Structure-based discovery of novel amide-containing nicotinamide phosphoribosyltransferase (Nampt) inhibitors

Article

pubs.acs.org/jmc

Structure-Based Discovery of Novel Amide-Containing Nicotinamide

Phosphoribosyltransferase (Nampt) Inhibitors

Xiaozhang Zheng,*^,†Paul Bauer,^†Timm Baumeister,^†Alexandre J. Buckmelter,^†Maureen Caligiuri,^†

Karl H. Clodfelter,^†Bingsong Han,^†Yen-Ching Ho,^†Nikolai Kley,^†Jian Lin,^†Dominic J. Reynolds,^†

Geeta Sharma,^†Chase C. Smith,^†Zhongguo Wang,^†Peter S. Dragovich,^‡Janet Gunzner-Toste,^‡

Bianca M. Liederer,^‡Justin Ly,^‡Thomas O’Brien,^‡Angela Oh,^‡Leslie Wang,^‡Weiru Wang,^‡Yang Xiao,^‡

Mark Zak,^‡Guiling Zhao,^‡Po-wai Yuen,^§and Kenneth W. Bair^†

^†Forma Therapeutics, Inc., 500 Arsenal Street, Watertown, Massachusetts 02472, United States

^‡Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States

^§Pharmaron Beijing, Co. Ltd., 6 Tai He Road, BDA Beijing, 100176, P. R. China

S

* Supporting Information

ABSTRACT: Crystal structures of several urea- and thiourea-derived compounds in complex with the nicotinamide

phosphoribosyltransferase (Nampt) protein were utilized to design a potent amide-containing inhibitor bearing an aza-indole

moiety (7, Nampt BC IC₅₀= 9.0 nM, A2780 cell proliferation IC₅₀= 10 nM). The Nampt−7 cocrystal structure was

subsequently obtained and enabled the design of additional amide-containing inhibitors which incorporated various other fused

6,5-heterocyclic moieties and biaryl sulfone or sulfonamide motifs. Additional modiﬁcations of these molecules aﬀorded many

potent biaryl sulfone-containing Nampt inhibitors which also exhibited favorable in vitro ADME properties (microsomal and

hepatocyte stability, MDCK permeability, plasma protein binding). An optimized compound (58) was a potent inhibitor of

multiple cancer cell lines (IC₅₀<10 nM vs U251, HT1080, PC3, MiaPaCa2, and HCT116 lines), displayed acceptable mouse PK

properties (F = 41%, CL = 52.4 mL/min/kg), and exhibited robust eﬃcacy in a U251 mouse xenograft model.

ribose polymerases (PARPs) and sirtuins (Figure 1).²Many

INTRODUCTION

■

tumors consume NAD at a higher rate than normal tissues due

Nicotinamide adenine dinucleotide (NAD) is a critical cofactor

to increased PARP activity.³Rapidly proliferating cancers have

for multiple enzymes involved in cell metabolism and

homeostasis.¹Mammalian cells utilize multiple biological

increased biosynthetic and homeostatic requirements that

utilize NAD as a cofactor and/or substrate.⁴This combination

pathways to generate NAD which initiate with diﬀerent

of increased NAD consumption and dependence upon NAD-

precursors, such as tryptophan (Trp), nicotinamide (NAM),

driven processes make the proliferation and/or survival of many

nicotinic acid (NA), and nicotinamide riboside (NR). In the

cancers highly dependent upon Nampt activity. Therefore,

NAM to NAD biosynthetic pathway, nicotinamide phosphor-

Nampt is an attractive target for the development of new cancer

ibosyltransferase (Nampt) is the rate-limiting enzyme. As

therapies.

shown in Figure 1, the homodimeric Nampt protein complex

uses an ATP-activated reaction to generate nicotinamide

mononucleotide (NMN, the immediate biosynthetic precursor

of NAD) via the condensation of NAM with 5-phosphoribosyl-

1-pyrophosphate (PRPP). The NAM employed in this process

can be obtained from exogenous sources but can also be

produced by NAD-consuming enzymes such as poly-ADP-

Several classes of Nampt inhibitors have been reported in the

scientiﬁc literature, and the two most advanced compounds, 1⁵

(GMX-1778) and 2⁶(APO-866), have been progressed to

Received: April 28, 2013

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Figure 1. Schematic representation of NAD biosynthetic pathways found in mammalian cells. Nampt catalyzes the conversion of NAM to NMN.

Figure 2. Examples of Nampt inhibitors.

Figure 3. Discovery of a novel amide-containing Nampt inhibitor.

human clinical trials (Figure 2).^7,8We recently reported the

structure-based identiﬁcation of urea-containing Nampt inhib-

itors, exempliﬁed by compounds 3 and 4 (Figure 2).^9,10In the

current work, we describe the continuation of our Nampt-

related research which led to the structure-enabled discovery of

novel and highly potent amide-containing Nampt inhibitors.¹¹

formed water-mediated hydrogen-bonds with the side chains of

the Nampt Asp219 and Ser241 residues (Figure 3).⁹Similar

water-mediated interactions were also crystallographically

noted between the urea present in compound 4 and the

Nampt protein.¹⁰Given these observations, we were intrigued

by the possibility of replacing the (thio)urea moieties contained

in 4 and 5 with an azaindole-derived amide (e.g., compound 6,

Figure 3). This modiﬁcation would ideally enable direct

formation of a hydrogen bond between the azaindole NH

group and the Nampt Asp219 side chain. In addition, the amide

NH moiety present in 6 was envisioned to mimic the

RESULTS AND DISCUSSION

■

Discovery of a Novel Amide-Containing Nampt

Inhibitor. In our previously disclosed cocrystal structure of

compound 5 with Nampt, we observed that the thiourea moiety

B

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Table 1. Linker SAR

a

b

c

Nampt biochemical inhibition. A2780 cell proliferation inhibition. This inhibition can be reversed by addition of 0.33 mM of NMN. Unless

d

otherwise noted, all biochemical and cell-based assay results are reported as the arithmetic mean of at least two separate runs (n = 2). Mouse liver

e

f

microsomal stability% remaining at 30 min. Human liver microsomal stability% remaining at 30 min. Racemic compound. See Supporting

Information for experimental details.

corresponding (thio)urea NH group contained in 4 and 5 and

thus favorably interact with the Asp219 and/or Ser241 side

chains either directly or in a water-mediated manner. The

particular azaindole isomer depicted in compound 6 was

purposefully selected via modeling to best position the N-atom

for condensation with the PRPP cofactor in the Nampt active

site (a possible requirement for obtaining potent cell activity

identiﬁed in our earlier studies).^9,12Disappointingly, however,

compound 6, was completely inactive in our biological assays.

We speculated that the rigidity of 6, combined with the

anticipated altered Nampt binding mode described above,

prevented its proper ﬁt into the Nampt binding site. Previous

structure−activity relationships (SAR) from the urea series

corresponding to 5 indicated that the addition of a methylene

group between the urea and the phenyl linker moiety was

tolerated.⁹We thus introduced such functionality into

compound 6, and the resulting (more ﬂexible) molecule (7)

encouragingly exhibited potent Nampt biochemical inhibition

Figure 4. Cocrystal structure of 7 (green) in complex with Nampt

activity as well as excellent potency in cell-based assays (Figure

(determined in this work; PBD code: 4KFN; monomers in gray and

3).¹³These properties compared favorably with those exhibited

cyan).¹⁴Water molecules are depicted as red spheres and possible

by the well-known Nampt inhibitors 1 and 2 in similar

assessments (Table 1).

hydrogen bonds are indicated with dashed yellow lines. The resolution

of the structure is 1.87 Å. For comparison, the previously published

crystal structure of a related thiourea-containing inhibitor (5) is shown

in magenta.⁹.

SAR Discussion. To enable further optimization of the

described amide-containing series of Nampt inhibitors, a

cocrystal structure of 7 in complex with Nampt was solved

(Figure 4). The bicyclic azaindole portion of the ligand was

observed in the NAM binding site of Nampt and was stacked

between the Tyr18′ and Phe193 side chains of the protein. As

envisioned in the design strategy depicted in Figure 3, the

azaindole did indeed displace the crystallographic water that

was previously observed near Asp219 in cocrystal structures of

related urea- and thiourea-containing Nampt inhibitors.^9,10As a

result of this displacement, the Asp219 side chain carboxyl was

located within hydrogen bonding distance of both the 7 amide

NH and the azaindole NH moiety. The direct interaction

between 7 and Asp219 shifted the ligand closer to this Nampt

residue relative to the binding location observed previously for

urea- and thiourea-containing inhibitors (e.g., 5; Figure 4).^9,10

This movement created space in the vicinity of Ser275, which

was occupied in the 7-Nampt cocrystal structure by a water

molecule (W1) that formed hydrogen bonds with the 7 amide

carbonyl and the Ser275 and Arg311 side chains. The Ph-SO₂-

piperidine portion of 7 was bound to the Nampt “tunnel”

C

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Table 2. Heterobiaryl SAR

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Table 2. continued

a

b

c

Nampt biochemical inhibition. A2780 cell proliferation inhibition. This inhibition can be reversed by addition of 0.33 mM of NMN. Unless

d

otherwise noted, all biochemical and cell-based assay results are reported as the arithmetic mean of at least two separate runs (n = 2). Mouse liver

e

f

g

microsomal stability% remaining at 30 min. Human liver microsomal stability% remaining at 30 min. Below limit of quantiﬁcation. Not

determined. See Supporting Information for experimental details.

region in a manner similar to that noted previously for related

urea and thiourea inhibitiors^9,10with water-mediated (W2, W3,

and W4) hydrogen bonds being formed to both sulfonamide

oxygen atoms. However, due to the shift in the position of 7

relative to 5 described above, the Ph-SO₂-piperidine and

azaindole-carboxamide moieties of 7 were spatially distant and

could not be directly linked (as was attempted in the inactive

compound 6, Figure 3). This observation clearly explained how

the nonobvious inclusion of the methylene group present in 7

enabled optimal binding of the linked Ph-SO₂-piperidine and

azaindole-carboxamide moieties to Nampt, thereby aﬀording

the dramatic potency improvements relative to 6.

With the preceding crystallographic information in hand, we

initiated activities to deﬁne various structure−activity relation-

ships within the amide inhibitor series. We ﬁrst conﬁrmed that,

consistent with our previous ﬁndings,⁹an inhibitor bearing a

E

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terminal phenyl sulfone was equipotent and more metabolically

stable than the corresponding piperidine-sulfonamide (compare

7 and 8, Table 1). With respect to the moiety linking the amide

and diaryl sulfone, we found that, consistent with the close

proximity of the methylene group present in 7 to various

Nampt residues, adding a methyl group to this portion of 7

caused a complete loss of activity (9; Table 1). Similarly,

replacing the methylene present in 8 with an ethylene linker

(compound 10) caused a 30-fold loss in biochemical activity

and an even more dramatic loss of cellular potency. This

unfavorable outcome was likely due to the inability to

appropriately reposition the diaryl sulfone moiety of 10 in

the Nampt tunnel region in order to accommodate the larger

linker group. Collectively, these results identiﬁed an unsub-

stituted methylene moiety as the preferred linker for obtaining

both potent biochemical and cellular anti-Nampt activity.

Accordingly, this functional group was retained in subsequent

inhibitor designs throughout the remainder of the SAR studies.

Encouraged by this discovery, we next investigated the SAR

surrounding the azaindole moiety present in compounds 7 and

8. Our previous modiﬁcations to the corresponding region of

related urea-containing inhibitors indicated that an appropri-

ately positioned nucleophilic nitrogen atom was often necessary

to provide potent, cell-active compounds and that subtle

positional alterations of this atom could have severe negative

eﬀects.^9,10These results are believed to be due to the ability of

certain inhibitors to function as NAM mimics and readily form

PRPP-derived phosphoribosylated adducts in the Nampt active

site.^12,15Consistent with this hypothesis, the PRPP adduct

corresponding to the potent cell-active inhibitor 8 was observed

by mass spectrometry in biochemical Nampt inhibition

experiments (see Supporting Information for more details).

As shown in Table 2, moving the pyrido nitrogen atom present

in 7 to the adjacent position (compound 11) caused a >100-

fold loss of biological activities. The weak biological activities

exhibited by 11 may result from its inability to form the

corresponding PRPP adduct, although we did not conﬁrm this

possibility via mass spectrometry.¹⁶Introducing an additional

nitrogen atom into the azaindole system drastically diminished

biological activity (12), likely due to ionization of the new

heterocycle under physiological conditions, thus causing an

electrostatically unfavorable interaction with Asp219 (cf., Figure

4; JChem predicts an NH pK_avalue of 7.7 for compound 12

and its tautomeric form as compared to 10.8 for compound 8).

Similarly, methylation of the pyrrole nitrogen of 8, which was

expected to disrupt a hydrogen bonding interaction with the

side chain of Asp219 (cf., Figure 4), led to signiﬁcant loss of

biological activities (compare to 13). In contrast, the N-

methylated compound 14 exhibited slightly improved bio-

chemical activity as compared with the unmethylated analog 11.

This result was consistent with an altered Nampt binding mode

for 11 relative to 8 which did not involve formation of a

hydrogen bond between the azaindole NH of 11 and Asp219

(cf., Figure 4).¹⁶Not unexpectedly, changing the amide

attachment location to the 3-position of the azaindole, and

thereby likely signiﬁcantly altering the positioning of this

moiety relative to that observed in the 7−Nampt cocrystal

structure, was also highly detrimental to biological activity

(compare 15 with 8).

interested in examining 6,5-fused-heterocycles which lacked

the NH present in azaindole-containing molecules such as 7

and 8. Such compounds would test whether loss of the

associated H-bond with Asp219 could be tolerated if additional

steric bulk was not simultaneously introduced into the inhibitor

(as in compound 13 above). Such toleration might be achieved

by the combination of proper steric ﬁt of the nonazaindole

heterocycles in the NAM binding region of Nampt and the

formation of an appropriate H-bond interaction between the

amide NH present in the new designs and Asp219 (cf., Figure

4).

As shown in Table 2, many compounds bearing furopyr-

idines, thienopyridines, and thiazolopyridines were identiﬁed

which exhibited potent Nampt inhibition properties in

biochemical assays (16−24). These results conﬁrmed that

appropriate 6,5-bicyclic heterocycles which were incapable of

H-bonding with Asp219 could nevertheless be tolerated in the

Nampt active site (cf., Figure 4). However, only a subset of

these biochemically active molecules displayed submicromolar

potency in Nampt-related cell-based assessments (17−20).

This discrepancy may partially result from the inability of the

less cell-potent compounds to form the corresponding PRPP

adducts, although we did not conﬁrm this possibility for all such

inhibitors.¹⁵Consistent with this hypothesis, the PRPP−ribose

adduct corresponding to 20, a potent Nampt inhibitor in cell

culture, was indeed detected by mass spectrometry in

biochemical Nampt inhibition experiments (see Supporting

Information for more details). Many of the disappointing cell

culture results might also be related to the relatively weak

Nampt biochemical inhibition exhibited by the corresponding

molecules (e.g., 16, 21, 23, and 24). However, consistent with

the PRPP−ribose adduct hypothesis, compound 22 displayed

drastically attenuated cell potency relative to its biochemically

equipotent structural isomer 19. The latter inhibitor likely

positions its thienopyridine nitrogen in a manner similar to that

observed crystallographically for the corresponding N-atom of

the cell-potent azaindole-containing compound (7) described

earlier in this work (cf., Figure 4). The proposed positioning

may reﬂect an optimal orientation for the formation of the

corresponding PRPP adducts in the Nampt active site and, due

to the diﬀerent location of the nitrogen atom in the

thienopyridine ring system, is not achievable by the structural

isomer 22. In addition, the suggested binding of 19 to Nampt

orients the compound’s thienopyridine sulfur atom away from

Asp219 and thus avoids an unfavorable electronic interaction

between these two moieties. Such binding contrasts with that

observed crystallographically for compound 7, in which the

inhibitor’s azaindole NH faces Asp219 and forms an H-bond

with the residue (cf., Figure 4). Compound 20 presumably

binds to Nampt in a manner analogous to that described for 19,

and similar orientations likely also apply to the potent

furopyridine-containing inhibitors 17 and 18. These optimal

positionings are not possible for the less potent isomeric

compounds 16 and 21, as overlap of the respective pyridine

nitrogens with the corresponding N-atoms present in 17−20

unfavorably orients their polar furopyridine oxygen and

thienopyridine sulfur moieties toward Asp219.

We also explored inhibitors containing other 6,5-bicyclic

heterocycles in which the amide group was attached to the 6-

membered aromatic ring of the fused system. As shown in

Table 2, all of these molecules inhibited Nampt to varying

degrees in biochemical assays (25−35) but only four, which

incorporated two closely related heterocycles, exhibited potent

Satisﬁed with the potent biochemical and cellular activity

exhibited by the azaindole-containing compounds 7 and 8, we

explored incorporation of other fused heterobiaryl systems into

the amide-based inhibitor design. We were particularly

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cell-based activity (27, 28, 32, and 33). For compounds 25−35,

the lack of strong cell potency correlated well with weak

biochemical Nampt inhibition (inhibitor 25 is a possible

exception), thus making it diﬃcult to determine whether the

ability of the molecules to form PRPP−ribose adducts

contributed to their cell culture activity. However, consistent

with the ribose adduct hypothesis described above, the PRPP−

ribose adducts corresponding to the cell-potent inhibitors 28

and 33 were observed in biochemical experiments by mass

spectrometry (see Supporting Information for more details). In

addition, the exposed imidazopyridine nitrogen atom contained

in an optimized compound related to 28 was crystallo-

graphically observed in a location very similar to that noted

for the corresponding N-atom of the azaindole-containing

molecule 7 (cf., Figure 5). Additional details regarding the

implications of this common binding mode are provided later

in this work.

liver microsomes consistently indicated that biaryl sulfones

exhibited improved stability properties relative to the

corresponding piperidine-derived phenyl-sulfonamides (e.g.,

compare 17 with 18). The biaryl sulfone motif was therefore

also incorporated into many subsequent inhibitor designs.

As a prelude to conducting in vivo eﬃcacy experiments, we

next focused our inhibitor optimization eﬀorts on identifying

highly potent cell-active compounds which also exhibited

attractive in vitro DMPK properties. This work was initially

conducted using the azaindole present in 8 with the expectation

that promising modiﬁcations so identiﬁed could be transferred

to compounds containing other fused heterocycles. Previous

work with related urea-containing molecules indicated that

alterations to the biarylsulfone portion of the inhibitors could

greatly impact potency and biopharmaceutical properties.^9,10

Similar modiﬁcations were expected to be tolerated in the

amide-containing Nampt inhibitor series, since the biarylsul-

fone terminus was anticipated to be highly solvent-exposed (cf.,

Figure 4). As shown in Table 3, derivitization of the terminal

phenyl ring contained in 8 aﬀorded a variety of compounds that

all displayed potent biochemical and cell-based anti-Nampt

properties (41−51). Among these molecules, those bearing

terminal 3-Me-Ph (42), 3-CF₃-Ph (44), 3,5-di-F-Ph (49), and

4-morpholino-3-pyridyl (51) substituents exhibited A2780 cell

IC₅₀values of less than 10 nM. The latter three compounds

also showed good in vitro stability toward both murine and

human liver microsomes. Those three moieties were therefore

chosen for inclusion in amide-containing Nampt inhibitors

bearing nonazaindole fused heterocycles. As shown in Table 4,

all such combinations successfully aﬀorded very potent cell-

active Nampt inhibitors, and most of these compounds

exhibited good microsomal stability properties (exceptions:

52, 57, 59, and 62). Crystallographic rationalizations for the

structure−activity relationships depicted in Tables 3 and 4 are

provided below.

To enable further optimization of the described amide-

containing Nampt inhibitors, a cocrystal structure of compound

58 in complex with Nampt was solved (Figure 5). The binding

mode of the ligand was very similar to that observed for

compound 7 with a hydrogen bond noted between the amide

NH and Asp219. A water-mediated (W1) hydrogen bond was

also observed between the amide carbonyl and Ser275. The

bicyclic imidazopyridine ring of 58 was stacked between the

side chains of Nampt residues Tyr18′ and Phe193 in the NAM

binding region with the imidazole nitrogen pointing toward the

PRPP binding site. Interestingly, this nitrogen atom was

positioned in a location that was very similar to that observed

for the pyridyl N-atom contained in 7 (Figure 5). This

positioning was maintained in spite of the diﬀering

connectivities between the bicyclic rings present in 7 and 58

and the associated carboxamide moieties of two molecules, and

it may reﬂect an optimal orientation for the formation of the

corresponding PRPP adducts in the Nampt active site.^15,17

Consistent with this hypothesis, the locations of the exposed

heteroaryl N-atoms present in 7 and 58 were also very similar

to those observed crystallographically for the pyridine nitrogen

of the NMN product that results from Nampt-catalyzed

condensation of NAM with PRPP (cf., Figure 1).¹⁸Many of

the atoms present in the bicyclic heterocycles contained in 7

and 58 were observed in close spatial proximity (<3.8 Å) to the

side chains of several Nampt residues [e.g., Asp219, Arg311,

and Asp16′ (not shown)]. Such proximity likely explains the

failure of compounds containing 6,6-bicyclic heterocycles to

Figure 5. Co-crystal structure of compound 58 (green) in complex

with Nampt (gray); PBD Code: 4KFO. Water molecules are depicted

as red spheres and possible hydrogen bonds are indicated with dashed

yellow lines. The resolution of the structure is 1.6 Å. For comparison,

the structure of compound 7 is shown in magenta.

Inclusion of 6,6-fused bicyclic heterocycles into the inhibitor

design was less successful, with the best compounds showing

weak biochemical inhibition and very poor cellular activity

(36−38). These results likely reﬂect the inability of the larger

bicyclic heterocycles to ﬁt properly in the NAM binding site of

Nampt, in contrast to the good ﬁt observed for many

compounds containing 6,5-bicyclic moieties (cf., Figure 4). In

addition, several biaryl-containing molecules exhibited relatively

potent biochemical Nampt inhibition properties but were

devoid of cell-based activity (39 and 40). It is unknown at this

time whether this discrepancy is related to the PRPP−ribose

adduct hypothesis outlined above or is due to some other

phenomenon (e.g., relatively weak biochemical Nampt

inhibition).

Collectively, the above SAR exploration identiﬁed the fused

heterocycles present in compounds 7−8, 17−20, 27−28, and

32−33 as those most likely to impart both potent biochemical

and cell-based Nampt inhibition properties to compounds

which contained them. These ﬁve fused heterocycles were

therefore employed in the subsequent SAR studies described in

the remainder of this work. In addition, in vitro assessments

conducted with the Table 2 compounds in murine and human

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Table 3. Biarylsulfone Substitution SAR

a

b

c

Nampt biochemical inhibition. A2780 cell proliferation inhibition. This inhibition can be reversed by addition of 0.33 mM of NMN. Unless

d

otherwise noted, all biochemical and cell-based assay results are reported as the arithmetic mean of at least two separate runs (n = 2). Mouse liver

e

microsomal stability% remaining at 30 min. Human liver microsomal stability% remaining at 30 min. See Supporting Information for

experimental details.

potently inhibit Nampt (e.g., 36−38, Table 2; may also apply to

compounds 13 and 14) but does not illuminate why many

close structural isomers of the heterocycles contained in the

most active inhibitors lost signiﬁcant potency (e.g., 29−31, 34−

35).

The diﬂuorophenyl moiety of 58 moved slightly closer to the

protein surface relative to the piperidine present in compound

7 and ﬁlled a groove which was deﬁned by His191, Tyr188,

Val242, and Tyr240 (not all residues shown in Figure 5). This

location oriented one of the meta-substituents (relative to the

sulfone moiety) of the terminal phenyl moiety toward the

protein surface and the other meta-substituent toward solvent.

The vector emerging from the para-position (relative to the

sulfone moiety) of the terminal phenyl group also pointed into

a solvent-exposed region. The observed binding mode was thus

consistent with the structure−activity relationships depicted in

Tables 3 and 4, with analogs bearing 3-substituents being the

most potent and a variety of 4-substituents being tolerated. A

water-mediated hydrogen bond involving the sulfone moiety of

58 was also observed, although this was not identical to those

noted in the 7−Nampt cocrystal structure described at the

beginning of this work.

The explorations described above identiﬁed multiple

compounds which exhibited potent anti-Nampt properties in

biochemical and cellular assays as well as good microsomal

stabilities. In anticipation of conducting in vivo animal

experiments with such molecules, we proﬁled them in various

additional in vitro ADME assessments designed to assess their

in vivo potential. As shown in Table 5, of the molecules proﬁled

in these in vitro ADME assessments, compound 58 exhibited

the most optimal combination of in vitro metabolic stability,

MDCK permeability, and protein binding. Collectively, these

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Table 4. Combined SAR

a

b

c

Nampt biochemical inhibition. A2780 cell proliferation inhibition. This inhibition can be reversed by addition of 0.33 mM of NMN. Unless

d

otherwise noted, all biochemical and cell-based assay results are reported as the arithmetic mean of at least two separate runs (n = 2). Mouse liver

e

microsomal stability% remaining at 30 min. Human liver microsomal stability% remaining at 30 min. See Supporting Information for

experimental details.

Table 5. In Vitro Stability, Permeability, and Plasma Protein Binding of Selected Nampt Inhibitors

a

b

c

d

e

entry

MH, CL_hep(mL/min/kg)

HH, CL_hep(mL/min/kg)

10⁶MDCK P_app,A‑B(cm/s)

mouse PPB (f_u)

human PPB (f_u)

1

21

67

60

70

57

25

48

78

73

42

43

74

69

7

15

10

7

10

15

5

0.005

0.033

0.007

0.028

0.015

0.011

0.017

0.011

0.002

0.067

0.025

0.133

0.120

0.005

0.007

0.006

0.028

0.011

0.010

0.005

0.009

0.004

0.060

0.012

0.122

0.028

2

44

49

51

53

54

55

56

58

60

61

63

12

1

13

10

14

16

14

10

6

13

15

8

12

3

5

6

0.3

a

b

Hepatic clearance predicted from mouse hepatocytes (stable, moderate, labile = <27, 27−63, >63 mL/min/kg, respectively). Hepatic clearance

c

predicted from human hepatocytes (stable, moderate, labile = <6.2, 6.2−15, >15 mL/min/kg, respectively). Apparent permeability coeﬃcients

d

e

(P_app,A‑B) across MDCK cell monolayers. Mouse plasma protein binding; fraction unbound (f_u). Human plasma protein binding; fraction unbound

(f_u). All the data reported in the table is the average of ≥2 runs. See Supporting Information for experimental details associated with each

assessment.

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a

Table 6. Antiproliferation Eﬀects Determined for Compound 58 in Various Cell Lines

b

c

U251 (glioblastoma)

HT1080 (ﬁbrosarcoma)

PC3 (prostate cancer)

MiaPaCa2 (pancreatic cancer)

HCT116 (colon cancer)

2.0 0.5

IC₅₀(nM)

1.8 0.6

2.1 0.2

2.7 1.0

7.4 2.7

a

b

c

All antiproliferation eﬀects were reversed by addition of 0.3 mM NMN. SRB end point. CyQuant end point. All the data reported in the table

reﬂect the average of three independent runs. See Supporting Information for experimental details.

properties compared favorably with those exhibited by

compounds 1 and 2 in similar evaluations (58 = signiﬁcantly

less protein bound than both 1 and 2 and more metabolically

stable than 2). In addition, compound 58 displayed potent

antiproliferative eﬀects in a variety of cell lines for which mouse

xenograft models were available (Table 6). The compound was

therefore examined in in vivo mouse PK studies and a xenograft

eﬃcacy experiment (employing a model derived from one

Table 6 cell line) to evaluate its oral exposure and antitumor

eﬀects.

In an eﬃcacy study, compound 58 exhibited signiﬁcant

antitumor eﬀects against a U251 human glioblastoma tumor

xenograft model without signiﬁcant body weight loss. In mice

bearing U251 xenografts, oral administration of the compound

for 7 days (25 mg/kg QD) produced rapid tumor regression

(Figure 7). The antitumor eﬀects of compound 58 were

durable, with the mean tumor volume of the treated group

failing to return to baseline levels by the end of study (day 13).

The described xenograft results conﬁrmed that an amide-

containing Nampt inhibitor with appropriate biological and

DMPK properties can function as a potent in vivo anticancer

agent.¹⁹

Chemistry. The amide derivatives were prepared by

utilizing the procedures shown in Schemes 1−8. As depicted

in Scheme 1, aromatic acids (65) or esters (66) were coupled

with appropriate amines (64) to give the amides 6−63 (details

are noted below). To speed up our SAR exploration, a three

step parallel synthesis protocol was also developed as shown in

Scheme 2. In this process, sulﬁnate (67) was coupled with

various aryl boronic acids (68a−68g) to provide the

corresponding biaryl sulfone intermediates (69). Removal of

the Boc protecting group from 69 led to the benzyl amines

(64a−64g). BOP facilitated coupling of the amines (64) with

azaindole-2-carboxylic acid (65a) provided the ﬁnal products

41−43, 45, 46, 48, and 50. The relatively low yields of the ﬁnal

products reﬂect the three-step reaction sequence in which the

intermediates 68 and 64 were not isolated and/or puriﬁed.

The syntheses of the aromatic acid methyl esters 66a and

66b are summarized in Schemes 3 and 4. Treatment of the

diamine (70) with imidate (71) gave the intermediate 72,

which was cyclized under basic conditions to yield compound

66a (Scheme 3). Cyclization of 5-bromopyrazin-2-amine (73)

with 2-bromo-1,1-dimethoxyethane (74) provided the imida-

zopyrazine intermediate (75). Coupling of 75 with zinc cyanide

aﬀorded the corresponding nitrile compound (76). Partial

Table 7. Mean Pharmacokinetic Parameters of Compound

58 in NCR Nude Mice (n = 3)

dose

route (mg/kg)

C

T_max

c

V_ss

(L/kg)

CL

F

(%)

a

^maxb

d

e

f

(μM)

(h)

(mL/min/kg)

IV

5

6.7

52.4

PO

10

1.21

0.25

41.1

a

Formulations: IV = 35% polyethylene glycol (PEG 400)/10%

ethanol/15% PG/40% H₂O (v/v/v); PO = 60% polyethylene glycol

b

(PEG 400)/10% ethanol/30% H₂O (v/v/v). Maximum plasma

c

concentration. Time at which maximum plasma concentration was

d

e

observed. Volume of distribution (steady-state). Clearance.

f

Bioavailibility. See Supporting Information for experimental descrip-

tions.

As summarized in Table 7 and shown in Figure 6, compound

58 demonstrated reasonable PK properties in mice. After IV

dose administration at 5 mg/kg, the plasma clearance was

moderate (52.4 mL/min/kg) and the volume of distribution

was 6.7 L/kg. Following a single oral dose of 10 mg/kg,

absorption of 58 was rapid (T_max= 0.25 h) and the compound

showed good oral exposure (AUC_inf= 3.06 μM h, C_max= 1.21

μM; %F = 41.1) with a free C_maxvalue ≥18-fold above the

cellular IC₅₀values (Table 6, 0.0018−0.0074 μM).

Figure 6. Mean ( standard deviation) plasma concentration−time proﬁles of compound 58 in NCR nude mice (n = 3) following intravenous and

oral administration.

J

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Figure 7. Left panel: compound 58 in vivo eﬃcacy results in U251 human glioblastoma tumor xenograft model. Right panel: changes in body

weight. Data reﬂect mean values ( standard deviation) of eight animals in each group.

a

Scheme 1

a

Reagents and conditions: (i) HATU, EtN(i-Pr)₂, DMF, 25 °C, 5−96%; (ii) EDCI, HOBt, EtN(i-Pr)₂, DMF, 80 °C, 4−76%; (iii) BOP, EtN(i-Pr)₂,

DMF, 25 °C, 40−50%; (iv) AlMe₃, toluene, 0−110 °C, 6−92%.

a

Scheme 2

a

Reagents and conditions: (i) Cu(OAc)₂, Et₃N, DMSO, 60 °C; (ii) HCl, 25 °C; (iii) BOP, Et₃N, DMA, 25 °C, 4−11% over three steps. All steps

were performed using a parallel synthesis approach, and intermediates 68 and 64 were not isolated and/or puriﬁed.

hydrolysis of 76 provided the ester compound 66b. The

methods employed to synthesize 66a and 66b reﬂect

unoptimized reaction conditions. As the described procedures

aﬀorded suﬃcient quantities of these intermediates for ﬁnal

compound preparation, no attempts were made to further

improve these processes.

Schemes 5−7 highlight the synthesis of various amines (64).

In Scheme 5, sulﬁnate (77) was coupled with aryl boronic acids

(68h−68j) to provide the corresponding biaryl sulfone

intermediates (78). Removal of the acetyl group from 78 led

to the benzyl amines 64h−64j. Synthesis of compound 64k is

summarized in Scheme 6. Nucleophlic reaction of 79 with 80

K

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a

Scheme 3

a

Reagents and conditions: (i) CF₃COOH, 0−25 °C, 98%; (ii) Na₂CO₃, H₂O/MeOH, reﬂux, 3%.

a

Scheme 4

a

Reagents and conditions: (i) EtOH/48% HBr, 80 °C, 15%; (ii) Zn(CN)₂, Pd(PPh₃)₄, DMF, 120 °C, 24%; (iii) HCl/MeOH, 80 °C, 12%.

a

Scheme 5

a

Reagents and conditions: (i) Cu(OCOCH₃)₂, K₂CO₃, DMSO, 25 °C, 16−40%; (ii) 12 M HCl/i-PrOH, 100 °C, 86−100%.

a

Scheme 6

a

Reagents and conditions: (i) DMSO, 120 °C, 30%; (ii) H₂−Raney Ni, 2 M NH₃/MeOH, 25 °C, 94%.

a

Scheme 7

a

Reagents and conditions: (i) EtN(i-Pr)₂, CH₂Cl₂, 25 °C, 100%; (ii) Ti(OEt)₄, t-BuSONH₂, THF, 25 °C, 59%; (iii) MeLi, Et₂O, −78 to 25 °C,

47%; (ii) 4.0 M HCl-1,4-dioxane, MeOH, 25 °C, 68%.

a

Scheme 8

a

Reagents and conditions: (i) Pd₂(dba)₃, Xantphos, EtN(i-Pr)₂, toluene, 100 °C, 94%; (ii) Oxone, H₂O−CH₃CN, 25 °C, 94%; (iii) NaOEt, MeOH,

50 °C, 97%.

gave the sulfone nitrile (81), which was subsequently reduced

to the corresponding benzyl amine 64k. In Scheme 7, sulfonyl

chloride (82) was reacted with piperidine (83) to give the

sulfonamide-aldehyde (84), which was then converted to the

L

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temperature. 4-(Piperidin-1-ylsulfonyl)aniline (64m, 120 mg, 0.50

mmol) was then added, and the reaction mixture was stirred at room

temperature for 16 h. The mixture was diluted with water (50 mL) and

extracted with EtOAc (3 × 20 mL). The organic layers were combined

and washed with water (3 × 20 mL) and brine (30 mL), dried with

anhydrous Na₂SO₄, ﬁltered, and concentrated under reduced pressure.

The residue was puriﬁed by Biotage with CH₂₁Cl₂/MeOH (100:0 to

90:10) to give the title product in 5% yield. H NMR (300 MHz,

DMSO-d₆) δ: 12.23 (s, 1H), 10.72 (s, 1H), 9.01 (s, 1H), 8.25 (d, J =

5.7 Hz, 1H), 8.55 (d, J = 8.7 Hz, 2H), 7.73 (d, J = 8.7 Hz, 2H), 7.62 (s,

1H), 7.39 (d, J = 5.1 Hz, 1H), 2.86 (br s, 4H), 1.53 (br s, 4H), 1.34 (br

s, 2H). LCMS (method LCMS1, ESI): RT = 1.28 min, m/z = 385.2

[M + H]⁺.

corresponding sulﬁnamide (85). Methyllithium addition to 85

yielded compound 86, and subsequent removal of the sulﬁnyl

group gave amine compound 64l (Scheme 7).

The synthesis of key intermediate 67 is shown in Scheme 8.

Bromide (87) was coupled with the thiol (88) to give the

thioether (89). Oxidation of the thioether (89) with oxone led

to compound 90 and treatment of this intermediate with base

provided compound 67.

CONCLUSIONS

■

In summary, using structure-guided design and leveraging key

information from our previously disclosed (thio)urea cocrystal

structures, we discovered a structurally novel amide-containing

Nampt inhibitor (7). Guided by the crystal structure of

compound 7 in complex with Nampt, further SAR optimization

ultimately yielded compound 58, which possessed single digit

nanomolar cellular IC₅₀against several human tumor cell lines

and excellent in vitro and in vivo ADME properties. Our results

suggest that (easily synthesized) compound 58 oﬀers a robust

and structurally distinct alternative to the well-known Nampt

inhibitors 1 and 2 for conducting in vitro and in vivo studies to

probe Nampt-related biology. In particular, the binding mode

of compound 58 to Nampt is crystallographically characterized

in the present work, thus enabling a good understanding of the

molecular interactions which contribute to its potent

biochemical inhibition of the protein as well as the SAR

associated with close analogs. In contrast, the crystal structure

of compound 1 in complex with Nampt has not been disclosed

in the literature. In addition, 58 exhibits very potent anti-

Nampt activity in cell culture against multiple xenograftable

lines. It also displays robust and durable eﬃcacy in a mouse

xenograft model derived from one such line (U251) at doses

that are comparable to those typically employed in related

Compounds 7−11, 16, 22−23, 25−26, 28, 31, 33, 35−38, 44, 47,

49, 51, 58, 60−61, and 63 were synthesized using conditions (HATU

coupling) similar to those employed to make compound 6.

N-{[4-(Piperidine-1-sulfonyl)phenyl]methyl}-1H-pyrrolo[3,2-

c]pyridine-2-carboxamide (7). Substituting the corresponding

reagents with 1H-pyrrolo[3,2-c]pyridine-2-carboxylic acid (65a) and

(4-(piperidin-1-ylsulfonyl)phenyl)methanamine (64n), the title prod-

1

uct was obtained in 24% yield. H NMR (300 MHz, DMSO-d₆) δ:

12.07 (s, 1H), 9.32 (t, J = 1.5 Hz, 1H), 8.92 (s, 1H), 8.21 (d, J = 6.0

Hz, 1H), 7.69 (d, J = 8.1 Hz, 2H), 7.56 (d, J = 8.1 Hz, 2H), 7.38−7.31

(m, 2H), 4.61 (d, J = 5.7 Hz, 1H), 2.86−2.81 (m, 4H), 1.51 (br s, 4H),

1.32 (br s, 2H). LCMS (method LCMS1, ESI): RT = 0.99 min, m/z =

399.1 [M + H]⁺.

N-{[4-(Benzenesulfonyl)phenyl]methyl}-1H-pyrrolo[3,2-c]-

pyridine-2-carboxamide (8). Substituting the corresponding

reagents with 1H-pyrrolo[3,2-c]pyridine-2-carboxylic acid (65a) and

(4-(phenylsulfonyl)phenyl)methanamine (64o), the title product was

1

obtained in 68% yield. H NMR (300 MHz, DMSO-d₆) δ: 12.0 (s,

1H), 9.25 (s, 1H), 8.92 (s, 1H), 8.20 (s, 1H), 8.00−7.90 (m, 4H),

7.73−7.50 (m, 5H), 7.35 (s, 1H), 7.22 (s, 1H), 4.60 (s, 2H). LCMS

(method LCMS1, ESI): RT = 1.50 min, m/z = 392.1 [M + H]⁺.

N-{1-[4-(Piperidine-1-sulfonyl)phenyl]ethyl}-1H-pyrrolo[3,2-

c]pyridine-2-carboxamide (9). Substituting the corresponding

reagents with 1H-pyrrolo[3,2-c]pyridine-2-carboxylic acid (65a) and

1-(4-(piperidin-1-ylsulfonyl)phenyl)ethanamine hydrochloride (64l),

studies with 1.⁵Importantly, these eﬀects are observed after

d

once-daily oral administration of 58, in contrast to the

1

the title product was obtained in 96% yield. H NMR (300 MHz,

intraperitoneal delivery frequently employed in xenograft

CDCl₃) δ: 8.98 (s, 1H), 8.39 (d, J = 6.0 Hz, 1H), 7.76−7.71 (m, 2H),

7.56−7.52 (m, 2H), 7.35−7.31 (m, 1H), 7.03 (s, 1H), 6.60−6.54 (m,

1H), 5.45−4.36 (m, 1H), 3.01−2.96 (m, 4H), 1.69−1.60 (m, 7 H),

1.45−1.37 (m, 2H). LCMS (method LCMS1, ESI): RT = 1.85 min,

m/z = 413.0 [M + H]⁺.

N-{2-[4-(Benzenesulfonyl)phenyl]ethyl}-1H-pyrrolo[3,2-c]-

pyridine-2-carboxamide (10). Substituting the corresponding

reagents with 1H-pyrrolo[3,2-c]pyridine-2-carboxylic acid (65a) and

2-(4-(phenylsulfonyl)phenyl)ethanamine (64p²⁰), the title product

was obtained in 57% yield. ¹H NMR (300 MHz, DMSO-d₆) δ: 12.0 (s,

1H), 8.95 (s, 1H), 8.8.85 (s, 1H), 8.20 (s, 1H), 8.00−7.97 (m, 4H),

7.73−7.54 (m, 3H), 7.43 (s, 1H), 7.35 (s, 1H), 7.18 (s, 1H), 3.70−

3.30 (m, 2H), 3.00−2.80 (m, 2H). LCMS (method LCMS1, ESI): RT

= 0.85 min, m/z = 406.1 [M + H]⁺.

studies conducted with 2.⁶As the described experiments are

c

somewhat limited in scope and do not directly compare

individual molecules, additional studies will be required to fully

deﬁne the biological characteristics of 58 relative to other

Nampt inhibitors, including compounds 1 and 2.

EXPERIMENTAL SECTION

■

General. Unless otherwise indicated, all reagents and solvents were

purchased from commercial sources and were used without further

puriﬁcation (including the following numbered compounds: 64n, 65a,

65d, 65e, 65f, 65g, 65h, 65i, 65k, 65l, 65m, 65n, 65p, 65r, 65s, and

66d). Moisture or oxygen sensitive reactions were conducted under an

atmosphere of argon or nitrogen gas. Unless otherwise stated, ¹H

NMR spectra were recorded at 300 or 400 MHz using Varian or

Bruker instruments operating at the indicated frequencies. Chemical

shifts are expressed in ppm relative to an internal standard;

tetramethylsilane (ppm = 0.00). The following abbreviations are

used: br = broad signal, s = singlet, d = doublet, dd = doublet of

doublets, t = triplet, q = quartet, p = pentet, m = multiplet. Puriﬁcation

by silica gel chromatography was carried out using Biotage systems

with prepacked cartridges. Chemical purities were >95% for all ﬁnal

compounds, as assessed by LCMS analysis at UV 220 nm. Details

regarding the LCMS conditions can be found in the Supporting

Information.

N-{[4-(Piperidine-1-sulfonyl)phenyl]methyl}-1H-pyrrolo[2,3-

c]pyridine-2-carboxamide (11). Substituting the corresponding

reagents with 1H-pyrrolo[2,3-c]pyridine-2-carboxylic acid (65b) and

(4-(piperidin-1-ylsulfonyl)phenyl)methanamine (64n), the title prod-

1

uct was obtained in 40% yield. H NMR (300 MHz, DMSO-d₆) δ:

12.13 (s, 1H), 9.38 (s, 1H), 8.78 (s, 1H), 8.11 (d, J = 5.1 Hz, 1H),

7.71−7.55 (m, 5H), 7.21 (s, 1H), 4.62 (d, J = 6.0 Hz, 1H), 2.86−2.81

(m, 4H), 1.51 (br s, 4H), 1.32 (br s, 2H). LCMS (method LCMS1,

ESI): RT = 1.02 min, m/z = 400.0 [M + H]⁺.

N-{[4-(Piperidine-1-sulfonyl)phenyl]methyl}furo[3,2-c]-

pyridine-2-carboxamide (16). Substituting the corresponding

reagents with furo[3,2-c]pyridine-2-carboxylic acid (65c²¹) and (4-

(piperidin-1-ylsulfonyl)phenyl)methanamine (64n), the title product

N-[4-(Piperidine-1-sulfonyl)phenyl]-1H-pyrrolo[3,2-c]-

pyridine-2-carboxamide (6). To a suspension of 1H-pyrrolo[3,2-

c]pyridine-2-carboxylic acid (65a, 81 mg, 0.50 mmol) in DMF (8 mL)

were added HATU (285 mg, 0.75 mmol) and EtN(i-Pr)₂(0.131 mL,

0.749 mmol), and the resulting mixture was stirred for 25 min at room

1

was obtained in 15% yield. H NMR (300 MHz, CDCl₃) δ: 9.05 (s,

1H), 8.62 (d, J = 6.0 Hz, 1H), 7.73−7.70 (m, 2H), 7.60 (s, 1H), 7.52−

7.45 (m, 3H), 7.20−7.13 (m, 1H), 4.76 (d, J = 6.0 Hz, 2H), 3.00−2.93

M

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(m, 4H), 1.68−1.58 (m, 4H), 1.45−1.38 (m, 2H). LCMS (method

LCMS1, ESI): RT = 1.89 min, m/z = 400.0 [M + H]⁺.

9.27 (m, 2H), 9.13−9.09 (m, 1H), 7.96−7.88 (m, 4H), 7.85−7.72 (m,

2H), 7.69−7.53 (m, 5H), 4.54 (d, J = 6.0 Hz, 2H). LCMS (method

LCMS1, ESI): RT = 1.64 min, m/z = 393.1 [M + H]⁺.

N-{[4-(Piperidine-1-sulfonyl)phenyl]methyl}thieno[2,3-b]-

pyridine-2-carboxamide (22). Substituting the corresponding

reagents with thieno[2,3-b]pyridine-2-carboxylic acid (65d) and (4-

(piperidin-1-ylsulfonyl)phenyl)methanamine (64n), the title product

was obtained in 62% yield. ¹H NMR (300 MHz, DMSO-d₆) δ: 9.52 (t,

J = 6.0 Hz, 1H), 8.66−8.63 (m, 1H), 8.37 (d, J = 3.3 Hz, 1H), 8.13 (s,

1H), 7.77−7.64 (m, 2H), 7.61−7.55 (m, 2H), 7.51−7.47 (m, 1H),

4.60 (d, J = 6.0 Hz, 2H), 2.87−2.82 (m, 4H), 1.52−1.47 (m, 4H),

1.35−1.31 (m, 2H). LCMS (method LCMS1, ESI): RT = 1.40 min,

m/z = 416.0 [M + H]⁺.

N-{[4-(Benzenesulfonyl)phenyl]methyl}isoquinoline-6-car-

boxamide (36). Substituting the corresponding reagents with

isoquinoline-6-carboxylic acid (65l) and (4-(phenylsulfonyl)phenyl)-

1

methanamine (64o), the title product was obtained in 96% yield. H

NMR (300 MHz, DMSO-d₆) δ: 9.40 (s, 2H), 8.58 (s, 1H), 8.47 (s,

1H), 8.22−8.18 (m, 1H), 8.05−8.02 (m, 1H), 7.98−7.82 (m, 5H),

7.70−7.50 (m, 5H), 4.60 (s, 2H). LCMS (method LCMS1, ESI): RT

= 1.15 min, m/z = 403.1 [M + H]⁺.

N-{[4-(Benzenesulfonyl)phenyl]methyl}isoquinoline-7-car-

boxamide (37). Substituting the corresponding reagents with

isoquinoline-7-carboxylic acid (65m) and (4-(phenylsulfonyl)phenyl)-

N-{[4-(Benzenesulfonyl)phenyl]methyl}thieno[3,2-b]-

pyridine-2-carboxamide (23). Substituting the corresponding

reagents with thieno[3,2-b]pyridine-2-carboxylic acid (65e) and (4-

(phenylsulfonyl)phenyl)methanamine (64o), the title product was

1

methanamine (64o), the title product was obtained in 37% yield. H

NMR (300 MHz, DMSO-d₆) δ: 9.40 (s, 2H), 8.68 (s, 1H), 8.57 (s,

1H), 8.20 (d, J = 8.6 Hz, 1H), 8.04 (d, J = 8.6 Hz, 1H), 7.98−7.82 (m,

5H), 7.70−7.50 (m, 5H), 4.60 (s, 2H). LCMS (method LCMS1, ESI):

RT = 1.16 min, m/z = 403.1 [M + H]⁺.

1

obtained with 22.1% yield. H NMR (300 MHz, CDCl₃) δ: 8.72 (d, J

= 4.5 Hz, 1H), 8.27−8.19 (m, 1H), 8.04 (s, 1H), 7.98−7.90 (m, 2H),

7.86−7.83 (m, 2H), 7.73−7.43 (m, 5H), 7.34−7.31 (m, 1H), 7.16 (t, J

= 5.4 Hz, 1H), 4.70 (d, J = 6.0 Hz, 2H). LCMS (LCMS10, ESI): RT =

1.74 min, m/z = 409.0 [M + H]⁺.

N-{[4-(Benzenesulfonyl)phenyl]methyl}quinazoline-6-car-

boxamide (38). Substituting the corresponding reagents with

quinazoline-6-carboxylic acid (65n) and (4-(phenylsulfonyl)phenyl)-

N-{[4-(Piperidine-1-sulfonyl)phenyl]methyl}-1,3-benzothia-

zole-6-carboxamide (25). Substituting the corresponding reagents

with benzo[d]thiazole-6-carboxylic acid (65f) and (4-(piperidin-1-

ylsulfonyl)phenyl)methanamine (64n), the title product was obtained

1

methanamine (64o), the title product was obtained in 15% yield. H

NMR (300 MHz, CD₃OD) δ: 9.61 (br s, 1H), 9.31 (s, 1H), 8.60 (d, J

= 2.1 Hz, 1H), 8.43 (d, J = 2.4 Hz, 1H), 8.40 (d, J = 2.4 Hz, 1H), 8.07

(d, J = 9.3 Hz, 1H), 7.95 (s, 2H), 7.92 (s, 2H), 7.62−7.51 (m, 5H),

4.69 (s, 2H). LCMS (method LCMS1, ESI): RT = 1.77 min, m/z =

404.1 [M + H]⁺.

1

in 83% yield. H NMR (300 MHz, CDCl₃) δ: 9.12 (s, 1H), 8.56 (s,

1H), 8.16 (d, J = 9.0 Hz, 1H), 7.99−7.94 (m, 1H), 7.65−7.60 (m,

2H), 7.49−7.43 (m, 2H), 7.18−7.10 (m, 1H), 4.76 (d, J = 6.0 Hz,

2H), 2.97−2.91 (m, 4H), 1.66−1.57 (m, 4H), 1.44−1.35 (m, 2H).

LCMS (method LCMS1, ESI): RT = 2.18 min, m/z = 416.0 [M +

H]⁺.

N-{[4-(Piperidine-1-sulfonyl)phenyl]methyl}-1H-1,3-benzo-

diazole-5-carboxamide (26). Substituting the corresponding

reagents with 1H-benzo[d]imidazole-6-carboxylic acid (65g) and (4-

(piperidin-1-ylsulfonyl)phenyl)methanamine (64n), the title product

was obtained in 77% yield. ¹H NMR (300 MHz, CDCl₃) δ: 8.18 (br s,

1H), 8.03 (s, 1H), 7.73−7.66 (m, 2H), 7.62−7.56 (m, 2H), 7.45−7.39

(m, 2H), 4.69 (d, J = 6.0 Hz, 2H), 2.94−2.87 (m, 4H), 1.63−1.53 (m,

4H), 1.42−1.32 (m, 2H). LCMS (method LCMS1, ESI): RT = 1.81

min, m/z = 399.0 [M + H]⁺.

N-{[4-(Benzenesulfonyl)phenyl]methyl}imidazo[1,2-a]-

pyridine-6-carboxamide (28). Substituting the corresponding

reagents with imidazo[1,2-a]pyridine-6-carboxylic acid (65h²²) and

(4-(phenylsulfonyl)phenyl)methanamine (64o), the title product was

obtained in 79% yield. ¹H NMR (300 MHz, DMSO-d₆) δ: 9.19 (t, J =

6.0 Hz, 1H), 9.12 (s, 1H), 8.04 (s, 1H), 7.95−7.89 (m, 4H), 7.69−7.52

(m, 8H), 4.53 (d, J = 6.0 Hz, 2H). LCMS (method LCMS1, ESI): RT

= 1.69 min, m/z = 391.9 [M + H]⁺.

N-{[4-(Piperidine-1-sulfonyl)phenyl]methyl}imidazo[1,2-a]-

pyridine-7-carboxamide (31). Substituting the corresponding

reagents with imidazo[1,2-a]pyridine-7-carboxylic acid (65i) and (4-

(piperidin-1-ylsulfonyl)phenyl)methanamine (64n), the title product

was obtained in 66% yield. ¹H NMR (300 MHz, CDCl₃) δ: 8.23−8.19

(m, 1H), 8.16 (s, 1H), 7.74−7.65 (m, 4H), 7.53−7.49 (m, 2H), 7.39−

7.35 (m, 2H), 4.78 (d, J = 6.0 Hz, 2H), 3.00−2.95 (m, 4H), 1.68−1.59

(m, 4H), 1.47−1.38 (m, 2H). LCMS (method LCMS1, ESI): RT =

1.81 min, m/z = 399.0 [M + H]⁺.

N-({4-[3-(Triﬂuoromethyl)benzenesulfonyl]phenyl}methyl)-

1H-pyrrolo[3,2-c]pyridine-2-carboxamide (44). Substituting the

corresponding reagents with 1H-pyrrolo[3,2-c]pyridine-2-carboxylic

acid (65a) and (4-((3-(triﬂuoromethyl)phenyl)sulfonyl)phenyl)-

1

methanamine (64i), the title product was obtained in 25% yield. H

NMR (300 MHz, DMSO-d₆) δ: 12.35 (s, 1H), 9.37 (t, J = 6.0 Hz,

1H), 9.05 (s, 1H), 8.28−8.21 (m, 3H), 8.10−8.01 (m, 3H), 7.85 (t, J =

6.0 Hz, 1H), 7.57 (d, J = 9.0 Hz, 2H), 7.48 (d, J = 6.0 Hz, 1H), 7.39 (s,

1H), 4.58 (d, J = 6.0 Hz, 2H). LCMS (method LCMS1, ESI): RT =

1.73 min, m/z = 460.2 [M + H]⁺.

N-{[4-(4-Fluorobenzenesulfonyl)phenyl]methyl}-1H-pyrrolo-

[3,2-c]pyridine-2-carboxamide (47). Substituting the correspond-

ing reagents with 1H-pyrrolo[3,2-c]pyridine-2-carboxylic acid (65a)

and (4-((4-ﬂuorophenyl)sulfonyl)phenyl)methanamine (64k), the

title product was obtained in 26% yield. ¹H NMR (300 MHz,

DMSO-d₆) δ: 9.40 (s, 1H), 9.10 (s, 1H), 8.29 (d, J = 5.9 Hz, 1H), 8.16

(s, 1H), 8.07−7.92 (m, 5H), 7.58−7.40 (m, 6H), 4.57 (s, 2H). LCMS

(method LCMS1, ESI): RT = 0.99 min, m/z = 410.17 [M + H]⁺.

N-{[4-(3,5-Diﬂuorobenzenesulfonyl)phenyl]methyl}-1H-

pyrrolo[3,2-c]pyridine-2-carboxamide (49). Substituting the

corresponding reagents with 1H-pyrrolo[3,2-c]pyridine-2-carboxylic

acid (65a) and (4-((3,5-diﬂuorophenyl)sulfonyl)phenyl)methanamine

1

(64h), the title product was obtained in 39% yield. H NMR (300

MHz, DMSO-d₆) δ: 12.0 (s, 2H), 9.24 (s, 1H), 8.90 (s, 1H), 8.20 (d, J

= 5.8 Hz, 1H), 8.04 (d, J = 8.0 Hz, 2H), 7.80−7.50 (m, 5H), 7.38−

7.23 (m, 2H), 4.60 (s, 2H). LCMS (method LCMS1, ESI) RT = 1.03

min, m/z = 428.0 [M + H]⁺.

N-[(4-{[6-(Morpholin-4-yl)pyridin-3-yl]sulfonyl}phenyl)-

methyl]-1H-pyrrolo[3,2-c]pyridine-2-carboxamide (51). Substi-

tuting the corresponding reagents with 1H-pyrrolo[3,2-c]pyridine-2-

carboxylic acid (65a) and (4-((6-morpholinopyridin-3-yl)sulfonyl)-

phenyl)methanamine (64j), the title product was obtained in 57%

N-{[4-(Benzenesulfonyl)phenyl]methyl}imidazo[1,2-a]-

pyrimidine-6-carboxamide (33). Substituting the corresponding

reagents with imidazo[1,2-a]pyrimidine-6-carboxylic acid (65j) and

(4-(phenylsulfonyl)phenyl)methanamine (64o), the title product was

obtained in 3% yield. ¹H NMR (300 MHz, DMSO-d₆) δ: 9.45 (s, 1H),

9.33 (t, J = 5.4 Hz, 1H), 8.91 (d, J = 2.4 Hz, 1H), 8.00 (s, 1H), 7.94−

7.90 (m, 4H), 7.79 (s, 1H), 7.68−7.56 (m, 5H), 4.55 (d, J = 5.7 Hz,

1H). LCMS (method LCMS1, ESI): RT = 0.99 min, m/z = 393.0 [M

+ H]⁺.

1

yield. H NMR (300 MHz, DMSO-d₆) δ: 12.10 (br s, 1H), 9.28 (s,

1H), 8.89 (d, J = 1.0 Hz, 1H), 8.56 (d, J = 2.2 Hz, 1H), 8.17 (d, J = 5.8

Hz, 1H), 7.90−7.84 (m, 3H), 7.52 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 5.8

Hz, 1H), 7.25 (s, 1H), 6.88 (d, J = 9.2 Hz, 1H), 4.54 (d, J = 4.7 Hz,

2H), 3.65−3.55 (m, 8H). LCMS (method LCMS3, ESI): RT = 4.78

min, m/z = 478.1 [M + H]⁺.

N-{[4-(Benzenesulfonyl)phenyl]methyl}-[1,2,4]triazolo[4,3-

a]pyridine-6-carboxamide (35). Substituting the corresponding

reagents with [1,2,4]triazolo[4,3-a]pyridine-6-carboxylic acid (65k)

and (4-(phenylsulfonyl)phenyl)methanamine (64o), the title product

N-{[4-(3,5-Diﬂuorobenzenesulfonyl)phenyl]methyl}imidazo-

[1,2-a]pyridine-6-carboxamide (58). Substituting the correspond-

ing reagents with imidazo[1,2-a]pyridine-6-carboxylic acid (65h²²)

and (4-((3,5-diﬂuorophenyl)sulfonyl)phenyl)methanamine (64h), the

title product was obtained in 57% yield. ¹H NMR (300 MHz, DMSO-

1

was obtained in 20% yield. H NMR (300 MHz, DMSO-d₆) δ: 9.35−

N

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d₆) δ: 9.20 (s, 1H), 9.10 (s, 1H), 8.06−7.96 (m, 3H), 7.75 (s, 2H),

7.75−7.50 (m, 5H), 4.60 (s, 2H). LCMS (method LCMS1, ESI): RT

= 1.03 min, m/z = 428.04 [M + H]⁺.

LCMS (method LCMS1, ESI): RT = 1.64 min, m/z = 399.2 [M +

H]⁺.

N-{[4-(Piperidine-1-sulfonyl)phenyl]methyl}imidazo[1,5-a]-

pyridine-7-carboxamide (30). Substituting the corresponding

reagents with imidazo[1,5-a]pyridine-7-carboxylic acid (65q) and (4-

(piperidin-1-ylsulfonyl)phenyl)methanamine (64n), the title product

was obtained in 11% yield. ¹H NMR (300 MHz, DMSO-d₆) δ: 9.15 (t,

J = 5.9 Hz, 1H), 8.46 (d, J = 7.2 Hz, 1H), 8.34 (d, J = 7.5 Hz, 1H),

8.18 (s, 1H), 7.76−7.51 (m, 5H), 7.08−7.05 (m, 1H), 4.53 (d, J = 6.0

Hz, 2H), 2.81 (t, J = 5.2 Hz, 4H), 1.59−1.45 (m, 4H), 1.40−1.19 (m,

2H). LCMS (LCMS4, ESI): RT = 1.43 min, m/z = 399.0 [M + H]⁺.

N-{[4-(Benzenesulfonyl)phenyl]methyl}-5-(pyridin-3-yl)-1H-

pyrazole-3-carboxamide (39). Substituting the corresponding

reagents with 3-(pyridin-3-yl)-1H-pyrazole-5-carboxylic acid (65r)

and (4-(phenylsulfonyl)phenyl)methanamine (64o), the title product

was obtained in 29% yield. ¹H NMR (300 MHz, DMSO-d₆) δ: 12.8 (s,

1H), 9.00 (s, 1H), 8.95 (s, 1H), 8.18 (s, 1H), 8.00−7.80 (m, 4H),

7.70−7.40 (m, 7H), 7.20 (s, 1H), 4.50 (s, 2H). LCMS (method

LCMS1, ESI): RT = 1.13 min, m/z = 419.1 [M + H]⁺.

N-[(4-{[6-(Morpholin-4-yl)pyridin-3-yl]sulfonyl}phenyl)-

methyl]imidazo[1,2-a]pyridine-6-carboxamide (60). Substitut-

ing the corresponding reagents with imidazo[1,2-a]pyridine-6-

carboxylic acid (65h²²) and (4-((6-morpholinopyridin-3-yl)sulfonyl)-

phenyl)methanamine (64j), the title product was obtained in 82%

1

yield. H NMR (300 MHz, DMSO-d₆) δ: 9.22−9.16 (m, 1H), 9.13−

9.11 (m, 1H), 8.56 (d, J = 2.3 Hz, 1H), 8.12 (s, 1H), 8.04 (s, 1H),

7.90−7.85 (m, 2H), 7.66−7.50 (m, 5H), 6.89 (d, J = 9.2 Hz, 1H), 4.52

(d, J = 5.9 Hz, 2H), 3.65−3.56 (m, 8H). LCMS (method LCMS3,

ESI): RT = 4.63 min, m/z = 478.1 [M + H]⁺.

N-{[4-(3,5-Diﬂuorobenzenesulfonyl)phenyl]methyl}imidazo-

[1,2-a]pyrimidine-6-carboxamide (61). Substituting the corre-

sponding reagents with imidazo[1,2-a]pyrimidine-6-carboxylic acid

(65j) and (4-((3,5-diﬂuorophenyl)sulfonyl)phenyl)methanamine

1

(64h), the title product was obtained in 66% yield. H NMR (300

1

MHz, DMSO-d₆) δ: H NMR (300 MHz, DMSO-d₆) δ: 9.48 (d, J =

N-{[4-(Benzenesulfonyl)phenyl]methyl}-3-(pyridin-3-yl)-1,2-

oxazole-5-carboxamidee (40). Substituting the corresponding

reagents with 3-(pyridin-3-yl)isoxazole-5-carboxylic acid (65s) and

(4-(phenylsulfonyl)phenyl)methanamine (64o), the title product was

obtained in 40% yield. ¹H NMR (300 MHz, CDCl₃): δ 9.05 (d, J = 2.1

Hz, 1H), 8.72−8.71 (m, 1H), 8.12−8.06 (m, 1H), 7.93 (dd, J = 7.5,

1.8 Hz, 4H), 7.57−7.47 (m, 6H), 7.37 (d, J = 6.0 Hz, 1H), 7.07 (s,

1H), 4.70 (d, J = 6.0 Hz, 2H). LCMS (method LCMS1, ESI): RT =

1.93 min, m/z = 420.06 [M + H]⁺.

N-({4-[3,5-Diﬂuorobenzenesulfonyl]phenyl}methyl)furo[2,3-

c]pyridine-2-carboxamide (52). Substituting the corresponding

reagents with furo[2,3-c]pyridine-2-carboxylic acid (65t) and (4-((3,5-

diﬂuorophenyl)sulfonyl)phenyl)methanamine (64h), the title product

was obtained in 51% yield. ¹H NMR (300 MHz, DMSO-d₆) δ: 9.65 (t,

J = 6.0 Hz, 1H), 9.09 (s, 1H), 8.50 (d, J = 6.0 Hz, 1H), 8.04 (d, J = 9.0

Hz, 2H), 7.62 (m, 7H), 4.59 (d, J = 6.0 Hz, 2H). LCMS (LCMS11,

ESI): RT = 1.92 min, m/z = 429.2 [M + H]⁺.

2.4 Hz, 1H), 9.38 (t, J = 6.0 Hz, 1H), 8.93 (d, J = 2.4 Hz, 1H), 8.03−

7.99 (m, 3H), 7.80 (d, J = 1.5 Hz, 1H), 7.77−7.72 (m, 2H), 7.69−7.60

(m, 3H), 4.58 (d, J = 5.7 Hz, 2H). LCMS (method LCMS3, ESI) RT

= 5.64 min, m/z = 429.0 [M + H]⁺.

N-[(4-{[6-(Morpholin-4-yl)pyridin-3-yl]sulfonyl}phenyl)-

methyl]imidazo[1,2-a]pyrimidine-6-carboxamide (63). Substi-

tuting the corresponding reagents with imidazo[1,2-a]pyrimidine-6-

carboxylic acid (65j) and (4-((6-morpholinopyridin-3-yl)sulfonyl)-

phenyl)methanamine (64j), the title product was obtained in 75%

1

yield. H NMR (300 MHz, DMSO-d₆) δ: 9.46 (d, J = 2.5 Hz, 1H),

9.34 (t, J = 5.6 Hz, 1H), 8.92 (d, J = 2.5 Hz, 1H), 8.57 (d, J = 2.6 Hz,

1H), 8.00 (d, J = 1.5 Hz, 1H), 7.91−7.86 (m, 3H), 7.79 (d, J = 1.5 Hz,

1H), 7.55 (d, J = 8.4 Hz, 2H), 6.88 (d, J = 9.3 Hz, 1H), 4.55 (d, J = 5.7

Hz, 2H), 3.65−3.55 (m, 8H). LCMS (method LCMS3, ESI): RT =

4.94 min, m/z = 478.9 [M + H]⁺.

N-{[4-(Benzenesulfonyl)phenyl]methyl}-1-methyl-1H-

pyrrolo[2,3-c]pyridine-2-carboxamide (14). A solution of 1-

methyl-1H-pyrrolo[2,3-c]pyridine-2-carboxylic acid (65o,²³200 mg,

1.14 mmol), EDCI (400 mg, 2.09 mmol), HOBt (300 mg, 2.22

mmol), (4-(phenylsulfonyl)phenyl)methanamine (64o, 500 mg, 2.02

mmol), and EtN(i-Pr)₂(0.8 g 12.5 mmol) in DMF (50 mL) was

stirred at room temperature for 2 days. The resulting mixture was

concentrated under vacuum. The residue was ﬁrst puriﬁed on a silica

gel column eluted with CH₂Cl₂/MeOH (10:1). The semipuriﬁed

product (300 mg) was repuriﬁed by Flash-Prep-HPLC using the

following conditions (IntelFlash-1): column, silica gel; mobile phase,

CH₃CN/H₂O = 1 5% increasing to CH₃CN/H₂O = 64% within 30

min; detector, UV 254 nm to yield the title product as an oﬀ-white

N-[(4-{[6-(Morpholin-4-yl)pyridin-3-yl]sulfonyl}phenyl)-

methyl]furo[2,3-c]pyridine-2-carboxamide (54). Substituting the

corresponding reagents with furo[2,3-c]pyridine-2-carboxylic acid

(65t) and (4-((6-morpholinopyridin-3-yl)sulfonyl)phenyl)-

1

methanamine (64j), the title product was obtained in 26% yield. H

NMR (300 MHz, DMSO-d₆): δ 9.60 (m, 1H), 9.01 (s, 1H), 8.48 (m,

1H), 8.44 (m, 1H), 7.90−7.79 (m, 4H), 7.61 (s, 1H), 7.55 (m, 1H),

6.85 (d, J = 8.4 Hz, 1H), 4.51 (d, J = 6.0 Hz, 2H), 3.64−3.48 (m, 8H).

LCMS (method LCMS3, ESI): RT = 1.63 min, m/z = 478.98 [M +

H]⁺.

N-({4-[3-(Triﬂuoromethyl)benzenesulfonyl]phenyl}methyl)-

imidazo[1,2-a]pyridine-6-carboxamide (59). Substituting the

corresponding reagents with imidazo[1,2-a]pyridine-6-carboxylic acid

(65h²²) and (4-((3-(triﬂuoromethyl)phenyl)sulfonyl)phenyl)-

1

solid in 4% yield. H NMR (300 MHz, DMSO-d₆) δ: 8.97 (s, 1H),

8.19 (d, J = 5.7 Hz, 1H), 7.96−7.93 (m, 4H), 7.68−7.56 (m, 6H), 7.15

(s, 1H), 4.55 (d, J = 6.0 Hz, 2H), 4.06 (s, 3H). LCMS (LCMS4, ESI):

RT = 1.48 min, m/z = 406.0 [M + H]⁺.

Compounds 15, 27, 30, 39−40, 52, 54, 59, and 62 were synthesized

using conditions (EDCI/HOBt coupling) similar to those employed

to make compound 14.

1

methanamine (64i), the title product was obtained in 46% yield. H

NMR (300 MHz, DMSO-d₆): δ 9.20 (t, J = 6.0 Hz, 1H), 9.13−9.12

(m, 1H), 8.28−8.23 (m, 2H), 8.09−8.00 (m, 4H), 7.86 (t, J = 7.8 Hz,

1H), 7.67−7.57 (m, 5H), 4.56 (d, J = 6.0 Hz, 2H). LCMS (method

LCMS3, ESI): RT = 5.72 min, m/z = 460.25 [M + H]⁺.

N-{[4-(Benzenesulfonyl)phenyl]methyl}-1H-pyrrolo[3,2-c]-

pyridine-3-carboxamide (15). Substituting the corresponding

reagents with 1H-pyrrolo[3,2-c]pyridine-3-carboxylic acid (65p) and

(4-(phenylsulfonyl)phenyl)methanamine (64o), the title product was

N-({4-[3-(Triﬂuoromethyl)benzenesulfonyl]phenyl}methyl)-

imidazo[1,2-a]pyrimidine-6-carboxamide (62). Substituting the

corresponding reagents with imidazo[1,2-a]pyrimidine-6-carboxylic

acid (65j) and (4-((3-(triﬂuoromethyl)phenyl)sulfonyl)phenyl)-

1

obtained in 15% yield. H NMR (300 MHz, DMSO-d₆): δ 11.96 (s,

methanamine (64i), the title product was obtained in 23% yield. H

1H), 9.31 (s, 1H), 8.74 (t, J = 6.0 Hz, 1H), 8.35−8.22 (m, 1H), 8.14

(s, 1H), 7.94 (d, J = 5.7 Hz, 4H), 7.70−7.56 (m, 5H), 7.43 (d, J = 5.7

Hz, 1H), 4.54 (d, J = 6.0 Hz, 2H). LCMS (method LCMS1, ESI): RT

= 1.55 min, m/z = 391.9 [M + H]⁺.

NMR (300 MHz, DMSO-d₆) δ: 9.46 (d, J = 2.4 Hz, 1H), 9.35 (t, J =

2.6 Hz, 1H), 8.92 (d, J = 2.4 Hz, 1H), 8.25−8.23 (m, 2H), 8.06−8.00

(m, 4H), 7.86−7.79 (m, 2H), 7.60 (d, J = 8.4 Hz, 2H), 4.57 (d, J = 6.0

Hz, 2H). LCMS (method LCMS1, ESI): RT = 5.88 min, m/z = 461.0

[M + H]⁺.

N-{[4-(Piperidine-1-sulfonyl)phenyl]methyl}furo[2,3-c]-

pyridine-2-carboxamide (17). A solution of (4-(piperidin-1-

ylsulfonyl)phenyl)methanamine (64n, 500 mg, 1.93 mmol), furo-

[2,3-c]pyridine-3-carboxylic acid (65t, 355 mg, 2.13 mmol), EtN(i-

Pr)₂(762 mg, 5.79 mmol), and BOP (1044 mg, 2.31 mmol) in DMF

(5 mL) was stirred overnight at room temperature. The reaction was

N-{[4-(Piperidine-1-sulfonyl)phenyl]methyl}imidazo[1,2-a]-

pyridine-6-carboxamide (27). Substituting the corresponding

reagents with imidazo[1,2-a]pyridine-6-carboxylic acid (65h²²) and

(4-(piperidin-1-ylsulfonyl)phenyl)methanamine (64n), the title prod-

1

uct was obtained in 76% yield. H NMR (300 MHz, DMSO-d₆) δ:

9.23 (s, 1H), 9.16 (s, 1H), 8.07 (s, 1H), 7.71−7.54 (m, 7H), 4.59 (d, J

= 6.0 Hz, 1H), 2.86−2.81 (m, 4H), 1.51 (br s, 4H), 1.32 (br s, 2H).

O

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quenched by the addition of 20 mL of water, and the precipitate was

collected by ﬁltration. The solid was puriﬁed by recrystallization from

ethanol to give the title product as a light brown solid in 40% yield. ¹H

NMR (300 MHz, DMSO-d₆) δ: 9.65 (t, J = 6.0 Hz, 1H), 9.06 (s, 1H),

8.49 (d, J = 3.0 Hz, 1H), 7.84 (t, J = 3.0 Hz, 1H), 7.70 (d, J = 4.0 Hz,

3H), 7.58 (d, J = 6.0 Hz, 2H), 4.60 (d, J = 6.0 Hz, 2H), 2.89−2.84 (m,

4H), 1.56−1.51 (m, 4H), 1.35−1.34 (m, 2H). LCMS (LCMS10, ESI):

RT = m/z = 400.0 [M + H]⁺.

7.65 (m, 1H), 7.51−7.45 (m, 2H), 7.39−7.34 (m, 2H), 4.69 (d, J = 6.0

Hz, 2H), 2.95−2.85 (m, 4H), 1.65−1.55 (m, 4H), 1.42−1.33 (m, 2H).

LCMS (method LCMS1, ESI) RT = 2.10 min, m/z = 416.0 [M + H]⁺.

N-{[4-(Benzenesulfonyl)phenyl]methyl}thieno[2,3-c]-

pyridine-2-carboxamide (20). Substituting the corresponding

reagents with methyl thieno[2,3-c]pyridine-2-carboxylate (66e²⁴) and

(4-(phenylsulfonyl)phenyl)methanamine (64o), the title product was

1

obtained in 85% yield. H NMR (300 MHz, DMSO-d₆) δ 9.60−9.55

(m, 1H), 9.29 (s, 1H), 8.50 (d, J = 6.0 Hz, 1H), 8.15 (s, 1H), 7.95−

7.89 (m, 5H), 7.68−7.54 (m, 5H), 4.55 (d, J = 6.0 Hz, 2H). LCMS

(method LCMS1, ESI) RT = 1.65 min, m/z = 409.0 [M + H]⁺.

N-{[4-(Piperidine-1-sulfonyl)phenyl]methyl}thieno[3,2-c]-

pyridine-2-carboxamide (21). Substituting the corresponding

reagents with methyl thieno[3,2-c]pyridine-2-carboxylate (66f²⁴) and

(4-(piperidin-1-ylsulfonyl)phenyl)methanamine (64n), the title prod-

uct was obtained in 39% yield. ¹H NMR (300 MHz, CDCl₃) δ 8.99 (s,

1H), 8.48 (d, J = 6.0 Hz, 1H), 8.00 (s, 1H), 7.82−7.74 (m, 2H), 7.54−

7.49 (m, 2H), 7.42−7.37 (m, 2H), 4.70 (d, J = 6.0 Hz, 2H), 2.94−2.88

(m, 4H), 1.65−1.55 (m, 4H), 1.43−1.34 (m, 2H). LCMS (method

LCMS1, ESI) RT = 2.07 min, m/z = 416.0 [M + H]⁺.

N-{[4-(Piperidine-1-sulfonyl)phenyl]methyl}imidazo[1,2-a]-

pyrimidine-6-carboxamide (32). This compound was synthesized

using conditions (BOP coupling) similar to those employed to make

compound 17. Substituting the corresponding reagents with imidazo-

[1,2-a]pyrimidine-6-carboxylic acid (65j) and (4-(piperidin-1-

ylsulfonyl)phenyl)methanamine (64n), the title product was obtained

1

in 50% yield. H NMR (300 MHz, DMSO-d₆) δ: 9.52 (d, J = 2.4 Hz,

1H), 9.39 (t, J = 5.6 Hz, 1H), 8.98 (d, J = 2.4 Hz, 1H), 8.05 (d, J = 1.2

Hz, 1H), 7.83 (s, 1H), 7.70 (d, J = 8.4 Hz, 2H), 7.60 (d, J = 8.1 Hz,

2H), 4.63 (d, J = 6.0 Hz, 2H), 2.88−2.85 (m, 4H), 1.53−1.51 (m,

4H), 1.36−1.34 (m, 2H). LCMS (LCMS5, ESI): RT = 1.88 min, m/z

= 400.0 [M + H]⁺.

N-{[4-(Benzenesulfonyl)phenyl]methyl}-[1,3]thiazolo[5,4-c]-

pyridine-2-carboxamide (2). Substituting the corresponding

reagents with methyl thiazolo[5,4-c]pyridine-2-carboxylate (66g) and

(4-(phenylsulfonyl)phenyl)methanamine (64o), the title product was

obtained in 48% yield. ¹H NMR (300 MHz, DMSO-d₆) δ 10.01 (t, J =

6.0 Hz, 1H), 9.50 (s, 1H), 8.73−8.70 (m, 1 H), 8.11−8.07 (m, 1H),

7.95−7.89 (m, 4H), 7.69−7.54 (m, 5 H), 4.55 (d, J = 6.0 Hz, 2H).

LCMS (method LCMS1, ESI) RT = 1.91 min, m/z = 410.2 [M + H]⁺.

N-{[4-(Benzenesulfonyl)phenyl]methyl}imidazo[1,5-a]-

pyridine-6-carboxamide (29). Substituting the corresponding

reagents with methyl imidazo[1,5-a]pyridine-6-carboxylate (66h²²)

and (4-(phenylsulfonyl)phenyl)methanamine (64o), the title product

was obtained with 28% yield. ¹H NMR (300 MHz, DMSO-d₆) δ: 8.71

(s, 1H), 8.26 (s, 1H), 7.92−7.84 (m, 4H), 7.60−7.45 (m, 7H), 7.05−

6.97 (m, 2H), 4.69 (d, J = 6.3 Hz, 2H). LCMS (LCMS4, ESI): RT =

1.36 min, m/z = 392.0 [M + H]⁺.

N-{[4-(Benzenesulfonyl)phenyl]methyl}-3H-imidazo[4,5-c]-

pyridine-2-carboxamide (12). To a cooled solution (water bath) of

(4-(phenylsulfonyl)phenyl)methanamine (64o, 154 mg, 0.621 mmol)

in toluene (20 mL) was added AlMe₃(1.13 mL, 2.26 mmol), and the

mixture was stirred for 40 min at room temperature. A solution of

methyl 3H-imidazo[4,5-c]pyridine-2-carboxylate (66a, 100 mg, 0.564

mmol) in toluene (10 mL) was then added via syringe. The ﬂask was

heated to 80 °C for 4 h, during which the solution became dark yellow

with heavy precipitate. The mixture was cooled to room temperature,

quenched with saturated Na−K tartrate (25 mL), and diluted with

EtOAc (25 mL) and water (20 mL). The mixture was stirred

vigorously for 1 h and then was ﬁltered. The biphasic ﬁltrate layers

were separated, and the organic layer was dried (MgSO₄), ﬁltered, and

concentrated to aﬀord a yellow solid. Puriﬁcation by preparative

HPLC [Waters Autopuriﬁcation MS-directed HPLC prep fraction

collection with the following conditions: Column, Xbridge C18 RP 18,

5 μm, 19*50 mm; ﬂow rate 20 mL/min; mobile phase, water with

0.1% ammonium hydroxide (A) and methanol with 0.1% ammonium

hydroxide (B) running the following gradient 0 to 2 min (15%B), 2 to

6 min (15−100%B); detector ZQ Mass Detector in electrospray

N-{[4-(Benzenesulfonyl)phenyl]methyl}imidazo[1,2-a]-

pyrazine-6-carboxamide (34). Substituting the corresponding

reagents with methyl imidazo[1,2-a]pyrazine-6-carboxylate (66b)

and (4-(phenylsulfonyl)phenyl)methanamine (64o), the title product

1

was obtained in 28% yield. H NMR (400 MHz, DMSO-d₆): δ 9.45

1

ionization mode] aﬀorded 14 mg (6.3%) of the title compound. H

(m, 1H), 9.26 (d, J = 1.2 Hz, 1H), 9.10 (s, 1H), 8.29 (s, 1H), 7.95−

7.91 (m, 5H), 7.66 (d, J = 7.2 Hz, 1H), 7.63−7.55 (m, 4H), 4.56, 4.56

(d, J = 6.4 Hz, 2H). LCMS (LCMS4, ESI): RT = 2.08 min, m/z =

393.0 [M + H]⁺.

N-(4-(3-(Triﬂuoromethyl)phenylsulfonyl)benzyl)furo[2,3-c]-

pyridine-2-carboxamide (53). Substituting the corresponding

reagents with ethyl furo[2,3-c]pyridine-2-carboxylate (66d) and (4-

((3-(triﬂuoromethyl)phenyl)sulfonyl)phenyl)methanamine (64i), the

title product was obtained in 60% yield. ¹H NMR (300 MHz, DMSO-

d₆) δ: 8.90 (s, 1H), 8.52−8.47 (m, 1H), 8.20 (s, 1H), 8.16−8.10 (m,

1H), 8.00−7.92 (m, 2H), 7.70−7.60 (m, 2H), 7.58−7.50 (m, 3H),

7.25 (s, 1H), 7.20 (s, 1H), 4.76−4.73 (m, 2H). LCMS (method

LCMS1, ESI): RT = 1.35 min, m/z = 460.99 [M + H]⁺.

N-{[4-(3,5-Diﬂuorobenzenesulfonyl)phenyl]methyl}thieno-

[2,3-c]pyridine-2-carboxamide (55). Substituting the correspond-

ing reagents with methyl thieno[2,3-c]pyridine-2-carboxylate (66e²⁴)

and (4-((3,5-diﬂuorophenyl)sulfonyl)phenyl)methanamine (64h), the

title product was obtained in 76% yield. ¹H NMR (300 MHz, DMSO-

d₆) δ: 9.60 (s, 1H), 9.30 (s, 1H), 8.50 (s, 1H), 8.17 (s, 1H), 8.00−7.82

(m, 2H), 7.90 (s, 1H), 7.75−7.64 (m, 2H), 7.64−7.52 (m, 3H), 4.60

(s, 2H). LCMS (method LCMS1, ESI): RT = 1.31 min, m/z = 445.02

[M + H]⁺.

NMR (300 MHz, DMSO-d₆) δ: 8.30 (s, 1H), 8.15 (s, 1H), 7.95−7.85

(m, 5H), 7.65−7.50 (m, 6H), 7.32−7.27 (m, 1H), 4.90 (s, 2H). LCMS

(method LCMS1, ESI): RT = 0.92 min, m/z = 393.02 [M + H]⁺.

Compounds 13, 18−21, 24, 29, 34, 53, and 55−57 were

synthesized using conditions (AlMe₃-mediated amide coupling)

similar to those employed to make compound 12.

N-{[4-(Benzenesulfonyl)phenyl]methyl}-1-methyl-1H-

pyrrolo[3,2-c]pyridine-2-carboxamide (13). Substituting the

corresponding reagents with methyl 1-methyl-1H-pyrrolo[3,2-c]-

pyridine-2-carboxylate (66c) and (4-(phenylsulfonyl)phenyl)-

1

methanamine (64o), the title product was obtained in 92% yield. H

NMR (300 MHz, DMSO-d₆) δ: 9.26 (s, 1H), 8.91 (s, 1H), 8.28 (s,

1H), 7.94−7.92 (m, 4H), 7.67−7.53 (m, 6H), 7.27 (s, 1H), 4.52 (s,

2H), 3.32 (s, 3H). LCMS (method LCMS1, ESI): RT = 0.93 min, m/z

= 406.01 [M + H]⁺.

N-{[4-(Benzenesulfonyl)phenyl]methyl}furo[2,3-c]pyridine-

2-carboxamide (18). Substituting the corresponding reagents with

ethyl furo[2,3-c]pyridine-2-carboxylate (66d) and (4-(phenylsulfonyl)-

phenyl)methanamine (64o), the title product was obtained in 68%

1

yield. H NMR (300 MHz, DMSO-d₆): δ 9.60 (s, 1H), 9.00 (s, 1H),

8.45−8.40 (m, 1H), 7.96−7.88 (m, 4H), 7.80−7.76 (m, 1H), 7.70−

7.50 (m, 6H), 4.52 (d, J = 5.24 Hz, 2H). LCMS (method LCMS1,

ESI): RT = 1.11 min, m/z = 392.97 [M + H]⁺.

N-{[4-(Piperidine-1-sulfonyl)phenyl]methyl}thieno[2,3-c]-

pyridine-2-carboxamide (19). Substituting the corresponding

reagents with methyl thieno[2,3-c]pyridine-2-carboxylate (66e²⁰) and

(4-(piperidin-1-ylsulfonyl)phenyl)methanamine (64n), the title prod-

uct was obtained in 37% yield. ¹H NMR (300 MHz, CDCl₃) δ 9.13 (s,

1H), 8.49 (d, J = 6.0 Hz, 1H), 7.93 (s, 1H), 7.83−7.75 (m, 1H), 7.70−

N-({4-[3-(Triﬂuoromethyl)benzenesulfonyl]phenyl}methyl)-

thieno[2,3-c]pyridine-2-carboxamide (56). Substituting the cor-

responding reagents with methyl thieno[2,3-c]pyridine-2-carboxylate

(66e²⁴) and ((4-((3-(triﬂuoromethyl)phenyl)sulfonyl)phenyl)-

1

methanamine (64i), the title product was obtained in 55% yield. H

NMR (300 MHz, DMSO-d₆): δ 9.58 (t, J = 6.0 Hz, 1H), 9.29 (s, 1H),

8.51 (d, J = 5.4 Hz, 1H), 8.27−8.22 (m, 2H), 8.15 (s, 1H), 8.08−8.01

(m, 3H), 7.92−7.83 (m, 2H), 7.59 (d, J = 8.7 Hz, 2H), 4.57 (d, J = 5.7

P

dx.doi.org/10.1021/jm4008664 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Hz, 2H). LCMS (method LCMS3, ESI): RT = 6.32 min, m/z =

477.22 [M + H]⁺.

corresponding reagent with (2-(triﬂuoromethyl)phenyl)boronic acid

(68c), the title product was obtained in 7% yield in 3 steps. LCMS

(method LCMS2, ESI): RT = 0.96 min, m/z = 460.1 [M + H]⁺.

N-({4-[4-(Triﬂuoromethyl)benzenesulfonyl]phenyl}methyl)-

1H-pyrrolo[3,2-c]pyridine-2-carboxamide (45). Substituting the

corresponding reagent with (4-(triﬂuoromethyl)phenyl)boronic acid

(68d), the title product was obtained in 11% yield in 3 steps. ¹H NMR

(400 MHz, DMSO-d₆): δ 12.61 (br s, 1H), 9.45 (t, J = 6.0 Hz, 1H),

9.16 (s, 1H), 8.31 (d, J = 6.4 Hz, 1H), 8.15 (d, J = 8.4 Hz, 2H), 8.00−

7.97 (m, 4H), 7.60−7.57 (m, 3H), 7.47 (s, 1H), 4.59 (d, J = 5.2 Hz,

2H). LCMS (method LCMS2, ESI): RT = 0.96 min, m/z = 460.3 [M

+ H]⁺.

N-[(4-{[6-(Morpholin-4-yl)pyridin-3-yl]sulfonyl}phenyl)-

methyl]thieno[2,3-c]pyridine-2-carboxamide (57). Substituting

the corresponding reagents with methyl thieno[2,3-c]pyridine-2-

carboxylate (66e²⁴) and (4-((6-morpholinopyridin-3-yl)sulfonyl)-

phenyl)methanamine (64j), the title product was obtained in 32%

yield. ¹H NMR (300 MHz, DMSO-d₆) δ 9.57 (t, J = 6.0 Hz, 1H), 9.30

(s, 1H), 8.57−8.55 (m, 1H), 8.51 (d, J = 6.0 Hz, 1H), 8.15 (s, 1H),

7.93−7.85 (m, 4 H), 7.53 (d, J = 6.0 Hz, 2H), 6.88 (d, J = 6.0 Hz, 1H),

4.54 (d, J = 6.0 Hz, 2 H), 3.65−3.55 (m, 8H). LCMS (method

LCMS3, ESI) RT = 5.15 min, m/z = 495.0 [M + H]⁺.

N-{[4-(3-Fluorobenzenesulfonyl)phenyl]methyl}-1H-pyrrolo-

[3,2-c]pyridine-2-carboxamide (46). Substituting the correspond-

ing reagent with 3-ﬂuorobenzeneboronic acid (68e), the title product

was obtained in 11% yield in 3 steps. ¹H NMR (400 MHz, DMSO-d₆)

δ 12.01 (s, 1H), 9.26 (t, J = 6.0 Hz, 1H), 8.93 (d, J = 1.2 Hz, 1H), 8.22

(d, J = 5.8 Hz, 1H), 8.02−7.95 (m, 2H), 7.83−7.76 (m, 2H), 7.71−

7.63 (m, 1H), 7.62−7.52 (m, 3H), 7.36 (dt, J = 5.8, 1.1 Hz, 1H), 7.30

(s, 1H), 4.59 (d, J = 5.9 Hz, 2H). LCMS (method LCMS2, ESI): RT =

0.95 min, m/z = 410.0 [M + H]⁺.

N-{[4-(3,4-Diﬂuorobenzenesulfonyl)phenyl]methyl}-1H-

pyrrolo[3,2-c]pyridine-2-carboxamide (48). Substituting the

corresponding reagent with 3,4-diﬂuorobenzeneboronic acid (68f),

the title product was obtained in 8% yield in 3 steps. LCMS (method

LCMS2, ESI): RT = 1.00 min, m/z = 428.1 [M + H]⁺.

N-{[4-(2-Methylbenzenesulfonyl)phenyl]methyl}-1H-

pyrrolo[3,2-c]pyridine-2-carboxamide (41). tert-Butyl 4-(2-

Methylbenzenesulfonyl)benzylcarbamate (69a). To a glass vial was

added sodium 4-(((tert-butoxycarbonyl)amino)methyl)-

benzenesulﬁnate (67, 0.2 M in DMSO, 400 μL, 80 μmol), 2-

methylbenzeneboronic acid (68a, 0.2 M in 1,4-dioxane, 600 μL, 120

μmol), Et₃N (50 μL, 360 μmol), and Cu(OAc)₂(0.2 M in DMSO,

500 μL, 100 μmol). The vial was capped and heated at 65 °C for 16 h

on a heater/shaker. The reaction was cooled to RT, and brine (2 mL)

was added followed by concentrated NH₄OH (100 μL) and 3:1

EtOAc/CH₂Cl₂(3 mL). The layers were separated, and the organic

layer was transferred to a new vial. The aqueous layer was extracted

twice with 3:1 EtOAc/CH₂Cl₂(2 mL), and the combined organic

layers were evaporated to dryness in a Genevac evaporator. The crude

title compound was obtained as a dark brown solid, which was used in

the next step without further puriﬁcation.

(4-(2-Methylbenzenesulfonyl)phenyl)methanamine Hydrochlor-

ide (64a). MeOH (500 μL) was added to the vial containing crude

69a (80 μmol), and the mixture was heated to 50 °C for 15 min to

dissolve the solid. The solution was cooled to RT, HCl (4.0 M in 1,4-

dioxane, 400 μL, 1.6 mmol) was added, and the vial was shaken at RT

for 30 min and then at 50 °C for 3 h. The solvent was evaporated to

dryness in a Genevac evaporator. The light brown solid thus obtained

was used in the next step without further puriﬁcation.

N-{[4-(2-Methylbenzenesulfonyl)phenyl]methyl}-1H-

pyrrolo[3,2-c]pyridine-2-carboxamide (41). To the vial contain-

ing crude 64a was added DMA (250 μL), Et₃N (67 μL, 480 μmol),

1H-pyrrolo[3,2-c]pyridine-2-carboxylic acid (65a, 0.2 M in DMA, 440

μL, 88 μmol), and BOP (0.2 M in DCE, 440 μL, 88 μmol). The

mixture was heated at 50 °C for 4 h and then was stirred at room

temperature for 16 h. Brine (1.5 mL), NaOH (1 M in water, 100 μL,

100 μmol), and 3:1 EtOAc/CH₂Cl₂(2 mL) were added, and the layers

were separated. The aqueous layer was extracted twice with 3:1

EtOAc/CH₂Cl₂(2 mL), and the combined organic layers were

evaporated to dryness in a Genevac evaporator. The residue was

puriﬁed by preparative HPLC [Waters Autopuriﬁcation MS-directed

HPLC prep fraction collection with the following conditions: column,

Xbridge C18 RP 18, 5 μm, 19*50 mm; ﬂow rate 20 mL/min; mobile

phase, water with 0.1% ammonium hydroxide (A) and methanol with

0.1% ammonium hydroxide (B) running the following gradient 0 to 2

min (15%B), 2 to 6 min (15−100%B); detector ZQ Mass Detector in

electrospray ionization mode] to give the title compound as a white

powder with 6.4% yield in 3 steps. LCMS (method LCMS2, ESI): RT

= 0.96 min, m/z = 406.1 [M + H]⁺.

Compounds 42−43, 45−46, 48, and 50 were synthesized using

conditions similar to those employed to make compound 41.

N-{[4-(3-methylbenzenesulfonyl)phenyl]methyl}-1H-

pyrrolo[3,2-c]pyridine-2-carboxamide (42). Substituting the

corresponding reagent with 3-methylbenzeneboronic acid (68b), the

title product was obtained in 4% yield in 3 steps. ¹H NMR (400 MHz,

DMSO-d₆) δ 11.99 (s, 1H), 9.24 (t, J = 6.0 Hz, 1H), 8.92 (d, J = 1.0

Hz, 1H), 8.22 (d, J = 5.8 Hz, 1H), 7.96−7.91 (m, 2H), 7.77−7.74 (m,

1H), 7.74−7.70 (m, 1H), 7.59−7.53 (m, 2H), 7.50−7.46 (m, 2H),

7.36 (dt, J = 5.8, 1.1 Hz, 1H), 7.29 (s, 1H), 4.57 (d, J = 5.9 Hz, 2H),

2.37 (s, 3H). LCMS (method LCMS2, ESI): RT = 0.98 min, m/z =

406.1 [M + H]⁺.

N-{[4-(pyridine-3-sulfonyl)phenyl]methyl}-1H-pyrrolo[3,2-c]-

pyridine-2-carboxamide (50). Substituting the corresponding

reagent with pyridin-3-ylboronic acid (68g), the title product was

obtained in 5% yield in 3 steps. LCMS (method LCMS2, ESI) RT =

0.69 min, m/z = 393.1 [M + H]⁺.

N-(3-Aminopyridin-4-yl)-2,2,2-trichloroacetamide (72). Pyr-

idine-3.4-diamine (70, 2.5 g, 22.91 mmol) was dissolved in CF₃COOH

(5.3 mL, 68.7 mmol). Methyl 2,2,2-trichloroacetimidate (71, 4.45 g,

25.2 mmol) was added over 30 min at 0 °C. The mixture was then

stirred at room temperature for 2 days. The mixture was ﬁltered, and

the solid was washed with diethyl ether and dried to aﬀord 5.7 g of

1

desired product as a white solid in 98% yield. H NMR (300 MHz,

DMSO-d₆) δ: 7.80 (d, J = 5.4 Hz, 1H), 7.70 (s, 1H), 7.06 (s, 1H), 6.60

(d, J = 5.4 Hz, 1H), 5.33 (s, 2H).

Methyl 3H-Imidazo[4,5-c]pyridine-2-carboxylate (66a). A

mixture of H₂O (1.21 mL, 67.2 mmol), Na₂CO₃(7.12 g, 67.2

mmol), and 72 (5.7 g, 22.40 mmol) in MeOH (80 mL) was heated at

reﬂux overnight. The mixture was cooled to room temperature, diluted

with EtOAc, washed with saturated NaHCO₃, dried, and concentrated.

Biotage puriﬁcation of the residue (10% MeOH/CH₂Cl₂) aﬀorded

100 mg of the title product in 3% yield. ¹H NMR (300 MHz, CD₃OD)

δ: 8.97 (s, 1H), 8.34 (s, 1H), 7.23 (s, 1H), 4.10 (s, 3H).

6-Bromoimidazo[1,2-a]pyrazine (75). To a solution of 5-

bromopyrazin-2-amine (73, 20 g, 114.9 mmol) and 2-bromo-1,1-

dimethoxyethane (74, 29 g, 171.6 mmol) in EtOH (500 mL)

maintained under argon was added 40% HBr (50 mL). The reaction

mixture was stirred at 80 °C for 12 h and then cooled to room

temperature. The mixture was concentrated under vacuum to remove

the excess EtOH. The pH value of the solution was adjusted to 9 with

1 N NaOH. The solution was extracted with 3 × 100 mL of EtOAc.

The organic layers were combined and then washed with 2 × 300 mL

of water and 1 × 500 mL of brine. The organic layer was dried over

anhydrous Na₂SO₄and concentrated under vacuum. The residue was

puriﬁed on a silica gel column eluted with 45% EtOAc in petroleum

ether to give the title product as a light-yellow solid in 15% yield.

LCMS (LCMS4, ESI): RT = 0.94 min, m/z = 197.9 [M + H]⁺.

Imidazo[1,2-a]pyrazine-6-carbonitrile (76). A mixture of 75 (2

g, 10.10 mmol), Zn(CN)₂(1.4 g, 12.2 mmol), and Pd(PPh₃)₄(700

mg, 0.61 mmol) in DMF (30 mL) was stirred under nitrogen at 120

°C for 12 h. The solid was removed by ﬁltration, and the ﬁltrate was

diluted with 200 mL of water. The resulting solution was extracted

with 3 × 100 mL of CH₂Cl₂. The combined organic layers were

washed with 2 × 300 mL of brine, were dried over anhydrous sodium

sulfate, and were concentrated under vacuum. The residue was puriﬁed

N-({4-[2-(Triﬂuoromethyl)benzenesulfonyl]phenyl}methyl)-

imidazo[1,2-a]pyrimidine-6-carboxamide (43). Substituting the

Q

dx.doi.org/10.1021/jm4008664 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

on a silica gel column eluted with 70% EtOAc in petroleum ether to

give the title product as a white solid in 24% yield. LCMS (LCMS5,

ESI): RT = 0.81 min, m/z = 145.0 [M + H]⁺.

4-(4-Fluorophenylsulfonyl)benzonitrile (81). To a solution of

4-ﬂuorobenzonitrile (79, 5.98 g, 49.4 mmol) in 20 mL of DMSO at

120 °C was added dropwise of sodium 4-ﬂuorobenzenesulﬁnate (80, 3

g, 16.47 mmol) in 20 mL of DMSO. The resulting mixture was stirred

at 120 °C overnight and then was cooled to room temperature. 400

mL of ice water was added into the reaction mixture, and the resulting

precipitate was ﬁltered, washed with water (2 × 30 mL), and dried.

Biotage puriﬁcation with EtOAc/hexanes aﬀorded 1.30 g of the title

Methyl Imidazo[1,2-a]pyrazine-6-carboxylate (66b). Hydro-

gen chloride gas was bubbled into a solution of 76 (200 mg, 1.39

mmol) in MeOH (20 mL) for 15 min. The resulting solution was

stirred at 80 °C for 2 h. The reaction mixture was concentrated under

vacuum. The residue was dissolved in 50 mL of EtOAc and then was

washed with 1 × 50 mL of water and 1 × 50 mL of brine. The organic

layer was dried over anhydrous sodium sulfate, was ﬁltered, and was

concentrated under vacuum. The residue was puriﬁed on a silica gel

column eluted with EtOAc/petroleum ether (1:4) to give the title

product as a white solid in 12% yield. LCMS (LCMS6, ESI): RT =

0.88 min, m/z = 178.0 [M + H]⁺.

1

product in 30% yield. H NMR (300 MHz, DMSO-d₆) δ: 8.04−8.26

(m, 6H), 7.44−7.52 (m, 2H).

(4-(4-Fluorophenylsulfonyl)phenyl)methanamine (64k). To a

solution of 81 (2.3 g, 8.8 mmol) in 150 mL of 2 N NH₃/MeOH was

added Raney Ni (500 mg), and the mixture was hydrogenated on a

Parr apparatus (45 psi) overnight. Nitrogen was bubbled through the

crude mixture to purge any residual H₂, and the mixture was ﬁltered

through Celite, rinsing with MeOH (2 × 30 mL). The ﬁltrate was

concentrated to dryness and triturated with Et₂O, and the solid was

collected by vacuum ﬁltration to give 2.2 g of the title product in 94%

yield which was used for next step without further puriﬁcation. LCMS

(method LCMS2, ESI): RT = 0.87 min, m/z = 266.0 [M + H]⁺.

4-(Piperidin-1-ylsulfonyl)benzaldehyde (84). In a 100 mL

round-bottomed ﬂask was added piperidine (83, 233 mg, 2.74 mmol)

and EtN(i-Pr)₂(0.85 mL, 4.88 mmol) in CH₂Cl₂(10 mL), followed

by a solution of 4-formylbenzene-1-sulfonyl chloride (82, 501 mg,

2.448 mmol) in 5 mL of CH₂Cl₂. The reaction mixture was stirred at

room temperature for 16 h and then was diluted with CH₂Cl₂(30 mL)

and was washed successively with water (10 mL) and brine (10 mL).

The organic layer was dried over MgSO₄, was ﬁltered through a short

plug of silica gel, and was concentrated to aﬀord the title product as a

colorless solid in 100% yield. ¹H NMR (300 MHz, CDCl₃) δ: 10.11 (s,

1H), 8.06−7.90 (m, 4H), 3.06−3.01 (m, 4H), 1.69−1.61 (m, 4H),

1.48−1.40 (m, 2H).

N-(4-(3,5-Diﬂuorophenylsulfonyl)benzyl)acetamide (78h). In

a 1 L round-bottomed ﬂask was added sodium 4-(acetamidomethyl)-

benzenesulﬁnate (77, 7.45 g, 31.7 mmol), 3,5-diﬂuorophenylboronic

acid (68h, 5 g, 31.7 mmol), copper(II) acetate (6.33 g, 34.8 mmol),

potassium carbonate (9.63 g, 67.9 mmol), and 100 mL of DMSO

followed by 10 g of regular 4 Å molecular sieves. The reaction mixture

was stirred at room temperature for overnight, and then the solution

was ﬁltered through Celite. The ﬁltrate was concentrated under

reduced pressure, diluted with EtOAc (300 mL), and washed with

water (3 × 50 mL). The organic layer was dried over Na₂SO₄, was

ﬁltered, and was concentrated. Biotage puriﬁcation of the residue (50%

1

EtOAc/hexanes) aﬀorded 4.07 g of the title product in 40% yield. H

NMR (300 MHz, DMSO-d₆) δ: 8.42 (t, J = 5.8 Hz, 1H), 7.99 (d, J =

8.4 Hz, 2H), 7.73−7.61 (m, 5H), 7.48 (d, J = 8.3 Hz, 1H), 4.29 (d, J =

6.0 Hz, 2H), 1.82 (s, 3H). LCMS (method LCMS3, ESI) RT = 5.79

min, m/z = 326.12 [M + H]⁺.

Compounds 78i − 78j were synthesized using conditions similar to

those employed to make compound 78h.

(E)-2-Methyl-N-(4-(piperidin-1-ylsulfonyl)benzylidene)-

propane-2-sulﬁnamide (85). To a 0.5 M solution of 84 (712 mg,

2.81 mmol) in THF (6 mL) under nitrogen was added titanium(IV)

ethoxide (1.2 mL, 5.62 mmol). tert-Butanesulﬁnamide (341 mg, 2.81

mmol) was then added, and the resulting mixture was stirred for 2 h at

room temperature. The reaction was poured into an equal volume of

sat. NaHCO₃with rapid stirring and immediately ﬁltered through

Celite. The ﬁlter cake was washed with EtOAc (2 × 30 mL), and the

aqueous layer was separated and washed once with EtOAc (20 mL).

The combined organic layers were dried (MgSO₄), ﬁltered, and

concentrated. The title product was obtained as a colorless solid by

quick puriﬁcation using silica gel chromatography (EtOAc/hexanes,

N-(4-((3-(Triﬂuoromethyl)phenyl)sulfonyl)benzyl)acetamide

(78i). Substituting the corresponding reagent with 3-(triﬂuoromethyl)-

phenylboronic acid (68i), the title product was obtained in 40% yield.

¹H NMR (300 MHz, DMSO-d₆) δ: 8.45−37 (m, 1H), 8.27−8.21 (m,

2H), 8.10−8.05 (m, 1H), 7.99 (d, J = 8.2 Hz, 2H), 7.86 (t, J = 7.8 Hz,

1H), 7.45 (d, J = 8.5 Hz, 2H), 4.29 (d, J = 5.9 Hz, 2H), 1.85 (s, 3H).

N-(4-((6-Morpholinopyridin-3-yl)sulfonyl)benzyl)acetamide

(78j). Substituting the corresponding reagent with 6-morpholinopyr-

idin-3-ylboronic acid (68j), the title product was obtained in 16%

1

yield. H NMR (300 MHz, DMSO-d₆) δ: 8.57−8.54 (m, 1H), 8.43−

8.36 (m, 1H), 7.89−7.82 (m, 3H), 7.41 (d, J = 8.2 Hz, 2H), 6.88 (d, J

= 9.2 Hz, 1H), 4.26 (d, J = 6.0 Hz, 2H), 3.65−3.55 (m, 8H), 1.84 (s,

3H).

1

40/60) in 59% yield (the product slowly hydrolyzes on silica gel). H

NMR (300 MHz, CDCl₃) δ: 8.64 (s, 1H), 8.01−7.84 (m, 4H), 3.05−

3.01 (m, 4H), 1.69−1.62 (m, 4H), 1.47−1.43 (m, 2H), 1.29 (s, 9H).

2-Methyl-N-(1-(4-(piperidin-1-ylsulfonyl)phenyl)ethyl)-

propane-2-sulﬁnamide (86). A solution of 85 (73 mg, 0.21 mmol)

was dissolved in 5:1 Et₂O/THF (6 mL) and cooled to −78 °C.

Methyllithium (1.6 M in Et₂O, 1.5 mL, 2.4 mmol) was added dropwise

via syringe and the mixture was stirred 1 h at −78 °C and then was

warmed to room temperature for 16 h. The mixture was neutralized

with a minimal amount of sat. Na₂SO₄, diluted with EtOAc (15 mL).

The organic layer was dried (MgSO₄) and concentrated. The title

product was obtained in 47% yield after silica gel chromatography

(EtOAc/hexanes, 40/60). ¹H NMR (300 MHz, CDCl₃) δ: 7.73 (d, J =

8.4 Hz, 2H), 7.50 (d, J = 8.4 Hz, 2H), 4.65−4.57 (m, 1H), 3.46 (d, J =

3.4 Hz, 1H), 2.99 (t, J = 5.4 Hz, 4H), 1.68−1.60 (m, 4H), 1.53 (d, J =

5.6 Hz, 3H), 1.47−1.39 (m, 2H), 1.24 (s, 9H).

1-(4-(Piperidin-1-ylsulfonyl)phenyl)ethanamine Hydrochlor-

ide (64l). A solution of 86 (36 mg, 0.1 mmol) in MeOH (0.5 mL) was

treated with 4 M HCl in 1,4-dioxane (0.5 mL), and the mixture was

stirred for 30 min before being concentrated to 30% of its original

volume. Et₂O (2 mL) was added, forming a precipitate. The mixture

was again concentrated to 30% of the original volume, and Et₂O (4

mL) was added. The title product was obtained as a colorless solid in

68% yield after vacuum ﬁltration and washing with cold Et₂O (2 mL).

This compound was used directly in the next step (synthesis of

compound 9) without further puriﬁcation.

(4-(3,5-Diﬂuorophenylsulfonyl)phenyl)methanamine (64h).

The mixture of 78h (4 g, 12.30 mmol) and 12 M HCl (102 mL, 3

M, 307 mmol) in 2-propanol was heated at 100 °C overnight. The

reaction mixture was cooled to room temperature, and the 2-propanol

was removed under reduced pressure to aﬀord a white solid. The solid

was then suspended in 3 M NaOH, ﬁltered, washed with water (60

1

mL), and dried to give 3.14 g of the title product in 90% yield. H

NMR (300 MHz, DMSO-d₆) δ: 7.96 (d, J = 8.5 Hz, 2H), 7.74−7.62

(m, 4H), 7.57 (d, J = 8.6 Hz, 2H), 3.76 (s, 2H). LCMS (method

LCMS1, ESI): RT = 0.91 min, m/z = 284.0 [M + H]⁺.

Compounds 64i−64j were synthesized using conditions similar to

those employed to make compound 64h.

(4-((3-(Triﬂuoromethyl)phenyl)sulfonyl)phenyl)-

methanamine (64i). Substituting the corresponding reagent with

78i, the title product was obtained in 86% yield. ¹H NMR (300 MHz,

CDCl₃) δ: 8.19 (s, 1H), 8.11 (d, J = 7.9 Hz, 1H), 7.91 (d, J = 8.4 Hz,

2H), 7.80 (d, J = 7.9 Hz, 1H), 7.64 (t, J = 7.9 Hz, 1H), 7.50 (d, J = 8.5

Hz, 2H), 3.94 (s, 2H), 1.48 (s, 2H).

(4-((6-Morpholinopyridin-3-yl)sulfonyl)phenyl)-

methanamine (64j). Substituting the corresponding reagent with

78j, the title product was obtained in 100% yield. ¹H NMR (300 MHz,

DMSO-d₆) δ: 8.57−8.55 (m, 1H), 7.90−7.81 (m, 3H), 7.51 (d, J = 8.5

Hz, 2H), 6.89 (d, J = 9.2 Hz, 1H), 3.73 (s, 2H), 3.65−3.56 (m, 8H),

1.85 (s, 2H).

R

dx.doi.org/10.1021/jm4008664 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Methyl 3-(4-((tert-Butoxycarbonylamino)methyl)-

phenylthio)propanoate (89). A 200 mL ﬂask equipped with a

magnetic stir bar and containing tert-butyl-4-bromobenzylcarbamate

(87, 6.72 g, 23.5 mmol) was charged with toluene (50 mL), EtN(i-Pr)₂

(8.21 mL, 47.0 mmol), Xantphos (0.680 g, 1.175 mmol), Pd₂(dba)₃

(0.538 g, 0.588 mmol), and methyl 3-mercaptopropanoate (88, 2.60

mL, 23.50 mmol). The mixture was heated to 100 °C under nitrogen

for 2.5 h. After cooling, the mixture was loaded directly on a short

silica gel column. Elution with 5:1 to 3:1 hexanes−EtOAc aﬀorded the

eﬃcacy data. We thank the DMPK group at Genentech for

their contribution to the generation of the DMPK data,

especially Erlie Marie Delarosa and Jonathan Cheong. We also

acknowledge Agilent Technologies (Woburn, MA) for

generating the Rapidﬁre LCMS data, Tandem Laboratories

(Woburn, MA) for performing the phosphoribosylated adduct

study, Yigong Shi’s group (Tsinghua University, Beijing, China)

for providing Nampt DNA construct and Nampt protein, and

James Kyranos and Jaime Escobedo for helpful discussions.

1

title product as a colorless oil in 94% yield. H NMR (400 MHz,

CDCl₃): δ 7.32 (d, J = 8.3 Hz, 2H), 7.21 (d, J = 8.2 Hz, 2H), 4.84 (br

s, 1H), 4.28 (d, J = 5.8 Hz, 2H), 3.67 (s, 3H), 3.14 (t, J = 7.4 Hz, 2H),

2.61 (t, J = 7.5 Hz, 2H), 1.45 (s, 9H). LCMS (method LCMS2, ESI)

m/z = 348.15 [M + Na]⁺.

ABBREVIATIONS USED

■

MLM, mouse liver microsome; HLM, human liver microsome;

MDCK, Madin−Darby canine kidney; SRB, sulforhodamine B;

CyQuant, cell counting via DNA content to assess proliferative

eﬀects; C_max, maximum concentration; t_1/2, half-life; T_max, time

to reach the maximum concentration; CL, clearance; V_ss,

volume of distribution; HATU, (O-(7-azabenzotriazol-1-yl)-

N,N,N′,N′-tetramethyluronium hexaﬂuorophosphate); HOBt,

1-hydroxybenzotriazole; DIAD, diisopropyl azodicarboxylate;

Xantphose, 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene;

BOP, (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium

hexaﬂuorophosphate; SCX, strong cationic exchange; THPP,

tris(hydroxypropyl)phosphine; D5W, 5% dextrose in water

Methyl 3-(4-((tert-Butoxycarbonylamino)methyl)-

phenylsulfonyl)propanoate (90). A 500 mL ﬂask equipped with

a magnetic stir bar was charged with Oxone (23.61 g, 38.4 mmol) and

water (82 mL). The mixture was stirred at RT for 5 min to dissolve the

Oxone, and a solution of 89 (5.0 g, 15.36 mmol) in CH₃CN (45 mL)

was then added at RT with rapid stirring. The mixture was stirred at

RT for 3 h. Water (60 mL) and EtOAc (100 mL) were added, and the

layers were separated. The aqueous layer was extracted with EtOAc

(100 mL). The combined organic layers were washed with half-

saturated brine (2 × 50 mL), dried (Na₂SO₄), and concentrated under

reduced pressure to aﬀord the title product as a white solid in 94%

1

yield. H NMR (400 MHz, CDCl₃): δ 7.86 (d, J = 8.4 Hz, 2H), 7.48

(d, J = 8.6 Hz, 2H), 5.03 (br s, 1H), 4.41 (d, J = 6.2 Hz, 2H), 3.64 (s,

3H), 3.41 (t, J = 7.7 Hz, 2H), 2.74 (t, J = 7.8 Hz, 2H), 1.46 (s, 9H).

LCMS (method LCMS2, ESI) m/z = 380.07 [M + Na]⁺.

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Sodium 4-(((tert-Butoxycarbonyl)amino)methyl)-

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solvent was removed under reduced pressure and the residue was dried

under high vacuum to give the title product as a light yellow solid in

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ASSOCIATED CONTENT

* Supporting Information

■

S

Details of computational methods; details of analytical LCMS

methods; in vitro ADME and experimental procedures;

experimental details for PK and eﬃcacy study of compound

58 in the mouse; protein expression/puriﬁcation and crystallo-

graphic methods and procedures for 7 and 58 (in complex with

Nampt); details of Nampt biochemical and cell-based assays.

This material is available free of charge via the Internet at

http://pubs.acs.org.

Accession Codes

PDB nos.: 4KFN for 7 complexed with Nampt; 4KFO for 58

complexed with Nampt.

AUTHOR INFORMATION

Corresponding Author

*Phone: 857-209-2382. E-mail: xzheng@formatherapeutics.

com.

■

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We acknowledge Forma’s high speed synthesis group and

George Luke for their synthetic contributions, ADME group for

generating the microsomal and solubility data, Rashida Garcia-

Dancey and Shohini Ganguly for helping generate the

biochemical and cellular data, and Lakshmanan Manikandan,

Saradhi Vijay, and Danilal C. Sharma for generating the in vivo

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Journal of Medicinal Chemistry

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dx.doi.org/10.1021/jm4008664 | J. Med. Chem. XXXX, XXX, XXX−XXX

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Structure-based discovery of novel amide-containing nicotinamide phosphoribosyltransferase (Nampt) inhibitors

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