Inhibitors of 15-Prostaglandin Dehydrogenase To Potentiate Tissue Repair

Article

pubs.acs.org/jmc

Inhibitors of 15-Prostaglandin Dehydrogenase To Potentiate Tissue

Repair

Monika I. Antczak,^†,⊥Yongyou Zhang,^‡,⊥Changguang Wang,^†Jennifer Doran,^†Jacinth Naidoo,^†

Sukesh Voruganti,^†Noelle S. Williams,^†Sanford D. Markowitz,*^,‡,§,∥and Joseph M. Ready*^,†

^†Department of Biochemistry, UT Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9038, United

States

^‡Department of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, United States

^§Seidman Cancer Center, University Hospitals of Cleveland, Cleveland, Ohio 44106, United States

^∥Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, Ohio 44106, United States

S

* Supporting Information

ABSTRACT: The enzyme 15-prostaglandin dehydrogenase

(15-PGDH) catalyzes the ﬁrst step in the degradation of

prostaglandins including PGE2. It is a negative regulator of

tissue repair and regeneration in multiple organs. Accordingly,

inhibitors of 15-PGDH are anticipated to elevate in vivo levels

of PGE2 and to promote healing and tissue regeneration. The

small molecule SW033291 (1) inhibits 15-PGDH with K_i=

0.1 nM in vitro, doubles PGE2 levels in vivo, and shows

eﬃcacy in mouse models of recovery from bone marrow

transplantation, ulcerative colitis, and partial hepatectomy. Here we describe optimized variants of 1 with improved solubility,

druglike properties, and in vivo activity.

has been shown to promote expansion of colonic stem cells in

culture,¹⁰and dmPGE2 has been shown to reduce disease

severity in a murine colitis model.¹¹Collectively, these

observations indicated that elevation of PGE2 levels in vivo

may potentiate tissue regeneration and repair.²

INTRODUCTION

■

Prostaglandin E2 (PGE2) is an endogenous signaling molecule

involved in pain, inﬂammation, and cell proliferation.¹It is

produced from arachidonic acid that is released from membranes

in response to stress, cytokines, or trauma (Figure 1). The

enzymes cyclooxygenase 1 or 2 (COX1/2) oxidize and cyclize

arachidonic acid to prostaglandin H2, which is then converted to

PGE2 by the action of prostaglandin E synthase (PGES). PGE2

is exported by dedicated transporters and can then activate one of

four G-protein-coupled receptors EP1−4. Binding of PGE2 to

these receptors activates second messengers including cyclic-

adenosine monophosphate and augments signaling through the

Wnt pathway.¹

Inhibitors of this pathway have been pursued as anti-

inﬂammatory, analgesic, and anticancer agents. However, we

were interested in developing strategies to increase rather than

decrease PGE2 levels in vivo. This objective emerged from the

observation that PGE2 promotes growth, diﬀerentiation, and

healing in a variety of cellular settings.²Accordingly, agents that

elevated PGE2 levels might aid healing and tissue regeneration.

In this context, PGE2 or the more metabolically stable analog

16,16-dimethyl-PGE2 (dmPGE2) augments hematopoiesis in

zebraﬁsh.^3,4Additionally, ex vivo exposure of murine bone

marrow or primate cord blood to dmPGE2 enhances their

eﬀectiveness in bone marrow transplantation assays.⁵⁻⁷A phase

1 study demonstrated that ex vivo treatment of human umbilical

cord blood with dmPGE2 may accelerate neutrophil recovery in

patients transplanted with the treated cells.^8,9Similarly, PGE2

PGE2 is degraded in vivo by the enzyme 15-prostaglandin

dehydrogenase (15-PGDH). This enzyme catalyzes the transfer

of the C15 hydride to NAD⁺, creating 15-keto-PGE2, which is

unable to bind to prostaglandin receptors.¹²We hypothesized

that inhibitors of 15-PGDH would block the degradation of

PGE2 and thereby elevate PGE2 levels in vivo. Encouragingly,

we found that the 15-PGDH knockout mouse has approximately

2-fold higher levels of PGE2 within the colon, lung, liver, and

bone marrow. Moreover, 15-PGDH-KO mice are completely

resistant to dextran sodium sulfate-induced colitis, display

increased hematopoietic capacity, and regrow liver tissue more

rapidly following partial resection compared to wild-type litter

mates.^13,14

Several research groups have disclosed inhibitors of 15-PGDH

(Figure 2A). For example, scientists at L’Oreal described a series

of tetrazoles¹⁵such as 2 that displayed partial enzyme inhibition

at 50 μM and aminooxyamides¹⁶including 3, which possessed an

IC₅₀of 6 μM against the puriﬁed enzyme (Figure 2). Cho and

colleagues have studied rhodanine alkylidenes such as compound

4.¹⁷This inhibitor was active against the enzyme in vitro (IC₅₀=

Received: February 21, 2017

Published: April 11, 2017

A

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Figure 1. Biological synthesis and degradation of prostaglandin E2 (PGE2).

Figure 2. (A) Inhibitors of 15-PGDH. (B) Potential binding model for 15-PGDH with PGE2 and inhibitor 1.

20 nM) and in A549 cells at 5 μM. Additionally, compound 4

showed activity in a cell-based model of wound healing. Finally, a

group from the NIH has disclosed several triazoles, exempliﬁed

by 5, and benzamidazoles, exempliﬁed by 6, with IC₅₀values as

low as 22 and 12 nM, respectively.¹⁸In a cell culture experiment,

these inhibitors displayed activities in the mid-nanomolar range.

While each of these lead compounds showed promising

inhibition in vitro, none of them have been reported to show

activity in any in vivo disease model.

BMT by reducing the risk of infection, minimizing bleeding, and

reducing the requirement for blood transfusion. Finally,

compound 1 showed no adverse eﬀects on weight, activity,

blood counts, or blood chemistry following 1 week of

administration of 20 mg/kg, which is 4-fold above the eﬃcacious

dose. Likewise, the 15-PGDH knockout mouse is healthy and

lives a normal lifespan.¹³These studies suggested that optimized

inhibitors of 15-PGDH would safely elevate PGE2 levels in vivo

and hasten recovery of multiple tissues.

We recently reported the discovery and characterization of the

sulfoxide SW033291 (1) as a tight binding inhibitor of 15-PGDH

with an apparent K_iof 0.1 nM.¹⁴In mice, 1 doubled PGE2 levels

in lungs, liver, colon, and bone marrow at 3 h after a dose of 10

mg/kg. Furthermore, we found that it (1) accelerated recovery of

neutrophils, platelets, and red blood cells following bone marrow

transplantation (BMT) in lethally irradiated mice, (2)

ameliorated the severity of colitis induced by dextran sodium

sulfate in mice, and (3) increased the rate and extent of liver

regeneration following partial liver resection in mice. In the

mouse BMT model, 15-PGDH inhibitor 1 accelerated

neutrophil recovery by approximately 1 week, with similar

eﬀects on platelets and erythrocytes. In humans, this activity is

anticipated to reduce morbidity and mortality associated with

Sulfoxide 1 is potent in vitro, active in cells, and eﬃcacious in

multiple mouse models of tissue regeneration. Nonetheless, we

recognized opportunities to improve its physicochemical

properties. For example, its high lipophilicity (cLogP = 5.8)¹⁹

was associated with low solubility and high plasma protein

binding. We were particularly eager to identify a highly soluble

inhibitor of 15-PGDH because intravenous (iv) administration is

preferred for drugs used to treat patients receiving bone marrow

or other hematopoietic stem cell transplants. We therefore

targeted discovery of a potent, safe inhibitor of 15-PGDH with

high aqueous solubility that would be suitable for iv

administration.

No crystal structure of 15-PGDH bound to any inhibitor or

PGE2 has been described, although the X-ray crystal structure of

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human 15-PGDH complexed with NAD⁺was solved by

Simeonov and co-workers.^18aConsistent with mutagenesis

studies, they proposed that the enzymatic processing of PGE2

involves deprotonation of the 15-OH by active site tyrosine 151

concurrent with hydride transfer to bound NAD⁺(Figure 2B).¹²

This mechanism is anticipated to form a partial negative charge

on the alcohol oxygen and a partial positive charge on the C15

carbon. In this context, we noted that the sulfoxide functionality

of 1 features a charge distribution that might mimic the charge

build-up in the transition state for oxidation of PGE2.

Additionally, the sulfoxide side chain might ﬁll a hydrophobic

binding site occupied by the C16−C20 alkyl chain of PGE2.

With this binding hypothesis in mind, we sought to decipher the

structural requirements for inhibition of 15-PGDH and to

discover optimized inhibitors of 15-PGDH suitable for use in

humans.

selected sulfoxides were accessed with preparative HPLC using a

chiral solid support.

Once installed, the sulfoxide group tended to dominate all

chemistry. For this reason, we frequently made changes to the

periphery of the inhibitors prior to oxidation to the sulfoxide. For

example, acetate 15 was hydrolyzed to the corresponding alcohol

and mesylated. Nucleophilic substitution proceeded oxidation to

the sulfoxide and ﬁnal ring closure (Scheme 1B). In this way the

terminal −CH₃of 1 was replaced with hydroxyl, halogens, or a

nitrile (16).

Methyl ester 17a also emerged as a versatile intermediate

(Scheme 2). Reduction with LiBH₄provided the primary alcohol

18, which could be diverted down several paths. First, azidation

with diphenylphosphoryl azide returned the benzylic azide 19.

Subsequent reduction and acylation led to amides and

carbamates 21. Alternatively, azide 19 could be oxidized and

cyclized prior to Staudinger reduction to form amine 24.

Sulfonylation provided the sulfonamide 25. A second set of

analogs derived from benzylic alcohol 18 was formed through

addition to isocyanates to form carbamates 22 after oxidation and

cyclization. Alternatively, sulfoxidation of 18 and base-mediated

cyclization yielded the primary alcohol 23a, which could be

acylated with carboxylic acids in the presence of a carbodiimide

to form the corresponding esters.

RESULTS AND DISCUSSION

■

Chemistry. The pyridine scaﬀold was assembled through an

annulation of 2-cyanothioacetamide with either β-diketones (7)

or enones (8, Scheme 1A). The reactions with enones were

Scheme 1. Synthesis of Pyridylthiophene Inhibitors of 15-

PGDH

A diverse set of amides arose from the para-methyl ester 17a

and meta-methyl ester 17b (Scheme 2B). In particular, following

formation of the thiophene ring (26), ester hydrolysis formed the

carboxylic acid 27, which could be converted to amides 28 using

HATU. Similarly, three alcohols (30−32) were accessible

through late-stage manipulation of the corresponding esters 29

(Scheme 2c). Speciﬁcally, addition of MeLi generated the tertiary

alcohol 30 while reduction with LiBH₄yielded primary alcohols

31 and 32.

An alternative synthesis of the thienopyridine scaﬀold is shown

in Scheme 3. This route proved particularly useful for analogs

featuring a methyl or hydrogen at the 4-position of the pyridine

(38, R′ = H or CH₃). In detail, an S_NAr/β-elimination sequence

between chloropyridine 33 and β-mercaptopropionate methyl

ester introduced the requisite sulfur atom in thiopyridone 36.

Alternatively, the same ring system could be accessed from

dichloropyridine 34. Thus, amination or Suzuki coupling proved

regioselective at the site distal to the nitrile to provide

chloropyridine 35. The sulfur was installed as before, and

alkylation, oxidation, and cyclization proceeded as described for

other thienopyridines to yield analogs 38.

Alkylidene malononitrile 39 served as the precursor to several

inhibitors featuring a pyrimidine ring (Scheme 4A). It underwent

a condensation with thioamides 40 to form thiopyrimidone 41.

Alkylation on sulfur, oxidation, and cyclization proceeded

analogously to what was observed in the pyridine series to

provide pyrimidine inhibitors of 15-PGDH (44). Alternatively,

commercially available dichloropyrimidine 45 provided the

opportunity to generate inhibitors lacking the exocyclic −NH₂

moiety. Thus, Suzuki coupling introduced two phenyl rings (46).

Lithiation and trapping with dibutyldithiane introduced the side

chain prior to ﬁnal oxidation to sulfoxide 48. Alternatively, S_NAr

reaction with piperdine introduced an aliphatic heterocycle (49).

Subsequent installation of the sulfoxide side chain and phenyl

ring generated the pyrimidine 51.

usually carried out under either air or oxygen balloon. Under an

inert atmosphere, the thiopyridone 9 was accompanied by a

thiopyridone lacking the nitrile. Additionally, we occasionally

observed conjugate addition of the thiopyridone to the enone.

Alkylation on sulfur with α-halocarbonyl compounds under basic

conditions led to cyclization onto the nitrile to yield the

thienopyridines 10. Similarly, alkylation of thiopyridone 9 with

chloromethyl thioethers yielded the dithianes 11. Mono-

oxidation to the sulfoxide followed by cyclization with hydroxide

or tert-butoxide base constructed the thienopyridine scaﬀold

(12). When the ﬁnal cyclization was performed on gram scale, it

was possible to isolate the corresponding sulfones 14 as minor

side products. Alternatively, the sulfoxide of 12 could be reduced

to a sulﬁde with TiCl₄and zinc dust (13). Single enantiomers of

To evaluate replacements for the thiophene ring within 1, we

synthesized two alternative scaﬀolds as shown in Scheme 5.

Known mercaptothiazole 52²⁰was alkylated with butyl bromide

and then subjected to Suzuki coupling conditions to introduce

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Scheme 2. Synthesis of Derivatives of 1 with Substitution on the Phenyl Ring

Biological Activity. Potential inhibitors were evaluated

using an in vitro enzyme assay, a cell based assay, and an in vivo

pharmacodynamic experiment. The in vitro experiment used

recombinant human 15-PGDH, assayed at an enzyme

concentration of approximately 3 nM. Sulfoxide 1 is a tight

binding inhibitor, and its IC₅₀approximates half the enzyme

concentration. Similar potencies were observed for many of the

optimized compounds shown below. In general, IC₅₀values less

than 3 nM are indistinguishable from each other, while inhibitors

with IC₅₀values greater than 3 nM are considered weaker

inhibitors than 1. Promising inhibitors of 15-PGDH were further

proﬁled in a cell-based assay using adenocarcinoma A549 cells. In

brief, addition of interleukin 1β (IL-1β) to these cells stimulates

PGE2 production. Inhibition of PGE2 metabolism leads to a

further increase in PGE2 levels in the culture media, which can be

quantiﬁed with an ELISA assay. The data are reported as a fold-

increase at a low dose (20 nM) and at a saturating dose (2.5 μM).

Illustrative results for inhibitor 1 are shown in Figure 3: at 20 nM,

sulfoxide 1 elevated PGE2 levels from 660 pg/mL to 1440 pg/

mL, a 2.2-fold increase. In all experiments in A549 cells, we

included 1 as a positive control. While the relative potency of

inhibitors was consistent across many experiments, the

magnitude of PGE2 induction varied. For this reason, in each

experiment we adjusted performance of compound 1 to its

average results from 12 independent experiments (1.9- and 2.7-

Scheme 3. Synthesis of 38

the thiophene. Final oxidation with hydrogen peroxide provided

thiazole sulfoxide 53. By use of a similar strategy, chloropyridine

54 was coupled with 2-thiophene boronic acid prior to reduction

of the nitro group to yield diaminopyridine 55. Condensation

with thiourea in the melt formed the thioimidazolone ring, while

alkylation and oxidation led to the ﬁnal imidazole sulfoxide 57.

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Scheme 4. Synthesis of Pyrimidines

Our ﬁrst experiments probed the requirement for a sulfoxide

side chain (Table 1). The initial characterization of 1 utilized the

racemic mixture, but we found that the enzyme activity resided

predominantly in the positive, R enantiomer.²¹While the single

enantiomer inhibitor (+)-1 showed greater potency in the cell-

based assay as expected, it showed markedly reduced solubility in

crystalline form compared to amorphous racemate. The

corresponding sulﬁde (13a) lost substantial activity, and the

sulfone (14a) was around 10-fold less active than the sulfoxide.

Attempts to replace the sulfoxide with carbonyls were not

successful, as a similarly sized ketone (10a), amide (10b), ester

(10c), and acid (10d) were all inactive against the recombinant

enzyme.

Scheme 5. Synthesis of Alternative Pyridyl-Fused

Heterocycles

The shape and composition of the sulfoxide side chain aﬀected

activity profoundly. For example, replacing the butyl chain with a

methyl group decreased activity approximately 100-fold (12a),

whereas an isopropyl, n-propyl, and n-pentyl (12b−d) side chain

showed similar activity as, although not better than, the n-butyl

side chain of 1. A larger n-hexyl replacement was deleterious

(12e). In an attempt to add polarity to the side chain to enhance

solubility, we explored addition of terminal methoxy (12f),

hydroxyl (16a), halogen (16b,c), and nitrile (16d) groups.

Encouragingly, we were able to incorporate an ether into the side

chain while maintaining robust activity both in vitro and in A549

cells (12f). Additionally, this modest chemical change was

accompanied by a roughly 10-fold increase in aqueous solubility.

By contrast, other polar groups on the sulfoxide side chain

decreased inhibitor potency in vitro and in cells.

Table 2 outlines studies aimed at identifying a more polar

replacement for the thiophene ring. We anticipated that N-

heterocycles might improve solubility and minimize the chance

of forming reactive metabolites such as epoxides. In this regard,

we discovered that the corresponding thiazole (12g) and oxazole

(12h) maintained full activity in vitro and were only slightly less

active than 1 in the cell-based PGE2 assay. Encouragingly,

thiazole 12g and oxazole 12h were roughly 4- and 9-fold more

soluble than 1, respectively. By contrast, the imidazole 12i was a

less potent inhibitor of 15-PGDH. As shown below, the phenyl

ring of 1 could be replaced with either a hydrogen or a methyl

group. In this context, the poor IC₅₀values of morpholine 38a

and unsubstituted pyridine 38b reveal the beneﬁt of a heteroaryl

substituent at the 6 position of the pyridine ring. Nonetheless,

the fact that the simpliﬁed analog 38b retains substantial

inhibition of 15-PGDH indicates that the thienopyridyl sulfoxide

Figure 3. Inhibitors of 15-PGDH elevate PGE2 levels in the medium of

IL-1β-treated A549 cells. Cells were treated with IL-1β and compound,

and PGE2 levels in the cell culture medium were determined after 16 h.

fold PGE2 induction at 20 nM and 2.5 μM, respectively) and

applied the same normalization factors to all other compounds

tested at the same time. Unadjusted data are presented in Table

S1 in the Supporting Information. Compounds showing

encouraging activity in vitro were further proﬁled for solubility

and for in vitro and in vivo ADME characteristics.

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Table 1. Structure−Activity Relationship for Sulfoxide Side Chain

a

Fold-increase in PGE2 levels in A549 cell culture medium relative to DMSO. Data are color-coded to indicate more active (blue), less active (red),

b

or equally active (white) compared with compound 1. Amorphous solid unless otherwise indicated. Solubility in pH 7 citrate buﬀer, 0.1 M.

c

d

Crystalline solid. Observed inhibition likely from trace 1.

scaﬀold contributes most of the binding energy. Taken together

with the des-phenyl derivative of 1 (i.e., 12j; see Table 4), these

results indicate that the heteroaryl ring at the 6-position increases

inhibitory activity by 2 orders of magnitude.

Table 2. Structure−Activity Relationship for 6-Pyridyl

Substituent

We investigated substitution on the phenyl ring of lead

compound 1 with the intention of identifying groups that would

improve solubility while maintaining high activity (Table 3).

Installing an ester or amide in the 4-position of the phenyl

decreased enzyme inhibition substantially (26a, 28a). The

corresponding carboxylic acid (27a) was a potent inhibitor and

displayed high aqueous solubility. However, it performed poorly

in the cell-based assay. More broadly, inhibitors carrying either a

positive or negative charge at physiological pH have not

performed as well as 1 in cell-based assays to date. The benzylic

alcohol (23a) and its corresponding ester (23b) were highly

active both in vitro and in the A549 cell-based assay. Moreover,

the alcohol showed approximately 50-fold improved solubility

compared to 1 and oﬀered the potential to form a prodrug.

Unfortunately, both inhibitors suﬀered from rapid degradation in

the presence of mouse liver S9 fractions, presumably due to ester

hydrolysis and oxidation to the corresponding aldehyde (23a, t_1/2

= 13 min; 23b, t_1/2= 2 min). We attempted to identify a more

stable inhibitor that maintained the activity proﬁle of 23a and

a

Fold-increase in PGE2 levels in A549 cell culture medium relative to

DMSO. Data are color-coded to indicate more active (blue), less active

(red), or equally active (white) compared with compound 1. pH 7

citrate buﬀer, 0.1 M.

b

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Table 3. Structure−Activity Relationship for the 4-Phenyl Ring

a

Fold-increase in PGE2 levels in A549 cell culture medium relative to DMSO. Data are color-coded to indicate more active (blue), less active (red),

b

or equally active (white) compared with compound 1. Amorphous solid unless otherwise indicated. Solubility in pH 7 citrate buﬀer, 0.1 M.

c

Solubility of racemate.

23b by synthesizing the analogous amine (24), amide (21a), urea

(21b), carbamate (22a), and sulfonamide (25). Among those,

the amide (21a) and urea (21b) maintained high enzyme

inhibitory properties, but both induced PGE2 only 80% as well as

1 at 2.5 μM, Similarly, a tertiary alcohol, 30, was a potent enzyme

inhibitor but was not as active as 1 in cells. Reasoning that the

rapid metabolism of 23a resulted in part from oxidation of the

benzylic alcohol, we prepared the ethanol derivative 31. This

compound displayed promising activity against the enzyme, but

it was less active than 1 in the cell-based assay. While the variation

between in vitro activity and cellular activity remains confusing,

we were gratiﬁed to ﬁnd that an inhibitor containing an ethylene

glycol moiety (32) was highly active both against the enzyme and

in cells. As we had observed with 1, the 15-PGDH inhibitor

activity resided predominantly in the (+)-enantiomer. Signiﬁ-

cantly, introduction of polar functionality on the phenyl ring

improved solubility >10-fold to 5 μg/mL and opened the

possibility of generating prodrugs.

In parallel with eﬀorts to add substituents to the phenyl ring of

1, we looked to replace this group with more polar substituents to

increase solubility. We were surprised to discover that removing

the phenyl ring altogether did not impact enzyme inhibition (12j,

Table 4). However, this inhibitor was nearly inactive in cells and

was rapidly metabolized in the presence of mouse liver S9

fractions (t_1/2= 13 min). Likewise replacing the phenyl ring with

a methyl group (38c) or an ethyl ester (12k) maintained good

potency against 15-PGDH but displayed lower activity than 1 in

cells. The corresponding carboxylic acid (12l) and tertiary amide

(12m) showed decreased inhibitory activity relative to the ester

(12k). We next replaced the phenyl ring of 1 with a variety of

heterocyclic rings. The bis-thiazole 12n showed excellent

potency against 15-PGDH and the highest activity we have

observed in cells. Surprisingly, however, its solubility was not

notably better than 1, despite a substantial increase in polarity

(cLogP = 3.3 vs 5.0).

Our ﬁrst clue toward developing highly soluble inhibitors of

15-PGDH came with compound 12o, which features a 3-pyridyl

appendage. It showed good activity both in vitro and in cells and

a ∼10-fold improvement in solubility relative to 1. More

signiﬁcantly, it showed pH-dependent solubility such that at pH

3 it was >50-fold more soluble than 1. Accordingly, we

incorporated a more basic heterocycle and obtained the 2-

Substitution on the phenyl ring meta to the pyridine was also

explored. The primary alcohol (23c), methyl ester (26b), and

tertiary amide (28b) were less active than 1, but the secondary

amide 28c showed good enzyme inhibition, robust activity in

cells, and excellent solubility.

G

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Table 4. Structure−Activity Relationship for the 4-Phenyl Ring

a

Fold-increase in PGE2 levels in A549 cell culture medium relative to DMSO. Data are color-coded to indicate more active (blue), less active (red),

b

c

or equally active (white) compared with compound 1. Amorphous solid unless otherwise indicated. Solubility in pH 7 citrate buﬀer, 0.1 M. n-Pr

sulfoxide in place of n-Bu sulfoxide. Solubility of the HCl salt in pH 7 water; ﬁnal pH = 5. Solubility of racemate.

d

e

substituted imidazole 12p. This inhibitor was highly active and

showed better solubility than 12o at both pH 7 and pH 3. An

isomeric imidazole, 12q, was even more soluble, exceeding 1 mg/

mL at pH 4. Additionally, the corresponding HCl salt was soluble

up to 4.3 mg/mL in neutral water. We attribute the improved

solubility of 12q vs 12p to an increase in the basicity of the

imidazole with a 2-methyl group (12q) compared to a 2-pyridyl

group (12p). With compound 1, the single enantiomer

crystalline form was >10-fold less soluble than the amorphous

racemate. Interestingly, however, optically active 12p showed a

similar solubility proﬁle as the racemic compound. Further

modifying 12q, we replaced the 2-methyl group with isopropyl

(2r), cyclopropyl (2s), and chloro groups (2t). The isopropyl

group appeared too big for the binding site, but both the

cyclopropyl and chloro groups were tolerated. However, only the

cyclopropyl-containing imidazole showed high activity in cells.

Finally, removing the N-methyl group from 12q decreased both

solubility and cellular activity compared to 1 (12u).

Incorporating either an imidazole or oxazole ring (44d, 44e)

greatly improved the solubility and provided active inhibitors.

Unfortunately, imidazole 44d had poor pharmacokinetic proper-

ties in mice, with only ¹/₃the total exposure of 1 following an ip

dose of 10 mg/kg body weight. Finally, the pyrimidine scaﬀold

allowed us to more fully probe the requirements for activity.

Removing the −NH₂from 44b to provide 48 was accompanied

by an approximately 20-fold loss in potency against the enzyme.

Replacement of the 4-phenyl ring with a saturated heterocycle

was further accompanied by a >10-fold loss in potency (51).

On the basis of the observations described above, we

synthesized several derivatives that incorporated multiple

structural changes from our previously reported 15-PGDH

inhibitor 1 (Scheme 6). Addition of a methyl group to the

thiazole ring of 12q provided 12v and only slightly decreased in

vitro and cell-based activity. Similarly, introducing an ether into

the sulfoxide side chain in (+)-12w or 12x maintained good

activity against 15-PGDH and in cells. Moreover, this change

dramatically improved solubility to >1 mg/mL at pH 7 for

(+)-12w and increased the in vitro metabolic stability relative to

12q: Both 12w and 12x had half-lives greater than 4 h in the

presence of mouse liver S9 fractions, whereas the half-life of

(+)-12q, featuring an n-butyl side chain, was 35 min under

identical conditions. The increased metabolic stability of the

ether side chain relative to the alkyl side chain could indicate that

We also explored inhibitors with a pyrimidine core (Table 5).

The direct analog to 1 (44a) retained good activity against the

enzyme and in cells, although solubility was not improved. While

replacing the thiophene appendage with a phenyl ring resulted in

decreased potency (44b), replacing the thiophene with a thiazole

led to 44c, an inhibitor with excellent activity in vitro and in cells.

This inhibitor also showed improved solubility relative to 1.

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(57) returned an inhibitor that was >1000X-fold less active

against the enzyme.

Table 5. Pyrimidine Inhibitors of 15-PGDH

The pharmacokinetic properties of selected inhibitors of 15-

PGDH were evaluated to identify compounds suitable for in vivo

experiments (Table 1). The active enantiomer of our initial lead

compound, (+)-1, was rapidly metabolized in the presence of

mouse liver S9 fractions and highly protein bound. Indeed, we

observed more rapid metabolism of the (+)-enantiomers with

several inhibitors. For example, the half-lives for the (R)-(+)-12q

and its enantiomer were 35 and 289 min, respectively. However,

in human liver S9 fractions, the more active R-enantiomer proved

to be at least as stable as the less active S-enantiomer. All of the

compounds shown in Table 6 achieved C_maxvalues in excess of

the in vitro IC₉₀values. Since these sulfoxides function as tight-

binding inhibitors, we anticipated that they would provide

substantial inhibition of 15-PGDH in vivo. Unfortunately, the

excellent in vitro stability of methyl ether 12w did not translate

into a longer in vivo half-life compared to (+)-1, potentially

because of the very low plasma protein binding.

Four of the most encouraging 15-PGDH inhibitors were

evaluated in vivo (Figure 4). Mice were treated with a single dose

of compound (2.5 mg/kg ip), and tissues were harvested at

various time points and analyzed for PGE2 levels with an ELISA

assay. Compared to untreated controls (t = 0 h), the inhibitors

(+)-1, (+)-12q, and (+)-44c doubled the PGE2 levels within the

bone marrow within 3 h of treatment. Levels decreased by 6 h

before returning to baseline by 12h. By contrast, (+)-32 was less

eﬀective than the other three analogs under these conditions.

Several aspects of these results are noteworthy. First, we

observed similar 2-fold elevation of PGE2 levels in the colon,

lung, and liver (see Figure S1). Second, this magnitude of PGE2

elevation appears maximal, as tissue PGE2 levels are consistently

2-fold higher in the 15-PGDH knockout mouse compared to

wild-type littermates. Third, the pharmacological eﬀects of (+)-1,

(+)-12q, and (+)-44c last much longer than the pharmacokinetic

half-lives might predict. For example, plasma levels of (+)-12q

drop below 10 nM within 3 h after an ip dose of 2.5 mg/kg body

weight. Nonetheless, tissue PGE2 levels remain elevated at 3 h.

a

Fold-increase in PGE2 levels in A549 cell culture medium relative to

DMSO. Data are color-coded to indicate more active (blue), less active

(red), or equally active (white) compared with compound 1.

b

Amorphous solid unless otherwise indicated. Solubility in pH 7

c

citrate buﬀer, 0.1 M. Solubility of racemate.

Scheme 6. Additional 15-PGDH Inhibitors

Since (+)-12q is a tight binding inhibitor of 15-PGDH (K_i^app

=

0.06 nM), we suspect that it remains bound to the enzyme even

after being cleared from the plasma. The levels of PGE2 continue

to increase, while the 15-PGDH is substantially inhibited, and

likely return to normal as more enzyme is synthesized. In an

important control experiment, the less active enantiomer

(−)-12q had no impact on tissue PGE2 levels (see Supporting

Information). Finally, while ethylene glycol 32 potently inhibited

15-PGDH in vitro and in cells and had excellent exposure and

stability in vivo, it only elevated PGE2 levels modestly in vivo.

Inhibitor (+)-12q displayed IC₅₀values of >10 μM against

important cytochrome P450 enzymes (CYP 1A, 2B6, 2C8, 2C9,

2C19, 2D6, 3A). The Safety44 panel of receptors, enzymes, and

channels (Cerep) indicated signiﬁcant binding only to the μ-

opioid channel and the adenosine A_2Areceptor at 10 μM.

Functional assays showed no activation or inhibition of the μ-

opioid channel up to 10 μM and an inconsequential IC₅₀of 900

nM against the A_2Areceptor. Finally, mice treated with 25 mg/kg

(+)-12q twice daily for 21 days, which is at least 10-fold higher

than the dose required for maximal elevation of PGE2 levels,

showed no ill eﬀect on behavior, weight, blood count, blood

chemistry, or initial liver pathology.

a

Fold-increase in PGE2 levels in A549 cell culture medium relative to

b

DMSO. Solubility of racemate.

oxidation of the n-butyl group is a dominant mode of

metabolism. Alternatively, the ether moiety could slow oxidation

of the sulfoxide to a sulfone through inductive eﬀects. As we

observed with 1, the sulfone version of 12q was about 30-fold less

active against 15-PGDH (14b). Finally, replacing the fused

thiophene ring of 12j with either a thiazole (53) or imidazole

I

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Table 6. ADME Properties of Selected 15-PGDH Inhibitors

compound

(S)-12q

(+)-1

(R)-12q

12w

32

83

44c

in vitro

a

mouse S9 (t_1/2, min)

13 (83)

35

289

224

>240

56

91

90

human S9 (t_1/2, min)

>240

78

a

plasma protein binding (mouse, %)

99.98

99

in vivo (mouse, 10 mg/kg ip)

_max(μM)

a

C

1.6 (1.5)

3.1

6.2

13.0

4.2

10.6

0.06

172

b

a

C

_3h(μM)

0.13 (0.26)

<0.01

47

0.06

116

a

AUC (min·μg/mL)

43 (83)

959

a

b

Data from racemic compound. Plasma concentration 3 h after dose.

signal (br s). Mass spectra (m/z) were recorded on an Agilent LC/MS

1100 or 1290 Inﬁnity using ESI ionization. All chemicals were used as

received unless otherwise noted.

Synthetic Methods. General Procedure for Synthesis of 2-

Sulﬁnylthieno[2,3-b]pyridine-3-amines As Exempliﬁed by the

Synthesis of 2-(Butylsulﬁnyl)-4-phenyl-6-(thiophen-2-yl)-

thieno[2,3-b]pyridin-3-amine (1). Step 1. 3-Phenyl-1-(thiophen-2-

yl)prop-2-en-1-one was prepared from benzaldehyde and 1-(thiophen-

2-yl)ethanone via aldol condensation using procedure described by

Azam.²²¹H NMR (400 MHz, CDCl₃) δ 7.88−7.80 (m, 2H), 7.67 (dd, J

= 4.9, 1.1 Hz, 1H), 7.66−7.59 (m, 2H), 7.47−7.34 (m, 4H), 7.18 (dd, J =

5.0, 3.8 Hz, 1H). ESI-MS (m/z): 215.1 [M + H]⁺.

Step 2. To a solution of 3-phenyl-1-(thiophen-2-yl)prop-2-en-1-one

(2.34 mmol, 500 mg) and cyanothioacetamide (7.0 mmol, 717 mg, 3.0

equiv) in ethanol (7 mL), a few drops of piperidine were added. The

reaction was stirred at reﬂux for 3 h. The solid that formed was collected,

suspended in acetic acid, and heated at 80 °C. After 30 min of heating,

the mixture was cooled to room temperature and ﬁltered to give 4-

phenyl-6-(thiophen-2-yl)-2-thioxo-1,2-dihydropyridine-3-carbonitrile

in 46% isolated yield. ¹H NMR (400 MHz, DMSO-d₆) δ 8.17 (d, J = 3.8

Hz, 1H), 7.96 (d, J = 5.0 Hz, 1H), 7.74−7.62 (m, 2H), 7.54 (dd, J = 5.1,

2.0 Hz, 3H), 7.31−7.19 (m, 1H), 7.01 (s, 1H). ESI-MS (m/z): 295.0 [M

+ H]⁺.

Step 3. A mixture of the thiopyridone from the previous step (0.34

mmol, 101 mg), butyl(chloromethyl)sulfane (0.34 mmol, 48 mg, 1.0

equiv), and Et₃N (0.51 mmol, 0.07 mL, 1.5 equiv) was stirred at reﬂux in

dry CH₃CN (0.35 mL) for 20 min. The reaction mixture was then

diluted with EtOAc and water. The organic phase was separated, and the

aqueous layer was extracted twice with EtOAc. The combined organic

phases were washed with saturated NaCl, dried over magnesium sulfate,

ﬁltered, and concentrated under reduced pressure. The residue was

puriﬁed by ﬂash chromatography to give 124 mg of 2-(((butylthio)-

methyl)thio)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile (92%). ¹H

NMR (400 MHz, CDCl₃) δ 7.70 (dd, J = 3.8, 1.1 Hz, 1H), 7.64−7.56

(m, 1H), 7.55−7.47 (m, 5H), 7.40 (d, J = 1.1 Hz, 1H), 7.14 (dd, J = 5.0,

3.8 Hz, 1H), 4.53 (s, 2H), 2.74 (t, J = 8.0 Hz, 2H), 1.72−1.57 (m, 2H),

1.49−1.34 (m, 2H), 0.90 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz,

CDCl₃) δ 163.0, 154.4, 153.7, 143.2, 135.9, 130.5, 130.1, 129.0, 128.6,

128.3, 127.2, 115.6, 114.1, 114.1, 103.2, 34.5, 32.1, 31.3, 22.0, 13.7. ESI-

MS (m/z): 397.0 [M + H]⁺.

Figure 4. Elevation of PGE2 levels in bone marrow following a single ip

dose of 2.5 mg/kg 15-PGDH inhibitor. C57BL/6J mice, n = 3 per time

point.

CONCLUSION

■

Taken together, these results reveal that heterocyclic sulfoxides

are potent inhibitors of 15-PGDH. Among these inhibitors,

sulfoxides provided the most potent activity, and nonpolar side

chains outperformed polar groups. The 2-thienyl ring in 1 could

be replaced with other aromatic and heteroaromatic rings, but

removal proved detrimental. By contrast, the phenyl substituent

from compound 1 could be removed or modiﬁed while retaining

in vitro activity, although metabolic stability was improved with

an aryl or heteroaryl ring at this position. Finally, the central ring

system of the inhibitors could feature either a thienopyridine or

thienopyimidine while retaining high potency.

Our results indicate that potent inhibition of 15-PGDH in

vitro is necessary but not suﬃcient for inhibition of the enzyme in

cells. Although we do not fully understand all the factors

involved, cell penetration is likely a key factor in determining

cellular activity. In this study, systematic modiﬁcation of our

initial inhibitor led to the discovery of two compounds with

improved solubility and druglike properties, (+)-12q and

(+)-44c. These inhibitors retain robust in vivo activity as

evidenced by their ability to elevate PGE2 levels in multiple

tissues. They appear to represent attractive lead compounds

toward the development of inhibitors of 15-PGDH for use in

accelerating recovery following bone marrow transplant and in

tissue regeneration more broadly.

Step 4. Acetic acid (0.90 mL) and hydrogen peroxide (0.57 mmol, 1.5

equiv, 30% solution in water) were added to the solution of 2-

(((butylthio)methyl)sulﬁnyl)-4-phenyl-6-(thiophen-2-yl)-

nicotinonitrile (0.38 mmol, 150 mg) in chloroform (0.90 mL). The

reaction mixture was stirred at 32 °C for 45 min. The reaction was then

diluted with EtOAc and washed with saturated NaHCO₃solution, dried

over magnesium sulfate, ﬁltered, and concentrated under reduced

pressure to give 153 mg of 2-(((butylthio)methyl)sulﬁnyl)-4-phenyl-6-

EXPERIMENTAL SECTION

■

General. All tested compounds have purity of >95% as judged by

HPLC analysis (UV detection at 210 nM). Compounds indicated as

optically active were >97% ee as judged by HPLC using the Chiralpak

ADH or Chiralcel ODH analytical HPLC columns. Chemical shifts δ are

in ppm, and spectra were referenced using the residual solvent peak. The

following abbreviations are used: singlet (s), doublet (d), triplet (t),

quartet (q), double doublet (dd), quintet (quin), multiplet (m), broad

1

(thiophen-2-yl)nicotinonitrile (98%). H NMR (400 MHz, CDCl₃) δ

7.75 (dd, J = 3.8, 1.1 Hz, 1H), 7.66−7.57 (m, 2H), 7.58−7.51 (m, 4H),

7.47 (s, 1H), 7.16 (dd, J = 5.0, 3.8 Hz, 1H), 4.74 (d, J = 13.0 Hz, 1H),

4.41 (d, J = 13.0 Hz, 1H), 2.97 (dt, J = 13.0, 8.2 Hz, 1H), 2.81 (dt, J =

12.9, 7.3 Hz, 1H), 1.94−1.76 (m, 2H), 1.53−1.38 (m, 2H), 0.94 (t, J =

7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 159.9, 154.7, 154.0, 142.4,

J

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135.4, 131.0, 130.4, 129.1, 128.8, 128.3, 127.8, 115.1, 115.0, 110.0, 103.1,

51.5, 490, 24.5, 22.5, 13.7. ESI-MS (m/z): 413.1 [M + H]⁺.

Ethyl 3-Amino-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]-

pyridine-2-carboxylate (10c). A mixture of 4-phenyl-6-(thiophen-

2-yl)-2-thioxo-1,2-dihydropyridine-3-carbonitrile (0.34 mmol, 100 mg),

ethyl 2-chloroacetate (0.54 mmol, 1.6 equiv), and EtONa (0.54 mmol,

1.6 equiv) in ethanol (1 mL) was stirred at reﬂux. When the reaction was

complete as judged by TLC, the reaction was diluted with EtOAc and

washed with 10% aq HCl. The organic phase was separated and aqueous

layer was extracted twice with EtOAc, dried over magnesium sulfate,

ﬁltered, and concentrated under reduced pressure to give 10c in 79%

yield. ¹H NMR (400 MHz, DMSO-d₆) δ 8.01 (d, J = 3.7 Hz, 1H), 7.87−

7.67 (m, 2H), 7.56 (d, J = 6.5 Hz, 5H), 7.36−6.90 (m, 1H), 5.73 (s, 2H),

4.23 (q, J = 7.1 Hz, 2H), 1.25 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz,

CD₂Cl₂) δ 165.3, 161.6, 161.4, 152.2, 147.6, 147.6, 143.9, 136.8, 129.2,

128.9, 128.8, 128.5, 128.3, 126.2, 120.5, 116.6, 60.50, 14.27. ESI-MS (m/

z): 381.1[M + H]⁺

3-Amino-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridine-2-

carboxylic Acid (10d). To a solution of 4-phenyl-6-(thiophen-2-yl)-2-

thioxo-1,2-dihydropyridine-3-carbonitrile (0.34 mmol, 100 mg) and

ethyl 2-chloroacetate (0.54 mmol, 1.6 equiv) in ethanol (1 mL), Et₃N

(0.54 mmol, 1.6 equiv) was added. The reaction was reﬂuxed for 20 min.

The reaction was then diluted with EtOAc and water. The organic phase

was separated, and aqueous layer was extracted twice with EtOAc. The

combined extractions were washed with saturated NaCl solution, dried

over magnesium sulfate, ﬁltered, and concentrated under reduced

pressure to aﬀord ethyl 2-((3-cyano-4-phenyl-6-(thiophen-2-yl)pyridin-

2-yl)thio)acetate, which was then dissolved in DMF and treated with 1

M aq NaOH at 50 °C to give 10d in 63% yield. ¹H NMR (400 MHz,

DMSO-d₆) δ 8.00 (dd, J = 3.7, 1.1 Hz, 1H), 7.79−7.64 (m, 2H), 7.55 (dt,

J = 7.6, 3.2 Hz, 5H), 7.16 (dd, J = 5.0, 3.7 Hz, 1H), 5.72 (s, 2H). ESI-MS

(m/z): 353.1[M + H]⁺.

Step 5. According to a procedure described by Kalugin,²³to the

solution of 2-(((butylsulﬁnyl)methyl)thio)-4-phenyl-6-(thiophen-2-

yl)nicotinonitrile (0.53 mmol, 220 mg) in DMF 2.10 mL/MeOH

1.05 mL was added KOH (0.32 mmol, 18 mg, 0.6 equiv, 1.7 M in water).

The reaction mixture was stirred at 35 °C for 30 min. Once complete,

the reaction was diluted with EtOAc and washed with 10% aq solution of

AcOH, the organic phase was separated, and the aqueous layer was

extracted twice with EtOAc, dried over magnesium sulfate, ﬁltered, and

concentrated under reduced pressure to give 211 mg of 2-

(butylsulﬁnyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-

1

amine (1) (96%). H NMR (400 MHz, CDCl₃) δ 7.8−7.29 (m, 7H),

7.32 (s, 1H), 7.10 (dd, J = 5.0, 3.7 Hz, 1H), 4.54 (s, 2H), 3.26 (ddd, J =

12.8, 9.1, 6.0 Hz, 1H), 3.09 (ddd, J = 12.8, 9.1, 6.6 Hz, 1H), 1.83−1.61

(m, 2H), 1.53−1.38 (m, 2H), 0.93 (t, J = 7.3 Hz, 3H). ¹³C NMR (101

MHz, CDCl₃) δ 161.4, 150.9, 146.6, 143.9, 143.6, 136.6, 129.3, 129.1,

128.8, 128.6, 128.4, 128.3, 126.0, 120.8, 117.1, 108.4, 55.3, 25.3, 21.9,

13.73. ESI-MS (m/z): 413.1 [M + H]⁺. Enantiomers of 1 were separated

on Chiralpak IC column using EtOH/Et₂NH 1000/1 with 0.5 mL/min

ﬂow rate. The ﬁrst peak was at 12.6 min and the second peak was at 14.3

23

min. Optical rotation: peak 1, [α]_D−90.04 (c 0.22, EtOH); peak 2,

23

[α]_D+104.18 (c 0.22, EtOH). The absolute stereochemistry was

assigned by X-ray crystallography. See Figure S3.

11-(3-Amino-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]-

pyridin-2-yl)pentan-1-one (10a). Step 1. A mixture of 4-phenyl-6-

(thiophen-2-yl)-2-thioxo-1,2-dihydropyridine-3-carbonitrile (0.068

mmol, 20 mg), Et₃N (0.11 mmol, 15 μL, 1.6 equiv), and 2-butyloxirane

(0.11 mmol, 11 mg, 1.6 equiv) in MeOH (500 μL) was stirred at rt.

When the reaction was completed as judged by TLC, the reaction

mixture was evaporated; the crude product was dissolved in CH₂Cl₂, and

the Dess−Martin periodinane (0.10 mmol, 1.5 equiv) was added at 0 °C.

The reaction mixture was stirred at rt for 2 h and then was quenched by

addition of 1:1 mixture of 20% Na₂S₂O₃/NaHCO₃solution. The

organic layer was separated, dried over magnesium sulfate, and the

solvent was removed under reduced pressure. The crude product was

puriﬁed by ﬂash chromatography to aﬀord 2-((2-oxohexyl)thio)-4-

2-(Methylsulﬁnyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]-

pyridin-3-amine (12a). 12a was prepared using the synthetic

1

procedures described for the preparation of 1. H NMR (500 MHz,

CDCl₃) δ 7.67−7.50 (m, 5H), 7.50−7.36 (m, 3H), 7.16−7.09 (m, 1H),

4.58 (s, 2H), 2.99 (s, 3H). ESI-MS (m/z): 371.1 [M + H]⁺.

4-Phenyl-2-(propylsulﬁnyl)-6-(thiophen-2-yl)thieno[2,3-b]-

pyridin-3-amine (12b). 12b was prepared using synthetic procedures

described for the preparation of analog 1. ¹H NMR (400 MHz, CDCl3)

δ 7.65 (dd, J = 3.8, 1.1 Hz, 1H), 7.61−7.49 (m, 4H), 7.49−7.41 (m, 3H),

7.12 (dd, J = 5.0, 3.7 Hz, 1H), 3.28 (ddd, J = 12.7, 8.4, 6.3 Hz, 1H), 3.07

(ddd, J = 12.7, 8.6, 7.0 Hz, 1H), 1.91−1.65 (m, 2H), 1.08 (t, J = 7.4 Hz,

3H). ¹³C NMR (101 MHz, CD₂Cl₂) δ 161.5, 150.9, 146.8, 143.8, 143.3,

136.8, 129.2, 128.8, 128.7, 128.6, 128.3, 126.1, 121.3, 117.1, 107.9, 57.1,

26.2, 16.8, 13.1. APCI-MS (m/z): 399.1 [M + H]⁺.

1

phenyl-6-(thiophen-2-yl)nicotinonitrile in 72% yield. H NMR (400

MHz, CDCl₃) δ 8.02 (s, 1H), 7.97 (d, J = 3.1 Hz, 1H), 7.71−7.59 (m,

2H), 7.55 (d, J = 3.2 Hz, 1H), 7.55−7.46 (m, 4H), 4.52 (s, 2H), 2.75 (t, J

= 7.8 Hz, 2H), 1.73−1.54 (m, 2H), 1.51−1.26 (m, 2H), 0.91 (t, J = 7.3

Hz, 3H). ESI-MS (m/z): 393.1 [M + H]⁺.

Step 2. To the solution of the ketone from step 1 (0.13 mmol, 50 mg)

in ethanol (0.5 mL) was added KOH (0.13 mmol, 2 mg, 1.0 equiv). The

reaction mixture was stirred at 50 °C for 30 min. Once complete, the

reaction was diluted with EtOAc and washed with 10% aq HCl. The

organic phase was separated and the aqueous layer was extracted twice

with EtOAc, dried over magnesium sulfate, ﬁltered, and concentrated

4-Phenyl-2-(isopropylsulﬁnyl)-4-phenyl-6-(thiophen-2-yl)-

thieno[2,3-b]pyridin-3-amine (12c). 12c was prepared using

1

synthetic procedures described for the preparation of analog 1. H

NMR (400 MHz, CDCl3) δ 7.64 (dd, J = 3.7, 1.1 Hz, 1H), 7.58−7.47

(m, 5H), 7.47−7.39 (m, 2H), 7.10 (dd, J = 5.0, 3.7 Hz, 1H), 4.59 (s, 2H),

3.38 (p, J = 6.8 Hz, 1H), 1.43 (d, J = 6.9 Hz, 3H), 1.25 (d, J = 6.8 Hz,

3H). ¹³C NMR (101 MHz, CD₂Cl₂) δ 161.8, 150.8, 146.7, 143.8, 143.7,

136.8, 129.2, 128.8, 128.7, 128.6, 128.3, 126.0, 121.5, 117.0, 105.9, 55.0,

15.9, 15.5. ESI-MS (m/z): 399.1 [M + H]⁺.

1

under reduced pressure to aﬀord ketone 10a in 98% yield. H NMR

(400 MHz, CDCl₃) δ 7.65 (dd, J = 3.8, 1.1 Hz, 1H), 7.62−7.56 (m, 2H),

7.55−7.48 (m, 4H), 7.40 (s, 1H), 7.13 (dd, J = 5.0, 3.8 Hz, 1H), 4.13 (s,

2H), 2.72 (t, J = 7.4 Hz, 2H), 1.72−1.56 (m, 2H), 1.42−1.25 (m, 2H),

0.88 (t, J = 7.3 Hz, 3H). ESI-MS (m/z): 393.1 [M + H]⁺.

2-(Pentylsulﬁnyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]-

pyridin-3-amine (12d). Hydrogen chloride gas was bubbled through

1-pentanethiol (10 mL, 8.1 mmol) cooled in a dry ice/acetone bath. At a

stabilized internal temperature of −74.2 °C, paraformaldehyde (3.02 g,

10.1 mmol) was added slowly via a solid addition funnel. Minimal

methylene chloride was used to rinse the clumped paraformaldehyde in

the funnel. Hydrogen chloride gas was bubbled for 3 h into the cold

stirring reaction, and then the reaction mixture was allowed to warm to

ambient temperature overnight. The aqueous layer was removed and the

organic layer was washed with brine, dried over Na₂SO₄, ﬁltered and

condensed to give (chloromethyl)(pentyl)sulfane with less than 2%

3-Amino-4-phenyl-N-propyl-6-(thiophen-2-yl)thieno[2,3-b]-

pyridine-2-carboxamide (10b). A mixture of 4-phenyl-6-(thiophen-

2-yl)-2-thioxo-1,2-dihydropyridine-3-carbonitrile (0.12 mmol, 35 mg),

2-chloro-N-propylacetamide (0.12 mmol, 16 mg, 1.0 equiv), and EtONa

(0.19 mmol, 1.6 equiv) in EtOH (1 mL) was stirred at 50 °C. When the

reaction was complete as judged by TLC, the reaction was diluted with

EtOAc and washed with 10% aq HCl. The organic phase was separated,

and the aqueous layer was extracted twice with EtOAc, dried over

magnesium sulfate, ﬁltered, and concentrated under reduced pressure to

aﬀord 10b in 61% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 7.98 (dd, J =

3.8, 1.2 Hz, 1H), 7.77 (t, J = 5.7 Hz, 1H), 7.73 (s, 1H), 7.70 (dd, J = 5.1,

1.1 Hz, 1H), 7.63−7.47 (m, 5H), 7.16 (dd, J = 5.1, 3.7 Hz, 1H), 5.80 (s,

2H), 3.12 (q, J = 6.7 Hz, 2H), 1.47 (h, J = 7.4 Hz, 2H), 0.83 (t, J = 7.4 Hz,

3H). ¹³C NMR (101 MHz, CD₂Cl₂) δ 165.4, 159.3, 159.2, 151.7, 147.4,

145.8, 143.9, 136.8, 129.1, 128.7, 128.6, 128.5, 128.3, 126.0, 121.5, 117.0,

41.3, 23.1, 11.7. ESI-MS (m/z): 394.1 [M + H]⁺.

1

dimer according to H NMR analysis. The crude product was used

without puriﬁcation. ¹H NMR (500 MHz, CDCl₃) δ 4.76 (d, J = 1.6 Hz,

2H), 2.75 (t, J = 7.5 Hz, 2H), 1.67 (p, J = 7.3 Hz, 2H), 1.47−1.26 (m,

4H), 0.92 (dd, J = 7.7, 6.2 Hz, 3H). Pentyl sulfoxide 12d was prepared

using (chloromethyl)(pentyl)sulfane and the synthetic procedures

described for the preparation of analog 1. ¹H NMR (500 MHz, CD₂Cl₂)

K

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Journal of Medicinal Chemistry

Article

δ 7.98−7.36 (m, 8H), 7.33−6.85 (m, 1H), 4.47 (s, 2H), 3.28−3.15 (m,

1H), 3.09−2.99 (m, 1H), 1.81−1.59 (m, 2H), 1.50−1.25 (m, 4H), 0.88

(t, J = 7.2 Hz, 3H). ESI-MS (m/z): 427.1 [M + H]⁺.

3.2 Hz, 1H), 7.65−7.39 (m, 6H), 4.63 (s, 2H), 3.28 (ddd, J = 12.8, 9.0,

6.2 Hz, 1H), 3.11 (ddd, J = 12.8, 9.0, 6.8 Hz, 1H), 1.85−1.63 (m, 2H),

1.56−1.42 (m, 2H), 0.94 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz,

CD₂Cl₂) δ 168.1, 161.2, 149.6, 147.3, 144.3, 142.9, 136.5, 129.3, 128.7,

128.6, 123.5, 122.2, 117.6, 108.9, 54.8, 25.0, 21.9, 13.5. ESI-MS (m/z):

414.1 [M + H]⁺.

2-(Hexylsulﬁnyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]-

pyridin-3-amine (12e). (Chloromethyl)(hexyl)sulfane (¹H NMR

(400 MHz, CDCl₃,) δ 4.76 (s, 2H), 2.76 (dd, J = 7.9, 6.9 Hz, 2H), 1.73−

1.59 (m, 2H), 1.51−1.20 (m, 6H), 0.97−0.82 (m, 3H)) was prepared

analogously to (chloromethyl)(pentyl)sulfane. It was used in the

synthesis of 12e using synthetic procedures described for the

2-(Butylsulﬁnyl)-6-(oxazol-2-yl)-4-phenylthieno[2,3-b]-

pyridin-3-amine (12h). (E)-1-(Oxazol-2-yl)-3-phenylprop-2-en-1-

one²⁶was used in the synthesis of oxazole 12h using procedures

1

preparation of analog 1. H NMR (500 MHz, CD₂Cl₂) δ 7.78−7.66

described for the synthesis of compound 1. H NMR (400 MHz,

(m, 1H), 7.63−7.46 (m, 7H), 7.27−7.02 (m, 1H), 4.11 (s, 2H), 3.43−

3.20 (m, 1H), 3.11 (ddd, J = 13.8, 9.4, 6.4 Hz, 1H), 1.89−1.63 (m, 2H),

1.58−1.39 (m, 4H), 1.40−1.21 (m, 2H), 0.91 (d, J = 6.8 Hz, 3H). ESI-

MS (m/z): 441.1 [M + H]⁺.

CDCl₃) δ 7.99 (s, 1H), 7.84 (d, J = 0.8 Hz, 1H), 7.58−7.41 (m, 5H),

7.33 (d, J = 0.8 Hz, 1H), 4.65 (s, 2H), 3.30 (ddd, J = 12.9, 8.8, 6.2 Hz,

1H), 3.10 (ddd, J = 12.8, 8.9, 6.9 Hz, 1H), 1.86−1.64 (m, 2H), 1.42−

1.54 (m, 2H), 0.93 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ

161.9, 160.2, 147.24, 143.8, 142.8, 140.3, 136.1, 129.5, 129.2, 128.8,

128.6, 123.5, 119.6, 109.1, 54.5, 25.0, 21.9, 13.7. ESI-MS (m/z): 398.1

[M + H]⁺.

2-((3-Methoxypropyl)sulﬁnyl)-4-phenyl-6-(thiophen-2-yl)-

thieno[2,3-b]pyridin-3-amine (12f). Step 1. 4-Phenyl-6-(thiophen-2-

yl)-2-thioxo-1,2-dihydropyridine-3-carbonitrile was alkylated with 4-

((chloromethyl)thio)butyl acetate as described for the synthesis of 1.

The crude mixture was condensed and puriﬁed by ﬂash chromatography

over SiO₂(0−30% EtOAc/hexanes to give 3-((((3-cyano-4-phenyl-6-

(thiophen-2-yl)pyridin-2-yl)thio)methyl)thio)propyl acetate in 26%

2-(Butylsulﬁnyl)-6-(1-methyl-1H-imidazol-2-yl)-4-phenyl-

thieno[2,3-b]pyridin-3-amine (12i). (E)-1-(1-Methyl-1H-imidazol-

2-yl)-3-phenylprop-2-en-1-one²⁷was used in the synthesis of imidazole

1

12i using procedures described for the synthesis of compound 1. H

1

yield. H NMR (400 MHz, CHCl₃) δ 7.72 (dd, J = 3.8, 1.1 Hz, 1H),

NMR (400 MHz, CDCl₃) δ 8.08 (s, 1H), 7.58−7.32 (m, 5H), 7.11 (d, J

= 1.1 Hz, 1H), 7.00 (d, J = 1.1 Hz, 1H), 4.58 (s, 2H), 4.19 (s, 3H), 3.27

(ddd, J = 12.7, 9.0, 6.0 Hz, 1H), 3.08 (ddd, J = 12.8, 9.1, 6.6 Hz, 1H),

1.79−1.60 (m, 2H), 1.56−1.37 (m, 2H), 0.92 (t, J = 7.3 Hz, 3H). ¹³C

NMR (101 MHz, CDCl₃) δ 160.7, 148.7, 146.8, 143.8, 143.2, 136.6,

129.2, 128.7, 128.6, 125.2, 121.5, 120.4, 107.3, 54.6, 36.8, 25.1, 21.9,

13.7. ESI-MS (m/z): 411.1 [M + H]⁺.

7.66−7.58 (m, 2H), 7.54 (dd, J = 4.2, 2.9 Hz, 4H), 7.44 (d, J = 1.3 Hz,

1H), 7.17 (dd, J = 5.0, 3.8 Hz, 1H), 4.55 (s, 2H), 4.18 (t, J = 6.3 Hz, 2H),

2.84 (t, J = 7.3 Hz, 2H), 2.05 (s, 3H), 2.05−1.97 (m, 2H). ¹³C NMR

(101 MHz, CDCl₃) δ 171.2, 162.3, 154.7, 153.9, 143.3, 136.0, 130.7,

130.3, 129.2, 128.7, 128.5, 127.4, 115.7, 114.4, 103.5, 63.1, 34.5, 28.9,

28.4, 21.1. ESI-MS (m/z): 441.0 [M + H]⁺.

Step 2. K₂CO₃(152 mg, 1.10 mmol) was added to a solution of the

acetate from step 1 (240.5 mg, 0.55 mmol) in methanol (4.0 mL) and

water (1.0 mL), and the reaction was stirred for 2.5 h. The mixture was

dried then diluted with EtOAc and washed twice with water and then

brine. The organic layer was dried over Na₂SO₄, ﬁltered, and

concentrated under reduce pressure to give 2-((((3-hydroxypropyl)-

thio)methyl)thio)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile in 98%

yield, which was used without further puriﬁcation. ¹H NMR (400

MHz, CHCl₃) δ 7.70 (dd, J = 3.8, 1.1 Hz, 1H), 7.63−7.58 (m, 2H), 7.53

(dd, J = 5.3, 1.7 Hz, 4H), 7.41 (s, 1H), 7.15 (dd, J = 5.0, 3.8 Hz, 1H), 4.54

(s, 2H), 3.76 (t, J = 6.0 Hz, 2H), 2.88 (t, J = 7.1 Hz, 2H), 1.93 (ddd, J =

13.2, 7.2, 6.1 Hz, 2H), 1.86−1.81 (m, 1H). ¹³C NMR (101 MHz,

CDCl₃) δ 162.8, 154.5, 153.8, 143.2, 135.9, 130.6, 130.2, 129.1, 128.7,

128.4, 127.3, 115.7, 114.3, 103.3, 61.5, 34.6, 31.8, 29.1 ESI-MS (m/z):

399.1 [M + H]⁺.

Step 3. A solution of the alcohol from step 2 (41.6 mg, 0.10 mmol) in

DMF (1.0 mL) was cooled in an ice bath before NaH (microspatula tip

full) was added. The suspension was stirred at room temperature for 15

min before the addition of MeI (34 μL, 0.55 mmol). The reaction was

stirred overnight, then diluted with EtOAc and washed several times

with water and then brine. The organic layer was dried over Na₂SO₄,

ﬁltered, and concentrated under reduce pressure. Puriﬁcation by ﬂash

chromatography on SiO2 (0−60% EtOAc/hexanes) provided 2-((((3-

methoxypropyl)thio)methyl)thio)-4-phenyl-6-(thiophen-2-yl)-

nicotinonitrile in 56% yield. ¹H NMR (400 MHz, CHCl₃) δ 7.72 (dd, J =

3.8, 1.1 Hz, 1H), 7.66−7.59 (m, 2H), 7.54 (h, J = 4.0, 3.6 Hz, 4H), 7.43

(s, 1H), 7.17 (dd, J = 5.0, 3.8 Hz, 1H), 4.55 (s, 2H), 3.49 (t, J = 6.1 Hz,

2H), 3.34 (s, 3H), 2.85 (t, J = 7.3 Hz, 2H), 2.02−1.88 (m, 2H). ¹³C

NMR (101 MHz, CDCl₃) δ 163.0, 154.6, 153.9, 143.3, 130.6, 130.3,

129.2, 128.7, 128.4, 127.3, 115.7, 114.3, 103.4, 71.1, 58.8, 34.7, 29.5,

29.3. ESI-MS (m/z): 413.1 [M + H]⁺.

2-(Butylsulﬁnyl)-6-(thiophen-2-yl)thieno[2,3-b]pyridin-3-

amine (12j). 6-(Thiophen-2-yl)-2-thioxo-1,2-dihydropyridine-3-car-

bonitrile²⁸was used in the synthesis of the 4-unsubstituted analog 12j

using procedures described for the synthesis of compound 1. ¹H NMR

(400 MHz, CDCl₃) δ 7.79 (d, J = 8.5, 1H), 7.65−7.49 (m, 2H), 7.39 (dt,

J = 5.1, 0.7 Hz, 1H), 7.06 (dd, J = 5.0, 3.7, Hz, 1H), 5.20 (s, 2H), 3.26

(ddd, J = 12.8, 9.0, 6.2 Hz, 1H), 3.10 (ddd, J = 12.8, 9.1, 6.6 Hz, 1H),

1.78−1.60 (m, 2H), 1.55−1.39 (m, 2H), 0.92 (t, J = 7.3 Hz, 3H). ESI-

MS (m/z): 337.1 [M + H]⁺.

Ethyl 3-Amino-2-(propylsulﬁnyl)-6-(thiophen-2-yl)thieno-

[2,3-b]pyridine-4-carboxylate (12k). Ethyl 2,4-dioxo-4-(thiophen-

2-yl)butanoate²⁹was used in the synthesis of ester 12k using procedures

1

described for the synthesis of compound 1. H NMR (400 MHz,

CDCl₃) δ 8.01 (s, 1H), 7.69 (dd, J = 3.8, 1.1 Hz, 1H), 7.46 (dd, J = 5.0,

1.1 Hz, 1H), 7.11 (dd, J = 5.0, 3.8 Hz, 1H), 6.09 (s, 2H), 4.49 (q, J = 7.1

Hz, 2H), 3.36−3.22 (m, 1H), 3.15−3.00 (m, 1H), 1.87−1.68 (m, 2H),

1.47 (t, J = 7.1 Hz, 3H), 1.07 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz,

CDCl₃) δ 166.5, 163.4, 151.5, 143.5, 142.9, 135.1, 129.4, 128.5, 126.6,

121.1, 116.8, 109.2, 63.1, 56.6, 16.8, 14.1, 13.3. ESI-MS (m/z): 394.9 [M

+ H]⁺.

3-Amino-2-(propylsulﬁnyl)-6-(thiophen-2-yl)thieno[2,3-b]-

pyridine-4-carboxylic Acid (12l). To a solution of ester 12k (20 mg,

0.051 mmol) in THF/MeOH/H₂O (0.11 mL:0.11 mL:0.036 mL) was

added LiOH (3.6 mg, 0.15 mmol, 3 equiv) at rt for 3 h to provide acid

12l in 40% yield following extraction into EtOAc and chromatographic

puriﬁcation with SiO₂. ¹H NMR (400 MHz, C₃D₇NO) δ 8.56 (s, 1H),

8.29 (d, J = 3.7 Hz, 1H), 8.19 (s, 1H), 7.99 (d, J = 5.0 Hz, 1H), 7.47−7.41

(m, 1H), 3.36 (ddd, J = 12.8, 8.4, 6.1 Hz, 1H), 3.24 (ddd, J = 12.8, 8.6, 6.8

Hz, 1H), 1.98−1.85 (m, 2H), 1.23 (t, J = 7.4 Hz, 3H). ¹³C NMR (101

MHz, DMF) δ 168.8, 162.6, 151.5, 143.3, 142.3, 137.7, 130.2, 129.1,

127.9, 121.3, 116.9, 111.7, 57.3, 16.7, 12.9. ESI-MS (m/z): 366.8 [M +

H]⁺.

3-Amino-2-(butylsulﬁnyl)-N,N-dimethyl-6-(thiophen-2-yl)-

thieno[2,3-b]pyridine-4-carboxamide (12m). Step 1. Ethyl 2-

(((butylthio)methyl)thio)-3-cyano-6-(thiophen-2-yl)isonicotinate was

prepared analogously to the corresponding intermediate thioether used

in the synthesis of compound 1. ¹H NMR (400 MHz, CDCl₃) δ 7.87 (s,

1H), 7.75 (dd, J = 3.8, 1.1 Hz, 1H), 7.55 (dd, J = 5.1, 1.1 Hz, 1H), 7.16

(dd, J = 5.0, 3.8 Hz, 1H), 4.50 (q, J = 7.2 Hz, 2H), 4.47 (s, 2H), 2.72 (t, J

= 7.2 Hz, 2H), 1.70−1.56 (m, 2H), 1.46 (t, J = 7.1 Hz, 3H), 1.43−1.33

(m, 2H), 0.89 (t, J = 7.3 Hz, 3H). ESI-MS (m/z): 393.1 [M + H]⁺.

Step 4. The sulﬁde from step 3 was oxidized with H₂O₂and cyclized

with KO^tBu as described for the synthesis of 1 to provide 12f. ¹H NMR

(400 MHz, CHCl₃) δ 7.64 (ddd, J = 7.2, 3.8, 1.7 Hz, 2H), 7.58−7.52 (m,

4H), 7.48−7.42 (m, 2H), 7.12 (qd, J = 3.7, 1.8 Hz, 1H), 4.57 (s, 2H),

3.50 (td, J = 6.1, 1.6 Hz, 2H), 3.40−3.29 (m, 4H), 3.26−3.13 (m, 1H),

2.09−1.95 (m, 2H). ESI-MS (m/z): 429.1 [M + H]⁺.

2-(Butylsulﬁnyl)-4-phenyl-6-(thiazol-2-yl)thieno[2,3-b]-

pyridin-3-amine (12g). 2-Bromo-1-(thiazol-2-yl)ethan-1-one²⁴was

converted to the corresponding Wittig reagent²⁵and then used to

prepare thiazole 12g using procedures described for the synthesis of

compound 1. ¹H NMR (400 MHz, CDCl3) δ 8.06 (s, 1H), 7.92 (d, J =

L

DOI: 10.1021/acs.jmedchem.7b00271

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Step 2. The ester from step 1 was hydrolyzed to 2-(((butylthio)-

methyl)thio)-3-cyano-6-(thiophen-2-yl)isonicotinic acid in 94% iso-

lated yield using conditions analogous to those described previously for

compound 12l. ¹H NMR (400 MHz, CDCl₃) δ 10.65 (s, 1H), 7.95 (s,

1H), 7.76 (dd, J = 3.8, 1.1 Hz, 1H), 7.57 (dd, J = 5.0, 1.0 Hz, 1H), 7.17

(dd, J = 5.0, 3.7 Hz, 1H), 4.46 (s, 2H), 2.72 (t, J = 7.2 Hz, 2H), 1.67−1.55

(m, 2H), 1.40 (h, J = 7.4 Hz, 2H), 0.90 (t, J = 7.3 Hz, 3H). ¹³C NMR

(101 MHz, CDCl₃) δ 167.1, 164.7, 154.8, 142.3, 140.1, 131.7, 128.9,

128.4, 114.3, 114.1, 102.8, 34.8, 32.1, 31.21, 22.0, 13.7. ESI-MS (m/z):

365.0 [M + H]⁺.

Step 3. Dimethylamine hydrochloride (14.8 mg, 0.18 mmol, 1.1

equiv) was added to a solution of the acid from step 2 (60 mg, 0.16

mmol), HATU (0.18 mmol, 69 mg, 1.1 equiv), and DMF (0.425 mL

followed by DIPEA (0.33 mmol, 0.057 mL, 2.0 equiv)). The solution

was stirred at room temperature for 3 h, then diluted with EtOAc and

washed with water. The organic layer was dried over Na₂SO₄, ﬁltered,

and concentrated under reduced pressure to give 2-(((butylthio)-

methyl)thio)-3-cyano-N,N-dimethyl-6-(thiophen-2-yl)isonicotinamide

in 53% yield following ﬂash chromatography on silica gel (20% EtOAc/

hexanes). ¹H NMR (400 MHz, CDCl₃) δ 7.67 (d, J = 3.8 Hz, 1H), 7.54

(d, J = 5.0 Hz, 1H), 7.34 (s, 1H), 7.15 (dd, J = 4.8, 3.9, 1H), 4.49 (s, 2H),

3.16 (s, 3H), 2.98 (s, 3H), 2.72 (t, J = 7.2 Hz, 2H), 1.62 (p, J = 7.7 Hz,

2H), 1.41 (h, J = 7.3 Hz, 2H), 0.90 (t, J = 7.3, 3H). ¹³C NMR (101 MHz,

CDCl₃) δ 165.4, 162.7, 154.7, 149.4, 142.6, 131.2, 128.7, 127.9, 113.9,

111.2, 100.9, 38.3, 35.0, 34.5, 32.1, 31.2, 21.9, 13.6. ESI-MS (m/z): 392.1

[M + H]⁺.

Steps 4 and 5. The amide from step 3 was oxidized and cyclized using

procedures described for the synthesis of compound 1 to provide 12m

in 17% isolated yield over two steps. ¹H NMR (400 MHz, CDCl₃) δ 7.66

(dd, J = 3.8, 1.1 Hz, 1H), 7.52−7.42 (m, 2H), 7.13 (dd, J = 5.0, 3.7 Hz,

1H), 3.34−3.23 (m, 1H), 3.21 (s, 3H), 3.15−3.02 (m, 1H), 2.96 (s, 3H),

1.79−1.62 (m, 2H), 1.55−1.36 (m, 2H), 0.93 (t, J = 7.3 Hz, 3H). ¹³C

NMR (101 MHz, CDCl₃) δ 168.0, 162.4, 151.4, 143.3, 141.6, 139.1,

129.3, 128.3, 126.4, 120.6, 112.7, 110.4, 55.3, 39.5, 35.3, 24.9, 21.9, 13.7.

ESI-MS (m/z): 408.1 [M + H]⁺.

2-(Butylsulﬁnyl)-4,6-di(thiazol-2-yl)thieno[2,3-b]pyridin-3-

amine (12n). (E)-1,3-Di(thiazol-2-yl)prop-2-en-1-one²⁵was con-

verted to bis-thiazole 12n using procedures described for the synthesis

of compound 1. ¹H NMR (400 MHz, CDCl₃) δ 8.48 (s, 1H), 8.01 (d, J =

3.1 Hz, 1H), 7.96 (d, J = 3.2 Hz, 1H), 7.61 (d, J = 3.3 Hz, 1H), 7.52 (d, J

= 3.1 Hz, 1H), 6.69 (s, 2H), 3.30 (ddd, J = 12.8, 9.2, 6.0 Hz, 1H), 3.14

(ddd, J = 12.8, 9.2, 6.4 Hz, 1H), 1.83−1.60 (m, 2H), 1.43−1.53 (m, 2H),

0.93 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 167.4, 166.0,

163.2, 149.8, 144.7, 144.3, 143.6, 138.0, 122.6, 122.5, 122.1, 117.5, 108.9,

54.5, 25.0, 21.9, 13.7. ESI-MS (m/z): 421.0 [M + H]⁺.

(s, 3H), 1.83−1.62 (m, 2H), 1.57−1.38 (m, 2H), 0.93 (t, J = 7.3 Hz,

3H). ¹³C NMR (101 MHz, CDCl₃) δ 167.6, 161.4, 149.9, 147.6, 144.3,

142.8, 134.7, 128.1, 126.6, 124.3, 122.4, 110.0, 118.7, 54.9, 31.3, 24.9,

21.9, 13.7, 13.6. ESI-MS (m/z): 432.1 [M + H]⁺. Enantiomers of 12q

were separated on a 1 cm Chiralpak AD column using 100% MeOH with

2.5 mL/min ﬂow rate. The (−)-(S)-enantiomer eluted at 13.4 min and

23

the (+)-(R)-enantiomer eluted at 19.9 min. Peak 1: [α]_D−63.86 (c

23

0.106, EtOH). Peak 2: [α]_D+64.75 (c 0.162, EtOH). The absolute

stereochemistry was assigned by X-ray crystallography. See Figure S4.

2-(Butylsulﬁnyl)-4-(2-isopropyl-1-methyl-1H-imidazol-5-yl)-

6-(thiazol-2-yl)thieno[2,3-b]pyridin-3-amine (12r). Imidazole 12r

was prepared using the procedures described for the synthesis of

compound 1. ¹H NMR (400 MHz, CDCl₃) δ 8.06 (s, 1H), 7.92 (d, J =

3.1 Hz, 1H), 7.51 (d, J = 3.2 Hz, 1H), 7.15 (s, 1H), 4.71 (s, 2H), 3.41 (s,

3H), 3.27 (ddd, J = 13.0, 8.5, 6.5 Hz, 1H), 3.19−2.98 (m, 2H), 1.83−

1.59 (m, 2H), 1.58−1.41 (m, 2H), 1.39 (d, J = 6.7 Hz, 6H), 0.94 (t, J =

7.3 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 167.7, 161.4, 155.7, 149.9,

144.3, 142.8, 134.8, 127.9, 126.4, 124.6, 122.5, 118.8, 109.7, 45.8, 30.0,

26.5, 24.9, 21.9, 21.2, 13.7. ESI-MS (m/z): 460.1 [M + H]⁺.

2-(Butylsulﬁnyl)-4-(2-cyclopropyl-1-methyl-1H-imidazol-5-

yl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-3-amine (12s). Imidazole

12s was prepared using the procedures described for the synthesis of

compound 1. ¹H NMR (400 MHz, CDCl₃) δ 8.04 (s, 1H), 7.91 (d, J =

3.1 Hz, 1H), 7.50 (d, J = 3.1 Hz, 1H), 7.07 (s, 1H), 4.77 (s, 2H), 3.51 (s,

3H), 3.27 (ddd, J = 12.9, 8.7, 6.4 Hz, 1H), 3.10 (ddd, J = 12.9, 8.8, 6.9 Hz,

1H), 1.95−1.78 (m, 1H), 1.81−1.62 (m, 2H), 1.58−1.37 (m, 2H),

1.17−0.98 (m, 4H), 0.93 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz,

CDCl₃) δ 167.7, 161.5, 152.4, 149.9, 144.3, 142.7, 134.6, 127.8, 126.6,

124.4, 122.5, 118.8, 109.6, 54.8, 30.9, 24.9, 21.9, 13.7, 7.5, 3.6. ESI-MS

(m/z): 458.1 [M + H]+.

2-(Butylsulﬁnyl)-4-(2-chloro-1-methyl-1H-imidazol-5-yl)-6-

(thiazol-2-yl)thieno[2,3-b]pyridin-3-amine (12t). Imidazole 12t

was prepared using the procedures described for the synthesis of

compound 1. ¹H NMR (400 MHz, CD₂Cl₂) δ 8.09 (s, 1H), 7.94 (d, J =

3.2 Hz, 1H), 7.56 (d, J = 3.2 Hz, 1H), 7.16 (s, 1H), 4.70 (s, 2H), 3.45 (s,

3H), 3.33−3.18 (m, 1H), 3.18−2.98 (m, 1H), 1.84−1.63 (m, 2H),

1.56−1.40 (m, 2H), 0.95 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz,

CD2Cl2) δ 167.5, 161.4, 150.0, 144.4, 142.2, 135.1, 133.5, 128.7, 128.5,

124.5, 122.6, 118.9, 110.0, 55.2, 32.0, 24.8, 21.9, 13.5. ESI-MS (m/z):

453.1 [M + H]⁺.

2-(Butylsulﬁnyl)-4-(2-methyl-1H-imidazol-5-yl)-6-(thiazol-2-

yl)thieno[2,3-b]pyridin-3-amine (12u). Imidazole 12u was prepared

1

using the procedures described for the synthesis of compound 1. H

NMR (400 MHz, CDCl₃) δ 10.51 (s, 1H), 8.10 (s, 1H), 7.89 (d, J = 3.2

Hz, 1H), 7.46 (d, J = 3.2 Hz, 1H), 7.40 (s, 1H), 3.31 (ddd, J = 12.8, 9.3,

5.8 Hz, 1H), 3.15 (ddd, J = 12.8, 9.3, 6.2 Hz, 1H), 2.42 (s, 3H), 1.79−

1.58 (m, 2H), 1.57−1.38 (m, 2H), 0.93 (t, J = 7.3 Hz, 3H). ESI-MS (m/

z): 418.1 [M + H]⁺.

2-(Butylsulﬁnyl)-4-(pyridin-3-yl)-6-(thiazol-2-yl)thieno[2,3-

b]pyridin-3-amine (12o). Pyridine 12o was prepared using the

1

procedures described for the preparation of analog 1. H NMR (400

MHz, CDCl₃) δ 8.80 (s, 1 H), 8.78 (dd, J = 4.9, 1.7 Hz, 1H), 8.04 (s,

1H), 7.91 (d, J = 3.2 Hz, 1H), 7.87−7.85 (m, 1H), 7.51 (d, J = 3.1 Hz,

1H), 7.47 (dd, J = 7.8, 4.8 Hz, 1H), 4.53 (s, 2H), 3.28 (ddd, J = 12.8, 8.8,

6.3 Hz, 1H), 3.11 (ddd, J = 12.8, 8.9, 6.9 Hz, 1H), 1.86−1.70 (m, 2H),

1.57−1.38 (m, 2H), 0.94 (t, J = 7.3 Hz, 3H). ESI-MS (m/z): 415.0 [M +

H]⁺.

2-(Butylsulﬁnyl)-4-(1,2-dimethyl-1H-imidazol-5-yl)-6-(4-

methylthiazol-2-yl)thieno[2,3-b]pyridin-3-amine (12v). Thiazole

12v was prepared using the procedures described for the synthesis of

compound 1. ¹H NMR (400 MHz, CD₂Cl₂) δ 8.04 (s, 1H), 7.12 (s, 1H),

7.08 (s, 1H), 4.75 (s, 2H), 3.40 (s, 3H), 3.26 (ddd, J = 13.1, 8.9, 6.2 Hz,

1H), 3.11 (ddd, J = 12.9, 8.9, 6.5 Hz, 1H), 2.50 (s, 3H), 2.47 (s, 3H),

1.79−1.64 (m, 2H), 1.57−1.42 (m, 2H), 0.95 (t, J = 7.3 Hz, 3H). ¹³C

NMR (101 MHz, CD₂Cl₂) δ 166.6, 161.4, 154.7, 150.0, 147.5, 142.7,

135.0, 128.0, 126.7, 124.4, 118.7, 117.2, 109.8, 55.1, 31.2, 24.9, 21.9,

17.0, 13.5, 13.3. ESI-MS (m/z): 446.1 [M + H]⁺.

4-(1,2-Dimethyl-1H-imidazol-5-yl)-2-((2-methoxyethyl)-

sulﬁnyl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-3-amine (12w).

Methyl ether 12w was prepared using the procedures described for

the synthesis of compound 1. ¹H NMR (400 MHz, CDCl₃) δ 8.05 (s,

1H), 7.92 (d, J = 3.2 Hz, 1H), 7.51 (d, J = 3.2 Hz, 1H), 7.11 (s, 1H), 4.73

(s, 2H), 3.88−3.82 (m, 1H), 3.75−3.62 (m, 1H), 3.57 (ddd, J = 13.1, 6.0,

3.9 Hz, 1H), 3.40 (s, 3H), 3.37 (s, 3H), 3.25 (ddd, J = 12.8, 8.0, 4.4 Hz,

1H), 2.48 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 167.6, 161.6, 150.0,

147.6, 144.3, 142.6, 134.8, 128.1, 126.6, 124.3, 122.5, 118.8, 110.0, 65.9,

59.1, 55.4, 31.3, 13.6. ESI-MS (m/z): 434.1 [M + H]⁺.

2-(Butylsulﬁnyl)-4-(1-methyl-1H-imidazol-2-yl)-6-(thiazol-2-

yl)thieno[2,3-b]pyridin-3-amine (12p). Imidazole 12p was prepared

1

using the procedures described for the synthesis of compound 1. H

NMR (400 MHz, CDCl₃) δ 8.13 (s, 1H), 7.91 (d, J = 3.2 Hz, 1H), 7.50

(d, J = 3.1 Hz, 1H), 7.24 (d, J = 1.2 Hz, 1H), 7.13 (d, J = 1.2 Hz, 1H),

5.78 (s, 2H), 3.80 (s, 3H), 3.26 (ddd, J = 12.8, 9.1, 6.0 Hz, 1H), 3.10

(ddd, J = 12.8, 9.2, 6.5 Hz, 1H), 1.82−1.57 (m, 2H), 1.56−1.35 (m, 2H),

0.92 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 167.6, 162.6,

149.4, 144.3, 143.8, 142.8, 134.4, 128.8, 123.9, 123.9, 122.5, 117.1, 110.3,

54.9, 35.0, 24.9, 21.9, 13.7. ESI-MS (m/z): 418.1 [M + H]⁺.

2-(Butylsulﬁnyl)-4-(1,2-dimethyl-1H-imidazol-5-yl)-6-(thia-

zol-2-yl)thieno[2,3-b]pyridin-3-amine (12q). Imidazole 12q was

prepared using the procedures described for the synthesis of compound

1. ¹H NMR (400 MHz, CDCl₃) δ 8.03 (s, 1H), 7.90 (d, J = 3.1 Hz, 1H),

7.50 (d, J = 3.2 Hz, 1H), 7.11 (s, 1H), 4.76 (s, 2H), 3.39 (s, 3H), 3.27

(ddd, J = 12.9, 8.7, 6.4 Hz, 1H), 3.09 (ddd, J = 12.8, 8.8, 6.9 Hz, 1H), 2.47

4-(1,2-Dimethyl-1H-imidazol-5-yl)-2-((3-methoxypropyl)-

sulﬁnyl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-3-amine (12x).

M

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Journal of Medicinal Chemistry

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Methyl ether 12x was prepared using the procedures described for the

synthesis of compound 1. ¹H NMR (400 MHz, acetone-d₆) δ 8.03 (s,

1H), 7.99 (d, J = 3.2 Hz, 1H), 7.82 (d, J = 3.2 Hz, 1H), 7.09 (s, 1H), 5.06

(s, 2H), 3.51 (s, 3H), 3.48 (t, J = 6.1 Hz, 2H), 3.26 (s, 3H), 3.26−3.18

(m, 1H), 3.18−3.12 (m, 1H), 2.43 (s, 3H), 2.00−1.89 (m, 2H). ESI-MS

(m/z): 448.1 [M + H]⁺.

6.4 Hz, 2H), 2.91−2.77 (m, 2H), 2.06 (d, J = 1.0 Hz, 3H), 2.03−1.94 (m,

2H).

Step 3. 4-Phenyl-6-(thiophen-2-yl)-2-thioxo-1,2-dihydropyridine-3-

carbonitrile (352.2 mg, 1.2 mmol) was alkylated with the chloromethyl

thioether from step 2 (602.3 mg, 3.1 mmol) using triethylamine (250

mL, 1.8 mmol) in acetonitrile (1.2 mL) as described for the synthesis of

compound 1. 3-((((3-Cyano-4-phenyl-6-(thiophen-2-yl)pyridin-2-yl)-

thio)methyl)thio)propyl acetate (15) was isolated in 26% yield

following ﬂash chromatography over SiO₂(0−30% EtOAc/hexanes).

¹H NMR (400 MHz, CHCl₃) δ 7.72 (dd, J = 3.8, 1.1 Hz, 1H), 7.66−7.58

(m, 2H), 7.54 (dd, J = 4.2, 2.9 Hz, 4H), 7.44 (d, J = 1.3 Hz, 1H), 7.17

(dd, J = 5.0, 3.8 Hz, 1H), 4.55 (s, 2H), 4.18 (t, J = 6.3 Hz, 2H), 2.84 (t, J =

7.3 Hz, 2H), 2.05 (s, 3H), 2.05−1.97 (m, 2H). ¹³C NMR (101 MHz,

CDCl₃) δ 171.2, 162.3, 154.7, 153.9, 143.3, 136.0, 130.7, 130.3, 129.2,

128.7, 128.5, 127.4, 115.7, 114.4, 103.5, 63.1, 34.5, 28.9, 28.4, 21.1. ESI-

MS (m/z): 441.0 [M + H]⁺.

Step 4. The thioether from step 4 was oxidized using procedures

described for the synthesis of compound 1 to provide 3-((((3-cyano-4-

phenyl-6-(thiophen-2-yl)pyridin-2-yl)thio)methyl)sulﬁnyl)propyl ace-

tate in 90% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.77 (dd, J = 3.8, 1.1

Hz, 1H), 7.67−7.61 (m, 2H), 7.57 (ddd, J = 6.9, 4.4, 2.1 Hz, 4H), 7.51 (s,

1H), 7.19 (dd, J = 5.0, 3.8 Hz, 1H), 4.81 (d, J = 13.1 Hz, 1H), 4.44 (d, J =

13.1 Hz, 1H), 4.22 (td, J = 6.4, 1.3 Hz, 2H), 3.09 (dt, J = 13.0, 8.1 Hz,

1H), 2.96−2.83 (m, 1H), 2.23 (tt, J = 7.3, 6.2 Hz, 2H), 2.04 (s, 3H). ¹³C

NMR (101 MHz, CDCl₃) δ 171.0, 159.8, 155.1, 154.2, 142.6, 135.6,

131.2, 130.6, 129.3, 129.0, 128.4, 128.0, 115.3, 115.2, 103.4, 62.9, 49.2,

48.4, 22.3, 21.0. ESI-MS (m/z): 457.1 [M + H]⁺

2-(Butylthio)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]-

pyridin-3-amine (13a). A ﬂask was charged with Zn dust (63 mg, 0.97

mmol, 8.0 equiv) and purged with nitrogen for 10 min. Dry THF (6 mL)

was then added, the gray suspension was cooled to 0 °C, and TiCl₄(0.48

mmol, 91 mg, 4.0 equiv) was added. After 10 min of stirring at 0 °C

sulfoxide 1 (50 mg, 0.12 mmol) in 2 mL of dry THF was added, and the

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

was quenched with 5 mL of 3 N NaOH and 5 mL of water. The aqueous

layer was extracted with EtOAc. The organic layer was separated, dried

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

crude product was puriﬁed by column chromatography. The puriﬁed

product was found to slowly oxidize back to sulfoxide 1 upon standing

exposed to air. ¹H NMR (400 MHz, CD₂Cl₂) δ 7.63 (dd, J = 3.7, 1.2 Hz,

1H), 7.57−7.50 (m, 3H), 7.50−7.46 (m, 2H), 7.43 (s, 1H), 7.41 (dd, J =

5.0, 1.1 Hz, 1H), 7.10 (dd, J = 5.0, 3.7 Hz, 1H), 3.98 (s, 2H), 2.76 (t, J =

7.3 Hz, 2H), 1.66−1.57 (m, 2H), 1.42 (h, J = 7.3 Hz, 2H), 0.90 (t, J = 7.4

Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 161.5, 149.5, 145.2, 144.3,

141.8, 137.5, 128.9, 128.8, 128.5, 128.1, 128.0, 125.3, 121.5, 116.6, 104.1,

36.9, 31.8, 21.7, 13.7. ESI-MS (m/z): 397.1 [M + H]⁺.

2-(Butylsulfonyl)-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]-

pyridin-3-amine (14a). Acetic acid (48.5 μL) and hydrogen peroxide

(0.031 mmol, 31.5 μL, 50% w/w) were added to the solution of

sulfoxide 1 (0.024 mmol, 10 mg) in chloroform (48.5 μL). The reaction

mixture was stirred at 32 °C for 4 h. After cooling, it was quenched with

saturated bicarbonate solution, diluted with chloroform and washed

with water, and dried over Na₂SO₄. The crude mixture was puriﬁed on a

short pipet column in 1% MeOH/DCM to provide sulfone 14a in 23%

Step 5. The sulfoxide from step 5 was cyclized using procedures

described for the synthesis of compound 1 to provide 3-((3-amino-4-

phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-2-yl)sulﬁnyl)propyl ace-

1

tate in 37% yield. H NMR (400 MHz, CDCl₃) δ 7.58 (m, 5H), 7.45

(dd, J = 5.0, 1.1 Hz, 2H), 7.41 (s, 1H), 7.10 (dd, J = 5.0, 3.7 Hz, 1H), 4.61

(s, 2H), 4.26−4.14 (m, 2H), 3.38−3.27 (m, 1H), 3.15 (dt, J = 13.0, 7.7

Hz, 1H), 2.16−2.06 (m, 2H), 2.05 (s, 3H). ¹³C NMR (101 MHz,

CDCl₃) δ 171.3, 171.0, 161.9, 151.3, 146.9, 143.82, 143.80, 136.8, 129.5,

129.1, 128.9, 128.83, 128.75, 128.4, 126.3, 121.3, 117.3, 106.4, 62.8, 60.5,

51.8, 22.8, 21.0, 14.3. ESI-MS (m/z): 457.1 [M + H]⁺.

Step 6. K₂CO₃(14.9 mg, 0.108 mmol) was added to a solution of 3-

((3-amino-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]pyridin-2-yl)-

sulﬁnyl)propan-1-ol (21.2 mg, 0.046 mmol) in methanol (0.4 mL) and

water (0.1 mL), and the reaction was stirred for 1 h. The methanol was

removed under reduced pressure, then the reaction mixture was

extracted with EtOAc and washed twice with water and then brine. The

organic layer was dried over Na₂SO₄, ﬁltered, and concentrated under

reduce pressure to give acetate 16a in 84% yield. ¹H NMR (400 MHz,

CHCl₃) δ 7.63 (dd, J = 3.8, 1.1 Hz, 1H), 7.55 (s, 4H), 7.46 (dd, J = 5.0,

1.1 Hz, 2H), 7.43 (s, 1H), 7.11 (dd, J = 5.0, 3.7 Hz, 1H), 4.60 (s, 2H),

3.77 (t, J = 5.8 Hz, 2H), 3.49−3.33 (m, 1H), 3.21 (dt, J = 13.4, 6.9 Hz,

1H), 2.17−1.96 (m, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 162.0, 151.3,

146.9, 143.8, 143.5, 136.8, 129.5, 129.0, 128.8, 128.4, 126.3, 121.4, 117.3,

106.4, 61.3, 52.8, 27.1, 14.3. ESI-MS (m/z): 415.1 [M + H]⁺.

2-((3-Chloropropyl)sulﬁnyl)-4-phenyl-6-(thiophen-2-yl)-

thieno[2,3-b]pyridin-3-amine (16b). Step 1. K₂CO₃(152 mg, 1.10

mmol) was added to a solution of acetate 15 (240.5 mg, 0.55 mmol) in

methanol (4.0 mL) and water (1.0 mL), and the reaction was stirred for

2.5 h. The mixture was dried and then diluted with EtOAc and washed

twice with water and then brine. The organic layer was dried over

Na₂SO₄, ﬁltered, and concentrated under reduce pressure to give 2-

((((3-hydroxypropyl)thio)methyl)thio)-4-phenyl-6-(thiophen-2-yl)-

nicotinonitrile in 98% yield, which was used without further puriﬁcation.

¹H NMR (400 MHz, CHCl₃) δ 7.70 (dd, J = 3.8, 1.1 Hz, 1H), 7.63−7.58

(m, 2H), 7.53 (dd, J = 5.3, 1.7 Hz, 4H), 7.41 (s, 1H), 7.15 (dd, J = 5.0, 3.8

Hz, 1H), 4.54 (s, 2H), 3.76 (t, J = 6.0 Hz, 2H), 2.88 (t, J = 7.1 Hz, 2H),

1.93 (ddd, J = 13.2, 7.2, 6.1 Hz, 2H), 1.86−1.81 (m, 1H). ¹³C NMR (101

MHz, CDCl₃) δ 162.8, 154.5, 153.8, 143.2, 135.9, 130.6, 130.2, 129.1,

128.7, 128.4, 127.3, 115.7, 114.3, 103.3, 61.5, 34.6, 31.8, 29.1 ESI-MS

(m/z): 399.1 [M + H]⁺.

1

yield. H NMR (400 MHz, CDCl3) δ 7.71 (dd, J = 3.8 1.2 Hz, 1H),

7.64−7.54 (m, 3H), 7.53−7.42 (m, 4H), 7.15 (dd, J = 5.0, 3.7 Hz, 1H),

5.09 (s, 2H), 3.38−3.02 (m, 2H), 1.92−1.67 (m, 2H), 1.52−1.28 (m,

2H), 0.92 (t, J = 7.4 Hz, 3H). ESI-MS (m/z): 428.8 [M + H]⁺.

2-(Butylsulfonyl)-4-(1,2-dimethyl-1H-imidazol-5-yl)-6-(thia-

zol-2-yl)thieno[2,3-b]pyridin-3-amine (14b). Sulfoxide 14b was

formed as a byproduct in the ﬁnal cyclization step in the synthesis of

analog 12q. ¹H NMR (400 MHz, CDCl₃) δ 8.08 (s, 1H), 7.95 (d, J = 3.2

Hz, 1H), 7.55 (d, J = 3.2 Hz, 1H), 7.16 (s, 1H), 5.31 (s, 2H), 3.41 (s,

3H), 3.24−3.18 (m, 2H), 2.51 (s, 3H), 1.87−1.73 (m, 2H), 1.49−1.36

(m, 2H), 0.95−0.85 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, CD₂Cl₂) δ

167.3, 161.2, 151.5, 148.0, 144.5, 144.2, 136.4, 127.9, 126.1, 123.4, 122.9,

119.0, 111.8, 56.6, 31.2, 24.6, 21.4, 13.3, 13.2. ESI-MS (m/z): 448.1 [M

+ H]⁺.

3-((3-Amino-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]-

pyridin-2-yl)sulﬁnyl)propan-1-ol (16a). Step 1. Porcine lipase (5.52

g) was added to a solution of 3-mercapto-1-propanol (5.03 g, 54.6

mmol) in ethyl acetate (70 mL).³⁰The reaction was heated at 28 °C for

12 days. Despite incomplete conversion, the mixture was ﬁltered and

condensed. Puriﬁcation was carried out on an automated ﬂash

chromatography system in 100% DCM to give 3-mercaptopropyl

acetate as an oil in 66% yield. ¹H NMR (400 MHz, CHCl₃) δ 4.17 (t, J =

6.2 Hz, 2H), 2.60 (q, J = 7.4 Hz, 2H), 2.05 (s, 3H), 1.93 (p, J = 6.6 Hz,

2H), 1.39 (t, J = 8.1 Hz, 2H).

Step 2. Hydrogen chloride gas was bubbled for 60 min into 3-

((chloromethyl)thio)propane-1-thiol (4.80 g, 35.7 mmol) in a dry ice/

acetone bath until the internal temperature stabilized before

paraformaldehyde (1.59 g, 53.3 mmol) was slowly added using a solid

addition funnel. The reaction was stirred at −78 °C for 1.5 h during

which hydrogen chloride bubbling was continued. HCl addition was

terminated, and the reaction was allowed to warm to ambient

temperature with stirring overnight. The crude mixture was diluted

with minimal DCM. The aqueous phase was removed and the organic

layer was washed with brine and dried over Na₂SO₄, ﬁltered, and

condensed to give 3-((chloromethyl)thio)propane-1-thiol as an oil in

80% yield of a 2.4:1 mixture of desired chloromethyl thioether and

dithiane dimer. ¹H NMR (400 MHz, CHCl₃) δ 4.74 (s, 2H), 4.17 (t, J =

Step 2. Triethylamine (180 μL, 1.32 mmol) was added to an ice-

cooled solution of the primary alcohol from step 1 (180.4 mg, 0.45

mmol) in CH₂Cl₂(4.5 mL), followed by the dropwise addition of

N

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methanesulfonyl chloride (81 mL, 1.0 mmol). The reaction mixture was

stirred in the ice batch for 30 min, after which all starting material had

been consumed. The reaction mixture was washed with brine, and the

organic layer was dried over Na₂SO₄, ﬁltered, and concentrated under

reduce pressure to give 3-((((3-cyano-4-phenyl-6-(thiophen-2-yl)-

pyridin-2-yl)thio)methyl)thio)propyl methanesulfonate in quantitative

yield. ¹H NMR (400 MHz, CHCl₃) δ 7.73 (dd, J = 3.6, 1.1 Hz, 1H), 7.62

(dd, J = 6.6, 2.9 Hz, 2H), 7.55 (ddd, J = 5.6, 4.2, 1.8 Hz, 4H), 7.45 (s,

1H), 7.18 (dd, J = 5.0, 3.8 Hz, 1H), 4.56 (s, 2H), 4.36 (t, J = 6.1 Hz, 2H),

3.03 (s, 3H), 2.90 (t, J = 7.0 Hz, 2H), 2.13 (p, J = 6.6 Hz, 2H). ¹³C NMR

(101 MHz, CDCl₃) δ 162.3, 154.47, 154.45, 153.8, 142.9, 135.7, 130.7,

130.2, 129.0, 128.7, 128.3, 127.4, 115.5, 114.32, 114.26, 68.19, 68.16,

68.1, 46.1, 46.09, 46.07, 37.3, 34.3, 28.6, 28.2. ESI-MS (m/z): 477.0 [M

+ H]⁺.

The organic layer was dried over Na₂SO₄, ﬁltered, and concentrated to

provide 2-((((3-cyanopropyl)thio)methyl)thio)-4-phenyl-6-(thiophen-

2-yl)nicotinonitrile in 89% yield, which was carried forward without

additional puriﬁcation. ¹H NMR (400 MHz, CHCl₃) δ 7.71 (dd, J = 3.4,

1.4 Hz, 1H), 7.60 (dt, J = 6.4, 1.9 Hz, 2H), 7.54 (ddt, J = 7.1, 4.2, 1.6 Hz,

4H), 7.44 (d, J = 1.4 Hz, 1H), 7.16 (ddd, J = 5.2, 3.8, 1.5 Hz, 1H), 4.53 (s,

2H), 2.92−2.84 (m, 2H), 2.52 (td, J = 7.1, 1.5 Hz, 2H), 2.08−2.00 (m,

2H). ESI-MS (m/z): 408.1 [M + H]⁺. Oxidation and cyclization were

performed as described for the synthesis of analog 1 to provide nitrile

16d in 45% yield. ¹H NMR (400 MHz, CDCl₃δ 7.65 (d, J = 3.7 Hz, 1H),

7.56 (d, J = 6.5 Hz, 4H), 7.52−7.43 (m, 3H), 7.13 (t, J = 4.4 Hz, 1H),

4.64 (s, 2H), 3.40 (dt, J = 13.2, 6.6, 5.9 Hz, 1H), 3.19 (dt, J = 13.0, 7.5 Hz,

1H), 2.59 (t, J = 7.1 Hz, 2H), 2.20 (p, J = 7.2 Hz, 2H). ESI-MS (m/z):

424.0 [M + H]⁺.

N-(4-(3-Amino-2-(butylsulﬁnyl)-6-(thiazol-2-yl)thieno[2,3-b]-

pyridin-4-yl)benzyl)acetamide (21a). Step 1. 2-Cyanothioacetamide

(275 mg, 2.74 mmol) and methyl (E)-4-(3-oxo-3-(thiazol-2-yl)prop-1-

en-1-yl)benzoate (250 mg, 0.915 mmol) were combined in a vial that

was evacuated and backﬁlled with O₂. Ethanol (2.75 mL) and piperdine

(2 drops) were added. The solution was sparged with O₂for a few

minutes and then stirred at 80 °C for 4 h. The solvent was evaporated

under reduced pressure, and the product was alkylated directly. Thus,

butyl(chloromethyl)sulfane (252.5 mg, 1.83 mmol) in acetonitrile (2

mL) was added to the thiopyridone followed by Et₃N (278 mg, 2.75

mmol). The solution was stirred at 80 °C for 20 min. The reaction

mixture was diluted with EtOAc and washed with water, dried over

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

solid was puriﬁed using automated ﬂash chromatography (20% EtOAc/

hexanes) to provide methyl 4-(2-(((butylthio)methyl)thio)-3-cyano-6-

Step 3. The mesylate from step 2 (48.2 mg, 0.10 mmol) was dissolved

in DMF (1.0 mL), and lithium chloride (67.3 mg, 1.59 mmol) was

added. The reaction mixture was stirred for 5 h, diluted with EtOAc, and

washed several times with water and then brine. The organic layer was

dried over Na₂SO₄, ﬁltered, and concentrated under reduce pressure.

The crude reaction mixture was puriﬁed via ﬂash chromatography over

silica gel (0−50% EtOAc/hexanes) to provide 2-((((3-chloropropyl)-

thio)methyl)thio)-4-phenyl-6-(thiophen-2-yl)nicotinonitrile in 69%

1

yield. H NMR (400 MHz, CHCl₃) δ 7.71 (dd, J = 3.8, 1.1 Hz, 1H),

7.64−7.58 (m, 2H), 7.56−7.52 (m, 4H), 7.43 (s, 1H), 7.16 (dd, J = 5.0,

3.8 Hz, 1H), 4.54 (s, 2H), 3.67 (t, J = 6.3 Hz, 2H), 2.92 (t, J = 7.0 Hz,

2H), 2.22−2.06 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 162.7, 154.6,

153.9, 143.2, 136.0, 130.7, 130.3, 129.2, 128.7, 128.4, 127.4, 115.6, 114.4,

103.4, 43.5, 34.5, 32.0, 29.6. ESI-MS (m/z): 417.0 [M + H]⁺.

Step 4. The thioether from step 3 was oxidized to 2-((((3-

chloropropyl)sulﬁnyl)methyl)thio)-4-phenyl-6-(thiophen-2-yl)-

nicotinonitrile using procedures described for the synthesis of

1

(thiazol-2-yl)pyridin-4-yl)benzoate (17a) as a solid in 24% yield. H

NMR (400 MHz, CDCl₃) δ 8.18 (d, J = 8.4 Hz, 2H), 8.02 (s, 1H), 7.98

(d, J = 3.1 Hz, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.58 (d, J = 3.2 Hz, 1H),

4.52 (s, 2H), 3.95 (s, 3H), 2.76 (t, J = 7.3 Hz, 2H), 1.64 (tt, J = 7.7, 6.3

Hz, 2H), 1.42 (h, J = 7.3 Hz, 2H), 0.91 (t, J = 7.3 Hz, 3H). ¹³C NMR

(101 MHz, CDCl₃) δ 166.9, 166.2, 163.6, 153.9, 152.4, 144.9, 139.6,

131.7, 130.3, 128.5, 123.7, 114.9, 105.7, 52.4, 34.8, 32.2, 31.2, 22.0, 13.7.

ESI-MS (m/z): 456.1 [M + H]⁺.

1

compound 1. H NMR (400 MHz, CHCl₃) δ 7.76 (dd, J = 3.9, 1.1

Hz, 1H), 7.62 (ddd, J = 5.1, 3.8, 2.1 Hz, 2H), 7.59−7.54 (m, 4H), 7.51 (s,

1H), 7.18 (dd, J = 5.0, 3.8 Hz, 1H), 4.74 (d, J = 13.1 Hz, 1H), 4.51 (d, J =

13.1 Hz, 1H), 3.80−3.63 (m, 2H), 3.29−3.16 (m, 1H), 2.96 (ddd, J =

13.0, 7.5, 6.4 Hz, 1H), 2.45−2.31 (m, 2H). ¹³C NMR (101 MHz,

CDCl₃) δ 159.8, 155.0, 154.2, 142.6, 135.6, 131.2, 130.6, 129.3, 129.0,

128.4, 127.9, 115.3, 115.2, 103.4, 49.6, 48.9, 43.6, 25.9. ESI-MS (m/z):

433.0 [M + H]⁺.

Step 5. The sulfoxide from step 4 was cyclized using procedures

described for the synthesis of compound 1 to provide chloride 16b in

88% yield. ¹H NMR (400 MHz, CHCl₃) δ 7.56 (m, 5H), 7.45 (m, 2H),

7.40 (s, 1H), 7.09 (t, J = 4.4 Hz, 1H), 4.61 (s, 2H), 3.66 (tt, J = 8.0, 4.0

Hz, 2H), 3.40 (dt, J = 14.1, 7.3 Hz, 1H), 3.24 (dt, J = 13.1, 7.6 Hz, 1H),

2.25 (p, J = 7.0 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ. 161.9, 151.4,

147.0, 143.9, 143.7, 136.8, 129.5, 129.0, 128.8, 128.4, 126.3, 121.3, 117.3,

106.2, 52.1, 43.4, 26.4. ESI-MS (m/z): 433.0 [M + H]⁺.

Step 2. To the solution of the ester from step 1 (336 mg, 0.74 mmol)

in THF (8.41 mL), LiBH₄(96.3 mg, 4.42 mmol) was added at 0 °C. The

reaction was stirred at room temperature for 36 h and then diluted with

EtOAc and water. The organic layer was dried over Na₂SO₄, ﬁltered, and

concentrated under reduced pressure to give 2-(((butylthio)methyl)-

thio)-4-(4-(hydroxymethyl)phenyl)-6-(thiazol-2-yl)nicotinonitrile

1

(18) in 96% yield, which was used without additional puriﬁcation. H

NMR (400 MHz, CDCl₃) δ 8.02 (s, 1H), 7.98 (d, J = 3.1 Hz, 1H), 7.65

(d, J = 8.1 Hz, 2H), 7.56 (d, J = 3.1 Hz, 1H), 7.52 (d, J = 8.0 Hz, 2H),

4.79 (d, J = 4.3 Hz, 2H), 4.52 (s, 2H), 2.75 (t, J = 7.4 Hz, 2H), 1.71−1.58

(m, 2H), 1.49−1.33 (m, 2H), 0.91 (t, J = 7.4 Hz, 3H). ¹³C NMR (101

MHz, CDCl₃) δ 167.3, 163.3, 154.8, 152.2, 144.8, 143.3, 134.6, 128.6,

127.3, 123.4, 115.3, 114.9, 105.6, 64.6, 34.7, 32.1, 31.2, 22.1, 13.6. ESI-

MS (m/z): 428.1 [M + H]⁺

Step 3. To a solution of alcohol 18 (70 mg, 0.16 mmol) in toluene was

added diphenylphosphoryl azide (55 mg, 0.20 mmol, 1.2 equiv) and 1,8-

diazabicyclo[5.4.0]undec-7-ene (39 mg, 0.26 mmol, 1.3 equiv), and the

reaction was stirred overnight at room temperature. Once complete as

judged by LC/MS, the reaction was diluted with EtOAc and water. The

organic phase was separated, and the aqueous layer was extracted twice

with EtOAc. The organic layer was dried over magnesium sulfate,

ﬁltered, and concentrated under reduced pressure. The residue was

puriﬁed by ﬂash chromatography to give 59 mg of 4-(4-(azidomethyl)-

phenyl)-2-(((butylthio)methyl)thio)-6-(thiazol-2-yl)nicotinonitrile

(19, 79% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.02 (s, 1H), 7.98 (d, J =

3.1 Hz, 1H), 7.67 (d, J = 7.5 Hz, 2H), 7.57 (d, J = 3.1 Hz, 1H), 7.48 (d, J

= 8.0 Hz, 2H), 4.52 (s, 2H), 4.44 (s, 2H), 2.75 (t, J = 7.4 Hz, 2H), 1.69−

1.58 (m, 2H), 1.50−1.35 (m, 2H), 0.91 (t, J = 7.3 Hz, 3H). ¹³C NMR

(101 MHz, CDCl₃) δ 167.1, 163.4, 154.3, 152.3, 144.9, 137.9, 135.3,

129.8, 128.9, 128.6, 123.5, 120.1, 115.2, 114.9, 110.0, 105.6, 54.2, 34.8,

32.15, 31.2, 21.9, 13.6 ESI-MS (m/z): 453.1 [M + H]⁺.

2-((3-Fluoropropyl)sulﬁnyl)-4-phenyl-6-(thiophen-2-yl)-

thieno[2,3-b]pyridin-3-amine (16c). Kryptoﬁx 222 (44.4 mg, 0.012

mmol), KF (6.1 mg, 0.10 mmol), and K₂CO₃(3.0 mg, 0.022 mmol)

were added to a vial containing the mesylate from step 2 in the synthesis

of 16b (54.2 mg, 0.11 mmol). DMF (1.1 mL) was added, and the

reaction was heated at 85 °C for 65 min. The cooled mixture was diluted

with EtOAc and washed several times with water and then brine. The

organic layer was dried over Na₂SO₄, ﬁltered, and condensed to provide

2-((((3-ﬂuoropropyl)thio)methyl)thio)-4-phenyl-6-(thiophen-2-yl)-

nicotinonitrile in 96%, which was carried forward without additional

puriﬁcation. Oxidation and cyclization using procedures described for

1

the synthesis of compound 1 provided ﬂuoride 16c in 32% yield. H

NMR (400 MHz, CHCl₃) δ 7.68 (dd, J = 3.8, 1.1 Hz, 1H), 7.56 (q, J =

2.7 Hz, 4H), 7.53−7.44 (m, 3H), 7.14 (dd, J = 5.1, 3.7 Hz, 1H), 4.65 (td,

J = 5.8, 3.2 Hz, 1H), 4.60 (s, 2H), 4.53 (td, J = 5.8, 3.1 Hz, 1H), 3.40 (dt, J

= 13.0, 7.3 Hz, 1H), 3.24 (dt, J = 13.1, 7.6 Hz, 1H), 2.19 (dtt, J = 26.4,

7.5, 5.7 Hz, 2H). ESI-MS (m/z): 417.1 [M + H]⁺.

4-((3-Amino-4-phenyl-6-(thiophen-2-yl)thieno[2,3-b]-

pyridin-2-yl)sulﬁnyl)butanenitrile (16d). DMF (1.1 mL) was added

to a vial containing the mesylate from step 2 of the synthesis of 16b (56.4

mg, 0.12 mmol) and potassium cyanate (76.9 mg, 1.18 mmol). The

reaction was heated to 85 °C for 4 h. The reaction mixture was cooled,

diluted with EtOAc, and washed several times with water, then brine.

Step 4. To a solution of azide 19 (407 mg, 0.89 mmol) in THF (8.5

mL) was added PPh₃(5.4 mmol, 1.41 g, 6.0 equiv), and the reaction

O

DOI: 10.1021/acs.jmedchem.7b00271

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Journal of Medicinal Chemistry

Article

mixture was stirred overnight at room temperature. Once complete,

water was added and reaction was stirred for additional 5 h at room

temperature and diluted with EtOAc. The organic phase was separated,

and the aqueous layer was extracted twice with EtOAc. The organic layer

was dried over magnesium sulfate, ﬁltered, and concentrated under

reduced pressure. The residue was puriﬁed by ﬂash chromatography to

give 4-(4-(aminomethyl)phenyl)-2-(((butylthio)methyl)thio)-6-(thia-

zol-2-yl)nicotinonitrile (20) in 62% yield. ¹H NMR (400 MHz, CDCl₃)

δ 8.01 (s, 1H), 7.97 (d, J = 3.1 Hz, 1H), 7.63 (d, J = 8.2 Hz, 2H), 7.55 (d,

J = 3.2 Hz, 1H), 7.47 (d, J = 8.2 Hz, 2H), 4.51 (s, 2H), 3.96 (s, 2H), 2.75

(t, J = 7.3 Hz, 2H), 1.72−1.52 (m, 2H), 1.42 (h, J = 7.3 Hz, 2H), 0.90 (t, J

= 7.3 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 163.3, 154.8, 152.2,

145.2, 144.8, 134.0, 128.7, 127.8, 123.41, 115.3, 114.9, 110.0, 105.6, 45.9,

34.7, 32.1, 31.2, 23.0, 13.6. ESI-MS (m/z): 427.1 [M + H]⁺.

Step 5. To a solution of amine 20 (30 mg, 0.07 mmol) in THF were

added acetic anhydride (21.4 mg, 0.21 mmol, 3.0 equiv) and pyridine

(16.6 mg, 0.21 mmol, 3.0 equiv), and the reaction was stirred at 50 °C

overnight. Upon completion, the reaction was diluted with EtOAc and

water. The organic phase was separated, and aqueous layer was extracted

twice with EtOAc. The organic layer was dried over magnesium sulfate,

ﬁltered, and concentrated under reduced pressure. The residue was

puriﬁed by ﬂash chromatography to give N-(4-(2-(((butylthio)methyl)-

thio)-3-cyano-6-(thiazol-2-yl)pyridin-4-yl)benzyl)acetamide in quanti-

tative yield. ¹H NMR (400 MHz, CDCl₃) δ 7.99 (s, 1H), 7.97 (d, J = 3.1

Hz, 1H), 7.61 (d, J = 8.2 Hz, 2H), 7.56 (d, J = 3.2 Hz, 1H), 7.43 (d, J =

8.1 Hz, 2H), 5.95 (t, J = 7.5 Hz, 1H), 4.51 (s, 2H), 4.49 (d, J = 7.4 Hz,

2H), 2.74 (t, J = 7.3 Hz, 2H), 2.04 (s, 3H), 1.72−1.54 (m, 2H), 1.49−

1.32 (m, 2H), 0.90 (t, J = 7.3 Hz, 3H). ESI-MS (m/z): 469.1 [M + H]⁺.

Step 6. N-(4-(2-(((Butylsulﬁnyl)methyl)thio)-3-cyano-6-(thiazol-2-

yl)pyridin-4-yl)benzyl)acetamide was prepared in 78% yield via

oxidation with hydrogen peroxide as described previously for the

synthesis of compound 1. ¹H NMR (400 MHz, CDCl₃) δ 8.07 (s, 1H),

7.98 (d, J = 3.1 Hz, 1H), 7.61 (d, J = 8.2 Hz, 2H), 7.58 (d, J = 3.1 Hz,

1H), 7.43 (d, J = 8.1 Hz, 2H), 6.04 (t, J = 6.3 Hz, 1H) 4.70 (d, J = 13.1

Hz, 1H), 4.50 (d, J = 5.9 Hz, 2H), 4.39 (d, J = 13.1 Hz, 1H), 2.96 (dt, J =

13.0, 8.1 Hz, 1H), 2.81 (dt, J = 12.9, 7.3 Hz, 1H), 2.05 (s, 3H), 1.91−

1.73 (m, 2H), 1.62−1.37 (m, 2H), 0.94 (t, J = 7.4 Hz, 3H). ESI-MS (m/

z): 485.1 [M + H]⁺.

4-(3-Amino-2-(butylsulﬁnyl)-6-(thiazol-2-yl)thieno[2,3-b]-

pyridin-4-yl)benzyl Ethylcarbamate (22a). Carbamate 22a was

prepared from alcohol 18 using synthetic procedures described for the

preparation of analog 21b. ¹H NMR (400 MHz, CD₃OD) δ 7.97−7.83

(m, 2H), 7.75−7.64 (m, 1H), 7.62−7.41 (m, 4H), 5.17 (s, 2H), 3.24

(ddd, J = 12.1, 5.9, 3.1 Hz, 1H), 3.15 (q, J = 7.5 Hz, 2H), 3.07 (ddd, J =

12.4, 6.5, 3.0 Hz, 1H), 1.78−1.55 (m, 2H), 1.56−1.38 (m, 2H), 1.11 (t, J

= 7.2 Hz, 3H), 0.94 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz, CD₃OD) δ

167.9, 167.1, 1615, 149.7, 147.8, 143.9, 143.4, 138.9, 135.7, 128.6, 127.5,

122.7, 122.7, 117.2, 108.7, 65.1, 54.5, 35.2, 24.9, 21.4, 13.9, 12.6. ESI-MS

(m/z): 515.1 [M + H]⁺.

(4-(3-Amino-2-(butylsulﬁnyl)-6-(thiazol-2-yl)thieno[2,3-b]-

pyridin-4-yl)phenyl)methanol (23a). Thioether 18 was oxidized

and cyclized to provide alcohol 23a in 16% isolated yield using

procedures described for the synthesis of compound 1. ¹H NMR (400

MHz, CDCl₃) δ 8.02 (s, 1H), 7.90 (d, J = 3.2 Hz, 1H), 7.59−7.40 (m,

5H), 4.80 (s, 2H), 4.63 (s, 2H), 3.27 (ddd, J = 12.8, 9.0, 6.1 Hz, 1H), 3.10

(ddd, J = 12.8, 9.1, 6.6 Hz, 1H), 1.78−1.61 (m, 2H), 1.55−1.40 (m, 2H),

0.93 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 168.2, 161.4,

149.6, 147.4, 144.2, 143.1, 142.3, 135.5, 128.8, 127.2, 123.5, 122.2, 117.8,

108.3, 64.5, 54.5, 25.0, 21.9, 13.7. ESI-MS (m/z): 444.1 [M + H]⁺.

4-(3-Amino-2-(butylsulﬁnyl)-6-(thiazol-2-yl)thieno[2,3-b]-

pyridin-4-yl)benzyl Dimethylglycinate (23b). N,N-Dimethylgly-

cine (3.5 mg, 0.034 mmol), 1-ethyl-3-(3-dimethylaminopropyl)-

carbodiimide (6.5 mg, 0.034 mmol), and DMAP (4.1 mg, 0.0334

mmol) were combined with (4-(3-amino-2-(butylsulﬁnyl)-6-(thiazol-2-

yl)thieno[2,3-b]pyridin-4-yl)phenyl)methanol23a (10 mg, 0.023

mmol) and dissolved in DMF (270 μL). The reaction mixture stirred

at room temperature overnight, then neutralized with 1 M NaOH,

washed with H₂O, and extracted with EtOAc. The organic layer was

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

The crude product was puriﬁed using ﬂash chromatography (7%

MeOH, 93% DCM) to give a quantitative yield of green solid product.

¹H NMR (400 MHz, CDCl₃) δ 8.02 (s, 1H), 7.90 (d, J = 3.2 Hz, 1H),

7.56−7.43 (m, 5H), 5.25 (s, 2H), 4.61 (s, 2H), 3.35−3.27 (m, 1H), 3.26

(s, 2H), 3.16−3.04 (m, 1H), 2.37 (s, 6H), 1.78−1.63 (m, 2H), 1.55−

1.39 (m, 2H), 0.93 (t, J = 7.3 Hz, 3H). ESI-MS (m/z): 529.1 [M + H]⁺.

(3-(3-Amino-2-(butylsulﬁnyl)-6-(thiazol-2-yl)thieno[2,3-b]-

pyridin-4-yl)phenyl)methanol (23c). Following the procedure for

the preparation of analog 23a, (E)-3-(3-oxo-3-(thiazol-2-yl)prop-1-en-

Step 7. The sulfoxide from step 6 was cyclized using the procedure

described for the synthesis of compound 1 to provide acetate 21a in 52%

yield. ¹H NMR (400 MHz, CD₂Cl₂) δ 8.01 (s, 1H), 7.89 (d, J = 3.1 Hz,

1H), 7.53 (d, J = 3.2 Hz, 1H), 7.47−7.41 (m, 4H), 6.25 (s, 1H), 4.59 (s,

2H), 4.50 (d, J = 6.1 Hz, 2H), 3.23 (ddd, J = 13.0, 9.2, 6.1 Hz, 1H), 3.08

(ddd, J = 12.9, 9.2, 6.5 Hz, 1H), 2.03 (s, 3H), 1.57−1.86 (m, 2H), 1.57−

1.35 (m, 2H), 0.93 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz, acetone) δ

169.1, 167.6, 161.2, 149.5, 147.4, 144.5, 142.1, 141.5, 135.1, 128.7, 127.7,

123.5, 122.7, 117.4, 110.9, 54.9, 42.5, 24.8, 22.1, 21.6, 13.1. ESI-MS (m/

z): 485.1 [M + H]⁺.

1

1-yl)benzoate was converted to alcohol 23c. H NMR (400 MHz,

CDCl₃) δ 8.01 (s, 1H), 7.88 (d, J = 3.1 Hz, 1H), 7.55−7.30 (m, 5H),

4.75 (s, 2H), 4.62 (s, 2H), 3.26 (ddd, J = 12.8, 9.1, 6.0 Hz, 1H), 3.09

(ddd, J = 12.8, 9.2, 6.5 Hz, 1H), 1.76−1.61 (m, 2H), 1.51−1.38 (m, 2H),

0.92 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 171.1, 168.1,

161.4, 149.6, 147.2, 144.2, 143.1, 136.5, 128.9, 128.8, 127.7, 127.1, 126.9,

123.2, 122.3, 117.7, 64.5, 54.5, 25.0, 21.9, 13.7. ESI-MS (m/z): 444.1 [M

+ H]⁺.

4-(4-(Aminomethyl)phenyl)-2-(butylsulﬁnyl)-6-(thiazol-2-yl)-

thieno[2,3-b]pyridin-3-amine (24). Azide 19 was oxidized and

cyclized as described for the synthesis of compound 1. To a solution of

the resultant 4-(4-(azidomethyl)phenyl)-2-(butylsulﬁnyl)-6-(thiazol-2-

yl)thieno[2,3-b]pyridin-3-amine (10 mg, 0.02 mmol) in THF was added

PPh₃(6 equiv), and the reaction mixture was stirred overnight at room

temperature. Once complete, water was added and reaction was stirred

for additional 5 h at room temperature and diluted with EtOAc. The

organic phase was separated, and the aqueous layer was extracted twice

with EtOAc. The organic layer was dried over magnesium sulfate,

ﬁltered, and concentrated under reduced pressure. The residue was

puriﬁed by ﬂash chromatography to give 7 mg of amine 24 (79% yield).

¹H NMR (400 MHz, acetone-d₆) δ 7.98 (s, 1H), 7.94 (d, J = 3.2 Hz, 1H),

7.78 (d, J = 3.2 Hz, 1H), 7.65−7.44 (m, 4H), 4.89 (s, 2H), 4.52 (s, 2H),

3.17 (ddd, J = 12.7, 8.9, 6.0 Hz, 1H), 3.11−2.99 (m, 1H), 1.78−1.59 (m,

2H), 1.60−1.36 (m, 2H), 0.91 (t, J = 7.3 Hz, 3H). ESI-MS (m/z): 443.1

[M + H]⁺.

N-(4-(3-Amino-2-(butylsulﬁnyl)-6-(thiazol-2-yl)thieno[2,3-b]-

pyridin-4-yl)benzyl)methanesulfonamide (25). Amine 24 (10 mg,

0.023 mmol) was suspended in DCM (300 μL) and Et₃N (3.5 μL,

0.025) and cooled to 0 °C under N₂. Methanesulfonyl chloride (1.8 μL,

0.023 mmol) was added to the reaction mixture, which was stirred at

1-(4-(3-Amino-2-(butylsulﬁnyl)-6-(thiazol-2-yl)thieno[2,3-b]-

pyridin-4-yl)benzyl)-3-ethylurea (21b). To the solution amine 20

(50 mg, 0.117 mmol) in THF was added ethyl isocyanate (13 mg, 0.23

mmol, 19 μL) at 0 °C. The reaction was stirred at room temperature for

1 h. During this time a solid formed, which was ﬁltered, washed with

small amount of EtOAc, and dried under reduced pressure to give 40 mg

of 1-(4-(2-(((butylthio)methyl)thio)-3-cyano-6-(thiazol-2-yl)pyridin-

1

4-yl)benzyl)-3-ethylurea (69% yield). H NMR (400 MHz, DMSO-

d₆) δ 8.13−8.02 (m, 2H), 7.91 (s, 1H), 7.73−7.61 (m, 2H), 7.49−7.37

(m, 2H), 6.40 (t, J = 6.1 Hz, 1H), 5.94 (t, J = 5.6 Hz, 1H), 4.62 (s, 2H),

4.27 (d, J = 6.0 Hz, 2H), 3.02 (q, J = 6.8 Hz, 2H), 2.68 (t, J = 7.5 Hz, 2H),

1.66−1.45 (m, 2H), 1.41−1.20 (m, 2H), 0.98 (t, J = 6.9 Hz, 3H), 0.82 (t,

J = 7.4 Hz, 3H). ESI-MS (m/z): 498.1 [M + H]⁺. The resulting thioether

was oxidized and cyclized using procedures described for the synthesis of

compound 1 to provide urea 21b in 37% yield over two steps. ¹H NMR

(400 MHz, CD₂Cl₂) 7.95 (s, 1H), 7.86 (d, J = 3.1 Hz, 1H), 7.50 (d, J =

3.1 Hz, 1H), 7.46−7.30 (m, 4H), 5.43 (s, 1H), 4.98 (s, 1H), 4.59 (s, 2H),

4.41 (d, J = 6.1 Hz, 2H), 3.32−3.13 (m, 3H), 3.14−3.00 (m, 1H), 1.92−

1.57 (m, 2H), 1.57−1.37 (m, 2H), 1.11 (t, J = 7.2 Hz, 1H), 0.93 (t, J = 7.3

Hz, 1H). ¹³C NMR (101 MHz, CD₃OD) δ 167.9, 161.5, 159.6, 149.7,

148.1, 143.9, 143.5, 142.1, 134.7, 128.5, 127.2, 122.8, 122.6, 117.2, 108.5,

54.5, 42.9, 34.5, 24.9, 21.4, 14.4, 12.6. ESI-MS (m/z): 514.1 [M + H]⁺.

P

DOI: 10.1021/acs.jmedchem.7b00271

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Journal of Medicinal Chemistry

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room temperature for 4 h before an additional methanesulfonyl chloride

(1 equiv). After stirring overnight, the reaction mixture was diluted with

EtOAc, washed with water and brine, and the organic layer was

separated. The combined organic layers were dried over sodium sulfate,

concentrated under reduced pressure and puriﬁed using automated

chromatography on silica gel (3% MeOH, 97% CH₂Cl₂) and then

preparative thin layer chromatography (100% EtOAc) to give 24 in 3.3%

isolated yield. ¹H NMR (400 MHz, acetone-d₆) δ 8.02 (s, 1H), 7.99 (d, J

= 3.1 Hz, 1H), 7.82 (d, J = 3.2 Hz, 1H), 7.70−7.58 (m, 4H), 4.84 (s, 1H),

4.46 (s, 2H), 3.23−3.13 (m, 1H), 3.12−3.02 (m, 1H), 2.93 (s, 3H),

1.76−1.63 (m, 2H), 1.55−1.41 (m, 2H), 0.92 (t, J = 7.3 Hz, 3H). ESI-

MS (m/z): 521.1 [M + H]⁺.

Methyl 4-(3-Amino-2-(butylsulﬁnyl)-6-(thiazol-2-yl)thieno-

[2,3-b]pyridin-4-yl)benzoate (26a). Methyl ester 26a was synthe-

sized from intermediate 17a according to the procedures described for

compound 1. ¹H NMR (400 MHz, CDCl₃) δ 8.19 (d, J = 7.5 Hz, 2H),

8.04 (s, 1H), 7.91 (d, J = 3.2 Hz, 1H), 7.67−7.54 (m, 2H), 7.50 (d, J = 3.2

Hz, 1H), 3.97 (s, 3H), 3.27 (ddd, J = 12.8, 8.9, 6.2 Hz, 1H), 3.10 (ddd, J

= 12.8, 9.0, 6.8 Hz, 1H), 1.81−1.63 (m, 2H), 1.54−1.39 (m, 2H), 0.93 (t,

J = 7.3 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 167.8, 166.2, 161.4,

149.6, 145.9, 144.2, 142.8, 140.9, 131.0, 129.9, 128.9, 122.9, 122.4, 117.4,

109.4, 54.7, 52.5, 25.0, 21.9, 13.7. ESI-MS (m/z): 472.1 [M + H]⁺.

Methyl 3-(3-Amino-2-(butylsulﬁnyl)-6-(thiazol-2-yl)thieno-

[2,3-b]pyridin-4-yl)benzoate (26b). Methyl ester 26b was synthe-

sized from intermediate 17b according to the procedures described for

compound 1. ¹H NMR (400 MHz, CDCl₃) δ 8.26−8.11 (m, 2H), 8.02

(s, 1H), 7.89 (d, J = 3.2 Hz, 1H), 7.76−7.56 (m, 2H), 7.49 (d, J = 3.1 Hz,

1H), 4.54 (s, 2H), 3.93 (s, 3H), 3.27 (ddd, J = 12.8, 9.0, 6.2 Hz, 1H), 3.09

(ddd, J = 12.8, 9.0, 6.7 Hz, 1H), 1.79−1.61 (m, 2H), 1.55−1.39 (m, 2H),

0.92 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 167.9, 166.1,

162.5, 161.4, 149.7, 145.9, 144.2, 142.7, 136.7, 133.0, 130.7, 130.4, 129.7,

128.8, 123.2, 122.4, 117.7, 54.7, 52.5, 25.1, 21.9, 13.7. ESI-MS (m/z):

472.1 [M + H]⁺.

4-(3-Amino-2-(butylsulﬁnyl)-6-(thiazol-2-yl)thieno[2,3-b]-

pyridin-4-yl)benzoic Acid (27a). To a solution of ester 26a (29 mg,

0.06 mmol) in THF (278 μL), MeOH (278 μL), and H₂O (93 μL),

LiOH (4.4 mg, 0.18 mmol) was added. After 3 h of stirring at room

temperature the reaction mixture was diluted with EtOAc and washed

with 1 M HCl. The organic layer was dried over Na₂SO₄, ﬁltered, and

concentrated under reduced pressure to give bright green solid in 84%

isolated yield. ¹H NMR (400 MHz, CDCl₃) δ 8.16 (d, J = 8.4 Hz, 2H),

8.05 (s, 1H), 7.95 (d, J = 3.2 Hz, 1H), 7.68−7.55 (m, 2H), 7.52 (d, J = 3.2

Hz, 1H), 3.40−3.24 (m, 1H), 3.24−3.04 (m, 1H), 1.83−1.65 (m, 2H),

1.55−1.37 (m, 2H), 0.93 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz,

CDCl₃) δ 168.7, 168.3, 161.6, 149.3, 146.2, 143.9, 143.3, 141.0, 131.1,

130.3, 129.0, 122.8, 122.6, 117.8, 108.9, 54.3, 25.1, 21.9, 13.7. ESI-MS

(m/z): 458.1 [M + H]⁺.

4-(3-Amino-2-(butylsulﬁnyl)-6-(thiazol-2-yl)thieno[2,3-b]-

pyridin-4-yl)-N,N-dimethylbenzamide (28a). Dimethylamine hy-

drochloride (4.82 mg, 0.059 mmol) was added to a solution of acid 27a

(24.6 mg, 0.05 mmol), HATU (22.5 mg, 0.06 mmol), and DMF (140

μL) followed by DIPEA (19 μL, 0.10 mmol). The solution was stirred at

room temperature for 3 h and then diluted with EtOAc and washed with

water. The organic layer was dried over Na₂SO₄, ﬁltered, and

concentrated under reduced pressure to give amide 28a in 59% yield.

¹H NMR (400 MHz, CDCl₃) δ 8.03 (s, 1H), 7.91 (d, J = 3.2 Hz, 1H),

for the synthesis of 28a. ¹H NMR (400 MHz, CDCl₃) δ 8.04−7.92 (m,

3H), 7.90 (d, J = 3.1 Hz, 1H), 7.64−7.51 (m, 3H), 7.50 (d, J = 3.2 Hz,

1H), 4.54 (s, 2H), 3.81 (t, J = 4.9 Hz, 2H), 3.62 (q, J = 4.7 Hz, 2H),

3.30−3.15 (m, 1H), 3.15−2.96 (m, 1H), 1.72−1.54 (m, 2H), 1.53−1.33

(m, 2H), 0.92 (t, J = 7.3 Hz, 3H). ESI-MS (m/z): 501.1 [M + H]⁺.

2-(4-(3-Amino-2-(butylsulﬁnyl)-6-(thiazol-2-yl)thieno[2,3-b]-

pyridin-4-yl)phenyl)propan-2-ol (30). To the solution of methyl

ester 26a (15 mg, 0.031 mmol) in THF (500 μL) at −78 °C, CH₃Li·LiBr

(1.5 M in ether, 104 μL, 0.156 mmol) was added, and the reaction

mixture was stirred for 3 h before an additional 2.0 equiv of CH₃Li·LiBr

was added. After an additional hour of stirring, the reaction mixture was

quenched with water and diluted with EtOAc. The organic layer was

separated, dried over sodium sulfate, and concentrated under reduced

pressure. The crude product was puriﬁed using automated chromatog-

raphy over silica gel (4% MeOH in CH₂Cl₂) to give tertiary alcohol 30 in

1

25% isolated yield. H NMR (400 MHz, (CD₃)₂CO) δ 8.03 (s, 1H),

7.98 (d, J = 3.2 Hz, 1H), 7.81 (d, J = 3.2 Hz, 1H), 7.77 (d, J = 8.6 Hz,

2H), 7.57 (d, J = 8.1 Hz, 2H), 4.87 (s, 1H), 4.84 (s, 1H), 3.25−3.13 (m,

1H), 3.11−3.01 (m, 1H), 1.76−1.63 (m, 2H), 1.59 (s, 6H), 1.53−1.40

(m, 2H), 0.92 (t, J = 7.3 Hz, 3H). ESI-MS (m/z): 472.1 [M + H]⁺.

2-(4-(3-Amino-2-(butylsulﬁnyl)-6-(thiazol-2-yl)thieno[2,3-b]-

pyridin-4-yl)phenyl)ethan-1-ol (31). Step 1. 2-(((Butylthio)-

methyl)thio)-4-(4-iodophenyl)-6-(thiazol-2-yl)nicotinonitrile was syn-

thesized from 1-(thiazol-2-yl)-2-(triphenyl-l5-phosphanylidene)ethan-

1-one (1.67 g, 4.31 mmol) and 4-iodobenzaldehyde (1.0 g, 4.3 mmol)

using the procedures described for the preparation of compound 1. ¹H

NMR (400 MHz, CDCl₃) δ 8.05 (d, J = 3.0 Hz, 1H), 7.94 (d, J = 16.1 Hz,

1H), 7.89 (d, J = 16.1 Hz, 1H), 7.75 (d, J = 8.4 Hz, 2H), 7.70 (d, J = 3.0

Hz, 1H), 7.41 (d, J = 8.4 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 181.4,

144.7, 144.4, 138.2, 134.1, 130.2, 126.6, 121.2, 97.5. ESI-MS (m/z):

341.9 [M + H]⁺.

Step 2. The aryl iodide from step 1 (114 mg, 0.218 mmol), Pd₂(dba)₃

(52 mg, 0.057 mmol), tri-(2-furyl)phosphine (46 mg, 0.20 mmol), and 4

Å molecular sieve (700 mg) were combined in a 4 mL vial, and this vial

was charged with argon. Dry and degassed diisopropylamine (1.5 mL)

and tert-butoxylacetylene (1.6 mL, 0.8 M in diethyl ether) were added to

the reaction vial sequentially at room temperature.³¹The iodide was

consumed as judged by TLC analysis after stirring overnight. The

reaction mixture was ﬁltered through an aluminum oxide column

(Brockmann I, basic, activated), and the desired fraction was eluted with

300 mL of EtOAc. The crude reaction product was concentrated under

reduced pressure, dissolved in hexanes/MeOH (1:10, 4 mL), and stirred

at 70 °C overnight. The reaction solvents were removed under reduced

pressure, and automated chromatography over silica gel (20% EtOAc,

80% Hex) provided methyl 2-(4-(2-(((butylthio)methyl)thio)-3-cyano-

6-(thiazol-2-yl)pyridin-4-yl)phenyl)acetate in 36% yield. ¹H NMR (400

MHz, CDCl₃) δ 8.01 (s, 1H), 7.97 (d, J = 3.1 Hz, 1H), 7.62 (d, J = 7.5

Hz, 2H), 7.55 (d, J = 3.1 Hz, 1H), 7.44 (d, J = 8.0 Hz, 2H), 4.51 (s, 2H),

3.71 (s, 3H), 3.70 (s, 2H), 2.74 (t, J = 7.5 Hz, 2H), 1.69−1.57 (m, 2H),

1.49−1.34 (m, 2H), 0.90 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz,

CDCl₃) δ 171.3, 167.2, 163.4, 154.6, 152.2, 144.9, 136.5, 134.2, 130.1,

128.7, 123.4, 115.3, 115.0, 105.6, 52.2, 40.9, 34.7, 32.1, 31.2, 22.0, 13.6,

11.7.

Step 3. The thioether from step 2 was oxidized and cyclized as

described for the synthesis of compound 1 to provide methyl 2-(4-(3-

amino-2-(butylsulﬁnyl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-4-yl)-

phenyl)acetate in 68% yield. ¹H NMR (400 MHz, CD₃)₂CO) δ 8.00 (s,

1H), 7.96 (d, J = 3.2 Hz, 1H), 7.79 (d, J = 3.2 Hz, 1H), 7.61−7.49 (m,

4H), 3.80 (s, 2H), 3.68 (s, 3H), 3.22−3.13 (m, 1H), 3.11−3.01 (m, 1H),

1.76−1.61 (m, 2H), 1.54−1.37 (m, 2H), 0.92 (t, J = 7.3 Hz, 3H). ESI-

MS (m/z): 486.1 [M + H]⁺.

Step 4. The methyl ester from step 3 was reduced with LiBH₄

following the procedure described previously for the preparation of

intermediate 18 to yield primary alcohol 31 in 30% yield following

chromatography on silica gel (EtOAc). ¹H NMR (400 MHz, CD₂Cl₂) δ

8.06 (s, 1H), 7.92 (d, J = 3.2 Hz, 1H), 7.53 (d, J = 3.2 Hz, 1H), 7.47−7.39

(m, 4H), 4.54 (m, 2H), 3.91 (t, J = 6.6 Hz, 2H), 3.28−3.20 (m, 1H),

3.15−3.05 (m, 1H), 2.96 (t, J = 6.6 Hz, 2H), 1.75−1.63 (m, 2H), 1.55−

1.42 (m, 2H), 0.94 (t, J = 7.3 Hz, 3H). ESI-MS (m/z): 458.1 [M + H]⁺.

7.66−7.44 (m, 5H), 3.36−3.21 (m, 1H), 3.14 (s, 3H), 3.13−3.06 (m,

1H), 3.02 (s, 3H), 1.81−1.64 (m, 2H), 1.55−1.41 (m, 2H), 0.93 (t, J =

7.3 Hz, 3H). ESI-MS (m/z): 485.1 [M + H]⁺.

3-(3-Amino-2-(butylsulﬁnyl)-6-(thiazol-2-yl)thieno[2,3-b]-

pyridin-4-yl)-N,N-dimethylbenzamide (28b). Amide 28b was

synthesized from ester 17b following the procedures described for the

1

synthesis of 28a. H NMR (400 MHz, chloroform-d) δ 8.02 (s, 1H),

7.90 (d, J = 3.1 Hz, 1H), 7.62−7.51 (m, 4H), 7.49 (d, J = 3.1 Hz, 1H),

4.59 (s, 2H), 3.27 (ddd, J = 12.8, 9.0, 6.1 Hz, 1H), 3.15−2.97 (m, 7H),

1.78−1.64 (m, 2H), 1.55−1.39 (m, 2H), 0.93 (t, J = 7.3 Hz, 3H). ESI-

MS (m/z): 485.1 [M + H]⁺.

3-(3-Amino-2-(butylsulﬁnyl)-6-(thiazol-2-yl)thieno[2,3-b]-

pyridin-4-yl)-N-(2-hydroxyethyl)benzamide (28c). Amide 28c

was synthesized from ester 17b following the procedures described

Q

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Article

2-(4-(3-Amino-2-(butylsulﬁnyl)-6-(thiazol-2-yl)thieno[2,3-b]-

pyridin-4-yl)phenoxy)ethan-1-ol (32). Step 1. 2-Acetylthiazole (534

μL, 5.15 mmol) was added to a solution of methyl 2-(4-formylphenoxy)-

acetate³²(1.0 g, 5.2 mmol) in MeOH (11 mL) under N₂. NaOMe (279

mg, 5.15 mmol) was added last and the reaction mixture stirred at room

temperature overnight. The reaction mixture was ﬁltered, and the

precipitate was washed with small amount of MeOH and then diluted

with CH₂Cl₂and washed with H₂O. The organic layer was separated,

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

provide solid methyl (E)-2-(4-(3-oxo-3-(thiazol-2-yl)prop-1-en-1-yl)

phenoxy)acetate in 21% isolated yield. ¹H NMR (400 MHz,

chloroform-d) δ 8.03 (d, J = 3.0 Hz, 1H), 7.95 (d, J = 15.9 Hz, 1H),

7.82 (d, J = 16.0 Hz, 1H), 7.70−7.61 (m, 3H), 6.92 (d, J = 8.8 Hz, 2H),

4.67 (s, 2H), 3.80 (s, 3H). ESI-MS (m/z): 304.1 [M + H].

(60% in mineral oil) was added. The reaction mixture was stirred at

reﬂux overnight and then allowed to cool to rt. EtOH (1.5 mL) was

added, and then volatile solvents were removed under reduced pressure.

Water (8 mL) was added and the solution was adjusted to pH 6 with

concentrated HCl before ﬁltration to give a crude solid 4-methyl-6-

morpholino-2-thioxo-1,2-dihydropyridine-3-carbonitrile that was used

directly in the next step without puriﬁcation. ESI (m/z): 236.1 [M +

H]⁺.

Step 4. The thiopyridone from step 3 was alkylated with

(chloromethyl)(butyl)sulfane, oxidized with hydrogen peroxide, and

cyclized with KO^tBu as described for the synthesis of compound 1 to

provide morpholine 38a. ¹H NMR (400 MHz, CDCl₃) δ 6.36 (s, 1H),

4.91 (s, 2H), 3.85−3.76 (m, 4H), 3.63−3.58 (m, 4H), 3.32−3.18 (m,

1H), 3.09−2.99 (m, 1H), 2.65 (s, 3H), 1.81−1.66 (m, 2H), 1.07 (t, J =

7.4 Hz, 3H). ESI (m/z): 340.1 [M + H]⁺.

2-(Butylsulﬁnyl)thieno[2,3-b]pyridin-3-amine (38b). 3-Cyano-

1,2-dihydropyridine-2-thione was alkylated with (chloromethyl)-

(butyl)sulfane, oxidized with hydrogen peroxide, and cyclized with

KO^tBu as described for the synthesis of compound 1 to provide the

unsubstituted analog 38b. ¹H NMR (400 MHz, CDCl₃) δ 8.61 (dd, J =

4.7, 1.6 Hz, 1H), 7.89 (dd, J = 8.1, 1.6 Hz, 1H), 7.33 (dd, J = 8.1, 4.6 Hz,

1H), 3.39−3.18 (m, 1H), 3.20−3.03 (m, 1H), 1.74 (p, J = 7.6 Hz, 2H),

1.63−1.38 (m, 2H), 0.94 (t, J = 7.3 Hz, 3H). ESI-MS (m/z): 255 [M +

H]⁺.

4-Methyl-2-(propylsulﬁnyl)-6-(thiazol-2-yl)thieno[2,3-b]-

pyridin-3-amine (38c). Step 1. 1,2-Dibromobutane (1.64 mmol, 310

mg, 143 μL) was added to a suspension of zinc powder (33.3 mmol, 2.18

g) in THF 2 mL. The mixture was heated to reﬂux for 5 min, and then

chlorotrimethylsilane (1.73 mmol, 188 mg, 220 μL) was added. Next, a

solution of 2-bromothiazole (11.1 mmol, 1.82 g, 1.0 mL) in THF (10

mL) was added dropwise, and the mixture was stirred at 50 °C for 1 h to

give a THF solution of 2-thiazolylzinc bromide.

Step 2. The enone from step 1 was condensed with cyanothioace-

tamide, alkylated with (chloromethyl)(butyl)sulfane, oxidized, and

cyclized as described for the synthesis of compound 1 to provide methyl

2-(4-(3-amino-2-(butylsulﬁnyl)-6-(thiazol-2-yl)thieno[2,3-b]pyridin-4-

yl)phenoxy)acetate. ¹H NMR (400 MHz, CDCl₃) δ 7.97 (s, 1H), 7.89−

7.86 (m, 1H), 7.47 (d, J = 3.1 Hz, 1H), 7.46−7.37 (m, 2H), 7.02 (d, J =

8.5 Hz, 2H), 4.70 (s, 2H), 4.66 (s, 2H), 3.82 (s, 3H), 3.34−3.18 (m, 1H),

3.16−3.01 (m, 1H), 1.77−1.64 (m, 2H), 1.51−1.38 (m, 2H), 0.92 (t, J =

7.3 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 168.9, 168.1, 161.4, 158.6,

149.6, 146.8, 144.2, 143.1, 130.2, 129.6, 123.4, 122.2, 117.9, 114.8, 110.0,

65.2, 54.6, 52.4, 25.0, 21.9, 13.7. ESI-MS (m/z): 502.1 [M + H]⁺.

Step 3. The methyl ester from step 2 was reduced with LiBH₄

following the procedure described in the synthesis of intermediate 18 to

1

provide primary alcohol 32. H NMR (400 MHz, CDCl₃) δ 8.01 (s,

1H), 7.90 (d, J = 3.2 Hz, 1H), 7.48 (d, J = 3.1 Hz, 1H), 7.46−7.35 (m,

2H), 7.07−6.99 (m, 2H), 4.69 (s, 2H), 4.18−4.11 (m, 2H), 4.04−3.96

(m, 2H), 3.34−3.23 (m, 1H), 3.17−3.04 (m, 1H), 1.80−1.61 (m, 2H),

1.53−1.40 (m, 2H), 0.93 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz,

CD₂Cl₂) δ 168.1, 161.3, 159.6, 149.6, 147.2, 144.2, 143.4, 130.1, 128.2,

123.7, 122.2, 117.8, 114.7, 108.6, 69.6, 61.2, 54.7, 24.25.0, 21.9, 13.5.

ESI-MS (m/z): 474.1 [M + H]⁺. Enantiomers were separated on a 1 cm

Chiralpak AD column using 50% EtOH in hexanes with a 2.5 mL/min

ﬂow rate monitoring absorbance at 254 and 315 nM. The positive

enantiomer eluted at 27.5−31 min, and the negative enantiomer eluded

Step 2. 2,6-Dichloro-4-methylnicotinonitrile (0.27 mmol, 50 mg) and

tetrakis(triphenylphosphine)palladium (0.013 mmol, 15 mg, 5 mol %)

were added sequentially to the THF solution of 2-thiazolylzinc bromide

(550 μL) from step 1, and the mixture was stirred at 60 °C for 12 h.³³

The reaction solution was poured into saturated aqueous sodium

bicarbonate solution and the aqueous layer was extracted with EtOAc.

The organic phases were dried with MgSO₄, ﬁltered, and concentrated

under reduced pressure. The crude compound was puriﬁed by ﬂash

chromatography on silica gel to give 2-chloro-4-methyl-6-(thiazol-2-

yl)nicotinonitrile in 45% yield. ESI-MS (m/z): 236.0 [M + H]⁺.

Step 3. The chloropyridine from step 2 was subjected to substitution

with methyl 3-mercaptopropionate, alkylated, oxidized, and cyclized as

23

at 32−36 min. Peak 1: [α]_D²³+75.46 (c 0.212, EtOH). Peak 2: [α]_D

−51.24 (c 0.263, EtOH).

4-Methyl-6-morpholino-2-(propylsulﬁnyl)thieno[2,3-b]-

pyridin-3-amine (38a). Step 1. Anhydrous MeOH (3.97 mL) was

added to 2,6-dichloro-4-methylnicotinonitrile (34, 500 mg, 2.67 mmol)

under a N₂atmosphere, and the mixture was cooled to 0 °C. Morpholine

(473.7 μL, 5.493 mmol) was added dropwise to the solution and the

solution stirred at room temperature overnight. The reaction mixture

was ﬁltered, and the precipitate was washed with MeOH (0.5 mL) and

H₂O (3−4 mL). The solid was dissolved in CH₂Cl₂and dried over

MgSO₄. The solution was ﬁltered and concentrated under reduced

pressure. The crude product was puriﬁed using automated ﬂash

chromatography to give 2-chloro-4-methyl-6-morpholinonicotinonitrile

(35a) in 85% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.26 (s, 1H), 3.81−

3.73 (m, 4H), 3.68−3.58 (m, 4H), 2.42 (d, J = 0.8 Hz, 3H). ¹³C NMR

(101 MHz, CDCl₃) δ 158.55, 153.80, 115.84, 104.32, 97.92, 94.99,

66.37, 44.80, 21.02. ESI-MS (m/z): 238.1 [M + H]⁺.

1

described for compound 38a to provide thiazole 38c. H NMR (400

MHz, CDCl₃) δ 7.95 (s, 1H), 7.93 (d, J = 3.2 Hz, 1H), 7.48 (d, J = 3.2

Hz, 1H), 5.16 (s, 2H), 3.37−3.23 (m, 1H), 3.16−3.05 (m, 1H), 2.85 (s,

3H), 1.83 (h, J = 7.5 Hz, 2H), 1.11 (t, J = 7.4 Hz, 3H). ¹³C NMR (101

MHz, CDCl₃) δ 168.3, 161.1, 154.2, 150.1, 148.6, 144.1, 126.3, 122.1,

118.1, 108.8, 56.9, 20.3, 16.7, 13.3. ESI-MS (m/z): 338.0 [M + H]⁺.

General Synthesis of Thieno[2,3-d]pyrimidines As Exempli-

ﬁed by the Synthesis of 6-(Butylsulﬁnyl)-4-phenyl-2-(thiophen-

2-yl)thieno[2,3-d]pyrimidin-5-amine (44a). Step 1. According to a

procedure described by Lorente,³⁴a mixture of NaO-i-Pr (1.75 mmol,

1.0 equiv, prepared in situ from sodium and dry i-PrOH), thiophene-2-

carbothioamide (1.75 mmol, 250 mg, 1.0 equiv), and 2-(ethoxy-

(phenyl)methylene)malononitrile (1.75 mmol, 346 mg, 1.0 equiv) in i-

PrOH (30 mL) was stirred for 5 h at rt. The reaction was then acidiﬁed

with concentrated HCl and stirred overnight. The solvent was

evaporated, and the resulting solid was suspended in acetic acid and

heated to 80 °C for 2 h. After cooling to room temperature, the mixture

was ﬁltered to give 315 mg of 4-phenyl-2-(thiophen-2-yl)-6-thioxo-1,6-

dihydropyrimidine-5-carbonitrile, which was used without additional

puriﬁcation (60%). ESI-MS (m/z): 296.1 [M + H]⁺.

Step 2. NaOMe (73.6 mg, 1.36 mmol) and methyl 3-mercaptopro-

pionate (151 μL, 1.363 mmol) were added to a solution of the

chloropyridine from step 1 (324 mg, 1.36 mmol) in DMF (4.10 mL),

and the reaction mixture was stirred at 80 °C for 1 h. The reaction

mixture was allowed to cool to rt, diluted with EtOAc, and washed with

H₂O. The organic layer was separated, dried over MgSO₄, ﬁltered, and

concentrated under reduced pressure to give a crude mixture of 1:1

starting material to desired thioether (methyl 3-((3-cyano-4-methyl-6-

morpholinopyridin-2-yl)thio)propanoate), which was used directly in

the next step without additional puriﬁcation. ESI (m/z): 322.1 [M +

H]⁺.

Step 2. A mixture of the pyrimidine from step 1 (0.68 mmol, 200 mg),

butyl(chloromethyl)sulfane (0.0.68 mmol, 94 mg, 1.0 equiv) and Et₃N

(1.02 mmol, 1.5 equiv) was stirred at reﬂuxed in dry CH₃CN (700 μL)

for 20 min. The reaction was allowed to cool to rt and diluted with

EtOAc and water. The organic phase was separated, and the aqueous

layer was extracted twice with EtOAc. The combined extractions were

Step 3. NaH (150.8 mg, 3.769 mmol, 60% in mineral oil) and THF

(10 mL) were added to a ﬂame-dried ﬂask under N₂, followed by the

crude reaction mixture from step 2 dissolved in THF (10 mL). The

reaction was stirred at reﬂux for 6 h, and an additional 2.0 equiv of NaH

R

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Journal of Medicinal Chemistry

Article

washed with saturated NaCl solution, dried over magnesium sulfate,

ﬁltered, and concentrated under reduced pressure. The residue obtained

was then puriﬁed by ﬂash chromatography to give 180 mg of 4-

(((butylthio)methyl)thio)-6-phenyl-2-(thiophen-2-yl)pyrimidine-5-

carbonitrile (68%). ¹H NMR (400 MHz, CDCl₃) δ 8.15 (dd, J = 3.7, 1.2

Hz, 1H), 8.12−8.03 (m, 2H), 7.61 (dd, J = 4.9, 1.2 Hz, 1H), 7.59−7.50

(m, 3H), 7.18 (dd, J = 4.9, 3.7 Hz, 1H), 4.54 (s, 2H), 2.75 (t, J = 7.3 Hz,

2H), 1.73−1.56 (m, 2H), 1.48−1.34 (m, 2H), 0.91 (t, J = 7.3 Hz, 3H).

398.1 [M + H]⁺.

7.63 (m, 2H), 7.63−7.49 (m, 3H), 7.29 (s, 1H), 7.07 (s, 1H), 4.85 (s,

2H), 4.18 (s, 3H), 3.29 (ddd, J = 12.8, 8.6, 6.3 Hz, 1H), 3.11 (ddd, J =

12.8, 8.7, 6.9 Hz, 1H), 1.83−1.65 (m, 2H), 1.59−1.39 (m, 2H), 0.94 (t, J

= 7.3 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 169.6, 162.3, 153.2,

143.0, 142.1, 136.6, 130.5, 129.4, 129.0, 128.9, 125.8, 119.32, 107.6, 54.7,

37.1, 25.0, 21.8, 13.7. ESI-MS (m/z): 412.1 [M + H]⁺.

6-(Butylsulﬁnyl)-2-(oxazol-4-yl)-4-phenylthieno[2,3-d]-

pyrimidin-5-amine (44e). Oxazole-4-carbothioamide and 2-(ethoxy-

(phenyl)methylene)malononitrile were converted to pyrimidine 44e

1

using procedures described for the synthesis of 44a. H NMR (400

Step 3. Acetic acid (860 μL) and hydrogen peroxide (0.54 mmol, 1.5

equiv, 30% solution in water) were added to a solution of the thioether

from step 2 (0.36 mmol, 140 mg) in chloroform (860 μL). The reaction

mixture was stirred at 32 °C for 45 min. Once complete, the reaction was

diluted with EtOAc and washed with saturated NaHCO₃solution, dried

over magnesium sulfate, ﬁltered, and concentrated under reduced

pressure to give 139 mg of 4-(((butylsulﬁnyl)methyl)thio)-6-phenyl-2-

MHz, CDCl₃) δ 8.51 (d, J = 1.1 Hz, 1H), 8.01 (d, J = 1.1 Hz, 1H), 7.75−

7.61 (m, 2H), 7.62−7.48 (m, 3H), 4.56 (s, 2H), 3.29 (ddd, J = 12.9, 8.8,

6.3 Hz, 1H), 3.09 (ddd, J = 12.9, 8.9, 6.9 Hz, 1H), 1.81−1.64 (m, 2H),

1.56−1.39 (m, 2H), 0.93 (t, J = 7.3 Hz, 3H). ESI-MS (m/z): 399.1 [M +

H]⁺.

6-(Butylsulﬁnyl)-2,4-diphenylthieno[2,3-d]pyrimidine (48).

Step 1. 2,4-Dichlorothieno[2,3-d]pyrimidine³⁵(100 mg, 0.50 mmol),

phenylboronic acid (242 mg, 2.0 mmmol, 4.0 equiv), potassium

carbonate (1.5 mmol, 3.0 equiv), Pd(OAc)₂(5 mol %), SPhos (10 mol

%) in CH₃CN/H₂O (1.5:1) were heated at 100 °C overnight. After

cooling to rt, the reaction mixture was diluted with EtOAc and washed

with water. The organic layer was dried over magnesium sulfate, and the

solvent was removed under reduced pressure. The crude product was

puriﬁed by ﬂash chromatography on silica gel to aﬀord designed product

2,4-diphenylthieno[2,3-d]pyrimidine in 89% yield. ¹H NMR (400 MHz,

CDCl3) δ 8.70−8.59 (m, 2H), 8.14−8.02 (m, 2H), 7.65−7.44 (m, 8H).

ESI-MS (m/z): 289.0 [M + H]⁺.

Step 2. To a solution of the diphenylpyrimidine from step 1 (53 mg,

0.28 mmol) in THF (1.0 mL) was added n-BuLi (0.56 mmol, 2.0 equiv,

225 μL of 2.5 M solution in hexanes) at −78 °C. The reaction mixture

was stirred for 5 min before 1,2-dibutyldisulfane (1.14 mmol, 4.0 equiv)

in THF was added. The reaction mixture was stirred for 1 h at −78 °C

and then quenched with water. The crude product was puriﬁed by ﬂash

chromatography on silica gel to aﬀord 6-(butylthio)-2,4-diphenyl-

thieno[2,3-d]pyrimidine in 61% yield. ¹H NMR (400 MHz, CDCl₃) δ

8.62−8.56 (m, 2H), 8.06−7.98 (m, 2H), 7.61−7.41 (m, 8H), 3.01 (t, J =

7.3, 2H), 1.76−1.62 (m, 2H), 1.55−1.38 (m, 2H), 0.92 (t, J = 7.4 Hz,

3H). ESI-MS (m/z): 377.1 [M + H]⁺.

1

(thiophen-2-yl)pyrimidine-5-carbonitrile (94%). H NMR (400 MHz,

CDCl₃) δ 8.21 (dd, J = 3.9, 1.3 Hz, 1H), 8.09 (dt, J = 6.8, 1.5 Hz, 2H),

7.64 (dd, J = 5.0, 1.3 Hz, 1H), 7.63−7.49 (m, 3H), 7.20 (dd, J = 5.0, 3.8

Hz, 1H), 4.71 (d, J = 13.1 Hz, 1H), 4.45 (d, J = 13.1 Hz, 1H), 2.94 (dt, J =

12.9, 8.1 Hz, 1H), 2.83 (dt, J = 13.0, 7.3 Hz, 1H), 1.84 (p, J = 7.6 Hz,

2H), 1.54−1.38 (m, 2H), 0.95 (t, J = 7.3 Hz, 3H). ESI-MS (m/z): 414

[M + H]⁺.

Step 4. To a solution of the sulfoxide from step 3 (0.12 mmol, 50 mg)

in DMF (480 μL) was added KOH (0.06 mmol, 3.4 mg in 34 μL of

water, 0.5 equiv). The reaction mixture was stirred at rt for 20 min. Once

the reaction was complete as judged by TLC analysis, the mixture was

diluted with EtOAc and washed with 5% aqueous acetic acid. The

organic phase was separated and aqueous layer was extracted twice with

EtOAc. The combined organic layers were dried over magnesium

sulfate, ﬁltered, and concentrated under reduced pressure to give crude

product, which was puriﬁed by ﬂash chromatography (EtOAc) on silica

gel to provide the pyrimidine analog 44a in 40% yield. ¹H NMR (400

MHz, CDCl₃) δ 8.10 (dd, J = 3.7, 1.3 Hz, 1H), 7.74−7.65 (m, 2H),

7.62−7.53 (m, 3H), 7.50 (dd, J = 5.0, 1.2 Hz, 1H), 7.14 (dd, J = 5.0, 3.7

Hz, 1H), 4.79 (s, 2H), 3.28 (ddd, J = 12.8, 9.0, 6.2 Hz, 1H), 3.11 (ddd, J

= 12.8, 9.0, 6.7 Hz, 1H), 1.84−1.63 (m, 2H), 1.54−1.41 (m, 2H), 0.94 (t,

J = 7.3 Hz, 3H). ¹³C NMR (101 MHz, CD₂Cl₂) δ 169.7, 162.5, 157.1,

142.8, 142.3, 136.8, 130.6, 130.4, 129.8, 128.9, 128.8, 128.4, 119.1, 106.9,

54.9, 24.94, 21.9, 13.5. ESI-MS (m/z): 414 [M + H]⁺.

Step 3. Acetic acid (250 μL) and hydrogen peroxide (20 μL, 30%

solution in water) were added to the solution of the thioether from step

3 (35 mg, 0.1 mmol) in chloroform (250 μL). The reaction mixture was

stirred at 32 °C for 45 min. Once complete, the reaction was diluted with

EtOAc and was washed with saturated NaHCO₃solution, dried over

magnesium sulfate, ﬁltered, and concentrated under reduce pressure to

6-(Butylsulﬁnyl)-2,4-diphenylthieno[2,3-d]pyrimidin-5-

amine (44b). Benzothioamide and 2-(ethoxy(phenyl)methylene)-

malononitrile were converted to diphenylpyrimidine 44b using

1

procedures described for the synthesis of 44a. H NMR (400 MHz,

1

give sulfoxide 48 in 79% yield. H NMR (400 MHz, CDCl₃) δ 8.69−

CDCl₃) δ 8.73−8.37 (m, 2H), 7.78−7.68 (m, 2H), 7.66−7.55 (m, 3H),

7.53−7.40 (m, 3H), 4.83 (s, 2H), 3.30 (ddd, J = 12.7, 8.9, 6.3 Hz, 1H),

3.21−3.01 (m, 1H), 1.87−1.66 (m, 2H), 1.57−1.41 (m, 2H), 0.95 (t, J =

7.3 Hz, 3H). ¹³C NMR (101 MHz, CD₂Cl₂) δ 170.0, 162.4, 160.2, 142.1,

137.2, 136.9, 131.0, 130.3, 129, 128.8, 128.6, 119.6, 107.3, 54.9, 24.9,

21.9, 13.5. ESI-MS (m/z): 408 [M + H]⁺.

8.59 (m, 2H), 8.09−7.99 (m, 2H), 7.95 (s, 1H), 7.65−7.56 (m, 3H),

7.56−7.45 (m, 3H), 3.18−3.02 (m, 2H), 1.87−1.64 (m, 2H), 1.54−1.42

(m, 2H), 0.94 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 172.1,

161.8, 160.8, 146.8, 137.4, 137.12, 131.0, 130.8, 129.3, 129.0, 128.7,

128.6, 125.3, 122.9, 57.4, 24.3, 21.8, 13.6. ESI-MS (m/z): 393.1 [M +

H]⁺.

6-(Butylsulﬁnyl)-4-phenyl-2-(thiazol-2-yl)thieno[2,3-d]-

pyrimidin-5-amine (44c). Thiazole-2-carbothioamide and 2-(ethoxy-

(phenyl)methylene)malononitrile were converted to pyrimidine 44c

6-(Butylsulﬁnyl)-2-phenyl-4-(piperidin-1-yl)thieno[2,3-d]-

pyrimidine (51). Step 1. 4,6-Dichlorothieno[2,3-b]pyrimidine³⁵(50

mg, 0.24 mmol) and piperidine (0.36 mmol, 1.5 equiv) were combined

in EtOH and stirred at room temperature overnight. The solvent was

evaporated under reduced pressure, and the mixture was puriﬁed by

ﬂash chromatography on silica gel to give 6-chloro-4-(piperidin-1-

1

using procedures described for the synthesis of 44a. H NMR (400

MHz, CDCl₃) δ 8.06 (dd, J = 3.1, 1H), 7.75−7.66 (m, 2H), 7.75−7.66

(m, 3H), 7.55 (dd, J = 3.1, 1H), 4.87 (s, 2H), 3.30 (ddd, J = 12.8, 8.4, 6.3

Hz, 1H), 3.12 (ddd, J = 12.8, 8.6, 6.9 Hz, 1H), 1.85−1.65 (m, 2H),

1.55−1.40 (m, 2H), 0.95 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz,

CD₂Cl₂) δ 169.7, 166.3, 162.8, 155.0, 145.2, 141.9, 136.4, 130.6, 128.9,

128.9, 123.5, 120.6, 108.9, 54.9, 24.9, 219, 13.5. ESI-MS (m/z): 415.1

[M + H]⁺. Enantiomers of 44c were separated on a 1 cm Chiralpak AD

HPLC column using 30% EtOH/hexanes with a 2.5 mL/min ﬂow rate,

monitoring absorption at 315 and 254 nm. Peak 1 eluted at 28−32 min,

and peak 2 eluted at 35−42 min. Peak 1: [α]_D²³−52.15 (c 0.39, EtOH).

Peak 2: [α]_D²³+65.36 (c 0.33, EtOH).

6-(Butylsulﬁnyl)-2-(1-methyl-1H-imidazol-2-yl)-4-

phenylthieno[2,3-d]pyrimidin-5-amine (44d). 1-Methyl-1H-imi-

dazole-2-carbothioamide and 2-(ethoxy(phenyl)methylene)-

malononitrile were converted to pyrimidine 44d using procedures

described for the synthesis of 44a. ¹H NMR (400 MHz, CDCl₃) δ7.75−

1

yl)thieno[2,3-b]pyrimidine in quantitative yield. H NMR (400 MHz,

CDCl₃) δ 7.28 (d, J = 6.1 Hz, 1H), 7.18 (d, J = 6.2 Hz, 1H), 4.01−3.67

(m, 4H), 1.92−1.63 (m, 6H). ESI-MS (m/z): 254.0 [M + H]⁺.

Step 2. To the solution of the thiophene from step 1 (52 mg, 0.20

mmol) in THF was added n-BuLi (0.4 mmol, 2.0 equiv, 1.6 M solution

in hexanes) at −78 °C. The reaction mixture was stirred for 5 min before

1,2-dibutyldisulfane (0.80 mmol, 4.0 equiv) in THF was added. The

reaction mixture was stirred for additional 1 h at −78 °C and then

quenched by the addition of water. The crude product was puriﬁed by

ﬂash chromatography on silica gel to aﬀord 2-(butylthio)-6-chloro-4-

1

(piperidin-1-yl)thieno[2,3-b]pyrimidine in 74% yield. H NMR (400

MHz, CHCl₃) δ 7.24 (s, 1 H), 3.93−3.74 (m, 4H), 2.83 (t, J = 7.3 Hz,

2H), 1.82−1.66 (m, 6H), 1.66−1.53 (m, 2H), 1.49−1.33 (m, 2H), 0.89

S

DOI: 10.1021/acs.jmedchem.7b00271

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 171.6, 157.6, 155.2,

132.5, 124.9, 115.0, 48.2, 37.5, 31.5, 25.8, 24.3, 21.6, 13.6. ESI-MS (m/

z): 342.1 [M + H]⁺.

analog 53 in 60% yield. ¹H NMR (400 MHz, CDCl₃) δ 8.39 (d, J = 8.4

Hz, 1H), 8.06 (d, J = 8.4 Hz, 1H), 7.74 (dd, J = 3.8, 1.1 Hz, 1H), 7.61

(dd, J = 5.0, 1.1 Hz, 1H), 7.19 (dd, J = 5.0, 3.8 Hz, 1H), 3.15 (ddd, J =

13.3, 9.8, 6.0 Hz, 1H), 2.96 (ddd, J = 13.3, 9.9, 4.9 Hz, 1H), 1.98−1.80

(m, 1H), 1.60−1.52 (m, 1H), 1.52−1.37 (m, 2H), 0.93 (t, J = 7.2 Hz,

3H). ¹³C NMR (101 MHz, CDCl₃) δ 163.5, 161.4, 158.1, 147.7, 136.8,

131.0, 130.6, 130.1, 128.4, 117.8, 54.6, 23.8, 21.9, 13.7. ESI-MS (m/z):

323.0 [M + H]⁺.

2-(Butylsulﬁnyl)-5-(thiophen-2-yl)-3H-imidazo[4,5-b]-

pyridine (57). Step 1. 2-Thiophene boronic acid (742 mg, 5.8 mmol, 2.0

equiv), 6-chloro-3-nitropyridin-2-amine (500 mg, 2.9 mmol, 1.0 equiv),

cesium carbonate (5.8 mmol, 2.0 equiv), PdCl₂dppf (10 mol %), CuCl

(2.9 mmol, 1.0 equiv) in DMF were heated at 100 °C for 12 h. After

cooling to rt, the reaction mixture was diluted with EtOAc and washed

with water and then brine. The organic layer was dried over magnesium

sulfate, and the solvent was removed under reduced pressure. The crude

product was puriﬁed by column chromatography (hexanes/EtOAc, 8/

2) to aﬀord 3-nitro-6-(thiophen-2-yl)pyridin-2-amine in 63% yield. ¹H

NMR (400 MHz, CDCl₃) δ 8.42 (d, J = 8.7 Hz, 1H), 7.70 (dd, J = 3.8,

1.1 Hz, 1H), 7.54 (dd, J = 5.0, 1.1 Hz, 1H), 7.15 (dd, J = 5.0, 3.8 Hz, 1H),

7.09 (d, J = 8.7 Hz, 1H). ESI-MS (m/z): 222.1 [M + H]⁺.

Step 2. The nitroarene from step 1 (1.20 mmol, 265.4 mg) was

dissolved in a 5:1 acetone/water mixture. Zinc powder (12.0 mmol, 784

mg, 10 equiv) and ammonium chloride (18 mmol, 962.5 mg, 15 equiv)

were added to the solution, which was stirred at room temperature for 1

h. The solution was then ﬁltered through a Celite pad and washed with

ethyl acetate. The ﬁltrate was washed twice with brine, and then the

aqueous layer was back-extracted with EtOAc. The combined organic

layers were dried over magnesium sulfate, ﬁltered, and concentrated

under reduced pressure. Further puriﬁcation by column chromatog-

raphy gave 118.2 mg of 6-(thiophen-2-yl)pyridine-2,3-diamine (52%

yield). ¹H NMR (400 MHz, CD₃OD) δ 7.34 (dd, J = 3.6, 1.1 Hz, 1H),

7.25 (dd, J = 5.1, 1.1 Hz, 1H), 7.00 (dd, J = 5.1, 3.6 Hz, 1H), 6.94 (d, J =

7.8 Hz, 1H), 6.90 (d, J = 7.8 Hz, 1H). ESI-MS (m/z): 192.1 [M + H]⁺.

Step 3 Thiourea (16.97 mmol, 223.0 mg, 5 equiv) was added to the

diamine from step 2. The solution was heated at 170 °C for 2 h. The

addition of ethanol at room temperature produced solid, which was

ﬁltered to give 112.5 mg of 5-(thiophen-2-yl)-1,3-dihydro-2H-imidazo-

[4,5-b]pyridine-2-thione (82% yield). ¹H NMR (400 MHz, DMSO-d₆)

δ 13.12 (s, 1H), 12.75 (s, 1H), 7.69 (dd, J = 3.7, 1.1 Hz, 1H), 7.66 (d, J =

8.2 Hz, 1H), 7.56 (dd, J = 5.0, 1.1 Hz, 1H), 7.47 (d, J = 8.3 Hz, 1H), 7.12

(dd, J = 5.0, 3.7 Hz, 1H). ESI-MS (m/z): 234.0 [M + H]⁺.

Step 4. A mixture of the thiourea from step 3 (0.39 mmol, 92 mg),

potassium carbonate (0.45 mmol, 61.9 mg, 1.1 equiv), 1-bromobutane

(0.39 mmol, 42.8 μL, 1 equiv), 18-crown-6 (0.039 mmol, 10.5 mg, 0.1

equiv), and DMF (2.67 mL) was heated at 80 °C for 3 h. After cooling to

room temperature, the reaction mixture was diluted with EtOAc and

washed with water. The organic layer was dried over magnesium sulfate,

ﬁltered, and concentrated under high pressure to give 74.4 mg of 2-

(butylthio)-5-(thiophen-2-yl)-3H-imidazo[4,5-b]pyridine (65% yield).

¹H NMR (400 MHz, CDCl₃) δ 7.95−7.83 (m, 1H), 7.61−7.53 (m, 2H),

Step 3. The chloride from step 2 (52 mg, 0.15 mmol), phenylboronic

acid (27 mg, 0.22 mmol, 1.5 equiv), potassium carbonate (0.3 mmol, 2.0

equiv), and PdCl₂dtbpf (10 mol %) in CH₃CN (1 mL) and H₂O (0.5

mL) were heated at 100 °C overnight. After cooling to rt, the reaction

mixture was diluted with EtOAc and washed with water. The organic

layer was dried over magnesium sulfate, and the solvent was removed

under reduced pressure. The crude product was puriﬁed by ﬂash

chromatography on silica gel to aﬀord 2-(butylthio)-6-phenyl-4-

1

(piperidin-1-yl)thieno[2,3-b]pyrimidine in 19% yield. H NMR (400

MHz, CHCl₃) δ 8.49−8.36 (m, 2H), 7.51−7.36 (m, 3H), 7.29 (s, 1H),

3.95−3.85 (m, 4H), 2.90 (t, J = 7.4 Hz, 2H), 1.76−1.73 (m, 6H), 1.70−

1.59 (m, 2H), 1.48−1.39 (m, 2H), 0.91 (t, J = 7.4 Hz, 3H). ESI-MS (m/

z): 384.0 [M + H]⁺.

Step 4. Acetic acid (50 μL) and hydrogen peroxide (5.0 μL, 30%

solution in water) were added to the solution of the thioether from step

3 (10 mg, 0.026 mmol) in chloroform (50 μL). The reaction mixture

was stirred at 32 °C for 45 min. Once complete, the reaction was diluted

with EtOAc and was washed with saturated NaHCO₃solution, dried

over magnesium sulfate, ﬁltered, and concentrated under reduce

pressure to give sulfoxide 51 in 76% yield. ¹H NMR (400 MHz,

CDCl₃) δ 8.54−8.34 (m, 2H), 7.73 (s, 1H), 7.55−7.36 (m, 3H), 4.09−

3.86 (m, 4H), 3.24−2.94 (m, 2H), 1.97−1.36 (m, 10H), 0.93 (t, J = 7.3

Hz, 3H). ESI-MS (m/z): 400.1 [M + H]⁺.

2-(Butylsulﬁnyl)-5-(thiophen-2-yl)thiazolo[5,4-b]pyridine

(53). Step 1. Potassium ethyl xanthate (1.9 g, 0.012 mol) and anhydrous

N-methyl-2-pyrrolidone (14.1 mL) were added to 2,6-dichloropyridin-

3-amine (1.0 g, 0.0061 mol) under N₂. The solution was heated at 170

°C for 3.5 h. The reaction mixture was allowed to cool to room

temperature, acidiﬁed to pH 5 using AcOH, diluted in EtOAc, and

washed several times with H₂O. The organic layer was separated, dried

over MgSO₄, and concentrated under reduced pressure. This gave 5-

chlorothiazolo[5,4-b]pyridine-2-thiol (52)²⁰as a red solid in 18% yield

1

which was used without further puriﬁcation. H NMR (400 MHz,

CDCl₃) δ 7.41 (d, J = 8.4 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H). ESI-MS (m/

z): 202.9.

Step 2. K₂CO₃(75 mg, 0.54 mmol), 1-bromobutane (53.3 μL, 0.493

mmol), 18-crown-6 (13.2 mg, 0.0493 mmol), and DMF (3.4 mL) were

added to the thiol from step 1, and the solution was heated at 80 °C for 3

h. The solution was diluted with EtOAc, washed with H₂O, and the

organic layer was separated, dried over MgSO₄, and concentrated under

reduced pressure. The crude product was puriﬁed using ﬂash

chromatography on silica gel to give 2-(butylthio)-5-chlorothiazolo-

1

[5,4-b]pyridine in 76% isolated yield. H NMR (400 MHz, CDCl₃) δ

7.93 (d, J = 8.5 Hz, 1H), 7.30 (d, J = 8.5 Hz, 1H), 3.31 (t, J = 7.3 Hz, 2H),

1.76 (p, J = 7.5 Hz, 2H), 1.52−1.39 (m, 2H), 0.93 (t, J = 7.3 Hz, 3H).

ESI-MS (m/z): 259.0 [M + H]⁺.

Step 3. 2-Thienylboronic acid (49.4 mg, 0.386 mmol), CsCO₃(126

mg, 0.386 mmol), Pd(dppf)Cl₂(15.8 mg, 0.0193 mmol), CuCl (19.1

mg, 0.193 mmol), and DMF (1 mL) were added to the pyridyl chloride

from step 2 (50 mg, 0.19 mmol) under N₂. The reaction mixture was

heated to 100 °C for 30 min. Then the N₂was disconnected and the vial

was capped and sealed with Teﬂon tape and allowed to stir at 100 °C

overnight. The reaction mixture was diluted in EtOAc, washed with

H₂O, and the organic layer was separated, dried over MgSO₄, ﬁltered,

and concentrated under reduced pressure. Chromatography on silica gel

provided 2-(butylthio)-5-(thiophen-2-yl)thiazolo[5,4-b]pyridine in

7.36 (d, J = 5.1, 1H), 7.16−7.06 (m, 1H), 3.28 (t, J = 7.3 Hz, 2H), 1.76−

1.62 (m, 2H), 1.48−1.32 (m, 2H), 0.90 (t, J = 7.4 Hz, 3H). ESI-MS (m/

z): 290.1 [M + H]⁺.

Step 5. Chloroform (450 μL), acetic acid (450 μL), and hydrogen

peroxide (0.37 mmol, 2.0 equiv, 40 μL) were added to the thioether

from step 4 (54.4 mg, 0.19 mmol) and heated at 45 °C for 2.5 h. The

solution was then diluted with EtOAc and washed with 10% acetic acid.

The organic layer was separated, dried with magnesium sulfate, ﬁltered,

concentrated, and puriﬁed via chromatography on silica gel to give 16.8

mg of sulfoxide 57 (30% yield). ¹H NMR (400 MHz, CDCl₃) δ 12.17 (s,

1H), 8.06 (d, J = 8.5 Hz, 1H), 7.84−7.60 (m, 2H), 7.46−7.29 (m, 1H),

7.16−7.05 (m, 1H), 3.35 (ddd, J = 13.4, 9.8, 5.8 Hz, 1H), 3.26 (ddd, J =

13.4, 9.8, 5.4 Hz, 1H), 2.16−1.89 (m, 2H), 1.59−1.39 (m, 2H), 0.93 (t, J

= 7.3 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 155.0, 149.3, 147.4,

144.4, 135.2, 128.7, 128.2, 127.9, 125.3, 115.7, 54.7, 23.6, 21.8, 13.6. ESI-

MS (m/z): 306.1 [M + H]⁺.

1

31% yield. H NMR (400 MHz, CDCl₃) δ 7.98 (d, J = 8.6 Hz, 1H),

7.63 (dd, J = 3.8, 1.1 Hz, 1H), 7.51 (dd, J = 5.1, 1.1 Hz, 1H), 7.23 (d, J =

8.6 Hz, 1H), 7.14 (dd, J = 5.0, 3.8 Hz, 1H), 3.24 (t, J = 7.3 Hz, 2H),

1.78−1.66 (m, 2H), 1.57−1.41 (m, 2H), 0.96 (t, J = 7.4 Hz, 3H). ESI-

MS (m/z): 307.0 [M + H]⁺.

Step 4. To a solution of the thioether from step 3 (18 mg. 0.059

mmol) in chloroform (142 μL) and AcOH (142 μL) was added H₂O₂

(12.0 μL, 0.118 mmol, 30% solution in H₂O). The reaction mixture was

heated at 35 °C for 2.5 h. The reaction was diluted with EtOAc and

washed with saturated NaHCO₃. The organic layer was separated, dried

over MgSO₄, ﬁltered, and concentrated under reduced pressure to give

Inhibition of 15-PGDH. As described previously,¹⁴test compounds

were dissolved in DMSO as 50 mM stock solutions. Reactions were

assembled with 5 nM 15-PGDH enzyme and 2-fold dilutions of

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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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inhibitor from 2500 to 0.15 nM, plus 150 μM NAD(+) and 25 μM

PGE2 in reaction buﬀer (50 mM Tris-HCl, pH 7.5, 0.01% Tween 20).

The reaction mix was incubated for 15 min at 25 °C in an Envision

Reader (PerkinElmer). Enzyme activity was determined by following

generation of NADH as assayed by recording ﬂuorescence at ex/em =

340 nm/485 nm every 30s for 3 min, commencing immediately after

addition of PGE2. IC₅₀values were calculated with GraphPad Prism 5

software using the sigmoidal dose−response function and plotted

against inhibitor concentration.

Elevation of PGE2 Levels in A549 Cell Culture. The A549 cell

line was maintained in F12K medium supplemented with 10% fetal calf

serum (FBS) and 50 μg/mL gentamicin in a humidiﬁed atmosphere

containing 5% CO₂at 37 °C. Cells were plated in duplicate in a 24-well

plate (1 mL per well) at 1 × 10⁵cells per well and grown for 24 h before

stimulation with IL-1β (1 ng/mL) overnight (16 h) to induce COX2

expression and PGE2 production. Cells were then treated for an

additional 8 h with fresh medium containing the indicated concentration

of 15-PGDH inhibitor. The medium was then collected and the level of

PGE2 analyzed using a PGE2 enzyme immunoassay Kit (R&D System).

Data were collected from four independent experiments. Results were

tabulated graphically with error bars corresponding to standard error of

the means and compared using two-tailed t tests.

Evaluation of PGE2 Levels in Mouse Tissue. Female CB57/L6

mice (8−12 weeks of age) were obtained from Jackson Laboratory. The

animals were housed in standard microisolator cages and maintained on

a deﬁned irradiated diet (Prolab Isopro RMH 3000) and autoclaved

water. Three to ﬁve mice/group were administered a 0.2 mL dose of

inhibitor corresponding to 2.5 mg/kg by ip injection formulated as

follows. (+)-1: 10% EtOH, 5% Cremophor EL, and 85% D5W. (+)-12q:

10% EtOH/10% PEG400/80% 50 mM citrate−phosphate buﬀer pH

3.6. (+)-32: 10% DMSO, 10% Cremophor, and 80% D₅W pH 7.4.

(+)-44c: 6% DMSO, 4% EtOH; 10% Cremophor EL, and 80% lactic

acid, pH 5.5. Solid tissues were harvested, rinsed in ice-cold PBS

containing indomethacin (5.6 μg/mL), and snap frozen in liquid

nitrogen. Marrow was ﬂushed in 1.5 mL of ice cold PBS with

indomethacin, pelleted at 2000 rpm for 5 min in an Eppendorf

centrifuge, and snap frozen in liquid nitrogen. Frozen samples were

pulverized in liquid nitrogen. The powder was transferred to an

Eppendorf tube with 500 μL of cold PBS with indomethacin and then

homogenized using a tissue homogenizer. The suspension was sonicated

in ice−water for 5 min using cycles of 20 s of sonication with 20 s of

cooling, followed by centrifugation for 10 min at 12 000 rpm. The PGE2

level of the supernatant was measured using a PGE2 ELISA Kit (R&D

Systems, catalog no. KGE004B) and normalized to protein concen-

tration measured by BCA assay (Thermo Scientiﬁc, catalog no. 23225)

and expressed as (ng of PGE2)/(mg of protein). Each sample was

assayed in triplicate.

Solubility Determination. An amount of 1 mL of citrate−

phosphate buﬀer (0.1 M citric acid/disodium phosphate buﬀer), at

pH 7 unless otherwise indicated, was added to a 20 mL scintillation vial.

Determination of the solubility of the salt form of s-12q was performed

in dH₂0. Compound was slowly added to buﬀer until supersaturated.

The solution was stirred overnight (16 h) protected from light at rt. The

solution was transferred to a Teﬂon microcentrifuge tube and spun at

16 100g for 5 min. The supernatant was transferred to a second clean

Teﬂon microcentrifuge tube and spun again. 0.1 mL of supernatant from

the top of the centrifuge tube was carefully removed and placed in a

standard microcentrifuge tube containing 0.2 mL of methanol or

acetonitrile containing 0.15% formic acid and either tolbutamide or n-

benzylbenzamide internal standard (organic crash). The solution was

vortexed for 15 s, incubated at rt for 10 min, centrifuged at 16 100g, and

transferred to a HPLC vial containing an insert. As necessary, dilutions

of compound were prepared prior to mixing with organic crash.

Compound concentration was determined by LC−MS/MS analysis in

comparison to a standard curve prepared by addition of varying amounts

of compounds to 0.3 mL of a 1:2 mixture of 0.1 M citrate−phosphate

buﬀer/organic crash identical to that used for the solubility

determination. Standard curve solutions were vortexed, centrifuged,

and processed in an identical fashion to solubility determination tubes.

Analytical methods were developed for each compound using a SCIEX

(Framingham, MA) 3200 or 4000-QTrap combination triple quadru-

pole/ion trap instrument. The parent ion and the two most prominent

daughter ions were followed to conﬁrm compound identity, although

only the most abundant daughter was used for quantitation. A Shimadzu

(Columbia, MD) Prominence LC with Agilent C18 XDB column (5 μm

packing; 50 mm × 4.6 mm) was used for chromatography.

Liver S9 Stability. Female ICR/CD-1 mouse S9 fractions were

purchased from BioreclamationIVT (Chestertown, MD). 0.025 mL (0.5

mg) of S9 protein was added to a 15 mL glass screw cap tube. 0.350 mL

of a 50 mM Tris, pH 7.5 solution, containing the compound of interest

was added on ice. The ﬁnal concentration of compound after addition of

all reagents was 2 μM. 0.125 mL of an NADPH-regenerating system (1.7

mg/mL NADP, 7.8 mg/mL glucose 6-phosphate, 6 U/mL glucose 6-

phosphate dehydrogenase in 2% w/v NaHCO₃/10 mm MgCl₂) was

added for analysis of phase I metabolism. The tube was then placed in a

37 °C shaking water bath. At varying time points after addition of phase I

cofactors, the reaction was stopped by the addition of 0.5 mL of

methanol containing 0.2% formic acid and either tolbutamide or n-

benzylbenzamide IS. The samples were incubated for 10 min at rt and

then spun at 16 100g for 5 min in a microcentrifuge. The supernatant

was analyzed by LC−MS/MS as described above. The method

described by McNaney et al.³⁶was used with modiﬁcation for

determination of metabolic stability half-life by substrate depletion. A

“% remaining” value was used to assess metabolic stability of a

compound over time. The LC−MS/MS peak area of the incubated

sample at each time point was divided by the LC−MS/MS peak area of

the time 0 (T₀) sample and multiplied by 100. The natural logarithm

(ln) of the % remaining of compound was then plotted versus time (in

min) and a linear regression curve plotted going through y-intercept at

ln(100). The metabolism of some compounds failed to show linear

kinetics at a later time point, so those time points were excluded. The

half-life (T_1/2) was calculated as T_1/2= 0.693/slope.

Mouse PK Analysis. Female CD-1 mice (5−6 weeks of age) were

obtained from Charles River. The animals were housed in standard

microisolator cages and were administered inhibitor compounds at 10

mg/kg in 0.2 mL by ip injection formulated as follows. 1: 10% EtOH, 5%

Cremophor EL, and 85% D5W. 12q: 10% EtOH/10% PEG400/80% 50

mM citrate−phosphate buﬀer, pH 3.6. 12w: 100% 50 mM citrate−

phosphate buﬀer pH 3.6. 32: 10% DMSO, 10% Cremophor, and 80%

D₅W, pH 7.4. 44c: 6% DMSO, 4% EtOH, 10% Cremophor EL, and 80%

lactic acid, pH 5.5. Animals were euthanized by inhalation overdose of

CO₂in groups of 3 at 10, 30, 90, 180, 360, 960, and 1440 min postdose

and blood collected by cardiac puncture, using acidiﬁed citrate dextrose

(ACD) as the anticoagulant. Plasma was isolated from blood by

centrifugation at 9600g for 10 min and stored at −80 °C until analysis.

0.1 mL of plasma was precipitated with 0.2 mL of an organic crash

solution containing either methanol or acetonitrile, 0.15% formic acid,

and an internal standard (either tolbutamide or n-benzylbenzamide).

Extraction conditions were optimized prior to PK analysis for eﬃcient

and reproducible recovery over a 3 log range of concentrations. The

solution was centrifuged twice at 16 100g twice for 5 min. The ﬁnal

supernatant was analyzed by LC−MS/MS as described above, and

compound concentrations were determined in reference to a standard

curve prepared by addition of the appropriate compound to blank

plasma. A value of 3× above the signal obtained in the blank plasma was

designated the limit of detection (LOD). The limit of quantitation

(LOQ) was deﬁned as the lowest concentration at which back

calculation yielded a concentration within 20% of the theoretical value

and above the LOD signal. The LOQ values were as follows: (+)-1, 1

ng/mL; (rac)-1, 10 ng/mL; 12q, 5 ng/mL; 12w, 1 ng/mL; 32, 5 ng/mL;

44c, 5 ng/mL. Pharmacokinetic parameters were determined using the

noncompartmental analysis tool in Phoenix WinNonlin (Certara, Corp.,

Princeton, NJ).

ASSOCIATED CONTENT

■

S

* Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.7b00271.

U

DOI: 10.1021/acs.jmedchem.7b00271

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

xenotransplants and shows long-term safety in preclinical nonhuman

primate transplant models. Cell Stem Cell 2011, 8, 445−458.

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enhances hematopoietic stem cell homing, survival, and proliferation.

Blood 2009, 113, 5444−5455.

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haematopoietic grafts to prostaglandin E2 in vitro facilitates engraftment

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Additional pharmacokinetic and pharmacodynamic data

in mice and spectral data for synthetic compounds (PDF)

(-)-12q·TsOH·acetone (CIF)

(+)-1·1/2 MeOH (CIF)

Molecular formula strings and some data (CSV)

AUTHOR INFORMATION

■

(9) Cutler, C.; Multani, P.; Robbins, D.; Kim, H. T.; Le, T.; Hoggatt, J.;

Pelus, L. M.; Desponts, C.; Chen, Y.-B.; Rezner, B.; Armand, P.; Koreth,

J.; Glotzbecker, B.; Ho, V. T.; Alyea, E.; Isom, M.; Kao, G.; Armant, M.;

Silberstein, L.; Hu, P.; Soiffer, R. J.; Scadden, D. T.; Ritz, J.; Goessling,

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H.; Batlle, E. Isolation and in vitro expansion of human colonic stem

cells. Nat. Med. 2011, 17, 1225−1227.

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Segi, E.; Tsuboi, K.; Sugimoto, Y.; Kobayashi, T.; Miyachi, Y.; Ichikawa,

A.; Narumiya, S. The prostaglandin receptor EP4 suppresses colitis,

mucosal damage and CD4 cell activation in the gut. J. Clin. Invest. 2002,

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Prostaglandin catabolizing enzymes. Prostaglandins Other Lipid

Mediators 2002, 68−69, 483−493. (b) Tai, H. H.; Cho, H.; Tong, M.;

Ding, Y. NAD⁺-linked 15-hydroxyprostaglandin dehydrogenase:

Structure and biological functions. Curr. Pharm. Des. 2006, 12, 955−962.

(13) Myung, S.-J.; Rerko, R. M.; Yan, M.; Platzer, P.; Guda, K.; Dotson,

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P.; Newman, R. A.; Willis, J.; Dawson, D.; Markowitz, S. D. 15-

hydroxyprostaglandin dehydrogenase is an in vivo suppressor of colon

tumorigenesis. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 12098−12102.

(14) (a) Zhang, Y.; Desai, A.; Yang, S. Y.; Bae, K. B.; Antczak, M. I.;

Fink, S. P.; Tiwari, S.; Willis, J. E.; Williams, N. S.; Dawson, D. M.; Wald,

D.; Chen, W.-D.; Wang, Z.; Kasturi, L.; Larusch, G. A.; He, L.;

Cominelli, F.; Di Martino, L.; Djuric, Z.; Milne, G. L.; Chance, M.;

Sanabria, J.; Dealwis, C.; Mikkola, D.; Naidoo, J.; Wei, S.; Tai, H.-H.;

Gerson, S. L.; Ready, J. M.; Posner, B.; Willson, J. K. V.; Markowitz, S. D.

Inhibition of the prostaglandin-degrading enzyme 15-pgdh potentiates

tissue regeneration. Science 2015, 348, aaa2340. (b) Markowitz, S.;

Willson, J. K. V.; Posner, B. A.; Ready, J.; Zhang, Y.; Tai, H.-H.; Moss,

M.; Antczak, M. Compositions and methods of modulating 15-hydroxy-

prostaglandin dehydrogenase. WO2013158649 A1, 2013. (c) Marko-

witz, S.; Willson, J. K. V.; Posner, B.; Ready, J.; Antczak, M.; Zhang, Y.;

Desai, A.; Gerson, S.; Greenlee, W. Preparation of thienopyridines and

similar bicyclic heterocyclic compounds as modulators of 15-hydroxy-

prostaglandin dehydrogenase type i for therapy. WO2015065716 A1,

2015. (d) Markowitz, S. D.; Ready, J.; Zhang, Y.; Antczak, M.; Willson, J.

K. V.; Posner, B. A.; Greenlee, W. Compositions and methods of

modulating short-chain dehydrogenase activity. WO2016168472 A1,

2016.

Corresponding Authors

*S.D.M.: e-mail, sxm10@case.edu.

*J.M.R.: e-mail, Joseph.ready@utsouthwestern.edu

ORCID

Joseph M. Ready: 0000-0003-1305-9581

Author Contributions

^⊥M.I.A. and Y.Z. contributed equally.

Notes

The authors declare the following competing ﬁnancial

interest(s): UTSW and Case Western Reserve have ﬁled patents

covering the material described herein. See reference 14.

ACKNOWLEDGMENTS

■

Financial support is from the NIH (Grants R01 GM102403, R01

CA216863, J.M.R.), the Welch Foundation (Grant I-1612,

J.M.R.), and CPRIT (Grant RP110708-C3, N.S.W.). The

Preclinical Pharmacology Core (N.S.W.) is supported in part

by funding from the Institute for Innovations in Medicine at

UTSW. Additional funding is from the NIH (Grant P50

CA150964, S.D.M.) and a Scholar Innovator Award from the

Harrington Discovery Institute (S.D.M.).

ABBREVIATIONS USED

■

ADME, adsorption, distribution, metabolism, and excretion;

BMT, bone marrow transplantation; COX, cyclooxygenase;

dmPGE2, 16,16-dimethyl-PGE2; ELISA, enzyme-linked immu-

nosorbent assay; HATU, 1-[bis(dimethylamino)methylene]-

1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexaﬂuorophos-

phate; iv, intravenous; NAD⁺, nicotinamide adenine dinucleo-

tide; 15-PGDH, 15-prostaglandin dehydrogenase; PGE2,

prostaglandin E2; PGES, prostaglandin E synthase

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J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Inhibitors of 15-Prostaglandin Dehydrogenase To Potentiate Tissue Repair

DOI: 10.1021/acs.jmedchem.7b00271

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