yl)ethanamine (0.030 mL, 0.251 mmol). The reaction was allowed

to stir overnight. The reaction was diluted with water and ethyl acetate.

The organic layer was washed with saturated sodium bicarbonate and

saturated sodium chloride. The ethyl acetate layer was dried over

magnesium sulfate, ﬁltered, and concentrated. The crude material was

triturated in diethyl ether/ethyl acetate to obtain a white solid. Yield:

1

ethyl acetate to give 29e. Yield: 50 mg (38%). H NMR (400 MHz,

DMSO-d₆) δ 8.49−8.42 (m, 2H), 7.94 (t, J = 5.6 Hz, 1H), 7.60 (d, J =

7.8 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.29−7.19 (m, 3H), 7.09 (t, J =

7.4 Hz, 1H), 6.62 (s, 1H), 4.40 (bs, 1H), 4.02 (bs, 1H), 3.74 (s, 3H),

3.32−3.29 (m, 2H), 3.04 (bs, 2H), 2.73 (t, J = 7.0 Hz, 2H), 2.41−2.36

(m, 1H), 1.71−1.66 (m, 2H), 1.57−1.46 (m, 2H). TOF ES+ MS:

391.1 (M + H). HPLC retention time = 4.68 min; purity >95%.

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1-(1-Benzyl-1H-indole-2-carbonyl)-N-(2-(pyridin-4-yl)ethyl)-

piperidine-4-carboxamide (29f). 24f (48.5 mg, 0.193 mmol), DMAP

(23.56 mg, 0.193 mmol), and EDCI (37.0 mg, 0.193 mmol) were

dissolved in THF (3.0 mL). The reaction remained a suspension after

30 min. Amine 16 (30 mg, 0.129 mmol) was added followed by DIEA

(0.034 mL, 0.193 mmol). The reaction was stirred overnight at room

temperature. The reaction was diluted with water and ethyl acetate.

The organic phase was washed with water and saturated sodium

chloride, then it was dried over sodium sulfate. The resulting crude

material was puriﬁed using SP1 Biotage using a gradient of 30% ethyl

acetate/hexanes to 100% ethyl acetate. The product was isolated as a

2.26 (m, 1H), 1.71−1.18 (m, 4H). TOF ES+ MS: 519.0 (M + H).

HPLC retention time = 5.83 min; purity >95%.

1-(1-(4-Chlorobenzyl)-5-ﬂuoro-1H-indole-2-carbonyl)-N-(2-(pyri-

din-4-yl)ethyl)piperidine-4-carboxamide (29i). 26i (190 mg, 0.429

mmol) and lithium hydroxide/H₂O (180 mg, 4.29 mmol) were

dissolved in 2/1 water/THF (4.5 mL). The reaction was stirred

overnight at room temperature. After 16 h, the reaction was

concentrated until a suspension formed. It was cooled by an ice

bath and diluted with minimal amount of water to allow a stir bar to

move freely. 2N HCl was added dropwise until pH 2 was reached. The

resulting suspension was diluted with ethyl acetate. The aqueous phase

was washed with another aliquot of ethyl acetate. The organic phases

were combined and washed with saturated sodium chloride, ﬁltered,

and concentrated. No further puriﬁcation was performed. The product

1-(1-(4-chlorobenzyl)-5-ﬂuoro-1H-indole-2-carbonyl)piperidine-4-car-

1

white solid. Yield: 20 mg (33%). H NMR (500 MHz, DMSO-d₆) δ

8.48−8.43 (m, 2H), 7.88 (t, J = 5.6 Hz, 1H), 7.62 (dt, J = 7.8 Hz, 1H),

7.58 (dd, J = 8.4, 0.9 Hz, 1H), 7.29−7.17 (m, 7H), 7.13−7.05 (m,

4H), 6.69 (s, 1H), 5.49 (s, 2H), 4.40 (bs, 1H), 3.92 (bs, 1H), 3.30 (m,

2H), 2.89 (bs, 2H), 2.73 (t, J = 6.9 Hz, 2H), 2.35−2.25 (m, 1H),

1.74−1.21 (m, 4H). TOF ES+ MS: 467.2 (M + H), 489.2 (M + Na).

HPLC retention time = 5.30 min; purity >95%.

1

boxylic acid was isolated as a white solid. Yield: 129 mg, 73%. H

NMR (400 MHz, DMSO-d₆) δ 12.26 (s, 1H), 7.59 (dd, J = 9.0, 4.4

Hz, 1H), 7.40 (dd, J = 9.6, 2.6 Hz, 1H), 7.35−7.30 (m, 2H), 7.12−

7.05 (m, 3H), 6.71 (s, 1H), 5.48 (s, 2H), 4.28 (bs, 1H), 3.84 (bs, 1H),

3.00 (bs, 2H), 2.48−2.41 (m, 1H), 1.53 (bm, 4H). HPLC retention

time = 5.51 min; purity >95%.

1-(1-(2,6-Diﬂuorobenzyl)-1H-indole-2-carbonyl)-N-(2-(pyridin-4-

yl)ethyl)piperidine-4-carboxamide (29g). 24g (104 mg, 0.361

mmol), HOBt (56.3 mg, 0.417 mmol), and EDCI (80 mg, 0.417

mmol) were dissolved in THF (volume, 3.0 mL). The reaction was

stirred for 30 min. Solid 16 (30 mg, 0.13 mmol) was added followed

by DIEA (0.073 mL, 0.417 mmol). The reaction was stirred overnight

at room temperature. The reaction was diluted with water and ethyl

acetate. The organic phase was washed with water and saturated

sodium chloride; it was dried over sodium sulfate. The resulting crude

material was puriﬁed using SP1 Biotage using a gradient of 30% ethyl

acetate/hexanes to 100% ethyl acetate. The product was isolated as a

1-(1-(4-Chlorobenzyl)-5-ﬂuoro-1H-indole-2-carbonyl)piperidine-4-

carboxylic acid (106 mg, 0.256 mmol), DMAP (62.4 mg, 0.51 mmol),

and EDCI (98 mg, 0.51 mmol) were dissolved in DCM (5.0 mL). The

reaction was stirred for 10 min before adding 2-(pyridin-4-

yl)ethanamine (0.061 mL, 0.51 mmol) and DIEA (0.089 mL, 0.51

mmol). The reaction was stirred overnight at room temperature. It was

diluted with water and ethyl acetate. The organic phase was washed

with water, saturated sodium carbonate, and saturated sodium

chloride. It was ﬁltered and concentrated. The resulting crude material

was triturated with ethyl acetate. The resulting white solid was ﬁltered

to obtain the desired product. Yield: 80 mg, 60%. ¹H NMR (500 MHz,

DMSO-d₆) δ 8.48−8.43 (m, 2H), 7.90 (t, J = 5.7 Hz, 1H), 7.57 (dd, J

= 9.1, 4.4 Hz, 1H), 7.40 (dd, J = 9.5, 2.5 Hz, 1H), 7.36−7.33 (m, 2H),

7.23−7.20 (m, 2H), 7.13−7.07 (m, 3H), 6.70 (s, 1H), 5.48 (s, 2H),

4.40 (bs, 1H), 3.90 (bs, 1H), 3.34−3.30 (m, 22H), 3.04−2.70 (m,

4H), 2.36−2.27 (m, 1H), 1.67−1.18 (m, 4H). TOF ES+ MS: 519.1

(M + H), 541.1 (M + Na). HPLC retention time = 5.51 min; purity

>95%.

1-(1-(4-Chlorobenzyl)-1H-pyrrolo[2,3-c]pyridine-2-carbonyl)-N-

(2-(pyridin-4-yl)ethyl)piperidine-4-carboxamide (29j). 24j (50 mg,

0.174 mmol) was dissolved into DCM (2 mL) at RT, followed by 16

(59 mg, 0.192 mmol) and TEA (122 μL, 0.872 mmol). Once all

material was in solution, EDCI (37 mg, 0.192 mmol) and HOBt (29

mg, 0.192 mmol) were added and the reaction was allowed to stir for

18 h at RT. At this time, the reaction was diluted with a solution of

ethyl acetate/diethyl ether (1:1) and washed with water (3×), 10% aq

sodium carbonate (3×), and brine (1×), then dried (magnesium

sulfate) and concentrated in vacuo. The resulting residue was then

puriﬁed by ﬂash chromatography (50 g silica, 5% 7.0 M methanolic

ammonia/95% ethyl acetate) to give the title compound as white solid.

Yield: 53 mg (61%). ¹H NMR (400 MHz, CDCl₃) δ 8.78 (s, 1H), 8.49

(d, J = 5.4 Hz, 2H), 8.27 (d, J = 5.5 Hz, 1H), 7.51 (d, J = 5.5 Hz, 1H),

7.21 (d, J = 8.2 Hz, 2H), 7.09 (d, J = 5.3 Hz, 2H), 7.05 (d, J = 8.2 Hz,

2H), 6.56 (s, 1H), 5.67 (t, J = 5.6 Hz, 1H), 5.48 (s, 2H), 4.57 (s, 1H),

3.91 (s, 1H), 3.53 (q, J = 6.6 Hz, 2H), 2.96−2.69 (m, 4H), 2.21 (tt, J =

11.1, 7.5, 3.7 Hz, 1H), 1.88−1.69 (m, 1H), 1.67−1.46 (m, 2H), 1.33−

1.22 (m, 1H). TOF ES+ MS: (M + H) 502.2, (M + Na) 524.2. HPLC

retention time = 4.01 min, >95% purity.

1

white solid. Yield: 68 mg (49%). H NMR (500 MHz, DMSO-d₆) δ

8.49−8.44 (m, 2H), 7.91 (t, J = 5.6 Hz, 1H), 7.61−7.55 (m, 2H),

7.41−7.31 (m, 1H), 7.28−7.20 (m, 3H), 7.12−7.01 (m, 3H), 6.67 (s,

1H), 5.63 (s, 4H), 4.42 (bs, 1H), 3.97 (bs, 1H), 3.37−3.29 (m, 3H),

3.03−2.69 (m, 4H), 2.37−2.27 (m, 1H), 1.73−1.31 (m, 4H). TOF ES

+ MS: 503.2 (M + H). HPLC retention time = 5.37 min; purity =

92%.

1-(1-(4-Chlorobenzyl)-6-ﬂuoro-1H-indole-2-carbonyl)-N-(2-(pyri-

din-4-yl)ethyl)piperidine-4-carboxamide (29h). 22h (140 mg, 0.316

mmol) and lithium hydroxide, H₂O (133 mg, 3.16 mmol) were

dissolved in 1/1 THF/water (4 mL). The reaction was stirred

overnight at room temperature. After 16 h, the reaction was

concentrated until a suspension formed. It was cooled by an ice

bath and diluted with minimal amount of water to allow a stir bar to

move freely. Then 2N HCl was added dropwise until pH 2 was

reached. The resulting suspension was diluted with ethyl acetate. The

aqueous phase was washed with another aliquot of ethyl acetate. The

organic phases were combined and washed with saturated sodium

chloride, ﬁltered, and concentrated. No further puriﬁcation was

performed. The product was isolated as a white solid. Yield: 106 mg,

1

81%. H NMR (400 MHz, DMSO-d₆) δ 12.28 (s, 1H), 7.64 (dd, J =

8.8, 5.5 Hz, 1H), 7.51 (dd, J = 10.5, 2.3 Hz, 1H), 7.37−7.29 (m, 2H),

7.12−7.05 (m, 2H), 7.03−6.93 (m, 1H), 6.75 (s, 1H), 5.47 (s, 2H),

4.26 (s, 1H), 3.87 (s, 1H), 3.00 (bs, 2H), 2.48−2.43 (m, 1H), 1.95−

1.21 (m, 4H). TOF ES+ MS: 415.0 (M + H), 437.0 (M + Na). HPLC

retention time = 7.02 min; purity >95%.

The crude acid from above (106 mg, 0.256 mmol), EDCI (98 mg,

0.511 mmol), and DMAP (62.4 mg, 0.511 mmol) were suspended in

DCM (volume, 5.0 mL). The reaction was stirred for 10 min before 2-

(pyridin-4-yl)ethanamine (0.061 mL, 0.511 mmol) and DIEA (0.089

mL, 0.511 mmol) were added. The reaction was stirred overnight. The

reaction was diluted with water and ethyl acetate. The organic phase

was washed with water, saturated sodium carbonate, and saturated

sodium chloride. It was dried over sodium sulfate, ﬁltered, and

concentrated. The crude material was triturated using ethyl acetate.

The white solid was ﬁltered to obtain the desired product 29h. Yield:

90 mg (68%). ¹H NMR (500 MHz, DMSO-d₆) δ 8.48−8.43 (m, 2H),

7.89 (t, J = 5.7 Hz, 1H), 7.64 (dd, J = 8.7, 5.5 Hz, 1H), 7.49 (dd, J =

10.4, 2.3 Hz, 1H), 7.38−7.32 (m, 2H), 7.24−7.18 (m, 2H), 7.13−7.08

(m, 2H), 7.01−6.93 (m, 1H), 6.74 (s, 1H), 5.47 (s, 2H), 4.37 (bs,

1H), 3.93 (bs, 1H), 3.34−3.28 (m, 2H), 3.04−2.70 (m, 4H), 2.36−

1-(1-(4-Chlorobenzyl)-4-ﬂuoro-1H-pyrrole-2-carbonyl)-N-(2-(pyri-

din-4-yl)ethyl)piperidine-4-carboxamide (29k). The following were

added sequentially to DCM (3 mL): 18k (50 mg, 0.20 mmol), TEA

(0.11 mL, 0.79 mmol), EDCI (45 mg, 0.24 mmol), HOBt (36 mg,

0.24 mmol), and then 16 (64 mg, 0.24 mmol). The reaction was

allowed to stir at room temperature for 14 h, at which time it was

diluted with ethyl acetate, washed with water (3×), 10% aq sodium

carbonate (3×), and brine (1×), dried with magnesium sulfate, and

concentrated in vacuo. The residue was the puriﬁed by ﬂash

chromatography (10 g silica, 1% 7 M methanolic ammonia/ethyl

acetate) to provide the desired material as a white powder. Yield: 48

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Journal of Medicinal Chemistry

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1

mg (52%). H NMR (400 MHz, CDCl₃) δ 8.54 (d, J = 4.3 Hz, 2H),

Author Contributions

^#S.D.L. and D.J.M. contributed equally to this work.

7.25−7.20 (m, 2H), 7.14 (d, J = 4.4 Hz, 2H), 7.06 (d, J = 6.9 Hz, 2H),

6.58−6.54 (m, 1H), 6.05 (s, 1H), 5.46−5.38 (m, 1H), 5.17 (s, 2H),

4.29 (d, J = 6.7 Hz, 2H), 3.55 (q, J = 7.3 Hz, 2H), 2.83 (dt, J = 17.9,

9.0 Hz, 4H), 2.27−2.15 (m, 1H), 1.77−1.63 (m, 2H), 1.38 (d, J = 13.4

Hz, 2H). TOF ES+ MS: (M + H) 469.2, (M + Na) 491.2. HPLC

retention time = 5.18 min, >95% purity.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

Kinetic Solubility Assay. The experimental media contained 1

mM ^L-glutamine, 10 U/mL penicillin, 10 μg/mL streptomycin, 0.1

mM nonessential amino acids, 110 μg/mL sodium pyruvate, and 5%

fetal bovine sera in high-glucose DMEM solution. Compounds were

dissolved in DMSO to generate 25 mM solutions. Then 20 μL of each

stock solution was serially diluted with DMSO through 12 50%

dilutions. Then 1 μL of each working solution was added to one well

of a clear, round-bottom 96-well plate containing 199 μL of media.

This resulted in 0.12−250 μM testing concentrations. The plate was

placed in a Molecular Devices SpectraMax Plus UV/vis spectropho-

tometer and shaken for 15 s. Measurement of the OD₆₀₀resulted in a

series of curves. The concentration at which the average (n = 3 for

each concentration and test compound) OD₆₀₀reading rose above

background was noted, and the aqueous solubility is reported as the

mean concentration between this point and the previous point.

Parallel Artiﬁcial Membrane Permeability Assay (PAMPA).

Compounds were dissolved in DMSO to generate 10 mM compound

solution. The experiment was performed using the Double Sink

protocol provided by pION, Inc. with the PAMPA explorer system. A

cosolvent system with 20% ACN and 5 μL of BBB-1 lipid solution

were used for the experiment.

This work was supported by an NIH Partnerships for

Biodefense Viral Pathogens grant (R01 AI089417, Principal

Investigator D.J.M.) and a UM Rackham Merit Scholarship for

S.J.B. The VMCC is grateful for ongoing support from the Ella

and Hans Vahlteich Fund and Beverly Vahlteich Delaney. We

thank Dr. Brian Shay of the UM Biomedical Mass Spec Facility

for assistance with the LC/MS/MS analysis of samples from

the MLM stability assays. The authors also wish to acknowl-

edge the UM PK Core for LC/MS/MS analyses of the plasma

and brain samples from the mouse PK study.

ABBREVIATIONS USED

■

CNS, central nervous system; CPE, cytopathic eﬀect; EDC, N-

(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride;

MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide; NSV, neuroadapted Sindbis virus; VEEV, Venezuelan

equine encephalitis virus; WEEV, western equine encephalitis

virus; PAMPA, parallel artiﬁcial membrane permeability assay;

MLM, mouse liver microsome; Pgp, P-glycoprotein; MDR1,

multiple drug resistance protein 1; rho123, rhodamine123;

FMV, Fort Morgan virus; ADME, absorption, distribution,

metabolism, excretion

Mouse Liver Microsomal (MLM T_1/2) Stability Assay. Half-lives

for compounds incubated with Balb-C mouse liver microsomes were

determined as previously described.⁷

In Vitro Antiviral and Cytotoxicity Assays. WEEV replicon,

MTT and cultured human BE(2)-C neuronal cell infection assays were

performed as previously described.⁷

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■

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AUTHOR INFORMATION

Corresponding Authors

*For SDL: phone, 734 615 0454; E-mail, sdlarsen@umich.edu.

*For DJM: phone, 734-763-0565; E-mail: milldavi@umich.edu.

■

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Journal of Medicinal Chemistry

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Optimization of novel indole-2-carboxamide inhibitors of neurotropic alphavirus replication

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