1,3-Diarylpyrazolyl-acylsulfonamides as Potent Anti-tuberculosis Agents Targeting Cell Wall Biosynthesis in Mycobacterium tuberculosis

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1,3-Diarylpyrazolyl-acylsulfonamides as Potent Anti-tuberculosis

Agents Targeting Cell Wall Biosynthesis in Mycobacterium

tuberculosis

Lutete Peguy Khonde, Rudolf Muller, Grant A. Boyle, Virsinha Reddy, Aloysius T. Nchinda,

̈

Charles J. Eyermann, Stephen Fienberg, Vinayak Singh, Alissa Myrick, Efrem Abay, Mathew Njoroge,

Nina Lawrence, Qin Su, Timothy G. Myers, Helena I. M. Bosho ﬀ , Clifton E. Barry, III,

Frederick A. Sirgel, Paul D. van Helden, Lisa M. Massoudi, Gregory T. Robertson, Anne J. Lenaerts,

Gregory S. Basarab, Sandeep R. Ghorpade,* and Kelly Chibale *

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ABSTRACT: Phenotypic whole cell high-throughput screening of

a ∼150,000 diverse set of compounds against Mycobacterium

tuberculosis (Mtb) in cholesterol-containing media identiﬁed 1,3-

diarylpyrazolyl-acylsulfonamide 1 as a moderately active hit.

Structure−activity relationship (SAR) studies demonstrated a

clear scope to improve whole cell potency to MIC values of <0.5

μM, and a plausible pharmacophore model was developed to

describe the chemical space of active compounds. Compounds are bactericidal in vitro against replicating Mtb and retained activity

against multidrug-resistant clinical isolates. Initial biology triage assays indicated cell wall biosynthesis as a plausible mode-of-action

for the series. However, no cross-resistance with known cell wall targets such as MmpL3, DprE1, InhA, and EthA was detected,

suggesting a potentially novel mode-of-action or inhibition. The in vitro and in vivo drug metabolism and pharmacokinetics proﬁles

of several active compounds from the series were established leading to the identiﬁcation of a compound for in vivo eﬃcacy proof-of-

concept studies.

in combination regimens aimed at treatment shortening for

drug-sensitive TB. Gratifyingly, a few recently approved drugs

with novel MoAs such as bedaquiline,²pretomanid,³and

delamanid⁴have been added to the arsenal of the available TB

drugs. Several follow-up drug candidates such as the DprE1

inhibitors macozinone (PBTZ-169)⁵and TBA-7371,⁶the

MmpL3 inhibitor SQ-109,⁷the QcrB inhibitor telacebec

(Q203),⁸and the leucyl-tRNA synthetase inhibitor GSK

3036656⁹are in the clinical development pipeline (Figure

1). Other known drugs such as linezolid have also shown great

promise in the treatment of MDR-TB and have encouraged

preclinical eﬀorts to discover safer analogues.¹⁰Considering

the signiﬁcant attrition rates in clinical development and the

empirical nature of the eﬀective combination therapy toward

shorter treatments, there is a continuous need to replenish the

global TB drug pipeline with new bactericidal agents that avoid

INTRODUCTION

■

Tuberculosis (TB) continues to be a global epidemic most

severely aﬀecting the underprivileged sectors of society

throughout the world. According to the World Health

Organization 2019 global TB report, 10 million people fell

sick with TB in 2018 and 1.5 million people lost their lives.¹

The most common drug regimen currently used for the

treatment of drug-sensitive TB is 40 years old and consists of

four drugsrifampicin (RIF), isoniazid (INH), pyrazinamide

(PZA), and ethambutol (EMB)taken over a minimum

period of 6 months. The long treatment duration along with

unpleasant side eﬀects often lead to patient noncompliance,

resulting in the development of drug-resistant TB, viz.,

multidrug resistant (MDR) TB that is refractory to two

frontline drugs RIF and INH and extensively drug resistant

(XDR) TB that develops additional resistance to any

ﬂuoroquinolone and at least one of the three injectable

second-line drugs (amikacin, kanamycin, or capreomycin).

Treatments for drug-resistant TB are fraught with limited

choices of poorly eﬃcacious medicines with severe side eﬀects

and lengthy treatment durations of 18−24 months. Hence,

there has been a strong global drive toward identifying safe

new drugs with novel mode-of-actions (MoAs), which would

be eﬀective for treatment of drug-resistant TB and be included

Received: May 8, 2021

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Figure 1. New approved drugs and clinical candidate for TB.

preexisting clinical drug resistance either through novel MoA

or novel mode-of-inhibition (MoI) of clinically validated drug

targets.

intermediates released from nitrite.^14,17Among the few hits

that were conﬁrmed from the screening campaign, compound

1 was identiﬁed as a singleton with moderate activity under

various media conditions; notably, it did not show any

previous literature reports of antitubercular activity (Table 1).

Mycobacterium tuberculosis (Mtb), the causative agent of TB,

is known to adapt metabolically to the various nutrients

available during its cycle of infection, persistence, and

reactivation.^11,12Fatty acids and cholesterol are important

carbon sources for in vivo survival and pathogenesis of

Mtb,^13,14even though this organism can utilize carbohydrates

and carbon dioxide as carbon sources and needs glucose for in

vivo persistence.¹⁵There have been eﬀorts to screen

compound libraries against Mtb surviving on fatty acid and

cholesterol media to identify compounds eﬃcacious against

various pathological forms of Mtb.¹⁶Herein, we report

structure−activity relationship (SAR) studies and preliminary

target identiﬁcation studies of 1,3-diarylpyrazolyl-acylsulfona-

mides identiﬁed from the phenotypic screening of a compound

library supplied by DuPont in cholesterol-containing media.

a

Table 1. Hit Triage of Compound 1

MIC 7H9/ADC/Tw (μM)

4.7

6.25

MIC 7H9/glucose/BSA/Tx (μM)

MIC 7H9/DPPC/cholesterol/BSA/Tx (μM)

MIC 7H9/cholesterol/BSA/Tx (μM)

IC₉₀in 7H9/2.5 mM butyrate/pH6/0.1 mM nitrite (μM)

HepG2 IC₅₀glucose (μM)

>50

RESULTS AND DISCUSSION

■

HepG2 IC₅₀galactose (μM)

solubility (μM)

>50

170

Phenotypic Hit with a Novel MoA. A high-throughput

phenotypic whole cell screen of a ∼150,000 diverse set of

compounds from DuPont Agrichemicals compound library

against Mtb was conducted at the National Institute of Allergy

and Infectious Diseases of the National Institutes of Health

(NIAID/NIH, U.S.) using Mtb in two diﬀerent media,

Middlebrook 7H9/dipalmitoylphosphatidylcholine (DPPC)/

cholesterol/bovine serum albumin (BSA)/tyloxapol (DPPC/

chol) and Middlebrook 7H9/butyrate pH 6.0/nitrite/BSA/

tyloxapol (butyrate pH6/nitrite). This screen identiﬁed ∼2000

primary hits. The activity of these hits was conﬁrmed by

determining the minimum inhibitory concentrations (MICs)

that lead to no visible growth from solid samples against Mtb

under three conditionsDPPC/chol, butyrate pH6/nitrite, as

well as 7H9/DPPC/casitone/tyloxapol (DPPC/cas). Of the

three media used, activity in DPPC/chol medium may closely

simulate in vivo nutritional ambience for replicating Mtb,

whereas DPPC/cas is a protein-free minimal media used to

identify weaker inhibitors that would be missed in standard

serum containing media due to the eﬀect of protein binding.

The low pH butyrate medium containing nitrite reﬂects some

of the environmental stresses encountered by Mtb residing in

macrophages in vivo, namely, acidic pH of the intraphagosomal

environment of the macrophage and reactive nitrite

a

MIC−minimum inhibitory concentration against Mtb H37Rv;

7H9−Middlebrook 7H9; ADC−albumin-dextrose-catalase supple-

ment; Tw−Tween 80; BSA−bovine serum albumin; Tx−tyloxapol;

DPPC−dipalmitoylphosphatidylcholine; IC₅₀−50% inhibitory con-

centration; solubility−aqueous solubility at pH 7.4.

Though 1 has a 1,3-diarylpyrazole core found in some known

anti-Mtb actives,^18,19it has a unique N-(methylsulfonyl)-

propanamide substituent at the pyrazole C4 position. It had

been reported as a potassium channel modulator for the

treatment of a variety of human disorders.²⁰Screening against

various tool strains of Mtb indicated a novel MoA as described

in the biology triage section of this manuscript. The compound

was noncytotoxic against HepG2 cell line under both glucose-

and galactose-containing media conditions at the highest

concentration tested, minimizing the possibility of mitochon-

drial cytotoxicity.²¹All these observations sparked further

interest in exploring the potential of 1 to deliver a novel drug

candidate for TB treatment. Detailed SAR and target

identiﬁcation studies were initiated as detailed in the following

sections.

Synthesis. As the hit compound 1 was identiﬁed as a

singleton from the screen, detailed SAR studies were needed to

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identify key pharmacophoric features and to expand the SAR

scope for increasing anti-Mtb activity. To this end, a systematic

exploration of each substituent around the central pyrazole

ring and variations of the pyrazole core were undertaken.

Compounds were speciﬁcally synthesized to explore the SAR

around substituents around the N1 (compounds 1−12, Table

2), C3 (compounds 13−25, Table 3), and C4 positions

(compounds 26−45, Table 4). Furthermore, additional

compounds were synthesized to explore replacements for the

N-(methylsulfonyl)propanamide substituent at the C4 position

Table 3. SAR at the C3 Position

a

Table 2. SAR at the N1-Substituent

a

MIC−measured in Middlebrook 7H9/Glu/BSA/tyloxapol media;

solubility−aqueous solubility at pH 7.

(compounds 46−50, Table 5) and to vary the central pyrazole

core (compounds 51−58, Table 6).

Compounds 1−25 and 35 were synthesized by modiﬁcation

of the reaction sequence described previously as depicted in

Scheme 1.²⁰In short, the key intermediates, 4-formylpyrazoles

(A), were synthesized by treating the appropriately substituted

a

MICs were measured in Middlebrook 7H9/Glu/BSA/tyloxapol

media; solubility was determined in aqueous pH 7.4 phosphate buﬀer

simulating thermodynamic conditions.

C

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Table 4. SAR at C4 N-Sulfonylpropanamide

Table 5. Heterocycles at C4

a

MIC−measured in Middlebrook 7H9/Glu/BSA/tyloxapol media at

day 14; solubility−aqueous solubility at pH 7.

hydrazones with POCl₃in a Vilsmeier−Haack formylation

̈

reaction, which was followed by a regioselective Knovenagel

condensation reaction with malonic acid to obtain the

corresponding trans-acrylic acids B. Selective reduction of

the acrylic acids in order to avoid concomitant halogen

hydrogenolysis gave the corresponding propanoic acid

intermediate C which was coupled with methanesulfonamide

in the presence of carbonyldiimidazole (CDI) and diazabicy-

cloundecene (DBU) or, in a few cases, in the presence of

dicyclohexylcarbodiimide (DCC) and dimethylaminopyridine

(DMAP) to yield target compounds. The detailed synthetic

schemes are described in Supporting Information (Schemes S1

and S2). Syntheses of compounds 26−41 with modiﬁed alkyl

linkers at C4 are described in Schemes S3 −S6. Urea and

carbamate derivatives 42−45 could be synthesized from

common intermediate A via alcohols D and amines E as

described in Scheme S7. Syntheses of compounds 46−50 with

replacement of acylsulfonamide functionality are described in

Schemes S8 and S9. Syntheses of compounds 51−58 with

variations to the central pyrazole core are described in

Schemes S10 −S14.

SAR at the N1-Substituent. Mtb activity measured as

MIC in Middlebrook 7H9/Glu/BSA/Tx media and aqueous

solubility were monitored as ﬁrst tier criteria toward selection

of compounds for further characterization (Table 2).

Compound 2 with an unsubstituted phenyl ring showed a

much higher MIC (>50 μM) relative to 1. Replacement of p-

bromo on the phenyl ring with p-ﬂuoro, as in 3, or moving

bromine to the meta or ortho positions as in 4 and 5,

respectively, led to deterioration of activity (MICs ≥ 50 μM).

Similarly, 6 with the phenyl ring replaced with 4-pyridyl and 7

with a cyclohexyl ring showed much weaker MICs (≥50 μM).

Compounds 11 and 12 with polar CN and methylsulfoxide

a

MIC−measured in Middlebrook 7H9/Glu/BSA/tyloxapol media;

solubility−aqueous solubility at pH 7.

D

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retained MICs similar to 1, albeit with lower aqueous solubility

(100 and 50 μM, respectively). Compound 10 with a 4-CF₃-3-

pyridyl ring at N1 showed a comparable MIC to 1 with

somewhat improved solubility (90 μM) compared to 9.

Overall, variations at the N1-phenyl ring (R₁) demonstrated

the essentiality of an aryl ring with larger hydrophobic para-

substituents for Mtb activity.

Table 6. SAR at the Pyrazole Core

SAR at the C3 Position. Compared to the N1 position,

substituents at the C3 position (R₂) of the pyrazole showed a

wider scope for structural variation leading to slightly lower

MICs (Table 3). Compound 13 with a hydrogen atom in place

of Cl on the C3 phenyl group retained a similar MIC to 1.

Compound 14 with m-Cl-phenyl also retained a similar MIC,

but compound 15 with o-Cl-phenyl showed a >10-fold higher

MIC. Compound 16 with a 4-pyridyl ring at C3 in place of Cl-

phenyl improved the MIC ∼4-fold, thus providing a more

potent tool compound for MoA studies (see below).

Compound 17 with a 3-pyridyl ring showed a MIC similar

to compound 1, and 18 with a pyrazine ring showed a 5-fold

higher MIC. Compounds 19 and 20 with saturated cyclohexyl

and tetrahydropyran rings, respectively, at the C3-position

showed MICs similar to 1, but 21 with a basic piperidine at C3

and its N-formyl derivative 22 showed much weaker activity

(MICs ≥ 50 μM) indicating an intolerance toward polar

functionality at this position for nonaromatic R₂substituents.

Similarly, higher (3−5-fold) MICs were observed with 23

having a p-NO₂-phenyl at the C3 position and the

corresponding aniline derivative 24. Compound 25 with a

C3 benzyl group also showed much weaker MIC, thus limiting

the SAR scope to explore directly attached aryl, heteroaryl, and

saturated rings.

SAR at C4 N-(Methylsulfonyl)propanamide. A few

analogues were synthesized to determine the essentiality of the

N-sulfonylpropanamide functionality linked to the pyrazole C4

position (R₃, Table 4). The corresponding carboxylic acid 26

and N-methyl amide derivative 27 showed much weaker

activity (MICs > 50 μM), establishing N-sulfonylpropanamide

as a critical functionality for potency. Methylation of the

sulfonamide NH led to 28 that showed a signiﬁcant decrease in

activity (MIC > 50 μM) along with low aqueous solubility (<5

μM). Replacement of the carbonyl group with methylene

(compound 29) led to a high MIC (>50 μM). The data point

to the need for an acidic functionality (the sulfonylpropana-

mide) that has speciﬁc binding interaction with the target not

seen with the carboxylic acid of 26. The speciﬁcity of the

sulfonylpropanamide was further supported by swapping

positions of the sulfonyl and carbonyl groups (compound

30), leading to high MIC (>50 μM). The length of the alkyl

chain linking the N-(methylsulfonyl)carbamoyl group to the

pyrazole core was also critical to maintain lower MICs as

demonstrated with 31 (MIC > 50 μM) and 32 (MIC 37 μM)

having shorter and longer linking alkyl chains to the pyrazole

C4 position. The ethyl sulfonyl analogue 33 showed a 4-fold

higher MIC, while the larger cyclopropylsulfonyl group was

even less active (MIC > 50 μM) indicating a limited scope

around the sulfonyl alkyl group. Hence, N-(methylsulfonyl)-

propanamide represents an optimal pharmacophore predicted

to engage in key interactions with the target as delineated later.

Modiﬁcations on the propanamide chain were then explored

to better understand SAR and continue compound optimiza-

tion, particularly to improve metabolic stability of the

acylsulfonamide while maintaining the potency of the

compounds [refer drug metabolism and pharmacokinetics

a

MIC−measured in Middlebrook 7H9/Glu/BSA/tyloxapol media;

solubility−aqueous solubility at pH 7.

groups, respectively, at the para position of the phenyl ring

showed 5−10-fold weaker activity indicating the essentiality of

hydrophobic substituents at this position. Only compounds 8

and 9 with p-chlorophenyl and p-CF₃-phenyl, respectively,

E

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Scheme 1. General Synthetic Scheme

a

Reagents and conditions: (i) KOAc, EtOH, 80 °C; (ii) POCl₃, DMF, 80 °C; (iii) malonic acid, piperidine, pyridine, 90 °C; (iv) method 1:

NH₂NH₂·H₂O, MeOH, 80 °C; method 2: NBSH, Et₃N, THF, 45 °C; (v) method 1: CH₃SO₂NH₂, CDI, DBU, DMF, 90 °C; method 2:

CH₃SO₂NH₂, DCC, DMAP, 0−45 °C; (vi) NaBH₄, MeOH, 25 °C; (vii) PPh₃, DIAD, NaN₃, H₂SO₄, DCM, 0 °C to 25 °C; (viii) PPh₃, THF/

H₂O, 60 °C; (ix) CDI, methanesulfonamide, DIPEA, DCM, dichloroethane, or DMF depending on the solubility of the substrate, 25−90 °C.

(DMPK) section]. Addition of a methyl group either on α- or

β-carbons of the propanamide chain led to compounds 36 and

37 with much weaker activity (MICs ≥ 50 μM) compared to

the unsubstituted analogue 35 (MIC 6.25 μM), indicating a

likely clash at the target level or perturbation of the preferred

side chain binding conformation. Comparatively, better MICs

were observed by constraining the methyl group into a

cyclopropane ring as in 40 and 41 (MICs 12.5−19 μM). The

trans-acrylamide derivatives 38 and 39 showed more potent

MICs (1.2−4.7 μM). Compounds 42, 43, and 44 with a

sulfonylurea tethered to C4 and a net replacement of CH₂with

NH showed lower MICs compared to the corresponding N-

(methylsulfonyl)propanamide derivatives, indicating a possible

H-bond donor interaction from the urea NH with the target.

Compound 45 with a sulfonylcarbamate functionality showed

an 8-fold higher MIC relative to 43, supporting the hypothesis

that there is a contribution to binding by the urea NH.

Further, we explored replacing the N-acylsulfonamide group

with another functionality envisioned to similarly position an

acidic group (Table 5). Compounds 46 and 47 with tetrazole

and 1,2,4-oxadiazolone moieties attached to C4 via an ethyl

linker and compound 48 with a pyrimidinetrione attached via a

methylene linker showed much higher MICs (≥50 μM). Only

49 with an 1,3,4-oxadiazolone attached via an ethyl linker at

the C4 position showed a lower MIC, about 4-fold higher than

1, albeit the low solubility (<5 μM) rendered the compound

less interesting. Overall, there is a limited scope for

replacement of the N-acylsulfonamide functionality. Com-

pound 50 wherein the propanamide was cyclized into a

dihydroisoxazole ring also retained an MIC within 4-fold of

compound 1. Further scope for optimization of MICs using C4

functionalities as in compounds 49 and 50 by varying other

substituents in the molecule remains to be explored.

of alternative isosteric heterocycles. Compounds 51 and 52

retained the same relative position of all three substituents and

the pyrazole nitrogen lone pair of 1 and 3 and, not surprisingly,

showed similar MICs. Analogues 53 and 54 with 1,4-

diarylpyrazole and 3,5-diarylpyrazole cores also display the

three substituents in the same relative orientation as 1 but shift

the position of the pyrazole nitrogen lone pair resulting in

much higher MICs (≥50 μM) and indicating an important

target interaction for the pyrazole N2 of 1. 1,5-Diarylpyrazole

55 had a much higher MIC (≥50 μM) further conﬁrming the

importance of relative positions of aryl rings in maintaining

potency. Replacement of the pyrazole with other ﬁve-

membered heterocycles as with 56 and 57 having oxazole

and thiazole cores, respectively, showed 5−8-fold weaker

MICs. Compound 58 with a furan core showed much more

potent activity with MIC below 0.5 μM. The above

observations indicate a smaller angle between the R1 and R2

substituents of the smaller pyrazole, and furan heterocycles

(150.3 and 125.5°, respectively) are favored over what would

be seen with the larger thiazole core (154.6°) in 57. Loss of

potency for oxazole 56 is consistent with the observation that

polar atoms are not well tolerated at the other position of the

ﬁve-membered ring as seen for 53 and 54. The furan oxygen is

favorable as is the 2-position N of the pyrazole of 1 and of 51.

Improved activity of furan analogue 58 could be attributed to

O being more electronegative than N, possibly making a

stronger hydrogen-bonding interaction with the target.

The above SAR demonstrates the speciﬁc structural

requirements of compound 1 and the analogues required to

maintain anti-Mtb activity alluding to speciﬁc target engage-

ment by the scaﬀold.

Representative Pharmacophore. We have accordingly

developed a pharmacophore hypothesis to summarize key

structural features of the scaﬀold that may aid in eﬀorts to

further optimize this series. To describe the chemical space of

the active acyl sulfonamide pyrazole compounds, a series of

SAR of the Pyrazole Core. Table 6 shows the analogues

synthesized to understand the importance of the relative

positions of substituents on the pyrazole ring and the inﬂuence

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Figure 2. Representations of the AARRR pharmacophore model. (A) 2D representation of the pharmacophore model with two of the ring features

(R₁and R₂) both shown to be about 4 Å from the central R feature forming an angle of 140° with each other. The ﬁrst acceptor feature (A₁) lies

5.65 Å from R with the acceptor feature A₂lying an additional 2.59 Å from A₁forming an angle of 158.4° with A₁and R. (B) 3D representation of

the pharmacophore model showing the A₁and A₂features to be out of the plane formed by R, R₁, and R₂with a −95.0° dihedral angle around a

line from R to A₁.

Figure 3. (A) Compound 43 ﬁtted to the ﬁve-feature AARRR pharmacophore hypothesis very closely. The three aromatic ring features are

occupied by the chlorophenyl, pyrazole, and pyridyl groups, while the deprotonated sulfonamide ﬁts the negative charge feature and sulfone O ﬁts

the acceptor feature; (B) an overlay of all active compounds in this set closely ﬁtting all ﬁve pharmacophore features. (C) Compound 34 is an

example of an inactive compound, i.e., an excellent ﬁt of the pharmacophore. This loss in activity is brought about by the bulky cyclopropyl group

extending into a region outside of the pharmacophore. This region could be deﬁned as an excluded volume. (D) Inactive compounds from this set

either ﬁt the pharmacophore hypothesis fully or partially with many occupying spaces outside the deﬁnition of the pharmacophore, suggesting

potential for the deﬁnition of excluded volumes past the A₂feature and between the A₁and R₁features.

pharmacophore hypotheses were generated from the set of

compounds using the phase module from the Schrodinger

molecular modeling suite.²²The activity cutoﬀ was deﬁned as

MIC < 25 μM in the 7H9/glucose/BSA/Tx assay among the

set of 58 compounds in this series. An AARRR (A-acceptor,

R−ring) pharmacophore hypothesis (Figure 2) consisting of

three aromatic rings (R, R₁, and R₂) and two hydrogen bond

acceptors was found to best describe the chemical space of

active compounds. A central aromatic ring feature (R)

corresponds to the central pyrazole core with two more

aromatic ring features corresponding to aromatic substitutions

at the 1 and 3 positions (R₁and R₂, respectively). The ﬁrst

acceptor feature (A₁) can be found approximately 5.65 Å from

the C4 position of the pyrazole core ﬁtting the negatively

charged N of the acyl sulfonamide moiety. At physiological pH,

the acyl sulfonamide NH is deprotonated, creating an anion

that could serve as a hydrogen bond acceptor. The second

acceptor (A₂) lies another 2.43 Å away forming a 158° angle

between A₁and the central ring feature (Figure 2A). A₁moves

out of the plane formed by the three ring features forming a

95° dihedral angle around a line connecting the R feature and

the A₁feature (Figure 2B). In this set of compounds, R₁often

seems, at a minimum, to require negatively charged acid

groups as only compounds with a pK_abelow physiological pH

have shown activity.

A representative active compound 43 ﬁts all ﬁve of these

features very closely (Figure 3A). The pharmacophore ﬁt of

each ligand was determined via phase docking with the ﬁt

evaluated using the phase ﬁtness score.²²All active compounds

featured in this manuscript ﬁt the ﬁve features (Figure 3B) very

closely.

A bioisostere replacement of the acyl sulfonamide with the

1,3,4-oxadiazolone of 49 introduces an acid center with a

predicted²³pK_aof 7.87 0.4 in contrast to the predicted pK_a

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Table 7. MICs Against Isogenic Single-Drug Resistant Mutant Strains of Mtb

MIC (μM)

Mtb strain

1

8

43

58

RIF

INH

ETH

15.6

H37RvMA

Δ cyd

4

2

4

2

4

0.5

0.24

0.5

1

2

0.12

<0.12

4

0.24

0.12

0.24

0.5

0.24

0.12

<0.13

1

0.002

0.005

0.004

0.005

0.002

0.001

0.003

0.002

7.8

QcrB^A317T

MmpL3^G253E

MmpL3^G758A

DprE1^Y314C

DprE1^P116S

DprE1^T314H

InhA-OE

3

0.5

0.4

15.6

8

31.2

>62.5

7.8

KatG^T198A

EthA^C253R

15.6

8

>125

2

0.5

a

InhA-OE: InhA overexpressor (H37Rv-LP:fabG1/inhA-c-15t); RIF: rifampicin; INH: isoniazid; ETH: ethionamide.

of the sulfonamide of 43 of 3.72 0.23, thus lowering the

propensity for an acceptor/negative charge to form at

physiological pH. Since the oxadiazolone has a pK_aclose to

physiological pH, both the protonated and deprotonated states

need to be considered. Only the deprotonated form ﬁts all ﬁve

features of the pharmacophore hypothesis. This poorer ﬁt

explains the weaker MIC (19 μM) of 49 compared to 43 (1.56

μM) (Figure S1c).

Compounds 28−31 were all inactive with poor pharmaco-

phore ﬁts for at least one reason as explained in the

supplementary section with Figure S1. Compounds 33 and

34 (Figure 3C) with their respective ethyl and cyclopropyl

attachments to the sulfone and 36 and 37 with methyl groups

in positions α and β to the pyrazole core all showed near

perfect ﬁts for the pharmacophore model despite a complete

loss of activity. These compounds contain additional bulk

occupying regions not deﬁned by the pharmacophore

suggesting potential excluded volumes. Excluded volumes

beyond the A₂feature and adjacent to the alkyl linker at the

sites of potential branching would penalize these compounds.

These excluded volumes would need more examples to be

properly deﬁned. An overlay of all inactive compounds

presented in the study (Figure 3D) demonstrates many of

them to occupy regions that could be considered for excluded

volumes.

Compound 58 with a furan core fulﬁlls all pharmacophore

features (Figure S1d) and showed potent activity, whereas

compounds 53, 54, and 56 with changes in the core ring (R)

are examples of weak actives with good ﬁts. Comparing the

matched pair of inactive 53 and active 51 would suggest that a

heteroaromatic acceptor N contributes to activity. However,

small changes to the π-characteristics of the core caused by

these rearrangements may also contribute to the lower activity.

More data from analogues containing rearrangements of this

core would be needed to conﬁrm the contribution of this

acceptor and warrant expanding the deﬁnition of this

pharmacophore feature to include an acceptor corresponding

to the 2-position of the pyrazole core.

Biology Triage. Compounds 1, 8, 43, and 58 were

screened against various tool strains of Mtb to deconvolute the

MoA for anti-Mtb activity. None of the compounds showed

modulation in MICs against a cytochrome bd oxidase

knockout mutant strain (Δcyd)^24,25nor against a QcrB mutant

(QcrBA317T), thereby eliminating the two targets of the

respiratory pathway as a likely target (Table 7). Neither did the

compounds elicit a positive response in the PrecA-LUX

bioluminescence reporter assay,²⁶which is designed to detect

modulation in the recA expression, an indicator of DNA

damage, hence ruling it out as a MoA for the compounds

(Figure S2 in the Supporting Information). However, the

compounds showed sustained signals in the PiniB-LUX

bioluminescence reporter assay that detects modulation in

the iniBAC operon expression,²⁶an indicator of cell wall

biosynthesis being disrupted (Figure S3). The prediction that

the compounds inhibited cell wall biosynthesis was further

conﬁrmed by transcriptional proﬁling studies that showed

upregulation in the genes involved in the cell wall biosynthesis,

including many of the genes upregulated by INH such as

acpM, fabD, kasA, kasB, accD6, rv2248, efpA, iniB, iniA, and

iniC (Tables S3 and S4 in the Supporting Information).

Despite these similarities, some of the genes that were

upregulated during INH treatment (ppsA, ppsB, ppsC, ppsD,

ppsE, and fbpC2) were downregulated during exposure to

compounds 8 and 16, indicating diﬀerences in transcriptional

responses. The compounds were screened against the mutant

strains of MmpL3 and DprE1 which frequently encountered

cell wall targets (Table 7). Two strains carrying a mutation in

MmpL3 (G253E or G758A) were not resistant, arguing against

MmpL3 as the target. Similarly, three strains carrying

mutations in DprE1 (Y314C, P116S, or T314H), which

confer resistance to other DprE1 inhibitors, were not resistant,

suggesting that DprE1 is not the target. In addition, other

isogenic single-drug resistant strains of known cell wall

targeting drugs such as INH and ethionamide (ETH) were

also not cross-resistant to the compounds (Table 7). Taken

together, these data suggest a MoA that most likely involves

interference with Mtb cell wall biogenesis in a manner that

does not lead to cross resistance to other cell wall inhibitors.

Studies to determine the speciﬁc MoA of this series are

underway.

Bactericidal Activity Against Clinical Isolates. The

bactericidal activity of representative compounds 1, 8, and 43

against replicating Mtb was evaluated through the exposure of

Mtb cultures to various concentrations of compounds for 8

days and allowing the treated cultures to regrow on fresh

media after washing out the excess free drug (Figure S4 in the

Supporting Information). All tested compounds showed 2−2.5

log CFU reduction at 1−4× MIC concentrations within 8

days, indicating the bactericidal nature of the compounds.

However, the compounds were not active against non-

replicating Mtb under nutrient starvation conditions. Com-

pound 16 retained activity against three drug-sensitive, two

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Table 8. MICs of Compound 16 Against Clinical Mtb Isolates

a

Mtb isolate

resistant proﬁles

MIC of compound 16 (μM)

H37RvMa

S2371

S1125

MD55

MD96

R88

susceptible

≤15

30

15−30

b

MDR: INH; RMP; EMB; PZA; SM

XDR: INH; RIF; AM; KM; CAP; OFLX; EMB; ETH; PZA; SM

INH mono-resistant

MDR: INH; RIF; EMB; ETH

RIF mono-resistant

60

b

≤30

b

30

60

b

R296

R3027

b

60

a

b

MICs were determined by the MIGIT method. Values within 4-fold of MICs against drug-susceptible isolate are considered sensitive to the test

compound; INH: isoniazid; RIF: rifampicin; AM: amikacin; KM: kanamycin; CAP: capreomycin; OFLX: oﬂoxacin; EMB: ethambutol; ETH:

ethionamide; PZA: pyrazinamide; SM: streptomycin.

a

Table 9. In Vitro and In Vivo DMPK Parameters

microsomal clearance H/R/M %

remaining

Human

PPB fu

t_1/2terminal

V_d

CL_b

CL_u

AUC_0−t

(min μmol/L)

compound TPSA log D

(h)

(L/kg) (mL/min/kg) (mL/min/kg)

1

8

81.1

93.9

90.3

93.9

2.4

2.0

90/74/71

98/>99/87

94/98/94

85/94/79

37/95/94

86/83/77

88/82/85

>99/92/96

99/98/91

97/97/98

>99/87/91

98/87/85

0.004

0.023

0.036

0.009

0.022

0.07

0.015

0.01

0.041

0.03

2.0

3.6

3.0

1.3

1.7

2.3

1.1

0.9

2.0

5.8

3.6

Nd

0.2

1.2

8.7

nd

28.1

29.3

nd

1.0

nd

1.18

2.56

67.4

>200

178

145

>200

295

111

1872

>20000

8091

2071

>20000

>4878

>20000

313

3527

4883

134

15

31

8.7

21

29

22

1359

20

10

16

17

20

38

40

43

45

51

58

1.87

0.93

0.84

0.55

0.98

1.06

0.81

2.73

2.1

106

103.2

81.1

0.01

0.007

3.13

>200

101.6

0.36

>20000

a

In vivo mouse PK parameters calculated from the noncompartmental analysis of intravenous dosing at 2 mg/kg; TPSA: total polar surface area; H/

R/M: human/rat/mouse; PPB: plasma protein binding; fu: fraction unbound; V_d: Volume of distribution; CL_b: total body clearance determined

from whole blood; CL_u: unbound blood clearance determined from CL_band plasma protein binding (assuming blood to plasma ratio of 1). AUC:

area under the curve; nd: not determined.

monodrug resistant, and three MDR/XDR clinical isolates of

Mtb, indicating potential utility of the compounds from the

series to treat drug-sensitive as well as drug-resistant TB (Table

8). The MIGIT (mycobacteria growth indicator tube) assay

uses higher inoculum in liquid broth designed for faster growth

of mycobacteria and involves longer incubation times relative

to the Alamar Blue MIC microtiter plate assay used for the

SAR investigation herein.²⁷Hence, MIC values between the

two assays will not be comparable. Nonetheless, it is gratifying

that the MIC shifts between MDR and susceptible Mtb strains

in the MIGIT assays were relatively small (less than 4-fold),

thereby suggesting broader clinical utility.

In Vitro and In Vivo DMPK Proﬁles. In general, the

compounds in the series showed moderate-to-good metabolic

stability in mouse, rat, and human microsomes as presented in

Table 9. The high plasma protein binding (PPB) observed for

the compounds may be attributed to high albumin binding

associated with the acidic acylsulfonamide functionality along

with the high degree of lipophilicity. Compound 20 showed

lower protein binding presumably due to lower lipophilicity

imparted by the tetrahydropyran ring attached to the C3

position. The compounds in Table 9 were also proﬁled for in

vivo pharmacokinetics (PK) following intravenous dosing in

mice.

clearance and consequently lower exposure. Rapid Cl_bvalues

were also observed for 16 and 17 with 4-pyridyl and 3-pyridyl

groups, respectively, at the C3 position. The rapid in vivo

clearance of these compounds was all the more problematic

due to the relatively high PPB leading to exceedingly high

unbound clearance (CL_u) values calculated by correcting Cl_b

for plasma-free fraction (assuming blood-to-plasma ratio of

1).²⁸In vivo metabolite identiﬁcation studies of 17 revealed

that the high clearances were in part due to hydrolysis of the

acylsulfonamide (data in Supporting Information). Subsequent

SAR exploration therefore focused on modiﬁcations that were

designed to increase stability of the acylsulfonamide while

maintaining the potency of the compounds. However, neither

addition of a double bond to the acyl chain (38) nor

cyclopropanation (40), replacement of the sulfonamide with a

sulfonylurea (43), or sulfonylcarbamate (45) improved the

clearances of the compounds. In addition, except for 10 which

had a N1 CF₃-3-pyridyl ring, all pyridyl compounds had total

clearances that exceeded hepatic blood ﬂow in mice (90 mL/

min/kg), which suggests that extra-hepatic metabolism may be

a signiﬁcant mechanism of clearance. The high total polar

surface area (TPSA) and the low log D of these pyridyl

derivatives (Table 7) suggest that the compounds have low

lipoidal permeability, and it is therefore likely that transporter-

mediated clearance was also a key contributor to the observed

total clearance.^29,30This was further validated by performing in

vivo metabolite identiﬁcation studies on 43. Following

intravenous dosing of the compound in mice, only unchanged

The hit compound 1 had a low in vivo mouse blood

clearance (Cl_b) and a low volume of distribution (V_d) that

translated to high in vivo exposure. Replacing the N1 aryl group

with a 4-CF₃-3-pyridyl ring in 10 led to a dramatic increase in

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compound was detected in blood, urine, and feces, suggesting

that metabolism was not a major contributor to its clearance.

Compounds, 1, 8, and 51 had high exposure on oral dosing

(data not shown), with 8 in particular showing almost

complete absorption (bioavailability > 95%) even at high

doses. This compound therefore had the best balance of in

vitro potency and in vivo PK and was therefore selected for

further in vivo proof-of-concept studies.

In Vivo Eﬃcacy. In vivo eﬃcacy testing was performed at

an oral dose of 200 mg/kg in the acute Balb/c mouse model to

evaluate the ability of 8 to inhibit replicating intracellular Mtb

in lungs.³¹⁻³⁴Treatment was initiated 7 days after a low-dose

aerosol infection with Mtb Erdman pFCA LuxAB strain³²and

continued for 12 consecutive days. The compound was well

tolerated during the treatment period but was not eﬃcacious in

reducing Mtb lung burdens in treated mice relative to the

untreated mice as measured by quantiﬁcation of relative light

units of the luciferase expressing Erdman strain from the lung

homogenate or by CFU counts obtained by plating serial

dilutions of the lung homogenate on agar plates. As a positive

control, EMB at 100 mg/kg once a day led to a 2-log CFU

reduction in this model. To explain the results, free oral

exposure of compound 8 in healthy mice at 200 mg/kg oral

dose was compared with the MIC against the Mtb Erdman

strain used for the eﬃcacy studies (9.1 μM) and the inhibitory

concentration against intracellular Mtb (IC₉₀= 11 μM)

(Figure 4). In-study PK performed on the last 2 days of

compounds against a panel of various tool strains of Mtb

ruled out the involvement of known mechanisms and/or

targets such as DNA damage and respiration but suggested

involvement of cell wall damage in the MoA. RNA microarray

studies of Mtb cultures treated with the compounds conﬁrmed

the upregulation of genes that are involved in lipid

metabolism/fatty acid biosynthesis. Isogenic single-drug

resistant mutant strains of MmpL3, DprE1, InhA, and EthA

were not cross-resistant with the compounds, indicating novel

MoA or perhaps an alternative MoI of the targets. The

compounds are bactericidal against replicating Mtb and retain

activity against MDR clinical isolates of Mtb. These features

make the series an attractive chemical matter for further target

identiﬁcation and drug discovery eﬀorts to potentially identify

a novel clinical candidate for treatment of TB. Further drug

optimization work is needed to improve upon both the Mtb

activity of the series and in vivo PK if compounds with eﬃcacy

in TB models of infection are to be realized.

EXPERIMENTAL SECTION

■

MIC Testing and Bioluminescence Reporter Assay. MIC

determinations were done as previously described.³⁵INH was

included as a positive control. Brieﬂy, Mtb H37Rv (ATCC 27294)

was grown in Middlebrook 7H9/Glu/BSA/tyloxapol consisting of

Middlebrook 7H9 broth base (4.7 g/L)/BSA fraction V (5 g/L)/

dextrose (4 g/L)/NaCl (0.81 g/L)/0.05% tyloxapol to an OD_650nmof

0.2, at which stage the cells were diluted 1000-fold in the same

medium. The compound was serially diluted in Middlebrook 7H9/

Glu/BSA/tyloxapol medium in round-bottom 96-well plates

(Nunclon) at 50 μL/well followed by addition of an equal volume

of diluted cell suspension. Plates were incubated in sealed bags at 37

°C, and growth was recorded after 1 and 2 weeks of growth using an

enlarged inverted mirror. The MIC was recorded as the compound

concentration that completely inhibited all visible growth. Bio-

luminescence reporter assays were carried out as described by Naran

et al.²⁶

MICs Against Mutant Strains. Alamar Blue ﬂuorescence-based

broth microdilution assay was used to assess the MICs of compounds

against Mtb mutant strains, as described previously.³⁶MICs were

determined after 7 days of growth in standard Middlebrook 7H9/

glycerol/ADC/Tween-80. For QcrB and cydA mutants, the growth

measurement was monitored by OD₆₀₀. MIC was deﬁned as the

concentration required to inhibit growth by 90%.

Figure 4. Free exposures of 8 (calculated using human PPB)

following a single oral dose of 200 mg/kg in mice.

RNA Extraction and Transcriptional Proﬁling. RNA extraction

from Mtb H37Rv (ATCC 27294) grown to an OD₆₅₀of 0.2 and

subsequently treated for 6 h with 1× and 10× MIC of compound or

vehicle control was performed using previously described methods.³⁷

RNA was also extracted from control cultures treated for 6 h with

INH (2 μM) or solvent control. RNA (4 mg) yielding an RNA

integrity number of 8 or higher, as determined by the Agilent 2100

Bioanalyzer, was subsequently used for ﬂuorescent-tagged cDNA

synthesis with a random hexamer (4.5 mg) (Invitrogen) in a ﬁnal

volume of 14.5 μL by sequential heat denaturation at 70 °C for 5 min,

cooling on ice, and addition of 5 μL of 5× First-Strand buﬀer, 1.25 μL

of 0.1 M DTT, 2.5 μL of dNTP mix (consisting of 5 mM each of

dATP, dGTP, dTTP, and 0.5 mM dCTP), 1 μL of 200 U/μL

SuperScriptIII (Invitrogen), 1 μL of 40 U/μL RNAseOut, and 1 μL of

Cy3 or Cy5-dCTP (GE) with incubation at 25 °C for 5 min and at 48

°C for 90 min. RNA was removed by adding 5 μL of 1 M NaOH

followed by incubation at 70 °C for 15 min. After neutralization with

5 μL of 1 M HCl, ﬂuorescent cDNA was puriﬁed on an Amicon Ultra-

0.5 column (Millipore) according to the manufacturer’s recommen-

dations. Fluorescent cDNA was analyzed with a NanoDrop ND-1000

spectrophotometer. Equal amounts (0.7 μg) of Cy3- and Cy5-labeled

cDNA were hybridized to the Agilent SurePrint G3 4 × 44K custom

oligonucleotide microarrays (design number 021966, 021362) in the

TECAN HS Pro 4800 hybridization station. Hybridization was

treatment matched closely with the healthy PK (data in the

Supporting Information). From the two analyses, it is evident

that compound 8 at 200 mg/kg failed to achieve drug

exposures necessary to exert an antimicrobial eﬀect in vivo.

While much higher or more frequent dosing may provide

suﬃcient exposure to demonstrate eﬃcacy, the underlying data

suggest that further optimization of antibacterial activity and/

or PK is required to do so at lower doses that would feasibly

predict human utility. Work toward achieving these improve-

ments is in progress and will be reported in a future

manuscript.

CONCLUSIONS

■

In summary, phenotypic screening of a DuPont compound

library against Mtb in cholesterol-containing media identiﬁed

1,3-diarylpyrazolyl-acylsulfonamide 1 as a moderately active hit

with a potentially novel MoA. The SAR studies described here

demonstrate a clear scope to improve the MICs of compounds

to <0.5 μM, and a plausible pharmacophore model was

developed to summarize SAR of the series and describe the

chemical space of active compounds. Screening of the

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performed using Agilent 2× Gene expression hybridization HI-RPM

buﬀer and 10× Blocking Reagent at 65 °C for 17 h. Arrays were

washed with Agilent Gene Expression Wash Buﬀer 1 at room

temperature and Gene Expression Wash Buﬀer 2 at 37 °C. Slides were

dried under nitrogen gas for 3 min at 30 °C and imaged using an

Agilent high-resolution DNA microarray scanner (model G2505C) at

5 μm resolution and 100/10% PMT dual scanning for XDR extended

dynamic range. Agilent Feature Extraction software was used for

image analysis.

Pharmacophore Hypothesis Generation. To generate the

pharmacophore hypothesis, all compounds in this set were prepared

using Maestro 12.4³⁸to generate minimized 3D structures and

determine the most likely protonation state at biological pH. A

pharmacophore model was then developed using the Phase Develop

Pharmacophore Model tool.²²The pharmacophore model was

created from multiple ligands with the active ligands deﬁned by an

activity cutoﬀ of MIC < 25 μM in the 7H9/glucose/BSA/Tx assay.

The ﬁnd best alignment method was chosen with the default

hypothesis settings of four to ﬁve features and all default

pharmacophore features allowed. The pharmacophore hypotheses

generated were then inspected, and the hypothesis that best

represented the dataset was selected and validated against a wider

set of compounds from this series.

EtOAc (3×). The combined organic layers were dried over Na₂SO₄

and concentrated in vacuo. The crude reaction mixture was puriﬁed by

ﬂash chromatography using ISCO Teledyne on a 12 g RediSep Rf

column and elution using a gradient of the appropriate solvent

mixtures to yield the desired products.

Method 2: DCC-Mediated Sulfonamide Coupling. To the

solution of the appropriate acid (1.00 equiv) and methanesulfonamide

(1.20 equiv) in DMF were added DCC (2.50 equiv) and DMAP

(1.50 equiv). The reaction mixture was stirred at 30−40 °C for 16−

48 h. The reaction mixture was quenched with water (5 mL), and pH

was adjusted to 6 with 1 N HCl. The aqueous phase was extracted

with EtOAc (3×). The organic phase was washed with brine, dried

over Na₂SO₄, and concentrated in vacuo. The residue was puriﬁed by

a silica gel column or preparative HPLC chromatography to yield the

appropriate compound.

Method 3: Acid Chloride Coupling with Sulfonamide. To a

solution of appropriate sulfonamide (5 equiv) in dichloromethane

(DCM) was added Et₃N (5−10 equiv). Then, the appropriate acyl

chloride (1 equiv) was added at 0 °C. The mixture was stirred at 15 to

25 °C for 16−24 h. The mixture was poured into 1 N HCl and

extracted with DCM. The combined organic phase was concentrated

and puriﬁed by prep-HPLC or by silica gel column chromatography.

3-(1-(4-Bromophenyl)-3-(4-chlorophenyl)-1H-pyrazol-4-yl)-N-

(methylsulfonyl)propanamide (1). Method 3. Yield: 19%. ¹H NMR:

(400 MHz, DMSO-d₆): δ 11.92−11.73 (m, 1H), 8.49−8.36 (m, 1H),

7.88−7.78 (m, 2H), 7.78−7.64 (m, 4H), 7.60−7.49 (m, 2H), 3.21 (s,

3H), 2.98−2.85 (m, 2H), 2.70−2.58 (m, 2H). ¹³C NMR (101 MHz,

DMSO-d₆): δ 171.8, 149.5, 138.6, 132.7, 132.4 (2C), 131.8, 129.1

(2C), 128.7 (2C), 127.8, 120.1, 119.9 (2C), 118.4, 41.0, 35.7, 19.2.

LC/MS (APCI⁺): calculated for C₁₉H₁₇BrClN₃O₃S, 480.99; observed

m/z [M + H]⁺482.0, 483.9; HPLC purity: >99%.

DMPK. All protocols for in vitro DMPK studies and mouse PK

studies are available in the Supporting Information. Animal studies

were conducted following guidelines and policies as stipulated in the

UCT Research Ethics Code for Use of Animals in Research and

Teaching after review and approval of the experimental protocol by

the UCT Senate Animal Ethics Committee (protocol FHS-AEC 013/

032).

Chemistry. All commercial reagents were purchased from Sigma-

Aldrich, Combi-Blocks, Enamine, or Fluorochem and were used

without further puriﬁcation. Solvents were used as received unless

otherwise stated. Analytical thin-layer chromatography was performed

on SiO₂plates on aluminum backing. Visualization was accomplished

by UV irradiation at 254 and 220 nm. Flash column chromatography

was performed using a Teledyne ISCO ﬂash puriﬁcation system with

SiO₂60 (particle size 0.040−0.055 mm, 230−400 mesh). Purity of all

ﬁnal derivatives for biological testing was conﬁrmed to be >95% as

determined using an Agilent 1260 Inﬁnity binary pump, Agilent 1260

Inﬁnity diode array detector (DAD), Agilent 1290 Inﬁnity column

compartment, Agilent 1260 Inﬁnity standard autosampler, and Agilent

6120 quadrupole (single) mass spectrometer equipped with APCI and

ESI multimode ionization source using a Kinetex Core C18 2.6 μm

column (50 mm × 3 mm), mobile phase B of 0.4% acetic acid, 10 mM

ammonium acetate in a 9:1 ratio of HPLC grade methanol, and type 1

water, mobile phase A of 0.4% acetic acid in 10 mM ammonium

acetate in HPLC grade (type 1) water with a ﬂow rate of 0.9 mL/min,

and DAD; or an Agilent UPLC−MS was used: Agilent Technologies

6150 quadrupole, ES ionization, coupled with an Agilent Tech-

nologies 1290 Inﬁnity II series UPLC system Agilent 1290 series

HPLC at two wavelengths 254 and 290 nm using the following

conditions: Kinetex 1.7 μm Evo C18 100A, LC column 50 mm × 2.1

mm, solvent A of 0.1% (formic acid) water, and solvent B of 0.1%

(formic acid) acetonitrile. The structures of the intermediates and

ﬁnal products were conﬁrmed by 1H NMR and mass spectrometry.

1H NMR spectra were recorded on a Bruker spectrometer at 300 or

400 MHz. Chemical shifts (δ) are given in ppm downﬁeld from TMS

as the internal standard. Coupling constants, J, are recorded in hertz

(Hz). Synthesis details and data of intermediates are supplied in

Supporting Information (Section 1).

3-(3-(4-Chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-N-

(methylsulfonyl)propanamide (2). Method 2. Yield: 32%. ¹H NMR:

(400 MHz, DMSO-d₆): δ 12.09−11.57 (m, 1H), 8.45−8.34 (m, 1H),

7.89−7.81 (m, 2H), 7.81−7.71 (m, 2H), 7.62−7.46 (m, 4H), 7.37−

7.29 (m, 1H), 3.18 (s, 3H), 3.00−2.86 (m, 2H), 2.64−2.57 (m, 2H).

LC/MS (APCI⁺): calculated for C₁₉H₁₈ClN₃O₃S, 403.08; observed

m/z [M + H]⁺404.1; HPLC purity: >99%.

3-(3-(4-Chlorophenyl)-1-(4-ﬂuorophenyl)-1H-pyrazol-4-yl)-N-

1

(methylsulfonyl)propanamide (3). Method 1. Yield: 14%. H NMR

(300 MHz, DMSO-d₆): δ 11.72 (s, 1H), 8.36 (s, 1H), 7.91−7.84 (m,

2H), 7.78−7.71 (m, 2H), 7.59−7.52 (m, 2H), 7.37 (t, J = 8.8 Hz,

2H), 3.21 (s, 3H), 2.93 (t, J = 7.5 Hz, 2H), 2.63 (t, J = 7.5 Hz, 2H).

LC/MS (ESI⁺): calculated for C₁₉H₁₇ClFN₃O₃S, 421.07; observed

m/z [M + H]⁺422.1; HPLC purity: 97%.

3-(1-(3-Bromophenyl)-3-(4-chlorophenyl)-1H-pyrazol-4-yl)-N-

1

(methylsulfonyl)propanamide (4). Method 1. Yield: 20%. H NMR

(300 MHz, DMSO-d₆): δ 11.72 (s, 1H), 8.48 (s, 1H), 8.09 (t, J = 1.9

Hz, 1H), 7.88 (dt, J = 7.2, 2.0 Hz, 1H), 7.81−7.73 (m, 2H), 7.60−

7.43 (m, 4H), 3.21 (s, 3H), 2.93 (t, J = 7.5 Hz, 2H), 2.64 (t, J = 7.5

Hz, 2H). LC/MS (APCI⁺): calculated for C₁₉H₁₇BrClN₃O₃S, 480.99;

observed m/z [M + H]⁺481.9, 483.9; HPLC purity: 97%.

3-(1-(2-Bromophenyl)-3-(4-chlorophenyl)-1H-pyrazol-4-yl)-N-

1

(methylsulfonyl)propanamide (5). Method 2. Yield: 31%. H NMR

(400 MHz, Chloroform-d): δ 7.74−7.65 (m, 5H), 7.60−7.58 (m,

3H), 7.45−7.43 (m, 1H), 3.27−3.25 (m, 3H), 3.13−3.07 (m, 2H),

2.64−2.58 (m, 2H). LC/MS (APCI⁺): calculated for

C₁₉H₁₇BrClN₃O₃S, 480.99; observed m/z [M + H]⁺482.0; 484.0;

HPLC purity: >99%.

3-(3-(4-Chlorophenyl)-1-(pyridin-4-yl)-1H-pyrazol-4-yl)-N-

1

(methylsulfonyl)propanamide (6). Method 2. Yield: 23%. H NMR

General Sulfonamide Coupling Procedure for the Synthesis of

Compounds 1−25, 27−29, 31−41, and 51−57. Method 1: CDI-

Mediated Sulfonamide Coupling. To a solution of the appropriate

acid (1 equiv) in N,N-dimethylformamide (DMF) was added CDI (2

equiv), and the resulting reaction mixture was stirred for 10 min at 50

°C. Then, the appropriate sulfonamide (1.2 equiv) and DBU (1.2

equiv) were added, and the resulting reaction mixture was heated to

90 °C for 16−48 h. The reaction mixture was then cooled to 25 °C,

water was added (30 mL), and the aqueous layer was extracted with

(400 MHz, DMSO-d₆): δ 8.67−8.62 (m, 2H), 8.62−8.57 (m, 1H),

8.22−8.14 (m, 1H), 7.89−7.85 (m, 2H), 7.81−7.76 (m, 2H), 7.60−

1

7.55 (m, 2H), 3.00−2.97 (m, 3H), 2.90−2.86 (m, 2H); H NMR

(400 MHz, DMSO-d₆+ D₂O): δ 8.64−8.56 (m, 2H), 8.51−8.45 (m,

1H), 8.29−8.12 (m, 1H), 7.86−7.79 (m, 2H), 7.78−7.68 (m, 2H),

7.58−7.50 (m, 2H), 3.03 (s, 3H), 2.90−2.81 (m, 2H). LC/MS

(APCI⁺): calculated for C₁₈H₁₇ClN₄O₃S, 404.07; observed m/z [M +

H]⁺405.0; HPLC purity: 98%.

K

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3-(3-(4-Chlorophenyl)-1-cyclohexyl-1H-pyrazol-4-yl)-N-

(400 MHz, chloroform-d): δ 7.76 (s, 1H), 7.62−7.60 (m, 2H), 7.57−

7.38 (m, 7H), 3.18 (s, 3H), 3.06−3.02 (t, J = 7.2 Hz, 2H), 2.51−2.48

(t, J = 7.2 Hz, 2H). LC/MS (APCI⁺): calculated for C₁₉H₁₈BrN₃O₃S,

447.03; observed m/z [M + H]⁺448.0; HPLC purity: 99%.

3-(1-(4-Bromophenyl)-3-(3-chlorophenyl)-1H-pyrazol-4-yl)-N-

(methylsulfonyl)propanamide (14). Method 2. Yield: 10%. ¹H NMR

(400 MHz, chloroform-d): δ 7.83 (s, 1H), 7.70 (s, 1H), 7.64−7.53

(m, 5H), 7.43−7.34 (m, 2H), 3.26 (s, 3H), 3.07 (t, J = 7.2 Hz, 2H),

2.58 (t, J = 7.2 Hz, 2H). LC/MS (APCI⁺): calculated for

C₁₉H₁₇BrClN₃O₃S, 480.99; observed m/z [M + H]⁺482.0, 484.0;

HPLC purity: >99%.

1

(methylsulfonyl)propanamide (7). Method 2. Yield: 37%. H NMR

(400 MHz, Chloroform-d): δ 7.74 (s, 1H), 7.57−7.55 (m, 2H), 7.42−

7.40 (m, 2H), 7.35 (s, 1H), 4.14−4.10 (m, 1H), 3.26 (s, 3H), 3.04−

3.00 (t, J = 7.2 Hz, 2H), 2.54−2.51 (t, J = 7.2 Hz, 2H), 2.20−2.17 (m,

2H), 1.94−1.91 (m, 2 H), 1.77−1.71 (m, 3H), 1.46−1.26 (m, 3H).

LC/MS (APCI⁺): calculated for C₁₉H₂₄ClN₃O₃S, 409.12; observed

m/z [M + H]⁺410.0; HPLC purity: 96%.

3-(1,3-Bis(4-chlorophenyl)-1H-pyrazol-5-yl)-N-(methylsulfonyl)-

propanamide (8). Method 1. Yield: 52%. ¹H NMR (300 MHz,

methanol-d₄): δ 8.14 (s, 1H), 7.84−7.75 (m, 2H), 7.76−7.66 (m,

2H), 7.54−7.46 (m, 4H), 3.21 (s, 3H), 3.10−2.98 (m, 2H), 2.64 (t, J

= 7.4 Hz, 2H). ¹³C NMR (151 MHz, methanol-d₄): δ 174.0, 151.9,

140.0, 135.1, 133.3, 132.9, 130.6 (2C), 130.5 (2C), 129.8 (2C), 129.0

(2C), 121.3 (2C), 41.4, 37.4, 20.4. LC/MS (ESI⁺): calculated for

C₁₉H₁₇Cl₂N₃O₃S, 437.04; observed m/z [M + H]⁺438.1; HPLC

purity: >99%.

3-(3-(4-Chlorophenyl)-1-(4-(triﬂuoromethyl)phenyl)-1H-pyrazol-

4-yl)-N-(methylsulfonyl)propanamide (9). Method 3. Yield: 11%. ¹H

NMR (400 MHz, chloroform-d): 7.92 (s, 1H), 7.85−7.83 (d, J = 8.4

Hz, 2H), 7.70−7.63 (m, 4H), 7.44−7.42 (d, J = 8.8 Hz, 2H), 3.21 (s,

3H), 3.05−3.03 (m, 2H), 2.61−2.57 (m, 2H). LC/MS (ESI⁺):

calculated for C₂₀H₁₇ClF₃N₃O₃S, 471.06; observed m/z [M + H]⁺

472.1, 474.1; HPLC purity: 98%.

3-(3-(4-Chlorophenyl)-1-(6-(triﬂuoromethyl)pyridin-3-yl)-1H-pyr-

azol-4-yl)-N-(methylsulfonyl)propanamide (10). Method 1. Yield:

22%. ¹H NMR (400 MHz, Methanol-d₄): δ 9.22 (d, J = 2.5 Hz, 1H),

8.42 (ddd, J = 8.6, 2.6, 0.8 Hz, 1H), 8.34 (d, J = 0.9 Hz, 1H), 7.92

(dd, J = 8.6, 0.7 Hz, 1H), 7.80−7.62 (m, 2H), 7.56−7.43 (m, 2H),

3.21 (s, 3H), 3.11−2.97 (m, 2H), 2.66 (t, J = 7.3 Hz, 2H). ¹³C NMR

(151 MHz, methanol-d₄): δ 174.2, 153.4, 145.6 (q, J = 35.4 Hz),

141.1, 139.7, 135.5, 132.8, 130.6 (2C), 129.9, 129.4, 127.7, 125.7,

123.9, 122.7 (d, J = 10.1 Hz), 122.1, 41.4, 37.3, 20.5. LC/MS

(APCI⁺): calculated for C₁₉H₁₆ClF₃N₄O₃S, 472.06; observed m/z [M

+ H]⁺473.0; HPLC purity: >99%.

3-(3-(4-Chlorophenyl)-1-(4-cyanophenyl)-1H-pyrazol-4-yl)-N-

(methylsulfonyl)propanamide (11). Method 3. Yield: 33%. ¹H NMR

(400 MHz, chloroform-d): δ 7.96 (s, 1H), 7.89−7.87 (m, 2H), 7.78−

7.76 (m, 2H), 7.67−7.65 (m, 2H), 7.49−7.47 (m, 2H), 3.31 (s, 3H),

3.14−3.10 (m, 2H), 2.63−2.59 (m, 2H). LC/MS (APCI⁺): calculated

for C₂₀H₁₇ClN₄O₃S, 428.07; observed m/z [M + H]⁺429.1; HPLC

purity: >99%.

3-(3-(4-Chlorophenyl)-1-(4-(methylsulﬁnyl)phenyl)-1H-pyrazol-

4-yl)-N-(methylsulfonyl)propanamide (12). Step 1. 3-(3-(4-Chlor-

ophenyl)-1-(4-(methylthio)phenyl)-1H-pyrazol-4-yl)-N-

(methylsulfonyl)propanamide from acid 63l following sulfonamide

coupling method 2 (Scheme S1). Yield: 40 mg (31%). ¹H NMR (400

MHz, chloroform-d): 7.83 (s, 1H), 7.67−7.65 (m, 4H), 7.47−7.46

(m, 3H), 7.45−7.34 (m, 2H), 3.28 (s, 3H), 3.12−3.08 (m, 2H),

2.62−2.58 (m, 2H), 2.54 (s, 3H). LC/MS (APCI⁺): calculated for

C₂₀H₂₀ClN₃O₃S₂, 449.06; observed m/z [M + H]⁺450.0; HPLC

purity: 95%.

3-(3-(2-Chlorophenyl)-1-(4-chlorophenyl)-1H-pyrazol-4-yl)-N-

1

(methylsulfonyl)propanamide (15). Method 1. Yield: 9%. H NMR

(300 MHz, methanol-d₄): δ 8.13 (d, J = 0.8 Hz, 1H), 7.73 (d, J = 8.9

Hz, 2H), 7.57−7.52 (m, 1H), 7.50−7.36 (m, 5H), 3.17 (s, 3H), 2.76

(t, J = 7.4 Hz, 2H), 2.58−2.43 (m, 2H). LC/MS (APCI⁺): calculated

for C₁₉H₁₇Cl₂N₃O₃S, 437.04; observed m/z [M + H]⁺437.9, 439.9

(chlorine isotopic pattern); HPLC purity: >99%.

3-(1-(4-Bromophenyl)-3-(pyridin-4-yl)-1H-pyrazol-4-yl)-N-

(methylsulfonyl)propanamide (16). Method 3. Yield: 19%. ¹H NMR

(400 MHz, DMSO-d₆): δ 8.74−8.63 (m, 2H), 8.51−8.47 (m, 1H),

7.88−7.83 (m, 2H), 7.74 (s, 4H), 3.10−3.07 (s, 3H), 2.99−2.93 (m,

2H), 2.59−2.55 (m, 2H). ¹³C NMR (151 MHz, DMSO-d₆): δ 172.3,

150.1 (2C), 147.9, 140.2, 138.5, 132.4 (2C), 128.4, 121.6 (2C),

121.2, 120.2 (2C), 118.8, 40.9, 36.1, 19.4. LC/MS (APCI⁺):

calculated for C₁₈H₁₇BrN₄O₃S, 448.02; observed m/z [M + H]⁺

449.0; HPLC purity: 97%.

3-(1-(4-Bromophenyl)-3-(pyridin-3-yl)-1H-pyrazol-4-yl)-N-

1

(methylsulfonyl)propanamide (17). Method 3. Yield: 6%. H NMR

(400 MHz, chloroform-d): δ 8.79 (s, 1H), 8.45−8.44 (d, J = 4.4 Hz,

1H), 8.04−8.02 (d, J = 7.6 Hz, 1H), 7.83 (s, 1H), 7.56−7.50 (m,

4H), 7.31−7.30 (m, 1H), 3.19 (s, 3H), 3.01 (m, 2 H), 2.59−2.56 (m,

2H). ¹³C NMR (151 MHz, chloroform-d): δ 171.8, 147.7, 147.1,

146.6, 138.7, 137.0, 132.7, 132.6, 130.4, 128.1, 127.0, 124.6, 120.5,

120.2, 119.9, 41.7, 37.3, 19.8. LC/MS (APCI⁺): calculated for

C₁₈H₁₇BrN₄O₃S, 448.02; observed m/z [M + H] ⁺449.0, 451.0 (1:1)

bromine isotopic pattern; HPLC purity: >99%.

3-(1-(4-Bromophenyl)-3-(pyrazin-2-yl)-1H-pyrazol-4-yl)-N-

(methylsulfonyl)propanamide (18). Method 2. Yield: 12%. ¹H NMR

(400 MHz, chloroform-d): δ 9.44 (s, 1H), 8.67 (br s, 1H), 8.58 (s,

1H), 7.91 (s, 1H), 7.63 (q, J = 9.0 Hz, 4H), 3.28−3.19 (m, 5H), 2.79

(t, J = 7.1 Hz, 2H). LC/MS (APCI⁺): calculated for C₁₇H₁₆BrN₅O₃S,

449.02; observed m/z [M + H]⁺449.8, 451.9 (1:1) bromine isotopic

pattern; HPLC purity: 91%.

3-(1-(4-Bromophenyl)-3-cyclohexyl-1H-pyrazol-4-yl)-N-

(methylsulfonyl)propanamide (19). Method 2. Yield: 40%. ¹H NMR

(400 MHz, chloroform-d): δ 8.17 (s, 1H), 7.65 (s, 1H), 7.52 (s, 4H),

3.30 (s, 3H), 2.93−2.82 (m, 2H), 2.60 (s, 3H), 1.88 (br s, 4H), 1.80−

1.62 (m, 3H), 1.35 (s, 3H). LC/MS (APCI⁺): calculated for

C₁₉H₂₄BrN₃O₃S, 453.07; observed m/z [M + H]⁺454.0; HPLC

purity: >99%.

3-(1-(4-Bromophenyl)-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-

4-yl)-N-(methylsulfonyl)propanamide (20). Method 2. Yield: 13%.

¹H NMR (400 MHz, DMSO-d₆): δ 8.18 (s, 1H), 7.71−7.62 (m, 4H),

Step 2. To a solution of 3-(3-(4-chlorophenyl)-1-(4-(methylthio)-

phenyl)-1H-pyrazol-4-yl)-N-(methylsulfonyl)propanamide (35 mg,

77.78 μmol) in MeOH (200 μL) and THF (200 μL) was added

NaIO₄(17 mg, 81.67 μmol, 5 μL). The mixture was stirred at 20 °C

for 15 h. The reaction mixture was diluted with aqueous Na₂SO₃(5

mL) and extracted with EtOAc (10 mL × 2). The combined organic

layers were dried over Na₂SO₄and concentrated. The residue was

puriﬁed by prep-HPLC (column: Phenomenex Synergi C18 150 × 25

× 10 μm; mobile phase: [water (0.225%FA)-ACN]; B %: 35−65%,

12 min) to give 3-(3-(4-chlorophenyl)-1-(4-(methylsulﬁnyl)phenyl)-

1H-pyrazol-4-yl)-N-(methylsulfonyl)propanamide (12). Yield: 21 mg

3.94−3.92 (m, 2H), 3.46−3.45 (m, 2H), 3.43−3.26 (m, 2H), 3.07 (s,

3H), 2.70−2.69 (m, 1H), 2.67−2.66 (m, 3H), 1.77−1.73 (m, 4H).

¹³C NMR (151 MHz, DMSO-d₆): δ 172.7, 155.6, 138.9, 132.2 (2C),

126.2, 119.7, 119.4 (2C), 117.4, 67.1 (2C), 40.9, 36.9, 32.6, 31.9

(2C), 18.4. LC/MS (APCI⁺): calculated for C₁₈H₂₂BrN₃O₄S, 455.05;

+

observed m/z [M + H] 456.0; HPLC purity: 98%.

3-(1-(4-Bromophenyl)-3-(piperidin-4-yl)-1H-pyrazol-4-yl)-N-

(methylsulfonyl)propanamide (21). Step 1. Tert-butyl 4-(1-(4-

bromophenyl)-4-(3-(methylsulfonamido)-3-oxopropyl)-1H-pyrazol-

3-yl)piperidine-1-carboxylate was obtained as a white solid. Yield: 60

mg (17%) from acid 67 h by sulfonamide coupling method 2

(Scheme S2). LC/MS (APCI⁺): calculated for C₂₃H₃₁BrN₄O₅S,

554.12; observed m/z [M + H-Boc]⁺455.0; HPLC purity: 83%.

Step 2. The mixture of tert-butyl 4-(1-(4-bromophenyl)-4-(3-

(methylsulfonamido)-3-oxopropyl)-1H-pyrazol-3-yl)piperidine-1-car-

boxylate (55 mg, 99.01 μmol) in TFA (100 μL) and DCM (1 mL)

1

(58%). H NMR (400 MHz, chloroform-d): δ 7.90−7.88 (m, 3H),

7.73−7.66 (m, 4H), 7.48−7.46 (m, 2H), 3.30 (s, 3H), 3.13−3.09 (m,

2H), 2.78 (s, 3H), 2.67−2.64 (m, 2H). LC/MS (APCI⁺): calculated

for C₂₀H₂₀ClN₃O₄S₂, 465.06; observed m/z [M + H]⁺466.0, 468.0;

HPLC purity: >99%.

3-(1-(4-Bromophenyl)-3-phenyl-1H-pyrazol-4-yl)-N-

(methylsulfonyl)propanamide (13). Method 2. Yield: 29%. ¹H NMR

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https://doi.org/10.1021/acs.jmedchem.1c00837

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Article

was stirred at 20 °C for 2 h. The reaction mixture was concentrated.

The obtained residue was puriﬁed by prep-HPLC (column:

Phenomenex Synergi C18 150 × 25 × 10 μm, mobile phase:

[water (0.225%FA)-ACN]; B %: 5−35%, 12 min) to give 3-(1-(4-

bromophenyl)-3-(piperidin-4-yl)-1H-pyrazol-4-yl)-N-

(methylsulfonyl)propanamide (21) as a white solid. Yield: 27 mg

chlorophenyl)-1H-pyrazol-4-yl)propanoyl chloride (68) (220 mg,

518.72 μmol) in DCM (2 mL) at −50 °C (Scheme S3). The mixture

was stirred at −30 °C for ∼3 h. The mixture was washed with water

(5 mL) and concentrated. The residue was puriﬁed by prep-HPLC to

give 3-(1-(4-bromophenyl)-3-(4-chlorophenyl)-1H-pyrazol-4-yl)-N-

methylpropanamide (27) as a light-yellow solid. Yield: 60 mg

1

(59%). H NMR (400 MHz, DMSO-d₆): δ 8.22 (s, 1H), 7.71−7.62

(27%). H NMR (300 MHz, DMSO-d₆): δ 8.42 (s, 1H), 7.85−7.81

(m, 4H),3.36−3.33 (m, 3H), 3.06 (s, 3H), 2.77−2.63 (m, 2H), 2.49−

2.27 (m, 2H), 2.01−1.91 (m, 4H). LC/MS (APCI⁺): calculated for

C₁₈H₂₃BrN₄O₃S, 454.07; observed m/z [M + H]⁺455.0; HPLC

purity: 99%.

(m, 2H), 7.77−7.75 (m, 2H), 7.72−7.70 (m, 2H), 7.56−7.54 (m,

2H), 2.89 (t, J = 7.6 Hz, 2H), 2.56 (d, J = 8.4 Hz, 3H), 2.43 (t, J = 7.6

Hz, 2H). LC/MS (APCI⁺): calculated for C₁₉H₁₇BrClN₃O, 417.02;

observed m/z [M + H]⁺418.0, 420.0; HPLC purity: 98%.

3-(1-(4-Bromophenyl)-3-(1-formylpiperidin-4-yl)-1H-pyrazol-4-

yl)-N-(methylsulfonyl)propanamide (22). Method 2. Yield: 32%. ¹H

NMR (400 MHz, DMSO-d₆): δ 8.21 (s, 1H), 8.03 (s, 1H), 7.71−7.63

(m, 4H), 4.26−4.22 (m, 1H), 3.79−3.76 (m, 1H), 3.19 (s, 3H),

3.00−2.80 (m, 1H), 2.79−2.59 (m, 6H), 2.34−1.54 (m, 4H). LC/MS

(APCI⁺): calculated for C₁₉H₂₃BrN₄O₄S, 482.06; observed m/z [M +

H]⁺483.1; HPLC purity: >99%.

3-(1-(4-Bromophenyl)-3-(4-nitrophenyl)-1H-pyrazol-4-yl)-N-

(methylsulfonyl)propanamide (23). Method 2. Yield: 71%. ¹H NMR

(400 MHz, methanol-d₄): δ 8.38−8.30 (m, 2H), 8.22 (d, J = 6.3 Hz,

1H), 8.06−7.98 (m, 2H), 7.82−7.74 (m, 2H), 7.69−7.61 (m, 2H),

3.16 (s, 3H), 3.09 (t, J = 7.5 Hz, 2H), 2.65 (t, J = 7.5 Hz, 2H). LC/

MS (ESI⁺): calculated for C₁₉H₁₇BrN₄O₅S, 492.01; observed m/z [M

+ H]⁺493.0, 495.0 (1:1) bromine isotopic pattern; HPLC purity:

93%.

3-(3-(4-Aminophenyl)-1-(4-bromophenyl)-1H-pyrazol-4-yl)-N-

(methylsulfonyl)propanamide (24). To a mixture of 3-(1-(4-

bromophenyl)-3-(4-nitrophenyl)-1H-pyrazol-4-yl)-N-

(methylsulfonyl)propanamide (23) (100 mg, 0.203 mmol) and iron

(53 mg, 0.953 mmol) in EtOH (2 mL) was added a saturated solution

of NH₄Cl (1 mL) and heated to 80 °C for 18 h. The reaction mixture

was cooled to 25 °C, and the organic layer was separated. The organic

layer was dried over Na₂SO₄, and the solvent was evaporated under

reduced pressure. The residue was puriﬁed by ﬂash silica gel column

using DCM: MeOH (0−5% gradient) as eluents to aﬀord 3-(3-(4-

aminophenyl)-1-(4-bromophenyl)-1H-pyrazol-4-yl)-N-

(methylsulfonyl)propanamide (24) as a gray solid. Yield: 5 mg (5%).

¹H NMR (400 MHz, methanol-d₄): δ 8.19 (s, 1H), 7.90 (d, J = 8.5

3-(1-(4-Bromophenyl)-3-(4-chlorophenyl)-1H-pyrazol-4-yl)-N-

methyl-N-(methylsulfonyl)propanamide (28). To a solution of N-

methylmethanesulfonamide (116 mg, 1.06 mmol) in THF (5.00 mL)

was added NaH 60% dispersion (45 mg, 1.13 mmol) at 0−10 °C.

After stirring at 15 °C for 1 h, a solution of 3-(1-(4-bromophenyl)-3-

(4-chlorophenyl)-1H-pyrazol-4-yl)propanoyl chloride (68) (300 mg,

707.35 μmol) in DCM (3.00 mL) was added at −10 °C via a syringe.

The mixture was stirred at 15 °C for another 2 h. The mixture was

washed with brine (5 mL) and concentrated. The residue was puriﬁed

by prep-HPLC to give 3-(1-(4-bromophenyl)-3-(4-chlorophenyl)-

1H-pyrazol-4-yl)-N-methyl-N-(methylsulfonyl)propanamide (28).

1

Yield: 65 mg (17%). H NMR (300 MHz, methanol-d₄): δ 8.25−

8.17 (m, 1H), 7.80−7.71 (m, 4H), 7.69−7.61 (m, 2H), 7.54−7.46

(m, 2H), 3.27 (s, 6H), 3.11−2.98 (m, 4H). LC/MS (APCI⁺):

calculated for C₂₀H₁₉BrClN₃O₃S, 495.00; Observed m/z [M + H]⁺

496.0, 498.0; HPLC purity: 98%.

N-(3-(1-(4-Bromophenyl)-3-(4-chlorophenyl)-1H-pyrazol-4-yl)-

propyl)methanesulfonamide (29). To a solution of 3-(1-(4-

bromophenyl)-3-(4-chlorophenyl)-1H-pyrazol-4-yl)propan-1-amine

(72) (90 mg, 230.36 μmol) in DCM (3 mL) were added Et₃N (93

mg, 921.44 μmol, 128 μL) and MsCl (36 μL) at 0−10 °C (Scheme

S3). The mixture was stirred at 10 °C for 1 h. The mixture was

quenched with water (2 mL) and extracted with DCM (5 mL × 2).

The organic phase was concentrated and puriﬁed by reverse-phase

HPLC to give N-(3-(1-(4-bromophenyl)-3-(4-chlorophenyl)-1H-

pyrazol-4-yl)propyl)methanesulfonamide (29) as a white solid.

1

Yield: 68 mg (63%). H NMR (400 MHz, DMSO-d₆): δ 8.54−8.41

(m, 1H), 7.91−7.81 (m, 2H), 7.81−7.74 (m, 2H), 7.74−7.66 (m,

2H), 7.60−7.48 (m, 2H), 7.12−7.00 (m, 1H), 3.10−2.96 (m, 2H),

2.89 (s, 3H), 2.76−2.69 (m, 2H), 1.87−1.73 (m, 2H). LC/MS

(APCI⁺): calculated for C₁₉H₁₉BrClN₃O₂S, 467.01; observed m/z [M

+ H]⁺468.0; HPLC purity: >99%.

Hz, 2H), 7.76 (d, J = 8.8 Hz, 2H), 7.66 (d, J = 8.8 Hz, 2H), 7.49 (d, J

= 8.5 Hz, 2H), 3.23 (s, 3H), 3.06 (t, J = 7.4 Hz, 2H), 2.68 (t, J = 7.4

Hz, 2H). LC/MS (APCI⁺): calculated for C₁₉H₁₉BrN₄O₃S, 462.04;

observed m/z [M + H]⁺463.1, 465.1 (1:1) bromine isotopic pattern;

HPLC purity: 92%.

N-((2-(1-(4-Bromophenyl)-3-(4-chlorophenyl)-1H-pyrazol-4-yl)-

ethyl)sulfonyl)acetamide (30). To the solution of 2-(1-(4-bromo-

phenyl)-3-(4-chlorophenyl)-1H-pyrazol-4-yl)ethane-1-sulfonamide

(74) (150 mg, 340.34 μmol) and Et₃N (69 mg, 680.68 μmol, 94 μL)

in THF (4 mL) was added acetyl chloride (40 mg, 510.51 μmol, 36

μL) drop-wise in ice bath (Scheme S4). The mixture was stirred at 40

°C for 16 h. The mixture was concentrated and separated between

water (3 mL) and EtOAc (3 mL). The organic phase was dried over

anhydrous Na₂SO₄and concentrated. The residue was puriﬁed by

prep-HPLC (column: Phenomenex Synergi C18150 × 25 × 10 μm;

mobile phase: [water (0.225%FA)-ACN]; B %: 57−87%, 10 min) and

then puriﬁed again by prep-HPLC (column: Phenomenex Gemini

150 × 25 mm × 10 μm; mobile phase: [water (0.05% ammonia

hydroxide v/v)-ACN]; B %: 22−52%, 10 min) to aﬀord N-((2-(1-(4-

bromophenyl)-3-(4-chlorophenyl)-1H-pyrazol-4-yl)ethyl)sulfonyl)-

3-(3-Benzyl-1-(4-bromophenyl)-1H-pyrazol-4-yl)-N-

(methylsulfonyl)propanamide (25). Method 3. Yield: 20%. ¹H NMR

(400 MHz, chloroform-d): δ 7.71 (s, 1H), 7.58 (s, 4H), 7.36−7.32

(m, 5H), 4.09 (s, 2H), 3.21 (s, 3H), 2.74−2.71 (m, 2H), 2.06−2.02

(m, 2H). LC/MS (APCI⁺): calculated for C₂₀H₂₀BrN₃O₃S, 461.04;

observed m/z [M + H]⁺462.0; HPLC purity: >99%.

3-(1-(4-Bromophenyl)-3-(4-chlorophenyl)-1H-pyrazol-4-yl)-

propanoic Acid (26). To a solution of (E)-3-(1-(4-bromophenyl)-3-

(4-chlorophenyl)-1H-pyrazol-4-yl)acrylic acid (62a) (1.66 g, 4.11

mmol) in MeOH (150 mL) was added NH₂NH₂·H₂O (16.47 g,

328.99 mmol, 16 mL), and the mixture was heated to 80 °C for 48 h

(Scheme S1). The mixture was cooled to room temperature, diluted

with ice water (100 mL) and adjusted to pH = 3 with 1 M HCl. The

aqueous layer was extracted with DCM (50 mL × 3). The organic

layer was washed with brine (50 mL), dried over anhydrous Na₂SO₄,

and concentrated. To the residue was added petroleum ether (20 mL)

slowly. The mixture was concentrated slowly to give some solid. The

solid collected to give 3-(1-(4-bromophenyl)-3-(4-chlorophenyl)-1H-

pyrazol-4-yl)propanoic acid (26) as a yellow solid. Yield: 1.80 g

1

acetamide (30) as a white solid. Yield: 29 mg (17%). H NMR (400

MHz, DMSO-d₆): δ 8.58 (s, 1H), 7.88−7.83 (m, 3H), 7.74−7.70 (m,

4H), 7.54−7.51 (m, 2H), 3.60−3.56 (m, 2H), 3.06−3.03 (m, 2H),

1.87 (s,3H). LC/MS (APCI⁺): calculated for C₁₉H₁₇BrClN₃O₃S,

480.99; observed m/z [M + H]⁺482.0; HPLC purity: >99%.

2-(1-(4-Bromophenyl)-3-(4-chlorophenyl)-1H-pyrazol-4-yl)-N-

(methylsulfonyl)acetamide (31). Method 2. Yield: 11%.¹H NMR

(400 MHz, chloroform-d): δ 8.05 (s, 1H), 7.88 (s, 1H), 7.67−7.60

(m, 6H), 7.49−7.47 (d, J = 8.4 Hz, 2H), 3.81 (s, 2H), 3.15 (s, 3H).

LC/MS (APCI⁺): calculated for C₁₈H₁₅BrClN₃O₃S, 466.97; observed

m/z [M + H]⁺467.9, 470.0; HPLC purity: 99%.

1

(99%). H NMR (400 MHz, chloroform-d): δ7.85−7.75 (m, 1H),

7.59 (d, J = 7.8 Hz, 6H), 7.48−7.38 (m, 2H), 3.02 (m, 2H), 2.67 (m,

2H). LC/MS (APCI⁺): calculated for C₁₈H₁₄BrClN₂O, 403.99;

observed m/z [M + H]⁺404.9, 407.0; HPLC purity: 92%.

3-(1-(4-Bromophenyl)-3-(4-chlorophenyl)-1H-pyrazol-4-yl)-N-

methylpropanamide (27). Method 3. To a solution of MeNH₂/THF

(2 M, 5 mL) was added the solution of 3-(1-(4-bromophenyl)-3-(4-

M

https://doi.org/10.1021/acs.jmedchem.1c00837

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

4-(1-(4-Bromophenyl)-3-(4-chlorophenyl)-1H-pyrazol-4-yl)-N-

Hz, 2H), 8.33−8.26 (m, 3H), 7.83−7.75 (m, 2H), 7.70−7.62 (m,

2H), 3.29 (s, 4H), 2.63 (ddd, J = 8.9, 6.6, 4.7 Hz, 1H), 1.88 (dt, J =

8.2, 4.7 Hz, 1H), 1.73 (dt, J = 9.2, 4.7 Hz, 1H), 1.51 (ddd, J = 8.2, 6.6,

4.7 Hz, 1H). ¹³C NMR (151 MHz, methanol-d₄): δ 173.6, 163.1,

162.9, 162.6, 148.5, 148.1, 148.0, 145.9, 145.6, 139.9, 130.1, 125.0,

124.6, 121.8, 121.7, 41.6, 25.8, 19.0, 17.0. LC/MS (ESI⁺): calculated

for C₁₉H₁₇BrN₄O₃S, 460.02; observed m/z [M + H]⁺460.9; HPLC

purity: >98%.

3-(3-(4-Bromophenyl)-1-(4-chlorophenyl)-1H-pyrazol-5-yl)-N-

(methylsulfonyl)propanamide (51). Method 2. Yield: 25%. ¹H NMR

(400 MHz, chloroform-d): δ 8.00 (s, 1H), 7.72−7.70 (m, 2H), 7.56−

7.45 (m, 6H), 6.53 (s, 1H), 3.30 (s, 3H), 3.11−3.07 (t, J = 7.2 Hz,

2H), 2.69−2.65 (t, J = 7.2 Hz, 2H). LC/MS (APCI⁺): calculated for

C₁₉H₁₇BrClN₃O₃S, 480.99; observed m/z [M + H]⁺482.0; HPLC

purity: 99%.

3-(3-(4-Chlorophenyl)-1-(4-ﬂuorophenyl)-1H-pyrazol-5-yl)-

N(methylsulfonyl)propanamide (52). Method 2. Yield: 20%. ¹H

NMR (300 MHz, methanol-d₄): δ 7.85−7.76 (m, 2H), 7.64−7.53 (m,

2H), 7.46−7.39 (m, 2H), 7.39−7.28 (m, 2H), 6.71 (s, 1H), 3.21 (s,

3H), 3.00 (t, J = 7.3 Hz, 2H), 2.71 (t, J = 7.3 Hz, 2H). LC/MS

(ESI⁺): calculated for C₁₉H₁₇ClFN₃O₃S₃, 421.07; observed m/z [M +

H]⁺422.1; HPLC purity: 96%.

3-(1-(4-Bromophenyl)-4-(4-chlorophenyl)-1H-pyrazol-3-yl)-N-

(methylsulfonyl)propanamide (53). Method 2. Yield: 15%. ¹H NMR

(400 MHz, chloroform-d): δ 10.70 (s, 1H), 8.00 (s, 1H), 7.64−7.45

(m, 4H), 7.43−7.41 (m, 2H), 7.34−7.32 (m, 2H), 3.32 (s, 3H),

3.19−3.16 (t, J = 6.4 Hz, 2H), 2.89−2.86 (t, J = 6.4 Hz, 2H). LC/MS

(APCI⁺): calculated for C₁₉H₁₇BrClN₃O₃S, 480.99; observed m/z [M

+ H]⁺482.0; HPLC purity: 95%.

1

(methylsulfonyl)butanamide (32). Method 2. Yield: 9%. H NMR

(300 MHz, methanol-d₄): δ 8.20 (s, 1H), 7.78−7.72 (m, 4H), 7.66−

7.64 (m, 2H), 7.50−7.48 (m, 2H), 3.17 (s, 3H), 2.79−2.75 (t, J = 7.6

Hz, 2H), 2.39−2.36 (t, J = 7.2 Hz, 2H), 1.99−1.95 (t, J = 7.6 Hz,

2H). LC/MS (APCI⁺): calculated for C₂₀H₁₉BrClN₃O₃S, 495.00;

observed m/z [M + H]⁺496.0; HPLC purity: 96%.

3-(1-(4-Bromophenyl)-3-(4-chlorophenyl)-1H-pyrazol-4-yl)-N-

1

(ethylsulfonyl) Propanamide (33). Method 1. Yield: 23%. H NMR

(300 MHz, methanol-d₄): δ 8.14 (s, 1H), 7.73 (ddt, J = 7.5, 4.7, 2.4

Hz, 4H), 7.69−7.61 (m, 2H), 7.53−7.46 (m, 2H), 3.42−3.35 (m,

2H), 3.05 (t, J = 7.3 Hz, 2H), 2.66 (t, J = 7.3 Hz, 2H), 1.25 (t, J = 7.4

Hz, 3H). LC/MS (ESI⁺): calculated for C₂₀H₁₉BrClN₃O₃S, 495.00;

observed m/z [M + H]⁺496.1; HPLC purity: 98%.

3-(1-(4-Bromophenyl)-3-(4-chlorophenyl)-1H-pyrazol-4-yl)-N-

(cyclopropylsulfonyl) Propanamide (34). Method 1. Yield: 25 mg

(10%). ¹H NMR (300 MHz, methanol-d₄): δ 8.15 (s, 1H), 7.80−7.67

(m, 4H), 7.68−7.62 (m, 2H), 7.54−7.46 (m, 2H), 3.05 (t, J = 7.3 Hz,

2H), 2.98−2.86 (m, 1H), 2.65 (t, J = 7.3 Hz, 2H), 1.26−1.13 (m,

2H), 1.08−0.98 (m, 2H). LC/MS (APCI⁺): calculated for

C₂₁H₁₉BrClN₃O₃S, 507.00; observed m/z [M + H]⁺508.1; HPLC

purity: >99%.

3-(1-(4-Chlorophenyl)-3-(pyridin-4-yl)-1H-pyrazol-4-yl)-N-

1

(methylsulfonyl)propanamide (35). Method 1. Yield: 8%. H NMR

(300 MHz, methanol-d₄): δ 8.68−8.58 (m, 2H), 8.21 (s, 1H), 7.89−

7.80 (m, 4H), 7.57−7.48 (m, 2H), 3.23 (s, 3H), 3.14 (t, J = 7.4 Hz,

2H), 2.71 (t, J = 7.3 Hz, 2H). LC/MS (ESI⁺): calculated for

C₁₈H₁₇ClN₄O₃S, 404.07; observed m/z [M + H]⁺405.1; HPLC

purity: >99%.

3-(1-(4-Chlorophenyl)-3-(pyridin-4-yl)-1H-pyrazol-4-yl)-2-meth-

3-(3,5-Bis(4-chlorophenyl)-1H-pyrazol-1-yl)-N-(methylsulfonyl)-

1

yl-N-(methylsulfonyl)propanamide (36). Method 1. Yield: 18%. H

propanamide (54). Method 1. Yield: 11%. H NMR (300 MHz,

NMR (300 MHz, methanol-d₄): δ 8.68−8.61 (m, 2H), 8.17 (s, 1H),

7.88−7.80 (m, 4H), 7.56−7.49 (m, 2H), 3.15 (s, 4H), 2.98−2.84 (m,

1H), 2.80−2.66 (m, 1H), 1.25 (d, J = 6.9 Hz, 3H). LC/MS (ESI⁺):

calculated for C₁₉H₁₉ClN₄O₃S, 418.09; observed m/z [M + H]⁺

419.1; HPLC purity: >99%.

Methanol-d₄): δ 7.89−7.79 (m, 2H), 7.60−7.49 (m, 4H), 7.45−7.36

(m, 2H), 6.73 (s, 1H), 4.45 (t, J = 6.5 Hz, 2H), 3.14 (s, 3H), 2.97 (t, J

= 6.5 Hz, 2H). LC/MS (ESI⁺): calculated for C₁₉H₁₇Cl₂N₃O₃S,

437.04; observed m/z [M + H]⁺438.0; HPLC purity: >99%.

3-(5-(4-Bromophenyl)-1-(4-chlorophenyl)-1H-pyrazol-3-yl)-N-

(methylsulfonyl)propanamide (55). Method 2. Yield: 34%. ¹H NMR

(400 MHz, chloroform-d): δ 10.71 (s, 1H), 7.50−7.49 (m, 2H),

7.38−7.36 (m, 2H), 7.27−7.25 (m, 2H), 7.11−7.09 (m, 2H), 6.37 (s,

1H), 3.30 (s, 3H), 3.13−3.11 (t, J = 6.0 Hz, 2H), 2.88−2.84 (t, J = 6.0

Hz, 2H). LC/MS (APCI⁺): calculated for C₁₉H₁₇BrClN₃O₃S, 480.99;

observed m/z [M + H]⁺482.0; HPLC purity: >99%.

3-(1-(4-Chlorophenyl)-3-(pyridin-4-yl)-1H-pyrazol-4-yl)-N-

1

(methylsulfonyl)butanamide (37). Method 1. Yield: 21%. H NMR

(300 MHz, methanol-d₄): δ 8.62 (d, J = 5.0 Hz, 2H), 8.28 (s, 1H),

7.94−7.76 (m, 4H), 7.51 (d, J = 8.9 Hz, 2H), 3.64 (q, J = 7.2 Hz,

1H), 3.10 (s, 3H), 2.75−2.54 (m, 2H), 1.35 (d, J = 7.2 Hz, 3H). LC/

MS (ESI⁺): calculated for C₁₉H₁₉ClN₄O₃S, 418.09; observed m/z [M

+ H]⁺419.1; HPLC purity: >99%.

(E)-3-(1-(4-Chlorophenyl)-3-(pyridin-4-yl)-1H-pyrazol-4-yl)-N-

(methylsulfonyl)acrylamide (38). Method 1. Isolated as hydro-

chloride salt. Yield: 21%. ¹H NMR (300 MHz, DMSO-d₆): δ 9.24 (s,

1H), 8.94 (d, J = 5.6 Hz, 2H), 8.10 (d, J = 5.7 Hz, 2H), 8.04−8.00

(m, 2H), 7.76−7.65 (m, 3H), 6.57 (d, J = 15.8 Hz, 1H), 3.32 (s, 3H).

¹³C NMR (101 MHz, DMSO-d₆): δ 164.4, 147.5, 145.5, 144.3 (2C),

3-(2,4-Bis(4-chlorophenyl)oxazol-5-yl)-N-(methylsulfonyl)-

1

propanamide (56). Method 3. Yield: 11%. H NMR (400 MHz,

methanol-d₄): δ 8.06 (d, J = 6.80 Hz, 2H), 7.76 (d, J = 6.80 Hz, 2H),

7.54 (d, J = 6.80 Hz, 2H), 7.49 (d, J = 6.80 Hz, 2H), 3.32 (t, J = 5.60

Hz, 2H), 3.22 (s, 3H), 2.85 (t, J = 7.20 Hz, 2H). LC/MS (APCI⁺):

calculated for C₁₉H₁₆Cl₂N₂O₄S, 439.31; observed m/z [M + H]⁺

441.0. HPLC purity: 96%.

3-(2,4-Bis(4-chlorophenyl)thiazol-5-yl)-N-(methylsulfonyl)-

propanamide (57). Yield: 20%. ¹H NMR (400 MHz, methanol-d₄): δ

7.98−7.90 (m, 2H), 7.73−7.62 (m, 2H), 7.55−7.45 (m, 4H), 3.29 (d,

J = 7.2 Hz, 2H), 3.20 (s, 3H), 2.73 (t, J = 7.2 Hz, 2H). LC/MS

(ESI⁺): calculated for C₁₉H₁₆Cl₂N₂O₃S, 454.00; observed m/z [M +

H]⁺455.0. HPLC purity: >99%.

General Procedure for the Synthesis of Carbamate 45 and

Ureas (42−44 and 58). To a solution of methanesulfonamide (1.5−

2.5 equiv) in DCM or DMF was added diisopropylethylamine

(DIPEA, 7.5 equiv) and CDI (0.5−2.5 equiv) at 0 °C. The resulting

mixture was stirred at 25 °C for 16 h. Then, the appropriate amine or

alcohol (1 equiv) dissolved in DCM or DMF (depending on the

solubility) was added and heated at 45−90 °C for 24−48 h. Solvents

were evaporated under reduced pressure. Crude product was puriﬁed

by preparative HPLC or silica gel column chromatography.

N-(((3-(Pyridin-4-yl)-1-(6-(triﬂuoromethyl)pyridin-3-yl)-1H-pyra-

zol-4-yl)methyl)carbamoyl)methanesulfonamide (42). Yield: 31%.

¹H NMR (400 MHz, methanol-d₄): δ 9.35 (d, J = 2.36 Hz, 1H), 8.85

137.4, 132.5, 132.1, 130.5, 129.7 (2C), 124.6 (2C), 120.9 (2C),

119.1, 118.6, 41.3. LC/MS (ESI⁺): calculated for C₁₈H₁₅ClN₄O₃S,

402.06; observed m/z [M + H]⁺403.0, 404.9; HPLC purity: 98%.

(E)-3-(1-(4-Bromophenyl)-3-(pyridin-4-yl)-1H-pyrazol-4-yl)-N-

1

(methylsulfonyl)acrylamide (39). Method 1. Yield: 10%. H NMR

(300 MHz, methanol-d₄): δ 9.02−8.90 (m, 3H), 8.49−8.43 (m, 2H),

7.97−7.87 (m, 3H), 7.81−7.71 (m, 2H), 6.61 (d, J = 15.5 Hz, 1H),

3.34 (s, 3H). LC/MS (ESI⁺): calculated for C₁₈H₁₇BrN₄O₃S, 448.02;

observed m/z [M + H]⁺448.8; HPLC purity: 99%.

2-(1-(4-Chlorophenyl)-3-(pyridin-4-yl)-1H-pyrazol-4-yl)-N-

(methylsulfonyl)cyclopropane-1-carboxamide (40). Method 1.

1

Yield: 39%. H NMR (300 MHz, methanol-d₄): δ 8.91−8.83 (m,

2H), 8.60−8.52 (m, 2H), 8.41 (s, 1H), 7.96−7.89 (m, 2H), 7.62−

7.55 (m, 2H), 3.35 (s, 3H), 2.78−2.68 (m, 1H), 1.96 (dt, J = 8.8, 4.7

Hz, 1H), 1.80 (dt, J = 9.3, 4.7 Hz, 1H), 1.58 (ddd, J = 8.1, 6.5, 4.3 Hz,

1H). ¹³C NMR (151 MHz, methanol-d₄): δ 172.1, 161.4, 161.1,

147.5, 147.1, 146.5, 144.1, 143.8, 138.5, 132.4, 128.7, 123.7, 123.3,

122.9, 120.4, 40.2, 24.3, 17.5, 15.6. LC/MS (ESI⁺): C₁₉H₁₇ClN₄O₃S,

416.07; observed m/z [M + H]⁺417.1; HPLC purity: >99%.

2-(1-(4-Bromophenyl)-3-(pyridin-4-yl)-1H-pyrazol-4-yl)-N-

(methylsulfonyl)cyclopropane-1-carboxamide (41). Method 1.

(d, J = 5.96 Hz, 2H), 8.66 (s, 1H), 8.56 (dd, J = 2.36, 8.42 Hz, 1H),

8.37 (d, J = 6.48 Hz, 2H), 8.02 (d, J = 8.60 Hz, 1H), 4.67 (s, 2H),

3.25 (s, 3H). LC/MS (APCI⁺): calculated for C₁₇H₁₅F₃N₆O₃S,

440.40; observed m/z [M + H]⁺441.1.

1

Yield: 22%. H NMR (600 MHz, methanol-d₄): δ 8.74 (d, J = 5.7

N

https://doi.org/10.1021/acs.jmedchem.1c00837

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

N-(((1-(4-Chlorophenyl)-3-(pyridin-4-yl)-1H-pyrazol-4-yl)-

methyl)carbamoyl)methanesulfonamide (43). Yield: 19%. ¹H NMR

(400 MHz, methanol-d₄): δ 8.88 (d, J = 5.44 Hz, 2H), 8.53 (d, J =

5.20 Hz, 2H), 8.49 (s, 1H), 7.93 (d, J = 7.72 Hz, 2H), 7.58 (d, J =

7.72 Hz, 2H), 4.68 (s, 2H), 3.26 (s, 3H). ¹³C NMR (151 MHz,

methanol-d₄): δ 154.5, 149.8 (2C), 148.9, 143.7, 139.7, 133.7, 130.9,

130.7, 123.9, 123.7, 121.7, 121.5, 121.2, 120.6, 41.8, 35.5. LC/MS

(APCI⁺): calculated for C₁₇H₁₆ClN₅O₃S, 405.86; observed m/z [M +

H]⁺406.1; HPLC purity: 97%.

concentrated and diluted with water (5 mL). The aqueous phase was

adjusted to pH = 7 with 1 N HCl and extracted with EtOAc (5 mL ×

3). The organic phase was concentrated and puriﬁed by prep-HPLC

(column: Phenomenex Synergi C18 150 × 25 × 10 μm; mobile

phase: [water (0.225%FA)-ACN]; B %: 65−95%, 11 min) to aﬀord 3-

(2-(1-(4-bromophenyl)-3-(4-chlorophenyl)-1H-pyrazol-4-yl)ethyl)-

1,2,4-oxadiazol-5(4H)-one (47) as a white solid. Yield: 58 mg (32%).

¹H NMR (400 MHz, chloroform-d): δ 9.41 (s, 1H), 7.86 (s, 1H),

7.66−7.54 (m, 6H), 7.47−7.40 (m, 2H), 3.17−3.02 (m, 2H), 2.88−

2.77 (m, 2H). LC/MS (APCI⁺): calculated for C₁₉H₁₄BrClN₄O₂,

444.00; observed m/z [M + H]⁺445.0; HPLC purity: >99%.

N-(((1,3-Bis(4-chlorophenyl)-1H-pyrazol-4-yl)methyl)-

1

carbamoyl)methanesulfonamide (44). Yield: 15%. H NMR (300

MHz, DMSO-d₆): δ 10.00 (s, 1H), 8.51 (s, 1H), 7.94−7.88 (m, 2H),

7.80−7.74 (m, 2H), 7.63−7.53 (m, 4H), 6.85 (t, J = 5.4 Hz, 1H),

4.38 (d, J = 5.4 Hz, 2H), 3.22 (s, 3H). LC/MS (APCI⁺): calculated

for C₁₈H₁₆Cl₂N₄O₃S, 438.03; observed m/z [M + H]⁺438.9, 440.9;

HPLC purity: 96%.

5-((1,3-Bis(4-chlorophenyl)-1H-pyrazol-4-yl)methyl)pyrimidine-

2,4,6(1H,3H,5H)-trione (48). To a solution of 1,3-bis(4-chlorophen-

yl)-1H-pyrazole-4-carbaldehyde (61 h) (0.150 g, 0.473 mmol) and

pyrimidine-2,4,6(1H,3H,5H)-trione (0.064 g, 0.500 mmol) was added

a mixture of EtOH (10 mL) and DMF (10 mL) (Scheme S9). The

reaction mixture was heated in a pressure tube to 80 °C for 16 h. The

resulting suspension was cooled to 25 °C, NaBH₄(0.038 g, 1.004

mmol) was added, and the resulting mixture was stirred for 1 h at 25

°C to yield a clear orange solution. The solvents were evaporated in

vacuo to aﬀord a residue. To the obtained residue were added water

(80 mL) and some NaCl and hexane (70 mL) and EtOAc (10 mL),

and the resulting suspension was acidiﬁed with HCl to pH = 2 and

stirred for 1 h. The resulting precipitate was ﬁltered oﬀ, yielding a

white solid. The water layer was reextracted with EtOAc (80 mL × 2),

and the combined organic phases were dried over Na₂SO₄,

evaporated, and puriﬁed by SiO₂ﬂash chromatography [CH₂Cl₂/

iPrOH 95/5 to 90/10] yielding 5-((1,3-bis(4-chlorophenyl)-1H-

pyrazol-4-yl)methyl)-5-hydroxypyrimidine-2,4,6(1H,3H,5H)-trione

(0.027 g, 0.059 mmol, 12%) as a slightly yellowish solid (byproduct).

The white precipitate showed broad signals in NMR [due to B(OH)₃

contamination] and was therefore dissolved in MeOH (80 mL) and

HCl conc (1 mL), and the solvent was evaporated. This procedure

was repeated to give 5-((1,3-bis(4-chlorophenyl)-1H-pyrazol-4-yl)-

methyl)pyrimidine-2,4,6(1H,3H,5H)-trione (48) as a yellow solid.

(1-(4-Chlorophenyl)-3-(pyridin-4-yl)-1H-pyrazol-4-yl)methyl

1

(methylsulfonyl)carbamate (45). Yield: 26%. H NMR (400 MHz,

DMSO-d₆): δ 11.81 (s, 1H), 8.81 (s, 1H), 8.71 (s, 2H), 7.95−7.98

(m, 2H), 7.79−7.80 (m, 2H), 7.62−7.64 (m, 2H), 5.29 (s, 2H), 3.24

(s, 3H). ¹³C NMR (101 MHz, DMSO-d₆): δ 152.4, 150.7, 150.5,

149.5, 139.7, 138.3, 132.0, 131.7, 130.1, 130.0 (2C), 122.3, 120.8,

120.5, 116.8, 58.4, 41.2. LC/MS (APCI⁺): calculated for

C₁₇H₁₅ClN₄O₄S, 406.84; observed m/z [M + H]⁺407.1; HPLC

purity: 98%.

N-(((5-(4-Bromophenyl)-2-(pyridin-4-yl)furan-3-yl)methyl)-

1

carbamoyl)methanesulfonamide (58). Yield: 29%. H NMR (400

MHz, DMSO-d₆): δ 10.41 (s, 1H), 8.76 (d, J = 6.1 Hz, 2H), 8.00 (d, J

= 6.1 Hz, 2H), 7.90−7.84 (m, 2H), 7.74−7.67 (m, 2H), 7.21 (s, 2H),

4.51 (d, J = 5.7 Hz, 2H), 3.24 (s, 3H). ¹³C NMR (151 MHz, DMSO-

d₆): δ 158.2, 157.9, 153.5, 152.6, 146.0, 143.9, 140.1, 132.1 (2C),

129.9, 128.0, 126.3 (2C), 122.1, 119.9, 110.9, 41.4, 35.3. LC/MS

(ESI⁺): calculated C₁₈H₁₆BrN₃O₄S, 449.00; observed m/z [M + H]⁺

450.0, 452.0 (1:1) bromine isotopic pattern; HPLC purity: 96%.

Synthesis of Compounds with Heterocyclic Rings at C4−

46−50. 5-(2-(1-(4-Bromophenyl)-3-(4-chlorophenyl)-1H-pyrazol-4-

yl)ethyl)-1H-tetrazole (46). To a solution of 3-(1-(4-bromophenyl)-

3-(4-chlorophenyl)-1H-pyrazol-4-yl)propanenitrile (93) (100 mg,

258.62 μmol) in isopropanol (1 mL) and water (500 μL) were

added ZnBr₂(58 mg, 258.62 μmol, 13 μL) and NaN₃(50 mg, 775.86

μmol, 27 μL) (Scheme S8). The mixture was stirred at 120 °C for 15

h. The reaction mixture was diluted with water (10 mL) and extracted

with EtOAc (10 mL × 2). The combined organic layers were dried

over Na₂SO₄and concentrated. The residue was puriﬁed by prep-

HPLC (column: Phenomenex Synergi C18 150 × 30 mm × 4 μm;

mobile phase: [water (0.225%FA)-ACN]; B %: 60−90%, 12 min) to

aﬀord 5-(2-(1-(4-bromophenyl)-3-(4-chlorophenyl)-1H-pyrazol-4-

yl)ethyl)-1H-tetrazole (46) as a white solid. Yield: 21 mg (19%).

¹H NMR (300 MHz, DMSO-d₆): δ 8.52 (s, 1H), 7.85−7.83 (m, 2H),

7.77−7.70 (m, 4H), 7.56−7.54 (m, 2H), 3.22−3.18 (m, 2H), 3.12−

3.10 (m, 2H). LC/MS (APCI⁺): calculated for C₁₈H₁₄BrClN₆,

428.02; observed m/z [M + H]⁺429.0; HPLC purity: >99%.

3-(2-(1-(4-Bromophenyl)-3-(4-chlorophenyl)-1H-pyrazol-4-yl)-

ethyl)-1,2,4-oxadiazol-5(4H)-one (47). Step 1. To a mixture of 3-(1-

(4-bromophenyl)-3-(4-chlorophenyl)-1H-pyrazol-4-yl)propanenitrile

(93) (1.00 g, 2.59 mmol) and NH₂OH·H₂O (270 mg, 3.89 mmol) in

water (5 mg, 259.00 μmol) and EtOH (10 mL) was added a solution

of NaOMe (168 mg, 3.11 mmol) in MeOH (4 mL) (Scheme S8).

The mixture was stirred at 90 °C for 6 h. The mixture was poured into

water (10 mL) and stirred for 15 min. The solid was collected to

aﬀord (Z)-3-(1-(4-bromophenyl)-3-(4-chlorophenyl)-1H-pyrazol-4-

yl)-N′-hydroxypropanimidamide (94) as a light-yellow solid. Yield:

1 g (crude). LC/MS (APCI⁺): calculated for C₁₈H₁₆BrClN₄O,

418.02; observed m/z [M + H]⁺419.0; HPLC purity: 69%.

1

Yield: 162 mg (78%). H NMR (300 MHz, DMSO-d₆): δ 11.77 (s,

2H), 8.18 (s, 1H), 7.86 (d, J = 8.8 Hz, 2H), 7.69 (d, J = 8.4 Hz, 2H),

7.57 (d, J = 8.8 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 3.66 (s, 2H), 3.39

(s, 1H). LC/MS (ESI⁺): calculated for C₂₀H₁₄Cl₂N₄O₃, 428.04;

observed m/z [M + H]⁺429.1, 431.0; HPLC purity: 98%.

5-(2-(1-(4-Bromophenyl)-3-(4-chlorophenyl)-1H-pyrazol-4-yl)-

ethyl)-1,3,4-oxadiazol-2(3H)-one (49). Step 1. The reaction mixture

of 3-(1-(4-bromophenyl)-3-(4-chlorophenyl)-1H-pyrazol-4-yl)-

propanoic acid (26) (500 mg, 1.23 mmol), Et₃N (36 mg, 356.70

μmol, 49 μL), and NH₂NH₂·H₂O (157 mg, 3.08 mmol, 153 μL) in

toluene was stirred at 130 °C for 3 h (Scheme S8). The mixture was

separated between water (3 mL) and EtOAc (3 mL). The aqueous

phase was extracted with EtOAc (3 mL × 2). The combined organic

phase was washed with brine (5 mL), dried with anhydrous Na₂SO₄,

ﬁltered, and concentrated to aﬀord crude 3-(1-(4-bromophenyl)-3-(4-

chlorophenyl)-1H-pyrazol-4-yl)propanehydrazide (95) as a yellow

solid. Yield: 250 mg (48%). LC/MS (APCI⁺): calculated for

C₁₈H₁₆BrClN₄O, 418.02; observed m/z [M + H]⁺419.0; HPLC

purity: 67%.

Step 2. To the solution of 3-(1-(4-bromophenyl)-3-(4-chlor-

ophenyl)-1H-pyrazol-4-yl)propanehydrazide (95) (250 mg, 595.66

μmol) in THF (5 mL) was added CDI (145 mg, 893.49 μmol). The

mixture was stirred at 60 °C for 4 h under a nitrogen atmosphere. The

mixture was poured into water (5 mL) and extracted with EtOAc (10

mL × 3). The organic phase was concentrated and puriﬁed by prep-

HPLC (column: Phenomenex Synergi C18 150 × 25 × 10 μm;

mobile phase: [water (0.225%FA)-ACN]; B %: 65−95%, 10 min) to

aﬀord 5-(2-(1-(4-bromophenyl)-3-(4-chlorophenyl)-1H-pyrazol-4-

yl)ethyl)-1,3,4-oxadiazol-2(3H)-one (49) as a white solid. Yield: 93

Step 2. To a solution of (Z)-3-(1-(4-bromophenyl)-3-(4-

chlorophenyl)-1H-pyrazol-4-yl)-N′-hydroxypropanimidamide (94)

(250 mg, 411.01 μmol) in DMF (800 μL) and EtOH (2 mL) was

added NaOMe (4 M, 206 μL). The mixture was stirred at 15 °C for

0.5 h, and diethylcarbonate (304 mg, 2.57 mmol, 310 μL) was added.

The mixture was stirred at 80 °C for another 16 h. The mixture was

1

mg (34%). H NMR (300 MHz, DMSO-d₆): δ 8.57 (s, 1H), 7.86−

7.82 (m, 2H), 7.76−7.69 (m, 4H), 7.59−7.51 (m, 2H), 3.05−2.96

(m, 2H), 2.94−2.87 (m, 2H). LC/MS (APCI⁺): calculated for

C₁₉H₁₄BrClN₄O₂, 444.00; observed m/z [M + H]⁺445.0; HPLC

purity: 98%.

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N-(5-(1,3-Bis(4-chlorophenyl)-1H-pyrazol-4-yl)-4,5-dihydroisoxa-

zol-3-yl)methanesulfonamide (50). To a solution of methanesulfo-

namide (250 mg, 2.63 mmol) in DMF (2 mL) was added NaH (100

mg, 2.71 mmol), and the reaction mixture was stirred for 3 min under

a nitrogen atmosphere. Then, a solution of 5-(1,3-bis(4-chlorophen-

yl)-1H-pyrazol-4-yl)-3-bromo-4,5-dihydroisoxazole (97) (0.100 g,

0.229 mmol) in DMF (2 mL) was added, and the mixture was

heated to 90 °C in a pressure tube for 16 h (Scheme S9). The cooled

solution was diluted with NaHCO₃solution (50 mL) and extracted

with EtOAc (2 × 50 mL). The organic phase was dried over Na₂SO₄

and evaporated, and the material was puriﬁed by ﬂash chromatog-

raphy (Et₂O/HCOOH 99.5/0.5 to 99/1) yielding N-(5-(1,3-bis(4-

chlorophenyl)-1H-pyrazol-4-yl)-4,5-dihydroisoxazol-3-yl)-

methanesulfonamide (50) after crystallization from CH₂Cl₂/diiso-

propylether. Yield: 28 mg (27%). ¹H NMR (300 MHz, methanol-d₄):

δ 8.50 (s, 1H), 7.86 (d, J = 9.0 Hz, 2H), 7.80 (d, J = 8.6 Hz, 2H),

7.55−7.47 (m, 4H), 5.71 (t, J = 10.0 Hz, 1H), 3.47 (dd, J = 16.7, 9.8

Hz, 1H), 3.31−3.24 (m, 1H), 3.21 (s, 3H). LC/MS (ESI⁺):

calculated for C₁₉H₁₆Cl₂N₄O₃S, 450.03; observed m/z [M + H]⁺

450.8, 452.8; HPLC purity: >99%.

Aloysius T. Nchinda − Drug Discovery and Development

Centre (H3D), Department of Chemistry, University of Cape

Town, Rondebosch 7701, South Africa

Charles J. Eyermann − Drug Discovery and Development

Centre (H3D), Department of Chemistry, University of Cape

Town, Rondebosch 7701, South Africa

Stephen Fienberg − Drug Discovery and Development Centre

(H3D), Department of Chemistry, University of Cape Town,

Rondebosch 7701, South Africa

Vinayak Singh − Drug Discovery and Development Centre

(H3D), Institute of Infectious Disease and Molecular

Medicine and South African Medical Research Council Drug

Discovery and Development Research Unit, Department of

Chemistry and Institute of Infectious Disease and Molecular

Medicine, University of Cape Town, Rondebosch 7701, South

Africa; orcid.org/0000-0001-9002-2489

^°Alissa Myrick − Drug Discovery and Development Centre

(H3D), Institute of Infectious Disease and Molecular

Medicine, University of Cape Town, Rondebosch 7701, South

Africa

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00837.

■

sı

Efrem Abay − Drug Discovery and Development Centre

(H3D), Division of Clinical Pharmacology, Department of

Medicine, University of Cape Town, Cape Town 7925, South

Africa

Mathew Njoroge − Drug Discovery and Development Centre

(H3D), Division of Clinical Pharmacology, Department of

Medicine, University of Cape Town, Cape Town 7925, South

Africa

Nina Lawrence − Drug Discovery and Development Centre

(H3D), Division of Clinical Pharmacology, Department of

Medicine, University of Cape Town, Cape Town 7925, South

Africa

Qin Su − Genomic Technologies Section, Research

Technologies Branch, National Institutes of Health, National

Institute of Allergy and Infectious Diseases, Bethesda,

Maryland 20892, United States

Timothy G. Myers − Genomic Technologies Section, Research

Technologies Branch, National Institutes of Health, National

Institute of Allergy and Infectious Diseases, Bethesda,

Maryland 20892, United States

Helena I. M. Boshoﬀ − Tuberculosis Research Section,

Laboratory of Clinical Infectious Diseases, National Institutes

of Health, National Institute of Allergy and Infectious

Diseases, Bethesda, Maryland 20892, United States;

orcid.org/0000-0002-4333-206X

Clifton E. Barry, III − Tuberculosis Research Section,

Laboratory of Clinical Infectious Diseases, National Institutes

of Health, National Institute of Allergy and Infectious

Diseases, Bethesda, Maryland 20892, United States

Frederick A. Sirgel − South African Medical Research Council

Centre for Tuberculosis Research / DST/NRF Centre of

Excellence for Biomedical Tuberculosis Research, Division of

Molecular Biology and Human Genetics, Faculty of Medicine

and Health Science, Stellenbosch University, Cape Town

7505, South Africa

Paul D. van Helden − South African Medical Research

Council Centre for Tuberculosis Research / DST/NRF

Centre of Excellence for Biomedical Tuberculosis Research,

Division of Molecular Biology and Human Genetics, Faculty

of Medicine and Health Science, Stellenbosch University,

Cape Town 7505, South Africa

Synthetic schemes, synthesis of intermediates, analytical

spectra for lead compounds, microbiology assays, in vitro

DMPK assays, mouse PK studies, and in vivo eﬃcacy

studies (PDF)

Data from RNA microarray experiment of compound 8

(XLSX)

Data from RNA microarray experiment of compound 16

(XLSX)

Molecular formula strings and some data (CSV)

AUTHOR INFORMATION

Corresponding Authors

■

Sandeep R. Ghorpade − Drug Discovery and Development

Centre (H3D), Department of Chemistry, University of Cape

Town, Rondebosch 7701, South Africa; orcid.org/0000-

0002-9311-1572; Phone: +27 21 650 1250;

Email: Sandeep.ghorpade@uct.ac.za

Kelly Chibale − Drug Discovery and Development Centre

(H3D), Department of Chemistry, University of Cape Town,

Rondebosch 7701, South Africa; South African Medical

Research Council Drug Discovery and Development Research

Unit, Department of Chemistry and Institute of Infectious

Disease and Molecular Medicine, University of Cape Town,

Rondebosch 7701, South Africa; orcid.org/0000-0002-

1327-4727; Phone: +27 21 650 2553;

Email: kelly.chibale@uct.ac.za

Authors

Lutete Peguy Khonde − Drug Discovery and Development

Centre (H3D), Department of Chemistry, University of Cape

Town, Rondebosch 7701, South Africa

Rudolf Muller − Drug Discovery and Development Centre

̈

(H3D), Department of Chemistry, University of Cape Town,

Rondebosch 7701, South Africa

Grant A. Boyle − Drug Discovery and Development Centre

(H3D), Department of Chemistry, University of Cape Town,

Rondebosch 7701, South Africa

Virsinha Reddy − Drug Discovery and Development Centre

(H3D), Department of Chemistry, University of Cape Town,

Rondebosch 7701, South Africa

Lisa M. Massoudi − Mycobacteria Research Laboratories,

Department of Microbiology, Immunology, and Pathology,

P

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Colorado State University, Fort Collins, Colorado 80523,

United States

Gregory T. Robertson − Mycobacteria Research Laboratories,

Department of Microbiology, Immunology, and Pathology,

Colorado State University, Fort Collins, Colorado 80523,

United States

Anne J. Lenaerts − Mycobacteria Research Laboratories,

Department of Microbiology, Immunology, and Pathology,

Colorado State University, Fort Collins, Colorado 80523,

United States

Gregory S. Basarab − Drug Discovery and Development

Centre (H3D), Department of Chemistry, University of Cape

Town, Rondebosch 7701, South Africa; Drug Discovery and

Development Centre (H3D), Division of Clinical

Pharmacology, Department of Medicine, University of Cape

Town, Cape Town 7925, South Africa; orcid.org/0000-

0001-5684-6046

ACKNOWLEDGMENTS

■

The authors acknowledge DuPont Chemicals, USA for

donating the compound library. The authors thank Dr. Sridevi

Bashayam, Dr. Jayanth Thiruvellore, and Rajkumar Dhinakaran

from Syngene, India and the chemistry team from WuXi

AppTec, China for profound chemistry support. The authors

thank Tando Ntsabo and Nicole Cardoso from TB biology,

H3D for performing MIC and biology triage assays. The

authors thank the technical staﬀ at in vitro DMPK and

parasitology divisions of H3D for their excellent technical

assistance in generating all in vitro DMPK and cytotoxicity

data. The authors thank Marianna de Kock and Claudia Spies,

SAMRC and Stellenbosch University, South Africa for their

excellent technical assistance in generating MICs against

clinical TB isolates.

ABBREVIATIONS

■

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00837

TB, tuberculosis; MIC, minimum inhibitory concentration;

SAR, structure−activity relationship; RT, room temperature;

MeOH, methanol; EtOH, ethanol; EtOAc, ethyl acetate;

DIPEA, diisopropylethylamine; THF, tetrahydrofuran; DMF,

N,N-dimethylformamide; BuOH, butanol; HATU, 1-[bis-

(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]-

pyridinium 3-oxide hexaﬂuorophosphate; POCl₃, phosphorus-

(V) oxychloride; SOCl₂, thionyl chloride; NaBH₄, sodium

borohydride; NaCNBH₃, sodium cyanoborohydride; LAH,

lithium aluminum hydride; mCPBA, meta-chloroperbenzoic

acid; AUC, area under the curve

Author Contributions

Chemical synthesis, compound design, and SAR expansion

were led by R.M. Chemical synthesis was performed by R.M.,

L.P.K., G.A.B., V.R., and A.T.N. Computational and

compound design support was provided by C.J.E. Pharmaco-

phore model was developed and written by S.F. Analysis and

interpretation of the biology data were done by V.S. and A.M.

In vivo PK experiments were performed by E.A. MetID studies

were performed by M.N. In vitro DMPK data were analyzed by

N.L. Agilent microarray protocols were developed by Q.S. and

T.G.M. The screening of DuPont compound library and the

RNA microarray experiment were performed by H.I.M.B. MIC

data were generated by H.I.M.B. High-throughput screening

was conceptualized and led by C.E.B. MICs against clinical

isolates were conducted by F.A.S. and P.D.v.H. Eﬃcacy studies

were planned and conducted by L.M. and G.R. Eﬃcacy studies

were guided by A.L. G.S.B. provided scientiﬁc and strategic

guidance for the work and edited the manuscript. S.R.G.

provided strategic support and gave inputs on the compound

design. Introduction, hit identiﬁcation, SAR, biology triage,

and conclusion sections of the manuscript were written by

S.R.G. Synthesis and chemistry experimental sections were

written by L.P·K. In vitro and in vivo DMPK and eﬃcacy

sections were written by M.N. and S.R.G. and were further

edited by G.R. Funding acquisition along with scientiﬁc and

strategic support were done by K.C. All authors have given

approval to the ﬁnal version of the manuscript.

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https://doi.org/10.1021/acs.jmedchem.1c00837

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

1,3-Diarylpyrazolyl-acylsulfonamides as Potent Anti-tuberculosis Agents Targeting Cell Wall Biosynthesis in Mycobacterium tuberculosis

DOI: 10.1021/acs.jmedchem.1c00837

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Full text of DOI:10.1021/acs.jmedchem.1c00837

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