5-Vinyl-3-pyridinecarbonitrile inhibitors of PKCθ: Optimization of enzymatic and functional activity

Bioorganic & Medicinal Chemistry 17 (2009) 7933–7948

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: w ww.elsevier.com/locate/bmc

5-Vinyl-3-pyridinecarbonitrile inhibitors of PKCh: Optimization

of enzymatic and functional activity

a

c

b

c

L. Nathan Tumey ^a,, Niala Bhagirath , Agnes Brennan , Natasja Brooijmans , Julie Lee ,

*

Xiaoke Yang ^c, Diane H. Boschelli ^a

^aWyeth Research, Medicinal Chemistry, 401 N. Middletown Rd., Pearl River, NY 10965, United States

^bWyeth Research, Structural Biology and Computational Chemistry, 401 N. Middletown Rd., Pearl River, NY 10965, United States

^cWyeth Research, Inﬂammation, 200 Cambridge Park Drive, Cambridge, MA 12140, United States

a r t i c l e i n f o

a b s t r a c t

Article history:

PKCh is a serine/threonine kinase involved in the regulation of IL2 production in T cells. It has recently

become an attractive therapeutic target for a variety of immunological disorders. We describe the opti-

mization of the enzymatic and cellular potency of a series of 5-vinyl-3-pyridinecarbonitrile inhibitors of

PKCh. A binding model was developed that explains much of the SAR observed for this series, including

the enzymatic potency observed for 19. An analysis of functional potency against various physiochemical

parameters suggests that cellular potency is correlated with Log D_7.4, but not with c Log P, PAMPA perme-

ability, or TPSA.

Received 31 August 2009

Revised 8 October 2009

Accepted 9 October 2009

Keywords:

PKC

PKC-theta

Kinase

Inhibitor

Inﬂammation

Pyridinecarbonitrile

1. Introduction

including the experimental autoimmune encephalomyelitis (EAE)

model of multiple sclerosis,^3,4the type II collagen-induced arthritis

Interleukin-2 (IL2) is a cytokine that plays an essential role in

the growth, survival, and differentiation of T cells. IL2 expression

in T cells is mediated by a variety of transcription factors which

target the CD28 response element promoter. The activation of

these transcription factors is carefully regulated and requires the

co-stimulation of the T cell receptor (TCR) signaling complex and

CD28. The CD28 mediated signal is thought to be transmitted by

the PI3K/AKT pathway which ultimately results in PDK1 dependent

phosphorylation and activation of protein kinase C theta (PKCh).

Activated PKCh, in turn, is recruited to the TCR complex where it

plays a critical role in the activation of the aforementioned tran-

scription factors. Thus, PKCh plays a unique role in the integration

of signaling from the TCR complex and CD28 which results in

immunostimulation via IL2 expression.¹

PKCh is a serine/threonine kinase which, as its role in IL2 pro-

duction would suggest, is expressed primarily in lymphocytes

and mast cells. Due to its important role in the activation and dif-

ferentiation of T cells, PKCh modulation has been extensively stud-

ied for its potential pharmacologic utility in various immunological

disorders.²PKCh knockout (KO) mice have been reported to have

diminished responses in various T cell mediated disease models

(CIA) model,⁵and the ovalbumin challenge (OVA) model of asth-

ma.^6,7PKCh KO mice have also showed signiﬁcantly increased sur-

vival following a cardiac allograft transplantation, suggesting a

potential utility of PKCh inhibitors as immunosuppressives follow-

ing transplantation.⁸In addition to lymphocytes and mast cells,

PKCh is also expressed in skeletal muscle where it is speculated

to play a role in free fatty acid induced insulin resistance.⁹As such,

PKCh may also be emerging as an attractive target for diabetes mel-

litus. Interestingly, in spite of its crucial role in a variety of immune

responses, PKCh KO mice have been shown to have normal Th1 dif-

ferentiation and normal viral clearance in spite of their defective T

cell activation pathway.^6,10This suggests that selective PKCh inhib-

itors may be able to modulate immunological disorders without

imparting severe immunodeﬁciency. Therefore, PKCh has recently

become an attractive target for pharmacological intervention of a

variety of diseases.

PKCs are a broad family of structurally homologous serine/threo-

nine kinases which play important roles in cell growth, differentia-

tion, and apoptosis.¹¹PKCs are generally grouped into three classes

based upon their requirements for activation: classical PKCs (

a, b,

c

), novel PKCs (d, , h), and atypical PKCs (f,

e

,

g

i

/k).¹²Novel PKCs re-

quire only diacylglycerol (DAG) for activation while classical PKCs

require both DAG and Ca²⁺. Atypical PKCs do not require DAG or

Ca²⁺for activation. Among the PKC isoforms, PKCh is most closely re-

* Corresponding author. Tel.: +1 845 602 1876; fax: +1 845 602 5561.

E-mail address: tumeyn@wyeth.com (L.N. Tumey).

doi:10.1016/j.bmc.2009.10.020

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L. N. Tumey et al. / Bioorg. Med. Chem. 17 (2009) 7933–7948

NH

HN

N

R

N

HN

( )_n

R

CN

N

R'

N

I

IV

NH

R

HN

N

CN

R'

R N

R'

S

N

III

II

Figure 1.

lated to PKCd,¹³another novel PKC isoform which is thought to

play a role in the regulation of B cell proliferation.^14,15The PKCh

active site differs from PKCd by only one amino acid: Tyr460 (Phe

in PKCd). While numerous inhibitors of PKCs have been de-

scribed,^16,17only a handful of h-isoform selective inhibitors are

known. These include 2,4-diaminopyridines,¹⁸thieno[2,3-b]pyri-

dine-5-carbonitriles,^19–21and 5-aryl-3-pyridinecarbonitrile.^22–25

While a 2.0 Å structure of the PKCh kinase domain with the pan-

kinase inhibitor staurosporine has been determined, no co-crystal

structure with a selective PKCh inhibitor has been reported.²⁶

to our report on the related thieno[2,3-b]pyridine scaffold in

2008,²¹this reaction failed to give any observable product under

a variety of reaction conditions. We suspected that the poor reac-

tivity of this boronic acid may be due to a facile

p-allyl insertion

of the catalytic palladium which prevents the oxidative addition

of the palladium into the aryl iodide. This being the case, we spec-

ulated that addition of a nucleophile should free the palladium

from the p-allyl complex thereby allowing the Suzuki coupling to

take place with the newly formed 3-amino substituted allylboronic

acid. Gratifyingly, treatment of 1 with boronic acid 2 (1.5 equiv) in

the presence of a slight excess of piperidine (2 equiv), Cs₂CO₃

(2 equiv) and Pd(PPh₃)₂Cl₂(0.15 equiv) gave the desired 5-allyla-

mino-3-pyridinecarbonitrile 4a. The examples herein (vide infra)

build upon our 2008 publication and further illustrate the versatil-

ity and attractiveness of this ‘three-component-coupling’ reaction

for the addition of solubilizing tails to aryl halides (see Scheme 1).

Compound 4a proved to have an IC₅₀value of 190 nM against

PKCh (Table 1). Having established a baseline level of enzymatic

activity for this new series, we set about exploring additional

amine substituents and extensions of the alkene linker. The ‘3-

component coupling’ reaction described above was easily adapted

to make a variety of amino-substituted prop-1-enyl derivatives

such as 11a–15a (Scheme 2, reaction 1). We next turned our atten-

tion to the extension of the alkene linker (Scheme 2, reactions 2

and 3) The above conditions were operationally attractive and

we therefore initially explored the analogous reaction with homo-

allyl tosylate 5.²⁸While the desired product was formed, the

appearance of two signiﬁcant byproducts (Scheme 2, reaction 2)

complicated the isolation of the product and adversely affected

2. Results

We recently reported a series of 5-aryl-3-pyridinecarbonitrile

PKCh inhibitors (I).^22–25We previously found that the analogous

aryl group on a series of 2-arylthieno[2,3-b]pyridine-5-carbonitr-

iles (II) could be replaced by a simple alkene tethered to an amine

‘tail’ that imparted improved solubility (III).²¹This resulted in

compounds that retained the potency of parent aryl derivatives,

but with signiﬁcantly reduced molecular weight. In this paper,

we report that the 5-aryl group of compounds (I) can be replaced

by an alkene linker to give a new class of selective PKCh inhibitors,

5-vinyl-3-pyridinecarbonitriles (IV) (Fig. 1). A detailed account of

the optimization of potency, selectivity, functional activity, and

pharmaceutical properties will be described.

We initially reasoned that 5-allylamino-substituted 3-pyridine-

carbonitriles could be easily obtained by a Suzuki coupling of the

previously reported 5-iodonicotinonitrile 1²³with commercially

available 3-chloroallylboronic acid 2 to give 3. However, similar

NH

HN

N

a

I

CN

Cl

N

X

Cl

B(OH)₂

N

1

N

3

4a

2

b

Cl

B(OH)₂

2

Scheme 1. Initial synthesis of 4a. Reagents and conditions: (a) Cs₂CO₃, Pd(PPh₃)₂Cl₂, DMSO, 90 °C, 16 h; (b) piperidine, Cs₂CO₃, Pd(PPh₃)₂Cl₂, DMSO, 90 °C, 16 h.

L. N. Tumey et al. / Bioorg. Med. Chem. 17 (2009) 7933–7948

7935

Table 1

Exploration of amine solubilizing group and linker length

NH

R

N

HN

N

CN

R'

( )_n

a

b

X

Compd

n

PKCh IC₅₀(nM)

PKCd IC₅₀(nM)

Fold selectivity

T cell IC₅₀(nM)

4a

4b

0

1

190 1.1

35 1.7

690 100

260 12

3.6

7.4

ND^c

ND

N

4c

2

19 4.5

94 6.1

4.9

ND

11a

11b

0

1

51 6.1

16 2.2

67 1.4

250 18

1.3

16

ND

O

N

11c

2

34 11

120 6.0

3.5

ND

12a

12b

0

1

31 2.4

2.2 0.66

43 5.9

2.5 0.08

1.4

1.2

330 11

49 29

N

12c

2

6.4 1.9

45 1.8

7.0

77 26

13a

13b

0

1

10 2.4

4.0 1.1

22 2.4

16 1.7

2.2

4.0

1600

2300

HN

N

13c

2

2.8 0.6

27 6.2

9.6

820 190

14a

14b

14c

0

1

2

3.6 0.55

0.88 0.04

5.2 0.75

8.0 1.9

5.8 0.6

18 3.4

2.2

6.6

3.4

2700

210 59

ND

H₂N

HO

N

15a

15b

0

1

140 5.8

6.0 0.09

760 25

180 16

5.4

30

ND

1600

N

15c

2

11 0.12

62 4.2

5.6

ND

a

b

c

Represents the average of at least two measurements, standard error of the mean.

Represents a single measurement, or the average of at least two measurements standard error of the mean.

ND = not determined.

the yield. The ﬁrst byproduct, (E)-5-(buta-1,3-dienyl)-4-(4-methyl-

1H-indol-5-ylamino)-3-pyridinecarbonitrile 6, presumably re-

sulted from simple elimination of the tosylate. The mechanism

for the formation of the second byproduct, a 4-carbamoyl-but-1-

enyl derivative, remains unclear.

activity against PKCh regardless of the identity of the amine solubi-

lizing group (NRR⁰). Based on this initial set of compounds, the

optimal combination of linker and amine appeared to be a two-car-

bon linker to a 4-aminopiperidine (14b). This combination resulted

in sub-nM potency against PKCh (0.88 nM), but only moderate

selectivity over PKCd (6.6ꢀ). As described previously, selectivity

over PKCd is considered desirable due to the role this enzyme plays

in the regulation of B cell proliferation. Interestingly, replacing the

primary amine of 14b with an alcohol (15b) resulted in a signiﬁ-

cant increase in selectivity over PKCd. A comparison of activity

and selectivity of the unsubstituted piperidine 4b with the 4-

substituted piperidines 14b and 15b clearly indicates that the

distal position of the piperidine plays an important role in PKCh

activity and selectivity. Therefore, a larger set of compounds was

prepared (Table 2, discussed below) in order to probe the impor-

tance of this position.

After an initial evaluation of PKCh and PKCd activity, com-

pounds of interest were assayed for functional activity in T cells

isolated from C57 mice. The T cells were stimulated with anti-

CD3 and anti-CD28 antibodies in order to elicit the production of

IL2. The functional activity of the compounds was determined as

the IC₅₀value for the reduction of IL2 release. Perhaps not surpris-

ingly, compounds of similar potency gave a wide range of func-

tional activity (Table 1). For example, compound 12b containing

a methylpiperazine tail had an IC₅₀value of 49 nM in T cells while

the related des methyl analog, 13b, had an IC₅₀value of 2300 nM.

Discrepancies such as this are frequently attributed to off-target

activity or physiochemical properties such as solubility and perme-

ability. Correlation of cellular potency with off-target activity and

physiochemical parameters will be discussed in detail below.

In spite of the excellent PKCh potency of this series of com-

pounds, we were unable to obtain a co-crystal structure. Therefore,

in order to better understand the emerging SAR, a PKCh binding

model of this series was developed by utilizing the published PKCh

While the above reaction conditions allowed for the prelimin-

ary exploration of 4-aminobut-1-enyl derivatives such as

compound 4b (Table 1), due to the potent activity of these 4-

aminobut-1-enyl derivatives (vide infra), a more efﬁcient synthesis

was targeted. We found that separation of the nucleophilic

displacement step from the Suzuki coupling step minimized the

formation of both byproducts. Speciﬁcally, homoallyl tosylate 5

was treated with excess amine in DMSO at 50 °C for 16 h then trea-

ted with 1, Cs₂CO₃, and Pd(PPh₃)₂Cl₂and heated to 90 °C for an

additional 16 h. Upon cooling, the reaction was generally ﬁltered

and the ﬁltrate was puriﬁed by HPLC or silica gel chromatography

to give the desired 4-aminobut-1-enyl pyridine. Only minimal

amounts of the above two byproducts were observed when this

two-step process was utilized. The same two-step, one-pot proce-

dure was utilized for the synthesis of 5-aminopent-1-enyl-pyri-

dines such as 4c, 9c–12c, and 15c from 5-chloropent-1-enyl

boronic ester 8, as shown in Scheme 2, reaction 2. Primary and sec-

ondary amines could be easily introduced by utilizing the previ-

ously described reactions with Boc-protected piperazines and

Boc-amino substituted piperidines followed by deprotection

(Scheme 3).

Table 1 illustrates the initial set of compounds that was synthe-

sized in order to probe the SAR of the alkene linked solubilizing

tail. As previously mentioned, compound 4a was a 190 nM inhibi-

tor of PKCh with minimal selectivity over PKCd. Changes in the al-

kyl amine (11a–15a) resulted in signiﬁcant modulation of PKCh

activity. Importantly, the addition of a secondary basic amine

(12a–14a) resulted in compounds with improved potency. Exten-

sion of the alkyl linker to two and three carbons served to increase

7936

L. N. Tumey et al. / Bioorg. Med. Chem. 17 (2009) 7933–7948

Reaction 1:

NH

a

HN

I

HN

CN

R'

CN

N

R

Cl

B(OH)₂

2

N

1

N

4a, 9a-12a, 15a

NH

Reaction 2:

b or c,d

R'

N

HN

N

R

HN

CN

HN

N

1

O

CN

+

N

CN

R

R'

TsO

O

B

O

6

N

5

O

4b, 9b-12b, 15b, 17, 28-37

Reaction 3:

NH

e

f, d

O

HN

N

Cl

B

R

CN

1

O

7

8

N

R'

4c, 9c-12c, 15c

Scheme 2. Synthesis of 4-aminoalk-1-enyl pyridines. Reagents and conditions: (a) RR⁰NH, Cs₂CO₃, Pd(PPh₃)₂Cl₂, DMSO, 90 °C, 16 h; (b) RR⁰NH, 5, Cs₂CO₃, Pd(PPh₃)₂Cl₂, DMSO,

90 °C, 16 h; (c) RR⁰NH, 5, DMSO, 16 h, rt; (d) Cs₂CO₃, Pd(PPh₃)₂Cl₂, 90 °C, 16 h; (e) 4,4,5,5-tetramethyl-1,3,2-dioxaborolane, Zr(cp)₂ClH, Et₃N, rt; (f) RR⁰NH, DMSO, 16 h, rt.

NH

a

Boc

N

HN

N

HN

N

CN

N

CN

( )_n

9a (n=0)

9b (n=1)

9c (n=2)

13a (n=0)

13b (n=1)

13c (n=2)

NH

a

BocHN

H₂N

HN

N

HN

N

CN

N

CN

( )_n

10a (n=0)

10b (n=1)

10c (n=2)

14a (n=0)

14b (n=1)

14c (n=2)

Scheme 3. Synthesis of piperazinyl and 4-aminopiperidine derivatives. Reagents and condition: (a) TFA/DCM, rt.

X-ray structure with staurosporine²⁶and various in-house struc-

position (see Section 4). This modiﬁcation resulted in a PKCh bind-

ing model which was consistent with the SAR observed around

both the indole ‘headpiece’ and alkenyl ‘tailpiece’ (vide infra).

The SAR shown in Table 1 indicates that the length of the linker

between the core and the saturated heterocycle plays an important

role in enabling the formation of additional favorable interactions

by the alkene tailpiece. Consistent with this SAR, docking studies

of compound 4a ( Fig. 2, left panel) suggest that the piperidine

nitrogen does not pick up any speciﬁc hydrogen bonding interac-

tions with any of the polar or charged amino acids in the binding

site. On the other hand, compound 14b (Fig. 2, right panel) is able

to form three additional hydrogen bonding interactions with

Asp508 and Asn509 through its charged terminal amine group.

These additional interactions result in a 200-fold enhancement of

the potency. Interestingly, SAR suggests that this same interaction

tures of 3-pyridinecarbonitriles with Lck, a related Src family ki-

nase. The binding modes of several speciﬁc compounds were

further optimized using QXP docking²⁹against this PKCh model.

These experiments indicate that the pyridine nitrogen forms the

critical hinge region hydrogen bond to Leu461 and the indole-NH

forms a hydrogen bonding interaction with Glu428, which is part

of Helix C (Fig. 2). The interaction of the indole-NH with this resi-

due is incompatible with the PKCh staurosporine X-ray structure,

but was observed in X-ray structures with Lck. SAR around the in-

dole ‘headpiece’^22,27agrees with the formation of the hydrogen

bonding interaction of the indole with the Glu428. Therefore, in or-

der to obtain the binding model described above, the PKCh X-ray

structure was optimized by utilizing the conformation of the gly-

cine-rich loop of Lck in order to accommodate the headpiece in this

L. N. Tumey et al. / Bioorg. Med. Chem. 17 (2009) 7933–7948

7937

Table 2

SAR of the cyclic amine tailpiece

NH

X

( )_n

N

HN

N

CN

( )_m

a

b

Compd

n

m

X

PKCh IC₅₀(nM)

PKCd IC₅₀(nM)

Fold selectivity

T cell IC₅₀(nM)

4b

14b

16

17

18

19

20

21

22

23

24

25

26

27

28

15b

29

30

31

32

33

34

35

2

1

2

1

2

3

1

2

3

H

NH₂

35 1.7

0.88 0.04

9.3 1.4

4.9 0.38

5.7 0.59

1.8 0.35

6.8 0.31

8.9 2.2

3.5 0.47

1.3 0.38

16 3.3

22 3.0

22 4.7

23 0.7

31 2.9

6.0 0.09

42 4.7

39 11

19 6.2

24 3.9

270 12

5.8 0.6

28 4.6

56 18

7.5

6.6

3.0

11

5.9

14

4.4

13

4.3

14

12

11

13

8.8

10

ND^c

210 59

ND

630 200

ND

170 34

ND

3700

ND

740 220

740 125

ND

900

ND

1600

ND

420 76

ND

NHMe

NMe₂

CH₂NH₂

(R)-NH₂

(S)-NH₂

NH₂

(R)-NH₂

(S)-NH₂

NHSO₂Me

NHSO₂Et

NHSO₂iPr

NHSO₂Ph

NHAc

33 3.6

26 5.1

29 5.0

120 33

15 1.1

18 3.8

200 11

240 42

290 86

200 67

310 39

180 16

220 58

140 11

77 6.7

120 2.7

120 11

1300 130

930 98

OH

OMe

30

5.2

3.5

4.0

5.1

16

11

13

CH₂OH

Ph

(S)-OH

(R)-OH

(S)-OH

(R)-OH

7.4 1.5

120 34

71 0.25

ND

a

b

c

Represents the average of at least two measurements, standard error of the mean.

Represents a single measurement, or the average of at least two measurements standard error of the mean.

ND = not determined.

is likely taking place in PKCd. It is important to reiterate that (based

on sequence homology) the only difference between the PKCh and

PKCd binding pockets is Tyr460, shown adjacent to the hinge bind-

ing region in Figure 2. Attempts to exploit this difference by cap-

turing a hydrogen bond from the terminal OH of Tyr460 have

been unsuccessful to-date. As illustrated in Figure 2, the location

of this residue in proximity to the hinge may make it challenging

to access Tyr460 while retaining the key hinge interaction. This dif-

ﬁculty in accessing Tyr460 is likely the underlying reason for the

relatively poor theta/delta selectivity of this series of compounds.

As explained above, the amino functionality of 14b is thought to

interact with Asp508/Asn509 resulting in a signiﬁcant boost in

Figure 2. Predicted binding modes for compound 4a (left) and 14b (right) with hybrid model of PKCh. The C Helix is shown in the back of the binding site and the hinge region

is shown on the left hand side. Hydrogen bonding interactions are indicated by blue dashed lines. The pyridine core and indole headpiece form critical interactions with the

enzyme.

7938

L. N. Tumey et al. / Bioorg. Med. Chem. 17 (2009) 7933–7948

NH

see Scheme 2

a

1

R

L

( )_n

N

R

L

( )_n

N

HN

N

HN

N

H

N

R

L

( )_n

NH

CN

N

Boc

( )_m

Boc

( )_m

16 (L = bond, R = Me)

18 (L = CH₂, R = H)

19-23 (L = bond, R = H)

NH

H

N

b

H₂N

HN

N

RO₂S

HN

CN

N

CN

N

14b

24-27

NH

Y

c

HN

N

HN

N

CN

N

CN

38-43

N

13b

Scheme 4. Synthesis of amino-substituted heterocycles and substituted piperazines. Reagents and conditions: (a) TFA, DCM, rt; (b) RSO₂Cl, Et₃N, DMF, rt; (c) electrophile,

Et₃N, DMF, rt.

PKCh potency over the parent piperidine (4b). With this in mind, a

further set of analogs were made containing functionalities amena-

ble to potential H-bond and ionic interactions with Asp508/

Asn509. These analogs were generally made by the previously de-

scribed route (Scheme 2) or by minor modiﬁcations of the previ-

ously described route (Scheme 4). Movement of the 4-amino

group of compound 14b to the 3 position (19–20) and extension

of the amino group (18) resulted in a slight to moderate loss of

activity. Likewise, methylation of the amine (16, 17) and contrac-

tion of the six-membered ring to a ﬁve- or four-membered ring

(21–23) resulted in a modest loss of PKCh activity. Enantiomer

pairs show that there is a slight preference for the (R) enantiomer

of 3-aminopiperidines (19, 20) while there is a slight preference for

the (S) enantiomer of the 3-aminopyrrolidines (22, 23). Sulfonyla-

tion or acylation of the amine (24–28) were signiﬁcantly deleteri-

ous to PKCh activity, possibly indicative of a salt bridge between

the amine moiety and Asp508. However, there appears to be no

major size constraint as both large (26, 27) and small (24, 28) sub-

stituents at this position have similar enzymatic activity. Various

other 4-substituted piperidines were also examined (29–35).

Replacement of the amino functionality described above with hy-

droxy generally resulted in compounds with 10–20ꢀ reduced PKCh

activity (15b, 32–35). These results are consistent with the loss of a

salt bridge interaction between the amino functionality and

Asp508. 4-Hydroxypiperidine 15b is the only non-amino substi-

tuted piperidine exhibiting potency under 10 nM. Interestingly,

for reasons that are unclear, this is the only compound of the series

that exhibits >25-fold selectivity over PKCd.

fold) over PKCd. Unexpectedly, replacement of the methyl group

of 12b with a phenyl (37) resulted in a compound with dramatic

loss of PKCh activity but retention of PKCd activity. As such, com-

pound 37 was unexpectedly ꢁ20-fold selective towards PKCd. How-

ever, replacement of the phenyl group with amides and small

sulfonamides (38–41) resulted in compounds with very weak po-

tency against PKCd, thereby possessing up to 39-fold selectivity

for PKCh. Unfortunately, these substituents also resulted in a

ꢁ10-fold loss of PKCh activity and poor microsomal stability (data

not shown). Increasing the size of the sulfonamide (42–43) re-

sulted in loss of selectivity over PKCd.

In the course of a related SAR study, we recently reported the

synthesis of 6-methyl-3-pyridinecarbonitriles. The compounds

were made via palladium mediated reactions with the intermedi-

ate 44.²⁷In order to probe the effect of 6-methyl substitution on

this series of compounds, 44 was treated under the aforemen-

tioned conditions to give compounds 45 and 47 (Scheme 5). Treat-

ment of compound 45 with methanesulfonyl chloride gave

sulfonamide 46. As illustrated in Table 4, substitution with a 6-

methyl had a notably detrimental effect on PKCh activity. No signif-

icant effects on selectivity over PKCd were observed. Reduction of

the alkene linker also resulted in a compound (48) with modestly

reduced PKCh activity as compared to the parent compound, 14b

(Scheme 6).

3. Discussion and conclusions

The modeling and SAR studies described above gave us a good

understanding of the structural features necessary for potent PKCh

inhibition. However, an examination of the data described in

Tables 1–3 illustrates that enzymatic activity alone is not sufﬁcient

to achieve functional (cellular) activity. Several compounds, partic-

ularly those containing primary and secondary amines such as 13b,

14a, and 21, possessed sub-10 nM potency against the target, but

The potent cellular activity of the methylpiperazine analog 12b

prompted further exploration of the piperazinyl moiety (Table 3).

It was previously shown (Table 1) that loss of the methyl group

from the piperazine resulted in retention of enzymatic potency

but loss of cellular potency. Likewise, expansion of the piperazine

to a homopiperazine (36) resulted in a compound with good PKCh

inhibitory activity (3.6 nM) but poor functional activity (1900 nM).

Interestingly, this compound possessed moderate selectivity (16-

gave only weak (lM) inhibition of IL2 release from T cells. First,

in order to rule out activity due to non-PKCh mediated effects

L. N. Tumey et al. / Bioorg. Med. Chem. 17 (2009) 7933–7948

7939

Table 3

SAR of piperazine tailpiece

NH

X

N

HN

N

CN

( )_n

a

b

Compd

n

X

PKCh IC₅₀(nM)

PKCd IC₅₀(nM)

Fold selectivity

T cell IC₅₀(nM)

12b

13b

36

37

38

39

40

41

42

1

2

1

Me

H

Ph

2.2 0.66

4.0 1.1

3.6 1.2

68 13

38 1.2

51 5.1

12 0.68

12 0.22

11 0.44

50 2.6

2.5 0.08

16 1.7

56 7.6

3.4 0.35

390 31

2000 390

240 18

270 100

81 25

1.2

4.0

16

0.05

10

39

20

23

7.4

8.2

49 29

2300

1900

ND^c

ND

840

ND

Ac

Pivalate

SO₂Me

SO₂Et

SO₂iPr

SO₂Ph

ND

43

410 140

a

b

c

Represents the average of at least two measurements, standard error of the mean.

Represents a single meausrement, or the average of at least two measurements standard error of the mean.

ND = not determined.

NH

H

N

HN

N

H₂N

MeO₂S

HN

N

d

N

CN

N

CN

NH

c

,

b

,

a

46

45

HN

I

CN

NH

N

HN

44

N

CN

N

47

Scheme 5. Synthesis of 6-methyl-5-vinyl-3-pyridinecarbonitriles. Reagents and conditions: (a) 4-(N-Boc-amino)piperidine, 5, 50 °C, DMSO, 16 h; (b) 44, Cs₂CO₃,

Pd(PPh₃)₂Cl₂, 90 °C, 16 h; (c) TFA, DCM; (d) MeSO₂Cl, Et₃N, DMF, rt; (e) N-Boc-piperazine, 5, 50 °C, DMSO, 16 h.

(‘off-target’ effects), the cellular efﬁcacy of a variety of compounds

was measured in T cells isolated from PKCh knockout (KO) mice.

Gratifyingly, the compounds were dramatically less potent in the

KO T cells than in the wild-type (WT) T cells (Table 5). This strongly

suggests that the functional activity in WT T cells is due to inhibition

of PKCh rather than to off-target effects mediated by other enzymes

in the IL2 pathway.

Having established the ‘functional selectivity’ of this series, it is

still necessary to address the exceptionally weak cellular activity of

some of the most potent PKCh inhibitors. In order to gain a more

quantitative assessment of this phenomenon, the T cell IC₅₀value

was ‘normalized’ by dividing by the PKCh IC₅₀thus giving a mea-

Cell_eff, therefore, is a measure of how effectively a particular

inhibitor of PKCh exerts functional effects on T cells. A low Cell_eff

indicates that an inhibitor efﬁciently inhibits functional activity

while a high Cell_effindicates that an inhibitor displays a large dis-

crepancy between enzymatic and functional activity. Other than

off-target effects, discrepancies between enzymatic and functional

activity are frequently attributed to poor physiochemical proper-

ties such as low permeability, low solubility, or high polarity.

Polarity can be evaluated a number of ways, including ‘total polar

surface area’ (TPSA), c Log P, and Log D. Table 5 illustrates several

calculated and measured physiochemical properties of 18 com-

pounds for which both enzymatic and functional activity were

measured. The entire set of compounds is highly soluble (typically

sure of ‘cellular efﬁciency’ (Cell_eff

)

>100 lg/mL) and clearly this parameter does not play a role in

Cell_eff¼ T cell IC₅₀ðnMÞ=PKCh IC₅₀ðnMÞ

Cell_eff. Figure 3 illustrates a plot of Cell_effagainst the remaining

physiochemical parameters: c Log P, Log D_7.4, PAMPA permeability,

and TPSA.

Parallel Artiﬁcial Membrane Permeability Assay (PAMPA) is a

common model of permeability which measures the ﬂux of a com-

pound across an artiﬁcial membrane.³⁰PAMPA permeability is fre-

Stimulation of PKCh KO T cells requires much higher concentrations of anti-CD3

and anti-28 in order to elicit an IL2 response.

7940

L. N. Tumey et al. / Bioorg. Med. Chem. 17 (2009) 7933–7948

Table 4

Activity of 6-methyl-5-vinyl-3-pyridinecarbonitriles

NH

HN

Cy

CN

R

N

a

Compd

Cy

R

PKCh IC₅₀(nM)

PKCd IC₅₀(nM)

Fold selectivity

14b

45

H

Me

0.88 0.04

46 0.21

5.8 0.6

ND^b

6.6

ND

H₂N

N

24

46

H

Me

16 3.3

57 5.1

200 11

250 26

12

4.4

H

N

MeO₂S

HN

N

13b

47

H

Me

4.0 1.1

17 2.0

16 1.7

120 12

4.0

7.0

N

a

Represents the average of at least two measurements, standard error of the mean.

ND = not determined.

b

NH

H

N

H₂N

Boc

HN

N

HN

CN

a,b

N

CN

N

48

10b

PKCθ IC₅₀= 5.2 0.42 nM

Scheme 6. Synthesis of 5-alkyl-3-pyridinecarbonitriles. Reagents and conditions: (a) H₂, Pd/C (10%), EtOH, rt; (b) TFA/DCM, rt.

Table 5

Calculated and measured physiochemical properties of key compounds

Compd PKCh IC₅₀(nM) T cell IC₅₀(nM) Cell_effPKCh KO T cell IC₅₀(nM) Permeability (ﬂux, 10⁶cm/s) TPSA (A²) Log D_7.4c log P Solubility (

l

g/mL)

12a

12b

12c

13a

13b

13c

14a

14b

15b

17

19

21

23

24

26

33

36

40

31

330

49

77

11

22

12

8900

4600

4300

>10,000

ND^a

0

71.0

79.8

93.8

88.0

71.0

93.8

113.9

88.0

79.8

105.1

2.7

3.3

3.7

0.8

0.0

2.1

1.1

1.3

2.7

3.2

2.1

1.3

1.8

2.4

3.4

3.1

0.2

3.8

2.8

3.1

3.6

3.3

3.6

4.2

2.9

3.3

3.2

3.8

4.1

3.9

3.6

3.1

3.7

3.5

3.8

4.0

>100

30

2.2

6.4

12

0.86

0.07

0.02

0.11

0.16

0.03

0.04

0.02

0.08

ND

0.06

ND

0.05

0.07

0.01

0.02

1600

2300

820

2700

210

1600

630

170

3700

740

900

420

1900

840

133

573

294

750

239

267

128

94

415

575

45

40

57

4.0

2.8

3.6

0.88

6.0

4.9

1.8

8.9

1.3

16

ND

>10,000

ND

>10,000

ND

>10,000

ND

22

7.4

3.6

12

>10,000

ND

>10,000

531

70

a

ND = not determined.

quently utilized to predict absorption through GI membranes, cell

membranes, and skin. Therefore, it is perhaps surprising that no dis-

cernable relationship was observed between Cell_effand PAMPA per-

meability. In fact, most compounds exhibited quite poor

permeability regardless of their cellular efﬁcacy. The reasons behind

this poor permeability are unclear. Likewise, TPSA shows no correla-

tion with cellular efﬁcacy. TPSA is a calculated property that has fre-

quently been shown to be correlated with passive membrane

permeability.³¹The TPSA of the molecules in Table 5 is considered

moderate (60–140 Å²), which is generally predictive of acceptable

permeability and absorption. It is clear from Figure 3 that PAMPA

and TPSA play essentially no role in the cellular activity of this series.

The calculated partition coefﬁcient between octanol and water

(c Log P) has become a ubiquitous measure of lipophilicity of small

L. N. Tumey et al. / Bioorg. Med. Chem. 17 (2009) 7933–7948

7941

Figure 3. Plots of Cell_effagainst various physiochemical parameters.

organic molecules. While the focus of c Log P discussions tends to

be on the upper limits of this parameter, it is also generally recog-

nized that organic molecules must posses a minimal amount of

lipophilicity (lower limit of c Log P) in order to facilitate membrane

permeation and absorption.³²For GI absorption, this lower limit of

c Log P has been shown to be ꢁ3.³³It is generally accepted that

c Log P accurately reﬂects the lipophilicity of neutral organic mol-

ecules. However, c Log P is of limited utility for ionizable com-

pounds because the property calculation is made, by deﬁnition,

on only the neutral form of the organic molecule and does not take

into account ionization of various functionalities such as amines

and carboxylic acids. Recent advances in computational chemistry

have enabled the accurate and timely prediction of Log D, which is

a partition coefﬁcient which takes into account the ratio of various

ionization states that are present a given pH.³⁴Compounds that

contain multiple ionizable amines, particularly primary and sec-

ondary amines, exhibit Log D_7.4<< c Log P (i.e., 13a–c, 14a–b and

36). However, compounds with only a single ionizable group such

as 12a and 40 show nearly identical Log D and c Log P values.

Therefore, within this data set, Log D_7.4appears to be a more rele-

vant measure of polarity than c Log P.

With this in mind, it is interesting to note that there is a reason-

able positive correlation between Log D_7.4and Cell_effwhile there is

no measurable correlation between c Log P and Cell_eff(Fig. 3). We

believe that compounds with a very low Log D_7.4such as 36 and

13b are too polar to effectively cross cellular membranes and inhi-

bit intracellular PKCh. The Pearson’s coefﬁcient (R²) for the Log D/

Cell_effcorrelation is 0.57 which suggests that, as expected, there

are factors other than Log D_7.4that play a role in cellular potency

(permeability, binding kinetics, etc.). Based on the correlation illus-

trated in Figure 3, it is our belief that optimal cellular efﬁcacy can

be achieved with molecules possessing Log D_7.4P 2.5.

As previously described, the PKCh inhibitors were routinely

screened against PKCd due to concerns about possible deleterious

effects upon B cells. In addition, select compounds of interest were

screened against a broader panel of PKC isoforms containing repre-

sentative examples of each of the three families of PKCs: novel,

atypical, and classical. PKCh and PKCd are both novel PKC isoforms

and, as such, it is hardly surprising that the compounds shown in

Table 6 also have signiﬁcant activity against the two remaining no-

vel isoforms, PKCe and PKCg. Only compounds 14b and 19 showed

modest selectivity against all three novel isoforms. Of the novel

isoforms, good selectivity (>100-fold) could only be achieved

against PKCg. Perhaps not surprisingly, the compounds did not

show signiﬁcant inhibition of one representative example of the

atypical and classical isoform families. The implications of activity

against PKCe are unclear at this time. Overexpression of PKCe is

associated with increased tumorogenesis in nude mice³⁵and its

inhibition is speculated to be a possible target for oncology

drugs.¹⁶

3-Quinolinecarbonitriles and related scaffolds are known to

inhibit Src family kinases.³⁶Due to concerns about structural

similarities between the 3-quinolinecarbonitriles and the 5-vinyl-

3-pyridinecarbonitriles, the compounds in Table 6 were screened

against two Src family kinases, Lck and Lyn. Lyn is of particular

concern due to reports that Lyn KO mice possess a hyperresponsive

B cell phenotype.³⁷Gratifyingly, the compounds described in Table

6 generally showed well over 1000-fold selectivity over both Lyn

and Lck. It is generally thought that kinase selectivity within the

3-quinolinecarbonitrile scaffold is largely driven by the group at

Table 6

Selectivity of select compounds against PKC isoforms and Src family kinases

Compd

Novel

PKC

Atypical

Classical

Src family

Lyn IC₅₀(nM) Lck IC₅₀(nM)

PKCh IC₅₀(nM)

PKCd IC₅₀(nM)

e

IC₅₀(nM)

PKCg

IC₅₀(nM)

PKCf IC₅₀(nM)

PKCb IC₅₀(nM)

13a

13b

14b

19

12

45

5.8

27

18

26

16

550

190

100

350

88

>100,000

>100

2500

2300

>100,000

1400

30,000

5100

9600

19,000

20,000

ND

1200

600

2900

47,000

4.0

0.88

1.8

16

2.5

6.5

14

24

8.0

7942

L. N. Tumey et al. / Bioorg. Med. Chem. 17 (2009) 7933–7948

the 4-position of the quinoline. This suggests that the 4-indolyl

headpiece is not tolerated within the Src family ATP pocket or,

alternatively, that the fused carbocyclic ring of the quinoline is

necessary for Src family activity (note that compound 19 is selec-

tive over 3 additional Src family kinases, vide infra).

pounds show excellent selectivity over a wide range of other ki-

nases, including atypical and classical PKCs. Additional studies

describing the advancement of this series of compounds will be

disclosed in due course.

Compound 19 was screened against a broader panel of kinases

as illustrated in Table 7. With the exception of modest activity

against Src and PKA, only minimal inhibition was observed. Selec-

tivity was generally well over 500-fold for 20 out of the 22 kinases

tested. The selectivity against Src and PKA was >250-fold.

4. Experimental section

4.1. Modeling and docking experiments

The hybrid PKCh model was developed with the PKCh-Stauro-

sporine X-ray structure as the starting point. The glycine-rich loop

was modeled after an X-ray structure of Lck in complex with a

compound from the related thienopyridine series using PRIME

1.2.^àThe hybrid model was subsequently optimized further using

the Induced Fit Docking protocol.^§

Based on enzymatic potency, kinase selectivity, and functional

activity, compound 19 was selected for further evaluation in order

to determine its suitability for advancement. The compound was

shown to have no activity against CYP2C9 and modest activity

against CYP3A4 and CYP2D6 (46% and 53% inhibition, respectively,

at 3 lM). The compound proved to be exceptionally stable in rat

Docking studies were conducted with the QXP docking algo-

rithm²⁹utilizing the mcdock+ or mcldock+ sampling and scoring

scheme. Generally, 5000 Monte Carlo steps were used and if neces-

sary a core constraint of the pyridine core and the indole headpiece

was used through the mcldock+ procedure in QXP. The PKCh hybrid

model was used throughout all docking studies.

and human microsomes (T_1/2>30 min) and moderately stable in

BALB/c mouse microsomes (T_1/2= 22 min).³⁹This suggests that

ﬁrst-pass metabolism is not likely to adversely affect oral expo-

sure. However, the CaCo₂permeability (A to B) for this compound

was poor (0.5 ꢀ 10^ꢂ6cm/s), suggesting poor intestinal absorption.

Moreover, the ratio of permeability (A to B vs B to A) was 16,

strongly suggesting that 19 may be subject to active efﬂux, possi-

bly mediated by PGP efﬂux pumps. PGP is widely expressed in

intestine, kidney, and liver barriers and is frequently associated

with poor intestinal absorption and biliary excretion.^40,41Consis-

tent with these observations, compound 19 was found to have high

clearance (128 mL/min/kg, 2 mpk IV) and poor oral bioavailability

(3%, 10 mpk PO) in BALB/c mice. The acceptable PK of related 5-

heteroaryl-3-pyridinecarbonitriles²⁵suggests that the poor charac-

teristics of 19 are related to the vinyl side chain rather than the

pyridine core or indole headpiece.

4.2. Calculated physiochemical properties

TPSA was calculated by the method described by Ertl et al.³⁸

Calculations assume sulfur and phosphorous are not polar atoms.

c Log P and Log D were calculated using the Biobyte 4.3 algorithm

as implemented in Daylight, version 4.81.

4.3. General experimental conditions

The following HPLC methods were utilized to evaluate com-

pound purity.

In summary, we have described 5-vinyl-3-pyridinecarbonitriles

that exhibit excellent PKCh inhibitory activity. SAR and molecular

modeling studies indicate that an H-bond donor extending from

the alkene side chain makes three key interactions with Asp508

and Asn509. Cellular activity of this series of compounds was

found to be poorly correlated with enzymatic activity. However,

a combination of good enzymatic activity and a high Log D_7.4re-

sulted in compounds with low nM cellular activity. Representative

members of this series of compounds were found to have modest

selectivity over other novel PKC isoforms. However, the com-

4.3.1. Method A

1.0 mL/min, 20 min gradient ACN (10–95%) in H₂O/TFA, Prodigy

ODS3, 0.46 ꢀ 15 cm column.

4.3.2. Method B

1.0 mL/min, 20 min gradient MeOH (10–95%) in H₂O/TFA, Prod-

igy ODS3, 0.46 ꢀ 15 cm column.

4.3.3. Method C

1.0 mL/min, 5 min gradient ACN (10–95%) in H₂O/formic acid,

waters Xterra MS C18 3.5

Table 7

Activity of 19 against a broad panel of kinases

l

m 2.1 ꢀ 30 mm column.

Kinase

IC₅₀(nM)

4.3.4. Method D

Abl

3400

1.0 mL/min, 5 min gradient ACN (10–95%) in H₂O/NH₄OH,

AuroraB

CDK1

CDK2

CHK1

CK1 gamma

ERK2

Fyn

41,000

26,000

17,000

4500

>50,000

1700

waters Xterra MS C18 3.5

l

m 2.1 ꢀ 30 mm column.

4.3.5. Method E

1.0 mL/min, 10 min gradient ACN (10–95%) in H₂O/TFA, Prodigy

ODS3, 0.46 ꢀ 15 cm column.

Gck

2600

Hck

5500

35,000

1000

34,000

22,000

32,000

10,000

490

4.4. Synthetic examples

IKKa

IKKb

Met

MK2

4.4.1. 4-[(4-Methyl-1H-indol-5-yl)amino]-5-[(1E)-3-piperidin-

1-ylprop-1-en-1-yl]nicotinonitrile (4a)

A mixture of 1²³(100 mg, 0.27 mmol), piperidine (70 mg,

0.81 mmol), trans-2-chloromethylvinylboronic acid (2) (64 mg,

0.53 mmol), cesium carbonate (173 mg, 0.53 mmol) and

P38

a

PDGFR

PKA

PKB

a

2400

ROCK1

RSK1

Src

>50,000

11,000

600

à

Prime, version 1.2, Schrödinger, LLC, New York, NY 2005.

Induced Fit Docking Using Glide and Prime; Glide version 3.5, Schrödinger, LLC,

§

VEGFR2

3800

New York, NY, 2005; Prime version 1.2, Schrödinger, LLC, New York, NY, 2005.

L. N. Tumey et al. / Bioorg. Med. Chem. 17 (2009) 7933–7948

7943

Pd(PPh₃)₂Cl₂(9.5 mg, 0.014 mmol) in 1 mL of DMSO was heated at

90 °C for 16 h, cooled to room temperature, and ﬁltered. The ﬁl-

trate was concentrated and puriﬁed by HPLC to give 97 mg (74%

yield) of 4a as its TFA salt. ¹H NMR (DMSO-d₆) d 11.25 (1H, s),

9.65 (1H, br s), 9.47 (1H, s), 8.59 (1H, s), 8.54 (1H, s), 7.39 (1H, t,

J = 4.2 Hz), 7.27 (1H, d, J = 6.0 Hz), 6.99 (1H, d, J = 11.4 Hz), 6.94

(1H, d, J = 6.3 Hz), 6.55 (1H, s), 6.33 (1H, dt, J = 5.5, 11.7 Hz), 3.82

(2H, t, J = 4.0 Hz), 3.49–3.44 (2H, m), 2.91 (2H, dd, J = 7.2,

16.2 Hz), 2.32 (3H, s), 1.88–1.35 (6H, m); HPLC 94.9% at 215 nm,

4.8 min (Method A); MS (ESI) m/z 372.2187 (M+H).

cesium carbonate (173 mg, 0.53 mmol) and Pd(PPh₃)₄(16 mg,

0.014 mmol) in 3 mL of DMSO was heated at 70 °C for 16 h, cooled

to room temperature, diluted with EtOAc and washed with water.

The crude material was puriﬁed by silica gel chromatography with

an EtOAc/hexane gradient to give 21 mg (26% yield) of 6; MS 301.2

(M+H).

4.4.6. (E)-2-(5-Chloropent-1-enyl)-4,4,5,5-tetramethyl-1,3,2-

dioxaborolane (8)

A mixture of 5-chloro-1-pentyne 7 (500 mg, 4.9 mmol), 4,4,5,5-

tetramethyl-1,3,2-dioxaborolane (947 mg, 7.4 mmol), bis(cyclo-

pentadienyl)-zirconium(IV) chloride hydride (191 mg, 0.74 mmol)

and triethylamine (49.5 mg, 0.49 mmol) were stirred neat at room

temperature for 24 h. The reaction was dissolved in DCM, ﬁltered

through a pad of silica gel and washed with DCM. The eluent

was removed to give 824 mg (73% yield) of 8 as a clear yellow li-

quid; ¹H NMR (DMSO-d₆) d 6.50 (1H, dt, J = 6.4, 18 Hz), 5.37 (1H,

dt, J = 1.6, 18 Hz), 3.61 (2H, t, J = 6.6 Hz), 2.29–2.18 (2H, m), 1.88–

1.78 (2H, m), 1.19 (12H, s); MS 231.2 (M+H).

4.4.2. 4-[(4-Methyl-1H-indol-5-yl)amino]-5-[(1E)-4-piperidin-

1-ylbut-1-en-1-yl]nicotinonitrile (4b)

A mixture of piperidine (104 mg, 1.2 mmol) and (E)-4-(4,4,5,5-tet-

ramethyl-1,3,2-dioxaborolan-2-yl)but-3-enyl 4-methylbenzenesulf-

onate 5 (200 mg, 0.57 mmol) was stirred in 2 mL of DMSO. After

stirring for 16 h at room temperature, 1 (108 mg, 0.29 mmol), ce-

sium carbonate (186 mg, 0.57 mmol) and Pd(PPh₃)₄(17 mg,

0.015 mmol) were added. After heating to 70°C for 16 h, the reac-

tion mixture was cooled to room temperature and ﬁltered. The ﬁl-

trate was concentrated and puriﬁed by HPLC to give 73 mg (50 %

yield) of 4b as its TFA salt; ¹H NMR (DMSO-d₆) d 11.25 (1H, s),

9.44 (1H, br s), 9.23 (1H, br s), 8.56 (1H, s), 8.38 (1H, s), 7.40 (1H,

t, J = 2.0 Hz), 7.28 (1H, d, J = 6.3 Hz), 9.95 (1H, d, J = 6.3 Hz), 6.68

(1H, d, J = 11.7 Hz), 6.55 (1H, s), 6.24 (1H, dt, J = 5.0, 12 Hz), 3.44

(2H, d, J = 9 Hz), 3.19–3.14 (2H, m), 2.88 (2H, q, J = 8.3 Hz), 2.61

(2H, q, J = 5.3 Hz), 2.32 (3H, s), 1.87–1.37 (6H, m); HPLC 96% at

254 nm, 4.2 min (Method E); MS (ESI) m/z 386.2340 (M+H).

4.4.7. tert-Butyl 4-[(2E)-3-{5-cyano-4-[(4-methyl-1H-indol-5-yl)-

amino]pyridine-3-yl}prop-2-en-1-yl]piperazine-1-carboxylate

(9a)

Compound 9a was prepared from 1 and tert-butyl piperazine-1-

carboxylate using the procedure described for 4a and puriﬁed by

silica gel chromatography using a MeOH/DCM gradient to give

239 mg (96% yield) as a solid. ¹H NMR (DMSO-d₆) d 11.14 (1H, s),

8.59 (1H, s), 8.37 (1H, s), 8.25 (1H, s), 7.34 (1H, t, J = 2.6 Hz), 7.22

(1H, d, J = 8.8 Hz), 6.89 (1H, d, J = 8.8 Hz), 6.73 (1H, d, J = 15.2 Hz).

6.50 (1H, s), 6.25–6.17 (m, 1H), 3.33–3.28 (4H, m), 3.08 (2H, d,

J = 6.4 Hz), 2.36–2.27 (4H, m), 2.28 (3H, s), 1.38 (9H, s); MS 473.4

(M+H).

4.4.3. 4-[(4-Methyl-1H-indol-5-yl)amino]-5-[(1E)-5-piperidin-

1-ylpent-1-en-1-yl]nicotinonitrile (4c)

A mixture of piperidine (90 mg, 1.06 mmol) and (E)-2-(5-chloro-

pent-1-enyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane 8 (122 mg,

0.53 mmol) was stirred in 1 mL of DMSO. After stirring for 16 h at

room temperature, 1 (100 mg, 0.27 mmol), cesium carbonate

(173 mg, 0.53 mmol) and Pd(PPh₃)₄(16 mg, 0.014 mmol) were

added to the reaction mixture. The resulting suspension was heated

at 70°C for 16 h. After cooling to room temperature and ﬁltering, the

ﬁltrate was concentrated and puriﬁed by HPLC to give 53 mg (38 %

yield) of 4c as its TFA salt; ¹H NMR (DMSO-d₆) d 11.25 (1H, s), 9.60

(1H, br s), 9.35 (br s, 1H), 8.60 (1H, s), 8.40 (1H, s), 7.40 (1H, t,

J = 2.8 Hz), 7.28 (1H, d, J = 8.4 Hz), 6.95 (1H, d, J = 8.4 Hz), 6.64 (1H,

d, J = 16 Hz), 6.55 (1H, s), 6.33 (1H, dt, J = 6.6, 15.6 Hz), 3.46–3.41

(2H, m), 3.09–3.05 (2H, m), 2.91–2.83 (2H, m), 2.32 (3H, s), 2.26

(2H, q, J = 6.8 Hz), 1.88–1.36 (8H, m); HPLC 94.5% at 215 nm,

5.1 min (Method A); MS (ESI) m/z 400.2500 (M+H).

4.4.8. tert-Butyl 4-[(3E)-4-{5-cyano-4-[(4-methyl-1H-indol-5-

yl)amino]pyridin-3-yl}but-3-en-1-yl]piperazine-1-carboxylate

(9b)

Compound 9b was prepared from 1 and tert-butyl piperazine-1-

carboxylate using the procedure described for 4b and puriﬁed by

HPLC to give 83 mg (40% yield) TFA salt; ¹H NMR (DMSO-d₆) d

11.22 (1H, s), 9.69 (1H, br s), 9.24 (1H, br s), 8.50 (1H, s), 8.38

(1H, s), 7.39 (1H, t, J = 2.6 Hz), 7.28 (1H, d, J = 8.4 Hz), 6.94 (1H, d,

J = 8.4 Hz), 6.70 (1H, d, J = 15.6 Hz), 6.55 (1H, s), 6.29–6.18 (1H,

m), 4.08–3.99 (4H, m), 3.55–2.90 (8H, m), 2.32 (3H, s), 1.42 (9H,

s); MS 487.4 (M+H).

4.4.9. tert-Butyl 4-[(4E)-5-{5-cyano-4-[(4-methyl-1H-indol-5-

yl)amino]pyridin-3-yl}pent-4-en-1-yl]piperazine-1-carboxylate

(9c)

4.4.4. (3E)-4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)but-

3-en-1-yl-4-methylbenzenesulfonate (5)

Compound 9c was prepared from 1 and tert-butyl piperazine-1-

carboxylate using the procedure described for 4c and puriﬁed by

silica gel chromatography using a MeOH/DCM gradient to give

130 mg (49% yield) of 9c as a solid. ¹H NMR (DMSO-d₆) d 11.12

(1H, s), 8.52 (1H, s), 8.30 (1H, s), 8.21 (1H, s) , 7.34 (1H, t,

J = 2.8 Hz) 7.22 (1H, d, J = 8.4 Hz), 5.88 (1H, d, J = 8.8 Hz), 6.59

(1H, d, J = 15.6 Hz), 6.50 (1H, s), 6.28–6.19 (1H, m), 3.32–3.26

(2H, m), 2.35–2.25 (8H, m), 2.18 (2H, q, J = 7.0 Hz). 1.60 (2H, quint,

J = 7.0 Hz), 1.39 (9H, s); MS 501.5 (M+H).

A mixture of but-3-ynyl 4-methylbenzenesulfonate (5 g, 22.3

mmol), 4,4,5-tetramethyl-1,3,2-dioxaborolane (4.3 g, 33.5 mmol),

bis(cyclopentadienyl)-zirconium(IV) chloride hydride (864 mg,

3.35 mmol) and triethylamine (225 mg, 2.23 mmol) were stirred

neat at room temperature for 24 h. The reaction was dissolved in

DCM, ﬁltered through a pad of silica gel and washed with DCM.

The eluent was removed to give 5.62 g (72% yield) of 5 as a white

solid; ¹H NMR (DMSO-d₆) d 11.28 (1H, s), 10.07 (1H, s), 8.69 (1H, s),

8.56 (1H, s), 7.41 (1H, t, J = 2.6 Hz), 7.29 (1H, d, J = 8.4 Hz), 7.08–

6.91 (3H, m), 6.59–6.49 (2H, m), 5.52 (1H, d, J = 17.2 Hz), 5.37

(1H, d, J = 11.2 Hz), 2.33 (3H, s); MS 353.2 (M+H).

4.4.10. tert-Butyl {1-[(2E)-3-{5-cyano-4-[(4-methyl-1H-indol-5-

yl)amino]pyridine-3-yl}prop-2-en-1-yl]piperidin-4-

yl}carbamate (10a)

4.4.5. 5-[(1E)-Buta-1,3-dien-yl]-4-[(4-methyl-1H-indol-5-

yl)amino]nicotinonitrile (6)

Compound 10a was prepared from 1 and tert-butyl piperidin-4-

ylcarbamate using the procedure described for 4a and puriﬁed by

silica gel chromatography using a MeOH/DCM gradient to give

110 mg (42% yield) of 10a as a solid. ¹H NMR (DMSO-d₆) d 11.13

(1H, s), 8.60 (1H, s), 8.37 (1H, s), 8.24 (1H, s), 7.34 (1H, t,

A

mixture of 1 (100 mg, 0.27 mmol), indoline (95 mg,

0.80 mmol), (E)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-

but-3-enyl 4-methylbenzenesulfonate (5) (187 mg, 0.53 mmol),

7944

L. N. Tumey et al. / Bioorg. Med. Chem. 17 (2009) 7933–7948

J = 2.6 Hz), 7.22 (1H, d, J = 8.4 Hz), 6.89 (1H, d, J = 8.4 Hz), 6.73 (1H,

d, J = 15.6 Hz), 6.50 (1H, s), 6.21 (1H, dt, J = 6.6, 15.6 Hz), 3.35–2.78

(7H), 2.28 (3H, s), 2.03–1.88 (2H, m), 1.73–1.65 (2H, m), 1.38 (9H,

s); MS 487.5 (M+H).

¹H NMR (DMSO-d₆) d 11.25 (1H, s), 10.03 (1H, br s), 9.58 (1H, s),

8.59 (1H, s), 8.40 (1H, s), 7.40 (1H, t, J = 2.8 Hz), 7.28 (1H, d,

J = 8.4 Hz), 6.95 (1H, d, J = 8.0 Hz), 6.65 (1H, d, J = 15.2 Hz), 6.55

(1H, s), 6.32 (1H, dt, J = 6.6, 15.6 Hz), 4.00 (2H, d, J = 11.6 Hz),

3.67 (2H, t, J = 1.8 Hz), 3.49–4.50 (2H, m), 3.19–3.01 (4H, m), 2.32

(3H, s), 2.28 (2H, q, J = 6.8 Hz), 1.90–1.80 (2H, m); HPLC 100% at

215 nm, 4.22 min (Method E); MS (ESI) m/z 402.2293 (M+H).

4.4.11. tert-Butyl {1-[(3E)-4-{5-cyano-4-[(4-methyl-1H-indol-5-

yl)amino]pyridin-3-yl}but-3-en-1-yl]piperidin-4-yl}carbamate

(10b)

Compound 9b was prepared from 1 and tert-butyl piperidin-4-

ylcarbamate using the procedure described for 4b and puriﬁed by

HPLC to give 122 mg (39% yield) of 10b as its TFA salt; ¹H NMR

(DMSO-d₆) d 11.24 (1H, s), 9.39 (1H, br s), 9.21 (1H, br s), 8.54 (1H,

8.38 (1H, s), 7.40 (1H, t, J = 2.8 Hz), 7.28 (1H, d, J = 8.4 Hz), 7.10

(1H, t, J = 5.6 Hz), 6.94 (1H, d, J = 8.4 Hz), 6.67 (1H, d, J = 15.6 Hz),

6.55 (1H, s), 6.23 (1H, dt, J = 6.8, 15.6 Hz), 3.51 (2H, d, J = 11.2 Hz)

3.23–2.93 (5H, m), 2.62–2.45 (2H, m), 2.32 (3H, s), 1.96 (2H, d,

J = 12 Hz), 1.60 (2H, q, J = 12.3 Hz), 1.39 (9H, s); MS 501.4 (M+H).

4.4.16. 4-[(4-Methyl-1H-indol-5-yl)amino]-5-[(1E)-3-(4-methyl-

piperazin-1-yl)prop-1-en-1-yl]nicotinonitrile (12a)

Compound 12a was prepared from 1 and 1-methylpiperazine

using the procedure described for 4a giving 54 mg (44% yield) as

its HCl salt; ¹H NMR (DMSO-d₆) d 11.30 (1H, s), 10.08 (1H, br s),

8.74 (1H, s), 8.54 (1H, s) 7.41 (1H, t, J = 2.8 Hz), 7.29 (1H, d,

J = 8.4 Hz), 7.08 (1H, d, J = 14.8 Hz), 6.98 (1H, d, J = 8.4 Hz), 6.56

(1H, s), 6.42 (1H, dt, J = 7.4, 15.6 Hz), 3.95–3.27 (10H, m), 2.83

(3H, s), 2.34 (3H, s); HPLC 100% at 215 nm, 3.90 min (Method E);

MS (ESI) m/z 387.2297 (M+H).

4.4.12. tert-Butyl {1-[(4E)-5-{5-cyano-4-[(4-methyl-1H-indol-5-

yl)amino]pyridin-3-yl}pent-4-en-1-yl]piperidin-4-

yl}carbamate (10c)

4.4.17. 4-[(4-Methyl-1H-indol-5-yl)amino]-5-[(1E)-3-(4-methyl-

piperazin-1-yl)but-1-en-1-yl]nicotinonitrile (12b)

Compound 10c was prepared from 1 and tert-butyl piperidin-4-

ylcarbamate using the procedure described for 4c and puriﬁed by

silica gel chromatography using a MeOH/DCM gradient to give

114 mg (29% yield) of 10c as a solid. ¹H NMR (DMSO-d₆) d 11.12

(1H, s), 8.53 (1H, s) 8.29 (1H, s), 8.21 (1H, s), 7.34 (1H, t,

J = 2.6 Hz), 7.22 (1H, d, J = 8.4 Hz), 6.89 (1H, d, J = 8.0 Hz), 6.75

(1H, br s), 6.59 (1H, d, J = 15.2 Hz), 6.50 (1H, s), 6.23 (1H, dt,

J = 6.4, 15.6 Hz), 3.36–3.13 (3H, m), 2.85–2.72 (2H, m), 2.28 (3H,

s), 2.16 (1H, q, J = 7.2 Hz), 1.89–1.52 (6H, m), 1.37 (9H, s); MS

515.5 (M+H).

A mixture of 1 (100 mg, 0.27 mmol), 1-methylpiperazine (80 mg,

0.80 mmol), (E)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-

but-3-enyl 4-methylbenzenesulfonate (5) (187 mg, 0.53 mmol), ce-

sium carbonate (173 mg, 0.53 mmol) and Pd(PPh₃)₄(16 mg,

0.014 mmol) in 3 mL of DMSO was heated at 70 °C for 16 h, cooled

to room temperature, and ﬁltered. The ﬁltrate was concentrated

and puriﬁed by HPLC to give 12b which was converted to its HCl

by addition of HCl/dioxane (13 mg, 10% yield); ¹H NMR (DMSO-d₆)

d 11.29 (1H, s), 9.99 (1H, br s), 8.69 (1H, s) 8.51 (1H, s), 7.41 (1H, t,

J = 2.8 Hz), 7.29 (1H, d, J = 8.4 Hz), 6.97 (1H, d, J = 8.4 Hz), 6.73 (1H,

d, J = 12.4 Hz), 6.55 (1H, s), 6.35 (1H, dt, J = 7.2, 15.6 Hz), 3.65–3.20

(10H, m), 2.82 (3H, s), 2.72–2.62 (2H, m), 2.33 (3H, s); HPLC 97.7%

at 215 nm, 5.1 min (Method B); MS (ESI) m/z 401.2450 (M+H).

4.4.13. 4-[(4-Methyl-1H-indol-5-yl)amino]-5-[(1E)-3-morpholin-

4-ylprop-1-en-1-yl]nicotinonitrile (11a)

Compound 11a was prepared from 1 and morpholine using the

procedure described for 4a and puriﬁed by HPLC. The HCl salt was

generated to give 33 mg (30% yield) of 11a as a solid; ¹H NMR

(DMSO-d₆) d 11.81 (1H, s), 11.34 (1H, s), 10.30 (1H, s), 8.79 (1H,

s), 8.57 (1H, s), 7.41 (1H, t, J = 2.6 Hz), 7.30 (1H, d, J = 8.8 Hz),

7.11 (1H, d, J = 15.6 Hz), 6.99 (1H, d, J = 8.8 Hz), 6.57 (1H, s), 6.48

(1H, dt, J = 7.2, 15.6 Hz), 4.02–3.78 (6H, m), 3.50–3.42 (2H, m),

3.17–3.07 (2H, m), 2.35 (3H, s); HPLC 97.4% at 215 nm, 3.95 min

(Method E); MS (ESI) m/z 394.1980 (M+H).

4.4.18. 4-[(4-Methyl-1H-indol-5-yl)amino]-5-[(1E)-5-(4-methyl-

piperazin-1-yl)pent-1-en-1-yl]nicotinonitrile (12c)

Compound 12c was prepared from 1 and 1-methylpiperazine

using the procedure described for 4c giving 13 mg (10% yield) as

its TFA salt; ¹H NMR (DMSO-d₆) d 11.26 (1H, s), 9.66 (1H, s), 8.61

(1H, s), 8.39 (1H, s), 7.40 (1H, t, J = 2.8 Hz), 7.29 (1H, d, J = 8.4 Hz),

6.96 (1H, d, J = 8.4 Hz), 6.64 (1H, d, J = 15.2 Hz), 6.55 (1H, s), 6.35

(1H, dt, J = 6.8, 15.2 Hz), 3.55–2.92 (10H, m), 2.80 (3H, s), 2.32 (3H,

s), 2.26 (2H, q, J = 6.8 Hz), 1.81–1.76 (2H, m); HPLC 92.9% at

215 nm, 5.9 min (Method B); MS (ESI) m/z 415.2608 (M+H).

4.4.14. 4-[(4-Methyl-1H-indol-5-yl)amino]-5-[(1E)-4-morpholin-

4-ylbut-1-en-1-yl]nicotinonitrile (11b)

A mixture of 1 (150 mg, 0.40 mmol), morpholine (104 mg,

1.2 mmol), (E)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)but-

3-enyl 4-methylbenzenesulfonate (5) (281 mg, 0.80 mmol), cesium

carbonate (261 mg, 0.80 mmol) and Pd(PPh₃)₄(23 mg, 0.02 mmol)

in 4 mL of DMSO was heated at 70 °C for 16 h, cooled to room temper-

ature, diluted with EtOAc and washed with water. The crude extracts

were puriﬁed by silica gel chromatography with a MeOH/DCM gradi-

ent to give 11b. The product was treated with excess HCl/dioxane giv-

ing 53 mg of the HCl salt of 11b (31% yield); ¹H NMR (DMSO-d₆) d

11.36 (1H, br s), 11.31 (1H, s), 10.07 (1H, s), 8.71 (1H, s), 8.57 (1H,

s), 7.41 (1H, t, J = 2.6 Hz), 7.29 (1H, d, J = 8.4 Hz), 6.98 (1H, d,

J = 8.4 Hz), 6.75 (1H, d, J = 15.2 Hz), 6.57 (1H, s), 6.37 (1H, dt, J = 6.8,

15.6 Hz), 3.99–3.82 (6H, m), 3.29–3.23 (2H, m), 3.25–3.01 (2H, m),

2.72 (2H, q, J = 6.8 Hz), 2.33 (3H, s); HPLC 100% at 215 nm, 4.14 min

(Method E); MS (ESI) m/z 386.2 (MꢂH).

4.5. General procedure for the synthesis of (13a–c) and (14a–c)

Boc-protected amines 9a–c and 10a–c were dissolved in 10%

TFA/DCM. After stirring at room temperature for 1–3 h, the sol-

vents were removed and the residue was puriﬁed by HPLC or ﬂash

chromatography.

4.5.1. 4-[(4-Methyl-1H-indol-5-yl)amino]-5-[(1E)-3-piperazin-

1-ylprop-1-en-1-yl]nicotinonitrile (13a)

33.6 mg (27% yield); ¹H NMR (DMSO-d₆) d 11.13 (1H, s), 8.58

(1H, s), 8.36 (1H, s), 8.24 (1H, s), 7.34 (1H, t, J = 2.6 Hz), 7.22 (1H,

d, J = 8.8 Hz), 6.89 (1H, d, J = 8.4 Hz), 6.50 (1H, s), 6.20 (1H, dt,

J = 6.8, 15.2 Hz), 3.02 (2H, d, J = 6.4 Hz), 2.68 (4H, t, J = 4.8 Hz),

2.42–2.25 (4H, m), 2.29 (3H, s); HPLC 98.7% at 215 nm, 3.7 min

(Method A); MS (ESI) m/z 373.2136 (M+H).

4.4.15. 4-[(4-Methyl-1H-indol-5-yl)amino]-5-[(1E)-5-morpholin-

4-ylpent-1-en-1-yl]nicotinonitrile (11c)

Compound 11c was prepared from 1 and morpholine using the

procedure described for 4c giving 86 mg (58% yield) as its TFA salt;

4.5.2. 4-[(4-Methyl-1H-indol-5-yl)amino]-5-[(1E)-4-piperazin-

1-ylbut-1-en-1-yl]nicotinonitrile (13b)

28 mg (41% yield) as its TFA salt; ¹H NMR (DMSO-d₆) d 11.25

(1H, s), 9.51 (1H, br s), 9.01 (1H, br s), 8.58 (1H, s), 8.37 (1H, s),

L. N. Tumey et al. / Bioorg. Med. Chem. 17 (2009) 7933–7948

7945

7.40 (1H, t, J = 2.8 Hz), 7.28 (1H, d, J = 8.4 Hz), 6.95 (1H, d,

4.5.9. 5-[(1E)-5-(4-Hydroxypiperidin-1-yl)pent-1-en-1-yl]-4-

J = 8.4 Hz), 6.69 (1H, d, J = 15.6 Hz), 6.28 (1H, dt, J = 6.6, 15.6 Hz),

3.36–3.05 (10H, m), 2.59–2.51 (2H, m), 2.32 (3H, s); HPLC 98.3%

at 215 nm, 2.8 min (Method A); MS (ESI) m/z 387.2298 (M+H).

[(4-methyl-1H-indol-5-yl)amino]nicotinonitrile (15c)

Compound 15c was prepared from 1 and piperidin-4-ol using

the procedure described for 4c and puriﬁed by HPLC to give

101 mg (71% yield) TFA salt; ¹H NMR (DMSO-d₆) d 11.27 (1H, s),

9.71 (1H, d, J = 7.6 Hz), 9.47 (1H, br s), 8.62 (1H, d, J = 2.0 Hz),

8.41 (1H, d, J = 3.6 Hz), 7.40 (1H, t, J = 2.8 Hz), 7.28 (1H, d,

J = 8.4 Hz), 6.95 (1H, dd, J = 2.0, 8.8 Hz), 6.65 (1H, d, J = 14.4 Hz),

6.55 (1H, s), 6.38–6.29 (1H, m), 3.66–2.89 (7H, m), 2.32 (3H, s),

2.27–1.52 (8H, m); HPLC 95.5% at 215 nm, 4.08 min (Method E);

MS (ESI) m/z 416.2452 (M+H).

4.5.3. 4-[(4-Methyl-1H-indol-5-yl)amino]-5-[(1E)-5-piperazin-

1-ylpent-1-en-1-yl]nicotinonitrile (13c)

71 mg (60% yield) as its TFA salt; ¹H NMR (DMSO-d₆) d 11.26

(1H, s), 9.67 (1H, s), 9.22 (1H, s), 8.62 (1H, s), 8.40 (1H, s), 7.40

(1H, t, J = 2.6 Hz), 7.28 (1H, d, J = 8.4 Hz), 6.96 (1H, d, J = 8.4 Hz),

6.65 (1H, d, J = 15.6 Hz), 6.56 (1H, s), 6.34 (1H, dt, J = 6.8,

15.6 Hz), 3.45–3.10 (10H, m), 2.32 (3H, s), 2.28 (2H, q, J = 6.8 Hz),

1.86–1.76 (2H, m); HPLC 100% at 215 nm, 4.01 min (Method E);

MS (ESI) m/z 401.2456 (M+H).

4.5.10. 5-{(1E)-4-[4-(Methylamino)piperidin-1-yl]but-1-en-1-

yl}-4-[(4-methyl-1H-indol-5-yl)amino]nicotinonitrile (16)

tert-Butyl methyl(piperidin-4-yl)carbamate (113 mg; 0.53 mmol)

and (E)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)but-3-enyl

4-methylbenzenesulfonate 5 (186 mg, 0.53 mmol) were stirred in

1 mL of DMSO. After 16 h at room temperature, 1 (100 mg,

0.27 mmol), cesium carbonate (173 mg, 0.53 mmol) and Pd(PPh₃)₄

(16 mg, 0.014 mmol) were added and the reaction was heated at

70 °C for 16 h. The mixture was cooled to room temperature, diluted

with EtOAc and washed with water to give the Boc-protected inter-

mediate. The crude organic extracts were concentrated and treated

with 10% triﬂuoroacetic acid/DCM for 2 h at room temperature.

Concentration and puriﬁcation by HPLC gave 10 mg (5.7% yield)

of 16 as its TFA salt. HPLC 100% at 215 nm, 1.26 min (Method D);

MS (ESI) m/z 401.2 (M+H).

4.5.4. 5-[(1E)-3-(4-Aminopiperidin-1-yl)prop-1-en-1-yl]-4-[(4-

methyl-1H-indol-5-yl)amino]nicotinonitrile (14a)

19 mg (37% yield); ¹H NMR (DMSO-d₆) d 11.13 (1H, s), 8.58 (1H,

s), 8.36 (1H, s), 8.23 (1H, s), 7.34 (1H, t, J = 2.6 Hz), 7.22 (1H, d,

J = 8.4 Hz), 6.89 (1H, d, J = 8.4 Hz), 6.72 (1H, d, J = 15.6 Hz), 6.50

(1H, s), 6.22 (1H, dt, J = 6.8, 15.6 Hz), 3.52–3.38 (1H, m), 3.04 (2H,

d, J = 6.0 Hz), 2.77 (2H, d, J = 11.6 Hz), 2.28 (3H, s), 1.91 (2H, t,

J = 10.2 Hz), 1.66 (2H, d, J = 8.0 Hz), 1.28–1.18 (2H, m); HPLC

96.2% at 215 nm, 2.9 min (Method A); MS (ESI) m/z 387.2293

(M+H).

4.5.5. 5-[(1E)-4-(4-Aminopiperidin-1-yl)but-1-en-1-yl]-4-[(4-

methyl-1H-indol-5-yl)amino]nicotinonitrile (14b)

4.5.11. 5-{(1E)-4-[4-(Dimethylamino)piperidin-1-yl]but-1-en-

1-yl}-4-[(4-methyl-1H-indol-5-yl)amino]nicotinonitrile (17)

Compound 15b was prepared from 1 and N,N-dimethylpiperi-

din-4-amine using the procedure described for 4b giving 57 mg

(32% yield) as its TFA salt; HPLC 97.5% at 215 nm, 3.56 min (Meth-

od E); MS (ESI) m/z 429.3 (M+H).

53 mg (46% yield) as its TFA salt; ¹H NMR (DMSO-d₆) d 11.25

(1H, s), 9.81 (1H, bsw), 9.43 (1H, br s), 8.56 (1H, s), 8.38 (1H, s),

8.14 (3H, br s), 7.40 (1H, t, J = 2.8 Hz), 7.28 (1H, d, J = 8.4 Hz),

6.95 (1H, d, J = 84 Hz), 6.68 (1H, d, J = 15.6 Hz), 6.55 (1H, s), 6.25

(1H, dt, J = 6.8, 15.6 Hz), 3.60 (2H, d, J = 12.4 Hz), 3.35–3.16 (3H,

m), 3.00 (2H, q, J = 8.4 Hz), 2.60 (2H, q, J = 6.8 Hz), 2.32 (3H, s),

2.12 (2H, d, J = 12.4 Hz), 1.76 (2H, q, J = 11.6 Hz); HPLC 94.7% at

215 nm, 2.3 min (Method A); MS (ESI) m/z 401.2452 (M+H).

4.5.12. 5-{(1E)-4-[4-(Aminomethyl)piperidin-1-yl]but-1-en-1-

yl}-4-[(4-methyl-1H-indol-5-yl)amino]nicotinonitrile (18)

Compound 18 was prepared from 1 and tert-butylpiperidin-4-

ylmethylcarbamate using the procedure described for 16 to give

53 mg (31% yield) as its TFA salt; HPLC 100% at 215 nm, 1.09 min

(Method C); MS (ESI) m/z 415.3 (M+H).

4.5.6. 5-[(1E)-5-(4-Aminopiperidin-1-yl)pent-1-en-1-yl]-4-[(4-

methyl-1H-indol-5-yl)amino]nicotinonitrile (14c)

84 mg (84% yield) as its TFA salt; ¹H NMR (DMSO-d₆) d 11.25

(1H, s), 9.88 (1H, br s), 9.56 (1H, s), 8.58 (1H, s), 8.39 (1H, s), 8.19

(3H, br s), 7.39 (1H, t, J = 2.8 Hz), 7.28 (1H, d, J = 8.4 Hz), 6.94

(1H, d, J = 8.4 Hz), 6.65 (1H, d, J = 16 Hz), 6.55 (1H, s,), 6.31 (1H,

dt, J = 6.6, 15.6 Hz), 3.55 (2H, d, J = 12 Hz), 3.30–3.27 (1H, m),

3.15–2.97 (4H, m), 2.32 (3H, s), 2.26 (2H, q, J = 6.7 Hz), 2.09 (2H,

d, J = 6.0 Hz), 1.89–1.71 (4H, m); HPLC 100% at 215 nm, 3.95 min

(Method E); MS (ESI) m/z 415.2610 (M+H).

4.5.13. 5-{(1E)-4-[(3R)-3-Aminopiperidin-1-yl]but-1-en-1-yl}-

4-[(4-methyl-1H-indol-5-yl)amino]nicotinonitrile (19)

Compound 19 was prepared from 1 and (R) tert-butyl piperidin-

3-ylcarbamate using the procedure described for 16 to give 65 mg

(38% yield) as its TFA salt; ¹H NMR (DMSO-d₆) d 11.13 (1H, s), 8.52

(1H, br s), 8.27 (1H, s), 8.21 (1H, s), 7.35 (1H, t, J = 2.8 Hz), 7.22 (1H,

d, J = 8.4 Hz), 6.88 (1H, d, J = 8.4 Hz), 6.58 (1H, d, J = 15.2 Hz), 6.50

(1H, s), 6.22–6.15 (1H, m), 2.73–2.27 (7H, m), 2.28 (3H, s), 1.97–

0.99 (6H, m); HPLC 100% at 215 nm, 1.19 min (Method C); MS

(ESI) m/z 401.2452 (M+H).

4.5.7. 5-[(1E)-3-(4-Hydroxypiperidin-1-yl)prop-1-en-1-yl]-4-

[(4-methyl-1H-indol-5-yl)amino]nicotinonitrile (15a)

Compound 15a was prepared from 1 and piperidine-4-ol using

the procedure described for 4a giving 19 mg (18% yield) as its HCl

salt; ¹H NMR (DMSO-d₆) d 11.27 (1H, s), 10.61 (1H, br s), 9.94 (1H,

br s), 8.70 (1H, s), 8.53 (1H, d, J = 7.6 Hz), 7.40 (1H, t, J = 2.8 Hz),

7.29 (1H, d, J = 7.6 Hz), 7.04 (1H, dd, J = 4.8, 10 Hz), 6.97 (1H, dd,

J = 2.8, 8.8 Hz), 6.56 (1H, s), 6.46–6.39 (1H, m), 3.96–2.95 (7H, m),

2.34 (3H, s), 2.01–1.66 (4H, m); HPLC 100% at 215 nm, 3.86 min

(Method E); MS (ESI) m/z 388.2132 (M+H).

4.5.14. 5-{(1E)-4-[(3S)-3-Aminopiperidin-1-yl]but-1-en-1-yl}-4-

[(4-methyl-1H-indol-5-yl)amino]nicotinonitrile (20)

Compound 20 was prepared from 1 and (S) tert-butyl piperidin-

3-ylcarbamate using the procedure described for 16 to give 58 mg

(34% yield) as its TFA salt; HPLC 100% at 215 nm, 3.58 (Method E);

MS (ESI) m/z 401.2 (M+H).

4.5.8. 5-[(1E)-4-(4-Hydroxypiperidin-1-yl)but-1-en-1-yl]-4-[(4-

methyl-1H-indol-5-yl)amino]nicotinonitrile (15b)

Compound 15b was prepared from 1 and piperidin-4-ol using

the procedure described for 4b giving 55 mg (40% yield) as its

TFA salt; HPLC 100% at 215 nm, 1.25 min (Method C); MS (ESI)

m/z 402.2 (M+H).

4.5.15. 5-[(1E)-4-(3-Aminoazetidin-1-yl)but-1-en-1-yl]-4-[(4-

methyl-1H-indol-5-yl)amino]nicotinonitrile (21)

Compound 21 was prepared from 1 and tert-butyl azetidin-3-

ylcarbamate using the procedure described for 16 to give 41 mg

(25% yield) as its TFA salt; HPLC 100% at 215 nm, 1.25 min (Method

D); MS (ESI) m/z 373.3 (M+H).

7946

L. N. Tumey et al. / Bioorg. Med. Chem. 17 (2009) 7933–7948

4.5.16. 5-{(1E)-4-[(3R)-3-Aminopyrrolidin-1-yl]but-1-en-1-yl}-

4-[(4-methyl-1H-indol-5-yl)amino]nicotinonitrile (22)

Compound 22 was prepared from 1 and (R)-tert-butyl pyrroli-

din-3-ylcarbamate using the procedure described for 16 to give

49 mg (30% yield) as its TFA salt; HPLC 100% at 215 nm, 1.04 min

(Method D); MS (ESI) m/z 387.2 (M+H).

4.5.23. 5-[(1E)-4-(4-Methoxypiperidin-1-yl)but-1-en-1-yl]-4-

[(4-methyl-1H-indol-5-yl)amino]nicotinonitrile (29)

Compound 29 was prepared from 1 and 4-methoxypiperidine

using the procedure described for 4b and puriﬁed by HPLC to give

45 mg (40% yield); ¹H NMR (DMSO-d₆) d 11.12 (1H, s), 8.52 (1H, s),

8.27 (1H, s), 8.81 (1H, s), 7.34 (1H, t, J = 2.8 Hz), 7.22 (1H, d,

J = 8.8 Hz), 6.88 (1H, d, J = 8.8 Hz), 6.59 (1H, d, J = 15.2 Hz), 6.50

(1H, s), 6.17 (1H, dt, J = 6.6, 15.6 Hz), 3.21 (3H, s), 3.13 (1H, sept.,

J = 4.4 Hz), 2.69–2.66 (2H, m), 2.38–2.25 (4H, m), 2.28 (3H, s),

2.05 (2H, t, J = 9.2 Hz), 1.80 (2H, d, J = 9.6 Hz), 1.41–1.33 (2H, m);

HPLC 97.6% at 215 nm, 4.8 min (Method A); MS (ESI) m/z

416.2446 (M+H).

4.5.17. 5-{(1E)-4-[(3S)-3-Aminopyrrolidin-1-yl]but-1-en-1-yl}-

4-[(4-methyl-1H-indol-5-yl)amino]nicotinonitrile (23)

Compound 23 was prepared from 1 and (S)-tert-butyl pyrroli-

din-3-ylcarbamate using the procedure described for 16 to give

47 mg (28% yield) TFA salt; HPLC 100% at 215 nm, 1.05 min (Meth-

od D); MS (ESI) m/z 387.2 (M+H).

4.5.24. 5-{(1E)-4-[4-(Hydroxymethyl)piperidin-1-yl]but-1-en-

1-yl}-4-[(4-methyl-1H-indol-5-yl)amino]nicotinonitrile (30)

Compound 30 was prepared from 1 and piperidin-4-ylmethanol

using the procedure described for 4b giving 64 mg (57% yield); ¹H

NMR (DMSO-d₆) d 11.12 (1H, s), 8.51 (1H, s), 8.26 (1H, s), 8.21 (1H,

s), 7.34 (1H, t, J = 2.8 Hz), 7.21 (1H, d, J = 8.0 Hz), 6.87 (1H, d,

J = 8.0 Hz), 6.59 (1H, d, J = 15.6 Hz), 6.50 (1H, s), 6.18 (1H, dt,

J = 6.4, 15.6 Hz), 4.37 (1H, t, J = 5.4 Hz), 3.23–3.17 (3H, m), 2.85

(2H, d, J = 11.2 Hz), 2.40–2.29 (4H, m), 2.28 (3H, s), 1.84 (2H, dt,

J = 2.4, 11.6 Hz), 1.60 (2H, d, J = 12 Hz), 1.07 (2H, dq, J = 3.2,

12 Hz); HPLC 97.8% at 215 nm, 3.97 min (Method E); MS (ESI) m/z

416.2450 (M+H).

4.5.18. N-{1-[(3E)-4-{5-Cyano-4-[(4-methyl-1H-indol-5-yl)amino]-

pyridin-3-yl}but-3-en-1-yl]piperidin-4-yl}methanesulfonamide

(24)

A mixture of 14b (75 mg, 0.10 mmol), methanesulfonyl chlo-

ride (15 mg, 0.13 mmol) and triethylamine (41 mg, 0.40 mmol)

in 1 mL of DMF were stirred at room temperature for 1 h. The

reaction mixture was ﬁltered and the ﬁltrate was puriﬁed by HPLC

to give 24 as its TFA salt; ¹H NMR (DMSO-d₆) d 11.25 (1H, s), 9.46

(1H, br s), 9.38 (1H, br s), 8.56 (1H, s), 8.38 (1H, s), 7.41–7.37

(2H, m), 7.28 (1H, d, J = 8.8 Hz), 6.94 (1H, d, J = 8.8 Hz), 6.67

(1H, d, J = 15.2 Hz), 6.55 (1H, s), 6.24 (1H, dt, J = 6.8, 16 Hz), 3.68

(2H, d, J = 4 Hz), 3.55–3.38 (2H, m), 3.27–3.12 (3H, m), 2.96 (3H,

s), 2.62–2.50 (2H, m), 2.32 (3H, s), 2.08–1.62 (4H, s); HPLC

96.7% at 216 nm, 4.4 min (Method A); MS (ESI) m/z 479.2223

(M+H).

4.5.25. 4-[(4-Methyl-1H-indol-5-yl)amino]-5-[(1E)-4-(4-phenyl-

piperidin-1-yl)but-1-en-1-yl]nicotinonitrile (31)

Compound 31 was prepared from 1 and 4-phenylpiperidine

using the procedure described for 4b and puriﬁed by HPLC to give

58 mg (37% yield) as its TFA salt; HPLC 100% at 215 nm, 1.56 min

(Method C); MS (ESI) m/z 462.3 (M+H).

4.5.19. N-{1-[(3E)-4-{5-Cyano-4-[(4-methyl-1H-indol-5-yl)amino]-

pyridin-3-yl}but-3-en-1-yl]piperidin-4-yl}ethanesulfonamide (25)

Compound 25 was prepared from 14b and ethanesulfonyl chlo-

ride using the procedure described for 24 to give 6.3 mg (8.3%

yield) as its TFA salt; HPLC 98.0% at 215 nm, 4.27 min (Method

E); MS (ESI) m/z 493.4 (M+H).

4.5.26. 5-{(1E)-4-[(3S)-3-Hydroxypyrrolidin-1-yl]but-1-en-1-

yl}-4-[(4-methyl-1H-indol-5-yl)amino]nicotinonitrile (32)

Compound 32 was prepared from 1 and (S)-pyrrolidin-3-ol

using the procedure described for 4b and puriﬁed by HPLC to give

42 mg (40% yield); ¹H NMR (DMSO-d₆) d 11.12 (1H, s), 8.53 (1H, s),

8.27 (1H, s), 8.21 (1H, s), 7.34 (1H, t, J = 2.8 Hz), 7.22 (1H, d,

J = 8.4 Hz), 6.88 (1H, d, J = 8.4 Hz), 6.60 (1H, d, J = 15.6 Hz), 6.51

(1H, s), 6.20 (1H, dt, J = 6.8, 16 Hz), 4.64 (1H, d, J = 4.4 Hz), 4.15

(1H, oct., J = 3.5 Hz), 2.73–2.68 (1H, m), 2.57–2.28 (6H, m), 2.28

(3H, s), 1.98–1.89 (1H, m), 1.54–1.47 (1H, m), 1.17–1.07 (1H, m);

HPLC 93% at 215 nm, 3.8 min (Method A); MS (ESI) m/z 388.2137

(M+H).

4.5.20. N-{1-[(3E)-4-{5-Cyano-4-[(4-methyl-1H-indol-5-yl)amino]-

pyridin-3-yl}but-3-en-1-yl]piperidin-4-yl}propane-2-sulfonamide

(26)

Compound 26 was prepared from 14b and propane-2-sulfonyl

chloride using the procedure described for 24 to give 14 mg (19%

yield) as its TFA salt; HPLC 97.9% at 215 nm, 1.45 min (Method

C); MS (ESI) m/z 507.2 (M+H).

4.5.21. N-{1-[(3E)-4-{5-Cyano-4-[(4-methyl-1H-indol-5-yl)amino]-

pyridin-3-yl}but-3-en-1-yl]piperidin-4-yl}benzenesulfonamide

(27)

Compound 27 was prepared from 14b and benzenesulfonyl

chloride using the procedure described for 24 to give 6.2 mg (8%

yield) as its TFA salt; HPLC 97.8% at 215 nm, 4.88 min (Method

E); MS (ESI) m/z 541.4 (M+H).

4.5.27. 5-{(1E)-4-[(3R)-3-Hydroxypyrrolidin-1-yl]but-1-en-1-

yl}-4-[(4-methyl-1H-indol-5-yl)amino]nicotinonitrile (33)

Compound 33 was prepared from 1 and (R)-pyrrolidin-3-ol

using the procedure described for 4b and puriﬁed by HPLC to give

37 mg (35% yield); ¹H NMR (DMSO-d₆) d 11.12 (1H, s), 8.53 (1H, s),

8.27 (1H, s), 8.21 (1H, s), 7.34 (1H, t, J = 2.8 Hz), 7.22 (1H, d,

J = 8.4 Hz), 6.88 (1H, d, J = 8.4 Hz), 6.60 (1H, d, J = 15.6 Hz), 6.51

(1H, s), 6.20 (1H, dt, J = 6.8, 16 Hz), 4.64 (1H, d, J = 4.4 Hz), 4.15

(1H, oct., J = 3.5 Hz), 2.73–2.68 (1H, m), 2.57–2.28 (6H, m), 2.28

(3H, s), 1.98–1.89 (1H, m), 1.54–1.47 (1H, m), 1.17–1.07 (1H, m);

HPLC 93.6% at 215 nm, 3.89 min (Method E); MS (ESI) m/z

388.2136 (M+H).

4.5.22. N-{1-[(3E)-4-{5-Cyano-4-[(4-methyl-1H-indol-5-yl)amino]-

pyridin-3-yl}but-3-en-1-yl]piperidin-4-yl}acetamide (28)

Compound 28 was prepared from 14b and acetyl chloride using

the procedure described for 24 to give 48 mg (55% yield) TFA salt;

¹H NMR (DMSO-d₆) d 11.26 (1H, s), 9.61 (1H, br s), 9.43 (1H, br s),

8.61 (1H, s), 8.39 (1H, s), 7.98 (1H, d, J = 7.6 Hz), 7.41 (1H, t,

J = 2.6 Hz), 7.28 (1H, d, J = 8.0 Hz), 6.96 (1H, d, J = 8.0 Hz), 6.66

(1H, d, J = 14.8 Hz), 6.56 (1H, s), 6.26 (1H, dt, J = 6.6, 15.6 Hz),

3.82–3.75 (1H, m), 3.53 (2H, d, J = 11.6 Hz), 3.25–2.98 (4H, m),

2.63–2.49 (2H, m), 2.32 (3H, s), 1.96 (2H, d, J = 11.6 Hz), 1.81 (3H,

s), 1.58 (2H, q, J = 12.4 Hz); HPLC 94.9% at 215 nm, 4.1 min (Method

A); MS (ESI) m/z 443.2558 (M+H).

4.5.28. 5-{(1E)-4-[(3S)-3-Hydroxypiperidin-1-yl]but-1-en-1-yl}-

4-[(4-methyl-1H-indol-5-yl)amino]nicotinonitrile (34)

Compound 34 was prepared from 1 and (S)-piperidin-3-ol using

the procedure described for 4b and puriﬁed by HPLC to give 8 mg

(5.7% yield); HPLC 100% at 215 nm, 1.33 min (Method C); MS (ESI)

m/z 402.2 (M+H).

L. N. Tumey et al. / Bioorg. Med. Chem. 17 (2009) 7933–7948

7947

4.5.29. 5-{(1E)-4-[(3R)-3-Hydroxypiperidin-1-yl]but-1-en-1-yl}-

4.5.35. 5-{(1E)-4-[4-(Ethylsulfonyl)piperazin-1-yl]but-1-en-1-

4-[(4-methyl-1H-indol-5-yl)amino]nicotinonitrile (35)

Compound 35 was prepared from 1 and (R)-piperidin-3-ol using

the procedure described for 4b and puriﬁed by HPLC to give 7 mg

(5.0% yield); HPLC 100% at 215 nm, 1.33 min (Method C); MS (ESI)

m/z 402.2 (M+H).

yl}-4-[(4-methyl-1H-indol-5-yl)amino]nicotinonitrile (41)

Compound 41 was prepared from 13b and ethanesulfonyl chlo-

ride using the procedure described for 39 and puriﬁed by HPLC to

give 30 mg (35% yield) as its TFA salt; ¹H NMR (DMSO-d₆) d 11.24

(1H, s), 9.95 (1H, br s), 9.47 (1H, br s), 8.57 (1H, s), 8.39 (1H, s), 7.40

(1H, t, J = 3.0 Hz), 7.28 (1H, d, J = 8.8 Hz), 6.95 (1H, d, J = 8.8 Hz),

6.70 (1H, d, J = 15.6 Hz), 6.55 (1H, s), 6.25 (1H, dt, J = 6.6,

15.6 Hz), 3.83–3.52 (4H, m), 3.35–3.05 (6H, m), 3.20 (2H, q,

J = 7.3 Hz), 2.62 (2H, q, J = 6.9 Hz), 2.32 (3H, s), 1.32 (3H, t,

J = 7.4 Hz); HPLC 100% at 215 nm, 4.34 min (Method E); MS (ESI)

m/z 479.2227 (M+H).

4.5.30. 5-[(1E)-4-(1,4-Diazepan-1-yl)but-1-en-1-yl]-4-[(4-methyl-

1H-indol-5-yl)amino]nicotinonitrile (36)

Compound 36 was prepared from 1 and tert-butyl 1,4-diaze-

pane-1-carboxylate using the procedure described for 16 to give

66 mg (39% yield) TFA salt; HPLC 100% at 215 nm, 1.06 min (Meth-

od D); MS (ESI) m/z 401.2 (M+H).

4.5.36. 5-{(1E)-4-[4-(Isopropylsulfonyl)piperazin-1-yl]but-1-

en-1-yl}-4-[(4-methyl-1H-indol-5-yl)amino]nicotinonitrile (42)

Compound 42 was prepared from 13b and propane-2-sulfonyl

chloride using the procedure described for 39 and puriﬁed by HPLC

to give 10 mg (12% yield) as its TFA salt; HPLC 94.2% at 215 nm,

1.42 min (Method C); MS (ESI) m/z 493.2 (M+H).

4.5.31. 4-[(4-Methyl-1H-indol-5-yl)amino]-5-[(1E)-4-(4-phenyl-

piperazine-1-yl)but-1-en-1-yl]nicotinonitrile (37)

Compound 37 prepared from 1 and 1-phenylpiperazine using

the procedure described for 11b to give 27 mg (22% yield); ¹H

NMR (DMSO-d₆) d 11.13 (1H, s), 8.54 (1H, s), 8.28 (1H, s), 8.22

(1H, s), 7.35 (1H, t, J = 2.6 Hz), 7.23–7.17 (3H, m), 6.91–6.88

(3H, m), 6.76 (1H, t, J = 7.4 Hz), 6.64 (1H, d, J = 15.6 Hz), 6.50

(1H, s), 6.21 (1H, dt, J = 6.6, 15.2 Hz), 3.11 (4H, t, J = 4.8 Hz),

2.53 (4H, t, J = 5.0 Hz), 2.49–2.34 (4H, m), 2.29 (3H, s); HPLC

98.9% at 215 nm, 6.9 min (Method A); MS (ESI) m/z 463.2612

(M+H).

4.5.37. 4-[(4-Methyl-1H-indol-5-yl)amino]-5-{(1E)-4-[4-(phenyl-

sulfonyl)piperazin-1-yl]but-1-en-1-yl}nicotinonitrile (43)

Compound 43 was prepared from 13b and benzenesulfonyl

chloride using the procedure described for 39 and puriﬁed by HPLC

to give 32 mg (42% yield) as its TFA salt; ¹H NMR (DMSO-d₆) d

11.24 (1H, s), 9.75 (1H, br s), 9.45 (br s, 1H), 8.56 (1H, s), 8.36

(1H, s), 7.83–7.70 (5H, m), 7.40 (1H, t, J = 2.8 Hz), 7.27 (1H, d,

J = 8.0 Hz), 6.92 (1H, d, J = 8.0 Hz), 6.67 (1H, d, J = 15.6 Hz), 6.56

(1H, s), 6.20 (1H, dt, J = 6.6, 15.2 Hz), 3.73–3.48 (6H, m), 3.35–

3.10 (4H, m), 2.54 (2H, q, J = 6.9 Hz), 2.28 (3H, s); HPLC 98.5% at

215 nm, 6.2 min (Method A); MS (ESI) m/z 527.2228 (M+H).

4.5.32. 5-[(1E)-4-(4-Acetylpiperazin-1-yl)but-1-en-1-yl]-4-[(4-

methyl-1H-indol-5-yl)amino]nicotinonitrile (38)

Compound 38 was prepared from 1 and 1-(piperazin-1-yl)etha-

none using the procedure described for 4b and puriﬁed by HPLC to

give 98 mg (67% yield); ¹H NMR (DMSO-d₆) d 11.24 (1H, s), 9.88

(1H, br s), 9.37 (1H, br s), 8.54 (1H, s), 8.39 (1H, s), 7.39 (1H, t,

J = 2.8 Hz), 7.28 (1H, d, J = 8.8 Hz), 6.94 (1H, d, J = 8.8 Hz), 6.70

(1H, d, J = 15.6 Hz), 6.55 (1H, s), 6.25 (1H, dt, J = 6.6, 16 Hz), 4.03–

4.00 (2H, m), 3.51–2.88 (10H, m), 2.63 (2H, q, J = 7.2 Hz), 2.31

(3H, s), 2.06 (3H, s); HPLC 95.4% at 215 nm, 4.2 min (Method A);

MS (ESI) m/z 429.2393 (M+H).

4.5.38. 5-[(1E)-4-(4-Aminopiperidin-1-yl)but-1-en-1-yl]-6-

methyl-4-[(4-methyl-1H-indol-5-yl)amino]nicotinonitrile (45)

A

mixture of tert-butyl piperidin-4-ylcarbamate (520 mg,

2.6 mmol) and (E)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-

yl)but-3-enyl 4-methylbenzenesulfonate (5) (454 mg, 1.29 mmol)

in 2.5 mL of DMSO was stirred for 60 h at room temperature. Com-

pound 44 (250 mg, 0.64 mmol), cesium carbonate (421 mg,

1.29 mmol) and Pd(PPh₃)₄(37 mg, 0.032 mmol) were added to

the same pot and the resulting suspension was heated at 70° for

16 h. After cooling to room temperature, the reaction was diluted

with EtOAc and washed with water. The organic extracts were con-

centrated and puriﬁed by silica gel chromatography with a MeOH/

DCM gradient to give 179 mg of the desired intermediate tert-butyl

{1-[(3E)-4-{5-cyano-2-methyl-4-[(4-methyl-1H-indol-5-yl)amino]-

pyridin-3-yl}but-3-en-1-yl]piperidin-4-yl}carbamate. A solution of

this intermediate (137 mg, 0.27 mmol) in 10% triﬂuoroacetic acid/

DCM was stirred for 1 h at room temperature. The reaction was

concentrated and the residue was puriﬁed by HPLC to give

140 mg (95% yield) of 45 as its TFA salt; ¹H NMR (DMSO-d₆) d

11.29 (1H, s), 10.18 (1H, br s), 9.52 (1H, s), 8.67 (1H, s), 8.22 (3H,

br s), 7.41 (1H, t, J = 2.8 Hz), 7.28 (1H, d, J = 8.4 Hz), 6.94 (1H, d,

J = 8.4 Hz), 6.55 (1H, s), 6.33 (1H, d, J = 16.4 Hz), 6.03 (1H, dt,

J = 6.6, 16 Hz), 3.60 (2H, d, J = 12.8 Hz), 3.37–3.21 (3H, m), 3.10–

2.08 (2H, m), 2.73–2.63 (2H, m), 2.47 (3H, s), 2.31 (3H, s), 2.12

(2H, d, J = 12.8 Hz), 1.80 (2H, q, J = 11.7 Hz); HPLC 100% at

215 nm, 3.54 min (Method E); MS (ESI) m/z 415.2610 (M+H).

4.5.33. 5-{(1E)-4-[4-(2,2-Dimethylpropanoyl)piperazin-1-yl]but-1-

en-1-yl}-4-[(4-methyl-1H-indol-5-yl)amino]nicotinonitrile (39)

A

solution of 13b (75 mg, 0.12 mmol), pivaloyl chloride

(19 mg, 0.16 mmol) and triethylamine (36 mg, 0.36 mmol) in

1 mL of DMF was stirred at room temperature for 1 h. The reac-

tion mixture was ﬁltered and the ﬁltrate was puriﬁed by HPLC

to give 25 mg (30% yield) of 39 as its TFA salt; ¹H NMR (DMSO-

d₆) d 11.24 (1H, s), 10.02 (1H, br s), 9.43 (1H, br s), 8.56 (1H, s),

8.40 (1H, s), 7.39 (1H, t, J = 2.8 Hz), 7.28 (1H, d, J = 8.4 Hz), 6.94

(1H, d, J = 8.4 Hz), 6.70 (1H, d, J = 15.6 Hz), 6.55 (1H, s), 6.26

(1H, dt, J = 6.6, 15.2 Hz), 4.49–4.36 (2H, m), 3.57–3.49 (2H, m),

3.31–2.91 (m, 6H), 2.68–2.53 (2H, m), 2.53 (3H, s), 1.21 (9H, s);

HPLC 98.9% at 215 nm, 5.4 min (Method A); MS (ESI) m/z

471.2870 (M+H).

4.5.34. 4-[(4-Methyl-1H-indol-5-yl)amino]-5-{(1E)-4-[4-(methyl-

sulfonyl)piperazin-1-yl]but-1-en-1-yl}nicotinonitrile (40)

Compound 40 was prepared from

1 and 1-(methylsulfo-

nyl)piperazine using the procedure described for 4b and puriﬁed

by HPLC to give 80 mg (48% yield) as its TFA salt; ¹H NMR

(DMSO-d₆) d 11.24 (1H, s), 9.88 (1H, br s), 9.37 (1H, br s), 8.54

(1H, s), 8.39 (1H, s), 7.40 (1H, t, J = 2.6 Hz), 7.28 (1H, d,

J = 8.8 Hz), 6.94 (1H, d, J = 8.4 Hz), 6.70 (1H, d, J = 15.2 Hz), 6.55

(1H, s), 6.25 (1H, dt, J = 6.6, 15.6 Hz), 3.82–3.53 (4H, m), 3.37–

3.05 (6H, m), 3.03 (3H, s), 2.66–2.59 (2H, m), 2.32 (3H, s); HPLC

98.1% at 215 nm, 4.16 min (Method E); MS (ESI) m/z 465.2065

(M+H).

4.5.39. N-{1-[(3E)-4-{5-Cyano-2-methyl-4-[(4-methyl-1H-indol-

5-yl)amino]pyridin-3-yl}but-3-en-1-yl]piperidin-4-

yl}methanesulfonamide (46)

A solution of compound 45 (92 mg, 0.22 mmol), methane sulfo-

nyl chloride (36 mg, 0.31 mmol) and triethylamine (89 mg,

0.88 mmol) in 1 mL DMF was stirred for 1 h at room temperature.

The reaction was ﬁltered and the ﬁltrate was puriﬁed by HPLC to

7948

L. N. Tumey et al. / Bioorg. Med. Chem. 17 (2009) 7933–7948

give 50 mg (32% yield) of 46 as its TFA salt; ¹H NMR (DMSO-d₆) d

11.25 (1H, s), 9.46 (1H, br s), 9.28 (1H, br s), 8.59 (3H, br s), 7.40

(1H, t, J = 2.8 Hz), 7.30 (1H, d, J = 8.0 Hz), 6.92 (1H, d, J = 8.0 Hz),

6.55 (1H, s), 6.34 (1H, d, J = 16 Hz), 6.01 (1H, dt, J = 6.8, 16.4 Hz),

3.53 (2H, d, J = 12 Hz), 3.31–2.96 (5H, m), 2.96 (3H, s), 2.66–2.60

(2H, m), 2.49 (3H, s), 2.31 (3H, s), 2.07 (2H, d, J = 12.4 Hz), 1.72–

1.62 (2H, m); HPLC 98.0% at 215 nm, 4.8 min (Method A); MS

(ESI) m/z 493.2384 (M+H).

3. Salek-Ardakani, S.; So, T.; Halteman, B. S.; Altman, A.; Croft, M. J. Immunol.

2005, 175, 7635.

4. Tan, S.-L.; Zhao, J.; Bi, C.; Chen, X. Y. C.; Hepburn, D. L.; Wang, J.; Sedgwick, J. D.;

Chintalacharuvu, S. R.; Na, S. J. Immunol. 2006, 176, 2872.

5. Healy, A. M.; Izmailova, E.; Fitzgerald, M.; Walker, R.; Hattersley, M.; Silva, M.;

Siebert, E.; Terkelsen, J.; Picarella, D.; Pickard, M. D.; LeClair, B.; Chandra, S.;

Jaffee, B. J. Immunol. 2006, 177, 1886.

6. Marsland, B. J.; Soos, T. J.; Spaeth, G.; Littman, D. R.; Kopf, M. J. Exp. Med. 2004,

200, 181.

7. Salek-Ardakani, S.; So, T.; Halteman, B. S.; Altman, A.; Croft, M. J. Immunol.

2004, 173, 6440.

8. Wang, L.; Xiang, Z.; Ma, L.-L.; Chen, Z.; Gao, X.; Sun, Z.; Williams, P.; Chari, R. S.;

Yin, D.-P. Transplantation 2009, 87, 507.

9. Rondinone, C. M. Curr. Med. Chem.: Immunol. Endocrinol. Metab. Agents 2005, 5,

529.

4.5.40. 6-Methyl-4-[(4-methyl-1H-indol-5-yl)amino]-5-[(1E)-4-

piperazin-1-ylbut-1-en-1-yl]nicotinonitrile (47)

Compound 47 was prepared from 44 and tert-butyl piperazine-

1-carboxylate using the procedure described for 45 and puriﬁed by

HPLC to give 300 mg (60% yield) as its TFA salt; ¹H NMR (DMSO-d₆)

d 11.29 (1H, s), 9.64 (1H, s), 9.13 (2H, br s), 8.73 (1H, s), 7.41 (1H, t,

J = 2.8 Hz), 7.29 (1H, d, J = 8.6 Hz), 6.94 (1H, d, J = 8.6 Hz), 6.56 (1H,

s), 6.31 (1H, d, J = 16 Hz), 6.06 (1H, dt, J = 6.8, 16 Hz), 3.28–3.05

(10H, m), 2.65–2.59 (2H, m), 2.49 (3H, s), 2.32 (3H, s); HPLC

100% at 215 nm, 3.69 min (Method E); MS (ESI) m/z 401.2455

(M+H).

10. Berg-Brown, N. N.; Gronski, M. A.; Jones, R. G.; Elford, A. R.; Deenick, E. K.;

Odermatt, B.; Littman, D. R.; Ohashi, P. S. J. Exp. Med. 2004, 199, 743.

11. Reyland, M. E. Front Biosci. 2009, 14, 2386.

12. Thebault, S. Prog. Immunol. Res. 2005, 1.

13. Baier, G.; Telford, D.; Giampa, L.; Coggeshall, K. M.; Baier-Bitterlich, G.; Isakov,

N.; Altman, A. J. Biol. Chem. 1993, 268, 4997.

14. Mecklenbrauker, I.; Saijo, K.; Zheng, N. Y.; Leitges, M. Nature 2002, 860.

15. Miyamoto, A.; Nakayama, K.; Imaki, H.; Hirose, S.; Jiang, Y.; Abe, M.;

Tsukiyama, T.; Nagahama, H.; Ohno, S.; Hatakeyama, S.; Nakayama, K. I.

Nature 2002, 416, 865.

16. Gonelli, A.; Mischiati, C.; Guerrini, R.; Voltan, R.; Salvadori, S.; Zauli, G. Mini-

Rev. Med. Chem. 2009, 9, 498.

17. Goekjian, P. G.; Jirousek, M. R. Exp. Opin. Invest. Drugs 2001, 10, 2117.

18. Cywin, C. L.; Dahmann, G.; Prokopowicz, A. S.; Young, E. R. R.; Magolda, R. L.;

Cardozo, M. G.; Cogan, D. A.; DiSalvo, D.; Ginn, J. D.; Kashem, M. A.; Wolak, J. P.;

Homon, C. A.; Farrell, T. M.; Grbic, H.; Hu, H.; Kaplita, P. V.; Liu, L. H.; Spero, D.

M.; Jeanfavre, D. D.; O’Shea, K. M.; White, D. M.; Woska, J. R.; Brown, M. L.

Bioorg. Med. Chem. Lett. 2007, 17, 225.

19. Boschelli, D. H.; Wu, B.; Barrios Sosa, A. C.; Chen, J.; Asselin, M.; Cole, D. C.; Lee,

J.; Yang, X.; Chaudhary, D. Bioorg. Med. Chem. Lett. 2008, 18, 2850.

20. Wu, B.; Boschelli, D. H.; Lee, J.; Yang, X.; Chaudhary, D. Bioorg. Med. Chem. Lett.

2009, 19, 766.

21. Tumey, L. N.; Boschelli, D. H.; Lee, J.; Chaudhary, D. Bioorg. Med. Chem. Lett.

2008, 18, 4420.

22. Cole, D. C.; Asselin, M.; Brennan, A.; Czerwinski, R.; Ellingboe, J. W.; Fitz, L.;

Greco, R.; Huang, X.; Joseph-McCarthy, D.; Kelly, M. F.; Kirisits, M.; Lee, J.; Li, Y.;

Morgan, P.; Stock, J. R.; Tsao, D. H. H.; Wissner, A.; Yang, X.; Chaudhary, D. J.

Med. Chem. 2008, 51, 5958.

23. Boschelli, D. H.; Wang, D.; Prashad, A. S.; Subrath, J.; Wu, B.; Niu, C.; Lee, J.;

Yang, X.; Brennan, A.; Chaudhary, D. Bioorg. Med. Chem. Lett. 2009, 19, 3623.

24. Subrath, J.; Wang, D.; Wu, B.; Niu, C.; Boschelli, D. H.; Lee, J.; Yang, X.; Brennan,

A.; Chaudhary, D. Bioorg. Med. Chem. Lett. 2009, 19, 5423.

25. Prashad, A. S.; Wang, D.; Chen, J.; Wu, B.; Lin, M.; Zhang, M.; Kagan, M.; Lee, J.;

Yang, X.; Brennan, A.; Chaudhary, D.; Xu, X.; Leung, L.; Wang, J.; Boschelli, D. H.

Bioorg. Med. Chem. Lett. 2009, 19, 5799.

4.5.41. 5-[4-(4-Aminopiperidin-1-yl)butyl]-4-[(4-methyl-1H-

indol-5-yl)amino]nicotinonitrile (48)

Compound 10b (200 mg, 0.40 mmol) and palladium (20 mg,

10%; wet) in 20 mL of EtOH was stirred rapidly under H₂at 1 atm

for 16 h at room temperature. The solvent and palladium were re-

moved to give tert-butyl 1-(4-(5-cyano-4-(4-methyl-1H-indol-5-

ylamino)pyridin-3-yl)butyl)piperidin-4-ylcarbamate. Ten percent

of TFA/DCM (5.5 mL) were added to the crude intermediate. After

stirring for 2 h at room temperature the reaction was concentrated

and the residue was puriﬁed by HPLC to give 201 mg (78% yield) of

48 as its TFA salt; ¹H NMR (DMSO-d₆) d 11.30 (1H, s), 9.93 (2H, br s),

8.74 (1H, s), 8.29 (1H, s), 8.20 (3H, br s), 7.42 (1H, t, J = 2.8 Hz), 7.30

(1H, d, J = 8.0 Hz), 6.99 (1H, d, J = 8.0 Hz), 6.57 (1H, s), 3.57 (2H, d,

J = 11.6 Hz), 3.32–3.29 (1H, m), 3.08–2.96 (4H, m), 2.83–2.77 (2H,

m), 2.34 (3H, s), 2.14 (2H, d, J = 9.2 Hz), 1.85–1.59 (6H, m); HPLC

95.3% at 254 nm, 3.00 min (Method E); MS (ESI) m/z 403.2612

(M+H).

26. Xu, Z.-B.; Chaudhary, D.; Olland, S.; Wolfrom, S.; Czerwinski, R.; Malakian, K.;

Lin, L.; Stahl, M. L.; Joseph-McCarthy, D.; Benander, C.; Fitz, L.; Greco, R.;

Somers, W. S.; Mosyak, L. J. Biol. Chem. 2004, 279, 50401.

[27]. Niu, C.; Boschelli, D. H.; Tumey, L. N.; Bhagirath, N.; Subrath, J.; Shim, J.;

Wang, Y.; Wu, B.; Eid, C.; Lee, J.; Yang, X.; Brennan, A.; Chaudhary, D. Bioorg.

Med. Chem. Lett. 2009, 19, 5829.

28. Wang, Y. D.; Kimball, G.; Prashad, A. S.; Wang, Y. Tetrahedron Lett. 2005, 46,

8777.

29. McMartin, C.; Bohacek, R. S. J. Comput.-Aided Mol. Des. 1997, 11, 333.

30. Faller, B. Curr. Drug Metab. 2008, 9, 886.

4.6. Assay conditions

The PKCh and PKCd IMAP assays and T cell IL2 production assays

were performed as previously described.²²

Acknowledgments

31. Clark, D. E.; Grootenhuis, P. D. J. Curr. Top. Med. Chem. 2003, 3, 1193.

32. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Delivery Rev.

2001, 46, 3.

33. Veber, D. F.; Johnson, S. R.; Cheng, H.-Y.; Smith, B. R.; Ward, K. W.; Kopple, K. D.

J. Med. Chem. 2002, 45, 2615.

34. Bhal, S. K.; Kassam, K.; Peirson, I. G.; Pearl, G. M. Mol. Pharm. 2007, 4, 556.

35. Cacace, A. M.; Uefﬁng, M.; Philipp, A.; Han, E. K.-H.; Kolch, W.; Weinstein, I. B.

Oncogene 1996, 13, 2517.

We thank the following Wyeth departments for their support of

compound scaleup, analytical proﬁling, PK and metabolism

studies, crystallography and selectivity screening: Pharmaceutical

Proﬁling, Chemical Technologies, Structural Biology and Computa-

tional Chemistry, Drug Safety and Metabolism, Discovery Synthetic

Chemistry, GVK Bio, Chemical Development and Screening

Sciences. We also thank Barry Press, Zhang Xu, Xin Xu, Divya Chau-

dhary and Tarek Mansour for their support.

36. Boschelli, D. H. Curr. Top. Med. Chem. 2008, 8, 922.

37. Hibbs, M. L.; Tarlinton, D. M.; Armes, J.; Grail, D.; Hodgson, G.; Maglitto, R.;

Stacker, S. A.; Dunn, A. R. Cell 1995, 83, 301.

38. Ertl, P.; Rohde, B.; Selzer, P. J. Med. Chem. 2000, 43, 3714.

39. Di, L.; Kerns, E.; Gao, N.; Li, S.; Huang, Y.; Bourassa, J.; Huryn, D. J. Pharm. Sci.

2004, 93, 1537.

References and notes

40. Terada, T.; Inui, K. Biotechnol. Pharm. Aspects 2008, 7, 559.

41. del Amo, E.; Heikkinen, A.; Moenkkoenen, J. Eur. J. Pharm. Sci. 2009, 36, 200.

1. Manicassamy, S.; Gupta, S.; Sun, Z. Cell. Mol. Immunol. 2006, 3, 263.

2. Chaudhary, D.; Kasaian, M. Curr. Opin. Invest. Drugs 2006, 7, 432.

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