The Discovery and Hit-to-Lead Optimization of Tricyclic Sulfonamides as Potent and Efficacious Potentiators of Glycine Receptors

Article

pubs.acs.org/jmc

The Discovery and Hit-to-Lead Optimization of Tricyclic

Sulfonamides as Potent and Eﬃcacious Potentiators of Glycine

Receptors

Howard Bregman,*^,†Jeﬀrey R. Simard,^∇,○Kristin L. Andrews,^§Shawn Ayube,^∥Hao Chen,^⊥

Hakan Gunaydin,^§,¶Angel Guzman-Perez,^†Jiali Hu,^⊥Liyue Huang,^#Xin Huang,^§Paul H. Krolikowski,^∥

Sonya G. Lehto,^∇Richard T. Lewis,^†,□Klaus Michelsen,^∥,◇Pamela Pegman,^⊥,+Matthew H. Plant,^∥

Paul L. Shaﬀer,^§Yohannes Teﬀera,^#Shuyan Yi,^†Maosheng Zhang,^∇Jacinthe Gingras,^‡

and Erin F. DiMauro^†,¶

‡

§

∥

⊥

^†Departments of Medicinal Chemistry, Neuroscience, Molecular Engineering, Discovery Attribute Sciences, Biologics, and

^#Pharmacokinetics and Drug Metabolism. Amgen, Inc., 360 Binney Street, Cambridge, Massachusetts 02142, United States

^∇Department of Neuroscience, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320, United States

S

* Supporting Information

ABSTRACT: Current pain therapeutics suﬀer from undesirable psychotropic and sedative side eﬀects, as well as abuse potential.

Glycine receptors (GlyRs) are inhibitory ligand-gated ion channels expressed in nerves of the spinal dorsal horn, where their

activation is believed to reduce transmission of painful stimuli. Herein, we describe the identiﬁcation and hit-to-lead optimization

of a novel class of tricyclic sulfonamides as allosteric GlyR potentiators. Initial optimization of high-throughput screening (HTS)

hit 1 led to the identiﬁcation of 3, which demonstrated ex vivo potentiation of glycine-activated current in mouse dorsal horn

neurons from spinal cord slices. Further improvement of potency and pharmacokinetics produced in vivo proof-of-concept tool

molecule 20 (AM-1488), which reversed tactile allodynia in a mouse spared-nerve injury (SNI) model. Additional structural

optimization provided highly potent potentiator 32 (AM-3607), which was cocrystallized with human GlyRα3_crystto aﬀord the

ﬁrst described potentiator-bound X-ray cocrystal structure within this class of ligand-gated ion channels (LGICs).

pain processing support targeting this receptor class.⁴The β-

INTRODUCTION

■

subunit is believed to localize the channel at the postsynaptic

Chronic, neuropathic pain represents a major unmet medical

need. Current and most eﬀective treatment plans include the

cleft by interacting with gephryn on the neuronal cell surface.⁵

Because of these factors, we targeted potentiators of GlyRα3β

heteropentameric channels.

Positive modulators of GlyRs have been suggested as

administration of agents with undesirable psychotropic and

physiological side eﬀects.¹The identiﬁcation of novel anagelsic

agents devoid of these side eﬀects would provide an immensely

potential therapeutic agents to suppress pain signaling at the

positive societal impact. Strychnine-sensitive glycine receptors

level of the spinal cord.^4,6,7Reported GlyR modulators include

have been investigated as key regulators of spinal inhibitory

volatile and intravenous anesthetics,⁸n-alcohols,⁸avermectins,⁸

tropeines,⁸cannabinoid ligands,^9,10bivalent cations,⁸glutama-

tergic ligands,¹¹gelsemine,^12,13mono and disaccharides,¹⁴and

neurotransmission in the adult CNS.^2,3GlyRs functionally exist

as homopentameric (GlyRα1 and GlyRα3) or heteropenta-

meric (GlyRα1β and GlyRα3β) channels with either a 3:2 or

2:3 ratio of α:β subunits. The reported enrichment of GlyRs in

the spinal dorsal horn, the terminal point of aﬀerent nociceptive

sensitization and the reported role of GlyRα3 in inﬂammatory

Received: October 12, 2016

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ginkolic acid.¹⁵Recent detailed functional behavioral pain

studies suggested that the mechanism of analgesia aﬀorded with

the cannabinoid class of compounds, and gelsemine was

primarily due to potentiation of GlyRα3.^9,10,12Unfortunately,

these reported modulators suﬀer from a lack of potency,

selectivity, and/or pharmacokinetic (PK) properties favorable

for in vivo proﬁling. Recently, Pﬁzer reported the identiﬁcation

of a few small molecule potentiator chemotypes via a high-

throughput screening (HTS) campaign, with one sulfonamide

displaying selectivity over GABA_A-R α1/3β3γ2 without the

disclosure of hit optimization eﬀorts.¹⁶We sought to identify

small molecule GlyRα3 positive modulators with selectivity

over other ligand-gated ion channels (LGICs) suitable for oral

dosing in a rodent pain model of neuropathic pain to establish

target proof-of-concept (POC).

conﬁrmation of glycine modulation was required for compound

advancement.²¹

Our HTS campaign led to the identiﬁcation of modestly

potent hit 1 (mEC₃= 3.1 μM) with high passive permeability

and low eﬄux ratios in breast cancer resistance protein (BCRP)

and multidrug resistance protein 1 (MDR1) overexpressed

Madin−Darby canine kidney (MDCK) cell lines (Table 1). Hit

1 suﬀered from high intrinsic clearance in mouse liver

microsomes (MLM CL_int= 901 μL/min/mg), likely con-

tributing to the observed low plasma exposure following

intraperitoneal dosing in mice (plasma C_maxunbound = 0.13

μM @ 30 mg/kg). Toward identifying a minimal structural

core, replacement of the imidazole ring of the tetracycle with a

methyl amide in 3 aﬀorded improved potentiation (mEC₃=

0.78 μM) as well as a moderate stereopreference, with the two

cis pyrrolidine enantiomers 3 and 4 exhibiting a >10-fold

potency diﬀerence. Like hit 1, tricycle 3 suﬀered from high

metabolic intrinsic clearance and poor in vivo exposure upon

intraperitoneal dosing in mice (plasma C_maxunbound = 0.07

μM @ 50 mg/kg). Despite the poor PK proﬁle of 3, it was

considered an attractive lead molecule as the compound

exhibited good cellular potency, permeability with low eﬄux

ratio, and physicochemical properties conducive to CNS

penetration.²²Potency, as measured in HEK293T cells, was

optimized for human GlyRα3β, which correlated well with

potency against human GlyRα3, GlyRα1β, and GlyRα1 as well

as rodent GlyR cell lines for all compounds described herein.²¹

An in vitro metabolite proﬁling study using mouse liver

microsomes (MLM) indicated that major metabolic pathways

of 3 were oxidative metabolism on the quinolinone core,

pyrrolidine ring, and benzodioxolane moiety.

Accordingly, initial eﬀorts focused on suppressing the

oxidative metabolism of the tricyclic aryl ring by increasing

polarity and/or introducing a substituent at the relatively

electron-rich C-6, which was believed to be a metabolic soft site

(Table 2). 6-Hydroxy analogue 5 had decreased MLM CL_int

and 2-fold increased potency (mEC₃= 0.39 μM). The more

polar 6-cyano analogue 6 had further reduced MLM CL_intbut

decreased potency (mEC₃= 12.5 μM). The nonpolar 6-ﬂuoro

analogue 7 had retained potency but high intrinsic clearance

(MLM CL_int= 761 μL/min/mg). An aza-walk around the

quinolinone ring produced analogues with retained or

improved potency coupled with markedly improved MLM

CL_int, such as, 5-aza (8, mEC₃= 0.78 μM, MLM CL_int= 136

RESULTS AND DISCUSSION

■

In light of the paucity of reported, lead-like chemical matter for

GlyRs, we initiated a HTS in search of potentiators with the

goal of demonstrating in vivo POC. Our HTS and subsequent

primary screening assay utilized HEK293T cells stably

expressing human GlyRα3β. Using a standard membrane

potential dye that reports on net changes in membrane

potential across the cell membrane due to GlyR-mediated ion

ﬂux, we designed an assay that enabled assessment of the ability

of compound to directly activate GlyRα3β in the presence of

low levels (EC₁₀) of the endogenous agonist glycine. The assay

could also be performed in the absence of glycine, allowing for

the detection of agonist activity. On-target activity was

conﬁrmed by reversal of activation with the known pan-GlyR

antagonist, strychnine.¹⁷⁻¹⁹Compounds identiﬁed as on-target

potentiators with suitable potency were advanced to a Schild

shift assay which measured the ability of the compound to

modulate the activity of the agonist glycine, i.e., individual

glycine dose−response curves were generated in the presence

of compound, applied at a range of concentrations (Figure 1).

μL/min/mg) and 6-aza (9, mEC3 = 0.20 μM, MLM CL_int

=

151 μL/min/mg). This marked an important achievement:

adding polarity and improving metabolic stability while

improving activity. A signiﬁcant loss of potency was observed

with the 7-aza and 8-aza analogues, 10 and 11 (mEC₃= 6.3 and

3.1 μM, respectively).

Figure 1. Representative glycine Schild shift plot. In this example, the

mEC₃(or minimum tested compound concentration to elicit a

leftward shift in the Gly EC₅₀value ≥3×) corresponds to a compound

concentration [cpd] of 6.3 μM.

As a ﬁrst step toward electrophysiological ex vivo validation

of our in vitro cell-based functional potentiator assay, we

assessed 9 and less active enantiomer (ent)-9 in an ex vivo

mouse spinal cord slice assay. In addition to good potency, 9

possessed high passive permeability (cell line Papp ave = 52

μcm/s) and solubility (PBS, FaSSIF = 235, 296 μM), rendering

it suitable for this study. Neurons located in the upper layer of

the dorsal horn (laminae I and II) were histologically identiﬁed

and glycinergic proﬁles were further conﬁrmed upon focal

application of glycine (∼EC₃₀₋₄₀= 20 μM) followed by

conﬁrmation of a reversal eﬀect with the application of

strychnine (10 μM). To further ensure that detected currents

were a result of glycinergic signaling only, neurons were

The minimum tested concentration of potentiator required to

left shift the glycine dose−response curve at least 3-fold

(hereon referred to as mEC₃), a statistically relevant shift, was

used to rank order potentiators (see Figure 1 for a

representative example). All of the compounds described

herein increased the glycine potency (eﬀects in α) but not

glycine maximum eﬃcacy (no impact on β) in the Schild shift

assay.²⁰Therefore, the latter parameter was not utilized in our

SAR analyses. Although the activities obtained in the Schild

shift platform correlated well with dose−response values,

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a

Table 1. Proﬁles of Hit and Lead Compounds

a

Compounds are enantiopure unless otherwise noted as racemic.

a

Table 2. SAR and in Vitro PK Proﬁles of Quinolinone Ring

a

Compounds are enantiopure unless otherwise noted as racemic.

pretreated with a cocktail of inhibitors composed of

tetrodotoxin (TTX), 6-cyano-7-nitroquinoxaline-2,3-dione

(CNQX), (2R)-amino-5-phosphonovaleric acid (APV), and

bicuculine to silence TTX-sensitive sodium channels, α-amino-

3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainite

receptors, N-methyl-^D-aspartic acid (NMDA) receptors, and γ-

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Figure 2. Potentiation of glycine evoked current in adult mouse spinal neurons. Representative postsynaptic current (PSC) amplitude (pA)

modulation recorded from glycinergic neurons in an isolated adult mouse spinal cord slice: (a) glycine (20 μM), (b) glycine (20 μM) + 9 (0.5 μM)

on the top and (ent)-9 (0.5 μM) on the bottom, (c) glycine (20 μM) + 9 (0.5 μM) + strychnine (10 μM). On average, application of 0.5 μM of 9

led to a 4.9-fold increase of the PSC amplitude. This response was fully reversed in the presence of strychnine (n = 5 neurons, scale bars apply to all

electrophysiological traces).

aminobutyric acid (GABA_A) receptors, respectively. Bath

application of 9 at 0.5 μM with and without focal glycine

application was assessed to conﬁrm the potentiation mecha-

nism. GlyR activation was assessed by calculating the mean

ratio of postsynaptic current (PSC) amplitudes of 0.5 μM 9 or

(ent)-9 in the presence of 20 μM glycine (Figure 2b)/20 μM

glycine application alone (Figure 2a), where a ratio of 1.0

equates to no potentiation. The activation was conﬁrmed as

glycine-dependent if the PSC amplitude could be fully reversed

by strychnine (Figure 2c). In the presence of 9 at 0.5 μM, the

mean PSC ratio calculated was 4.9 3.7 (mean ratio STD; n

= 5). This data conﬁrmed that endogenous neuronal glycinergic

receptors can be positively modulated by 9 and served to

further validate our in vitro cell-based functional potentiator

assays. Less active enantiomer (ent)-9 (0.5 μM) did not evoke

increased current in the presence of 20 μM glycine, which

provided further support that the observed action of 9 was on

mechanism via GlyR potentiation.

substitution SAR was guided by these trends and focused on

small, systematic modiﬁcations relative to lead 9 with the goal

of further improving potency and PK (Table 3).

Toward replacing the benzodioxolane moiety, a known

structural alert,²⁵other 6,5-bicyclic heteroaromatics were

initially investigated. Benzimidazole and N-methyl benzimida-

zole replacements 12 and 13 suﬀered ∼10-fold reduced

potency. Compound 12 had insuﬃcient brain coverage over

muGlyRα3 despite improved microsomal stability, likely as a

result of poor passive permeability, and 13 suﬀered from high

eﬄux. Benzoxazole 14 had retained potency and improved

microsomal stability relative to 9 but was found to be unstable

in mouse plasma and assay buﬀer.²⁶Benzothiazole 15 suﬀered

decreased potency (hGlyRα3β mEC₃= 1.56 μM) and oral

coverage over muGlyRα3 and imidazopyridine 16 had reduced

potency (hGlyRα3β mEC₃= 3.1 μM). The importance of each

oxygen atom of the benzodioxolane was assessed by preparing

dihydrobenzofurans 17 and 18. Removal of either oxygen

aﬀorded a 5-fold drop in potency as well as slightly suppressed

oral coverage. Interestingly, benzofuran analogue 20 (AM-

1488)²⁷had retained potency relative to 9 while its regioisomer

19 had reduced activity. Additionally, suitable coverage levels of

20 were achieved at lower doses relative to benzodioxolane 9

(mouse brain C_max(u) /muGlyRα3 EC₅₀: 1.2 @ 10 mg/kg vs

0.62 for 9 @ 30 mg/kg). It is likely that the improved exposure

observed for 20 despite modest MLM stability was a result of

saturation of metabolism, supported by greater than dose

proportion increases in area under the curve (AUC) exposure

when increasing dose from 5 to 10 mg/kg (po) and well over

100% calculated oral bioavailability. As was the case with our

initial lead tricyclic sulfonamide, the enantiomer of 20, (ent)-20,

was signiﬁcantly less active (hGlyRα3β EC₅₀> 20 μM, mEC₃>

25 μM). Monocyclic sulfonamides, such as 21, were identiﬁed

with modest activity but with relatively high instrinsic clearance

(MLM CL_int= 432 μL/min/mg).

To identify compounds suitable for investigation in mouse

models of neuropathic pain, compounds with favorable potency

and microsomal stability such as 8 and 9 were advanced to

mouse PK studies. Target coverage values were calculated using

potentiation of mouse GlyRα3 rather than mouse GlyRα3β

activities due to lack of heteropentamer protein availability.²³At

a 30 mg/kg oral dose, 8 had poor target coverage (mouse brain

C

_max(u)/muGlyRα3 EC₅₀= 0.006), likely attributed to

solubility limited absorption (FaSSIF, PBS solubility = 28, 7

μM). In contrast, 9 had good solubility (FaSSIF, PBS = 296,

235 μM) and improved oral coverage at 30 mg/kg (mouse

brain C_max(u)/muGlyRα3 EC₅₀= 0.62). Brain concentrations

were measured due to target localization in the CNS. We next

directed our eﬀorts toward removing the oxidative metabolism

liabilities imparted by the benzodioxolane sulfonamide moiety

with the 6-aza substituted tricyclic core in place.

To guide our optimization of the sulfonamide portion of the

molecule, we utilized emerging trends relating physiochemical

properties with CL_int, Pgp, and BCRP eﬄux ratios. Analysis of

correlation plots (Figure 3) indicated that by targeting cLogD

(pH = 7.4)²⁴≤ 2, there was a high likelihood of achieving

MLM CL_int< 200 μL/min/mg, and that by maintaining PSA <

90, Pgp and BCRP eﬄux ratios <3 were likely to be attained.

Analogues containing hydrogen bond donors (depicted with

green squares) tended to have poor passive permeability and

high eﬄux. Our strategy toward exploring sulfonamide

Proﬁling of 20 across a range of CYS loop family receptors,

voltage-gated ion channels, kinases, and GPCRs indicated a

high degree of oﬀ-target selectivity (see Supporting Information

Part 4). Additionally, 20 was selective over hERG, BSEP, CYPs,

and PXR. Furthermore, 20 potentiated glycine-evoked current

in adult mouse spinal neurons at 0.5 μM (4.4-fold increase in

postsynaptic current amplitude relative to glycine alone).²⁷

With suitable mouse PK and a favorable expanded selectivity

proﬁle, 20 was advanced as a potential in vivo proof-of-concept

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Table 3. Sulfonamide Substitutions: SAR and PK Proﬁles

a

Fraction unbound (f_u) in mouse brain was determined as described

in Berry et al, 2010.²⁸Mouse brains were collected at multiple time

points following IP or PO administration. Drug concentrations in the

brain were determined according to the method described by Huang

et al.²⁹brain C_u= total brain concentration × f_u‑brain

.

tibial nerves can generate tactile allodynia that can be reversed

with a therapeutic agent.³⁰In this model, active enantiomer 20

demonstrated signiﬁcant reversal of tactile allodynia at an oral

dose of 20 mg/kg (mouse brain C(u)/muGlyRα3 EC₅₀: 1.9),

while the inactive enantiomer 21 did not elicit a signiﬁcant

eﬀect at the same dose and with similar exposure C(u) plasma

= 2.3 μM (est. C(u) brain = 0.61 μM). Notably, 20 did not

̈

signiﬁcantly aﬀect basic movements in naive mice at an oral

dose of 20 mg/kg.²⁷These results suggest that the reversal of

tactile allodynia was via potentiation of GlyRs and further

validates GlyRs as a therapeutic target.

With the goal of improving metabolic stability and with

guidance from metabolism identiﬁcation studies, we pursued

substitution of the pyrrolidine ring. Further, substitution at the

pyrrolidine 1- or 2-position may impact both the vector and

rotational freedom of the sulfonamide moiety potentially

impacting activity. Exploration of pyrrolidine ring SAR was

conducted concomitantly with the previously described

sulfonamide SAR exploration and thus retained the benzodiox-

olane sulfonamide moiety (Table 4).

First, substitution at ring fusion carbon “3” was investigated.

Both polar and nonpolar substitution at this position (22−24)

resulted in markedly reduced potentiation (mEC₃> 25 μM).

Similarly, methyl substitution at the ring fusion carbon “4” as in

25 was not tolerated (mEC₃> 25 μM). The chemistry

employed en route to the majority of tricyclic core

modiﬁcations was also suitable for rapidly accessing all eight

diastereomeric α-methyl pyrrolidines. In fact, the initial

synthesis aﬀorded a nonstatistical mixture of all eight possible

Figure 3. Physicochemical properties and in vitro PK trends. Pink,

compounds described herein; gray, structurally related tricyclic

sulfonamides (compounds not disclosed herein); green squares,

analogues containing an H-bond donor. (A) MLM CL_intvs Log D

(pH = 7.4). (B) MDCK human Pgp eﬄux ratio vs binned PSA. (C)

MDCK human BCRP eﬄux ratio vs binned PSA.

tool and examined in the mouse SNI model of neuropathic pain

(Figure 4). In SNI model, surgical ligation of the peroneal and

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Figure 4. Reversal of SNI-induced tactile allodynia by 20 when dosed at 20 mg/kg (po). At 60 min post oral dosing, 20 dosed at 20 mg/kg (po)

produced a signiﬁcant increase in von Frey threshold (1.53 0.31 g) relative to vehicle (0.82 0.19 g) by ANOVA with Dunnett’s Multiple

Comparisons; F_2,35= 4.7, p < 0.05. The less active enantiomer (ent)-20 also dosed at 20 mg/kg (po) did not reverse tactile allodynia. The positive

control, pregabalin, dosed at 30 mg/kg (po), was not included in the statistical analysis because of low n but appeared eﬀective. Exposure values were

generated at 90 min post dose.

a

Table 4. Pyrrolidine Modiﬁcations: SAR

a

*Tricycle aryl contains C−H rather than aza at 6-position.

stereoisomers at positions “1” and “2”, which were elaborated

to a mixture of ﬁnal compounds which were separated by

chromatography, providing compounds 26−33. Only one

minor isomer out of the eight isolated (32, AM-3607),²⁷

representing 3% of the isomeric mixture, aﬀorded improved

potency, while the other seven isomers suﬀered a dramatic loss

of potency. Furthermore, the potency of active α-methyl isomer

32 was almost an order of magnitude improved relative to its

des-methyl congener 9 (hGlyRα3β mEC₃= 0.025 vs 0.2 μM)

with an in vitro PK proﬁle of 32 similar to that of 9 (MDCK

P

_app(av), 43.1; MDR1 ER, 1.6; HLM, MLM CL_int, 83, 254 μL/

min/mg). Two-dimensional NMR studies (ROESY and

HMBC) indicated that the active analogue possessed a methyl

β to the carbonyl which projected into the concave face of the

tricyclic ring system.³¹Molecular modeling studies indicated

that in addition to reducing the rotational freedom of the

sulfonamide, the α-methyl substitution caused the structure to

favor a diﬀerent orientation of the tricycle, which we

hypothesized to be the active binding conformation. For the

des-methyl analogue 9, the analogous conformation was found

to be 3.08 kcal above its global minima (Figure 5).^32,33

Figure 5. Overlay of global minima conformations of 32 (magenta)

and 9 (cyan). Figures generated using PyMOL (The PyMOL

Molecular Graphics System, version 1.7.05, Schrodinger, LLC).

̈

were in good agreement with our measured cell-based assay

potency for active leads and their less active isomers (Table 5),

supporting that the compounds act through direct binding to

the GlyR receptors. Furthermore, a 17-fold improved K_Dis

observed for 32 over 20, consistent with the measured GlyRa3

EC₅₀values.

Toward enhancing our understanding of the binding

properties of our molecules, we developed a surface plasmon

resonance (SPR) assay using the crystallographic construct of

human GlyRα3 channels.^27,34The binding values we obtained

The X-ray cocrystal structure of 32 in complex with human

GlyRα3 in the presence of the native agonist glycine was solved

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with S_NAr of 4-chloronicotinaldehyde with methyl amine

providing 4-methylamino-pyridine 36. Subsequent Horner−

Wadsworth−Emmons oleﬁnation with the ylide generated

upon deprotonation of ethyl 2-(diethoxyphosphoryl)-2-ﬂuo-

roacetate followed by intramolecular cyclization of the E-oleﬁn

isomer provided 37.

Table 5. SPR Data for Active Leads and Their Less Active

Isomers

a

compd

20

hGlyRα3 EC₅₀(μM)

SPR hGlyRα3 K_D(μM)

0.45

>25

0.19 0.03

>20

(ent)-20

32

0.050

>25

0.011 0.005

>20

The general sequence utilized to access tricyclic pyrrolidine

cores is described in Scheme 2 and was inspired by established

routes for the known tricycle core of 41a.^37,38Substituted and

modiﬁed quinolinones (34 or 35) were N-methylated with

iodomethane in the presence of cesium carbonate in DMF to

give 38. The key step was a [3 + 2] dipolar cycloaddition of 38

with the azo-methine ylide generated from the treatment of N-

benzyl-1-methoxy-N-((trimethylsilyl)methyl)methanamine

with TFA. This reliably provided tricyclic N-benzyl pyrrolidines

39 as the racemic syn isomers in generally good yields. In the

synthesis of 39c, the [3 + 2] dipolar cycloaddition and amide

N-methylation steps were reversed.²⁷Ester hydrolysis and

decarboxylation under acidic conditions aﬀorded 40, and benzyl

hydrogenolysis with catalytic palladium hydroxide furnished the

penultimate tricyclic pyrrolidine core structure 41. Because of

the sensitivity of the nitrile functionality (R³= CN) under

reductive conditions, a two-step, one pot debenzylation

sequence was employed. Urethane formation by treatment

with vinyl carbonochloridate and subsequent debenzylation of

the formed salt was followed by urethane hydrolysis under

acidic conditions to provide the α-cyano tricyclic core 41f.

The syntheses of the α-methyl ring fusion, 4-cyano, and 4-

hydroxy modiﬁed pyrrolidine cores employed late stage

functionalization of the ring system (Scheme 3). Pyrrolidine

nitrogen protecting group swap from benzyl to tert-butyl

carbonate (Boc) under standard conditions provided 42.

Treatment with sodium hydride and excess iodomethane

aﬀected methylation of both the quinolinone nitrogen and

the ring fusion carbon α to the carbonyl, aﬀording α-

methylated core tricycle 44, following acid-mediated N-boc

cleavage. Toward 4-substituted tricyclic core structures, the N-

methylated tricycle of 42 was then regiospeciﬁcally brominated

in the presence of N-bromosuccinimide to give 43, which was

cyanated under palladium-mediated conditions to furnish cyano

substituted core 45. The brominated intermediate 43 was

alternatively converted to boronate ester 46 and subsequently

oxidized with hydrogen peroxide and sodium hydroxide to

hydroxylated core tricycle, then treated with HCl in dioxane to

cleave the N-boc group and aﬀord tricyclic amine intermediate

47.

30

a

Values are an average of three measurements with SEM values.

at high resolution (2.6 Å) (Figure 6).²⁷This represented the

ﬁrst public disclosure of a potentiator-bound X-ray cocrystal

structure with a human glycine receptor. 32 is bound in a novel

induced-pocket and adopts a conformation very close to its

calculated global minima conformation (0.05 kcal/mol diﬀer-

ence).³⁵The structure indicates H-bonding interactions with

the sulfonamide and amide carbonyl groups (water-mediated)

as well as hydrophobic interactions with the benzodioxolane,

pyrrolidine−naphthyridinone core, and methyl groups. The

ligand is bound at the interface between each of the ﬁve α-

subunit protomers in the extracellular domain and in close

proximity to bound glycine (10 Å away), which is in the

canonical orthosteric ligand binding pocket. It was shown that

upon binding of 32, the GlyRα3 protein adopts a conformation

which stabilizes glycine, thereby increasing binding aﬃnity. The

binding interactions identiﬁed in the X-ray crystal structure

supported our SAR observations and design hypotheses. For

example, the aza-naphthyridone carbonyl is engaged in a water-

mediated hydrogen bond and removal of the carbonyl

abolished activity (data not shown). Further, the nitrogen of

the pyridine ring of the aza-naphthyridone is pointed toward

the solvent, supporting the substitutional tolerance for both

polar (aza (32), hydroxy (5)) and nonpolar substituents

(ﬂuoro, 7). The pyridine ring nitrogen of aza-naphthyridones

10 and 11 are exposed to the hydrophobic binding pocket,

explaining their signiﬁcantly diminished activities.

CHEMISTRY

■

For the majority of the analogues described, a general route

toward the tricyclic amine penultimate core was employed. The

sequence began with either commercial or prepared quinoli-

nones or naphthyridones (Scheme 1). The noncommercial 3-

ethyl-carboxyl-quinolone and naphthyridone intermediates 34

were accessed by Friedlander synthesis of 2-formyl or 2-acetyl

anilines with ethyl malonate.³⁶The 3-cyano naphthyridone

variant (35) was prepared under similar conditions with the

anion of methyl 2-cyanoacetate as the nucleophile. The

preparation of 3-ﬂuoro-N-methyl-naphthyridone 37 began

The syntheses of the 4-ﬂuoro and 5-aza tricyclic pyrrolidine

cores began with Still−Gennari modiﬁed Horner−Wads-

Figure 6. (A) Potentiator binding pocket in human GlyRα3. The bound conformation of 32 (green) is overlaid with the calculated minimum energy

conformation (magenta). (B) X-ray of potentiator 32 and glycine bound to GlyRα3 (PDB codes: 5TIO and 5TIN).

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a

Scheme 1. Preparation of Quinolinone and Naphthyridone Intermediates

a

(a) piperidine, EtOH, reﬂux, 50−98%; (b) NH₃Me (40% in H₂O), MeOH−EtOH, 120 °C, sealed tube, 48%; (c) (i) NaH, ethyl 2-

(diethoxyphosphoryl)-2-ﬂuoroacetate, THF, (ii) aldehyde 36, 21%.

a

Scheme 2. Preparation of Tricyclic Pyrrolidine Cores

a

(a) Cs₂CO₃, MeI, DMF, 36−100%; (b) TFA, CH₂Cl₂, 52−100%, 18% for 39e; (c) HCl (aq), 100 °C, 78−100%; (d) Pd(OH)₂, H₂, EtOH:H₂O (5

or 10:1), 40 psi, 40−60 °C, 83−100%; (e) (i) vinyl carbonochloridate, DCM, 0 °C−rt, (ii) EtOH, 6 N HCl, 100 °C, 70%.

worth−Emmons oleﬁnation of 2-nitro-4-ﬂuoro benzaldehyde,

aﬀording oleﬁn 48 as a mixture of E and Z isomers (4:1).

Dipolar [3 + 2] cycloaddition of 48 with N-benzyl azo-methine

ylide aﬀorded N-benzyl pyrrolidine ester 49 (Scheme 4). Nitro

reduction and intramolecular cyclization aﬀorded tricyclic cores

50, which were either methylated at the quinolone nitrogen via

a protection, methylation, deprotection sequence to furnish

penultimate 4-ﬂuoro tricyclic core 51 or ﬁrst sulfonylated at the

secondary amine, then N-methylated to aﬀord 5-aza analogue

10.

prepared following a published two-step protocol.³⁹Alkylation

of neat benzyl amine with (1-chloroethyl)trimethylsilane at 180

°C provided ethyl trimethylsilyl secondary amine 52, which was

then converted to an imminium salt by treatment with

formaldehyde in the presence of potassium carbonate and

trapped by methanol to aﬀord 53. This sensitive intermediate

was used crude and in the presence of TFA decomposed to an

azo-methine ylide which underwent dipolar [3 + 2] cyclo-

addition with naphthyridone 38g to provide α-methyl-N-benzyl

pyrrolidines as an 1:8:8:1 mixture of racemic stereoisomers.

Cleavage of the N-benzyl group under standard hydrogenolysis

conditions provided α-methyl-pyrrolidine tricyclic intermediate

54 as a mixture of stereoisomers. As described earlier, only one

of the minor isomeric intermediates ((rac)-cis-54d) of this

The initial route toward α-methyl-pyrrolidine tricyclic

pyrrolidine cores followed a very similar synthetic route as

described for des-methyl pyrrolidine cores (Scheme 5). The

known methylated azo-methine ylide precursor 53 was

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a

Scheme 3. Preparation of α-Methyl Ring Fusion and 4-Cyano and 4-Hydroxyl Tricyclic Pyrrolidine Cores

a

(a) Pd/C (10%), Pd(OH)₂, (Boc)₂O, H₂, EtOH, 50 psi, 96%; (b) (i) NaH, MeI, DCE, 59%, (ii) NBS, DCM, 92%; (c) (i) NaH, MeI, (ii) HCl in

MeOH; (d) (i) Pd(PPh₃)₄, Zn(CN)₂, DMF, 100 °C, (ii) HCl/dioxane, 100%; (e) (Bpin)₂, PdCl₂(dppf), KOAc, dioxane, 90 °C, 100%; (f) (i) H₂O₂,

NaOH, THF/H₂O, (ii) HCl/dioxane, 60%.

a

Scheme 4. Preparation of 4-Fluoro Tricyclic Core and 5-Aza Tricyclic Core Analogue 10

a

(a) K₂CO₃, 1,4-dioxane, 93% for 48a, 24% for 48b; (b) TFA, DCM, 87% for 49a, 100% for 49b; (c) (i) Pd(OH)₂or Pd/C, H₂, EtOH, 60 °C, 55

psi, 100%, (ii) AcOH, 80 °C (for 49b only), 99% (d) (i) (Boc)₂O, Et₃N, EtOH, (ii) NaH, MeI, DMF, (iii) HCl/dioxane, 56%; (e) (i)

benzo[d][1,3]dioxole-5-sulfonyl chloride, Et₃N, DCM, (ii) NaH, MeI, DMF, 2%.

a

Scheme 5. Initial Route Towards Isomeric Mixture of α-Methyl Pyrrolidine Tricyclic Core

a

(a) Neat, 180 °C; (b) CH₂O (aq), K₂CO₃, MeOH; (c) (i) 38g, TFA, DCM, 33%, (ii) 3 N HCl:dioxane (2:3), 90 °C, (iii) Pd(OH)₂, H₂,

EtOH:H₂O (10:1), 40 °C, 40 psi, 96%, (over 2 steps).

mixture led to highly potent sulfonamide 32. A revised

synthesis was therefore developed to improve the relative

yield of the active diastereomer (rac)-cis-54d (Scheme 6).

The complex isomeric mixture of methylated pyrrolidines

was a result of both constitutional isomers (methyl group at

either α-pyrrolidine position) and stereoisomers (methyl group

projected into either the concave or convex face of tricyclic ring

system). The active isomer contained a methyl group proximal

to the carbonyl group and projected into the concave face of

the ring system. By starting with a surrogate for the methyl

group already present at the desired position, the regioisomer

of choice would ultimately be prepared. Accordingly,

commercial 3-acetyl-1,6-naphthyridin-2(1H)-one was N-methy-

lated, then cyclopropanated under Corey−Chaykovsky⁴⁰

conditions with the ylide of trimethylsulfoxonium iodide to

aﬀord 55 (Scheme 6). Cyclopropane ring opening at the least

sterically hindered carbon by sodium azide provided azide 56.

The crude azide was reduced with triphenylphosphine and the

liberated amine condensed intramolecularly with the acetyl

ketone to provide imine 57 as a racemic mixture of cis-

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Scheme 6. Improved Route Towards Isomeric Mixture of α-Methyl Pyrrolidine Tricyclic Core

a

(a) (i) MeI, Cs₂CO₃, DMF, 51%, (ii) NaH, trimethylsulfoxonium iodide, THF, DMSO; (b) NaN₃, Et₃N, AcOH, DMF, 70 °C; (c) PPh₃, acetone,

60 °C; (d) NaBH₄, MeOH/DCE, (30% over 4 steps).

a

Scheme 7. Synthesis of Sulfonamides

a

(a) Sulfonyl chloride, triethylamine, CH₃CN; (b) triethylamine, CH₃CN, 54%; (c) (i) triethylamine, CH₃CN, 75%, (ii) chiral SFC puriﬁcation.

pyrrolidines. Compound 57 was susceptible to isomerization

and oxidation to the pyrrole upon prolonged exposure to air.

Reduction of the 57 with sodium borohydride aﬀorded a 1:1

mixture of concave:convex methyl diastereomers, providing the

penultimate racemic tricyclic intermediates ((rac)-cis-54c and

(rac)-cis-54d). This route aﬀorded improved relative ratio of

the desired methylated tricyclic core with 25% of the desired

single enantiomer as compared to 3% for the initial route.

The described tricyclic sulfonamides were prepared under

the same general conditions, as generically depicted in Scheme

7. Sulfonylation was accomplished with sulfonyl chloride in the

presence of triethylamine in acetonitrile. Either speciﬁc isomers

or isomeric mixtures of tricyclic penultimate core intermediates

were used in analogue synthesis. For example, the single active

enantiomer of 41c was used in the synthesis of 20 and the

mixture of (rac)-cis-54c and (rac)-cis-54d was used toward the

synthesis of 32, which was obtained as a single enantiomer

following chiral SFC puriﬁcation. The relative and absolute

conﬁgurations of the active species are as drawn in the schemes

and were determined by two-dimensional NMR (ROESY and

HMBC) and the described X-ray cocrystal structure,

respectively.

CONCLUSION

■

Herein, we have detailed the discovery and synthesis of a novel

class of orally available and CNS-penetrant tricyclic sulfona-

mides as selective allosteric potentiators of glycine receptors.

Our property-guided hit-to-lead eﬀorts delivered proof-of-

concept ex vivo tool compound 3, which potentiated

postsynaptic currents in mouse spinal neurons of the dorsal

horn, and in vivo tool molecule 20, which demonstrated oral

eﬃcacy in a mouse spared-nerve injury model of neuropathic

pain. Concurrent systematic exploration of the eﬀects of core

methylation on functional activity and binding aﬃnity delivered

highly potent ligand 32, which facilitated the generation of the

ﬁrst potentiator-bound hGlyRα3 X-ray cocrystal structure to

date. An eﬃcient and scalable synthetic route to provide the

penultimate methylated tricyclic intermediate (rac)-cis-54d was

developed. This work provided methods and tools suitable for

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Animal Care and Use Committee. For protocol see Huang, Shaﬀer,

DiMauro, Gingras et al.²⁷

CL_intDeterminations. CL_intwas determined by spiking drug into

a mixture of 0.25 mg/mL mouse liver microsomes, 1 mM reduced

nicotinamide adenine dinucleotide phosphate, and 5 mM MgCl in 100

mM potassium phosphate buﬀer, pH 7.4, and incubating for various

time points up to 40 min.

Passive Permeability Determinations. Bidirectional transport

across Madin−Darby canine kidney cells transfected with control

vector (VC-MDCK), human MDR1 gene (MDR1-MDCK) and

human ABCG2 gene (BCRP-MDCK) was measured in triplicate at

5 μM. Average apparent permeability value (P_app) of both directions

from VC-MDCK was used as passive permeability.

the further investigation of the biology of GlyRs and the

generation of GlyR modulators with improved drug-like

properties.

EXPERIMENTAL SECTION

■

Functional Testing in FLIPR Assay. HEK293T cells expressing

human GlyRα3β were cultured in T 225 cm²culture ﬂasks under

standard cell culture conditions of 37 °C, 5% v/v CO₂, and 95%

humidity until 80−90% conﬂuent. Prior to the assay, cells are

harvested by brieﬂy washing with DPBS followed by addition of

TrypLE cell dissociation reagent for 2 min. Cells were then quantiﬁed

using a ViCell and subsequently diluted in Cell Plating Media to

achieve a plated cell density of ∼12000 cells/well. Using a Multidrop

Combi, 25 μL of cell suspension was dispensed into Corning

CellBIND 384-well ViewPlates and then incubated at 37 °C overnight

under the standard cell culture conditions described above. The next

day (∼18−24 h after plating), 5 μL of 6× Membrane Potential (MP)

blue dye was dispensed into each cell culture plate using a Multidrop

Combi. The cell plates were then incubated at 37 °C for 30 min and

then allowed to equilibrate to room temperature for an additional 30

min prior to running the assay.

General Chemistry. Unless otherwise noted, all materials were

obtained from commercial suppliers and used without further

puriﬁcation. Anhydrous solvents were obtained from Aldrich and

used directly. All reactions involving air- or moisture-sensitive reagents

were performed under a nitrogen or argon atmosphere. Purity and

reaction analyses were measured using Agilent 1100 series high

performance liquid chromatography (HPLC) systems with UV

detection at 254 and 215 nm (System A: Agilent Zorbax SB-C18

3.0 mm × 50 mm, 3.5 μm, 5−95% CH₃CN in H₂O with 0.1% TFA for

3.6 min at 1.5 mL/min or Halo Phenyl−Hexyl, 3 mm × 50 mm, 2.7

μm, 5−95% CH₃CN in H₂O with 0.1% TFA for 1.01 min at 2.0 mL/

min. System B: Waters Xbridge C18, 3 mm × 50 mm, 3.5 μm, 5−95%

CH₃CN in H₂O with 0.1% formic acid for 3.6 min at 1.5 mL/min.).

Purities for ﬁnal compounds were >95%. Exact mass conﬁrmation was

performed on an Agilent 1200 series high performance liquid

chromatography (HPLC) system (Santa Clara, CA, U.S.) by ﬂow

injection analysis, eluting with a binary solvent system A and B (A,

water with 0.1% FA; B, ACN with 0.1% FA) under isocratic conditions

(50% A/50% B) at 0.2 mL/min with MS detection by an Agilent

6510-Q time-of-ﬂight (TOF) mass spectrometer (Santa Clara, CA,

U.S.). Silica gel chromatography was generally performed with

prepacked silica gel cartidges (Biotage, Teledyne-Isco or Interchim).

Chiral method development performed on an analytical Thar SFC/

MS. Preparative chiral separations performed on Thar SFC Prep 80 or

SFC Prep 350 instruments. Library puriﬁcation methods: Preparative

LC/MS, Waters autopuriﬁcation system; liquid transfer system, Tecan;

drying system, Genevac; prep. column: Xbridge (19 mm × 100 mm,

C18, 10 μm); ﬂow rate, 40 mL/min; general gradient, 5−95% B, 0.1%

additive in both A and B; 10 min gradient. Mobile phase A, water;

mobile phase B, acetonitrile; additive, TFA or NH₄OH. ¹H NMR

spectra were recorded on a Bruker AV-400 (400 MHz) spectrometer

or a Varian 400 MHz spectrometer at ambient temperature, or the

NMR spectra were collected with a Bruker Avance III spectrometer

operating at a proton frequency of 500.13 MHz using a 10 mL Protasis

CapNMR ﬂow probe. NMR samples were delivered to the ﬂow probe

using a Protasis One-Minute NMR automation system comprised of a

Discovery Tower Sample Manager and a Waters Liquid Handler is

made by CTC, Switzerland (model 2777). All observed protons are

reported as parts per million (ppm) downﬁeld from tetramethylsilane

(TMS) or other internal reference in the appropriate solvent indicated.

Data are reported as follows: chemical shift, multiplicity (s = singlet, d

= doublet, t = triplet, q = quartet, br = broad, m = multiplet), coupling

constants, and number of protons. Low-resolution mass spectral (MS)

data were determined on an Agilent 1100 series LCMS with UV

detection at 254 and 215 nm and a low resonance electrospray mode

(ESI).

The membrane potential dye assay was performed on the FLIPR

Tetra. The net membrane potential of the cells changes upon

activation of GlyRα3β, resulting in the increased ﬂux of Cl⁻ions out of

the cell down a concentration gradient and a robust increase in

ﬂuorescence signal. All measurements were made using an exposure

time of 0.4 s, 510−545 nm/565−625 nm excitation/emission ﬁlter set,

excitation intensity of 40%, and camera gain of 50.

To examine the ability of compounds to directly activate GlyRα3β

as agonists, dose−response plates of compound were prepared at 4×

in assay buﬀer (10 mM Hepes, 60 mM NaCl, 5 mM KCl, 2 mM

MgCl₂, 1 mM CaCl₂, 10 mM D-glucose, 160 mM D-mannitol, pH 7.4)

using a 1:2 stepwise dilution series of compounds in standard 384-well

polypropylene plates. In a ﬁrst addition step, 10 μL of 4× compound

was transferred to a cell plate containing MP blue dye and ﬂuorescence

response (agonism) was measured for 2 min. Potentiation of GlyRα3β

was measured in a subsequent addition step by transferring 10 μL of

5× EC₁₀glycine to the cell plate and monitoring ﬂuorescence changes

for 4 min. The maximum concentration of compound in this assay is

20 μM.

Schild assays were used to visualize the modulation of glycine

potency by each compound. Glycine dose−response plates were

prepared at 4× in assay buﬀer using a 1:2 stepwise dilution series in

standard 384-well polypropylene plates. In this assay format, glycine

and compound were premixed, simultaneously delivered to cells, and

responses measured for 4 min. Glycine potency was then determined

in the presence of varying concentrations of compound.

All FLIPR kinetic traces were processed using an area under the

curve relative to baseline (AUC-BL) algorithm, where the baseline was

the ﬁrst 10 s of the kinetic measurement prior to a ﬂuorescence

change. Compound potencies were determined by normalizing

responses to percent of control (POC) using the maximum achievable

glycine response and baseline EC₁₀glycine response as references.

POC normalized data were then plotted against log [compound], and

data were ﬁt to a nonlinear regression four-parameter Hill ﬁt to

determine the EC₅₀. All curve ﬁtting was performed with the

GraphPad Prism 6 software. Schild assay dose response curves for

glycine were analyzed in a similar manner.

Chirality Naming Convention. Racemic mixtures are denoted

with a “rac” chemical name preﬁx and refer to racemic mixtures of “cis”

5,6-ring fusions.

For human and mouse GlyRα3 dose response assay protocols,

surface plasmon resonance, oﬀ-target proﬁling, and X-ray crystallog-

raphy protocols, see Huang et al.²⁷

General Procedure for Sulfonylation. To a solution of the

amine in DMF or MeCN was added the sulfonyl chloride at 0 °C

followed by dropwise addition of triethylamine at 0 °C. The reaction

mixture was stirred at room temperature for 12 h. The resulting DMF

suspension was ﬁltered through a 0.45 μm frit. (When MeCN was the

reaction solvent, the mixture was concentrated and the residue was

suspended in DMF before ﬁltration.) The ﬁltrate was puriﬁed with RP-

HPLC ramping ACN in H₂O, NH₄OH 0.1% to aﬀord the

Mouse Spared Nerve Injury Model. Adult male C57BL/6 mice

weighing 20−30 g were cared for in accordance to the Guide for the

Care and Use of Laboratory Animals, eighth edition (National

Research Council Committee for the Update of the Guide for the Care

and Use of Laboratory Animals, 2011). Animals were housed at an

Association for Assessment and Accreditation of Laboratory Animal

Committee accredited facility in nonsterile ventilated microisolator

housing. All research protocols were approved by the Institutional

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corresponding sulfonamide. Normal phase MPLC puriﬁcation was

alternately employed in some cases using EtOAc:EtOH (76:24) or

EtOAc in heptane.

[3,4-c]quinoline-2(9bH)-carboxylate (2.0730 g, 6.86 mmol) and N-

bromosuccinimide (0.584 mL, 6.86 mmol) in dichloroethane (34.3

mL). The resulting yellow solution was stirred at room temperature

for 36 h. The mixture became a yellow suspension. The solvent was

removed under reduced pressure. The resulting residue was

partitioned between EtOAc and 2 N NaOH. The aqueous layer was

extracted with EtOAc twice. The combined organics were washed with

brine, dried over MgSO₄, and ﬁltered, and the ﬁltrate was concentrated

under reduced pressure to provide a yellow solid. The crude material

was adsorbed onto a plug of silica gel and puriﬁed by chromatography

through a Redi-Sep prepacked silica gel column 40g, eluting with a

gradient of 10−40% EtOAc in heptane, to provide 43 (2.406 g, 6.31

mmol, 92% yield) as an oﬀ-white solid. ¹H NMR (400 MHz, DMSO-

d₆) δ 7.57 (d, J = 2.28 Hz, 1H), 7.49 (dd, J = 2.15, 8.68 Hz, 1H), 7.10

(d, J = 8.71 Hz, 1H), 3.90−4.08 (m, 1H), 3.58−3.77 (m, 2H), 3.49

(dt, J = 6.63, 10.65 Hz, 1H), 3.29 (s, 3H), 2.81−2.94 (m, 1H), 1.32−

1.46 (m, 10H). MS (ESI) m/z 325.2 (M − tBu + H)^+l.

Step 2: rac-tert-Butyl 5-Methyl-4-oxo-8-(4,4,5,5-tetramethyl-

1,3,2-dioxaborolan-2-yl)-3,3a,4,5-tetrahydro-1H-pyrrolo[3,4-c]-

quinoline-2(9bH)-carboxylate (46). A reaction vessel was charged

with 43 (0.518 g, 1.359 mmol), bis(pinacolato) diboron (0.690 g, 2.72

mmol), 1,1′-bis(diphenylphosphino)ferrocene palladium(II)dichloride

dichloromethane adduct (0.044 g, 0.054 mmol), and potassium acetate

(0.400 g, 4.08 mmol), then sealed. The vial was evacuated and

backﬁlled with nitrogen twice prior to the addition of 1,4-dioxane

(6.79 mL). The reaction mixture was stirred at 90 °C for 3.5 h. The

resulting mixture was diluted with EtOAc and ﬁltered. The ﬁltrate was

concentrated to give a dark oil. The crude material was puriﬁed with

silica gel chromatography eluting with a gradient of 15−30% EtOAc in

heptane, to provide 46 (0.712 g, 1.662 mmol, quantitative) as a white

solid. MS (ESI) m/z 372.2 (M − tBu + H)⁺.

Step 3: rac-(3aS,9bS)-8-Hydroxy-5-methyl-3,3a,5,9b-tetrahydro-

1H-pyrrolo[3,4-c]quinolin-4(2H)-one (47). A solution of 46 (0.205 g,

0.479 mmol) in THF (8 mL), water (2.3 mL), and 1 N NaOH (2.39

mL, 2.393 mmol) was treated with hydrogen peroxide solution (30%,

w/w) (0.543 g, 4.79 mmol) at 0 °C. The resulting mixture was allowed

to stir and warm to room temperature over 2 h. The resulting mixture

was quenched by the addition of saturated NH₄Cl. Then 1 mL of 1 N

HCl was added to adjust the pH to 4. The mixture was extracted with

EtOAc 3 times. The combined organics were washed with brine, dried

over MgSO₄, ﬁltered, and dried under reduced pressure to aﬀord rac-

tert-butyl 8-hydroxy-5-methyl-4-oxo-3,3a,4,5-tetrahydro-1H-pyrrolo-

[3,4-c]quinoline-2(9bH)-carboxylate (0.209 g, 0.656 mmol, quantita-

tive) as a colorless oil. MS (ESI) m/z 263.1 (M − tBu + H)^+l. rac-tert-

Butyl 8-hydroxy-5-methyl-4-oxo-3,3a,4,5-tetrahydro-1H-pyrrolo[3,4-

c]quinoline-2(9bH)-carboxylate (105 mg) was dissolved in MeOH

(1 mL), and 4 N HCl in dioxane (1 mL, 4 mmol) was added. The

reaction mixture was stirred at room temperature for 1h. The solvent

was removed under reduced pressure to aﬀord the HCl salt of 47

(0.073 g, 0.287 mmol, 59.9% yield). MS (ESI) m/z (ESI) 219.3 (M +

H)⁺

Step 4: (3aS,9bS)-2-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-8-hydroxy-

5-methyl-3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c]quinolin-4(2H)-one

(5). To 47 (64 mg, 0.25 mmol) in DMF (2 mL) was added

triethylamine (0.11 mL, 0.750 mmol) and benzo[d][1,3]dioxole-5-

sulfonyl chloride (0.055 g, 0.250 mmol) following the general

sulfonylation procedure to aﬀord the racemic mixture of 5 (24 mg,

24% yield) which was subjected to chiral resolution. Chiral SFC

conditions: Chiralpak AD-H, 2 cm × 15 cm, 45% methanol, 80 mL/

min (column back pressure = 78 bar), 100 bar BPR, 255 nm;

dissolution, 4 mL 1:1 MeOH:DCM. Peak assignment was determined

by SFC on Column, AD-H; solvent, 45% methanol. Absolute

conﬁguration assigned by analogy based on in vitro activity and the

known stereochemistry of active isomer 32 as determined by the X-ray

cocrystal structure. Peak 2 = 5 (19 mg): ¹H NMR (500 MHz, DMSO-

d₆) δ 7.30−7.36 (m, 1H), 7.25−7.29 (m, 1H), 7.07−7.12 (m, 1H),

6.87−6.91 (m, 1H), 6.64−6.70 (m, 2H), 6.15−6.21 (m, 2H), 3.88−

3.93 (m, 1H), 3.61−3.66 (m, 1H), 3.41−3.46 (m, 1H), 3.32−3.39 (m,

1H), 3.06−3.13 (m, 4H), 2.73−2.81 (m, 1H). MS (ESI) m/z (ESI)

Synthesis of Intermediate (3aR,9bR)-5-Methyl-3,3a,5,9b-

tetrahydro-1H-pyrrolo[3,4-c][1,6]naphthyridin-4(2H)-one

(41c).²⁷Chiral separation conditions of 41c. Note: This chiral

separation was conducted on the free-base. The sample was processed

using the following SFC conditions: column, Chiralpak AD, 5 μm, 2

cm i.d. × 15 cm length; ﬂow rate, 80 mL/min; mobile phase, 60%

methanol w/0.2% diethylamine, 40% CO₂; back pressure setting, 100

bar; sample dissolution, 60 g dissolved in 800 mL of methanol;

injection volume, 1.75 mL. Analysis: column, Chiralpak AD, 5 μm, 4.6

mm × 100 mm; ﬂow rate, 5 mL/min; mobile phase, 50% methanol w/

0.2% diethylamine, 50% CO₂; back pressure setting, 100 bar. Peak 1 =

(3aS,9bS)-41c.

(3aS,9bS)-2-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-5-methyl-

3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c]quinolin-4(2H)-one (3)

and (3aR,9bR)-2-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-5-methyl-

3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c]quinolin-4(2H)-one (4).

To a solution of 41a^37,38(0.204 g, 0.855 mmol) in DMF (8.55 mL)

was added triethylamine (0.119 mL, 0.855 mmol). The mixture was

stirred at rt for 5 min. Then 1,3-benzodioxole-5-sulfonyl chloride

(0.207 g, 0.940 mmol) was added at 0 °C followed by dropwise

addition of triethylamine (0.238 mL, 1.709 mmol) at 0 °C. The

reaction mixture was stirred at room temperature for 4 h. The resulting

mixture was diluted with water and extracted with DCM twice. The

combined organic layers were washed with brine, dried over MgSO₄,

ﬁltered, and dried under reduced pressure. The crude material was

adsorbed onto a plug of silica gel and puriﬁed by chromatography,

eluting with a gradient of 0−50% EtOAc in heptane, to provide the

racemic mixture of 3 and 4 (0.283 g, 0.732 mmol, 86% yield) as white

solid. This solid was subjected to chiral resolution. Chiral SFC

conditions: Chiralcel OJ, 2 cm × 15 cm, 30% methanol w/0.2% DEA,

80 mL/min, pressure drop = 54 bar, 254 nm detection, Sample

dissolved in 20 mL of DCM:MeOH (1:1). Sample processed with 1

mL injection volume and cycle time of 2.5 min. Peak assignment

determined by SFC on Chiralcel OJ with 30% methanol w/0.2%

diethylamine modiﬁer. Absolute conﬁguration assigned by analogy

based on in vitro activity and the known stereochemistry of active

isomer 32 as determined by the X-ray cocrystal structure. Peak 1 = 3

1

(98 mg) as a white solid. H NMR (400 MHz, DMSO-d₆) δ 7.25−

7.37 (m, 4H), 7.07−7.12 (m, 2H), 7.07−7.14 (m, 2H), 7.01−7.06 (m,

1H), 6.15−6.25 (m, 2H), 3.89−3.96 (m, 1H), 3.63−3.71 (m, 1H),

3.44−3.52 (m, 2H), 3.18 (s, 4H), 2.78−2.89 (m, 1H). MS (ESI) m/z

387.0 (M + H)⁺HRMS (ESI) m/z calcd for C₁₉H₁₈N₂O₆S (M + H)⁺

1

387.1015; found 387.1021. Peak 2 = 4 (148 mg) as a white solid. H

NMR (400 MHz, DMSO-d₆) δ 7.24−7.38 (m, 4H), 7.08−7.12 (m,

2H), 7.01−7.07 (m, 1H), 6.16−6.23 (m, 2H), 3.86−3.95 (m, 1H),

3.62−3.70 (m, 1H), 3.45−3.53 (m, 2H), 3.18 (s, 4H), 2.80−2.88 (m,

1H). MS m/z (ESI) 387.0 (M + H)⁺. HRMS (ESI) m/z calcd for

C₁₉H₁₈N₂O₆S (M + H)⁺387.1015; found 387.1012.

(3aS,9bS)-2-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-8-hydroxy-5-

methyl-3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c]quinolin-4(2H)-

one (5). Step 1: rac-(3aR,9bR)-tert-Butyl 8-Bromo-5-methyl-4-oxo-

3,3a,4,5-tetrahydro-1H-pyrrolo[3,4-c]quinoline-2(9bH)-carboxylate

(43). To a solution of 42 (1.005 g, 3.49 mmol) in DMF (17.43 mL) at

0 °C was added sodium hydride, 60% suspension in mineral oil (0.137

g, 3.42 mmol). The mixture was stirred at rt for 30 min. Iodomethane

(0.217 mL, 3.49 mmol) was added. The reaction mixture was stirred at

room temperature for 1 h. The reaction mixture was diluted with satd

NH₄Cl at 0 °C, then stirred at rt for 30 min and extracted with EtOAc

3 times. The combined organics were washed with brine, dried over

MgSO₄, and ﬁltered, and the ﬁltrate was concentrated on vacuo to give

1.1 g of an oﬀ-white solid. The crude material was absorbed onto a

plug of silica gel and puriﬁed by chromatography through a Redi-Sep

prepacked silica gel column 40g, eluting with a gradient of 0−35%

EtOAc in heptane, to provide rac-(3aR,9bR)-tert-butyl 5-methyl-4-oxo-

3,3a,4,5-tetrahydro-1H-pyrrolo[3,4-c]quinoline-2(9bH)-carboxylate

(0.617 g, 2.041 mmol, 59% yield) as colorless solid; m/z (ESI) 325.2

(M + Na)⁺. To a 100 mL round-bottom ﬂask was added rac-

(3aR,9bR)-tert-butyl 5-methyl-4-oxo-3,3a,4,5-tetrahydro-1H-pyrrolo-

L

DOI: 10.1021/acs.jmedchem.6b01496

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Journal of Medicinal Chemistry

Article

403.0 (M + H)⁺. HRMS (ESI) m/z calcd for C₁₉H₁₈N₂O₆S (M + H)⁺

403.0964; found 403.0959.

mmol), di-tert-butyl dicarbonate (7.68 g, 35.2 mmol), and triethyl-

amine (6.53 mL, 46.9 mmol) in EtOH (235 mL) was stirred at rt

overnight. The mixture was concentrated under reduced pressure, and

the crude material was puriﬁed with silica gel chromatography eluting

with a gradient of 0−50% EtOAc in heptane to provide rac-tert-butyl

8-ﬂuoro-4-oxo-3,3a,4,5-tetrahydro-1H-pyrrolo[3,4-c]quinoline-2-

(9bH)-carboxylate (4.07 g, 13.29 mmol, 56.6% yield) as light-yellow

solid. To a solution of rac-tert-butyl 8-ﬂuoro-4-oxo-3,3a,4,5-tetrahydro-

1H-pyrrolo[3,4-c]quinoline-2(9bH)-carboxylate (1 g, 3.26 mmol) in

DMF (16.32 mL) was added sodium hydride, 60% dispersion in

mineral oil (0.131 g, 3.26 mmol) at 0 °C. The mixture was stirred at rt

for 1 h prior to the addition of iodomethane (0.203 mL, 3.26 mmol).

The reaction mixture was stirred at room temperature overnight. The

resulting mixture was diluted with water and extracted with EtOAc

three times. The combined organics were washed with brine, dried

over MgSO₄, ﬁltered, and concentrated under reduced pressure to

aﬀord rac-tert-butyl 8-ﬂuoro-5-methyl-4-oxo-3,3a,4,5-tetrahydro-1H-

pyrrolo[3,4-c]quinoline-2(9bH)-carboxylate (1.3 g, 4.06 mmol, 124%

yield) as a yellow oil which was dissolved in MeOH (10 mL).

Hydrochloric acid, 4.0 M solution in 1,4-dioxane (10 mL, 40.0 mmol)

was added. The reaction mixture was stirred at room temperature for 1

h. The resulting mixture was concentrated to aﬀord 51 (0.897 g, 3.49

mmol, quantitative) as a yellow solid which was used without further

puriﬁcation. MS (ESI) m/z 221.0 (M + H)⁺.

Step 5: rac-(3aR,9bR)-2-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-8-ﬂuo-

ro-5-methyl-3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c]quinolin-4(2H)-

one (7). Compound 51 (0.072 g, 0.280 mmol) was dissolved in DMF

(2 mL) and treated with 1,3-benzodioxole-5-sulfonyl chloride (0.062 g,

0.280 mmol) and triethylamine (0.117 mL, 0.841 mmol) following the

general sulfonylation procedure to aﬀord 7 (77 mg, 0.19 mmol, 68%

yield). ¹H NMR (500 MHz, DMSO-d₆) δ 7.33 (dd, J = 1.69, 8.17 Hz,

1H), 7.27 (d, J = 1.62 Hz, 1H), 7.17−7.23 (m, 1H), 7.04−7.17 (m,

3H), 6.18 (d, J = 8.76 Hz, 2H), 3.87 (dd, J = 1.98, 10.02 Hz, 1H),

3.60−3.75 (m, 1H), 3.41−3.60 (m, 2H), 3.13−3.22 (m, 4H), 2.89 (t, J

= 9.93 Hz, 1H). MS (ESI) m/z 405.0 (M + H)⁺. HRMS (ESI) m/z

calcd for C₁₉H₁₇FN₂O₅S (M + H)⁺405.0920; found 405.0921.

(6aS,9aR)-8-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-5-methyl-

7,8,9,9a-tetrahydro-5H-pyrrolo[3,4-c][1,5]naphthyridin-6-

(6aH)-one (8). Step 1: Ethyl 2-Oxo-1,2-dihydro-1,5-naphthyridine-

3-carboxylate (34b). A mixture of 3-amino-pyridine-2-carbaldehyde

(2 g, 16.38 mmol), diethyl malonate (4.97 mL, 32.8 mmol) and

piperidine (0.404 mL, 4.09 mmol) in EtOH (27.3 mL) was reﬂuxed

for 18 h. The mixture was slowly cooled to rt with stirring and then

ﬁltered. The solid collected was washed with a small amount of EtOH

and then with heptane to aﬀord 34b (2.246 g, 10.29 mmol, 62.9%

yield) as a light-yellow solid. MS (ESI) m/z 219.2 (M + H)⁺.

Step 2: Ethyl 1-Methyl-2-oxo-1,2-dihydro-1,5-naphthyridine-3-

carboxylate (38b). To a solution of 34b (4.7 g, 21.54 mmol) in DMF

(150 mL) was added sodium hydride, 60% dispersion in mineral oil

(1.034 g, 25.8 mmol) portionwise at 0 °C. The reaction mixture was

stirred at room temperature for 30 min. Iodomethane (1.405 mL,

22.62 mmol) was added dropwise via syringe. The reaction mixture

was stirred at room temperature overnight. Additional iodomethane

(0.1 mL) was added. After stirring at rt for 2 h, 5 mL of water was

added and the resulting mixture stirred for 10 min. The solvents were

removed under reduced pressure, and the resulting residue was

suspended in water and extracted with DCM three times. The

combined organics were washed with brine, dried over MgSO₄,

ﬁltered, and dried under reduced pressure to aﬀord 12 g of a green

solid which was recrystallized from EtOAc to aﬀord 38b (3.55 g, 15.29

mmol, 71.0% yield). MS (ESI) m/z 233.1 (M + H)⁺.

rac-2-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-5-methyl-4-oxo-

2,3,3a,4,5,9b-hexahydro-1H-pyrrolo[3,4-c]quinoline-8-carbon-

itrile (6). Step 1: rac-5-Methyl-4-oxo-2,3,3a,4,5,9b-hexahydro-1H-

pyrrolo[3,4-c]quinoline-8-carbonitrile (45). To a 5 mL microwave

vial was added 43 (0.165 g, 0.433 mmol), zinc cyanide (0.027 mL,

0.433 mmol), tetrakis(triphenylphosphine)palladium (0.050 g, 0.043

mmol), and DMF (2.164 mL). The solvent was purged with nitrogen

for 5 min and sealed. The vessel was irradiated at 100 °C in a

microwave oven for 1 h. The mixture was diluted with EtOAc and

ﬁltered. The ﬁltrate was concentrated, and the crude material was

puriﬁed with silica gel chromatography, eluting with a gradient of 0−

40% EtOAc in heptane to provide rac-tert-butyl 8-cyano-5-methyl-4-

oxo-3,3a,4,5-tetrahydro-1H-pyrrolo[3,4-c]quinoline-2(9bH)-carboxy-

late as a yellow solid which was suspended in MeOH (4.5 mL) to

which 4 N HCl in dioxane (2 mL, 8 mmol) was added. The

suspension was stirred at room temperature overnight. The resulting

mixture was concentrated to aﬀord 45 as a hydrochloride salt (0.139 g,

0.527 mmol, quantitative) as a yellow solid which was used without

further puriﬁcation. MS (ESI) m/z 228.3 (M + H)⁺

Step 2: rac-2-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-5-methyl-4-oxo-

2,3,3a,4,5,9b-hexahydro-1H-pyrrolo[3,4-c]quinoline-8-carbonitrile

(6). 45 (0.0685 g, 0.260 mmol) in DMF (2 mL) was treated with 1,3-

benzodioxole-5-sulfonyl chloride (0.057 g, 0.260 mmol) and triethyl-

amine (0.072 mL, 0.519 mmol) following the general sulfonylation

procedure to aﬀord 6 (17 mg, 16% yield). ¹H NMR (500 MHz,

DMSO-d₆) δ 7.73−7.80 (m, 2H), 7.73−7.80 (m, 2H), 7.30−7.36 (m,

1H), 7.22−7.29 (m, 2H), 7.04−7.12 (m, 1H), 6.15−6.21 (m, 2H),

3.84−3.90 (m, 1H), 3.67−3.74 (m, 1H), 3.53−3.60 (m, 1H), 3.45−

3.51 (m, 1H), 3.24−3.29 (m, 1H), 3.20 (s, 3H), 2.88−2.98 (m, 1H).

MS (ESI) m/z (ESI) 412.1 (M + H)⁺. HRMS (ESI) m/z calcd for

C₂₀H₁₇N₃O₅S (M + H)⁺412.0967; found 412.0968.

rac-2-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-8-ﬂuoro-5-methyl-

3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c]quinolin-4(2H)-one (7).

Step 1: Methyl 3-(5-Fluoro-2-nitrophenyl)acrylate (48a). A mixture

of methyl p,p-bis(2,2,2-triﬂuoroethyl)-phosphonoacetate (10.74 mL,

29.6 mmol), 5-ﬂuoro-2-nitrobenzaldehyde (5.0 g, 29.6 mmol), and

potassium carbonate (8.19 g, 59.2 mmol) in 1,4-dioxane (233 mL) was

reﬂuxed overnight. The mixture was ﬁltered, and the ﬁltrate was

concentrated. The residue was partitioned between water and EtOAc

and extracted with EtOAc 3 times. The combined organics were

washed with brine, dried over MgSO₄, ﬁltered, and dried under

reduced pressure to aﬀord 48a (6.1 g, 93% yield) as a black solid. MS

(ESI) m/z 226.1 (M + H)⁺

Step 2: Methyl 1-Benzyl-4-(5-ﬂuoro-2-nitrophenyl)pyrrolidine-3-

carboxylate (49a). To a solution of 48a (6.1 g, 27.1 mmol) in DCM

(201 mL) at 0 °C under nitrogen ﬂow was added triﬂuoroacetic acid

(0.805 mL, 10.84 mmol) and N-(methoxymethyl)-N-

(trimethylsilylmethyl)benzylamine (20.79 mL, 81 mmol). The

resulting solution was stirred at rt overnight. The resulting mixture

was diluted with satd aq NaHCO₃and extracted with DCM twice. The

combined organics were washed with brine, dried over MgSO₄,

ﬁltered, and concentrated under reduced pressure. The crude material

was puriﬁed by silica gel chromatography eluting with a gradient of

10− 50% EtOAc/EtOH/NH₄OH (73:27:2) in heptane to provide 49a

(8.41g, 23.47 mmol, 87% yield) as a yellow oil. MS (ESI) m/z 359.1

(M + H)⁺.

Step 3: rac-8-Fluoro-3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c]-

quinolin-4(2H)-one (50a). A mixture of 49a (8.41 g, 23.47 mmol),

palladium hydroxide, 20 wt % Pd (dry basis) on carbon, and wet (0.8

g, 5.70 mmol) in EtOH (100 mL) was stirred at 60 °C under 55 psi of

H₂for 48 h. Additional palladium hydroxide, 20 wt % Pd (dry basis)

on carbon, and wet (0.8 g, 5.70 mmol) was added. The mixture was

stirred at 60 °C under 55 psi of H₂for 3 additional days. The resulting

mixture was ﬁltered through Celite, and the ﬁltrate was concentrated

to provide 50a (4.84 g, 23.47 mmol, quantitative) as a yellow solid,

which was used without further puriﬁcation. MS (ESI) m/z 207.2 (M

+ H)⁺.

Step 3: rac-Ethyl 8-Benzyl-5-methyl-6-oxo-6,6a,7,8,9,9a-hexahy-

dro-5H-pyrrolo[3,4-c][1,5]naphthyridine-6a-carboxylate (39b). To a

solution of 38b (3.55 g, 15.29 mmol) in DCM (113 mL) at 0 °C

under N₂was added triﬂuoroacetic acid (0.454 mL, 6.11 mmol) and

N-(methoxymethyl)-N-(trimethylsilylmethyl)benzylamine (19.55 mL,

76 mmol). The resulting solution was stirred at rt overnight. The

reaction mixture was diluted with satd aq NaHCO₃and extracted with

DCM twice. The combined organics were washed with brine, dried

over MgSO₄, ﬁltered, and concentrated under reduced pressure. The

Step 4: rac-8-Fluoro-5-methyl-3,3a,5,9b-tetrahydro-1H-pyrrolo-

[3,4-c]quinolin-4(2H)-one (51). A mixture of 50a (4.84 g, 23.47

M

DOI: 10.1021/acs.jmedchem.6b01496

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Journal of Medicinal Chemistry

Article

crude material was puriﬁed by silica gel chromatography eluting with a

gradient of 10−50% EtOAc/EtOH/NH₄OH (73:27:2) in heptane to

provide 39b (5.70 g, 15.60 mmol, 102% yield) as light-yellow oil. MS

(ESI) m/z 366.3 (M + H)⁺.

Step 4: rac-8-Benzyl-5-methyl-7,8,9,9a-tetrahydro-5H-pyrrolo-

[3,4-c][1,5]naphthyridin-6(6aH)-one (40b). A solution of 39b (5.70

g, 15.60 mmol) in hydrochloric acid 6.0 N (130 mL, 780 mmol) was

reﬂuxed for 12 h. The mixture was concentrated under reduced

pressure to aﬀord 40b (5.7 g, 15.56 mmol, quantitative) as an oﬀ-white

solid. MS (ESI) m/z 294.4 (M + H)⁺.

mL, 0.644 mmol) and N-(methoxymethyl)-N-(trimethylsilylmethyl)-

benzylamine (2.059 mL, 8.05 mmol), and the resulting solution stirred

at rt overnight. The mixture was diluted with satd NaHCO₃and

extracted with DCM twice. The combined organics were washed with

brine, dried over MgSO₄, ﬁltered, and concentrated under reduced

pressure. The crude material was puriﬁed by silica gel chromatography

eluting with a gradient of 0% to 40% EtOAc in heptane to provide 49b

(0.563 g, 1.649 mmol, quantitative) as yellow oil. MS (ESI) m/z 342.3

(M + H)⁺.

Step 3: rac-3,3a,5,9b-Tetrahydro-1H-pyrrolo[3,4-c][1,7]-

naphthyridin-4(2H)-one (50b). To a 100 mL pressure vessel was

added 49b (0.563 g, 1.649 mmol), EtOH (15 mL), and palladium 10

wt % on activated carbon (0.100 g, 0.940 mmol). The reaction mixture

was stirred at 60 °C under 55 psi of H₂overnight. Additional

palladium 10 wt % on activated carbon (0.100 g, 0.940 mmol) was

added. The reaction mixture was stirred at 60 °C under 55 psi of H₂

for an additional 48 h. The mixture was ﬁltered through Celite and

concentrated to aﬀord 307 mg of a colorless oil. LCMS indicated

complete hydrogenolysis but incomplete cyclization. The material was

dissolved in AcOH (5 mL) and was stirred at 80 °C for 2 days. The

resulting mixture was concentrated to provide a brown oil which was

puriﬁed by strong cation exchange (SCX) catch and release by washing

with MeOH, then eluting product with 2 M NH₃in MeOH to aﬀord

50b (0.310 g, 1.638 mmol, 99% yield) as a yellow solid. MS (ESI) m/z

190.3 (M + H)⁺.

Step 5: rac-5-Methyl-7,8,9,9a-tetrahydro-5H-pyrrolo[3,4-c][1,5]-

naphthyridin-6(6aH)-one (41b). A mixture of 40b (5.7 g, 15.56

mmol) and palladium hydroxide, 20 wt % pd (dry basis) on carbon,

wet (0.57 g, 0.812 mmol) in EtOH (50 mL), and water (10 mL) was

stirred at 60 °C under 42 psi of H₂for 12 h. The mixture was ﬁltered

through Celite and washed with water. The ﬁltrate was concentrated

to aﬀord 41b (4.32 g, 15.64 mmol, 101% yield) as an oﬀ-white solid

which was used without further puriﬁcation. MS (ESI) m/z 204.1 (M

+ H)⁺.

Step 6: (6aS,9aR)-8-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-5-methyl-

7,8,9,9a-tetrahydro-5H-pyrrolo[3,4-c][1,5]naphthyridin-6(6aH)-one

(8). 41b (0.3 g, 1.086 mmol) in MeCN (3.62 mL) was treated with

benzo[d][1,3]dioxole-5-sulfonyl chloride (0.240 g, 1.086 mmol) and

triethylamine (0.606 mL, 4.35 mmol) following the general

sulfonylation procedure to aﬀord a racemic mixture of 8 (0.2 g,

0.516 mmol, 47.5% yield). This was subjected to chiral resolution.

Chiral SFC conditions: Chiralpak OJ-H, 2 cm × 25 cm, 25% methanol

(0.1% DEA)/CO₂, 60 mL/min, 100 bar, 220 nm, inj vol 1.5 mL, 4

mg/mL 2:1 DCM/methanol. Peak assignment determined by SFC:

Chiralpak OJ-H, 25% methanol w/0.2% diethylamine. Absolute

conﬁguration assigned by analogy based on in vitro activity and the

known stereochemistry of active isomer 32 as determined by the X-ray

cocrystal structure. Peak 1 = 8 (115 mg): ¹H NMR (400 MHz,

DMSO-d₆) δ 8.12−8.16 (m, 1H), 7.46−7.50 (m, 1H), 7.30−7.35 (m,

2H), 7.25−7.26 (m, 1H), 7.06−7.09 (m, 1H), 6.18−6.21 (m, 2H),

3.58−3.64 (m, 3H), 3.53−3.57 (m, 1H), 3.46−3.52 (m, 1H), 3.34−

3.38 (m, 1H), 3.18−3.21 (m, 3H), 3.18−3.20 (m, 3H). MS (ESI) m/z

388.1 (M + H)⁺. HRMS calcd for C₁₈H₁₇N₃O₅S (M + H)⁺388.0967;

found 388.0970.

Step 4: rac-2-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-5-methyl-

3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,7]naphthyridin-4(2H)-one

(10). To a solution of 50b (0.103 g, 0.544 mmol) in DCM (4 mL) was

added 1,3-benzodioxole-5-sulfonyl chloride (0.120 g, 0.544 mmol) and

triethylamine (0.151 mL, 1.089 mmol) at 0 °C. The reaction mixture

was stirred at room temperature overnight. The resulting mixture was

diluted with water and extracted with EtOAc three times. The

combined organics were washed with brine, dried over MgSO₄,

ﬁltered, and concentrated under reduced pressure. The crude material

was puriﬁed with silica gel chromatography eluting with a gradient of

0−80% EtOAc/EtOH/NH₄OH (73:17:2) in heptane to provide 2-

(benzo[d][1,3]dioxol-5-ylsulfonyl)-3,3a,5,9b-tetrahydro-1H-pyrrolo-

[3,4-c][1,7]naphthyridin-4(2H)-one (0.027 g, 0.072 mmol, 13% yield)

as a tan solid. To this solid (0.027 g, 0.072 mmol) in DMF (1.45 mL)

at 0 °C was added sodium hydride, 60% in mineral oil (2.89 mg, 0.072

mmol). The reaction mixture was stirred at room temperature for 30

min prior to the addition of iodomethane (4.49 μL, 0.072 mmol). The

reaction mixture was stirred at room temperature for 2 h. The reaction

was quenched by the addition of a couple of drops of MeOH. The

mixture was ﬁltered through a 0.45 μm frit and puriﬁed with RP-

HPLC ramping ACN in H₂O, with NH₄OH 0.1% to aﬀord compound

(3aS,9bS)-2-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-5-methyl-

3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,6]naphthyridin-

4(2H)-one (9). To (3aS,9bS)-41c (0.073 M solution in MeCN (53

mL, 3.87 mmol) was added 1,3-benzodioxole-5-sulfonyl chloride

(0.854 g, 3.87 mmol) followed by triethylamine (1.076 mL, 7.74

mmol). The reaction mixture was stirred at room temperature over 3

days. The mixture was concentrated under reduced pressure. The

resulting residue was suspended in water and stirred for 30 min then

ﬁltered. The solid was washed with water and heptane successively.

The solid was dried on vacuo to aﬀord 9 (1.320 g, 3.41 mmol, 88%

1

10 (4 mg, 14% yield). H NMR (500 MHz, DMSO-d₆) δ 8.34−8.38

(m, 1H), 8.20−8.25 (m, 1H), 7.26−7.35 (m, 3H), 7.25−7.35 (m, 3H),

7.06−7.11 (m, 1H), 6.16−6.21 (m, 2H), 3.80−3.85 (m, 1H), 3.66−

3.72 (m, 1H), 3.53−3.59 (m, 1H), 3.45−3.51 (m, 1H), 3.45−3.51 (m,

1H), 3.23 (s, 4H), 2.95−3.00 (m, 1H), 2.92−3.03 (m, 1H). MS (ESI)

m/z 388.0 (M + H)⁺. HRMS calcd for C₁₈H₁₇N₃O₅S (M + H)⁺

388.0967; found 388.0970.

1

yield) as an oﬀ-white solid. H NMR (400 MHz, DMSO-d₆) δ 8.32−

8.44 (m, 2H), 7.28−7.41 (m, 2H), 7.04−7.17 (m, 2H), 6.16−6.24 (m,

2H), 3.87−3.93 (m, 1H), 3.68−3.75 (m, 1H), 3.52−3.62 (m, 1H),

3.46−3.51 (m, 1H), 3.24−3.31 (m, 1H), 3.17 (s, 3H), 2.83−2.94 (m,

1H). MS (ESI) m/z 388.1 (M + H)⁺. HRMS calcd for C₁₈H₁₇N₃O₅S

(M + H)⁺388.0967; found 388.0963.

rac-2-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-5-methyl-3,3a,5,9b-

tetrahydro-1H-pyrrolo[3,4-c][1,7]naphthyridin-4(2H)-one (11).

Step 1: Ethyl 1-Methyl-2-oxo-1,2-dihydro-1,8-naphthyridine-3-car-

boxylate (38d). A mixture of ethyl 2-oxo-1,2-dihydro-1,8-naphthyr-

idine-3-carboxylate (5.0566 g, 23.17 mmol) and cesium carbonate

(11.33 g, 34.8 mmol) in DMF (232 mL) was stirred at rt for 2 h with

overhead stirring. Iodomethane (1.583 mL, 25.5 mmol) was added

dropwise, and the resulting reaction mixture was stirred at room

temperature for 16 h. The solvent was removed under reduced

pressure and water was added and the mixture stirred for 30 min,

providing a suspension which was ﬁltered. The solid was washed with

water and heptane and dried to aﬀord 4.410 g of solid. The ﬁltrate was

extracted with DCM three times. The combined organics was washed

with brine, dried over MgSO₄, ﬁltered, and concentrated to aﬀord

1.025 g of yellow solid. The solids were combined to aﬀord 38d (5.435

g, 23.40 mmol, quantitative). MS (ESI) m/z 233.0 (M + H)⁺.

rac-2-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-5-methyl-3,3a,5,9b-

tetrahydro-1H-pyrrolo[3,4-c][1,7]naphthyridin-4(2H)-one (10).

Step 1: (E)-Methyl 3-(3-nitropyridin-4-yl)acrylate (48b). A mixture of

3-nitroisonicotinaldehyde (1 g, 6.57 mmol), methyl p,p-bis(2,2,2-

triﬂuoroethyl)-phosphonoacetate (2.385 mL, 6.57 mmol) and

potassium carbonate (1.817 g, 13.15 mmol) in 1,4-dioxane (51.8

mL) was reﬂuxed overnight. The resulting mixture was concentrated

under reduced pressure. The residue was dissolved in EtOAc and

washed with satd aq NaHCO₃and brine, dried with MgSO₄, ﬁltered,

and dried under reduced pressure. The crude material was puriﬁed

with silica gel chromatography, eluting with a gradient of 20−50%

EtOAc in heptane to provide 48b (0.335 g, 1.609 mmol, 24.5% yield)

as yellow solid. MS (ESI) m/z 209.1 (M + H)⁺.

Step 2: Methyl 1-Benzyl-4-(3-nitropyridin-4-yl)pyrrolidine-3-car-

boxylate (49b). To a solution of 48b (0.335 g, 1.609 mmol) in DCM

(11.92 mL) at 0 °C under N₂was added triﬂuoroacetic acid (0.048

N

DOI: 10.1021/acs.jmedchem.6b01496

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Article

Step 2: rac-Ethyl 2-Benzyl-5-methyl-4-oxo-2,3,3a,4,5,9b-hexahy-

dro-1H-pyrrolo[3,4-c][1,8]naphthyridine-3a-carboxylate (39d). To a

solution of 38d (5.435 g, 23.40 mmol) in DCM (173 mL) at 0 °C

under N₂was added triﬂuoroacetic acid (0.695 mL, 9.36 mmol) and

N-(methoxymethyl)-N-(trimethylsilylmethyl)benzylamine (29.9 mL,

117 mmol). The resulting solution was stirred at rt overnight. The

resulting mixture was diluted with satd aq NaHCO₃and extracted with

DCM twice. The combined organics were washed with brine, dried

over MgSO₄, ﬁltered, and concentrated under reduced pressure. The

crude material was puriﬁed with silica gel chromatography eluting with

a gradient of 10−50% EtOAc/EtOH/NH₄OH (73:27:2) in heptane to

provide 39d (9.08 g, 24.7 mmol, quantitative) as light-yellow oil. MS

(ESI) m/z 366.2 (M + H)⁺.

DCM/MeOH (3:2), 0.25 mL injected, and 40% methanol, 186 bar 1

mL injected. Peak assignment was determined by SFC on column OJ-

H solvent, 20% methanol with ammonia. Absolute conﬁguration

assigned by analogy based on in vitro activity and the known

stereochemistry of active isomer 32 as determined by the X-ray

1

cocrystal structure. Peak 1 = 13 (53 mg) as a white solid: H NMR

(400 MHz, DMSO-d₆) δ 8.41−8.47 (m, 1H), 8.35−8.39 (m, 1H),

8.30−8.35 (m, 1H), 8.06−8.11 (m, 1H), 7.80−7.84 (m, 1H), 7.71−

7.77 (m, 1H), 7.02−7.05 (m, 1H), 7.01−7.06 (m, 1H), 3.93 (s, 4H),

3.72−3.80 (m, 1H), 3.44−3.53 (m, 2H), 3.32 (s, 3H), 3.23−3.28 (m,

1H), 2.86−2.93 (m, 1H), 2.86−2.95 (m, 1H). MS (ESI) m/z 398.1

(M + H)⁺. HRMS calcd for C₁₉H₁₉N₅O₃S (M + H)⁺398.1287; found

398.1290.

Step 3: rac-2-Benzyl-5-methyl-3,3a,5,9b-tetrahydro-1H-pyrrolo-

[3,4-c][1,8]naphthyridin-4(2H)-one (40d). A solution of 39d (9.08 g,

24.85 mmol) in 6 N hydrochloric acid (207 mL, 1242 mmol) was

reﬂuxed for 16 h. The resulting mixture was concentrated to aﬀord

40d (9.02 g, 24.63 mmol, 99% yield) as a tan solid which was used

without further puriﬁcation. MS (ESI) m/z 294.3 (M + H)⁺.

Step 4: rac-5-Methyl-3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,8]-

naphthyridin-4(2H)-one (41d). A mixture of 40d (9.02 g, 24.63

mmol) and palladium hydroxide, 20 wt % pd (dry basis) on carbon,

wet (0.9 g, 1.282 mmol) in EtOH (50 mL) and water (10 mL) were

stirred at 60 °C under 42 psi of H₂for 12 h. The mixture was

concentrated under reduced pressure to aﬀord 41d (6.65 g, 24.08

mmol, 98% yield) as an oﬀ white solid which was used without further

puriﬁcation. MS (ESI) m/z 204.1 (M + H)⁺.

(3aS,9bS)-2-(Benzo[d]oxazol-5-ylsulfonyl)-5-methyl-

3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,6]naphthyridin-

4(2H)-one (14). Step 1: rac-2-((4-Hydroxy-3-nitrophenyl)sulfonyl)-

5-methyl-3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,7]naphthyridin-

4(2H)-one. To a suspension of rac-41c (0.380 g, 1.376 mmol) in

MeCN (9.17 mL) was added triethylamine (0.383 mL, 2.75 mmol).

The reaction mixture was stirred at room temperature for 5 min prior

to the addition of 4-hydroxy-3-nitrobenzene-1-sulfonyl chloride (0.327

g, 1.376 mmol). The resulting mixture was stirred at rt for 2 h. The

mixture was concentrated to aﬀord crude 2-((4-hydroxy-3-

nitrophenyl)sulfonyl)-5-methyl-3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-

c][1,7]naphthyridin-4(2H)-one (1.24 g) as a yellow solid which was

used without further puriﬁcation. MS (ESI) m/z 405.1 (M + H)⁺.

Step 2: rac-2-((3-Amino-4-hydroxyphenyl)sulfonyl)-5-methyl-

3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,7]naphthyridin-4(2H)-one.

A mixture of the 2-((4-hydroxy-3-nitrophenyl)sulfonyl)-5-methyl-

3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,7]naphthyridin-4(2H)-one

(1.24 g) and palladium hydroxide, 20 wt % pd (dry basis) on carbon,

wet (0.124 g, 0.177 mmol) in acetic acid (1 mL, 17.32 mmol), EtOH

(10 mL), and water (10 mL) was stirred at 40 °C under 42 psi of

hydrogen gas overnight. The mixture was ﬁltered through Celite and

washed with EtOH and water. The ﬁltrate was concentrated, and the

residue was dissolved in MeOH and concentrated to facilitate removal

of acetic acid. This was repeated 3 times until the residue became a

dark solid. The material was further dried under high vacuo to provide

1.24 g of crude 2-((3-amino-4-hydroxyphenyl)sulfonyl)-5-methyl-

3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,7]naphthyridin-4(2H)-one

which was used without further puriﬁcation. MS (ESI) m/z 375.2 (M

+ H)⁺.

Step 5: rac-2-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-5-methyl-

3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,7]naphthyridin-4(2H)-one

(11). 41d (0.066 g, 0.203 mmol) in DMF (2 mL) was treated with

benzo[d][1,3]dioxole-5-sulfonyl chloride (0.045 g, 0.203 mmol) and

triethylamine (0.121 mL, 0.869 mmol) following the general

1

sulfonylation procedure to aﬀord 11 (0.088 mmol, 47% yield). H

NMR (500 MHz, DMSO-d₆) δ 8.27 (dd, J = 1.47, 4.86 Hz, 1H), 7.70

(d, J = 6.11 Hz, 1H), 7.33 (dd, J = 1.55, 8.11 Hz, 1H), 7.28 (d, J = 1.53

Hz, 1H), 7.00−7.12 (m, 2H), 6.19 (d, J = 4.64 Hz, 2H), 3.86 (dd, J =

2.15, 10.18 Hz, 1H), 3.62−3.75 (m, 1H), 3.40−3.58 (m, 2H), 3.15−

3.29 (m, 4H), 2.91 (t, J = 9.87 Hz, 1H). MS (ESI) m/z 388.0 (M +

H)⁺. HRMS calcd for C₁₈H₁₇N₃O₅S (M + H)⁺388.0967; found

388.0961.

(3aS,9bS)-2-((1H-Benzo[d]imidazol-5-yl)sulfonyl)-5-methyl-

3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,6]naphthyridin-

4(2H)-one (12). rac-41c (0.156 g, 0.565 mmol) in DMF (3.77 mL)

was treated with triethylamine (0.393 mL, 2.82 mmol) and 1H-

benzo[d]imidazole-5-sulfonyl chloride hydrochloride (0.143 g, 0.565

mmol) following the general sulfonylation procedure to aﬀord the

racemic mixture of 12 (0.199 g, 0.52 mmol, 92% yield). Chiral SFC

conditions: Chiralcel OJ-H solvent, 35% methanol w/0.2% diethyl-

amine. Absolute conﬁguration assigned by analogy based on in vitro

activity and the known stereochemistry of active isomer 32 as

determined by the X-ray cocrystal structure. Peak 1 = 12 (49 mg),

Step 3: (3aS,9bS)-2-(Benzo[d]oxazol-5-ylsulfonyl)-5-methyl-

3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,6]naphthyridin-4(2H)-one

(14). A mixture of 2-((3-amino-4-hydroxyphenyl)sulfonyl)-5-methyl-

3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,7]naphthyridin-4(2H)-one

in ethyl orthoformate (10 mL, 60.1 mmol) was stirred at 120 °C for 5

h, and the resulting mixture was dried under reduced pressure. The

residue was partitioned between water and EtOAc and extracted with

EtOAc three times. The combined organics were washed with brine,

dried over MgSO₄, ﬁltered, and dried under reduced pressure. The

crude material was puriﬁed with silica gel chromatography eluting with

a gradient of 20−100% EtOAc/EtOH/NH₄OH (73:27:2) in heptane

to provide 0.2 g of the racemic product as white solid. Chiral SFC

conditions: column OJ-H (2 cm × 25 cm), 40% methanol (0.1%)/

CO₂, 100 bar, 60 mL/min, 220 nm, inj vol 2 mL, 10 mg/mL, 1:1

DCM:methanol. Peak assignment was determined by SFC on column

OJ-H 40% methanol. Peak 1 contained the desired enantiomeric

product (86 mg). Absolute conﬁguration assigned by analogy based on

in vitro activity and the known stereochemistry of active isomer 32 as

determined by the X-ray cocrystal structure. This material was further

puriﬁed with RP-HPLC ramping ACN in H₂O, NH₄OH 0.1% to

1

>96% ee: H NMR (400 MHz, DMSO-d₆) δ 8.47−8.50 (m, 1H),

8.36−8.39 (m, 1H), 8.32−8.35 (m, 1H), 8.00−8.08 (m, 1H), 7.76−

7.83 (m, 1H), 7.58−7.70 (m, 1H), 7.00−7.07 (m, 1H), 3.92−3.98 (m,

1H), 3.72−3.78 (m, 1H), 3.47−3.55 (m, 2H), 3.22−3.28 (m, 1H),

3.03 (s, 3H), 2.84−2.92 (m, 1H). MS (ESI) m/z 384.2 (M + H)⁺.

HRMS calcd for C₁₈H₁₇N₅O₃S (M + H)⁺384.1130; found 384.1127.

(3aS,9bS)-5-Methyl-2-((1-methyl-1H-benzo[d]imidazol-5-yl)-

sulfonyl)-3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,6]-

naphthyridin-4(2H)-one (13). To a suspension of rac-41c (0.159 g,

0.576 mmol) in MeCN (3.84 mL) was added triethylamine (0.320

mL, 2.303 mmol). The reaction mixture was stirred at room

temperature for 5 min prior to the addition of 1-methyl-1H-

benzo[d]imidazole-5-sulfonyl chloride (0.133 g, 0.576 mmol). The

resulting mixture was stirred at rt for 2 h then concentrated under

reduced pressure. The resulting residue was suspended in water and

ﬁltered. The solid was washed with water and heptane successively and

dried to give the racemic mixture of 13 (0.108 g, 0.27 mmol, 47%

yield) as a white solid which was subjected to chiral puriﬁcation

conditions. Chiral SFC conditions: Chiralcel OJ-H solvent, 35%

methanol, 70 mL/min, 220 nm, 179 bar, 108 mg dissolved in 6 mL of

1

aﬀord 14 (36 mg). H NMR (500 MHz, DMSO-d₆) δ 8.95−9.02 (m,

1H), 8.35−8.39 (m, 1H), 8.30−8.34 (m, 1H), 3.92−3.99 (m, 1H),

3.75−3.82 (m, 1H), 3.50−3.60 (m, 2H), 3.34−3.44 (m, 1H).). MS

(ESI) m/z 385.0 (M + H)⁺. HRMS calcd for C₁₈H₁₆N₄O₄S (M + H)⁺

385.0971; found 385.0970.

(3aS,9bS)-2-(Benzo[d]thiazol-6-ylsulfonyl)-5-methyl-

3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,6]naphthyridin-

4(2H)-one (15). (3aS,9bS)-41c (0.0736 M solution in MeCN, 6 mL,

0.442 mmol) was treated with 1,3-benzothiazole-6-sulfonyl chloride

O

DOI: 10.1021/acs.jmedchem.6b01496

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Journal of Medicinal Chemistry

Article

(103 mg, 0.442 mmol) and triethylamine (0.123 mL, 0.883 mmol)

sulfonyl chloride (3.36 g, 15.50 mmol), respectively. The mixture was

stirred at room temperature overnight, aﬀording a white suspension

(solution pinkish). To the mixture was added silica gel, and the

mixture dried under reduced pressure. The adsorbed material was

puriﬁed on MPLC ramping EtOAc:EtOH (76:24) in heptane (50−

100%, 10% DCM throughout), aﬀording racemic product (3.14 g,

55%). Chiral separation SFC conditions: Regis Whelk-O (s,s), 2 cm ×

25 cm, 55% methanol w/0.2% DEA 80 mL/min (column back

pressure = 117 bar), 100 bar BPR, 252 nm detection, dissolution 250

mL of 1:1 MeOH:DCM, sample processed with 4.0 mL injections and

4 min cycle time. Analytical data: Column dimensions, 4.6 mm × 100

mm; ﬂow rate, 5 mL/min; instrument, SFC/MS-1. Column oven, 40

°C. Absolute conﬁguration assigned by analogy based on in vitro

activity and the known stereochemistry of active isomer 32 as

determined by the X-ray cocrystal structure. Peak 1 = ent-20: ¹H NMR

(400 MHz, DMSO-d₆) δ 8.37 (d, J = 5.8 Hz, 1H), 8.34 (s, 1H), 8.19

(dd, J = 2.0, 8.7 Hz, 2H), 7.83 (d, J = 8.6 Hz, 1H), 7.75 (d, J = 8.8 Hz,

1H), 7.15 (dd, J = 0.8, 2.2 Hz, 1H), 7.03 (d, J = 5.7 Hz, 1H), 3.94 (dd,

J = 1.7, 10.2 Hz, 1H), 3.79−3.70 (m, 1H), 3.60−3.45 (m, 2H), 3.26

(d, J = 6.6 Hz, 1H), 3.03 (s, 3H), 2.90 (t, J = 10.1 Hz, 1H). MS (ESI)

m/z 384.1 (M + H)⁺. HRMS calcd for C₁₉H₁₇N₃O₄S 384.1018, found

384.102. Peak 2 = 20: ¹H NMR (400 MHz, DMSO-d₆) δ 8.37 (d, J =

5.8 Hz, 1H), 8.34 (s, 1H), 8.19 (dd, J = 2.0, 8.7 Hz, 2H), 7.83 (d, J =

8.6 Hz, 1H), 7.75 (d, J = 8.8 Hz, 1H), 7.15 (dd, J = 0.8, 2.2 Hz, 1H),

7.03 (d, J = 5.7 Hz, 1H), 3.94 (dd, J = 1.7, 10.2 Hz, 1H), 3.79−3.70

(m, 1H), 3.60−3.45 (m, 2H), 3.26 (d, J = 6.6 Hz, 1H), 3.03 (s, 3H),

2.90 (t, J = 10.1 Hz, 1H). MS (ESI) m/z 384.1 (M + H)⁺. HRMS

calcd for C₁₉H₁₇N₃O₄S 384.1018, found 384.1016.

following the general sulfonylation procedure to aﬀord 15 (0.121 g,

1

0.30 mmol, 68% yield). H NMR (400 MHz, DMSO-d₆) δ 9.66 (s,

1H), 8.74−8.80 (m, 1H), 8.32−8.42 (m, 2H), 8.24−8.31 (m, 1H),

7.89−7.97 (m, 1H), 6.98−7.08 (m, 1H), 3.94−4.02 (m, 1H), 3.75−

3.84 (m, 1H), 3.51−3.63 (m, 2H), 3.25−3.30 (m, 1H), 3.25−3.30 (m,

1H), 2.94−3.03 (m, 4H). MS (ESI) m/z 401.0 (M + H)⁺. HRMS

calcd for C₁₈H₁₆N₄O₃S₂(M + H)⁺401.0742; found 401.0738.

(3aS,9bS)-2-(Imidazo[1,2-a]pyridin-7-ylsulfonyl)-5-methyl-

3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,6]naphthyridin-

4(2H)-one (16). (3aS,9bS)-41c (0.05 g, 0.246 mmol) in MeCN (3

mL) was treated with imidazo[1,2-a]pyridine-7-sulfonyl chloride

(0.107 g, 0.492 mmol) and triethylamine (0.068 mL, 0.492 mmol)

following the general sulfonylation procedure to aﬀord 16 (0.027 g,

0.07 mmol, 29% yield). ¹H NMR (500 MHz, DMSO-d₆) δ 8.74 (d, J =

7.07 Hz, 1H), 8.30−8.44 (m, 2H), 8.21 (s, 1H), 7.99 (s, 1H), 7.84 (s, J

= 6.42 Hz, 1H), 7.22 (d, J = 7.20 Hz, 1H), 7.05 (d, J = 5.64 Hz, 1H),

3.99 (d, J = 10.25 Hz, 1H), 3.75−3.89 (m, 1H), 3.52−3.70 (m, 2H),

3.17−3.29 (m, 1H), 2.98−3.09 (m, 4H). MS (ESI) m/z 384.2 (M +

H)⁺. HRMS calcd for C₁₈H₁₇N₅O₃S (M + H)⁺384.1130; found

384.1128.

(3aS,9bS)-2-((2,3-Dihydrobenzofuran-6-yl)sulfonyl)-5-meth-

yl-3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,6]naphthyridin-

4(2H)-one (17). (3aS,9bS)-41c (0.0736 M solution in MeCN, 18.64

mL, 1.372 mmol) was treated with 2,3-dihydrobenzofuran-6-sulfonyl

chloride (0.3 g, 1.372 mmol) and triethylamine (0.382 mL, 2.74

mmol) following the general sulfonylation procedure to aﬀord 17

(0.310 g, 0.804 mmol, 59% yield). ¹H NMR (500 MHz, DMSO-d₆) δ

8.37−8.41 (m, 1H), 8.31−8.37 (m, 1H), 8.31−8.37 (m, 1H), 7.44 (d, J

= 7.66 Hz, 1H), 7.22−7.29 (m, 1H), 7.03−7.15 (m, 2H), 4.57−4.70

(m, 2H), 3.87−3.94 (m, 1H), 3.66−3.74 (m, 1H), 3.51−3.59 (m, 1H),

3.44−3.50 (m, 1H), 3.25−3.29 (m, 3H), 3.24−3.28 (m, 2H), 3.16 (s,

3H), 2.88 (t, J = 10.06 Hz, 1H). MS (ESI) m/z 386.0 (M + H)⁺.

HRMS calcd for C₁₉H₁₉N₃O₄S (M + H)⁺386.1175; found 386.1176.

(3aS,9bS)-2-((2,3-Dihydrobenzofuran-5-yl)sulfonyl)-5-meth-

yl-3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,6]naphthyridin-

4(2H)-one (18). (3aS,9bS)-41c (0.0736 M solution in MeCN, 6 mL,

0.442 mmol) was treated with 2,3-dihydro-1-benzofuran-5-sulfonyl

chloride (0.100 g, 0.442 mmol) and triethylamine (0.123 mL, 0.883

mmol) following the general sulfonylation procedure to aﬀord 18

(3aS,9bS)-2-((3-Chloro-4-ﬂuorophenyl)sulfonyl)-5-methyl-

3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,6]naphthyridin-

4(2H)-one (21). (3aS,9bS)- 41c (0.0736 M solution in MeCN, 5 mL,

0.368 mmol) was treated with 3-chloro-4-ﬂuorobenzenesulfonyl

chloride (0.084 g, 0.368 mmol) and triethylamine (0.102 mL, 0.736

mmol) following the general sulfonylation procedure to aﬀord 21

1

(0.091 g, 62% yield). H NMR (500 MHz, DMSO-d₆) δ 8.40 (d, J =

6.22 Hz, 1H), 8.36−8.37 (m, 1H), 8.01 (dd, J = 2.14, 6.81 Hz, 1H),

7.84 (ddd, J = 2.24, 4.46, 8.61 Hz, 1H), 7.65 (t, J = 8.61 Hz, 1H), 7.07

(d, J = 5.64 Hz, 1H), 3.89−4.00 (m, 1H), 3.70−3.81 (m, 1H), 3.59−

3.69 (m, 1H), 3.55 (dd, J = 6.49, 10.38 Hz, 1H), 3.24−3.29 (m, 1H),

3.12 (s, 3H), 2.94 (t, J = 10.12 Hz, 1H). m/z 418.0 (M + Na)⁺. HRMS

calcd for C₁₇H₁₆ClFN₃O₃S 396.0585, found 396.0578.

rac-2-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-3a-ﬂuoro-5-methyl-

3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,6]naphthyridin-

4(2H)-one (22). Step 1: 4-(Methylamino)nicotinaldehyde (36). To a

vial charged with 4-chloronicotinaldehyde (0.884 g, 6.24 mmol) was

added 2-methoxyethanol (3.57 mL) and methylamine, 40 wt %

solution in water (13.51 mL, 156 mmol), respectively. The vessel was

sealed and heated at 120 °C for 6 h. The orange solution was dried

under reduced pressure and 1 N HCl (15 mL) was added, and the

resulting mixture heated at 50 °C for 2 h. The mixture was cooled in

an ice water bath and basiﬁed by the addition of satd aq NaHCO₃

(∼10 mL). The solution was extracted 2× with EtOAc. The combined

organics were dried with Na₂SO₄, ﬁltered, and dried under reduced

pressure, aﬀording 36 as a yellow oil which was used without further

puriﬁcation (0.408g, 48%). MS (ESI) m/z 137.1 (M + H)⁺.

Step 2: 3-Fluoro-1-methyl-1,6-naphthyridin-2(1H)-one (37). A

ﬂask charged with dry THF (9.08 mL) was cooled in an ice−water

bath prior to the addition of NaH (60% in mineral oil) (0.180 g, 4.50

mmol). To the suspension was added 2-ﬂuoro-2-phosphonoacetic acid

triethyl ester (0.912 mL, 4.50 mmol), faster than dropwise. The

mixture was stirred for 30 min, then cooled in an ice/salt bath prior to

the dropwise addition of a suspension of 36 (0.408 g, 3.00 mmol) in

THF (5 mL), faster than dropwise while still stirring at ∼−10 °C. The

mixture was allowed to stir for 1.5 h with slow warming. To the

mixture was added water (∼5 mL) and the resulting orange solution

dried under reduced pressure. The resulting residue was triturated with

water, the ﬁltrate was dried under reduced pressure and loaded onto a

reverse phase column (55g, C18) and puriﬁed ramping ACN in H₂O

(0−100%, 0.1% NH₄OH) with product eluting very early. This

material was repuriﬁed by catch and release with a 10g SCX column,

1

(0.081 g, 0.21 mmol, 48% yield). H NMR (500 MHz, DMSO-d₆) δ

8.28−8.49 (m, 2H), 7.66 (s, 1H), 7.55 (dd, J = 1.56, 8.37 Hz, 1H),

7.07 (d, J = 5.45 Hz, 1H), 6.92 (d, J = 8.37 Hz, 1H), 4.66 (dt, J = 2.01,

8.82 Hz, 2H), 3.88 (dd, J = 1.75, 10.19 Hz, 1H), 3.61−3.75 (m, 1H),

3.38−3.58 (m, 2H), 3.27 (t, J = 8.50 Hz, 3H), 3.15 (s, 3H), 2.87 (t, J =

10.02 Hz, 1H). MS (ESI) m/z 386.0 (M + H)⁺. HRMS calcd for

C₁₉H₁₉N₃O₄S (M + H)⁺386.1175; found 386.1174.

(3aS,9bS)-2-(Benzofuran-6-ylsulfonyl)-5-methyl-3,3a,5,9b-

tetrahydro-1H-pyrrolo[3,4-c][1,6]naphthyridin-4(2H)-one (19).

To a solution of 17 (100 mg, 0.259 mmol) in THF (1 mL) was added

ceric ammonium nitrate (CAN) (284 mg, 0.519 mmol) and the

resulting suspension shaken at 60 °C. After 3 h, additional CAN (4

equiv, 568 mg) was added and the mixture was shaken over three

nights at room temperature. The resulting material was loaded directly

onto a 25g ultra snap column and puriﬁed ramping DCM:MeOH

(90:10) in DCM (0−100%, monitoring at 215 nm) aﬀording product

along with impurities. Repuriﬁcation with RP-HPLC (ACN in H₂O,

5−95%, 0.1% NH₄OH modiﬁer) provided 19 as a colorless ﬁlm (10

mg, 10%). ¹H NMR (500 MHz, DMSO-d₆) δ 8.56 (br s, 2H), 8.29 (d,

J = 2.1 Hz, 1H), 8.06 (s, 1H), 7.90 (d, J = 8.2 Hz, 1H), 7.70 (d, J = 8.2

Hz, 1H), 7.35 (br s, 1H), 7.16 (s, 1H), 3.96 (d, J = 10.2 Hz, 1H),

3.82−3.75 (m, 1H), 3.71−3.63 (m, 1H), 3.55 (dd, J = 6.7, 10.1 Hz,

1H), 3.34 (t, J = 6.7 Hz, 1H), 3.12 (s, 3H), 3.05 (t, J = 10.1 Hz, 1H)

MS (ESI) m/z 384.0 (M + H)⁺. HRMS calcd for C₁₉H₁₇N₃O₄S (M +

H)⁺384.1018; found 384.1022.

(3aR,9bR)-2-(Benzofuran-5-ylsulfonyl)-5-methyl-3,3a,5,9b-

tetrahydro-1H-pyrrolo[3,4-c][1,6]naphthyridin-4(2H)-one (20)

and (3aS,9bS)-2-(Benzofuran-5-ylsulfonyl)-5-methyl-3,3a,5,9b-

tetrahydro-1H-pyrrolo[3,4-c][1,6]naphthyridin-4(2H)-one (ent-

20). To a vial charged with rac-41c (3.00 g, 14.76 mmol) was added

MeCN (98 mL), Et₃N (8.23 mL, 59.0 mmol), and benzofuran-5-

P

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Journal of Medicinal Chemistry

Article

loading with DCM/MeOH, washing with MeOH and eluting product

with 2 M NH₃in MeOH, aﬀording 37 (110 mg, 21%) as a light-orange

solid. ¹H NMR (400 MHz, chloroform-d) δ 8.78 (s, 1H), 8.62 (d, J =

5.96 Hz, 1H), 7.46 (d, J = 8.29 Hz, 1H), 7.22 (d, J = 6.01 Hz, 1H),

3.75 (s, 3H). MS (ESI) m/z 179.1 (M + H)⁺.

washed with brine, dried over MgSO₄, ﬁltered, and dried under

reduced pressure. To this residue was added 8 mL of 4 M HCl in

MeOH (made from AcCl and MeOH), and the resulting solution was

stirred at room temperature overnight. The resulting mixture was dried

under reduced pressure and the residue puriﬁed by catch and release

with an SCX column washing with MeOH, then 2 M NH₃in MeOH.

The basic wash was dried under reduced pressure to provide 44 (0.365

g, 1.688 mmol, 97% yield) as a thick brown oil. MS (ESI) m/z 217.2

(M + 1)⁺.

Step 3: rac-2-Benzyl-3a-ﬂuoro-5-methyl-3,3a,5,9b-tetrahydro-

1H-pyrrolo[3,4-c][1,6]naphthyridin-4(2H)-one (39g). To a vial

charged with 37 (0.11 g, 0.617 mmol) was added DCM (1.764 mL)

and TFA (0.019 mL, 0.247 mmol) and the resulting yellow suspension

cooled in an ice−water bath prior to the addition of N-

(methoxymethyl)-N-(trimethylsilylmethyl)benzyl-amine (0.237 mL,

0.926 mmol), faster than dropwise. The mixture was stirred and

allowed to slowly warm to room temperature (ice melt). After 3 h,

additional N-(methoxymethyl)-N-(trimethylsilylmethyl)benzyl-amine

(3 eq, 474 μL) was added and stirring continued at room temperature

for 3 h then overnight at 40 °C. The mixture was dried under reduced

pressure and puriﬁed by catch and release with a 10g SCX column,

washing with MeOH, and then 2 M NH₃in MeOH. The basic wash

was dried under reduced pressure, aﬀording 39g (0.154g, 80%). MS

(ESI) m/z 312.0 (M + H)⁺.

Step 4: rac-3a-Fluoro-5-methyl-3,3a,5,9b-tetrahydro-1H-pyrrolo-

[3,4-c][1,6]naphthyridin-4(2H)-one (41g). To a pressure vessel

charged with 39g (0.154 g, 0.495 mmol) as a turbid solution in

EtOH (2.248 mL) was added water (0.225 mL) and palladium

hydroxide, 20 wt % Pd (dry basis) on carbon, wet, Degussa type e101

ne/w (0.035 g, 0.049 mmol). The resulting mixture was heated to 40

°C under 40 psi of H₂for 3 h. The mixture was ﬁltered through Celite

and dried under reduced pressure. The mixture was puriﬁed by catch

and release with a 10g SCX column washing with MeOH, then 2 M

NH₃in MeOH to aﬀord 41g which was used without further

puriﬁcation, (95 mg, 0.429 mmol) (95 mg, 87%).

Step 3: (3aR,9bS)-2-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-3a,5-di-

methyl-3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c]quinolin-4(2H)-one

(23). A solution of 44 (0.1 g, 0.462 mmol) in DMF (2 mL) was treated

with 1,3-benzodioxole-5-sulfonyl chloride (0.112 g, 0.509 mmol) and

triethylamine (0.193 mL, 1.387 mmol) following the general

sulfonylation procedure to aﬀord the racemate of 23 (0.143 g, 0.358

mmol, 77% yield). Then 50 mg of this material was subjected to chiral

resolution by SFC on column (S,S) Whelk-O, 5 μ, 2 cm × 25 cm, 45%

methanol w/0.2% DEA, 80 mL/min, 81 bar pressure drop, 100 bar

BPR, 251 nm, sample dissolved in DCM:MeOH (1:1, 10 mL). Sample

processed with 1.0 mL injection volume and cycle time of 6 min. Peak

assignment determined by SFC on (S,S) Whelk-O with 45% methanol

w/0.2% DEA modifer. Absolute conﬁguration assigned by analogy

based on in vitro activity and the known stereochemistry of active

isomer 32 as determined by the X-ray cocrystal structure. Peak 1 =

compound 23 (20 mg). ¹H NMR (400 MHz, DMSO-d₆) δ 7.25−7.38

(m, 4H), 7.03−7.16 (m, 3H), 6.15−6.22 (m, 2H), 4.10−4.16 (m, 1H),

3.63−3.70 (m, 1H), 3.21−3.28 (m, 1H), 3.10−3.18 (m, 4H), 2.77−

2.87 (m, 1H), 0.95 (s, 3H). MS (ESI) m/z 401.1 (M + H)⁺. HRMS

calcd for C₂₀H₂₀N₂O₅S (M + H)⁺401.1171; found 401.1170.

rac-2-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-5-methyl-4-oxo-

2,3,3a,4,5,9b-hexahydro-1H-pyrrolo[3,4-c][1,6]naphthyridine-

3a-carbonitrile (24). Step 1: 2-Oxo-1,2-dihydro-1,6-naphthyridine-

3-carbonitrile (35). To a vial charged with 4-amino-3-formylpyridine

(0.90 g, 7.37 mmol) was added EtOH (12.28 mL), cyanoacetic acid

methyl ester (1.312 mL, 14.74 mmol), and piperidine (0.182 mL,

1.842 mmol). The vial was sealed and heated with shaking to 120 °C

overnight, aﬀording a yellow precipitate. The mixture was cooled to

room temperature and the solid collected via vacuum ﬁltration and

washed with heptane to aﬀord 35 as a yellow solid (1.237 g, 7.23

mmol, 98% yield). ¹H NMR (400 MHz, DMSO-d₆) δ = 12.70 (br s, 1

H), 8.89 (d, J = 0.7 Hz, 1 H), 8.92 (d, J = 0.7 Hz, 1 H), 8.59 (d, J = 5.9

Hz, 1 H), 7.26 (td, J = 0.7, 5.8 Hz, 1 H). MS (ESI) m/z 172.1 (M +

H)⁺.

Step 2: 1-Methyl-2-oxo-1,2-dihydro-1,6-naphthyridine-3-carbon-

itrile (38f). To a vial charged with 35 (1.237 g, 7.23 mmol) was added

DMF (20.65 mL) and Cs₂CO₃(2.59 g, 7.95 mmol). The mixture was

stirred at room temperature for 15 min prior to the addition of

iodomethane (0.565 mL, 9.03 mmol). The resulting orange

suspension was stirred for 1 h at room temp. The mixture was

added to ice and the resulting solid collected via vacuum ﬁltration and

washed with water and the solid dried under a nitrogen/vacuum

sweep, aﬀording 38f as a tan solid (1.121 g, 6.05 mmol, 84% yield). ¹H

NMR (400 MHz, DMSO-d₆) δ = 8.97 (d, J = 0.5 Hz, 1 H), 8.91 (s, 1

H), 8.72 (d, J = 6.1 Hz, 1 H), 7.60 (d, J = 6.2 Hz, 1 H), 3.62 (s, 3 H).

MS (ESI) m/z 186.0 (M + H)⁺.

Step 3: rac-2-Benzyl-5-methyl-4-oxo-2,3,3a,4,5,9b-hexahydro-

1H-pyrrolo[3,4-c][1,6]naphthyridine-3a-carbonitrile (39f). To a vial

charged with 38f (1.12 g, 6.05 mmol) was added DCM (17.28 mL)

and TFA (0.186 mL, 2.419 mmol) and the resulting yellow suspension

cooled in an ice−water bath prior to the addition of N-

(methoxymethyl)-N-(trimethylsilylmethyl)benzyl-amine (2.321 mL,

9.07 mmol), faster than dropwise. The mixture was stirred and

allowed to slowly warm to room temperature (ice melt). After 4 h,

additional N-(methoxymethyl)-N-(trimethylsilylmethyl)benzyl-amine

(1 equiv) was added and stirring at room temperature continued for

1 h. The reaction mixture was dried under reduced pressure, and the

material was dissolved in MeOH/H₂O, ﬁltered through a 0.45 μm frit,

and loaded onto a Redisep load column and puriﬁed with a 120g RP

Interchim C18, 30 μm, column ramping ACN in H₂O (0−100%, 0.1%

Step 5: rac-2-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-3a-ﬂuoro-5-

methyl-3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,6]naphthyridin-

4(2H)-one (22). To a vial charged with 41g (95 mg, 0.429 mmol) was

added MeCN (2863 μL), Et₃N (239 μL, 1.718 mmol), and

benzo[d][1,3]dioxole-5-sulfonyl chloride (95 mg, 0.429 mmol),

respectively. The mixture was stirred at room temp for 1 h then

dried under reduced pressure and puriﬁed with a 55g RP C-18 column,

directly loading with IPA/DMSO and ramping with ACN in H₂O

(0.1% in NH₄OH), providing product with minor close impurities.

The material was repuriﬁed with RP-HPLC ramping ACN in H₂O

1

(5−75%, 0.1% TFA), providing 22 as a white solid (85 mg, 49%). H

NMR (500 MHz, DMSO-d₆) δ 8.51 (d, J = 7.59 Hz, 2H), 7.22−7.35

(m, 3H), 7.02 (d, J = 8.11 Hz, 1H), 6.19 (s, 2H), 4.05 (d, J = 11.74 Hz,

2H), (broad water peak over 3 aliphatic H), 3.29 (s, 3H). MS (ESI)

m/z 405.9 (M + H)⁺. HRMS calcd for C₁₈H₁₆FN₃O₅S = 406.0873,

found 406.0875.

(3aR,9bS)-2-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-3a,5-dimeth-

yl-3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c]quinolin-4(2H)-one

(23). Step 1: rac-tert-Butyl 4-Oxo-3,3a,4,5-tetrahydro-1H-pyrrolo-

[3,4-c]quinoline-2(9bH)-carboxylate (42). To a mixture of rac-2-

benzyl-3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c]quinolin-4(2H)-one (12

g, 0.0431 mmol), Pd/C (1.2 g, 10% on carbon), and Pd(OH)₂(1.2 g)

in EtOH (120 mL) was added (Boc)₂O (14.09 mL, 0.064 mol, 1.5

equiv) and the mixture stirred at 50 psi under H₂for 24 h. The

resulting mixture was ﬁltered and concentrated and the crude material

was puriﬁed by column chromatography using petroleum ether:ethyl

1

acetate (1:1) to aﬀord 42 as an oﬀ-white solid (12 g, 96% yield). H

NMR (400 MHz, DMSO-d₆) δ 10.36 (s, 1H), 7.27 (d, J = 7.5 Hz,

1H), 7.19 (td, J = 7.7, 1.4 Hz, 1H), 7.00−6.87 (m, 2H), 3.93 (t, J = 9.8

Hz, 1H), 3.60 (d, J = 8.5 Hz, 2H), 3.46 (ddd, J = 16.8, 10.5, 6.7 Hz,

1H), 3.14 (t, J = 6.7 Hz, 1H), 2.81 (td, J = 12.2, 10.4, 6.9 Hz, 1H), 1.38

(d, J = 14.5 Hz, 9H). MS (ESI) m/z 189.4 (M + H − Boc)⁺.

Step 2: rac-3a,5-Dimethyl-3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-

c]quinolin-4(2H)-one (44). To a solution of 42 (0.5 g, 1.734 mmol) in

DMF (8.67 mL) at 0 °C was added sodium hydride 60% suspension in

mineral oil (0.173 g, 4.34 mmol). The mixture was stirred at rt for 30

min prior to the addition of iodomethane (0.269 mL, 4.34 mmol). The

resulting reaction mixture was stirred at room temperature for 2 h then

quenched by the addition of satd aq NH₄Cl with ice bath cooling then

extracted with diethyl ether three times. The combined organics were

Q

DOI: 10.1021/acs.jmedchem.6b01496

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Journal of Medicinal Chemistry

Article

NH₄OH), providing 39f as a yellow sticky foam (996 mg, 52%). MS

(ESI) m/z 319.2 (M + H)⁺.

with 39e (0.134 g, 0.353 mmol) was added 6 N HCl (0.177 mL). The

vial was sealed and shaken at 130 °C. After 2 h, additional 6 N HCl (2

mL) was added and the mixture heated to 130 °C, under nitrogen.

After 2 more hours of concentration, HCl (2 mL) was added and the

solution was stirred for 20 h at 130 °C. The resulting mixture was

cooled in an ice−water bath while stirring and brought to basic pH by

the addition of 6N NaOH. The resulting slurry was dried under

reduced pressure and the solid (containing salts) shaken with DCM/

MeOH and ﬁltered to remove most of the salts. The ﬁltrate was dried

under reduced pressure and puriﬁed by catch and release with a 5g

SCX column, loading with MeOH/DCM and washing with MeOH,

water, MeOH, then 2 M NH₃in MeOH, aﬀording product elution.

The solution was dried under reduced pressure and the resulting

material (40e) used without further puriﬁcation, obtained as a brown

oil (85 mg, 78%). MS (ESI) m/z 308.1 (M + H)⁺.

Step 5: rac-5,9b-Dimethyl-3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-

c][1,6]naphthyridin-4(2H)-one (41e). To a pressure vessel charged

with 40e (0.085 g, 0.277 mmol) as a turbid solution in EtOH (1.257

mL) was added water (0.126 mL) and palladium hydroxide, 20 wt %

Pd (dry basis) on carbon, wet, Degussa type e101 ne/w (0.019 g,

0.028 mmol). The resulting mixture was heated to 40 °C under 40 psi

of H₂for 4 h. The mixture was ﬁltered through Celite and dried under

reduced pressure. The crude material was puriﬁed by catch and release

with a 5g SCX column washing with MeOH, then 2 M NH₃in MeOH

to aﬀord 41e with some minor impurities (∼75% pure, 50 mg, 83%

yield). The material was used without further puriﬁcation. MS (ESI)

m/z 218.1 (M + H)⁺.

Step 6: rac-2-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-5,9b-dimethyl-

3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,6]naphthyridin-4(2H)-one

(25). To a vial charged with 41e (50 mg, 0.230 mmol) was added

MeCN (1534 μL), Et₃N (128 μL, 0.921 mmol), and benzo[d][1,3]-

dioxole-5-sulfonyl chloride (50.8 mg, 0.230 mmol), respectively. The

mixture was stirred at room temperature overnight. The yellow

solution was dried under reduced pressure, dissolved in DMSO,

ﬁltered through a 0.25 μm frit, and puriﬁed with RP-HPLC ramping

ACN in H₂O (0−100%, 0.1% NH₄OH), aﬀording 25 as a pale-yellow

ﬁlm (25 mg, 0.062 mmol, 27.1% yield). ¹H NMR (500 MHz, DMSO-

d₆) δ 8.40 (br s, 2H), 7.27 (dd, J = 1.72, 8.14 Hz, 1H), 7.20 (d, J = 1.62

Hz, 1H), 6.94−7.12 (m, 2H), 6.18 (d, J = 4.41 Hz, 2H), 3.58−3.70

(m, 2H), 3.46 (dd, J = 6.78, 10.09 Hz, 1H), 3.20 (s, 3H), 3.17 (s, 1H),

3.05 (t, J = 7.27 Hz, 1H), 1.22 (s, 3H). MS (ESI) m/z 402.1 (M +

H)⁺, HRMS calcd for C₁₉H₁₉N₃O₅S 402.1124, found 402.1119.

Synthesis of (rac)-cis-52a, (rac)-cis-52b, (rac)-cis-52c, and

(rac)-cis-52d via Initial Route, and Sulfonamides 26−33. Step 1:

N-Benzyl-1-(trimethylsilyl)ethanamine (52). See Manzano et al., ref

39.

Step 4: rac-5-Methyl-4-oxo-2,3,3a,4,5,9b-hexahydro-1H-pyrrolo-

[3,4-c][1,6]naphthyridine-3a-carbonitrile (41f). To a vial charged

with 39f (0.10 g, 0.314 mmol) was added DCM (1.256 mL) and the

resulting solution cooled in an ice−water bath prior to the addition of

carbonochloridate (0.068 mL, 0.754 mmol) which was stirred for 10

min at 0 °C then overnight at 40 °C. The mixture was dried under

reduced pressure and EtOH (1.5 mL) and 6 N HCl (0.3 mL) were

added and the mixture shaken at 100 °C overnight. The mixture was

cooled to room temperature and added to IPA (∼30 mL), aﬀording a

light-brown precipitate which was collected by vacuum ﬁltration,

rinsing with minimal IPA, providing 41f as a brown solid (50 mg,

0.219 mmol, 69.7% yield). MS (ESI) m/z 229.2 (M + H)⁺.

Step 5: rac-2-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-5-methyl-4-oxo-

2,3,3a,4,5,9b-hexahydro-1H-pyrrolo[3,4-c][1,6]naphthyridine-3a-

carbonitrile (24). To a vial charged with 41f (48 mg, 0.210 mmol) was

added MeCN (1402 μL), Et₃N (117 μL, 0.841 mmol), and 1,3-

benzodioxole-5-sulfonyl chloride (46.4 mg, 0.210 mmol), respectively.

The mixture was shaken at room temperature for 90 min. The

resulting mixture was dried under reduced pressure, dissolved in

DMSO, ﬁltered through a 0.45 μm frit, and puriﬁed with RP-HPLC

ramping ACN in H₂O, NH₄OH 0.1%. Under these basic conditions,

∼50% of the product decomposed to the des-CN compound according

to LC-MS. Repuriﬁcation with RP-HPLC ramping ACN in H₂O, TFA

1

0.1% aﬀorded 24 (18 mg, 16%). H NMR (500 MHz, CD₃CN) δ =

3.33−3.39 (m, 4 H) 3.87−3.93 (m, 2 H) 4.18 (t, J = 8.17 Hz, 1 H)

4.24 (d, J = 10.77 Hz, 1 H) 6.15 (s, 2 H) 7.01 (d, J = 8.17 Hz, 1 H)

7.25 (d, J = 1.82 Hz, 1 H) 7.33−7.43 (m, 2 H) 8.55 (s, 1 H) 8.62 (d, J

= 6.36 Hz, 1 H). MS (ESI) m/z 413.0 (M + H)⁺, HRMS calcd for

C₁₉H₁₆N₄O₅S = 412.424, found 413.092.

rac-2-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-5,9b-dimethyl-

3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,6]naphthyridin-

4(2H)-one (25). Step 1: Ethyl 4-Methyl-2-oxo-1,2-dihydro-1,6-

naphthyridine-3-carboxylate (34f). To a microwave vial was added

1-(4-aminopyridin-3-yl)ethanone (1.00 g, 7.34 mmol), diethyl

malonate (5.58 mL, 36.7 mmol), and piperidine (0.182 mL, 1.836

mmol), respectively. The mixture was irradiated at 180 °C for 15 min,

aﬀording a tan precipitate. To the vessel was added water and the solid

collected by vacuum ﬁltration and washed with water to provide 34f as

a tan solid (0.848 g, 3.65 mmol, 49.7% yield), which was used without

further puriﬁcation. MS (ESI) m/z 233.1 (M + H)⁺.

Step 2: Ethyl 1,4-Dimethyl-2-oxo-1,2-dihydro-1,6-naphthyridine-

3-carboxylate (38e). To a vial charged with 34f (0.675 g, 2.91 mmol)

was added Cs₂CO₃(1.326 g, 4.07 mmol) and DMF (8.30 mL), and

the resulting suspension was cooled in an ice−water bath prior to the

addition of iodomethane (0.182 mL, 2.91 mmol) dropwise. The

mixture was allowed to stir while slowly warming to room temperature

(ice melt) over 2 h. The mixture was diluted with water and puriﬁed

by catch and release with a 10g SCX column, washing with MeOH,

then 2 M NH₃in MeOH to elute 38e, obtained as a yellow solid

(0.550 g, 2.233 mmol, 77% yield). ¹H NMR (400 MHz, DMSO-d₆) δ

= 9.07 (s, 1 H), 8.68 (d, J = 5.9 Hz, 1 H), 7.54 (d, J = 6.0 Hz, 1 H),

4.35 (q, J = 7.2 Hz, 2 H), 3.60 (s, 3 H), 2.50 (s, 3 H), 1.31 (t, J = 7.1

Hz, 3 H). MS (ESI) m/z 247.1 (M + H)⁺.

Step 3: rac-Ethyl 2-Benzyl-5,9b-dimethyl-4-oxo-2,3,3a,4,5,9b-

hexahydro-1H-pyrrolo[3,4-c][1,6]naphthyridine-3a-carboxylate

(39e). To a vial charged with 38e (0.500 g, 2.030 mmol) was added

DCM (5.80 mL) and the resulting solution cooled in an ice−water

bath prior to the addition of TFA (0.063 mL, 0.812 mmol) and N-

(methoxymethyl)-N-(trimethylsilylmethyl)benzyl-amine (2.60 mL,

10.15 mmol), faster than dropwise. The mixture was stirred and

allowed to warm to room temperature (ice melt) over 4 h. The

mixture was puriﬁed by catch and release by loading directly onto a

10g SCX column which was washed with MeOH, then 2 M NH₃in

MeOH. The basic wash was dried under reduced pressure and the

mixture puriﬁed with a 55g C-18 RP column ramping ACN in H₂O

(0−100%, 0.1% NH₄OH), aﬀording 39e as a yellow oil (0.134 g, 0.353

mmol, 17.39% yield). MS (ESI) m/z 380.1 (M + H)⁺.

Step 2: N-Benzyl-N-(methoxymethyl)-1-(trimethylsilyl)-

ethanamine (53). See Manzano et al., ref 39.

Step 3: Ethyl 1-Methyl-2-oxo-1,2-dihydro-1,6-naphthyridine-3-

carboxylate (38g). To a ﬂask charged with 34c (5.08 g, 23.28 mmol)

was added DMF (46.6 mL) and cesium carbonate (2.79 mL, 34.9

mmol). The resulting mixture was stirred for 15 min at room

temperature, aﬀording a thick yellow suspension which was cooled in

an ice−water bath and placed under nitrogen prior to the addition of

MeI (1.601 mL, 25.6 mmol), faster than dropwise. The resulting

mixture was allowed to stir and warm slowly to room temperature (ice

melt). After 2 h, the reaction was quenched by the addition of ice. The

aqueous solution was extracted with EtOAc two times. The combined

organics were dried with Na₂SO₄, ﬁltered, and dried under reduced

pressure. The organic layer was puriﬁed by catch and release with a

25g SCX column washing with MeOH, then 2 M NH₃in MeOH,

aﬀording 38g as a light-yellow solid (2.8 g, 36%). m/z (ESI) 233.1 (M

+ H)⁺

Step 4: Diastereomeric Mixture of cis-Ring Fusion Isomers, rac-

Ethyl 2-Benzyl-1,5-dimethyl-4-oxo-2,3,3a,4,5,9b-hexahydro-1H-

pyrrolo[3,4-c][1,6]naphthyridine-3a-carboxylate and rac-Ethyl 2-

Benzyl-3,5-dimethyl-4-oxo-2,3,3a,4,5,9b-hexahydro-1H-pyrrolo[3,4-

c][1,6]naphthyridine-3a-carboxylate. To a ﬂask charged with 38g

(0.6 g, 2.58 mmol) was added DCM (7.38 mL) and TFA (0.080 mL,

1.033 mmol). The mixture was cooled to 0 °C prior to the addition of

a solution of N-benzyl-N-(methoxymethyl)-1-(trimethylsilyl)-

Step 4: rac-2-Benzyl-5,9b-dimethyl-3,3a,5,9b-tetrahydro-1H-

pyrrolo[3,4-c][1,6]naphthyridin-4(2H)-one (40e). To a vial charged

R

DOI: 10.1021/acs.jmedchem.6b01496

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

ethanamine (1.624 g, 6.46 mmol) in DCM (1 mL), faster than

dropwise. The mixture was stirred overnight at room temperature. The

mixture was diluted with DCM, adsorbed onto silica gel, and puriﬁed

with a 100g ultra snap column ramping EtOAc:EtOH (76:24) in

heptane (0−30%, isocratic at 30%, then 30%−70%), aﬀording product

as a sticky yellow oil as a nonstatistical mixture of four racemic

diastereomers (0.327 g, 0.862 mmol, 33.4% yield). MS (ESI) m/z

380.1 (M + H)⁺.

Step 5: Diastereomeric Mixture of cis-Ring Fusion Isomers of 2-

Benzyl-1,5-dimethyl-3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,6]-

naphthyridin-4(2H)-one and 2-Benzyl-3,5-dimethyl-3,3a,5,9b-tetra-

hydro-1H-pyrrolo[3,4-c][1,6]naphthyridin-4(2H)-one. To a vial

charged with rac-ethyl 2-benzyl-1,5-dimethyl-4-oxo-2,3,3a,4,5,9b-hex-

ahydro-1H-pyrrolo[3,4-c][1,6]naphthyridine-3a-carboxylate/rac-ethyl

2-benzyl-3,5-dimethyl-4-oxo-2,3,3a,4,5,9b-hexahydro-1H-pyrrolo[3,4-

c][1,6]naphthyridine-3a-carboxylate (291 mg, 0.767 mmol) was added

1,4-dioxane (4.18 mL) and 3 N HCl (2.8 mL), respectively. The vessel

was sealed and heated at 90 °C over three nights. The resulting yellow

solution was dried under reduced pressure and was used directly

without further puriﬁcation in the next step (yield not determined).

MS (ESI) m/z 308.1 (M + H)⁺.

Step 6: rac-3,5-Dimethyl-3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c]-

[1,6]naphthyridin-4(2H)-one and 1,5-Dimethyl-3,3a,5,9b-tetrahy-

dro-1H-pyrrolo[3,4-c][1,6]naphthyridin-4(2H)-one/1,5-Dimethyl-

3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,6]naphthyridin-4(2H)-one,

(rac)-cis-54a−d. To a pressure vessel charged with rac-2-benzyl-1,5-

dimethyl-3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,6]naphthyridin-

4(2H)-one/rac-2-benzyl-3,5-dimethyl-3,3a,5,9b-tetrahydro-1H-

pyrrolo[3,4-c][1,6]naphthyridin-4(2H)-one (0.261 g, 0.849 mmol

(crude) as a turbid solution in EtOH (3.86 mL) was added water

(0.386 mL) and palladium hydroxide, 20 wt % pd (dry basis) on

carbon, wet, Degussa type e101 ne/w (0.060 g, 0.085 mmol). The

resulting mixture was heated to 40 °C under 40 psi of H₂for 3 h. The

mixture was ﬁltered through Celite and dried under reduced pressure.

The crude residue was further puriﬁed by catch and release with a 5g

SCX column washing with MeOH then 2 M NH₃in MeOH, aﬀording

(rac)-cis-54a−d as pale-yellow upon drying under reduced pressure

(160 mg, 0.736 mmol, 96% yield, 2 steps). MS (ESI) m/z 218.1 (M +

H)⁺.

Step 7: Sulfonamides 26−33. To a vial charged with (rac)-cis-54a−

d (160 mg, 0.736 mmol) was added MeCN (4.9 mL), Et₃N (0.4 mL,

2.95 mmol), and benzo[d][1,3]dioxole-5-sulfonyl chloride (162 mg,

0.736 mmol), respectively. The mixture was stirred at room temp for 2

h. The resulting mixture was dried under reduced pressure and puriﬁed

with a 50g ultra snap column ramping EtOAc:EtOH (76:24) in

heptane (0−100%, isocratic at 100%), aﬀording desired product as a

mixture of diastereomers light-yellow foam (211 mg, 71%). See the

Supporting Information for chiral separation conditions and NMR

structural conﬁrmations.

(1R,3aS,9bS)-2-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-1,5-dimethyl-

3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,6]naphthyridin-4(2H)-one

(28) (Absolute Stereochemistry Arbitrarily Assigned). Obtained 28

1

mg, 9.5%. H NMR (400 MHz, DMSO-d₆) δ 8.42 (d, J = 5.60 Hz,

1H), 8.32 (s, 1H), 7.44 (dd, J = 1.87, 8.19 Hz, 1H), 7.40 (d, J = 1.66

Hz, 1H), 7.16 (d, J = 8.09 Hz, 1H), 7.13 (d, J = 5.70 Hz, 1H), 6.21

(dd, J = 1.04, 4.56 Hz, 2H), 3.99−4.11 (m, 2H), 3.40 (d, J = 8.71 Hz,

1H), 3.32 (m, 2H), 3.28 (s, 3H), 0.69 (d, J = 6.74 Hz, 3H). MS (ESI)

m/z 402.0 (M + H)⁺. HRMS calcd for C₁₉H₁₉N₃O₅S 402.1124, found

402.1123.

(1S,3aR,9bR)-2-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-1,5-dimethyl-

3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,6]naphthyridin-4(2H)-one

(29) (Absolute Stereochemistry Arbitrarily Assigned). Obtained 31

1

mg, 10.4%. H NMR (400 MHz, DMSO-d₆) δ 8.42 (d, J = 5.70 Hz,

1H), 8.32 (s, 1H), 7.44 (dd, J = 1.87, 8.19 Hz, 1H), 7.40 (d, J = 1.66

Hz, 1H), 7.16 (d, J = 8.19 Hz, 1H), 7.13 (d, J = 5.70 Hz, 1H), 6.21

(dd, J = 0.93, 4.56 Hz, 2H), 3.99−4.12 (m, 2H), 3.38−3.45 (m, 1H),

3.32 (m, 2H), 3.28 (s, 3H), 0.69 (d, J = 6.74 Hz, 3H). MS (ESI) m/z

402.2 (M + H)⁺. HRMS calcd for C₁₉H₁₉N₃O₅S 402.1124, found

402.1121.

(3R,3aS,9bS)-2-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-3,5-dimethyl-

3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,6]naphthyridin-4(2H)-one

(30) (Absolute Stereochemistry Arbitrarily Assigned). Obtained 34

1

mg, 11.5%. H NMR (400 MHz, DMSO-d₆) δ 8.35−8.41 (m, 2H),

8.35−8.41 (m, 1H), 7.25 (dd, J = 1.87, 8.19 Hz, 1H), 7.18 (d, J = 1.87

Hz, 1H), 6.99−7.05 (m, 2H), 6.17 (dd, J = 0.93, 11.71 Hz, 2H), 4.29

(dq, J = 1.61, 6.51 Hz, 1H), 3.90 (td, J = 7.55, 10.08 Hz, 1H), 3.69

(dd, J = 7.72, 9.59 Hz, 1H), 3.02 (s, 3H), 2.96 (dd, J = 1.35, 7.26 Hz,

1H), 2.83 (t, J = 10.00 Hz, 1H), 1.38 (d, J = 6.63 Hz, 3H). MS (ESI)

m/z 402.2 (M + H)⁺. HRMS calcd for C₁₉H₁₉N₃O₅S 402.1124, found

402.1125.

(3S,3aR,9bR)-2-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-3,5-dimethyl-

3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,6]naphthyridin-4(2H)-one

(31) (Absolute Stereochemistry Arbitrarily Assigned). Obtained 46

1

mg, 15.5%. H NMR (400 MHz, DMSO-d₆) δ 8.36−8.40 (m, 2H),

7.25 (dd, J = 1.87, 8.19 Hz, 1H), 7.18 (d, J = 1.66 Hz, 1H), 6.99−7.04

(m, 2H), 6.17 (dd, J = 0.88, 11.77 Hz, 2H), 4.29 (dq, J = 1.71, 6.58 Hz,

1H), 3.90 (td, J = 7.48, 10.13 Hz, 1H), 3.65−3.73 (m, 1H), 3.02 (s,

3H), 2.96 (dd, J = 1.45, 7.26 Hz, 1H), 2.83 (t, J = 10.05 Hz, 1H), 1.38

(d, J = 6.63 Hz, 3H). MS (ESI) m/z 402.2 (M + H)⁺, HRMS calcd for

C₁₉H₁₉N₃O₅S 402.1124, found 402.1118.

(3S,3aS,9bS)-2-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-3,5-dimethyl-

3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,6]naphthyridin-4(2H)-one

(32). Obtained 5 mg, 1.7%. ¹H NMR (400 MHz, DMSO-d₆) δ 8.53 (s,

1H), 8.43 (d, J = 5.49 Hz, 1H), 7.42 (dd, J = 1.87, 8.09 Hz, 1H), 7.39

(d, J = 1.66 Hz, 1H), 7.16 (d, J = 8.19 Hz, 1H), 7.09 (d, J = 5.60 Hz,

1H), 6.21 (s, 2H), 4.08 (dd, J = 1.76, 10.78 Hz, 1H), 3.89−3.99 (m,

1H), 3.69 (t, J = 7.46 Hz, 1H), 3.54 (dd, J = 6.58, 10.83 Hz, 1H), 3.26

(s, 3H), 3.05 (t, J = 9.07 Hz, 1H), 0.84 (d, J = 6.74 Hz, 3H). MS (ESI)

m/z 402.2 (M + H)⁺, HRMS calcd for C₁₉H₁₉N₃O₅S 402.1124, found

402.1127.

Yields and analytical data for the individual diastereomers:

(1S,3aS,9bS)-2-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-1,5-dimethyl-

3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,6]naphthyridin-4(2H)-one

(26) (Absolute Stereochemistry Arbitrarily Assigned). Obtained 5

(3R,3aR,9bR)-2-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-3,5-dimethyl-

3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,6]naphthyridin-4(2H)-one

1

(33). Obtained 6 mg, 2%. H NMR (400 MHz, DMSO-d₆) δ 8.53 (s,

mg, 1.7% yield. H NMR (400 MHz, DMSO-d₆) δ 8.41 (d, J = 5.29

1H), 8.43 (d, J = 5.60 Hz, 1H), 7.42 (dd, J = 1.87, 8.09 Hz, 1H), 7.39

(d, J = 1.76 Hz, 1H), 7.16 (d, J = 8.19 Hz, 1H), 7.09 (d, J = 5.70 Hz,

1H), 6.21 (s, 2H), 4.08 (dd, J = 1.87, 10.78 Hz, 1H), 3.94 (qd, J =

6.74, 9.42 Hz, 1H), 3.69 (t, J = 7.31 Hz, 1H), 3.54 (dd, J = 6.58, 10.83

Hz, 1H), 3.26 (s, 3H), 3.05 (t, J = 9.07 Hz, 1H), 0.84 (d, J = 6.74 Hz,

3H). MS (ESI) m/z 402.1 (M + H)⁺. HRMS calcd for C₁₉H₁₉N₃O₅S =

401.44, found 402.1125

Synthesis of (rac)-cis-52c and (rac)-cis-52d via Improved

Route (through Intermediates 55, 56, 57).²⁷Chiral separation

conditions for 54: column, Chiralpak AD-H, 2 cm × 15 cm; modiﬁer

and percentage, 35% IPA w/0.2% DEA; ﬂow rate (mL/min), 80;

pressure drop (bar), 79; BPR (bar), 100; detection (nm), 257. Sample

dissolution: ∼80 mL of 1:1:1 dichloromethane:IPA:MeOH. Test

Injections: 0.25 mL (∼9 mg), 0.5 mL (∼18 mg). Then 3 mL ofDEA

was added to sample solution to free base. Sample processing: volume

(mL), 1; cycle time (min), 6. Desired peak was peak 3.

Hz, 1H), 8.32 (s, 1H), 7.24 (dd, J = 1.66, 8.19 Hz, 1H), 7.18 (d, J =

1.55 Hz, 1H), 6.98−7.06 (m, 2H), 6.17 (d, J = 11.71 Hz, 2H), 3.99 (d,

J = 11.51 Hz, 1H), 3.69 (dd, J = 6.27, 11.25 Hz, 1H), 3.27−3.30 (m,

1H), 3.11−3.22 (m, 2H), 2.98 (s, 3H), 1.39 (d, J = 5.91 Hz, 3H). MS

(ESI) m/z 402.2 (M + H)⁺. HRMS calcd for C₁₉H₁₉N₃O₅S 402.1124,

found 402.1118.

(1R,3aR,9bR)-2-(Benzo[d][1,3]dioxol-5-ylsulfonyl)-1,5-dimethyl-

3,3a,5,9b-tetrahydro-1H-pyrrolo[3,4-c][1,6]naphthyridin-4(2H)-one

(27) (Absolute Stereochemistry Arbitrarily Assigned). Obtained 5

1

mg, 1.7% yield. H NMR (400 MHz, DMSO-d₆) δ 8.41 (d, J = 5.60

Hz, 1H), 8.32 (s, 1H), 7.24 (dd, J = 1.92, 8.14 Hz, 1H), 7.18 (d, J =

1.76 Hz, 1H), 6.98−7.06 (m, 2H), 6.17 (dd, J = 0.93, 11.92 Hz, 2H),

3.99 (dd, J = 1.55, 11.30 Hz, 1H), 3.69 (dd, J = 6.32, 11.20 Hz, 1H),

3.29 (s, 1H), 3.11−3.23 (m, 2H), 2.98 (s, 3H), 1.39 (d, J = 6.01 Hz,

3H). MS (ESI) m/z 402.2 (M + H)⁺. HRMS calcd for C₁₉H₁₉N₃O₅S

402.1124, found 402.1122.

S

DOI: 10.1021/acs.jmedchem.6b01496

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Peak 1: (3R,3aR,9bR)-3,5-Dimethyl-3,3a,5,9b-tetrahydro-1H-

cell line validation work, Virginia Berry, Loren Berry, and Brett

Janosky for PKDM support, Dr. Benjamin C. Milgram for

useful chemistry discussions, and Dr. Margaret Chu-Moyer for

critical review of the manuscript.

pyrrolo[3,4-c][1,6]naphthyridin-4(2H)-one (54-d-Enantiomer of Ac-

1

tive Intermediate). Obtained 428 mg. H NMR (400 MHz, DMSO-

d₆) δ 8.35 (d, J = 0.62 Hz, 1H), 8.33 (dd, J = 0.88, 5.65 Hz, 1H), 7.02

(d, J = 5.70 Hz, 1H), 3.54−3.65 (m, 2H), 3.43 (dd, J = 7.26, 11.51 Hz,

1H), 3.25 (s, 3H), 3.10−3.21 (m, 2H), 0.74 (d, J = 6.84 Hz, 3H).

Peak 2: (3S,3aR,9bR)-3,5-Dimethyl-3,3a,5,9b-tetrahydro-1H-

pyrrolo[3,4-c][1,6]naphthyridin-4(2H)-one (54-c-Less Active Dia-

stereomer Peak 1 (Enantiomer Arbitrarily Assigned)). Obtained

ABBREVIATIONS USED

■

μW, microwave; CL, clearance; CL_int, intrinsic clearance; PPB,

plasma protein binding; DRG, dorsal root ganglion neuron;

HLM, human liver microsomes; RLM, rat liver microsomes;

MLM, mouse liver microsomes; MDR1, multidrug resistance

protein 1; MDCK, Madin−Darby canine kidney; BCRP, breast

cancer resistance protein; P_app, apparent passive permeability;

LE, ligand eﬃciency; LipE, lipophilic eﬃciency; V_d, volume of

distribution; TTX, tetrodotoxin; CNQX, 6-cyano-7-nitro-

quinoxaline-2,3-dione); APV, (2R)-amino-5-phosphonovaleric

acid; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic

acid; NMDA, N-methyl-^D-aspartic acid; GABA, γ-aminobutyric

acid; S_NAr, nucleophilic aromatic substitution

1

304 mg. H NMR (400 MHz, DMSO-d₆) δ 8.31−8.38 (m, 2H), 7.04

(d, J = 5.60 Hz, 1H), 3.37−3.53 (m, 2H), 3.24 (s, 4H), 2.63 (dd, J =

6.06, 9.90 Hz, 1H), 2.52−2.57 (m, 1H), 1.26 (d, J = 6.43 Hz, 3H).

Peak 3: (3S,3aS,9bS)-3,5-Dimethyl-3,3a,5,9b-tetrahydro-1H-

pyrrolo[3,4-c][1,6]naphthyridin-4(2H)-one (54-d-Active Intermedi-

ate). Obtained 267 mg. ¹H NMR (400 MHz, DMSO-d₆) δ 8.35 (d, J =

0.62 Hz, 1H), 8.33 (dd, J = 0.88, 5.65 Hz, 1H), 7.02 (d, J = 5.70 Hz,

1H), 3.54−3.65 (m, 2H), 3.43 (dd, J = 7.26, 11.51 Hz, 1H), 3.25 (s,

3H), 3.10−3.21 (m, 2H), 0.74 (d, J = 6.84 Hz, 3H).

Peak 4: (3R,3aS,9bS)-3,5-Dimethyl-3,3a,5,9b-tetrahydro-1H-

pyrrolo[3,4-c][1,6]naphthyridin-4(2H)-one (54-c-Less Active Dia-

stereomer Peak 2 (Enantiomer Arbitrarily Assigned). Obtained 298

mg. ¹H NMR (400 MHz, DMSO-d₆) δ 8.31−8.38 (m, 2H), 7.04 (d, J

= 5.60 Hz, 1H), 3.37−3.53 (m, 2H), 3.24 (s, 4H), 2.63 (dd, J = 6.06,

9.90 Hz, 1H), 2.52−2.57 (m, 1H), 1.26 (d, J = 6.43 Hz, 3H).

REFERENCES

■

27

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(32). See Huang et al., ref 27.

ASSOCIATED CONTENT

* Supporting Information

■

S

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.6b01496.

26−33, detailed chiral separation conditions; AM-3607, a

detailed suite of 1D and 2D NMR data (PDF)

Molecular formula strings (CSV)

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Reinold, H.; Smart, T. G.; Harvey, K.; Schutz, B.; Abo-Salem, O. M.;

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Corringer, P. J.; Zeilhofer, H. U. Phosphorylation state-dependent

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AUTHOR INFORMATION

Corresponding Author

*Phone: 617-444-5351. E-mail: hbregman@amgen.com.

■

ORCID

Howard Bregman: 0000-0001-8062-9770

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receptors. Br. J. Pharmacol. 2011, 164, 224−236.

(9) Xiong, W.; Cheng, K.; Cui, T.; Godlewski, G.; Rice, K. C.; Xu, Y.;

Zhang, L. Cannabinoid potentiation of glycine receptors contributes to

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One 2011, 6, e23886.

Present Addresses

^○For J.R.S.: C4 Therapeutics, 675 West Kendall Street,

Cambridge, Massachusetts 02142, United States.

^◇For K.M.: Biogen Idec., 225 Binney Street, Cambridge,

Massachusetts 02142, United States.

^¶For H.G. and E.F.D.: Merck & Co., Inc.; 33 Avenue Louis

Pasteur, Boston, Massachusetts 02115, United States.

⁺For P.P.: Pﬁzer, Inc.; 610 Main Street, Cambridge,

Massachusetts 02139, United States.

^□For R.T.L.: MD Anderson Cancer Center; 1515 Holcombe

Boulevard, Houston, Texas 77030, United States.

Author Contributions

All authors have contributed and have given approval to the

ﬁnal version of the manuscript.

Notes

The authors declare no competing ﬁnancial interest.

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ACKNOWLEDGMENTS

■

We thank the following people for their contributions: Grace Bi

and Larry Miller for chiral resolution and compound

puriﬁcation support, Iain Campuzano for the generation of

high-resolution mass spectroscopy data, Stephen Altmann for

T

DOI: 10.1021/acs.jmedchem.6b01496

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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U

DOI: 10.1021/acs.jmedchem.6b01496

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

The Discovery and Hit-to-Lead Optimization of Tricyclic Sulfonamides as Potent and Efficacious Potentiators of Glycine Receptors

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