Synthesis and Biological Evaluation of N-((1-(4-(Sulfonyl)piperazin-1-yl)cycloalkyl)methyl)benzamide Inhibitors of Glycine Transporter-1

Article

pubs.acs.org/jmc

Synthesis and Biological Evaluation of N‑((1-(4-(Sulfonyl)piperazin-

1-yl)cycloalkyl)methyl)benzamide Inhibitors of Glycine Transporter‑1

Christopher L. Cioﬃ,*^,†,⊥Shuang Liu,*^,†,#Mark A. Wolf,^†Peter R. Guzzo,^†,#Kashinath Sadalapure,^‡,∇

Visweswaran Parthasarathy,^‡David T. J. Loong,^‡,○Jun-Ho Maeng,^‡Edmund Carulli,^‡Xiao Fang,^‡

Kalesh Karunakaran,^‡Lakshman Matta,^‡Sok Hui Choo,^‡Shailijia Panduga,^‡Ronald N. Buckle,^†

Randall N. Davis,^†Samuel A. Sakwa,^†Priya Gupta,^‡Bruce J. Sargent,^†Nicholas A. Moore,^†,◆

Michele M. Luche,^∥Grant J. Carr,^∥Yuri L. Khmelnitsky,^§Jiﬀry Ismail,^§Mark Chung,^‡Mei Bai,^‡

Wei Yee Leong,^‡Nidhi Sachdev,^‡Srividya Swaminathan,^‡and Andrew J. Mhyre^∥,¶

^†Department of Medicinal Chemistry, AMRI, East Campus, 3 University Place, Rensselaer, New York 12144, United States

^‡Discovery Research and Development Chemistry, Singapore Research Center, AMRI, 61 Science Park Road, Science Park III,

117525, Singapore

^§Drug Metabolism and Pharmacokinetics, AMRI, East Campus, 17 University Place, Rensselaer, New York 12144, United States

^∥Bothell Research Center, AMRI, 22215 26th Ave SE, Bothell, Washington 98021-4425, United States

S

* Supporting Information

ABSTRACT: We previously disclosed the discovery of

rationally designed N-((1-(4-(propylsulfonyl)piperazin-1-yl)-

cycloalkyl)methyl)benzamide inhibitors of glycine transporter-1

(GlyT-1), represented by analogues 10 and 11. We describe

herein further structure−activity relationship exploration of

this series via an optimization strategy that primarily focused on the sulfonamide and benzamide appendages of the scaﬀold.

These eﬀorts led to the identiﬁcation of advanced leads possessing a desirable balance of excellent in vitro GlyT-1 potency and

selectivity, favorable ADME and in vitro pharmacological proﬁles, and suitable pharmacokinetic and safety characteristics.

Representative analogue (+)-67 exhibited robust in vivo activity in the cerebral spinal ﬂuid glycine biomarker model in both

rodents and nonhuman primates. Furthermore, rodent microdialysis experiments also demonstrated that oral administration of

(+)-67 signiﬁcantly elevated extracellular glycine levels within the medial prefrontal cortex (mPFC).

followed by expulsion of a magnesium block from the channel

pore via membrane depolarization.⁴

INTRODUCTION

■

The neurotransmitter glycine (1) (Figure 1) plays a key role in

both inhibitory and excitatory neurotransmission within the

central nervous system (CNS). Glycine acts as an endogenous

agonist at the strychnine-sensitive glycine-A binding site of

ionotropic glycine receptors (GlyRs), which induce inhibitory

post synaptic potentials (IPSPs) via Cl⁻inﬂux and membrane

hyperpolarization.^1,2GlyRs are largely expressed within the

hindbrain and spinal cord where their respective interneurons

facilitate sensory motor learning, process sensory stimuli such

as pain, relay reﬂex responses, and modulate respiratory rates

and rhythm patterns.³

Homeostatic glycine levels within the CNS are maintained via

two high aﬃnity transporters; GlyT-1 and glycine transporter-2

(GlyT-2).⁶Both transporters are of the Na⁺/Cl⁻solute carrier

family 6 (SLC6), share an approximate 50% sequence homol-

ogy, and present overlapping expression patterns within caudal

areas of the CNS (e.g., cerebellum, brainstem, and spinal cord).

In addition, GlyT-1 is also expressed within the forebrain (e.g.,

hippocampus, striatum, and prefrontal cortex (PFC))⁷and on

amacrine, ganglion, and Muller cells within mammalian and

nonmammalian retinae.⁸

Within the GlyR-rich hindbrain, GlyT-1 is primarily

expressed on glial astrocytes and is colocalized with GlyT-2,

where it serves to modulate inhibitory neurotransmission by

clearing glycine from the synaptic space surrounding the GlyRs.

Thus, inhibition of GlyT-1 may lead to increased extracellu-

lar glycine levels with concomitant enhanced GlyR activity and

inhibitory neurotransmission.⁹At excitatory glutamatergic

synapses, GlyT-1 is highly colocalized with NMDA receptors

Glycine, along with D-serine, also serves as an obligatory

coagonist that binds to the strychnine-insensitive glycine-B

site located on the GluN1 subunit of N-methyl-^D-aspartate

(NMDA) receptors.⁴NMDA receptors are ligand and voltage-

gated calcium permeable ionotropic glutamate receptors

(iGluRs) involved in excitatory neurotransmission and CNS

processes that underlie executive function, including long-term

potentiation (LTP), long-term depression (LTD), and synap-

tic plasticity.⁵Activation of NMDA receptors relies on simul-

taneous binding of ^L-glutamate at the GluN2 subunit and

obligatory coagonist glycine or ^D-serine at the GluN1 subunit

Received: June 17, 2016

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Figure 1. Representative GlyT-1 inhibitors.

on both glial and neuronal cells where it tightly maintains syn-

aptic glycine concentrations at subsaturation levels.¹⁰Inhibition

of GlyT-1 in these areas may lead to increased synaptic glycine

levels and glycine-B site occupancy resulting in a potentiation

of NMDA receptor function.¹¹Thus, GlyT-1 inhibition can

potentially lead to enhanced GlyR and/or NMDA receptor

activity, and the approach has been under investigation to treat

various CNS disorders that may be ameliorated by modula-

tion of either inhibitory glycinergic or excitatory glutamatergic

neurotransmission.

Parkinson’s disease.³³Lastly, recent preclinical studies suggest

that GlyT-1 inhibition may prevent hypoxia-induced neuronal

degeneration of the retina³⁴as well as provide a novel approach

for the treatment of hematological disorders such as sickle cell

anemia.³⁵

We previously disclosed a novel series of N-((1-(4-(propyl-

sulfonyl)piperazin-1-yl)cycloalkyl)methyl)benzamide GlyT-1

inhibitors derived from rationally designed hit 10 (Figure 2).³⁶

Numerous structurally diverse GlyT-1 inhibitors have been

disclosed, and representatives of this class (e.g., 2,¹²3,¹³4,¹⁴

5,¹⁵6,¹⁶7,¹⁷8,¹⁸9¹⁹) are highlighted in Figure 1. Due to the

emergence of the NMDA receptor hypofunction hypothesis

for schizophrenia (i.e., the glutamate hypothesis),²⁰selective

inhibition of GlyT-1 became an attractive approach to enhance

NMDA receptor activity by increasing local synaptic concen-

trations of glycine. Indeed, many of these agents were reported

to be eﬃcacious in several in vivo models predictive of anti-

psychotic and pro-cognitive activity.²¹Several GlyT-1 inhibitors

entered clinical trials for the treatment of schizophrenia, and

the approach enjoyed proof-of-concept in a series of Phase II

trials;²²however, a GlyT-1 inhibitor has yet to emerge as a

novel antipsychotic available to treat patients.^21,23

GlyT-1 inhibition has been studied in proof-of-concept

clinical trials for other CNS disorders including depression,²⁴

obsessive compulsive disorder (OCD),²⁵and addiction.^26,27

Furthermore, GlyT-1 inhibitors have demonstrated eﬃcacy in

animal models of neuropathic pain,^26,28anxiety,²⁹and epilepsy.³⁰

In addition, preclinical studies have shown that the approach may

also promote neuroprotection,³¹provide a therapeutic strategy

for autism spectrum disorders (ASDs),³²and potentially present

utility as an adjuvant treatment for symptomology associated

Figure 2. Previously reported N-((1-(4-(propylsulfonyl)piperazin-1-yl)-

cycloalkyl)methyl)benzamide GlyT-1 inhibitors 10 and 11.

Subsequent SAR campaigns led to the identiﬁcation of com-

pound 11, which possesses a favorable balance of in vitro GlyT-1

potency and selectivity, ADME and in vitro pharmacological

properties, and pharmacokinetic (PK) characteristics in rat.

Inhibitor 11 also provided in vivo proof-of-concept for the

series by producing a dose-dependent increase in rat cerebral

spinal ﬂuid (CSF) glycine levels upon oral administration.³⁶

We report herein further reﬁnement to our series whereby

optimization eﬀorts focused on the sulfonamide and benzamide

regions of the scaﬀold. This strategy led to the identiﬁcation of

advanced lead compounds that exhibited exquisite in vitro

GlyT-1 potency, favorable ADME proﬁles, desirable binding

characteristics, and robust in vivo glycine elevation activity

within the CNS of both rodents and nonhuman primates.

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a

Scheme 1

a

Reagents and conditions: (a) 1-propanesulfonyl chloride, Et₃N, CH₂Cl₂, 0 °C to rt, 3 h; (b) TFA, CH₂Cl₂, 0 °C to rt, 16 h; (c) 4,4-diﬂuoro-

cyclohexanone, Et₂AlCN, Ti(i-PrO)₄, toluene, 0 °C to rt for 16 h; (d) LiAlH₄, Et₂O, 0 °C to rt, 12 h; (e) substituted benzoyl chloride, Et₃N, CH₂Cl₂,

0 °C to rt, 4 h; or substituted benzoic acid, EDCI·HCl, HOBt, Et₃N, DMF, rt, 16 h; (f) 1.0 M HCl in H₂O, CH₃CN, 0 °C to rt, 30 min.

a

Scheme 2

a

Reagents and conditions: (a) cyclohexanone, PTSA, KCN, H₂O, rt, 16 h; (b), LiAlH₄, Et₂O, 0 °C to rt, 12 h; (c) triﬂuoroactetic anhydride, Et₃N,

CH₂Cl₂, 0 °C to rt, 16 h; (d) (i) 10% Pd/C, NH₄HCO₂, CH₃OH, 65 °C, 3 h; (ii) substituted sulfonyl chloride, Et₃N, CH₂Cl₂, 0 °C to rt, 1 h;

(iii) K₂CO₃, H₂O, CH₃OH, reﬂux, 16 h; (e) 2-amino-6-chlorobenzoic acid, HBTU, Et₃N, DMF, rt, 16 h; (f) 1.0 M HCl in Et₂O, CH₂Cl₂, 0 °C to rt,

30 min; (g) 2,4-dichlorobenzoyl chloride, Et₃N, CH₂Cl₂, 0 °C to rt, 16 h; (h) 10% HCl in H₂O, CH₃CN, 0 °C to rt, 30 min; or 1.0 M HCl in Et₂O,

Et₂O, 0 °C to rt, 30 min.

The desired ﬁnal compounds were readily manufactured via

acylation of 16 with either the corresponding benzoyl chloride

or benzoic acid, followed by treatment with aqueous HCl to

give the corresponding hydrochloride salts.³⁷The synthesis of

4-ﬂuoro-2-methoxy-6-methylbenzoic acid, which was used to

manufacture analogue 23, was achieved following the route

reported by Coulton and co-workers.³⁸

Scheme 2 highlights the synthetic routes used to access ana-

logues 10 and 29−39. A PTSA-mediated Strecker reaction

between 1-benzylpiperazine (24) and cyclohexanone in the

CHEMISTRY

■

The synthesis of n-propyl sulfonamide analogues 11 and

17−23 presented in Scheme 1 began with the sulfonylation of

tert-butyl piperazine-1-carboxylate (12) followed by Boc-depro-

tection to aﬀord n-propyl sulfonamide 14 in good yield.^36,37

Strecker condensation between piperazine 14 and 4,4-diﬂuoro-

cyclohexanone in the presence of Et₂AlCN and Ti(i-PrO)₄

furnished quaternary amino nitrile 15, which was subsequently

reduced with LiAlH₄to provide aminomethyl intermediate 16.

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a

Scheme 3

a

Reagents and conditions: (a) 1-methyl-1H-imidazole-4-sulfonyl chloride, Et₃N, CH₂Cl₂, 0 °C to rt, 5 h; (b), 12 N HCl, 1,4-dioxane, 0 °C to rt, 2 h;

(c) 4,4-diﬂuorocyclohexanone, TMSCN, ZnI₂, toluene, CH₃OH, 0 °C then reﬂux for 4 h followed by rt for 16 h; (d) LiAlH₄, THF, 0 °C to rt, 5 h;

(e) substituted benzoyl chloride, Et₃N, CH₂Cl₂, 0 °C to rt, 4 h; or substituted benzoic acid, EDCI·HCl, HOBt, Et₃N, DMF, rt, 16 h; (f) 1.0 M HCl

in H₂O, CH₃CN, 0 °C to rt, 30 min.

a

Scheme 4

a

Reagents and conditions: (a) 1-methyl-1H-1,2,3-triazole-4-sulfonyl chloride, Et₃N, CH₂Cl₂, 0 °C to rt, 5 h; (b), TFA, CH₂Cl₂, 0 °C to rt, 16 h;

(c) 4,4-diﬂuorocyclohexanone, TMSCN, ZnI₂, toluene, CH₃OH, 0 °C then reﬂux for 4 h followed by rt for 16 h; (d) LiAlH₄, THF, 0 °C to rt, 5 h;

(e) substituted benzoyl chloride, Et₃N, CH₂Cl₂, 0 °C to rt, 4 h; or substituted benzoic acid, EDCI·HCl, HOBt, Et₃N, DMF, rt, 16 h; (f) 1.0 M HCl

in H₂O, CH₃CN, 0 °C to rt, 30 min.

presence of KCN aﬀorded amino nitrile 25 in good yield, which

underwent smooth reduction with LiAlH₄to provide amino-

methyl intermediate 26. Treatment of 26 with triﬂuoroactetic

anhydride gave the triﬂuoroacetamide 27. Advanced amino-

methyl intermediates 28a−28k were obtained via a three-step

process that involved initial hydrogenolysis of 27 followed by

sulfonylation of the resulting debenzylated piperazine with the

corresponding sulfonyl chlorides and subsequent hydrolysis

of the triﬂuoroacetamide with K₂CO₃and H₂O in reﬂuxing

CH₃OH. Analogues 10 and 29−33 were then synthesized via

an HBTU-mediated peptide coupling between precursor inter-

mediates 28a−28f and 2-amino-6-chlorobenzoic acid, followed

by conversion to the hydrochloride salts in the presence

of HCl. Compounds 34−39 were prepared via acylation of

28g−28k with 2,4-dichlorobenzoyl chloride, followed by treat-

ment with HCl to give the corresponding hydrochloride salts.³⁷

The synthesis of N-((4,4-diﬂuoro-1-(4-((1-methyl-1H-imida-

zol-4-yl)sulfonyl)piperazin-1-yl)cyclohexyl)methyl)benzamide

analogues 44−49 is depicted in Scheme 3. Sulfonylation of 12

with 1-methyl-1H-imidazole-4-sulfonyl chloride followed by

Boc-deprotection aﬀorded sulfonamide 41, which provided

amino nitrile 42 using the aforementioned Strecker conditions.

LiAlH₄reduction of 42 aﬀorded aminomethyl intermediate 43,

which was used to manufacture 44−49 via acylation with the

corresponding benzoyl chloride or benzoic acid using standard

peptide coupling conditions followed by treatment with HCl to

give the corresponding hydrochloride salts.³⁷

Construction of N-((4,4-diﬂuoro-1-(4-((1-methyl-1H-1,2,3-

triazol-4-yl)sulfonyl)piperazin-1-yl)cyclohexyl)methyl)benzamide

analogues 54−60 was readily achieved following the route

presented in Scheme 4. Sulfonylation of 12 with 1-methyl-1H-

1,2,3-triazole-4-sulfonyl chloride³⁷followed by subsequent Boc-

deprotection and Strecker condensation with 4,4-diﬂuorocy-

clohexanone in the presence of TMSCN and ZnI₂provided

amino nitrile 52. Reduction of 52 with LiAlH₄provided amino-

methyl intermediate 53, which was used to manufacture

54−60 via acylation with the corresponding benzoyl chloride or

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a

Scheme 5

a

Reagents and conditions: (a) 4,4-diﬂuorocyclohexanone, TMSCN, ZnI₂, Et₂O, CH₃OH, toluene, rt for 4 h then reﬂux for 12 h; (b) (i) CH₃Li

(3.0 M in DME), reﬂux, 6 h; (ii) NaBH₄, CH₃OH, rt, 2 h; (iii) Boc₂O, Et₃N, CH₂Cl₂, rt, 5 h; (c) 10% Pd/C, NH₄HCO₂, CH₃OH, reﬂux, 4 h;

(d) 1-methyl-1H-1,2,3-triazole-4-sulfonyl chloride, Et₃N, CH₂Cl₂, 0 °C to rt, 2 h; (e) (i) 12 N HCl, CH₃OH, 0 °C to rt, 5 h; (ii) 2-chloro-

4-(triﬂuoromethyl)benzoic acid, HOBt, EDCI·HCl, Et₃N, DMF, rt, 16 h; (f) (i) resolution of the enantiomers via preparative chiral HPLC (Daicel

5 cm I.D. × 50 cm L, eluting with an isocratic mobile phase of 70% heptanes and 30% i-PrOH); (ii) 1.0 M HCl in H₂O, CH₃CN, 0 °C to rt, 30 min.

benzoic acid followed by treatment with HCl to give the cor-

responding hydrochloride salts.³⁷

that this region of the scaﬀold provided potential metabolic

soft-spots for CYP-mediated oxidation. Thus, installation of a

4,4-gem-difuoro moiety onto the central cyclohexyl ring led to

the discovery N-((4,4-diﬂuoro-1-(4-(propylsulfonyl)piperazin-

1-yl)cyclohexyl)methyl)-2,6-diﬂuorobenzamide 11, which exhib-

ited a favorable balance of potency (GlyT-1 IC₅₀= 67.5 nM),

selectivity (GlyT-2 IC₅₀> 75 μM), and signiﬁcantly improved

HLM stability.³⁶Compound 11 also provided in vivo proof-of-

concept for the series by inducing a dose-dependent increase in

rat CSF glycine levels upon acute oral administration of 20.8,

62.5, and 156.4 μmol/kg (10, 30, and 75 mg/kg).³⁶The work

reported herein describes follow-up optimization eﬀorts on the

sulfonamide and benzamide regions of analogues 10 and 11,

respectively. Key ﬁndings from both SAR campaigns converged

to provide optimized lead compounds, which were further

assessed for in vivo activity in rodents and nonhuman primates.

Structure−Activity Relationships. Guided by our pre-

viously reported SAR trends for the original N-((1-(4-(propy-

lsulfonyl)piperazin-1-yl)cycloalkyl)methyl)benzamide series,³⁶

we sought to enhance the potency of analogue 11 while

maintaining or improving its favorable HLM metabolic stability

by initially varying the benzamide component of the scaﬀold

and keeping the propyl sulfonamide in place. GlyT-1 potency

and in vitro human and rat microsomal intrinsic clearance data

(CL_int) for representatives of this series are shown in Table 1.

The SAR for this series revealed that potency trends mirrored

that of the previously reported N-((1-(4-(propylsulfonyl)-

piperazin-1-yl)cycloalkyl)methyl)benzamide series; the most

potent analogues featured two substituents on the benzamide

phenyl ring with at least one substituent positioned ortho to the

benzamide carbonyl, and there was no potency diﬀerentiation

between substituents possessing electron-donating (23) or

electron-withdrawing (11, 17−22) character. Furthermore, this

sample set (and the series as a whole) continued to demon-

strate excellent selectivity for GlyT-1 versus GlyT-2 (GlyT-2

IC₅₀> 75 μM). This campaign led to the discovery of key

analogues 21 and 22, which exhibited signiﬁcantly improved

GlyT-1 potency with comparable CL_intvalues relative to 11.

Concurrent with our benzamide SAR eﬀorts, we explored

alternative sulfonamide appendages starting from benchmark

analogues 2-amino-6-chloro-N-((1-(4-(propylsulfonyl)piperazin-

1-yl)cyclohexyl)methyl)benzamide (10, GlyT-1 IC₅₀= 15.1 nM)

and 2,4-dichloro-N-((1-(4-(propylsulfonyl)piperazin-1-yl)

We also explored the potential impact that substitution at the

methylene alpha to the benzamide NH would have on GlyT-1

potency and ADME properties for the series. The preparation

of the methyl-substituted enantiomers (−)-66 and (+)-67 is

shown in Scheme 5. Strecker condensation between 24 and

4,4-diﬂuorocyclohexanone in the presence of Et₂AlCN and

Ti(i-PrO)₄aﬀorded amino nitrile 61 in good yield. Nitrile 61

was subsequently treated with CH₃Li followed by NaBH₄

reduction, and the resulting racemic amine was Boc-protected

to give carbamate ( )-62.³⁷Hydrogenolysis of ( )-62 pro-

vided debenzylated piperazine ( )-63, which was sulfonylated

with 1-methyl-1H-1,2,3-triazole-4-sulfonyl chloride to aﬀord sul-

fonamide ( )-64 in good yield. HCl-promoted Boc-deprotection

of ( )-64 followed by HOBt-mediated peptide coupling of

the resulting amine with 2-chloro-4-(triﬂuoromethyl)benzoic

acid aﬀorded racemic benzamide ( )-65. Racemic ( )-65 was

resolved via chiral preparatory HPLC to give the corresponding

enantiomers (−)-66 and (+)-67.³⁷

RESULTS AND DISCUSSION

■

We previously conducted a rational design eﬀort with the

objective of identifying novel chemical matter that incorporated

benzamide and sulfonamide functionality positioned within a

pharmacophoric proximity relative to several reported GlyT-1

inhibitors. These eﬀorts led to the discovery of 2-amino-

6-chloro-N-((1-(4-(propylsulfonyl)piperazin-1-yl)cyclohexyl)-

methyl)benzamide 10, which exhibited good in vitro potency

(GlyT-1 IC₅₀= 15.1 nM) and excellent selectivity versus

GlyT-2 (GlyT-2 IC₅₀> 75 μM).³⁶Initial exploration of the

benzamide SAR from hit 10 revealed that (1) the benzamide

NH was critical for potency (methylation of the benzamide

nitrogen signiﬁcantly diminished potency), (2) at least two

substituents were required on the benzamide phenyl ring for

appreciable potency, (3) optimal GlyT-1 potency required at

least one substituent to be positioned ortho to the benzamide

carbonyl, and (4) monosubstitution at the meta position was

highly disfavored. These eﬀorts led to the discovery of highly

potent GlyT-1 inhibitors, yet this initial series containing

diverse benzamide analogues suﬀered from poor metabolic

stability in the presence of human liver microsomes (HLM).

Subsequent modiﬁcations to the central cyclohexyl ring suggested

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Table 1. SAR of Representative N-((4,4-Diﬂuoro-1-(4-(pro-

Table 2. Sulfonamide SAR for Representative N-((1-(4-(Sul-

pylsulfonyl)piperazin-1-yl)cyclohexyl)methyl)benzamide

fonyl)piperazin-1-yl)cyclohexyl)methyl)benzamide

a

Analogues

a

In vitro inhibitory GlyT-1 activity was determined using a whole-cell

scintillation proximity assay (SPA).³⁷The potency of each compound

was assessed in inhibiting the uptake of radiolabeled glycine

([¹⁴C]glycine) using the human choriocarcinoma cell line, JAR cells

(ATCC#HTB-144), which endogenously express human GlyT-1

b

(hGlyT-1b). For those compounds that were only tested twice

(n = 2), the IC₅₀data is shown as the mean of two independent

experiments. For compounds tested more than two times (n > 2), the

a

In vitro inhibitory GlyT-1 activity was determined using a whole-cell

scintillation proximity assay (SPA).³⁷The potency of each compound

was assessed in inhibiting the uptake of radiolabeled glycine

([¹⁴C]glycine) using the human choriocarcinoma cell line, JAR cells

(ATCC#HTB-144), which endogenously express human GlyT-1

IC₅₀data is represented as the mean

standard deviation (SD).

c

Microsomes were incubated at 37 °C with the necessary cofactor

regeneration system and individual test compounds at 3 μM. Reactions

were terminated at 0, 5, 15, 30, 45, and 60 min time intervals in

duplicate, and the amount of test compound remaining in the system

was determined using LCMS quantitation. The residual compound

remaining (%R) was determined by comparison to a zero time-point

and the ln(%R) plotted vs time. The slope was normalized to the

protein content in the incubation reaction to determine the intrinsic

clearance (CL_int) value. Testosterone incubated at 10 μM was used as

b

(hGlyT-1b). For those compounds that were only tested twice

(n = 2), the IC₅₀data is shown as the mean of two independent

experiments. For compounds tested more than two times (n > 2), the

IC₅₀data is represented as the mean standard deviation (SD).

heteroaromatic systems as potential sulfonamide appendages

within our series. Installation of 1-methyl-1H-pyrazole sul-

fonamide (37) led to ∼6-fold loss in potency relative to 34;

however, incorporation of 1-methyl-1H-imidazole (38) or

1-methyl-1H-1,2,3-triazole (39) led to an approximate 10- to

20-fold improvement in GlyT-1 potency values, respectively.

Interestingly, 2,6-diﬂuororobenzamide analogues containing

a regioisomeric 2-methyl-2H-1,2,3-triazole- or a des-methyl

1H-1,2,3-triazole-4-sulfonamide appendage exhibited a signiﬁ-

cant loss of potency relative to 1-methyl-1H-1,2,3-triazole 39

(GlyT-1 IC₅₀= 906.9 nM and 21.96 μM, respectively; struc-

tures not shown). The encouraging GlyT-1 potency results

obtained for 38 and 39 were tempered by very poor in vitro

HLM stability (5% remaining at 60 min and 0% remaining at

15 min, respectively). Thus, we explored a series of 1-methyl-

1H-imidazole and 1-methyl-1H-1,2,3-triazole sulfonamide

analogues that incorporated a central 4,4-gem-diﬂuoro cyclo-

hexyl ring while varying the benzamide region of the scaﬀold.

d

a standard. ND = not determined.

cyclohexyl)methyl)benzamide (34, GlyT-1 IC₅₀= 22.7 nM).

The results from this campaign revealed very narrow SAR for

this region of the scaﬀold, which is highlighted in Table 2.

Truncating the sulfonamide n-propyl chain to ethyl (30) led to

a 5-fold decrease in potency relative to 10, whereas further

truncation to methyl (29) led to a more signiﬁcant diminish-

ment in potency. Introduction of bulkier iso-butyl (31), phenyl

(32), or benzyl (33) sulfonamides was not well tolerated and

also led to signiﬁcant losses of potency relative to parent 10.

Interestingly, incorporation of a cyclopropylmethyl sulfon-

amide (35) resulted in a modest 2-fold loss in potency relative

to n-propyl comparator 34, whereas cyclobutyl (36) induced

a >10-fold loss in potency. Acknowledging previously dis-

closed GlyT-1 inhibitors,³⁹we also explored ﬁve-membered

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Table 3. SAR of Representative 1-Methyl-1H-imidazole and 1-Methyl-1H-1,2,3-triazole Sulfonamide GlyT-1 Inhibitors

a

In vitro inhibitory GlyT-1 activity was determined using a whole-cell scintillation proximity assay (SPA).³⁷The potency of each compound was

assessed in inhibiting the uptake of radiolabeled glycine ([¹⁴C]glycine) using the human choriocarcinoma cell line, JAR cells (ATCC#HTB-144),

which endogenously express human GlyT-1 (hGlyT-1b). For those compounds that were only tested twice (n = 2), the IC₅₀data is shown as the

b

mean of two independent experiments. For compounds tested more than two times (n > 2), the IC₅₀data is represented as the mean standard

c

d

deviation (SD). Compound concentration was 3 μM, and incubation time with the liver microsomes was 15 min. HLM = human liver microsomes.

e

RLM = rat liver microsomes.

Table 3 captures GlyT-1 potency, HLM, and rat liver micro-

somal (RLM) metabolic stability data for a series of 1-methyl-

1H-imidazole and 1-methyl-1H-1,2,3-triazole sulfonamide

analogues that feature a central 4,4-gem-diﬂuoro cyclohexyl

ring and varying benzamides. Strategic benzamide SAR for these

series incorporated substituents and substitution patterns that

provided the most potent analogues within the aforementioned

N-((4,4-diﬂuoro-1-(4-(propylsulfonyl)piperazin-1-yl)cyclohexyl)-

methyl)benzamide series. Nearly all of these analogues displayed

excellent GlyT-1 potency, selectivity over GlyT-2 (IC₅₀> 75 μM)

and improved HLM metabolic stability relative to parent com-

pounds 38 and 39. Analogues 45−60 exhibited low single digit

nanomolar to subnanomolar potency, with the 1-methyl-1H-

1,2,3-triazole sulfonamides 54−59 exhibiting an approximate

2-fold improvement in potency values relative to their 1-methyl-

1H-imidazole sulfonamide congeners 44−49. In addition, intro-

duction of a methyl group alpha to the benzamide nitrogen was

well tolerated, with racemic ( )-65 exhibiting a GlyT-1 IC₅₀

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Table 4. In Vitro ADMET Proﬁle for GlyT-1 Inhibitor (+)-67

Caco-2

permeability

(P_app(×10⁻⁶

cm/s))

CL_int

b

d

(μL/mL/min)

%PPB

R

a

c

e

solubility

H

4

R

6

cyno CYP inhibition (IC₅₀) 3A4, 2C9, 2D6, 2C19 hERG (IC₅₀)

H

monkey

81

A-B

23.9

B-A

47.4

tPSA

94.03

cLogP

3.40

22.2 μM

12 all > 7.2 μM 20.2 μM

88

87

a

b

c

Thermodynamic (shake ﬂask) solubility measured in PBS (pH = 7.4). Intrinsic clearance. Patch-Xpress’ patch-clamp assay; compounds were

d

e

tested (n = 3) in a ﬁve-point concentration−response on HE293 cells stably expressing the hERG channel. %PPB = plasma protein binding. tPSA

and cLogP values were determined by ChemDraw Ultra 12; H = human, R = rat, cyno = cynomolgus monkey.

a

Table 5. Rat and Cynomolgus Monkey PK Parameters of (+)-67

b

c

d

e

f

g

h

species

dose

CL

C_max(ng/mL)

t_max(h)

t_1/2(h)

4.33

V_ss

AUC_last(h·ng/mL)

%F

i

rat

3.1 μmol/kg (IV)

15.7 μmol/kg (PO)

23.3 2.83

(mL/min/kg)

352 113

2.67 2.89

1.75 0.08

(L/kg)

1955 611

27.2 8.5

j

cyno

1.5 μmol/kg (IV)

7.8 μmol/kg (PO)

1494 219

(mL/h/kg)

105.0 43.9

1.67 0.58

6.27 4.19 1929 592

(mL/kg)

690.0 395.1

31.0 21.1

a

̈

Dosing groups consisted of three drug naıve adult male Sprague−Dawley rats or three female cynomolgus monkeys. Data represented as mean

b

c

d

SD. Total body clearance. Maximum observed concentration of compound in plasma. Time of maximum observed concentration of compound in

plasma. Apparent half-life of the terminal phase of elimination of compound from plasma. Volume of distribution at steady state. Area under the

plasma concentration versus time curve from 0 to the last time point compound was quantiﬁable in plasma. Bioavailability; F = (AUC_INFpo

Dose_iv)/(AUC_INFiv× Dose_po). IV formulation = 5% DMSO and 10% Solutol in saline; IV dosing volume = 5 mL/kg; PO formulation = 5% Solutol

e

f

g

h

×

i

and 10% Captisol in 25 mM phosphate buﬀered saline (PBS; composition = 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.47 mM KH₂PO₄),

j

pH adjusted to 2 with HCl. IV formulation = 5% DMSO and 10% Solutol in saline, pH = 6.5; IV dosing volume = 2 mL/kg. PO formulation = 5%

Solutol in 20% Captisol, 25 mM PBS, pH = 2; PO dosing volume = 5 mL/kg.

a

Table 6. Glycine Elevation in CSF and (+)-67 Concentrations in CSF, Brain, and Plasma 2 h Post Oral Dosing in Rat

concentration of (+)-67 (nM)

b

dose (μmol/kg)

CSF glycine (ng/mL)

% vehicle control CSF glycine

CSF

brain

plasma

d

0.4

470 38.9

558 33.7

751 44.0

1528 168.9

100.4 8.3

119.3 7.2

bql

3.2 0.8

3.6 0.4

3.2 0.5

11.4 2.2

52.2 7.4

317.9 49

1.5

0.1 0.002

0.5 0.1

c

4.7

160.5 9.4

12.5

4

c

15.7

326.4 36.1

1.7 0.3

32.2 1.9

a

b

̈

Dosing groups consisting of drug naıve adult male Sprague−Dawley rats (n = 4−5/group). CSF glycine is summarized by treatment group

mean % vehicle control CSF glycine standard error of the mean (SEM); p < 0.05 vs vehicle control. The CSF glycine level taken as 100% = 468.2

27.3 ng/mL, which was obtained 30 min post vehicle dosing. Data were analyzed by 2-way ANOVA followed by Tukey’s posthoc test using

JMP v.6.0 statistical software. *Statistical signiﬁcance; p < 0.05 versus vehicle control. Test article vehicle = 5% Solutol and 10% Captisol in 25 mM

c

phosphate buﬀered saline (PBS; composition = 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.47 mM KH₂PO₄), pH adjusted to 2 with HCl.

d

bql = below quantitation limit (1.0 ng/mL).

value = 1.03 nM. Interestingly, an enantiopreference was

observed as chiral analogue (+)-67 (GlyT-1 IC₅₀= 1.06 nM)

was approximately 30-fold more potent than its corresponding

enantiomer (−)-66 (GlyT-1 IC₅₀= 33.1 nM).

(10 mg/kg) PO and with cynomolgus monkeys at 1.5 μmol/kg

(1 mg/kg) IV and 7.8 μmol/kg (5 mg/kg) PO. Compound

(+)-67 exhibited moderate CL and V_ssvalues with good half-

lives (t_1/2) for both species. Maximal plasma concentrations

(C_max) of 352 ng/mL for rat and 105 ng/mL for cynomolgus

monkey were achieved at 2.67 and 1.67 h post oral dose,

respectively. The observed plasma exposures (AUC_last) were

good for both species, ranging between 1955 h·ng/mL for rat

and 690 h·ng/mL for cynomolgus monkey, with resulting oral

bioavailabilities (%F) of 27.2 and 31.0%, respectively.

Due to its exquisite potency and favorable microsomal sta-

bility, emerging analogue (+)-67 was chosen for further

evaluation, and the respective ADME and in vitro pharmaco-

logical proﬁle is captured in Table 4. Compound (+)-67

exhibited moderate thermodynamic (shake ﬂask) solubility,

favorable CL_intvalues (human, rat, and cynomolgus monkey),

and no signiﬁcant oﬀ-target activity at the hERG channel,

CYPs, or within a standard panel of 69 GPCRs, ion channels,

enzymes, and transporters (data not shown). Lastly, (+)-67

exhibited favorable lipophilicity (cLogP = 3.40), and the com-

pound was classiﬁed as highly permeable in a standard Caco-2

permeability assay (eﬄux ratio = 2).

In Vivo Properties: PK Characteristics in Rat and

Cynomolgus Monkeys. The overall favorable in vitro

attributes of (+)-67 prompted us to further investigate its PK

proﬁle in rat and cynomolgus monkey (Macaca fascicularis)

(Table 5). Single dose PK studies were conducted with Sprague−

Dawley rats at 3.1 μmol/kg (2 mg/kg) IV and 15.7 μmol/kg

In Vivo Activity: Rat CSF Glycine Biomarker Model.

Inhibition of GlyT-1 leads to elevated levels of extracellular

glycine throughout the CNS, which spills over and pools within

the CSF where it can be readily measured. The rat CSF glycine

biomarker model allows for facile assessment of both in vivo

GlyT-1 engagement and potential exposure−response relation-

ships by providing both CSF glycine concentrations as well as

drug exposure levels in plasma, brain, and CSF from a single

study.⁴⁰Encouraged by the suitable PK characteristics observed

for (+)-67, we initially studied the compound’s eﬀect on CSF

glycine levels in an acute dose−response study with rats. Drug

̈

naive Sprague−Dawley rats were orally administered vehicle or

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(+)-67 at four diﬀerent doses (0.4, 1.5, 4.7, and 15.7 μmol/kg;

0.3, 1, 3, and 10 mg/kg), and CSF glycine levels were sub-

sequently measured 2 h post-dose. The rats were euthanized by

CO₂asphyxiation and a hypodermic needle was inserted into

the cisterna magna to withdraw 50−100 μL of CSF. The CSF

was diluted with deuterated-glycine as an internal standard, and

glycine levels were quantiﬁed by LC−MS/MS. As shown in

Table 6, oral administration of (+)-67 produced increases in rat

CSF glycine levels relative to vehicle that were statistically

signiﬁcant at the 4.7 and 15.7 μmol/kg doses, suggesting that

the compound is engaging GlyT-1 in vivo. In addition, con-

centrations of (+)-67 in plasma, brain, and CSF increased in

a dose-dependent manner. Noteworthy, compound (+)-67

exhibited statistically signiﬁcant pharmacodynamic (PD)

activity in this assay by inducing CSF glycine increases at the

4.7 and 15.7 μmol/kg dose levels despite relatively low total

brain-to-plasma (B/P) (0.2−0.1) and CSF-to-unbound plasma

concentration (C_CSF/C_u,p) (>0.1) ratios. The observed low

CNS exposure of (+)-67 may be attributed to a combination of

relatively high topological polar surface area (tPSA = 94.03)

and molecular weight (MW = 599.01 g/mol), which could be

hindering passive permeability (P_app) across the blood−brain

barrier (BBB). However, due to the high potency of the com-

pound, the projected free drug concentrations in the CNS

(C_u,band C_CSF) at the 4.7 and 15.7 μmol/kg doses appear to

equal or slightly exceed that required to signiﬁcantly inhibit

GlyT-1 as measured by the whole cell SPA assay (GlyT-1 IC₅₀

= 1.06 nM), leading to the observed CSF glycine level elevation

trend.⁴¹Furthermore, the dissociative half-life (residence time)

of (+)-67 at GlyT-1 has not been established; thus, it is unclear

to what extent this factor may also be contributing to the

observed CSF glycine elevation trends.⁴²Transporter occu-

pancy data has also not yet been determined.

Figure 3. (A) Eﬀects of compound 59 on the % increase of CSF

glycine from vehicle control for both acute (0.4, 1.6, 4.8, 16.1, 48.2,

and 160.9 μmol/kg, PO; represented as blue) and subchronic dosing

(1.6, 4.8, and 16.1 μmol/kg, PO, QD, 5 days; repesented as magenta).

CSF glycine is summarized by treatment group mean % vehicle control

CSF glycine SEM; p < 0.05 vs vehicle control. The CSF glycine level

taken as 100% for the acute study = 406.0 17.6 ng/mL, which was

obtained 30 min post vehicle dosing. The CSF glycine level taken as

Analogous inhibitor 59, which possesses an in vitro pharma-

cological and rodent in vivo CNS exposure proﬁle similar to

that of (+)-67 (data not shown), was concurrently analyzed in

the rat CSF glycine model in both an acute dose−response and

subsequent 5-day subchronic oral dosing study in an eﬀort to

understand the potential eﬀects of chronic exposure on PD.

Figure 3 presents CSF glycine elevation (as % versus vehicle)

and drug exposure levels of 59 (CSF, brain, and plasma)

obtained from both studies. In the acute dose−response study,

100% for the subchronic study = 521.2

21.5 ng/mL, which was

obtained 2 h post last-dose after QD dosing of vehicle over 5 days. All

dose−response curves were plotted using the software program

SigmaPlot, version 11.0. Data were analyzed by 2-way ANOVA

followed by Tukey’s posthoc test using JMP v.6.0 statistical software.

Data are expressed as mean

SEM; n = 5/group for each study.

̈

drug naive Sprague−Dawley rats were administered oral dose

(B) Exposure levels of 59 in CSF, brain, and plasma for both acute and

of vehicle or 59 (0.4, 1.6, 4.8, 16.1, 48.2, and 160.9 μmol/kg;

0.3, 1, 3, 10, 30, and 100 mg/kg) and then sacriﬁced 2 h post

dose. As shown in Figure 3A, 59 produced increases in CSF

glycine levels that were statistically signiﬁcant from vehicle

control in the 1.6 to 160.9 μmol/kg dose range. The study

demonstrated that, similar to (+)-67, analogue 59 also pos-

sesses excellent in vivo potency, as the eﬀective oral dose

required to double CSF glycine levels relative to vehicle

(ED₂₀₀) lies between 1.6 and 4.8 μmol/kg. The CSF, brain,

and plasma exposure levels of 59 also increased in the 0.4 to

16.1 μmol/kg dose group (Figure 3B). Drug exposure levels

did not increase in a dose-dependent fashion from the 48.2 to

160.9 μmol/kg doses, which may be attributed to oral absorp-

tion limitations due to the moderate solubility of the compound

(shake ﬂask solubility = 9.9 μM). In a subsequent subchronic

subchronic dosing (data acquired at the 2 h time-point post last-dose).

Data are expressed as mean

SEM; n = 5/group. *Statistical

signiﬁcance; p < 0.05 vs vehicle control. Vehicle preparation; 20 g of

Captisol was added to 100 mL of 25 mM PBS. The pH was lowered to

2 via addition of HCl. The mixture was stirred for 30 min, or until

dissolved and stored at 4 °C after use. Test article preparation; test

compound was weighed and dissolved in Solutol (5% of total volume).

Once dissolved, the solution is quickly stirred with 20% Captisol in

25 mM PBS, pH = 2 as stipulated above to the ﬁnal compound

concentration. The solution is then sonicated at room temperature for

20 min prior to dosing.

comparable to those observed for the same dose levels in the

acute dose−response study (1.6, 4.8, and 16.1 μmol/kg),

indicating that no tolerance for 59 had occurred. In addition,

drug exposure levels for the animals sacriﬁced 2 h post last-dose

were within a 2-fold range of those observed for the same dose

levels in the acute study, suggesting that there was no sig-

niﬁcant accumulation of 59 over the 5-day QD dosing period.

CSF glycine levels returned to baseline 48 h post last-dose for

̈

dosing study, drug naive Sprague−Dawley rats were adminis-

tered vehicle or an oral dose of 59 (1.6, 4.8, or 16.1 μmol/kg)

once daily (QD) over a 5-day period. The animals were then

sacriﬁced 2 or 48 h post last-dose. The observed CSF glycine

elevations for the cohorts sacriﬁced 2 h post last-dose were

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all three dose groups, demonstrating that the eﬀect of 59

was reversible (data not shown). Lastly, no gross behavioral

abnormalities with the animals were noted for either the acute

dose−response study or throughout the duration of the sub-

chronic dosing study.

In Vivo Activity: Cynomolgus Monkey CSF Glycine

Biomarker Model. Analogue (+)-67 was subsequently studied

in an acute dose−response CFS glycine study in cynomolgus

monkeys. All of the animals used in the study were surgically

prepared with indwelling cannulae inserted into the cisterna

magna and connected to a subcutaneous port to permit cere-

brospinal ﬂuid sampling. CSF samples (approximately 0.30 mL)

were obtained using a sterile Huber needle inserted into the

subcutaneous ports at the following time-points in relation to

dosing: predose (0), 0.5, 1, 2, 3, 4, 8, 12, and 24 h post dosing.

Blood samples were also taken at these time points to assess

drug exposure levels in the plasma.

Compound (+)-67 was orally administered over a dose range

of 0.4, 1.5, 4.7, and 15.7 μmol/kg, and statistically signiﬁcant

increases in CSF glycine levels were observed for the 4.7 and

15.7 μmol/kg doses (Figure 4A). Notably, the 15.7 μmol/kg

dose produced a robust increase in glycine levels that exceeded

300% relative to vehicle control. Similar to rat, (+)-67 exhibited

highly potent PD activity in the cynomolgus monkey with a

CSF glycine ED₂₀₀residing between 4.7 and 15.7 μmol/kg. The

highest CSF glycine elevation levels occurred between the

2 and 4 h time-points, which were trending back to baseline

over the remainder of the 24 h time-course. In addition,

the time-points correlating with observed peak CSF glycine

elevation track well with the (+)-67 T_maxobtained from the

cynomolgus monkey PK study (1.67 h). Drug plasma expo-

sure levels obtained in the CSF glycine study for the 4.7 and

15.7 μmol/kg doses also increased in a dose-dependent manner

(Figure 4B), whereas drug levels for the 0.4 and 1.5 μmol/kg

doses were below detection limits (<1.0 ng/mL). CSF drug

exposure levels for the 15.7 μmol/kg dose group were also

measurable; however, exposure levels for the 0.4, 1.5, and

4.7 μmol/kg dose groups were below detection limits. Similar

to rat, a good correlation between (+)-67 exposure levels

(plasma and CSF) and CSF glycine elevation was observed for

the 15.7 μmol/kg dose throughout the study time-course.

Lastly, no gross behavioral abnormalities with the animals were

observed throughout the duration of the study.

In Vivo Activity: Rat Medial Prefrontal Cortex Micro-

dialysis Experiments. We next examined if (+)-67 could also

elevate extracellular glycine levels within the rat medial pre-

frontal cortex (mPFC). Freely moving Sprague−Dawley rats

were given an oral gavage of 1.5, 4.7, and 15.7 μmol/kg of

(+)-67, and dialysate samples were taken from the mPFC over

a 5 h time-course, which were analyzed by HPLC and tandem

mass spectrometry. As shown in Figure 5, the mean normalized

glycine levels within the rat mPFC were signiﬁcantly increased

compared to basal levels (t = −80 to 0 min) from time-points

t = 80 to 300 min for the 4.7 and 15.7 μmol/kg dose groups

(p < 0.001). Notably, the 4.7 and 15.7 μmol/kg doses increased

the mean normalized glycine levels in a robust manner (>150%

of vehicle control basal levels) and glycine elevation was sus-

tained out to 5 h.

Figure 4. (A) Eﬀects of compound (+)-67 at 0.4, 1.5, 4.7, and

15.7 μmol/kg on CSF glycince levels in cynomolgus monkeys. Data

are expressed as mean SD; n = 3/group. (B) Corresponding plasma

exposure levels of (+)-67 for the 4.7 and 15.7 μmol/kg dosing groups

and CSF exposure levels for the 15.7 μmol/kg dosing group. Plasma

a

concentration data were analyzed with WinNonlin 5.2 software. nc =

b

noncalculable. bql = below quantitation limit (1.0 ng/mL). %CV =

Coeﬃcient of Variation. Vehicle preparation; 20 g of Captisol was

added to 100 mL of 25 mM PBS. The pH was lowered to 2 via

addition of HCl. The mixture was stirred for 30 min or until dissolved

and stored at 4 °C after use. Test article preparation; test compound

was weighed and dissolved in Solutol (5% of total volume). Once

dissolved, the solution is quickly stirred with 20% Captisol in 25 mM

PBS, pH = 2 as stipulated above to the ﬁnal compound concentration.

The solution is then sonicated at room temperature for 20 min prior

to dosing.

Thus, competitive inhibitors may provide more impactful inhi-

bition in areas of the CNS where physiological glycine con-

centrations are lower (i.e., forebrain). In addition, competitive

inhibition is potentially surmountable, which may circumvent

potential mechanism-based adverse events due to excessive and

prolonged glycine elevation.⁴³Employing similar Michaelis−

Menten saturation binding experiments reported by Mezler and

co-workers,⁴³we assessed the mechanism of binding for (+)-67

at GlyT-1. A series of glycine transport experiments using a

whole cell SPA assay with transfected HEK-293 cells expressing

GlyT-1 were conducted whereby changes in B_maxand K_dvalues

were measured by increasing [³H]-glycine concentrations in

the presence of varying concentrations of (+)-67 (Figure 6).

Increasing concentrations of inhibitor (+)-67 exhibited no

eﬀect on the B_maxvalues for glycine whereas its K_dvalues

increased, indicating that the compound was competing with

glycine for the same binding site at GlyT-1. Several additional

Mechanism of Binding. It has been suggested that com-

petitive GlyT-1 inhibitors might oﬀer potential therapeutic advan-

tages over noncompetitive inhibitors as the degree of competitive

inhibition could potentially depend on physiological glycine

concentrations, whereas noncompetitive inhibition would not.⁴³

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Figure 5. Eﬀects of compound (+)-67 on the extracellular levels of

̈

glycine in the mPFC of freely moving drug naive adult male Sprague−

Dawley rats (n = 5) administered by oral gavage of 1.5, 4.7, and

15.7 μmol/kg (1, 3, and 10 mg/kg) at time = 0. Data are expressed as

mean SE; n = 5 or 6/group. The mean normalized glycine levels

were signiﬁcantly increased compared to basal levels (t = −80 to

0 min) from time points t = 80 to 300 min (p < 0.001), and glycine

elevation was sustained over the 5 h time-course. Statistical analysis

was performed using Sigmastat 3.2 for Windows (SPSS Corporation).

Compound eﬀects were compared, using two-way (time × dose)

ANOVA for repeated measurements followed by Student−Newman−

Keuls posthoc test. Vehicle preparation; 20 g of Captisol was added to

100 mL of 25 mM PBS. The pH was lowered to 2 via addition of HCl.

The mixture was stirred for 30 min or until dissolved and stored at

4 °C after use. Test article preparation; test compound was weighed

and dissolved in Solutol (5% of total volume). Once dissolved, the

solution is quickly stirred with 20% Captisol in 25 mM PBS, pH = 2 as

stipulated above to the ﬁnal compound concentration. The solution is

then sonicated at room temperature for 20 min prior to dosing.

Figure 6. Determination of the mechanism of binding for compound

(+)-67. (A) Glycine transport experiments conducted whereby

B_maxand K_dvalues were obtained by increasing concentration of

[³H]-glycine in the presence of varying concentrations of (+)-67.

(B) Glycine transport experiments conducted whereby B_maxand K_d

values were obtained by increasing concentration of [³H]-glycine in

the presence of varying concentrations of 3.

CSF glycine levels for both species. In an acute dose−response

rat CSF glycine study, analogues 59 and (+)-67 induced dose-

dependent and robust increases in glycine levels that correlated

well with increasing plasma, brain, and CSF drug exposure

levels. A subsequent 5-day subchronic dosing study with 59

revealed that CSF glycine elevation levels mirrored those

observed in the acute dosing study at the same doses,

suggesting that tolerance to the inhibitor had not occurred.

In addition, exposures of 59 in the subchronic dosing study

were also similar to those observed in the acute study for the

same doses, providing evidence that there was no accumulation

of the compound over the 5-day, QD dosing period.

Furthermore, CSF glycine levels returned to baseline after a

48 h washout period for all dose groups, demonstrating that the

eﬀect of 59 was reversible.

The results obtained for the CSF glycine experiments

prompted us to examine whether analogues from our series

were also capable of elevating glycine levels in the mPFC,

which is regarded as the putative site of action for neuro-

psychiatric disorders such as schizophrenia. Indeed, micro-

dialysis experiments showed that inhibitor (+)-67 produced

robust increases in extracellular glycine levels within the mPFC

of freely moving Sprague−Dawley rats, verifying that the

compound is capable of engaging GlyT-1 in the forebrain.

Saturation binding experiments conﬁrmed that compound

(+)-67 (in addition to several other analogues within our

series) is competitive with glycine for binding at GlyT-1,

demonstrating that the series possesses a diﬀerentiating and

potentially preferable mechanism of binding relative to

noncompetitive inhibitors such as NFPS (ALX-5407) (3). In

conclusion, we believe that analogues from our series of

analogues within our series were also studied in these exper-

iments, and all of them were conﬁrmed to be competitive

inhibitors of GlyT-1 (data not shown). In contrast, increasing

concentrations of the known noncompetitive GlyT-1 inhibitor

NFPS (ALX-5407) (3), which has been reported to induce

mechanism-based toxicity,⁴⁴led to changes in B_maxvalues but

did not alter the K_dvalues for glycine. These results conﬁrm

that 3 is not competing with glycine for binding at GlyT-1, and

these ﬁndings were consistent with previously reported data.⁴⁴

CONCLUSIONS

■

Lead optimization eﬀorts for our GlyT-1 inhibitor series began

with benzamide SAR exploration starting from previously

reported N-((4,4-diﬂuoro-1-(4-(propylsulfonyl)piperazin-1-yl)-

cyclohexyl)methyl)benzamide 11. These eﬀorts led to the

discovery of key inhibitors 21 and 22, which were found to

possess superior GlyT-1 potency and comparable in vitro

microsomal metabolic stability relative to parent 11. Con-

current with this campaign, sulfonamide SAR exploration of

benchmark N-((1-(4-(propylsulfonyl)piperazin-1-yl)cyclohexyl)-

methyl)benzamide inhibitors 10 and 34 was conducted, which

lead to the discovery of key N-methyl imidazole and triazole

sulfonamide analogues 38 and 39. Our sulfonamide SAR

ﬁndings were converged with the benzamide SAR campaign to

provide standout analogues 59 and (+)-67.

Advanced lead (+)-67 possesses a favorable balance of

potency, ADME, and in vitro pharmacological proﬁles. The

compound exhibited suitable rat and nonhuman primate PK

characteristics and produced statistically signiﬁcant elevations of

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isocratic mobile phase of 70% heptanes and 30% i-PrOH, ﬂow rate =

1 mL/min, to give enantiopure (−)-66 and (+)-67.

N-((4,4-diﬂuoro-1-(4-(sulfonyl)piperazin-1-yl)cyclohexyl)-

methyl)benzamide GlyT-1 inhibitors possess favorable in vitro

and in vivo proﬁles that may prove beneﬁcial for the treatment

of the various neurological and neuropsychiatric disorders for

which the ﬁeld has been reported to show promise.

Enantiopurity analysis for chiral compounds (−)-66 and (+)-67 was

performed using a Shimadzu LC-20A HPLC system with a binary

solvent system A and B using an isocratic elusion [30% A, i-PrOH;

70% B, heptanes] and ﬂow rate = 1 mL/min, with UV detection at

254 nm (Method E). Optical rotation for (−)-66 and (+)-67 was

measured using a PerkinElmer polarimeter model 341. Measurements

were performed at 20 °C using a Na source lamp (589 nm) and

CH₃OH as the solvent.

EXPERIMENTAL SECTION

■

General Chemistry. All reactions were performed under a dry

atmosphere of nitrogen unless otherwise speciﬁed. Indicated reaction

temperatures refer to the reaction bath, while room temperature (rt) is

noted as 25 °C. Commercial grade reagents and anhydrous solvents

were used as received from vendors, and no attempts were made to

purify or dry these components further. Removal of solvents under

reduced pressure was accomplished with a Buchi rotary evaporator at

approximately 28 mmHg pressure using a Teﬂon-linked KNF vacuum

pump. Thin layer chromatography was performed using 1″ × 3″

AnalTech No. 02521 silica gel plates with ﬂuorescent indicator.

Visualization of TLC plates was made by observation with either short

wave UV light (254 nm lamp), 10% phosphomolybdic acid in ethanol,

or in iodine vapors. Preparative thin layer chromatography was

performed using Analtech, 20 × 20 cm, 1000 μm preparative TLC

plates. Flash column chromatography was carried out using a Teledyne

Isco CombiFlash Companion Unit with RediSepRf silica gel columns.

If needed, products were puriﬁed by reverse phase chromatography,

using a Teledyne Isco CombiFlash Companion Unit with RediS-

epGold C18 reverse phase column. Proton NMR spectra were

obtained either on a 300, 400, or 500 MHz Bruker Nuclear Magnetic

Resonance Spectrometer and chemical shifts (δ) are reported in parts

per million (ppm), coupling constant (J) values given in Hz, and with

the following spectral pattern designations: s, singlet; d, doublet;

t, triplet, q, quartet; dd, doublet of doublets; m, multiplet; br, broad.

Tetramethylsilane (TMS) was used as an internal reference. Melting

points are uncorrected and were obtained using a MEL-TEMP

Electrothermal melting point apparatus. Mass spectroscopic analyses

were performed using the following: (1) ESI ionization on a Varian

ProStar LCMS with a 1200L quadrapole mass spectrometer; (2) ESI,

APCI, or DUIS ionization on a Shimadzu LCMS-2020 single

quadrapole mass spectrometer. High pressure liquid chromatography

(HPLC) purity analysis was performed using the following: (1) Varian

Pro Star HPLC system with a binary solvent system A and B using a

gradient elusion [A, H₂O with either 0.05% or 0.1% triﬂuoroacetic acid

(TFA); B, CH₃CN with either 0.05% or 0.1% TFA] and ﬂow rate =

1 mL/min, with UV detection at either 223 or 254 nm; (2) Shimadzu

LC-20A HPLC system with a binary solvent system A and B using a

gradient elusion [A, H₂O with either 0.05% TFA; B, CH₃CN with

either 0.05% or 0.1% TFA] and ﬂow rate = 1 mL/min, with UV

detection at either 223 or 254 nm. All ﬁnal compounds were puriﬁed

to ≥95% purity, and these purity levels were measured by using the

following HPLC methods:

In Vitro GlyT-1 Inhibition Assessment. In vitro inhibitory

GlyT-1 activity was determined using a whole-cell scintillation prox-

imity assay (SPA).³⁷The potency of each compound was assessed in

inhibiting the uptake of radiolabeled glycine ([¹⁴C]glycine) using the

human choriocarcinoma cell line, JAR cells (ATCC#HTB-144), which

endogenously express human GlyT-1 (hGlyT-1b). In brief, 50,000 JARS

cells were plated per well of tissue culture treated 96-well Cytostar-T

plate (PerkinElmer) in RPMI media supplemented with 10% heat

inactivated FBS and allowed to attach overnight at 37 °C and 5% CO₂.

The compounds were then diluted into Uptake Buﬀer (10 mM Hepes,

120 mM NaCl, 2 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, and 5 mM

alanine, pH 7.4) with a ﬁnal DMSO concentration of 0.75% (v/v).

Following the overnight incubation, the media was replaced with

Uptake Buﬀer in the presence or absence of diluted compound and

preincubated at room temperature for 10 min prior to the addition of

[¹⁴C]glycine at a ﬁnal concentration of 5 mM. Following a 3 h

incubation period at 37 °C and 5% CO₂, the plates were sealed and

placed at room temperature in the dark for 15 min prior to

quantiﬁcation of incorporated radioactivity using a MicroBeta Trilux

scintillation plate reader (PerkinElmer). Compounds were serially

diluted 3-fold in DMSO generating a nine data point response curve

for each compound. A minimum of two replicates were performed per

determination. Data were plotted using XLﬁt software (IDBS) and ﬁt

into a four-parameter logistic model to determine the inhibitor

concentration at half-maximal response (IC₅₀). Plate statistics tracked

included average, standard deviation (SD), and CV of the positive

(max) and negative control wells (min); signal ratio; z′ of the plate;

IC₅₀value of the positive control, its r²and hill slope. The following

was tracked for each compound: IC₅₀values (in nM); % inhibition

at maximum concentration; r²and hill slopes of the ﬁtted response

curves. Calculations were based on the following formulas: signal

ratio = (positive control)/(negative control) z′ = 1 − (((3 × SD nega-

tive control) + (3 × SD positive control))/(average of positive con-

trol − average of negative control)); % inhibition of sample = (100 −

((sample − average of negative control)/(average of positive control −

average of negative control)) × 100. The assay window was established

by control wells incubated with [¹⁴C]glycine in the presence or

absence of 10 mM nonlabeled glycine. A dose response curve for a

synthesized reference standard was generated on every plate as a posi-

tive control.³⁷Plate performances were assessed using a combination

of assay window, z′, and IC₅₀of positive control.

In Vitro GlyT-2 Inhibition Assessment. Selectivity for GlyT-1

versus GlyT-2 was established using a GlyT-2 transfected cell model.³⁷

The cDNA for the human gene of GlyT-2 (SLC6A5, solute carrier

family 6, member 5; Accession #NM_004211) was synthesized

(Enzymax LLC) and subcloned into mammalian expression vector,

pcDNA3.1 (Invitrogen Corp.) using standard molecular biology

techniques. HEK293 cells (ATCC #CRL-1573) transfected with

GlyT-2 modiﬁed pcDNA3.1 (Invitrogen Corp.) were selected for

stable incorporation of the construct by resistance to the selective

pressure of Geneticin (450 mg/mL; Invitrogen Corp.). A polyclonal

HEK-GlyT2 cell population was used in the [¹⁴C]glycine uptake assay

previously described to evaluate the activity of the compounds of the

invention with the following protocol modiﬁcations. The HEK-GlyT-2

cells were plated on poly-^L-lysine coated Cytostar-T plates in DMEM

media supplemented with 10% heat inactivated FBS. After an

overnight incubation at 37 °C and 5% CO₂, the media was removed,

and the assay was conducted as described above. Compounds of the

invention were diluted in Uptake Buﬀer and tested against JAR or

(A) Varian Pro Star HPLC system; Phenomenex Luna C18(2)

5 μm column (4.6 × 150 mm), mobile phase, A = H₂O with

0.05% TFA and B = CH₃CN with 0.05% TFA; gradient

10−90% B (0.0−15.0 min), UV detection at 223 nm.

(B) Shimadzu LC-20A HPLC system; Phenomenex Luna C18(2)

5 μm column (4.6 × 250 mm), mobile phase, A = H₂O with

0.05% TFA and B = CH₃CN with 0.05% TFA; gradient

10−90% B (0.0−15.0 min), UV detection at 223 or 254 nm.

(C) Shimadzu LC-20A HPLC system; Xterra MS C18 5 μm

column (4.6 × 150 mm); mobile phase, A = H₂O with 0.05%

TFA and B = CH₃CN with 0.05% TFA; gradient 10−90%

B (0.0−15.0 min), UV detection at 223 or 254 nm.

(D) Varian Pro Star HPLC system; Inertsil ODS C18 5 μm column

(4.6 × 250 mm), mobile phase, A = H₂O with 0.1% TFA and

B = CH₃CN with 0.1% TFA; gradient 10−90% B (0.0−

15.0 min), UV detection at 254 nm.

Racemic ( )-65 was resolved by preparative chiral HPLC using a

Daicel 5 cm I.D. × 50 cm L chiral preparative column, eluting with an

L

DOI: 10.1021/acs.jmedchem.6b00914

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Journal of Medicinal Chemistry

Article

HEK-GlyT-2 cells in parallel to evaluate relative potency and selec-

tivity for GlyT-1 versus GlyT-2.³⁷

Step E. A mixture of (1-(4-(propylsulfonyl)piperazin-1-yl)-

cyclohexyl)methanamine (28a, 0.630 g, 2.07 mmol), 2-amino-6-

chlorobenzoic acid (0.374 g, 2.17 mmol), Et₃N (0.86 mL, 6.21 mmol),

and HBTU (1.17 g, 3.10 mmol) in DMF (20 mL) was stirred at rt for

16 h. The mixture was diluted with H₂O (100 mL) and extracted with

EtOAc (3 × 80 mL). The combined organic extracts were washed with

H₂O (3 × 80 mL) and brine (80 mL), dried over Na₂SO₄, ﬁltered, and

concentrated under reduced pressure. The resulting residue was

chromatographed over silica gel (0−50% EtOAc in hexanes) to give

2-amino-6-chloro-N-((1-(4-(propylsulfonyl)piperazin-1-yl)-

cyclohexyl)methyl)benzamide as a white foam (0.756 g, 80%) ¹H

NMR (500 MHz, DMSO-d₆) δ 10.03 (bs, 1H), 7.56 (d, J = 8.1 Hz,

1H), 7.38 (d, J = 7.1 Hz, 1H), 7.22 (m, 2H), 5.13 (dd, J = 14.3 Hz,

1.0 Hz, 2H), 4.71 (m, 1H), 3.83 (s, 3H), 3.73 (m, 1H), 3.48 (m, 1H),

3.31−3.19 (m, 3H), 2.42 (m, 1H), 2.18 (m 1H), 1.99−1.97 (m, 3H);

ESI MS m/z 296 [M + H]⁺.

Step F. To a 0 °C solution of 2-amino-6-chloro-N-((1-(4-(pro-

pylsulfonyl)piperazin-1-yl)cyclohexyl)methyl)benzamide (0.750 g,

1.64 mmol) in CH₂Cl₂(5 mL) was added a 1.0 M solution of HCl

in Et₂O (4.92 mL, 4.92 mmol). The mixture was concentrated under

reduced pressure to give 2-amino-6-chloro-N-((1-(4-(propylsulfonyl)-

piperazin-1-yl)cyclohexyl)methyl)benzamide hydrochloride (10),

which was lyophilized from CH₃CN and H₂O (0.790 g, quantitative):

¹H NMR (500 MHz, DMSO-d₆) δ 10.03 (bs, 1H), 7.15 (d, J = 8.1 Hz,

Syntheses. 2-Amino-6-chloro-N-((1-(4-(propylsulfonyl)-

piperazin-1-yl)cyclohexyl)methyl)benzamide Hydrochloride (10).

Step A. Cyclohexanone (5.0 g, 50.9 mmol) was added to a solu-

tion of 1-benzylpiperazine (24, 6.30 g, 34.0 mmol) and PTSA (7.76 g,

40.8 mmol) in H₂O (40 mL). The mixture stirred at rt for 30 min

followed by addition of a solution of KCN (2.88 g, 44.2 mmol) in H₂O

(20 mL). The mixture stirred at rt for 16 h and was ﬁltered to aﬀord

1-(4-benzylpiperazin-1-yl)cyclohexanecarbonitrile (25) as an oﬀ-white

1

solid (9.05 g, 94%) H NMR (300 MHz, CDCl₃) δ 7.32−7.24 (m,

5H), 3.50 (s, 2H), 2.79−2.60 (m, 4H), 2.60−2.38 (m, 4H), 2.14−2.09

(m, 2H), 1.79−1.72 (m, 2H), 1.59−1.51 (m, 5H), 1.31−1.24 (m, 1H).

Step B. A solution of 1-(4-benzylpiperazin-1-yl) cyclohexanecarbo-

nitrile (25, 5.0 g, 17.6 mmol) in Et₂O (90 mL) was added drop-

wise over 30 min to a 0 °C cooled suspension of LiAH₄(1.40 g,

35.2 mmol) in Et₂O (100 mL), and the resulting mixture stirred at rt

for 12 h. The reaction was quenched by careful addition of H₂O

(5 mL) and 1 N aq NaOH (2 mL). The mixture was ﬁltered through a

pad of Celite, and the ﬁltrate was extracted with Et₂O (100 mL). The

combined organic extracts were washed with H₂O (50 mL) and brine

(50 mL). The organic layer was dried over Na₂SO₄, ﬁltered, and

concentrated under reduced pressure to aﬀord the (1-(4-benzylpiper-

azin-1-yl)cyclohexyl)methanamine (26) as a colorless oil (5.0 g, quan-

titative): ¹H NMR (300 MHz, CDCl₃) δ 7.30−7.23 (m, 5H), 3.47 (s,

2H), 2.68 (s, 2H), 2.65−2.56 (m, 4H), 2.52−2.35 (m, 4H), 1.68 (s,

2H), 1.59−1.29 (m, 10H).

1H), 6.81−6.68 (m, 3H), 3.74 (m, 8H), 3.34 (m, 2H), 3.08 (m, 2H),

2.11−1.98 (m, 4H), 1.74−1.57 (m, 8H), 1.17 (m, 2H), 1.01 (t, J =

7.5 Hz, 3H); ESI MS m/z 296 [M + H]⁺; HPLC > 99% (AUC)

(Method A), t_R= 12.5 min.

Step C. To a 0 °C solution of (1-(4-benzylpiperazin-1-yl)cyclohexyl)-

methanamine (26, 5.0 g, 17.4 mmol) and Et₃N (7.3 mL, 52.3 mmol)

in CH₂Cl₂(80 mL) was added triﬂuoroacetic anhydride (3.0 mL,

21.2 mmol). The resulting mixture stirred at rt for 16 h and was then

washed with saturated aqueous NaHCO₃solution (50 mL), H₂O

(50 mL) and brine (50 mL). The organic layer was dried over Na₂SO₄,

ﬁltered, and concentrated under reduced pressure, and the resulting

residue was chromatographed over silica gel (0−50% EtOAc in

hexanes) to give N-((1-(4-benzylpiperazin-1-yl)cyclohexyl)methyl)-

2,2,2-triﬂuoroacetamide (27) as a colorless oil (4.74 g, 71%). ¹H NMR

(300 MHz, CDCl₃) δ 7.34−7.21 (m, 5H), 3.48 (s, 2H), 2.40 (bs, 2H),

2.67−2.54 (m, 4H), 2.52−2.33 (m, 4H), 1.71−1.30 (m, 9H), 1.20−

1.05 (m, 1H).

Step D. A mixture of N-((1-(4-benzylpiperazin-1-yl)cyclohexyl)-

methyl)-2,2,2-triﬂuoroacetamide (27, 10.0 g, 26.1 mmol), Pd/C

(10% w/w, 1.50 g), and NH₄HCO₂(4.93 g, 78.3 mmol) in CH₃OH

(200 mL) was heated at 65 °C for 3 h. The reaction was allowed to

cool to rt and was ﬁltered through a pad of Celite, which was rinsed

with CH₃OH (100 mL). The ﬁltrate was concentrated under reduced

pressure, and the resulting residue was dissolved in CH₂Cl₂(100 mL)

and washed with saturated aqueous NaHCO₃(3 × 100 mL) and brine

(100 mL). The organic layer was dried over Na₂SO₄, ﬁltered, and

concentrated under reduced pressure to aﬀord 2,2,2-triﬂuoro-N-((1-

(piperazin-1-yl)cyclohexyl)methyl)acetamide as a clear oil (5.50 g,

72%). To a 0 °C solution of 2,2,2-triﬂuoro-N-((1-(piperazin-

1-yl)cyclohexyl)methyl)acetamide (5.50 g, 18.8 mmol) and Et₃N

(8.7 mL, 56.3 mmol) in CH₂Cl₂(80 mL) was added a solution of

propane-1-sulfonyl chloride (2.94 g, 20.6 mmol) in CH₂Cl₂(30 mL)

dropwise. The reaction mixture stirred at rt for 1 h and was then

washed with H₂O (100 mL). The organic layer was dried over Na₂SO₄,

ﬁltered, and concentrated under reduced pressure. The resulting

residue was dissolved in CH₃OH (5 mL) and H₂O (0.5 mL) to which

K₂CO₃(3.71 g, 26.8 mmol) was added. The reaction mixture stirred at

reﬂux for 16 h and was then allowed to cool to rt and diluted with

H₂O (30 mL). The aqueous mixture was extracted with CH₂Cl₂(3 ×

30 mL), and the organic extracts were dried over Na₂SO₄, ﬁltered

and concentrated under reduced pressure to aﬀord the (1-(4-(propy-

lsulfonyl)piperazin-1-yl)cyclohexyl)methanamine (28a) as a clear oil

(0.39 g, 68%) ¹H NMR (500 MHz, DMSO-d₆) δ 10.03 (bs, 1H), 7.56

(d, J = 8.1 Hz, 1H), 7.38 (d, J = 7.1 Hz, 1H), 7.22 (m, 2H), 5.13 (dd, J

= 14.3 Hz, 1.0 Hz, 2H), 4.71 (m, 1H), 3.83 (s, 3H), 3.73 (m, 1H),

3.48 (m, 1H), 3.31−3.19 (m, 3H), 2.42 (m, 1H), 2.18 (m 1H), 1.99−

1.97 (m, 3H); ESI MS m/z 296 [M + H]⁺.

N-((4,4-Diﬂuoro-1-(4-(propylsulfonyl)piperazin-1-yl)cyclohexyl)-

methyl)-2,6-diﬂuorobenzamide Hydrochloride (11). Step A. To a

0 °C solution of tert-butyl piperazine-1-carboxylate (12, 10.0 g,

53.69 mmol) and Et₃N (22.4 mL, 161 mmol) in CH₂Cl₂(200 mL)

was slowly added 1-propanesulfonyl chloride (7.65 g, 53.6 mmol). The

mixture stirred at rt for 3 h, then washed with 2 N aq NaOH (50 mL),

2 N aq HCl (50 mL), H₂O (50 mL), and brine (50 mL). The organic

extracts were dried over Na₂SO₄, ﬁltered, and concentrated under

reduced pressure to aﬀord tert-butyl 4-(propylsulfonyl)piperazine-

1-carboxylate (13) as an oﬀ-white solid (15.7 g, quantitative): ¹H

NMR (500 MHz, CDCl₃) δ 7.91 (d, J = 1.7 Hz, 1H), 7.48 (d, J =

1.6 Hz, 1H), 7.21 (m, 1H), 7.14 (s, 1H), 7.09 (s, 1H), 3.96 (s, 3H),

3.75 (s, 3H); ESI MS m/z 293 [M + H]⁺.

Step B. To a 0 °C solution of tert-butyl 4-(propylsulfonyl)-

piperazine-1-carboxylate (13, 15.7 g, 53.6 mmol) in CH₂Cl₂(200 mL)

was slowly added TFA (21.4 mL, 268 mmol). The mixture was stirred

at rt for 16 h and washed with 2 N aq NaOH (3 × 50 mL), H₂O

(50 mL), and brine (50 mL). The organic layer was dried over

Na₂SO₄, ﬁltered, and concentrated under reduced pressure to aﬀord

1-(propylsulfonyl)piperazine (14) as a crystalline solid (10.3 g, quan-

titative): ¹H NMR (500 MHz, CDCl₃) δ 7.91 (d, J = 1.7 Hz, 1H), 7.48

(d, J = 1.6 Hz, 1H), 7.21 (m, 1H), 7.14 (s, 1H), 7.09 (s, 1H), 3.96

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

Step C. To a 0 °C solution of 4,4-diﬂuorocyclohexanone (0.200 g,

1.49 mmol) in toluene (2 mL) was added a solution of piperazine-4-

propyl sulfonamide (14, 0.287 g, 1.49 mmol) in toluene (2 mL),

followed by dropwise addition of Ti(i-PrO)₄(0.87 mL, 2.98 mmol).

The reaction mixture stirred at rt for 2 h, then cooled back down to

0 °C. To this was added a 1.0 M solution of Et₂AlCN in toluene (2.98

mL, 2.98 mmol) dropwise. The mixture then stirred at rt for 16 h.

H₂O (10 mL) was then carefully added, and the resulting precipitate

was ﬁltered. To the ﬁltrate was added CH₂Cl₂(20 mL) and H₂O

(20 mL), and the aqueous layer was separated and extracted with

CH₂Cl₂(3 × 20 mL). The combined organic extracts were dried over

Na₂SO₄, ﬁltered, and concentrated under reduced pressure. The

resulting residue was chromatographed over silica gel (0−30% EtOAc

in hexanes) to give 4,4-diﬂuoro-1-(4-(propylsulfonyl)piperazin-1-yl)

cyclohexanecarbonitrile (15) as a white foam (0.233 g, 50%): ¹H

NMR (300 MHz, CDCl₃) δ 3.38−3.34 (m, 4H), 2.93−2.90 (m, 2H),

2.75−2.72 (m, 4H), 2.18−2.02 (m, 8H), 1.90−1.83 (m, 2H), 1.07 (t, J

= 4.9 Hz, 3H).

M

DOI: 10.1021/acs.jmedchem.6b00914

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Journal of Medicinal Chemistry

Article

Step D. To a 0 °C solution of 4,4-diﬂuoro-1-(4-(propylsulfonyl)-

piperazin-1-yl) cyclohexanecarbonitrile (15, 2.95 g, 8.79 mmol) in

Et₂O (60 mL) was added LiAlH₄(0.67 g, 18 mmol). The mixture

stirred at rt for 12 h, cooled back to 0 °C, then quenched by the

sequential addition of H₂O (0.87 mL), 4 N aq NaOH (0.87 mL),

and H₂O (4 mL). The resulting mixture was stirred at rt for 30 min,

then ﬁltered. The ﬁltrate was concentrated under reduced pressure,

and the resulting residue was chromatographed over silica gel (0−10%

CH₃OH in CH₂Cl₂) to give (4,4-diﬂuoro-1-(4-(propylsulfonyl)pipe-

razin-1-yl)cyclohexyl)methanamine (16) as a colorless oil (2.2 g,

74%): ¹H NMR (300 MHz, CDCl₃) δ 3.23 (t, J = 4.8 Hz, 4H), 2.94−

2.85 (m, 2H), 2.76 (t, J = 4.8 Hz, 4H), 2.72 (s, 2H), 2.15−1.75 (m,

10H), 1.70−1.45 (m, 2H), 1.07 (t, J = 7.5 Hz, 3H); ESI MS m/z 340

[M + H]⁺.

benzamide hydrochloride (18) as a white solid (0.058 g, quantitative):

mp 200−205 °C; H NMR (300 MHz, DMSO-d₆) δ 10.55 (bs, 1H),

9.15 (m, 0.3H), 8.77 (m, 0.7H), 7.62−7.31 (m, 3H), 4.00−3.00

(m, 9H), 2.80−2.68 (m, 3H), 2.29−1.60 (m, 10H), 1.00 (t, J = 7.5 Hz,

3H); ESI MS m/z 562 [M + H]⁺; HPLC > 99% (AUC) (Method C),

t_R= 20.4 min.

1

2,4-Dichloro-N-((4,4-diﬂuoro-1-(4-(propylsulfonyl)piperazin-1-yl)-

cyclohexyl)methyl)benzamide Hydrochloride (19). Compound 19

was prepared according to a similar procedure described for the syn-

thesis of 11. mp 124−126 °C; ¹H NMR (300 MHz, DMSO-d₆)

δ 10.66 (bs, 1H), 8.96 (bs, 0.5H), 8.47 (bs, 0.5H), 7.80−7.35 (m, 3H),

3.95−3.00 (m, 9H), 2.82 (m, 3H), 2.30−1.50 (m, 10H), 0.99 (t, J =

7.5 Hz, 3H); ESI MS m/z 512 [M + H]⁺; HPLC 98.2% (AUC)

(Method B), t_R= 23.5 min.

2-Chloro-N-((4,4-diﬂuoro-1-(4-(propylsulfonyl)piperazin-1-yl)-

cyclohexyl)methyl)-4-ﬂuorobenzamide Hydrochloride (20). Com-

pound 20 was prepared according to a similar procedure described for

the synthesis of 18. mp 220−225 °C; ¹H NMR (300 MHz, DMSO-d₆)

δ 10.91 (s, 1H), 9.00 (m, 0.7H), 8.66 (m, 0.3H), 7.61−7.43 (m, 2H),

7.42−7.31 (m, 1H), 4.00 (m, 4H), 3.82−3.59 (m, 4H), 3.31−3.22

(m, 2H), 3.26−3.01 (m, 3H), 2.75 (bs, 1H), 2.30−1.99 (m, 6H), 1.75

(m, 2H), 0.98 (t, J = 7.5 Hz, 3H); ESI MS m/z 496 [M + H]⁺; HPLC

98.2% (AUC) (Method C), t_R= 18.8 min.

2-Chloro-N-((4,4-diﬂuoro-1-(4-(propylsulfonyl)piperazin-1-yl)-

cyclohexyl)methyl)-4-(triﬂuoromethyl)benzamide Hydrochloride

(21). Compound 21 was prepared according to a similar procedure

described for the synthesis of 18. mp 115−120 °C; ¹H NMR (400 MHz,

DMSO-d₆) δ 11.20 (bs, 1H), 9.12 (bs, 1H), 8.00 (m, 1H), 7.62−7.59

(m, 2H), 3.90−3.61 (m, 6H), 3.30 (m, 2H), 3.21−3.00 (m, 3H), 2.76

(m, 1H), 2.31−1.72 (m, 10H), 1.05 (t, J = 7.3 Hz, 3H); ESI MS m/z

546 [M + H]⁺; HPLC > 99% (AUC) (Method B), t_R= 29.6 min.

N-((4,4-Diﬂuoro-1-(4-(propylsulfonyl)piperazin-1-yl)cyclohexyl)-

methyl)-2,4-bis(triﬂuoromethyl)benzamide Hydrochloride (22).

Compound 22 was prepared according to a similar procedure described

for the synthesis of 18. mp 124−131 °C; ¹H NMR (300 MHz,

DMSO-d₆) δ 11.20 (bs, 1H), 9.14 (bs, 1H), 8.20−8.17 (m, 2H),

7.98−7.73 (m, 1H), 4.24−4.13 (m, 3H), 3.94−3.67 (m, 4H), 3.30−

3.02 (m, 4H), 2.73−2.50 (m, 1H), 2.20−1.53 (m, 10H), 0.97 (t, J =

7.3 Hz, 3H). ESI MS m/z 580 [M + H]⁺; HPLC > 99% (AUC)

(Method C), t_R= 21.6 min.

N-((4,4-Diﬂuoro-1-(4-(propylsulfonyl)piperazin-1-yl)cyclohexyl)-

methyl)-4-ﬂuoro-2-methoxy-6-methylbenzamide (23).³⁸To a stir-

ring suspension of 4-ﬂuoro-2-hydroxybenzoic acid (10.0 g, 64.0 mmol)

and K₂CO₃(22.0 g, 160 mmol) in DMF (200 mL) was slowly added

CH₃I (20.0 g, 140 mmol) at rt. The mixture was then heated at 90 °C

for 16 h. After allowing to cool to rt, the mixture was concentrated

under reduced pressure, and the resulting white solid rinsed over

a plug of silica gel (CH₂Cl₂eluent) to aﬀord methyl 4-ﬂuoro-

2-methoxybenzoate as clear oil (10.0 g, 92%). The intermediate was

taken into the next step without any further analysis.

Step E. To a 0 °C solution of (4,4-diﬂuoro-1-(4-(propylsulfonyl)-

piperazin-1-yl)cyclohexyl)methanamine (16, 0.15 g, 0.44 mmol) in

CH₂Cl₂(5 mL) was added Et₃N (0.12 mL, 0.88 mmol) and 2,6-diﬂuo-

robenzoyl chloride (0.05 mL, 0.44 mmol). The resulting mixture

stirred at rt for 4 h was diluted with CH₂Cl₂, then washed with

saturated aqueous NaHCO₃(10 mL). The organic layer was dried

over Na₂SO₄, ﬁltered, and concentrated under reduced pressure. The

resulting residue was chromatographed over silica gel (0−50% EtOAc

in hexanes) to give N-((4,4-diﬂuoro-1-(4-(propylsulﬁnyl)pipe-

razin-1-yl)cyclohexyl)methyl)-2,6-diﬂuorobenzamide as a white solid

1

(0.072 g, 46%): H NMR (300 MHz, CDCl₃) δ 7.42−7.36 (m, 1H),

6.96 (t, J = 8.1 Hz, 2H), 6.13 (bs, 1H), 3.55 (d, J = 3.15 Hz, 2H),

3.34−3.27 (m, 4H), 2.91−2.85 (m, 2 H), 2.79−2.72 (m, 4 H), 2.02−

1.82 (m, 8H), 1.71−1.56 (m, 2H), 1.06 (t, J = 4.9 Hz, 3H); ESI MS

m/z 480 [M + H]⁺.

Step F. To a 0 °C solution of N-((4,4-diﬂuoro-1-(4-(propy-

lsulfonyl)piperazin-1-yl)cyclohexyl)methyl)-2,6-diﬂuorobenzamide

(0.065 g, 0.135 mmol) in CH₃CN (1 mL) was added a 1.0 M solution

of HCl in H₂O (1.5 mL). The mixture was stirred at rt for 30 min and

lyophilized to give N-((4,4-diﬂuoro-1-(4-(propylsulfonyl)piperazin-

1-yl)cyclohexyl)methyl)-2,6-diﬂuorobenzamide hydrochloride (11) as

an amorphous white solid (0.068 g, quantitative): mp 165−169 °C; ¹H

NMR (300 MHz, DMSO-d₆) δ 10.68 (bs, 1H), 9.32 (m, 0.4H), 8.78

(m, 0.6H), 7.62−7.42 (m, 1H), 7.30−7.13 (m, 2H), 4.00−3.68

(m, 7H), 3.56−2.94 (m, 6H), 2.79 (m, 2H), 2.30−1.54 (m, 7H), 0.99

(t, J = 4.8 Hz, 3H); APCI MS m/z 480 [M + H]⁺; HPLC > 99%

(AUC) (Method B), t_R= 22.3 min.

2,6-Dichloro-N-((4,4-diﬂuoro-1-(4-(propylsulfonyl)piperazin-1-yl)-

cyclohexyl)methyl)benzamide Hydrochloride (17). Compound 17

was prepared according to a similar procedure described for the

1

synthesis of 11. mp 220−223 °C; H NMR (300 MHz, DMSO-d₆)

δ 10.48 (bs, 1H), 9.12 (bs, 0.3H), 8.63 (bs, 0.7H), 7.68−7.38 (m, 3H),

3.92−2.99 (m, 9H), 2.80−2.68 (m, 3H), 2.30−1.58 (m, 10H), 0.99

(t, J = 7.5 Hz, 3H); APCI MS m/z 512 [M + H]⁺; HPLC 95.7%

(AUC) (Method B), t_R= 23.1 min.

2-Chloro-N-((4,4-diﬂuoro-1-(4-(propylsulfonyl)piperazin-1-yl)-

cyclohexyl)methyl)-6-(triﬂuoromethoxy)benzamide Hydrochloride

(18). Step A. (4,4-Diﬂuoro-1-(4-(propylsulfonyl)piperazin-1-yl)-

cyclohexyl)methanamine (16, 0.15 g, 0.44 mmol), 2-chloro-6-(triﬂuo-

romethoxy)benzoic acid (0.105 g, 0.44 mmol), EDCI·HCl (0.09 g,

0.44 mmol), HOBt (0.07 g, 0.44 mmol), and Et₃N (0.3 mL,

1.91 mmol) in DMF (10 mL) stirred at rt for 16 h. The mixture was

diluted with H₂O (20) mL and extracted with EtOAc (3 × 20 mL).

The combined organic extracts were washed with H₂O (3 × 20 mL)

and brine (20 mL). The organic layers were then dried over Na₂SO₄,

ﬁltered, and concentrated under reduced pressure. The resulting

residue was chromatographed over silica gel (0−50% EtOAc in

hexanes) to give 2-chloro-N-((4,4-diﬂuoro-1-(4-(propylsulfonyl)-

piperazin-1-yl)cyclohexyl)methyl)-6-(triﬂuoromethoxy)benzamide as

a white solid (0.155 g, 63%): ESI MS m/z 562 [M + H]⁺.

Step B. To a 0 °C solution of 2-chloro-N-((4,4-diﬂuoro-1-

(4-(propylsulfonyl)piperazin-1-yl)cyclohexyl)methyl)-6-(triﬂuoromethoxy)-

benzamide (0.050 g, 0.08 mmol) in CH₃CN (5 mL) was added a 1.0 M

solution of HCl in H₂O (1.0 mL, 1.0 mmol). The mixture stirred at rt for

30 min and was lyophilized to give 2-chloro-N-((4,4-diﬂuoro-1-

(4-(propylsulfonyl)piperazin-1-yl)cyclohexyl)methyl)-6-(triﬂuoromethoxy)

To a solution of 4-ﬂuoro-2-methoxybenzoate (10.0 g, 58.8 mmol)

in CH₃OH (50 mL) was slowly added 6 N aq NaOH (30 mL) over a

period of 15 min. The mixture was stirred at rt for 1 h and was

acidiﬁed to pH = 2 with 6 N aq HCl. The resulting precipitate was

collected via vacuum ﬁltration to aﬀord 4-ﬂuoro-2-methyl benzoic acid

(9.2 g, 85%). H NMR (300 MHz, CDCl₃) δ 8.21 (dd, J = 9.0 Hz,

6.9 Hz, 1H), 6.82−6.88 (m, 1H), 6.79 (dd, J = 7.8 Hz, 2.1 Hz, 1H),

4.07 (s, 1H).

To a −78 °C cooled solution of TMEDA (1.95 mL, 19.2 mmol) in

THF (8 mL) was slowly added sec-BuLi (1.4 M solution in cyclo-

hexane, 18.5 mL, 25.8 mmol), followed by a solution of 4-ﬂuoro-

2-methyl-benzoic acid (1.0 g, 5.88 mmol) in THF (2 mL). The mix-

ture stirred at −78 °C for 2 h before addition of a solution of CH₃I

(1.46 mL, 23.5 mmol) in THF (2 mL). The mixture stirred at −78 °C

for 1 h and was then allowed to warm to rt and quenched with H₂O

(10 mL). The crude mixture was diluted with H₂O (10 mL) and

extracted with EtOAc (50 mL). The aqueous layer was collected and

acidiﬁed to pH = 2 with 2 N aq HCl then extracted with EtOAc (2 ×

20 mL). The combined organic extracts were dried over Na₂SO₄,

ﬁltered, and concentrated under reduced pressure. The resulting

1

N

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residue was chromatographed over silica gel (0−50% EtOAc in

hexanes) to give 4-ﬂuoro-2-methoxy-6-methyl benzoic acid as a white

solid (0.18 g, 16%). ¹H NMR (300 MHz, CDCl₃) δ 6.53−6.62

(m, 2H), 3.90 (s, 1H), 2.48 (s, 1H); MS (ESI⁺) m/z 185 (M + H).

Step E. To a 0 °C solution of 2-methoxy-6-methyl-4-ﬂuorobenzoic

acid (0.08 g, 0.44 mmol) in CH₂Cl₂(4 mL) was added oxalyl chloride

(0.08 mL, 0.88 mmol) followed by 2 drops of DMF, and the mixture

was stirred at rt for 1.5 h to generate 2-methoxy-6-methyl-4-ﬂuoro-

benzoyl chloride. This mixture was concentrated under reduced

pressure and dissolved in CH₂Cl₂(1 mL), which was then added to

a 0 °C solution of (4,4-diﬂuoro-1-(4-(propylsulfonyl)piperazin-

1-yl)cyclohexyl)methanamine (16, 0.15 g, 0.44 mmol) and Et₃N

(0.18 mL, 1.31 mmol) in CH₂Cl₂(4 mL). The resulting mixture

stirred at rt for 4 h and was then diluted with CH₂Cl₂and washed with

saturated aqueous NaHCO₃solution (5 mL). The organic layer was

dried over Na₂SO₄, ﬁltered, and concentrated under reduced pressure,

and the resulting residue was chromatographed over silica gel (0−50%

EtOAc in hexanes) to give N-((4,4-diﬂuoro-1-(4-(propylsulfonyl)pip-

erazin-1-yl)cyclohexyl)methyl)-4-ﬂuoro-2-methoxy-6-methylbenza-

mide (23) as a white solid (0.13 g, 58%): mp 160−163 °C; ¹H NMR

(300 MHz, DMSO-d₆) δ 10.65 (bs, 1H), 8.71 (bs, 0.6H), 8.15 (bs,

0.4H), 6.90−6.58 (m, 2H), 3.76 (s, 3H), 3.74−3.35 (m, 5H), 3.25−

2.60 (m, 7H), 2.19 (s, 3H), 2.05−1.52 (m, 10H), 0.98 (t, J = 7.5 Hz,

3H); APCI MS m/z 506 [M + H]⁺; HPLC > 99% (AUC) (Method B),

t_R= 22.3 min.

2,4-Dichloro-N-((1-(4-(propylsulfonyl)piperazin-1-yl)cyclohexyl)-

methyl)benzamide Hydrochloride (34). Step G. To a 0 °C solution of

(1-(4-(propylsulfonyl)piperazin-1-yl)cyclohexyl) methanamine (28a,

0.30 g, 0.99 mmol) and Et₃N (0.41 mL, 2.96 mmol) in CH₂Cl₂(3 mL)

was added 2,4-dichloro benzoyl chloride (0.30 g, 1.48 mmol). The

mixture stirred at rt for 16 h. The mixture was diluted with CH₂Cl₂

and washed with 1 N aq NaOH (10 mL), H₂O (10 mL), and brine

(10 mL). The organic layers were dried over Na₂SO₄, ﬁltered, and

concentrated under reduced pressure. The resulting residue was

chromatographed over silica gel (0−50% EtOAc in hexanes) to give

2,4-dichloro-N-((1-(4-(propylsulfonyl)piperazin-1-yl)cyclohexyl)-

methyl)benzamide (34) as an oﬀ-white solid (0.27 g, 57%); ¹H NMR

(300 MHz, CDCl₃) δ (d, J = 8.4 Hz, 1H), 7.41 (d, J = 2.0 Hz, 1H),

7.33 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 6.89 (bs, 1H), 3.60−3.52 (d, J = 5.1

Hz, 2H), 3.28−3.20 (m, 4H), 2.90−2.80 (m, 2), 2.79−2.69 (m, 4H),

1.90−1.78 (m, 2H), 1.72−1.40 (m, 10H), 1.08−1.03 (t, J = 7.4 Hz,

3H); ESI MS m/z 476 [M + H]⁺.

Step H. To a 0 °C solution of 2,4-dichloro-N-((1-(4-(propylsul-

fonyl)piperazin-1-yl)cyclohexyl)methyl)benzamide (0.27 g, 0.56 mmol)

in CH₃CN (1 mL) was added 10% HCl in H₂O (0.5 mL). The mix-

ture stirred at rt for 30 min and was then lyophilized to give

2,4-dichloro-N-((1-(4-(propylsulfonyl)piperazin-1-yl)cyclohexyl)

methyl)benzamide hydrochloride (34) as a white solid (0.28 g, quan-

1

titative): mp 202−208 °C; H NMR (500 MHz, DMSO-d₆) δ 10.83

(bs, 1H), 8.91 (bs, 1H), 7.72 (s, 1H), 7.53 (m, 2H), 3.75−3.59 (m,

8H), 3.28 (m, 2H), 3.11 (m, 2H), 2.08 (m, 2H), 1.93 (m, 2H), 1.69−

1.50 (m, 7H), 1.16 (m, 1H), 1.02 (t, J = 7.4 Hz, 3H); ESI MS m/z 476

[M + H]⁺; HPLC 98.7% (AUC) (Method A), t_R= 22.3 min.

2,4-Dichloro-N-((1-(4-((cyclopropylmethyl)sulfonyl)piperazin-1-

yl)cyclohexyl)methyl)benzamide Hydrochloride (35). Compound 35

was prepared according to a similar procedure described for the

2-Amino-6-chloro-N-((1-(4-(methylsulfonyl)piperazin-1-yl)-

cyclohexyl)methyl)benzamide Hydrochloride (29). Compound 29

was prepared according to a similar procedure described for the syn-

thesis of 10 using methylsulfonyl chloride. mp 178−181 °C; ¹H NMR

(300 MHz, DMSO-d₆) δ 8.80 (bs, 1H), 7.08 (t, J = 8.1 Hz, 1H), 6.72−

6.64 (m, 2H), 6.23 (bs, 2H), 3.84−3.27 (m, 10H), 2.91 (s, 3H), 2.17−

2.01 (m, 2H), 1.80−1.44 (m, 7H), 1.25−1.02 (m, 1H); ESI MS m/z

429 [M + H]⁺; HPLC 96.6% (AUC) (Method B), t_R= 19.6 min.

2-Amino-6-chloro-N-((1-(4-(ethylsulfonyl)piperazin-1-yl)-

cyclohexyl)methyl)benzamide Hydrochloride (30). Compound 30

was prepared according to a similar procedure described for the syn-

1

synthesis of 34. mp 217−219 °C; H NMR (300 MHz, DMSO-d₆) δ

11.01 (bs, 1H), 8.92 (t, J = 6.0 Hz, 1H), 7.72 (d, J = 1.2 Hz, 1H),

7.57−7.50 (m, 2H), 3.83−3.40 (m, 10H), 3.11 (d, J = 6.9 Hz, 2H),

2.12−1.85 (m, 4H), 1.77−1.46 (m, 5H), 1.22−0.95 (m, 2H), 0.62−

0.55 (m, 2H), 0.42−0.35 (m, 2H); ESI MS m/z 488 [M + H]⁺; HPLC

> 99% (AUC) (Method B), t_R= 20.5 min.

1

thesis of 10 using ethylsulfonyl chloride. mp 170−173 °C; H NMR

Cyclobutanesulfonyl Chloride. Step A. To a suspension of magne-

sium turnings (0.79 g, 32.0 mmol) in Et₂O (20 mL) was added a

solution of cyclobutyl bromide (1.8 mL, 19.1 mmol) in Et₂O (20 mL)

portion-wise. After the initial exothermic reaction had ceased, the

mixture was heated at reﬂux for 1 h. The suspension was allowed to

cool to rt, and the supernatant was collected and added portion-wise

to a 0 °C solution of sulfuryl chloride (4.6 mL, 57.1 mmol) in CH₂Cl₂

(30 mL). The mixture stirred at rt for 1 h and was concentrated under

reduced pressure. The residue was taken up in hexanes (150 mL), and

the resulting precipitate was ﬁltered. The ﬁltrate was concentrated

under reduced pressure to give cyclobutylsulfonyl chloride 2 (3.1 g,

crude, >99%) as a pale brown oil, which was carried into the next step

without puriﬁcation.

(300 MHz, CD₃OD) δ 7.46 (d, J = 2.0 Hz, 1H), 7.18 (s, 1H), 7.09 (s,

1H), 4.04−3.82 (m, 6H), 3.55−3.33 (m, 4H), 3.15 (q, J = 7.4 Hz,

2H), 2.18 (d, J = 11.8 Hz, 2H), 1.93−1.72 (m, 5H), 1.70−1.54 (m,

2H), 1.34 (t, J = 7.6 Hz, 3H), 1.30−1.25 (m, 1H), 1.01 (t, J = 7.5 Hz,

3H); ESI MS m/z 443 [M + H]⁺; HPLC > 99% (AUC) (Method B),

t_R= 19.8 min.

2-Amino-6-chloro-N-((1-(4-(isobutylsulfonyl)piperazin-1-yl)-

cyclohexyl)methyl)benzamide Hydrochloride (31). Compound 31

was prepared according to a similar procedure described for the syn-

thesis of 10 using isobutylsulfonyl chloride. mp 195−198 °C; ¹H

NMR (300 MHz, DMSO-d₆) δ 8.79 (s, 1H), 7.07 (t, J = 7.8 Hz, 1H),

6.66 (q, J = 8.1 Hz, 2H), 5.78 (bs, 2H), 3.88−3.18 (m, 10H), 2.98 (d, J

= 6.6 Hz, 2H), 2.25−2.06 (m, 3H), 1.98−1.39 (m, 7H), 1.23−1.08 (m,

1H), 1.03 (d, J = 6.6 Hz, 6H); APCI MS m/z 471 [M + H]⁺; HPLC

95.1% (AUC) (Method B), t_R= 21.1 min.

2-Amino-6-chloro-N-((1-(4-(phenylsulfonyl)piperazin-1-yl)-

cyclohexyl)methyl)benzamide Hydrochloride (32). Compound 32

was prepared according to a similar procedure described for the syn-

thesis of 10 using phenylsulfonyl chloride. mp 230−232 °C; ¹H NMR

(300 MHz, DMSO-d₆) δ 8.77 (bs, 1H), 7.80−7.66 (m, 5H), 7.12−

7.06 (m, 1H), 6.73−6.65 (m, 2H), 3.88−3.28 (m, 9H), 3.30−2.76 (m,

3H), 2.16−1.91 (m, 3H), 1.86−1.37 (m, 7H), 1.15−1.00 (m, 1H);

APCI MS m/z 491 [M + H]⁺; HPLC 96.8% (AUC) (Method B), t_R=

14.4 min.

2,4-Dichloro-N-((1-(4-(cyclobutylsulfonyl)piperazin-1-yl)-

cyclohexyl)methyl)benzamide Hydrochloride (36). Compound 36

was prepared according to a similar procedure described for the syn-

1

thesis of 34 using cyclobutylsulfonyl chloride. mp 249−252 °C; H

NMR (300 MHz, DMSO-d₆) δ 10.57 (bs, 1H), 8.87 (t, J = 5.7 Hz,

1H), 7.73 (s, 1H), 7.53 (d, J = 0.6 Hz, 2H), 4.10 (quintet, J = 8.4 Hz,

1H), 3.80−3.51 (m, 8H), 3.30−3.05 (m, 2H), 2.40−2.18 (m, 4H),

2.12−1.45 (m, 12H); ESI MS m/z 488 [M + H]⁺; HPLC > 99%

(AUC) (Method B), t_R= 21.5 min.

2,4-Dichloro-N-((1-(4-((1-methyl-1H-pyrazol-4-yl)sulfonyl)-

piperazin-1-yl)cyclohexyl)methyl)benzamide Hydrochloride (37).

Compound 37 was prepared according to a similar procedure described

for the synthesis of 34 using 1-methyl-1H-pyrazole-4-sulfonyl chloride.

2-Amino-N-((1-(4-(benzylsulfonyl)piperazin-1-yl)cyclohexyl)-

methyl)-6-chlorobenzamide Hydrochloride (33). Compound 33 was

prepared according to a similar procedure described for the synthesis

1

mp 164−165 °C; H NMR (400 MHz, DMSO-d₆) δ 9.40 (bs, 1H),

8.82 (bs, 1H), 8.44 (s, 1H), 7.86 (s, 1H), 7.74 (s, 1H), 7.53 (s, 2H),

3.92 (s, 3H), 3.79−3.59 (m, 6H), 2.84−2.63 (m, 4H), 2.10−1.99 (m,

2H), 1.74−1.45 (m, 7H), 1.21−0.97 (m, 1H); APCI MS m/z 514 [M

+ H]⁺; HPLC 98.2% (AUC) (Method B), t_R= 18.9 min.

1

of 10. mp 215−217 °C; H NMR (300 MHz, DMSO-d₆) δ 8.81 (bs,

1H), 7.44−7.33 (m, 5H), 7.10 (t, J = 8.1 Hz, 1H), 6.77−6.68 (m, 2H),

4.50 (s, 2H), 3.72−3.51 (m, 8H), 3.35−3.16 (m, 2H), 2.14−1.85 (m,

5H), 1.78−1.43 (m, 6H), 1.24−1.01 (m, 1H); ESI MS m/z 505 [M +

H]⁺; HPLC 96.3% (AUC), (Method B), t_R= 21.4 min.

2,4-Dichloro-N-((1-(4-((1-methyl-1H-imidazol-4-yl)sulfonyl)-

piperazin-1-yl)cyclohexyl)methyl)benzamide Hydrochloride (38).

O

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Compound 38 was prepared according to a similar procedure described

for the synthesis of 34 using 1-methyl-1H-imidazole-4-sulfonyl chloride.

mp 172−173 °C; ¹H NMR (300 MHz, DMSO-d₆): δ 10.14 (bs, 1H),

8.86 (s, 1H), 7.89 (d, J = 8.4 Hz, 2H), 7.73 (s, 1H), 7.52 (s, 2H),

3.79−3.58 (m, 9H), 3.42−3.07 (m, 4H), 2.11−1.94 (m, 2H), 1.87−

1.41 (m, 7H), 1.21−0.99 (m, 1H); ESI MS m/z 514 [M + H]⁺; HPLC

97.5% (AUC) (Method B), t_R= 20.5 min.

1-Methyl-1H-1,2,3-triazole-4-sulfonyl Chloride. Step A. To a 10 °C

solution of sodium 1H-1,2,3-triazole-4-thiolate (115 g, 0.93 mol) in

EtOH (1.1 L) was added benzyl bromide (111 mL, 0.93 mol)

dropwise over a period of 20 min. The mixture stirred at rt for 20 min

and was then diluted with EtOAc (3 L). The organic layer was washed

with H₂O (2 × 500 mL) and brine (500 mL), dried over Na₂SO₄,

ﬁltered, and concentrated to aﬀord crude 4-(benzylthio)-1H-1,2,

3-triazole as oﬀ white solid (165 g, 92%); ¹H NMR (300 MHz,

CDCl₃) δ 13.37 (bs, 1H), 7.48 (s, 1H), 7.21 (m, 5H), 4.09 (s, 2H);

ESI MS m/z 192 [M + H]⁺.

pressure to give a crude solid. The solids were suspended in Et₂O

and stirred for 30 min at rt then ﬁltered, washed with cold Et₂O, and

dried under vacuum to aﬀord tert-butyl 4-(1-methyl-1H-imidazol-

4-ylsulfonyl)piperazine-1-carboxylate (40) as an oﬀ-white solid

(16.8 g, 95%): ¹H NMR (300 MHz, CDCl₃) δ (dd, J = 1.5 Hz,

14.5 Hz, 2H), 3.76 (s, 3H), 3.55−3.45 (m, 4H), 3.20−3.10 (m, 4H),

1.43 (s, 9H); ESI MS m/z 331 [M + H]⁺.

Step B. To a 0 °C solution of tert-butyl 4-(1-methyl-1H-imidazol-

4-ylsulfonyl)piperazine-1-carboxylate (40, 6.0 g, 18.1 mmol) in

1,4-dioxane (30 mL) was slowly added 12 N aq HCl (30 mL) drop-

wise over a period of 30 min. The reaction mixture stirred at rt for 2 h

and was concentrated under reduced pressure. The residue was

azeotroped with toluene (3 × 100 mL), and the resultant solids were

dissolved in H₂O (100 mL). The aqueous mixture was washed

with CH₂Cl₂(2 × 100 mL) then carefully neutralized with solid

K₂CO₃(15 g). The aqueous mixture was then extracted with CH₂Cl₂

(4 × 100 mL), and the combined organic extracts were washed with

brine (50 mL), dried over Na₂SO₄, ﬁltered, and concentrated under

reduced pressure to aﬀord 1-(1-methyl-1H-imidazol-4-ylsulfonyl)pip-

Step B. To a solution of 4-(benzylthio)-1H-1,2,3-triazole (140 g,

0.73 mol) in DMF (1.4 L) was added K₂CO₃(202 g, 1.46 mol), and

the resulting mixture stirred at rt for 30 min. The reaction mixture was

then cooled to 0 °C, and dimethyl sulfate (104.5 mL, 1.09 mol) was

added dropwise over a period of 15 min. The mixture stirred at rt for

16 h and was then diluted with H₂O (3 L). The aqueous mixture was

extracted with EtOAc (5 × 500 mL), and the combined organic

extracts were dried over Na₂SO₄, ﬁltered, and concentrated under

reduced pressure. The resulting residue was chromatographed over

silica gel (0−50% EtOAc in hexanes) to give 4-(benzylthio)-1-methyl-

1H-1,2,3-triazole as oﬀ-white solid (34.1 g, 23%); ¹H NMR (400 MHz,

CD₃OD) δ 7.65 (s, 1H), 7.24−7.16 (m, 5H), 4.04 (s, 2H), 4.0 (s,

3H); ESI MS m/z 206 [M + H]⁺. The regioisomer was conﬁrmed

1

erazine (41) as oﬀ-white foam (3.8 g, 90%): H NMR (400 MHz,

CDCl₃) δ (dd, J = 1.1 Hz, 16.8 Hz, 2H), 3.80−3.75 (m, 5H), 3.74−

3.65 (m, 2H), 3.37−3.1 (m, 2H), 3.0−3.25 (m, 2H); ESI MS m/z 231

[M + H]⁺.

Step C. To a 0 °C solution of 4,4-diﬂuorocyclohexanone (3.0 g,

22.3 mmol) in toluene (20 mL) was added TMSCN (3.3 mL,

24.8 mmol) dropwise, followed by ZnI₂(0.360 g, 1.13 mmol). The

mixture was stirred at rt for 15 min followed by the addition of a

solution of 1-(1-methyl-1H-imidazol-4-ylsulfonyl)piperazine (41,

5.12 g, 22.2 mmol) in CH₃OH (90 mL). The mixture was then

heated at reﬂux for 4 h followed by stirring at rt for an additional 16 h.

The mixture was concentrated under reduced pressure, and the

resulting residue was dissolved in CH₂Cl₂(100 mL), washed with H₂O

(2 × 50 mL), dried over Na₂SO₄, ﬁltered, and concentrated under

reduced pressure to give 4,4- diﬂuoro-1-(4-(1-methyl-1H-imidazol-

4-ylsulfonyl)piperazin-1-yl)cyclohexanecarbonitrile (42) as an oﬀ-white

solid (3.10 g, 37%): ¹H NMR (300 MHz, CDCl₃) δ 7.52 (d, J = 1.2 Hz,

1H), 7.45 (d, J = 1.2 Hz, 1H), 3.77 (s, 3H), 3.31 (m, 4H), 2.71 (m,

4H), 2.2−1.85 (m, 8H); APCI MS m/z 374 [M + H]⁺.

Step D. To a 0 °C solution of 4,4-diﬂuoro-1-(4-(1-methyl-

1H-imidazol-4-ylsulfonyl)piperazin-1-yl)cyclohexanecarbonitrile (42)

(3.79 g, 10.2 mmol) in THF (80 mL) was added LiAlH₄(1.32 g,

34.8 mmol) portion-wise over a period of 15 min. The mixture stirred

at rt for 5 h and was then cooled to 0 °C and carefully quenched with

H₂O (5 mL) and 4 N aq NaOH (1.4 mL). The resulting mixture was

stirred at rt for 15 min, followed by addition of Na₂SO₄(5 g). The

mixture was ﬁltered, and the ﬁltrate was concentrated under reduced

pressure. The resulting residue was triturated with hexanes, and

the solids were ﬁltered and dried under reduced pressure to give

(4,4-diﬂuoro-1-(4-((1-methyl-1H-imidazol-4-yl)sulfonyl)piperazin-1-

yl)cyclohexyl)methanamine (43) (3.36 g, 87%): ¹H NMR (300 MHz,

CDCl₃) δ 7.52 (d, J = 1.2 Hz, 1H), 7.45 (d, J = 1.2 Hz, 1H), 3.77 (s,

3H), 3.18 (m, 4H), 2.71 (m, 4H), 2.2−1.85 (m, 10H); APCI MS m/z

378 [M + H]⁺.

1

via a H NMR NOESY experiment, which exhibited a positive NOE

enhancement between the methyl group hydrogens and the triazole

1

hydrogen. Furthermore, H NMR NOESY experiments for the cor-

responding regioisomers 4-(benzylsulfonyl)-2-methyl-2H-1,2,3-triazole

(34.1 g isolated, 23%) and 5-(benzylsulfonyl)-1-methyl-1H-1,2,3-triazole

(34.1 g isolated, 23%) did not exhibit an NOE enhancement between

the methyl group hydrogens and the triazole hydrogen.

Step C. To a solution of 4-(benzylthio)-1-methyl-1H-1,2,3-triazole

(12.0 g, 58.5 mmol) in CH₃CN (600 mL) was added HOAc (20 mL)

and H₂O (15 mL). The mixture was cooled to 5 °C, and 1,3-dichloro-

5,5-dimethylimidazolidine-2,4-dione (23.0 g, 117.0 mmol) was added

over a period of 20 min. The mixture continued to stir at 5 °C for

an additional 2 h. The mixture was cooled to 0 °C and diluted with

CH₂Cl₂(500 mL). To this was added a solution of 5% aqueous

NaHCO₃(300 mL), and the mixture stirred for 10 min. The separated

organic layer was washed with brine (300 mL), dried over Na₂SO₄,

ﬁltered, and concentrated to aﬀord crude 1-methyl-1H-1,2,3-triazole-

4-sulfonyl chloride (14.7 g), which was used as is in the next step.

2,4-Dichloro-N-((1-(4-((1-methyl-1H-1,2,3-triazol-4-yl)sulfonyl)-

piperazin-1-yl)cyclohexyl)methyl)benzamide Hydrochloride (39).

Compound 39 was prepared according to a similar procedure described

for the synthesis of 34 using 1-methyl-1H-1,2,3-triazole-4-sulfonyl

1

chloride. H NMR (500 MHz, CDCl₃) δ 7.95 (s, 1H), 7.71 (d, J =

Step E. To a 0 °C solution of (4,4-diﬂuoro-1-(4-((1-methyl-

1H-imidazol-4-yl)sulfonyl)piperazin-1-yl)cyclohexyl)methanamine

(43, 0.50 g, 1.32 mmol) in CH₂Cl₂(10 mL) was added Et₃N (0.54 mL,

3.97 mmol) and 2,6-diﬂuorobenzoyl chloride (0.233 g, 1.32 mmol). The

resulting mixture was stirred at rt for 4 h, diluted with CH₂Cl₂, and

then washed with saturated aqueous NaHCO₃solution (10 mL). The

organic layer was dried over Na₂SO₄, ﬁltered, and concentrated under

reduced pressure. The resulting residue was chromatographed over

silica gel (0−50% EtOAc in hexanes) to give N-((4,4-diﬂuoro-1-(4

-((1-methyl-1H-imidazol-4-yl)sulfonyl)piperazin-1-yl)cyclohexyl)-

methyl)-2,6-diﬂuorobenzamide as a white solid (0.409 g, 60%): ESI

MS m/z 518 [M + H]⁺.

Step F. To a 0 °C solution of N-((4,4-diﬂuoro-1-(4-((1-methyl-1H-

imidazol-4-yl)sulfonyl)piperazin-1-yl)cyclohexyl)methyl)-2,6-diﬂuoro-

benzamide (0.100 g, 0.19 mmol) in CH₃CN (1 mL) was slowly added

a 1.0 M solution of HCl in H₂O (1.5 mL). The mixture was lyophili-

zed to give N-((4,4-diﬂuoro-1-(4-((1-methyl-1H-imidazol-4-yl)sulfonyl)

8.5 Hz, 1H), 7.41 (d, J = 2.0 Hz, 1H), 7.33 (dd, J = 8.5, 2.0 Hz, 1H),

6.79 (bs, 1H), 4.19 (s, 3H), 3.57 (d, J = 5.0 Hz, 2H), 3.29−3.22 (m,

4H), 2.79−2.73 (m, 4H), 1.68−1.55 (m, 4H), 1.50−1.40 (m, 5H),

1.28−1.21 (m, 1H); ESI MS m/z 515 [M + H]⁺; HPLC 96.6% (AUC)

(Method D), t_R= 5.3 min.

N-((4,4-Diﬂuoro-1-(4-((1-methyl-1H-imidazol-4-yl)sulfonyl)-

piperazin-1-yl)cyclohexyl)methyl)-2,6-diﬂuorobenzamide Hydro-

chloride (44). Step A. To a 0 °C solution of tert-butyl piperazine-

1-carboxylate (12, 10.0 g, 53.7 mmol) in CH₂Cl₂(50 mL) was added

Et₃N (22.6 mL, 161 mmol) and a solution of 1-methyl-1H-imidazole-

4-sulfonyl chloride (11.6 g, 64.5 mmol) in CH₂Cl₂(50 mL) dropwise

over 30 min while maintaining the temperature of the reaction mixture

below 5 °C. The reaction mixture was stirred at rt for an additional 5 h

and was then quenched with a saturated aqueous solution of NaHCO₃

(50 mL). The aqueous mixture was extracted with CH₂Cl₂(3 × 100

mL), and the combined organic extracts were washed with brine (1 ×

50 mL), dried over Na₂SO₄, ﬁltered, and concentrated under reduced

P

DOI: 10.1021/acs.jmedchem.6b00914

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Journal of Medicinal Chemistry

Article

piperazin-1-yl)cyclohexyl)methyl)-2,6-diﬂuorobenzamide hydrochlor-

ide (44) as an amorphous white solid (0.067 g, quantita-

chloride (54). Step A. To a 0 °C solution of tert-butyl piperazine-

1-carboxylate (12, 10.0 g, 53.7 mmol) and Et₃N (22.6 mL, 161.2 mmol)

in CH₂Cl₂(50 mL) was added a solution of 1-methyl-1H-1,2,

3-triazole-4-sulfonyl chloride (11.7 g, 64.5 mmol) in CH₂Cl₂(50 mL)

dropwise over a period of 30 min. The reaction mixture stirred at rt

for 5 h and was then washed with a saturated solution of NaHCO₃

(50 mL) and brine (50 mL). The organic layer was dried over Na₂SO₄,

ﬁltered, and concentrated under reduced pressure to give tert-butyl

4-((1-methyl-1H-1,2,3-triazol-4-yl)sulfonyl)piperazine-1-carboxylate

(50) as a crude solid, which was used as-is in the next step.

1

tive): mp 198−199 °C; H NMR (300 MHz, DMSO-d₆) δ 10.68

(bs, 1H), 9.25 (bs, 1H), 7.88 (m, 2H), 7.52 (m, 1H), 7.22 (m, 2H),

4.00−3.12 (m, 11H), 2.35−2.03 (m, 10H); APCI MS m/z 519 [M +

H]⁺; HPLC > 99% (AUC) (Method B), t_R= 20.6 min.

2,6-Dichloro-N-((4,4-diﬂuoro-1-(4-((1-methyl-1H-imidazol-4-yl)-

sulfonyl)piperazin-1-yl)cyclohexyl)methyl)benzamide Hydrochlor-

ide (45). Compound 45 was prepared according to a similar procedure

described for the synthesis of 44 using 2,6-dichlorobenzoyl chloride.

1

mp 174−175 °C; H NMR (300 MHz, DMSO-d₆) δ 10.56 (s, 1H),

Step B. tert-Butyl 4-((1-methyl-1H-1,2,3-triazol-4-yl)sulfonyl)-

piperazine-1-carboxylate (50) was dissolved in CH₂Cl₂(50 mL).

This solution was cooled to 0 °C and TFA (30.6 g, 268.5 mmol) was

added. The mixture stirred at rt for 16 h and was then neutralized with

a saturated aqueous solution of NaHCO₃. The separated organic layer

was washed with brine (50 mL), dried over Na₂SO₄, ﬁltered, and

concentrated under reduced pressure to give 1-((1-methyl-1H-1,2,

3-triazol-4-yl)sulfonyl)piperazine (51) as crude solid, which was used

as-is in the next step (8.4 g, 68%): ESI MS m/z 232 [M + H]⁺.

Step C. To a 0 °C solution of 4,4-diﬂuorocyclohexanone (2.0 g,

14.9 mmol) in toluene (20 mL) was added TMSCN (2.18 mL,

16.3 mmol) dropwise, followed by ZnI₂(0.236 g, 0.74 mmol). The

mixture stirred at rt for 15 min followed by addition of a solution of

1-((1-methyl-1H-1,2,3-triazol-4-yl)sulfonyl)piperazine (51, 3.43 g,

14.8 mmol) in CH₃OH (90 mL). The mixture was heated at reﬂux

for 4 h, then at rt for 16 h. The mixture was concentrated under

reduced pressure. The resulting residue was dissolved in CH₂Cl₂

(100 mL), washed with H₂O (2 × 50 mL), dried over Na₂SO₄,

ﬁltered, and concentrated under reduced pressure to give 4,

4-diﬂuoro-1-(4-((1-methyl-1H-1,2,3-triazol-4-yl)sulfonyl)piperazin-1-

yl)cyclohexanecarbonitrile (52) as an oﬀ-white solid (1.89 g, 34%):

ESI MS m/z 375 [M + H]⁺.

9.16 (s, 1H), 7.89 (m, 2H), 7.58−7.46 (m, 3H), 3.73 (s, 3H), 3.62 (m,

2H), 3.41 (m, 4H), 3.25 (m, 4H), 2.40−2.00 (m, 8H); APCI MS m/z

551 [M + H]⁺; HPLC > 99% (AUC) (Method B), t_R= 19.0 min.

2-Chloro-N-((4,4-diﬂuoro-1-(4-((1-methyl-1H-imidazol-4-yl)-

sulfonyl)piperazin-1-yl)cyclohexyl)methyl)-6-(triﬂuoromethoxy)-

benzamide Hydrochloride (46). Step A. A mixture of (4,4-diﬂuoro

-1-(4-((1-methyl-1H-imidazol-4-yl)sulfonyl)piperazin-1-yl)cyclohexyl)-

methanamine (43, 0.150 g, 0.39 mmol), 2-chloro-6-(triﬂuoromethoxy)-

benzoic acid (0.09 g, 0.39 mmol), EDCI·HCl (0.07 g, 0.39 mmol),

HOBt (0.05 g, 0.39 mmol), and Et₃N (0.3 mL, 1.91 mmol) in DMF

(10 mL) stirred at rt for 16 h. The mixture was diluted with H₂O

(20 mL) and extracted with EtOAc (3 × 20 mL). The combined

organic extracts were washed with H₂O (3 × 20 mL) and brine

(20 mL). The organic layers were then dried over Na₂SO₄, ﬁltered,

and concentrated under reduced pressure. The resulting residue was

chromatographed over silica gel (0−50% EtOAc in hexanes) to give

2-chloro-N-((4,4-diﬂuoro-1-(4-((1-methyl-1H-imidazol-4-yl)sulfonyl)-

piperazin-1-yl)cyclohexyl)methyl)-6-(triﬂuoromethoxy)benzamide as

a white solid (0.057 g, 25%): ESI MS m/z 601 [M + H]⁺.

Step B. To a 0 °C solution of 2-chloro-N-((4,4-diﬂuoro-1-(4-((1-

methyl-1H-imidazol-4-yl)sulfonyl)piperazin-1-yl)cyclohexyl)methyl)-

6-(triﬂuoromethoxy)benzamide (0.050 g, 0.08 mmol) in CH₃CN

(5 mL) was added a 1.0 M solution of HCl in H₂O (1.0 mL, 1.0 mmol).

The mixture stirred at rt for 30 min and was lyophilized to give

2-chloro-N-((4,4-diﬂuoro-1-(4-((1-methyl-1H-imidazol-4-yl)sulfonyl)-

piperazin-1-yl)cyclohexyl)methyl)-6-(triﬂuoromethoxy)benzamide hy-

drochloride (46) as a white solid (0.021 g, 50%): mp 185−188 °C; ¹H

NMR (300 MHz, DMSO-d₆) δ 10.09 (s, 1H), 9.14 (s, 1H), 7.89 (m,

2H), 7.55 (m, 3H), 3.73 (s, 3H), 3.62 (m, 2H), 3.41 (m, 4H), 3.25 (m,

4H), 2.40−2.00 (m, 8H); ESI MS m/z 600 [M + H]⁺; HPLC > 99%

(AUC) (Method B), t_R= 22.8 min.

2,4-Dichloro-N-((4,4-diﬂuoro-1-(4-((1-methyl-1H-imidazol-4-yl)-

sulfonyl)piperazin-1-yl)cyclohexyl)methyl)benzamide Hydrochlor-

ide (47). Compound 47 was prepared according to a similar procedure

described for the synthesis of 44 using 2,4-dichlorobenzoyl chloride.

mp 235 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 10.27 (s, 1H), 8.97 (s,

1H), 7.89 (m, 2H), 7.74 (m, 1H), 7.55 (m, 2H), 7.58−7.46 (m, 3H),

3.73 (s, 3H), 3.62 (m, 3H), 3.41 (m, 2H), 3.25 (m, 2H), 2.40−2.00

(m, 8H); ESI MS m/z 550 [M + H]⁺; HPLC 97.8% (AUC) (Method

B), t_R= 19.5 min.

2-Chloro-N-((4,4-diﬂuoro-1-(4-((1-methyl-1H-imidazol-4-yl)-

sulfonyl)piperazin-1-yl)cyclohexyl)methyl)-4-ﬂuorobenzamide Hy-

drochloride (48). Compound 48 was prepared according to a similar

procedure described for the synthesis of 46 using 2-chloro-4-ﬂuoro-

benzoic acid. mp 232 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 10.15 (s,

1H), 8.91 (s, 1H), 7.86 (m, 2H), 7.80−7.34 (m, 3H), 3.73 (s, 3H),

3.62 (m, 2H), 3.41 (m, 4H), 3.25 (m, 4H), 2.40−2.00 (m, 8H);

ESI MS m/z 534 [M + H]⁺; HPLC 97.1% (AUC) (Method B), t_R=

19.6 min.

Step D. To a 0 °C solution of 4,4-diﬂuoro-1-(4-((1-methyl-1H-

1,2,3-triazol-4-yl)sulfonyl)piperazin-1-yl)cyclohexanecarbonitrile (52,

2.0 g, 5.34 mmol) in THF (80 mL) was added LiAlH₄(0.691 g,

18.2 mmol) portion-wise over 15 min. The mixture was stirred at rt for

5 h then cooled to 0 °C and carefully quenched by with H₂O (5 mL)

and 4 N aq NaOH (1.4 mL). The resulting mixture was stirred at rt

15 min followed by addition of Na₂SO₄(5 g). The mixture was stirred

for an additional 30 min and was ﬁltered. The ﬁltrate was concentrated

under reduced pressure and the resulting residue was triturated with

hexanes to give a white solid, which was ﬁltered and dried under

reduced pressure to give (4,4-diﬂuoro-1-(4-((1-methyl-1H-1,2,

3-triazol-4-yl)sulfonyl)piperazin-1-yl)cyclohexyl)methanamine (53)

1

as a white foam (1.75 g, 87%): H NMR (300 MHz, CDCl₃) δ 7.94

(s, 1H), 4.19 (s, 3H), 3.22 (s, 4H), 2.79−2.80 (m, 4H), 2.71 (s, 2H),

1.75−1.95 (m, 6H), 1.55−1.65 (m, 2H); ESI MS m/z 379 [M + H]⁺.

Step E. To a 0 °C solution of (4,4-diﬂuoro-1-(4-((1-methyl-1H-

1,2,3-triazol-4-yl)sulfonyl)piperazin-1-yl)cyclohexyl)methanamine (53,

0.500 g, 1.32 mmol) in CH₂Cl₂(10 mL) was added Et₃N (0.54 mL,

3.97 mmol) and 2,6-diﬂuorobenzoyl chloride (0.233 g, 1.32 mmol).

The resulting mixture was stirred at rt for 4 h, diluted with CH₂Cl₂,

and then washed with saturated aqueous NaHCO₃solution (10 mL).

The organic layer was dried over Na₂SO₄, ﬁltered, and concentrated

under reduced pressure. The resulting residue was chromatographed

over silica gel (0−50% EtOAc in hexanes) to give N-((4,4-diﬂuoro

-1-(4-((1-methyl-1H-1,2,3-triazol-4-yl)sulfonyl)piperazin-1-yl)cyclohexyl)-

1

methyl)-2,6-diﬂuorobenzamide as a white solid (0.465 g, 68%): H

NMR (300 MHz, DMSO-d₆) δ 7.96 (s, 1H), 7.39 (m, 1H), 6.96 (m,

2H), 6.08 (m, 1H), 4.19 (s, 3H), 3.55 (d, J = 6.2 Hz, 2H), 3.28 (m,

4H), 2.79 (m, 4H), 1.93 (m, 6H), 1.67 (m, 2H), 1.26 (m, 4H); ESI

MS m/z 518 [M + H]⁺.

2-Chloro-N-((4,4-diﬂuoro-1-(4-((1-methyl-1H-imidazol-4-yl)-

sulfonyl)piperazin-1-yl)cyclohexyl)methyl)-4-(triﬂuoromethyl)-

benzamide Hydrochloride (49). Compound 49 was prepared

according to a similar procedure described for the synthesis of 46

Step F. To a 0 °C solution of N-((4,4-diﬂuoro-1-(4-(1-methyl-1H-

1,2,3-triazol-4-ylsulfonyl)piperazin-1-yl)cyclohexyl)methyl)-2,6-di-

ﬂuorobenzamide (0.153 g, 0.295 mmol) in CH₃CN (1 mL) was added

a 1.0 M solution of HCl in H₂O (5 mL). The mixture stirred at rt for

30 min and was lyophilized to give N-((4,4-diﬂuoro-1-(4-(1-methyl-

1H-1,2,3-triazol-4-ylsulfonyl)piperazin-1-yl)cyclohexyl)methyl)-2,6-di-

ﬂuorobenzamide hydrochloride (54) as a white solid (0.163 g,

1

using 2-chloro-4-(triﬂuoromethyl)benzoic acid. mp 181−183 °C; H

NMR (300 MHz, DMSO-d₆) δ 10.78 (s, 1H), 9.07 (s, 1H), 7.89 (m,

4H), 7.71 (m, 1H), 3.73 (s, 3H), 3.62 (m, 2H), 3.41 (m, 4H), 3.25 (m,

4H), 2.40−2.00 (m, 8H); APCI MS m/z 585 [M + H]⁺; HPLC 98.8%

(AUC) (Method B), t_R= 20.3 min.

N-((4,4-Diﬂuoro-1-(4-((1-methyl-1H-1,2,3-triazol-4-yl)sulfonyl)-

piperazin-1-yl)cyclohexyl)methyl)-2,6-diﬂuorobenzamide Hydro

1

quantitative): mp 202−205 °C; H NMR (300 MHz, DMSO-d₆) δ

Q

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Journal of Medicinal Chemistry

Article

9.24 (m, 1H), 8.71 (m, 2H), 7.53 (m, 1H), 7.20 (m, 2H), 4.14 (s, 3H),

3.86 (m, 2H), 3.39 (m, 2H), 3.12 (m, 3H), 2.73 (m, 2H), 2.11 (m,

2H), 1.91 (m, 2H), 1.78 (m, 2H), 1.53 (m, 2H); APCI MS m/z 519

[M + H]⁺; HPLC > 99% (AUC) (Method C), t_R= 18.0 min.

2,6-Dichloro-N-((4,4-diﬂuoro-1-(4-((1-methyl-1H-1,2,3-triazol-4-

yl)sulfonyl)piperazin-1-yl)cyclohexyl)methyl)benzamide Hydro-

chloride (55). Compound 55 was prepared according to a similar

procedure described for the synthesis of 54 using 2,6-dichlorobenzoyl

(m, 1H), 7.52−8.05 (m, 3H), 4.13 (s, 3H), 3.75−3.80 (m, 2H), 3.33−

3.73 (m, 2H), 2.89−3.33 (m, 4H), 2.78−2.89 (m, 2H), 1.51−2.44 (m,

8H); ESI MS m/z 585 [M + H]⁺. %CHNCl for C₂₂H₂₆ClF₅N₆O₃S·

HCl: calcd %C = 42.52, %H = 4.38, %N = 13.52, %Cl = 11.41; found

%C = 42.38, %H = 4.31, %N = 13.37, %Cl = 11.24; HPLC 98.0%

(AUC) (Method C), t_R= 20.2 min.

N-((4,4-Diﬂuoro-1-(4-((1-methyl-1H-1,2,3-triazol-4-yl)sulfonyl)-

piperazin-1-yl)cyclohexyl)methyl)-2,4-bis(triﬂuoromethyl)-

benzamide Hydrochloride (60). Compound 60 was prepared

according to a similar procedure described for the synthesis of 56

using 2,4-bis(triﬂuoromethyl)benzoic acid. mp 180−182 °C; ¹H NMR

(300 MHz, DMSO-d₆) δ 8.75 (s, 1H), 8.62 (s, 1H), 8.18−8.12 (m,

2H), 7.74−7.71 (m, 1H), 4.15 (s, 3H), 3.31−3.30 (m, 2H), 3.10−2.90

(m, 4H), 2.85−2.70 (m, 4H), 2.00−1.40 (m, 8H); ESI MS m/z 619

[M + H]⁺; HPLC > 99% (AUC) (Method C), t_R= 20.9 min.

2-Chloro-N-(1-(4,4-diﬂuoro-1-(4-((1-methyl-1H-1,2,3-triazol-4-yl)-

sulfonyl)piperazin-1-yl)cyclohexyl)ethyl)-4-(triﬂuoromethyl)-

benzamide Hydrochloride ((−)-66 and (+)-67). Step A. ZnI₂(2.53 g,

7.95 mmol) was added to a solution of 1-benzylpiperazine (24, 14.0 g,

79.5 mmol) and 4,4-diﬂuorocyclohexanone (10.6 g, 79.5 mmol) in a

1:1 mixture of toluene and CH₃OH (160 mL), and the resulting

mixture stirred at rt for 1 h. The reaction mixture was cooled to 0 °C

and TMSCN (11.0 mL, 87.4 mmol) was added dropwise over a period

of 15 min. The reaction mixture stirred at rt for 4 h then at reﬂux for

12 h. The reaction mixture was allowed to cool to rt and was

concentrated under reduced pressure. The resulting residue was

dissolved in EtOAc (300 mL), and the organic layer was washed with

H₂O (2 × 200 mL) and brine (1 × 100 mL). The organic layer was

dried over Na₂SO₄, ﬁltered, and concentrated under reduced pressure,

and the resulting residue was chromatographed over silica gel (0−10%

EtOAc in hexanes) to give 1-(4-benzylpiperazin-1-yl)-4,4-diﬂuorocy-

clohexanecarbonitrile (61) as white amorphous solid (24.6 g, 97%):

¹H NMR (300 MHz, CDCl₃) δ 7.37−7.27 (m, 5H), 3.50 (s, 2H), 2.72

1

chloride. mp 265−270 °C; H NMR (300 MHz, DMSO-d₆) δ 10.13

(bs, 1H), 8.70 (bs, 1H), 8.95−8.55 (m, 1H), 7.67−7.37 (m, 3H), 4.13

(s, 3H), 3.80−3.75 (m, 2H), 3.73−3.33 (m, 2H), 3.33−2.89 (m, 4H),

2.78 (m, 2H), 2.44−1.51 (m, 8H); ESI MS m/z 551 [M + H]⁺; HPLC

97.8% (AUC) (Method C), t_R= 18.5 min.

2-Chloro-N-((4,4-diﬂuoro-1-(4-((1-methyl-1H-1,2,3-triazol-4-yl)-

sulfonyl)piperazin-1-yl)cyclohexyl)methyl)-6-(triﬂuoromethoxy)-

benzamide Hydrochloride (56). Step A. A mixture of (4,4-diﬂuoro-1

-(4-(1-methyl-1H-1,2,3-triazol-4-ylsulfonyl)piperazin-1-yl)cyclohexyl)-

methanamine (53, 0.150 g, 0.39 mmol), 2-chloro-6-(triﬂuoro-

methoxy)benzoic acid (0.09 g, 0.39 mmol), EDCI·HCl (0.07 g,

0.39 mmol), HOBt (0.050 g, 0.39 mmol), and Et₃N (0.37 mL,

1.93 mmol) in DMF (10 mL) was stirred at rt for 16 h. The mixture

was diluted with H₂O (20 mL) and extracted with EtOAc (3 ×

20 mL). The combined organic extracts were washed with H₂O (3 ×

20 mL) and brine (20 mL). The organic layers were then dried over

Na₂SO₄, ﬁltered, and concentrated under reduced pressure. The

resulting residue was chromatographed over silica gel (0−50% EtOAc

in hexanes) to give 2-chloro-N-((4,4-diﬂuoro-1-(4-(1-methyl-

1H-1,2,3-triazol-4-ylsulfonyl)piperazin-1-yl)cyclohexyl)methyl)-6-

(triﬂuoromethoxy)benzamide as a white solid (0.051 g, 20%): ¹H

NMR (300 MHz, CDCl₃) δ 8.12 (s, 1H), 7.95 (s, 1H), 7.36−7.39 (m,

2H), 5.82−5.89 (m, 1H), 4.19 (s, 3H), 3.56 (d, J = 6.2 Hz, 2H), 3.25−

3.33 (m, 4H), 2.74−2.82 (m, 4H), 1.81−1.99 (m, 6H), 1.72−1.80 (m,

2H); ESI MS m/z 601 [M + H]⁺.

Step B. To a 0 °C solution of 2-chloro-N-((4,4-diﬂuoro-1-(4-(1-

methyl-1H-1,2,3-triazol-4-ylsulfonyl)piperazin-1-yl)cyclohexyl)-

methyl)-6-(triﬂuoromethoxy)benzamide (0.051 g, 0.08 mmol) in

CH₃CN was added a 1.0 M solution of HCl in H₂O (1.0 mL,

1.0 mmol). The mixture was stirred at rt for 30 min and was lyophilized

to give 2-chloro-N-((4,4-diﬂuoro-1-(4-(1-methyl-1H-1,2,3-triazol-4-

ylsulfonyl)piperazin-1-yl)cyclohexyl)methyl)-6-(triﬂuoromethoxy)-

benzamide hydrochloride (56) as a white solid (0.021 g, 50%): mp

260−265 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 10.30 (bs, 1H), 9.15

(bs, 1H), 8.95−8.55 (m, 1H), 7.77−7.32 (m, 3H), 4.13 (s, 3H), 3.80−

3.75 (m, 2H), 3.73−3.33 (m, 2H), 3.32−2.89 (m, 4H), 2.87−2.74 (m,

2H), 2.44−1.51 (m, 8H); ESI MS m/z 601 [M + H]⁺; HPLC 98.3%

(AUC) (Method C), t_R= 20.1 min.

(m, 4H), 2.53 (m, 4H), 2.21−1.93 (m, 8H).

Step B. To a 0 °C solution of 1-(4-benzylpiperazin-1-yl)-4,4-

diﬂuorocyclohexanecarbonitrile (61, 16.0 g, 50.14 mmol) in anhydrous

Et₂O (160 mL) was added a 3.0 M solution of CH₃Li in DME (25 mL,

75.2 mmol) dropwise. The mixture was then heated at reﬂux for 6 h

and then cooled to 0 °C. To this mixture was added NaBH₄(5.68 g,

15.0 mmol) followed by dropwise addition of CH₃OH (160 mL). The

mixture stirred at rt for 2 h, followed by dropwise addition H₂O (25

mL). The mixture was concentrated under reduced pressure, and the

resulting residue was dissolved in EtOAc (300 mL). The organic layer

was washed with H₂O (100 mL) and brine (100 mL), dried over

Na₂SO₄, ﬁltered, and concentrated to aﬀord 1-(1-(4-benzylpiperazin-

1-yl)-4,4-diﬂuorocyclohexyl)ethanamine as an oil (17.8 g, crude,

>99%). To a 0 °C solution of 1-(1-(4-benzylpiperazin-1-yl)-4,4-diﬂuo-

rocyclohexyl)ethanamine (17.8 g, crude, 52.9 mmol) and Et₃N

(14.8 mL, 105 mmol) in CH₂Cl₂(100 mL) was added a solution of

di-tert-butyl dicarbonate (12.71 g, 58.2 mmol) in CH₂Cl₂(80 mL)

dropwise over a period of 15 min. The mixture stirred at rt for 5 h and

was washed with H₂O (2 × 150 mL) and brine (150 mL). The organic

layer was dried over Na₂SO₄, ﬁltered, and concentrated under reduced

pressure. The resulting residue was chromatographed over silica gel

(0−30% EtOAc in hexanes) to aﬀord ( )-tert-butyl (1-(1-(4-benzyl-

piperazin-1-yl)-4,4-diﬂuorocyclohexyl)ethyl)carbamate (( )-62) as

2,4-Dichloro-N-((4,4-diﬂuoro-1-(4-((1-methyl-1H-1,2,3-triazol-4-

yl)sulfonyl)piperazin-1-yl)cyclohexyl)methyl)benzamide Hydro-

chloride (57). Compound 57 was prepared according to a similar

procedure described for the synthesis of 55 using 2,4-dichlorobenzoyl

1

chloride. mp 175−180 °C; H NMR (300 MHz, DMSO-d₆) δ 10.31

(bs, 1H), 8.70−8.92 (m, 1H), 8.33−8.45 (m, 1H), 7.62−7.71 (m, 1H),

7.32−7.57 (m, 2H), 4.13 (s, 3H), 3.75−3.80 (m, 2H), 3.33−3.73 (m,

2H), 2.89−3.33 (m, 4H), 2.78−2.89 (m, 2H), 1.51−2.44 (m, 8H); ESI

MS m/z 551 [M + H]⁺; HPLC 97.4% purity (Method A).

2-Chloro-N-((4,4-diﬂuoro-1-(4-((1-methyl-1H-1,2,3-triazol-4-yl)-

sulfonyl)piperazin-1-yl)cyclohexyl)methyl)-4-ﬂuorobenzamide Hy-

drochloride (58). Compound 58 was prepared according to a similar

procedure described for the synthesis of 56 using 2-chloro-

1

white solid (18.4 g, 80%): H NMR (300 MHz, CDCl₃) δ 7.35−

7.28 (m, 5H), 4.35 (m, 1H), 3.87 (m, 1H), 3.48 (s, 2H), 2.78 (m, 4H),

2.41 (m, 4H), 1.77 (m, 5H), 1.42 (m, 10 H), 1.05 (d, J = 4.2 Hz, 3H).

Step C. A mixture of ( )-tert-butyl (1-(1-(4-benzylpiperazin-1-yl)-

4,4-diﬂuorocyclohexyl)ethyl)carbamate (( )-62, 11.3 g, 25.8 mmol),

10% Pd/C (10 wt %/wt loading, 2.26 g) and ammonium formate

(16.2 g, 258 mmol) in CH₃OH (180 mL) was heated at reﬂux for 4 h.

The mixture was allowed to cool to rt and was ﬁltered through a pad of

Celite. The ﬁltrate was concentrated under reduced pressure to aﬀord

( )-tert-butyl (1-(4,4-diﬂuoro-1-(piperazin-1-yl)cyclohexyl)ethyl)-

carbamate (( )-63) (8.0 g, crude) as colorless foam, which was

used as-is in the next step.

1

4-ﬂuorobenzoic acid. mp 230−232 °C; H NMR (300 MHz, DMSO-

d₆) δ 10.13 (bs, 0.4H), 8.91 (bs, 0.6H), 9.95 (bs, 1H), 8.35−8.51 (m,

1H), 7.21−7.67 (m, 3H), 4.13 (s, 3H), 3.75−3.80 (m, 2H), 3.33−3.73

(m, 3H), 2.89−3.33 (m, 4H), 2.78−2.89 (m, 2H), 1.51−2.44 (m, 8H);

ESI MS m/z 535 [M + H]⁺; HPLC 98.7% (AUC) (Method C), t_R=

18.4 min.

2-Chloro-N-((4,4-diﬂuoro-1-(4-((1-methyl-1H-1,2,3-triazol-4-yl)-

sulfonyl)piperazin-1-yl)cyclohexyl)methyl)-4-(triﬂuoromethyl)-

benzamide Hydrochloride (59). Compound 59 was prepared

according to a similar procedure described for the synthesis of 56

using 2-chloro-4-(triﬂuoromethyl)benzoic acid. mp 190−192 °C; ¹H

NMR (300 MHz, DMSO-d₆) δ 10.31 (bs, 1H), 9.15 (bs, 1H), 8.55−8.95

Step D. Crude ( )-tert-butyl (1-(4,4-diﬂuoro-1-(piperazin-1-yl)-

cyclohexyl)ethyl)carbamate (( )-63, 8.0 g, 23.0 mmol) was dissolved

R

DOI: 10.1021/acs.jmedchem.6b00914

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

in CH₂Cl₂(50 mL) and cooled to 0 °C. To this solution was added

Et₃N (16.2 mL, 115.2 mmol) followed by dropwise addition of a

solution of 1-methyl-1H-1,2,3-triazole-4-sulfonyl chloride (6.25 g,

34.5 mmol) in CH₂Cl₂(50 mL). The mixture was stirred at rt for 2 h

and was diluted with CH₂Cl₂(250 mL) and washed with saturated

aqueous NaHCO₃(100 mL). The organic layer was washed with brine

(100 mL), dried over Na₂SO₄, ﬁltered, and concentrated under

reduced pressure to aﬀord ( )-tert-butyl (1-(4,4-diﬂuoro-1-(4-((1-

methyl-1H-1,2,3-triazol-4-yl)sulfonyl)piperazin-1-yl)cyclohexyl)ethyl)-

carbamate (( )-64) as oﬀ-white solid (8.1 g, over two steps 71%): ¹H

NMR (300 MHz, CDCl₃) δ 8.00 (s, 1H), 4.41 (m, 1H), 3.89 (m, 1H),

3.21 (m, 3H), 2.85 (m, 4H), 1.90−1.71 (m, 6H), 1.42 (m, 10 H), 1.05

(d, J = 4.2 Hz, 3H).

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge on the ACS

Publications website at DOI: 10.1021/acs.jmedchem.6b00914.

■

S

In vivo protocols (PDF)

IC₅₀data (CSV)

AUTHOR INFORMATION

Corresponding Authors

*Tel: 518-283-6451. E-mail: christopher.cio ﬃ @acphs.edu.

*Tel: 518-878-6470. E-mail: shuang@consynance.com.

■

Step E. To a to 0 °C solution of ( )-tert-butyl (1-(4,4-diﬂuoro-1-(4-

((1-methyl-1H-1,2,3-triazol-4-yl)sulfonyl)piperazin-1-yl)cyclohexyl)-

ethyl)carbamate (( )-64, 8.0 g, 16.2 mmol) in CH₃OH (40 mL) was

slowly added 12 N aq HCl (40 mL) over a period of 30 min. The

mixture stirred at rt for 5 h and was concentrated under reduced

pressure. The resulting residue was dissolved in H₂O (50 mL) and

neutralized with solid NaHCO₃. The aqueous mixture was extracted

with EtOAc (2 × 100 mL), and the combined organic extracts were

washed with brine (100 mL), dried over Na₂SO₄, ﬁltered, and concen-

trated under reduced pressure to aﬀord ( )-1-(4,4-diﬂuoro-1-(4-((1-

methyl-1H-1,2,3-triazol-4-yl)sulfonyl)piperazin-1-yl)cyclohexyl)ethana-

mine as white solid (( )-64, 6.2 g, 97%): ESI MS m/z 393 [M + H]⁺.

To a solution of ( )-1-(4,4-diﬂuoro-1-(4-((1-methyl-1H-1,2,3-

triazol-4-yl)sulfonyl)piperazin-1-yl)cyclohexyl)ethanamine (( )-64,

4.7 g, 11.9 mmol) in DMF (50 mL) was added 2-chloro-4-(triﬂuoro-

methyl)benzoic acid (2.95 g, 13.1 mmol), HOBt (2.42 g, 17.9 mmol),

EDCI·HCl (3.43 g, 17.9 mmol), and Et₃N (5.0 mL, 35.9 mmol). The

resulting mixture stirred a rt for 16 h and was diluted with H₂O

(300 mL). The aqueous mixture was extracted with EtOAc (2 ×

100 mL), and the combined organic extracts were washed with H₂O

(3 × 100 mL) and brine (150 mL). The organic layer was dried over

Na₂SO₄, ﬁltered, and concentrated under reduced pressure. The

resulting residue was chromatographed over silica gel (0−10% EtOAc

in hexanes) to give ( )-2-chloro-N-(1-(4,4-diﬂuoro-1-(4-((1-methyl-

1H-1,2,3-triazol-4-yl)sulfonyl)piperazin-1-yl)cyclohexyl)ethyl)-

4-(triﬂuoromethyl)benzamide as an oﬀ-white solid (( )-65, 5.0 g,

69%); ¹H NMR (300 MHz, DMSO-d₆) δ 8.79 (s, 1H), 8.48 (m, 1H),

7.95 (s, 1H), 7.80 (m, 1H), 7.55 (m, 1H), 4.26 (m, 1H), 4.13 (s, 3H),

3.20−2.30 (m, 7H), 2.00−1.51 (m, 9H), 1.10 (m, 3H); APCI MS m/z

599 [M + H]⁺.

Present Addresses

^⊥Albany College of Pharmacy and Health Sciences, 106 New

Scotland Ave, Albany, New York 12208, United States.

^#ConSynance Therapeutics, Inc., Schenectady, New York

12303, United States.

^∇Pharma Inventor, Inc., University of British Columbia

Campus, 3800 Wesbrook Mall, Suite #215, Vancouver, British

Columbia, V6S 2L9, Canada.

^○Hummingbird Bioscience, PTE, Ltd., 28 Seah Street, #03-01,

188384, Singapore.

^◆Brains Online, Inc., 7000 Shoreline Court, South San Francisco,

California 94080, United States.

^¶Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N,

Seattle, Washington 98109, United States.

Notes

The authors declare the following competing ﬁnancial

interest(s): ConSynance Therapeutics, Inc. has executed an

exclusive license with AMRI that covers the intellectual

property for all of the GlyT-1 inhibitors disclosed within.

ACKNOWLEDGMENTS

■

The authors acknowledge the following for conducting the

various in vivo studies described within: 1) Pharmaron Beijing,

Co. Ltd. for conducting rat PK studies; 2) Covance

Laboratories Inc. for conducting rat in vivo CSF glycine studies;

3) Maccine Pte Ltd. for conducting the in-life portion of PK

and CSF glycine studies with cynomolgus monkeys, and 4)

Brains On-Line LLC., for conducting rat microdialysis

experiments.

Step F. ( )-2-Chloro-N-(1-(4,4-diﬂuoro-1-(4-((1-methyl-1H-1,2,3-

triazol-4-yl)sulfonyl)piperazin-1-yl)cyclohexyl)ethyl)-4-(triﬂuoromethyl)-

benzamide (( )-65, 0.289 g) was resolved by preparative chiral HPLC to

give (−)-66 (after treatment with 1.0 M solution of HCl in H₂O in

CH₃CN to give the corresponding HCl salt) as a white solid (0.121 g,

41%) and (+)-67 (after treatment with 1.0 M solution of HCl in H₂O

in CH₃CN to give the corresponding HCl salt) as a white solid (0.112

g, 38%).

ABBREVIATIONS USED

■

NMDA receptor, N-methyl-D-aspartate receptor; GlyT-1,

glycine transporter-1; GlyT-2, glycine transporter-2; iGluRs,

ionotropic glutamate receptors; LTP, long-term potentiation;

LTD, long-term depression; GlyR, glycine receptor; CSF,

cerebral spinal ﬂuid; SAR, structure−activity relationship; SPR,

structure−property relationship; ADME, absorption, distribu-

tion, metabolism, elimination; PPB, plasma protein binding;

OCD, obsessive compulsive disorder; SPA, scintillation

proximity assay; HLM, human liver microsomes; RLM, rat

liver microsomes; mPFC, medial prefrontal cortex; n-BuLi,

n-butyl lithium; THF, tetrahydrofuran; DMF, N,N-dimethyl-

formamide; DME, dimethoxyethane; CH₂Cl₂, dichlorome-

thane; CH₃CN, acetonitrile; Et₂AlCN, diethylaluminum

cyanide; Ti(i-PrO)₄, titanium isopropoxide; LiAlH₄, lithium

aluminum hydride; PTSA, para-toluene sulfonic acid; Et₂O,

diethyl ether; Boc₂O, di-tert-butyl dicarbonate; TFA, triﬂuoro-

acetic acid; TMS, tetramethylsilane; LiHMDS, lithium bis-

(trimethylsilyl)amide; NH₄HCO₂, ammonium formate;

i-Pr₂NEt, N,N-diisopropylethylamine; KCN, potassium cyanide;

1

Spectral data for (−)-66: mp 134−136 °C; H NMR (300 MHz,

DMSO-d₆) δ 8.79 (s, 1H), 8.48 (m, 1H), 7.95 (s, 1H), 7.80 (m, 1H),

7.55 (m, 1H), 4.26 (m, 1H), 4.13 (s, 3H), 3.20−2.30 (m, 7H), 2.00−

1.51 (m, 9H), 1.10 (m, 3H); APCI MS m/z 599 [M + H]⁺; HPLC >

99% (AUC) (Method B), t_R= 26.0 min; [α]²⁰= −9.78° (c = 0.47,

D

CH₃OH); chiral HPLC > 99% (AUC) (Method E), t_R= 7.45 min.

1

Spectral data for (+)-67: mp 134−136 °C; H NMR (300 MHz,

DMSO-d₆) δ 8.79 (s, 1H), 8.48 (m, 1H), 7.95 (s, 1H), 7.80 (m, 1H),

7.55 (m, 1H), 4.26 (m, 1H), 4.13 (s, 3H), 3.20−2.30 (m, 7H), 2.00−

1.51 (m, 9H), 1.10 (m, 3H); APCI MS m/z 599 [M + H]⁺; %CHNCl

for C₂₃H₂₈ClF₅N₆O₃S·HCl·0.75H₂O: calcd %C = 42.57, %H = 4.74, %

N = 12.95, %Cl = 10.93; found %C = 42.48, %H = 5.07, %N = 12.51,

%Cl = 11.03; HPLC > 99% (AUC) (Method B), t_R= 26.0 min; [α]²⁰

D

= +8.0° (c = 0.87, CH₃OH); chiral HPLC > 99% (AUC) (Method E),

t_R= 16.2 min.

S

DOI: 10.1021/acs.jmedchem.6b00914

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Lepeleire, I.; Van Laere, K.; Sur, C.; Hamill, T. G. Characterization of

the novel GlyT1 PET tracer [¹⁸F]MK-6577 in humans. Synapse 2015,

69, 33−40. (i) Borroni, E.; Zhou, Y.; Ostrowitzki, S.; Alberti, D.;

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D. F. Pre-clinical characterization of [¹¹C]RO5013853 as a novel

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Et₃N, triethylamine; HBTU, O-(benzotriazol-1-yl)-N,N,N′,N′-

tetramethyluronium hexaﬂuorophosphate; EDCI·HCl, 1-ethyl-3-

(3-(dimethylamino)propyl)carbodiimide hydrochloride; HOBt,

hydroxybenzotriazole; Gly, glycine; CYP, cytochrome P450;

CYP2C9, cytochrome P450 2C9; CYP2C19, cytochrome P450

2C19; CYP2D6, cytochrome P450 2D6; CYP3A4, cytochrome

̀

P450 3A4; hERG, human ether-a-go-go-related gene; PK,

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pharmacokinetics; PD, pharmacodynamics; IV, intravenous;

PO, oral; QD, once daily; CL_int, intrinsic clearance; Cl, clearance;

Vss, volume of distribution at steady state; AUC, area under the

curve; %F, % bioavailability

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Richter, D. W.; Betz, H. Inactivation of the glycine transporter 1 gene

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