Synthesis and biological evaluation of novel (4 or 5-aryl)pyrazolyl-indoles as inhibitors of interleukin-2 inducible T-cell kinase (ITK)

Article abstract of DOI:10.1016/j.bmc.2010.04.056

Interleukin-2 inducible T-cell kinase (ITK) is one of five kinases that belong to the Tec kinase family that plays an important role in T-cell and mast cell signaling. Various reports point to a role of ITK in the treatment of allergic asthma. For example, it was shown that mice lacking ITK have reduced airway hyperresponsiveness, inflammation and tracheal responses in an allergic asthma model. In this article, we disclose novel ITK inhibitors based on (4 or 5-aryl)pyrazolyl-indole scaffold that were also found to be selective for ITK over other kinases like IRK, CDK2, GSK3ss and PKA.

Full text of DOI:10.1016/j.bmc.2010.04.056

Bioorganic & Medicinal Chemistry 18 (2010) 4547–4559
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of novel (4 or 5-aryl)pyrazolyl-indoles
as inhibitors of interleukin-2 inducible T-cell kinase (ITK)
b
a
b,
Avdhoot D. Velankar a, , Gianluca Quintini , Arati Prabhu , Alexander Weber , Gundula Hunaeus b,
Britta Voland b, Monika Wuest b, Christian Orjeda b, Dipak Harel a, Shaji Varghese a, Vikas Gore a,
Meenal Patil a, Deepak Gayke a, Matthias Herdemann b,à, Isabelle Heit b,§, Andrea Zaliani b
*
a Global Discovery, Nycomed Pharma Pvt. Ltd, Plot number 29-30-31, Suren Road, Andheri (East), Mumbai 400 093, Maharashtra, India
b Global Discovery, Nycomed GmbH, Byk-Gulden-Str 2, 78467 Konstanz, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Interleukin-2 inducible T-cell kinase (ITK) is one of five kinases that belong to the Tec kinase family that
plays an important role in T-cell and mast cell signaling. Various reports point to a role of ITK in the treat-
ment of allergic asthma. For example, it was shown that mice lacking ITK have reduced airway hyperre-
sponsiveness, inflammation and tracheal responses in an allergic asthma model. In this article, we
disclose novel ITK inhibitors based on (4 or 5-aryl)pyrazolyl-indole scaffold that were also found to be
selective for ITK over other kinases like IRK, CDK2, GSK3ß and PKA.
Received 22 January 2010
Revised 19 April 2010
Accepted 20 April 2010
Available online 24 April 2010
Keywords:
ITK
Ó 2010 Elsevier Ltd. All rights reserved.
Interleukin-2 inducible T-cell kinase
Tec kinase
Pyrazolyl-indole
1. Introduction
the viral LTR, and virion assembly or release.5 Although a number
of companies have disclosed potent inhibitors of ITK,6,7 no ITK
Interleukin-2 inducible T-cell kinase (ITK) is one of five kinases
that belong to the Tec kinase family, which is primarily expressed
in hematopoietic cells and participates in major immunological
processes.1 ITK plays an important role in T-cell and mast cell sig-
naling.1 Number of reports point to a role of ITK in the treatment of
allergic asthma. For example, it was shown that mice lacking ITK
have reduced airway inflammation in an allergic asthma model,2
reduced airways hyperresponsiveness to allergen challenge and re-
duced tracheal responses to cholinergic challenge.3 In response to
allergen challenge with OVA (ovalbumin), ITK knockout mice
showed reduced lung inflammation, eosinophils infiltration and
mucous production.4 A new dimension of the contribution of ITK
to T-cell activation in HIV replication has been investigated re-
cently. Studies using siRNA specific for ITK as well as a reported
ITK inhibitor BMS509744 in primary human CD4+ T cells suggested
that inhibition of ITK blocks HIV replication by affecting multiple
stages of the HIV life cycle, including viral entry, transcription from
inhibitor has been tested in any clinical trials. Pyrazolyl-indole
derivatives have been reported as aurora kinase inhibitors.8 Our
hit finding activities along with molecular modeling studies pre-
dicted that (4 or 5-aryl)pyrazolyl-indole would serve as a good
scaffold for designing inhibitors of ITK. In this article, we wish to
disclose novel and selective ITK inhibitors based on (4 or 5-
aryl)pyrazolyl-indole scaffold.
2. Chemistry
Target compounds were synthesized as shown in Schemes 1–5.
As illustrated in Scheme 1, the synthesis of 4-phenylpyrazolyl-in-
dole scaffold began with benzyl cyanide, which on treatment with
N,N-dimethylformamide dimethyl acetal gave the acrylonitrile 1,
which on reflux with hydrazine hydrochloride furnished the de-
sired 3-amino-4-phenyl pyrazole. Diazotization of 2 with aqueous
NaNO2 under acidic conditions followed by treatment with aque-
ous KI provided the iodopyrazole 3, which was treated with di-t-
butyl dicarbonate to yield the Boc-protected iodopyrazole 4. React-
ing 4 with 1-(t-butoxycarbonyl)-6-(methoxycarbonyl)-1H-indol-2-
ylboronic acid under Suzuki coupling conditions furnished the es-
ter 5, which was subsequently hydrolyzed to acid 6. The acid 6 was
coupled with various amines using standard peptide coupling con-
ditions to give target compounds 7–13.
* Corresponding author. Tel.: +91 22 6646 3930; fax: +91 22 6646 3860.
E-mail address: avdhoot.velankar@nycomed.com (A.D. Velankar).
Present address: Boehringer Ingelheim Pharma GmbH & Co. KG, Germany, 88400
Biberach, Germany.
à
Present address: EPO, Patentlaan 3-9, 2288 EE The Hague, The Netherlands.
Present address: Pipeline Sourcing Konstanz, Nycomed GmbH, Byk-Gulden-Str. 2,
§
78467 Konstanz, Germany.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.056

4548
A. D. Velankar et al. / Bioorg. Med. Chem. 18 (2010) 4547–4559
O
(iii)
(i)
N
(ii)
CN
+
O
N
NH2
CN
N
N
1
H
2
(iv)
(v)
(vi)
I
I
COOH
N
N
H
N
COOMe
N
N N
N
N
boc
N N
boc
H
H
4
3
6
boc
5
(vii)
X
N
H
O
N N
H
7-13
Table 1
Scheme 1. Reagents and conditions: (i) DMF, 100 °C (75%); (ii) H2NNH2ꢁHCl, ethanol, reflux (65%); (iii) aq NaNO2, KI, concd H2SO4, MeOH (41%); (iv) (Boc)2O, Et3N, DCM
(99%); (v) 1-(t-butoxycarbonyl)-6-(methoxycarbonyl)-1H-indol-2-ylboronic acid, Pd(dppf)Cl2, dioxane, Cs2CO3, 33%; (vi) KOH, ethanol, reflux (64%); (vii) amine, EDCI, HOBt,
NaHCO3, DMF.
I
(i)
(ii)
(iii)
O
O
N
N
N
N
N
N
H
14
15
X
O
COOH
COOMe
N
H
(iv)
(v)
N
H
N
N
N
H
boc
N
N
H
N
N
18-24
Table 2
17
O
16
Scheme 2. Reagents and conditions: (i) DHP, TFA, toluene, reflux (98%); (ii) n-BuLi, I2, THF, ꢀ78 °C (61%); (iii) Pd(dppf)Cl2, dioxane, 1-(t-butoxycarbonyl)-6-
(methoxycarbonyl)-1H-indol-2-ylboronic acid, Cs2CO3, 32% (iv) KOH, ethanol, reflux, then acid (aq HCl) workup (55%); (v) amine, EDCI, HOBt, NaHCO3, DMF.
N
(iii)
(vi)
N
(ii)
(i)
N
N
N
N
O
N
H
N
O
O
27
26
25
COOMe
COOMe
(v)
(iv)
I
N
N
boc
H
N
N
N
N
O
N
N
H
30
O
29
28
X
COOH
(vii)
O
(viii)
N
H
N
H
N
N
N
H
N
H
32-37
Table 3
31
Scheme 3. Reagents and conditions: (i) DHP, TFA, toluene, reflux (98%); (ii) n-BuLi, BnBr, THF, ꢀ78 °C (38%); (iii) ethereal HCl, then DHP, TFA, toluene, reflux, 83%; (iv) n-BuLi,
I2, THF, ꢀ78 °C (41%); (v) Pd(dppf)Cl2, dioxane, Cs2CO3, 1-(t-butoxycarbonyl)-6-(methoxycarbonyl)-1H-indol-2-ylboronic acid, 31% (vi) 4 M HCl in dioxane, rt, then K2CO3/
MeOH, 85%; (vii) aqueous KOH, EtOH, reflux, 89%; (viii) amine, EDCI, HOBt, NaHCO3, DMF.
Commercially available 3-phenyl-1H-pyrazole was regioselec-
tively protected as THP aminal derivative 14 that was iodinated
to furnish the iodopyrazole 15, which on coupling with 1-(t-butox-
ycarbonyl)-6-(methoxycarbonyl)-1H-indol-2-ylboronic acid gave
the ester 16 (Scheme 2). Hydrolysis of the ester 16 provided acid
17, which was coupled with various amines as before to furnish
target compounds 18–24.
As shown in Scheme 3, pyrazole was converted to its THP deriv-
ative 25, which on treatment with n-BuLi and benzyl bromide fur-
nished the benzyl pyrazole 26 in moderate yield. Removal of the

A. D. Velankar et al. / Bioorg. Med. Chem. 18 (2010) 4547–4559
4549
H
O
Ph
Ph
(ii)
(i)
Ph
COOMe
COOMe
COOMe
39
N
H
N
boc
N N
N
H
N N
N N
H
boc
H
5
38
H
O
H
O
Ph
R1
(iii)
(v)
Ph
(iv)
Ph
COOH
N
O
N
N
H
N N
N
H
N N
N
H
H
40
O
N N
H
41
42-43
H
Table 4
Scheme 4. Reagents and conditions: (i) HCl in dioxane (97%); (ii) DMF, POCl3, (67%); (iii) KOH, ethanol, reflux (45%); (iv) N-isobutyl-N-methylamine, EDCI, NaHCO3, HOBt,
DMF; (v) amine, Et3N, AcOH, THF, NaBH4.
OHC
(ii)
(i)
PhH2C
COOMe
PhH2C
PhH2C
COOMe
30
N
H
N
H
N N
N N
H
H
45
R1
OHC
OHC
(iv)
(iii)
X
NR1R2
COOH
PhH2C
PhH2C
N
H
N N
H
N
H
N
H
O
O
N N
N N
H
46
H
50-53
Table 5
47: X = 3-Methyl-3H-imidazol-4-ylmethylamino
48: X = isobutylmethylamino
49: X = methylamino
Scheme 5. Reagents and conditions: (i) DMF, POCl3, (69%); (ii) KOH, ethanol, reflux (69%); (iii) amine, EDCI, HOBt, NaHCO3, DMF; (iv) amine, Et3N, AcOH, THF, NaBH4.
THP group, followed by reflux with DHP under acidic conditions
furnished the other regioisomeric benzyl pyrazole 27.9 A similar
sequence of events as before, which included iodination, Suzuki
coupling, hydrolysis and finally amide formation, provided the tar-
get compounds 32–37.
Suzuki coupling product 5 from Scheme 1 was treated with 4 M
HCl in dioxane to remove the Boc groups (Scheme 4). The resulting
ester 38 was formylated at the indole-3 position selectively using
POCl3/DMF complex to furnish the aldehyde 39, which on hydroly-
sis followed by coupling the acid 40 with N-methyl-N-isobutyl
amine afforded the amide 41. This amide was then subjected to
reductive amination with various primary amines using standard
conditions to afford indole-3-substituted derivatives 42–43
(Scheme 4, Table 4). On the other hand, amide 41 on reduction
with NaBH4 in CH3OH furnished the alcohol 44.
Scheme 5 depicts a similar sequence of reactions to afford indole-
3-substituted derivatives in the 5-benzylpyrazole series. Thus, the
ester 30 from Scheme 3 was formylated regioselectively at indole
3-C position. The ester group in the resulting aldehyde 45 was
hydrolyzed to give the acid 46 that was further coupled with three
different amines to yield the amides 47–49. Each of these amides
was then subjected to reductive amination with different amines
to afford the target compounds 50–53 (Scheme 5, Table 5). Reduc-
tion of 47 with NaBH4 in CH3OH afforded the alcohol 54. A common
feature of some of the final compounds in Schemes 1–5 is the
appearance of NMR signals due to tautomerism in the pyrazole ring.
atives with ITK indicated a common mode of binding which in-
volved three hydrogen bonds in the hinge region of ITK between:
(i) N–H of the pyrazole ring with the carbonyl of Glu436 of ITK,
(ii) N of pyrazole and –NH of Met438 of ITK and (iii) N–H of the in-
dole ring with the carbonyl of Met438 of ITK. Additionally the
amide chain points towards the solvent accessible region. In ITK,
Phe435 acts as a gatekeeper residue by guarding access to a hydro-
phobic pocket that is located adjacent to the ITK active site.10
Molecular modeling studies predicted that arylpyrazolyl-indoles
or arylmethylpyrazolyl-indoles, which have an aryl ring on the
pyrazole ring, would have an additional binding interaction:
p–p
stacking between Phe435 and the aryl ring on the pyrazole. It
was also apparent from docking that among the three series exam-
ined, 5-benzylpyrazolyl-indoles show the best binding to ITK, and
was followed in the decreasing order by 4-phenylpyrazolyl-indoles
and 5-phenylpyrazolyl-indoles. Compounds 13, 24 and 34 have
same amide residue (N-methyl-N-isobutyl) but belong to the 4-
phenyl, 5-phenyl and 5-benzyl series, respectively. Figures 1a
and c illustrate the comparative docking of 13, 24 and 34, respec-
tively onto ITK. The docking score of 24 (5-phenyl series) was the
lowest followed by that of 13 (4-phenyl series) while the docking
score of 34 (5-benzyl series) was the highest. As shown in Figures
1a and b, the orientation of the corresponding phenyl ring with re-
spect to Phe435 of ITK is different in 13 and 24. As depicted in Fig-
ure 1a for compound 13, as the 4-phenyl ring on the pyrazole is too
far to engage in effective p–p stacking interaction with Phe435 of
ITK, the molecule prefers to form three H-bonding interactions
with the hinge region of ITK as described before. In case of 24
(Fig. 1b), it seems that the phenyl ring is closer and oriented favor-
3. Results and discussion
ably for a possible p–p stacking interaction with Phe435 of ITK and
3.1. Molecular modeling studies
in doing so loses one of the three potential H-bonding interactions
with the hinge region. On the other hand, Figure 1c clearly shows
that in case of 34 which contains a benzyl group on 5-C atom of the
Our hit finding activities showed that pyrazolyl-indoles are
inhibitors of ITK. Docking studies of various pyrazolyl-indole deriv-

4550
A. D. Velankar et al. / Bioorg. Med. Chem. 18 (2010) 4547–4559
Figure 1c. Compound 34 docked into the ATP-binding pocket of ITK. The ligand
makes three hydrogen-bonding interactions (dashed yellow lines) with the
backbone atoms of Glu 436 and Met 438 of the ITK hinge. The 5-benzyl ring of
Figure 1a. Compound 13 docked into the ATP-binding pocket of ITK showing three
potential H-bonding interactions with hinge region and unfavorable orientation of
the phenyl ring for a p–p interaction with Phe435.
34 makes a favorable p–p stack with the gatekeeper Phe435.
pyrazole nucleus, the methylene group imparts some degree of
flexibility so that the phenyl ring of the benzyl group can orient it-
Increases in potencies of ITK inhibitors have been achieved by
designing inhibitors that by going around the gatekeeper residue
bind to the amino acid residues (e.g., Lys391 and Asp500) in the ki-
nase specificity pocket (KSP).6b We reasoned that incorporation of
functional motifs of suitable length and H-bonding potential in the
3-C position of the indole ring of our inhibitors should offer access
to the amino acid residues in the KSP and that this may result in
increase in potency. Accordingly, a number of analogues of the
three series discussed above were designed that had diverse types
of substitutions of varying lengths and functionality. Introduction
of bulky substituent in the immediate vicinity of indole-3-C atom
was avoided as it might have changed the torsion angle between
the indole and pyrazole rings due to steric repulsion, which in turn
could have affected the H-bonding interaction with the hinge
region. In the 5-benzyl series, presence of flexible benzyl group al-
lowed introduction of various groups in the 3-C position while
self for a favorable
p–p stacking interaction with Phe435 of ITK
while still engaging in the three possible H-bonding interactions
with the hinge region. When the above docking exercise was re-
peated for compounds 10, 21 and 32 which belong to the 4-phenyl,
5-phenyl and 5-benzyl series, respectively, and also have the same
amide substituent, similar modes of binding were predicted with
32 showing the best docking score followed by 10 and 21 in
decreasing order. Armed with these docking predictions, it was
decided to synthesize members from all three series and measure
their potencies for inhibition of ITK.
maintaining the
p–p stacking with the gatekeeper residue and
the three hinge interactions. The torsion angle as discussed above
changed minimally, with 4.3° as the maximum change predicted.
As depicted in Figure 2, docking studies of 53 with ITK revealed a
torsion angle of 8.1° when compared to the value of 6°, the torsion
angle in the unsubstituted derivative 36. Docking of remaining
analogues in Table 5 with ITK predicted similar results. Interest-
ingly the presence of –OH group in 53 provides for a favorable
H-bonding interaction with Lys391.
In case of 5-phenyl series, the rigidity imparted by presence of
5-phenyl group allowed only smaller aliphatic residues similar to
those present in compounds 43 and 44 (docking data not shown),
with minimal change in the torsion angle as discussed above. How-
ever, there is a loss of one H-bonding interaction in the hinge re-
gion compared to two such interactions for the unsubstituted
compound. In contrast, introduction of substituent in the 3-C posi-
tion of 4-phenyl series results in a flipped dock pose within the ac-
tive pocket, loss of
p–p stacking and all potential hinge
interactions (docking data not shown). Changes in torsion angle
by as much as 151° were predicted.
Insulin receptor tyrosine kinase (IRK) is structurally homolo-
gous to ITK but whereas ITK has phenylalanine in the gatekeeper
Figure 1b. Compound 24 docked into the ATP-binding pocket of ITK. While the
phenyl ring is positioned properly for a favorable
p–p stack with the gatekeeper
Phe435, only two possible H-bonding interactions with the hinge region are
predicted.

A. D. Velankar et al. / Bioorg. Med. Chem. 18 (2010) 4547–4559
4551
Table 1
Inhibitory activity of 4-phenyl-1H-pyrazol-3-yl-indole series
X
HN
N
H
N
O
Compound
X
ITK
IC50
M)
IRK
IC50
CDK2
IC50
GSK3ß PKA
IC50 IC50
M) M)
(
l
(
lM)
(
lM)
(l
(l
HN
7
8
2.51
2.00
OMe
F
N
F
F
HN
HN
N
N
9
10
11
1.1
N
HN
HN
0.79
0.63
7.08
9.33
0.63
0.16
0.54
1.26
10.0
N
Me
N
N
Me
7.08
Figure 2. Compound 53 docked into the ATP-binding pocket of ITK. Note the H-
bonding interaction with Lys391.
N
O
HN
12
13
0.63
0.33
5.75
>10
0.4
2.29
6.76
5.01
position, in IRK this role is played by a methionine residue. Hence it
was hypothesized that selectivity for ITK over IRK can be achieved
by designing molecules that have an aryl ring in close proximity to
Phe435 of ITK. In fact, it has been suggested in literature that
improvement in potency and selectivity can be achieved by modu-
lating interactions with the gatekeeper residue, as this residue var-
ies in different kinases.10,11 On the other hand, in the case of ITK
recent reports have detailed efforts to achieve increased potency
and selectivity by maximizing interactions with amino acid resi-
dues present in KSP.6b For therapeutic reasons selectivity over ki-
nases controlling the cell cycle or the cell metabolism is required
and for this reason we measured inhibitory activity on IRK,
CDK2, GSK3ß and PKA.
Me
Me Me
Not
active
3.16
N
Me
Table 2
Inhibitory activity of 5-phenyl-1H-pyrazol-3-yl-indole series
X
HN
N
H
N
O
Compound
X
IC50
3.16
10.0
(lM)
3.2. In vitro activity
HN
18
OMe
F
Compounds 7–13 were evaluated for their inhibitory activity
against recombinant full length ITK using an IMAP-based assay
and the IC50 values obtained are summarized in Table 1. Some of
these compounds inhibited ITK at sub-micromolar level (com-
pounds 10–13) and compound 13 is the most potent member in
N
19
20
21
22
F
F
HN
HN
N
N
4.36
1.26
3.16
N
this series with an IC50 value of 0.33 lM. Next, compounds 18–
HN
HN
N
Me
24 were assayed against ITK as described before to determine the
effect of changing the position of the phenyl ring from C-4 to C-5
of the pyrazole ring. As seen from Table 2, this resulted in dimin-
ished activity in all cases, with as much as fivefold loss in potency
(compare compounds 8 with 19, and 11 with 22). When the 5-ben-
zylpyrazolyl-indole scaffold, obtained by replacing the phenyl
group on C-5 of the pyrazole ring with benzyl group, was subjected
to docking studies with ITK, better binding was predicted. Thus, as
illustrated in Table 3, compounds 32 and 34 showed submicromo-
lar inhibition of ITK and the most potent member in this series was
N
N
Me
N
O
HN
23
24
1.82
0.85
Me
Me Me
N
Me
compound 34 with IC50 value of 0.56 lM. The 5-phenylpyrazolyl-
the kinase specificity pocket6b (KSP) of ITK. In the 4-phenylpyraz-
olyl-indole series, compounds 42–44 were synthesized (Scheme
4); but when tested against ITK showed appreciable reduction in
the activity against ITK (Table 4; compare compounds 42–44 with
13). This was in line with the predictions of docking studies. In the
5-phenyl series, analogues with similar 3-C substitution as in 44
were synthesized but contrary to docking predictions were found
to be inactive (data not shown).When similar substitutions were
carried out in the 3-C position of the indole moiety in the 5-ben-
zylpyrazoly-indole series (Scheme 5), almost no improvement in
indole derivatives were least potent against ITK in accordance with
the predictions of the docking studies, whereas, as opposed to the
predictions, 4-phenylpyrazolyl-indoles were generally more po-
tent than 5-benzylpyrazolyl-indoles. It should also be noted that
in each series the most potent compound was an amide with N-
isobutylmethylamine (compounds 13, 24 and 34). As there was
no improvement in potency by changing the position of the phenyl
group or by substitution with benzyl group, further substitution at
the indole 3-C atom was explored. Molecular modeling studies
with 3-indole substituted derivatives predicted interactions in

4552
A. D. Velankar et al. / Bioorg. Med. Chem. 18 (2010) 4547–4559
Table 3
potency was observed (Table 5, compare compounds 50 and 54
with 32 and compound 51 with 34). Rather, in the 3-C position
of indole, introduction of polar straight chain residue led to 30-fold
loss in potency (compare compound 53 with 36), whereas, aro-
matic residues did not affect the potency appreciably. In case of
53, this directly contradicts the prediction based on modeling stud-
ies that showed potential interaction with Lys391 and the reason
behind this discrepancy is unclear at this point.
Inhibitory activity of 5-benzyl-1H-pyrazol-3-yl-indole series
X
HN
N
N
H
O
Compound
X
ITK
IC50
IRK
IC50
M)
CDK2
IC50
GSK3ß
IC50
PKA
IC50
(lM)
Some of the most potent members from 4-phenylpyrazolyl-in-
dole and 5-benzylpyrazolyl-indole scaffolds (Tables 1 and 3) were
tested against kinases such as IRK, GSK3ß, CDK2 and PKA. As
shown in Table 1, compounds 10–12 that belong to the 4-phe-
nyl-pyrazolyl-indole series are equipotent towards ITK, CDK2 or
GSK3ß, but are comparatively less active against IRK and PKA. Only
compound 13 from the same series is selective for ITK when com-
pared to the four other kinases. Pleasingly three compounds from
the 5-benzyl-pyrazolyl-indole series show much greater prefer-
ence for ITK (Table 3, compounds 32–34). Of particular note is
compound 34, which is about 200-fold less active towards IRK,
while being completely inactive against CDK2, GSK3ß, PKA and
AKT1. It is instructive to note that the most potent compounds
from the 4- or 5-phenylpyrazolyl-indole as well as 5-benzylpyraz-
olyl-indole series that have the N-isobutyl-N-methylamine group
as part of the amide were the most selective against other kinases
(compounds 13, 24 and 34, respectively). Comparison of the amide
chains present in 13, 24 and 34 (N-isobutyl-N-methylamine) with
those in the rest of the compounds in Tables 1–3 reveal an impor-
tant difference: tertiary amide group in 13, 24 and 34 versus sec-
ondary amide groups in rest of the compounds in Tables 1–3. As
noted before, the amide group is directed towards the solvent
accessible region. The tertiary amide group in compounds 13, 24
and 34 has a methyl group on its amide nitrogen atom and hence
can form hydrogen bonds with the solvent molecules through its
carbonyl oxygen only whereas a secondary amide group should
be able to utilize both its carbonyl oxygen as well as the amide
hydrogen for hydrogen bonding with solvent. It should be noted
that most potent compounds (13, 24 and 34) in each series have
more lipophilic group on the amide nitrogen, although this may in-
cur a price by a decrease in solubility. Based on this, it seems that
the binding to the water-accessible region is detrimental to the po-
tency. Further, presence or absence of a glycine residue immedi-
ately after the hinge region in kinases has been reported to be
responsible for difference in the shape of the solvent accessible re-
gion influencing selectivity.12 However, both ITK and IRK have the
corresponding glycine residue present in the solvent accessible re-
gion, but differ in the gatekeeper residue. ITK has phenylalanine as
a gatekeeper residue whereas methionine plays the same role in
IRK. Comparison of selectivity of 13 and 34 for ITK inhibition over
IRK reveals that although both have the same amide residue which
occupies the water-accessible region, these compounds differ in
the corresponding group that will interact with the gatekeeper res-
idue. Docking studies predicted that by design the phenyl group in
(l
M)
(
l
(l
M)
(
lM)
N
Not
active
Not
active
HN
HN
32
0.63
5.01
31.6
12.6
N
Me
N
N
Me
Not
active
Not
active
33
34
1.00
0.56
20.0
Me Me
Not
active
Not
active
Not
active
>100
N
Me
N
35
36
2.06
1.63
HN
N
HNMe
N
O
HN
37
2.04
Me
Table 4
Inhibitory activity of 3-indole substituted 4-phenyl-1H-pyrazol-3-yl-indole series
R1
Me
Me
N
N
N
H
N
Me
O
Compound
R1
IC50 (lM)
OMe
42
20.4
NH
43
44
NH(CH2)3OH
>100
>100
OH
Table 5
Inhibitory activity of 3-indole substituted 5-benzyl-1H-pyrazol-3-yl-indole series
R1
X
HN
N
N
H
O
Compound
X
R1
IC50 (lM)
the 5-benzyl series is better placed for effective p–p stacking with
N
N
O
the gatekeeper residue (Phe435) in ITK than the corresponding
phenyl group in the 4-phenyl series. This fact along with the find-
ing that 34 is a more selective inhibitor (>178-fold selectivity over
IRK) of ITK than 13 (30-fold selectivity over IRK) supports our
hypothesis that interaction with the gatekeeper residue is energet-
ically more relevant than the interaction in the solvent accessible
region.
HN
50
0.63
0.50
N
Me
Me Me
F
N
Me
51
NH
NH
N
52
53
54
HNMe
5.0
4. Conclusion
HNMe
NH(CH2)4OH
50.1
1.58
N
N
Me
In summary, two novel series containing aryl substituted pyraz-
olyl-indoles that act as selective inhibitors of ITK were identified.
These compounds were designed with a phenyl group on the
HN
OH

A. D. Velankar et al. / Bioorg. Med. Chem. 18 (2010) 4547–4559
4553
pyrazole ring of the inhibitors so that the phenyl ring can engage into
interaction with the gatekeeper residue Phe435 in ITK. The 4-
H2O) and the reaction mixture was stirred at 0 °C for 1 h. An aque-
ous solution of KI (5.83 g, 35.12 mmol) was added dropwise, the
reaction was further stirred at 0 °C for 1 h, diluted with water
and extracted with EtOAc. The combined extracts were washed
with brine, dried over sodium sulfate and concentrated in vacuo.
The residue was purified by column chromatography with hex-
anes–EtOAc (70:30 v/v) as eluent to afford the title compound
(1.4 g, 41%). 1H NMR (DMSO-d6) d 13.41 (s, 1H), 7.97 (d, 1H,
J = 1.8 Hz), 7.57–7.54 (m, 2H), 7.42 (t, 2H, J = 7.5 Hz), 7.31 (tt, 1H,
J = 7.5, 1.8 Hz); GC–MS (EI) m/z 269.88 [M+].
a p–p
phenylpyrazolyl-indole series had generally the most potent com-
pounds (compound 13), whereas the 5-benzylpyrazolyl-indole ser-
ies displayed more selectivity for ITK (compound 34). Sub-
micromolar inhibitors of ITK (compounds 13 and 34) which are
selective for ITK over IRK were discovered. In fact, compound 34 is
completely inactive against kinases like CDK2, GSK3ß, PKA and
AKT1. Although substitution in the 3-C position of the indole part
did not improve potency or selectivity, there is still scope for substi-
tution in the phenyl group on the pyrazole ring that extends new
groups beyond the gatekeeper residue Phe435. Investigations to
probe whether these modifications result in enhanced potency
and or selectivity are in progress and will be reported in due course.
5.5. 3-Iodo-4-phenyl-pyrazole-1-carboxylic acid tert-butyl ester
(4)
To a reaction mixture containing 3 (0.5 g, 1.85 mmol) and Et3N
(0.30 ml, 0.22 g, 2.17 mmol) in CH2Cl2 (10 ml) was added di-tert-
butyl dicarbonate (0.48 g, 2.2 mmol). The reaction mixture was
stirred at room temperature for 24 h, quenched with water and ex-
tracted with EtOAc. The combined extracts were washed with
brine, dried over sodium sulfate and concentrated in vacuo. The
residue was purified by column chromatography with hexanes–
EtOAc (70:30 v/v) as eluent to furnish the title compound (0.67 g,
99%). 1H NMR (DMSO-d6) d 8.42 (s, 1H), 7.59 (dt, 2H, J = 6.9,
1.8 Hz), 7.49–7.44 (m, 2H), 7.41 (dt, 1H, J = 6.9, 1.5 Hz), 1.60 (s,
9H); GC–MS (EI) m/z 269.95 [MꢀBoc+H]+.
5. Experimental
5.1. General
All reagents were obtained from commercial sources and used
without further purification unless stated otherwise. THF was dis-
tilled from sodium-benzophenone. Flash column chromatography
was performed with Rankem Silica Gel (230–400 mesh size or
40–63 lm particle size). Thin layer chromatography (TLC) was per-
formed on Merck pre-coated TLC aluminum sheets with Silica Gel
60 F254. The 1H NMR spectra were recorded on a Bruker Avance
300 MHz spectrometer, and chemical shifts (d) are given in ppm
and are referenced to the corresponding solvent residual peak.
The spectral splitting patterns are indicated as follows: s, singlet;
d, doublet; t, triplet; q, quartet; dd, doublet of doublets; dt, doublet
of triplets; m, multiplet, br, broad. The LC/ES-MS analysis was per-
formed using a Thermo Finnigan LCQ Advantage Max spectrome-
ter. GC–MS analysis was carried out on a Thermo Focus GC
spectrometer. High-resolution mass spectra were obtained using
electrospray ionization (ESI) technique on a LC-MSD/TOF system
by Agilent.
5.6. 2-(1-tert-Butoxycarbonyl-4-phenyl-1H-pyrazol-3-yl)-
indole-1,6-dicarboxylic acid 1-tert-butyl ester 6-methyl ester
(5)
A pressure bottle containing a solution of 4 (1.3 g, 3.51 mmol)
and 1-(t-butoxycarbonyl)-6-(methoxycarbonyl)-1H-indol-2-ylbo-
ronic acid (1.68 g, 5.26 mmol) in 1,4-dioxane (40 ml) was purged
with nitrogen for 3 min. To it was added in succession Cs2CO3
(3.43 g, 10.52 mmol) and Pd(dppf)Cl2 (0.29 g, 0.35 mmol), the reac-
tion mixture was purged with nitrogen again for 3 min and the
reaction bottle was sealed. The reaction mixture was heated at
80 °C for 6 h, cooled to room temperature, diluted with water
and extracted with EtOAc. The combined extracts were washed
with brine, dried over sodium sulfate and concentrated in vacuo.
The residue was purified by column chromatography with hex-
anes–EtOAc as eluent to yield the title compound (0.6 g, 33%). 1H
NMR (DMSO-d6) d 8.79 (t, 1H, J = 0.6 Hz), 8.76 (s, 1H), 7.92 (dd,
1H, J = 8.1, 1.5 Hz), 7.80 (d, 1H, J = 8.1 Hz), 7.29–7.23 (m, 5H),
7.06 (d, 1H, J = 0.9 Hz), 3.90 (s, 3H), 163 (s, 9H), 1.20 (s, 9H); LC–
ESMS m/z 517.80 [M+H]+.
5.2. (Z)-3-Dimethylamino-2-phenyl-acrylonitrile (1)
A solution of benzyl cyanide (10 g, 85.36 mmol) and N,N-
dimethylformamide dimethyl acetal (17 ml, 15.25 g, 128 mmol)
in DMF (40 ml) was stirred at 100 °C for 12 h. The cooled reaction
mixture was diluted with water and the resulting precipitate was
filtered and dried to give the title compound (11.0 g, 75%). 1H
NMR (DMSO-d6) d 7.43 (s, 1H), 7.33–7.25 (m, 4H), 7.10–7.04 (m,
1H), 3.19 (s, 6H); GC–MS (EI) m/z 172.10 [M+].
5.3. 4-Phenyl-1H-pyrazol-3-ylamine (2)
5.7. 2-(4-Phenyl-1H-pyrazol-3-yl)-1H-indole-6-carboxylic acid
(6)
To a solution of 1 (2.0 g, 11.61 mmol) in EtOH (10 ml) was
added hydrazine monohydrochloride (6 g, 87.6 mmol) and the
reaction mixture was refluxed overnight. The reaction mixture
was concentrated, neutralized with aqueous saturated NaHCO3
and extracted with EtOAc. The combined extracts were washed
with brine, dried over sodium sulfate and concentrated in vacuo.
The residue was purified by column chromatography with hex-
anes–EtOAc (50:50 v/v) as eluent to yield the title compound
(1.2 g, 65%). 1H NMR (DMSO-d6) d 7.79 (s, 1H), 7.51 (dt, 2H,
J = 8.1, 1.8 Hz), 7.35 (t, 2H, J = 7.8 Hz), 7.16 (dt, 1H, J = 7.8, 0.6 Hz),
3.17 (s, 2H); GC–MS (EI) m/z 159.11 [M+].
A solution of 5 (0.32 g, 0.62 mmol) and KOH (0.14 g, 2.5 mmol)
in MeOH–H2O (14 ml, 6:1 v/v) was refluxed for 12 h. The reaction
mixture was concentrated in vacuo, acidified with aqueous satu-
rated NH4Cl solution and the resulting precipitate was filtered
and dried to furnish the title compound (0.12 g, 64%). 1H NMR
(DMSO-d6) d 13.35 (s, 1H), 11.74 (s, 1H), 8.04 (s, 1H), 7.97 (s,
1H), 7.55 (d, 1H, J = 8.7 Hz), 7.47–7.38 (m, 5H), 7.36–7.32 (m,
1H), 6.33 (s, 1H); LC–ESMS m/z 304.20 [M+H]+.
5.8. General procedure for amide formation
5.4. 3-Iodo-4-phenyl-1H-pyrazole (3)
To the solution of a carboxylic acid (0.23 mmol) and amine
(0.46 mmol) in DMF (2.5 ml) were added EDC (0.35 mmol), HOBt
(0.35 mmol) and NaHCO3 (0.8 mmol). The reaction mixture was
stirred at room temperature for 24 h, diluted with water and ex-
tracted with EtOAc. The combined extracts were washed with
To an ice-cooled solution of 2 (2.0 g, 12.56 mmol) in MeOH
(20 ml) was added aqueous H2SO4 (40 ml, 50% v/v). To the reaction
mixture was then added aqueous NaNO2 (0.87 g, 12.6 mmol, 12 ml

4554
A. D. Velankar et al. / Bioorg. Med. Chem. 18 (2010) 4547–4559
water, brine, dried over Na2SO4 and concentrated in vacuo. The
resulting residue was purified by column chromatography (typi-
cally MeOH–CH2Cl2, 5:95 v/v) to afford the desired amide.
3,4-dihydro-2H-pyran (0.60 ml, 0.55 g, 6.58 mmol). After stirring
the reaction mixture overnight at 80 °C, the toluene was evapo-
rated. The resulting residue was diluted with water and extracted
with EtOAc. The combined extracts were washed with brine, dried
over sodium sulfate and concentrated in vacuo. The residue was
purified by column chromatography with hexanes–EtOAc (80:20
v/v) to afford the title compound (1.4 g, 98%). 1H NMR (DMSO-d6)
d 7.93 (d, 1H, J = 2.4 Hz), 7.82 (d, 1H, J = 1.2 Hz), 7.80–7.79 (m,
1H), 7.43–7.3 (m, 2H), 7.30 (tt, 1H, J = 7.2, 1.8 Hz), 6.77 (d, 1H,
J = 2.7 Hz), 5.43 (dd, 1H, J = 9.9, 2.4 Hz), 3.98–3.91 (m, 1H), 3.68–
3.60 (m, 1H), 2.19–2.07 (m, 1H), 2.00–1.90 (m, 2H), 1.75–1.65
(m, 1H), 1.59–1.51 (m, 2H); LC–ESMS m/z 228.93 [M+H]+.
5.8.1. 2-(4-Phenyl-1H-pyrazol-3-yl)-1H-indole-6-carboxylic acid
(2-methoxy-ethyl)-amide (7)
1H NMR (DMSO-d6) d 13.28 (s, 1H), 11.63 (s, 1H), 8.36 (s, 1H),
8.00 (s, 1H), 7.92 (s, 1H), 7.43–7.33 (m, 7H,), 6.27 (s, 1H), 3.48–
3.40 (m, 4H), 3.27 (s, 3H); LC–HRMS (ESI+) m/z 361.16574 [M+H]+
(Calcd for C21H21N4O2: 361.1659).
5.8.2. 2-(4-Phenyl-1H-pyrazol-3-yl)-1H-indole-6-carboxylic acid
(6-trifluoromethyl-pyridin-3-ylmethyl)-amide (8)
1H NMR (DMSO-d6) d 13.30 (s, 1H), 11.69 (s, 1H), 9.06 (t, 1H,
J = 5.7 Hz), 8.75 (s, 1H), 8.02 (d, 1H, J = 1.5 Hz), 8.00 (s, 1H), 7.97
(s, 1H), 7.90 (s, 1H), 7.87 (s, 1H), 7.49–7.34 (m, 6H), 6.28 (s, 1H),
4.60 (d, 2H, J = 5.7 Hz); LC–HRMS (ESI+) m/z 462.15376 [M+H]+
(Calcd for C25H19F3N5O: 462.15362).
5.10. 3-Iodo-5-phenyl-1-(tetrahydro-pyran-2-yl)-1H-pyrazole
(15)
To a stirred solution of 14 (1.8 g, 7.88 mmol) in anhydrous THF
(12 ml) at ꢀ78 °C was added 1.6 M n-BuLi (5.4 ml, 8.64 mmol) and
the reaction mixture was stirred at ꢀ78 °C for 30 min. A solution of
I2 (2.2 g, 8.66 mmol) in THF (12 ml) was added dropwise at ꢀ78 °C,
the reaction mixture was warmed to room temperature over 2 h
and stirred for 3 h. The reaction mixture was quenched at 0 °C by
a saturated aqueous solution of NaHSO3 and extracted with EtOAc.
The combined extracts were washed with brine, dried over sodium
sulfate and concentrated in vacuo. The residue was purified by col-
umn chromatography with hexanes–EtOAc (80/20, v/v) as eluent
to yield the title compound (1.7 g, 61%). 1H NMR (DMSO-d6) d
7.80 (dt, 2H, J = 6.6, 1.5 Hz), 7.44–7.38 (m, 2H), 7.36–7.30 (m,
1H), 7.07 (s, 1H), 4.43 (dd, 1H, J = 9.6, 2.7 Hz), 3.97–3.93 (m, 1H),
3.67–3.59 (m, 1H), 2.45–2.30 (m, 1H), 2.06–1.99 (m, 1H), 1.95–
1.88 (m, 1H), 1.79–1.63 (m, 1H), 1.60–1.51 (m, 2H); GC–MS (EI)
m/z 353.92 [M+].
5.8.3. 2-(4-Phenyl-1H-pyrazol-3-yl)-1H-indole-6-carboxylic acid
(pyrazin-2-ylmethyl)-amide (9)
1H NMR (DMSO-d6) d 13.29 (s, 1H), 11.68 (s, 1H), 9.05 (s, 1H),
8.62 (d, 1H, J = 1.5 Hz), 8.59 (dd, 1H, J = 2.1, 1.5 Hz), 8.53 (d, 1H,
J = 2.7 Hz), 7.99 (s, 1H), 7.97 (s, 1H), 7.52 (d, 1H, J = 8.4 Hz), 7.46–
7.39 (m, 5H), 7.35–7.33 (m, 1H), 6.28 (s, 1H), 4.62 (d, 2H,
J = 5.7 Hz); LC–HRMS (ESI+) m/z 395.16114 [M+H]+ (Calcd for
C23H19N6O: 395.16149).
5.8.4. 2-(4-Phenyl-1H-pyrazol-3-yl)-1H-indole-6-carboxylic acid
(3-methyl-3H-imidazol-4-ylmethyl)-amide (10)
1H NMR (DMSO-d6) d 13.31 (s, 1H), 11.66 (s, 1H), 8.72 (t, 1H,
J = 6.0 Hz), 8.00 (s, 1H), 7.95 (s, 1H), 7.55 (s, 1H), 7.49–7.47 (m,
1H), 7.46–7.42 (m, 3H), 7.42–7.38 (m, 2H), 7.37–7.34 (m, 1H),
6.84 (s, 1H), 6.27 (s, 1H), 4.47 (d, 2H, J = 5.4 Hz), 3.64 (s, 3H); LC–
HRMS (ESI+) m/z 397.17709 [M+H]+ (Calcd for C23H21N6O:
397.17714).
5.11. 2-[5-Phenyl-1-(tetrahydro-pyran-2-yl)-1H-pyrazol-3-yl]-
indole-1,6-dicarboxylic acid 1-tert-butyl ester 6-methyl ester
(16)
5.8.5. 2-(4-Phenyl-1H-pyrazol-3-yl)-1H-indole-6-carboxylic acid
(2-methyl-2H-pyrazol-3-ylmethyl)-amide (11)
A pressure bottle containing a solution of 15 (2.0 g, 5.65 mmol)
and 1-(t-butoxycarbonyl)-6-(methoxycarbonyl)-1H-indol-2-ylbo-
ronic acid (2.13 g, 6.67 mmol) in 1,4-dioxane (20 ml) was purged
with nitrogen for 3 min. To it was added in succession Cs2CO3
(4.62 g, 14.18 mmol) and Pd(dppf)Cl2 (0.46 g, 0.56 mmol), the reac-
tion mixture was purged with nitrogen again for 3 min and the
reaction bottle was sealed. The reaction mixture was heated at
80 °C for 6 h, cooled to room temperature, diluted with water
and extracted with EtOAc. The combined extracts were washed
with brine, dried over sodium sulfate and concentrated in vacuo.
The residue was purified by column chromatography with hex-
anes–EtOAc (80/20, v/v) as eluent to yield the title compound
(0.9 g, 32%). 1H NMR (DMSO-d6) d 8.87 (t, 1H, J = 0.6 Hz), 7.90 (dt,
2H, J = 8.4, 1.5 Hz), 7.86–7.81 (m, 2H), 7.47–7.41 (m, 2H), 7.37–
7.31 (m, 1H), 7.04 (d, 1H, J = 0.9 Hz), 7.02 (s, 1H), 5.17 (dd, 1H,
J = 9.6, 2.1 Hz), 3.91 (s, 3H), 3.88–3.84 (m, 1H), 3.42–3.37 (m, 1H),
2.33–2.26 (s, 1H), 1.98–1.83 (m, 2H), 1.65–1.40 (m, 3H), 1.30 (s,
9H); LC–ESMS m/z 501.87 [M+H]+ (Calcd for C29H32N3O5).
1H NMR (DMSO-d6) d 13.30 (s, 1H), 11.68 (s, 1H), 8.85 (t, 1H,
J = 5.7 Hz), 8.00 (d, 1H, J = 1.5 Hz), 7.95 (s, 1H), 7.47 (d, 1H,
J = 1.5 Hz), 7.46–7.39 (m, 4H), 7.37–7.32 (m, 2H), 7.31 (d, 1H,
J = 1.5 Hz), 6.28 (d, 1H, J = 1.2 Hz), 6.16 (d, 1H, J = 1.8 Hz), 4.52 (d,
2H J = 5.7 Hz), 3.85 (s, 3H); LC–HRMS (ESI+) m/z 397.17699
[M+H]+ (Calcd for C23H21N6O: 397.17714).
5.8.6. 2-(4-Phenyl-1H-pyrazol-3-yl)-1H-indole-6-carboxylic acid
(5-methyl-isoxazol-4-ylmethyl)-amide (12)
1H NMR (DMSO-d6) d 13.29 (s, 1H), 11.66 (s, 1H), 8.75 (t, 1H,
J = 5.7 Hz), 8.40 (s, 1H), 8.00 (s, 1H), 7.92 (s, 1H), 7.45–7.41 (m,
5H), 7.38–7.33 (m, 2H), 6.26 (d, 1H, J = 1.5 Hz), 4.25 (d, 2H,
J = 5.4 Hz), 2.44 (s, 3H); LC–HRMS (ESI+) m/z 398.16087 [M+H]+
(Calcd for C23H20N5O2: 398.16115).
5.8.7. Phenyl-1H-pyrazol-3-yl)-1H-indole-6-carboxylic acid
isobutyl-methyl-amide (13)
1H NMR (CDCl3) d 10.34 (s, 1H), 7.61 (s, 1H), 7.53 (s, 1H), 7.49–
7.47 (m, 2H), 7.44–7.37 (m, 3H), 7.05 (d, 1H, J = 6.9 Hz), 6.48 (s,
1H), 3.45–3.30 (m, 2H), 3.15 (s, 3H), 1.93 (m, 1H), 1.01 (s, 3H),
0.72 (s, 3H); LC–ESMS m/z 373.20 [M+H]+.
5.12. 2-(5-Phenyl-1H-pyrazol-3-yl)-1H-indole-6-carboxylic acid
(17)
A solution of 16 (0.6 g, 1.20 mmol) and KOH (0.21 g, 3.74 mmol)
in EtOH–H2O (14 ml, 6:1 v/v) was refluxed for 12 h. The reaction
mixture was concentrated in vacuo and to the residue at 0 °C
was added ethereal HCl. After 3 h at room temperature, the reac-
tion mixture was concentrated, neutralized by aqueous saturated
NaHCO3 solution and extracted with EtOAc. The combined layers
were dried over Na2SO4 and concentrated to give the title
5.9. 5-Phenyl-1-(tetrahydro-pyran-2-yl)-1H-pyrazole (14)
To a solution of 3-phenyl-1H-pyrazole (0.9 g, 6.24 mmol) in tol-
uene (6 ml) was added trifluoroacetic acid (0.025 ml, 0.037 g,
0.32 mmol). The mixture was heated to 80 °C and to it was added

A. D. Velankar et al. / Bioorg. Med. Chem. 18 (2010) 4547–4559
4555
compound (0.2 g, 55%). 1H NMR (DMSO-d6) d 12.01 (br, 1H), 8.1 (s,
5.13.7. 2-(5-Phenyl-1H-pyrazol-3-yl)-1H-indole-6-carboxylic
1H), 7.86 (s, 1H), 7.83 (s, 1H), 7.64 (d, 1H, J = 8.1 Hz), 7.58 (t, 1H,
J = 8.1 Hz), 7.49 (t, 2H, J = 8.1 Hz), 7.37 (t, 1H, J = 7.2 Hz), 7.19 (s,
1H), 6.88 (s, 1H); LC–ESMS m/z 304.13 [M+H]+ (Calcd for
C18H14N3O2).
acid isobutyl-methyl-amide (24)
Prepared by the general procedure as in Section 5.8.
1H NMR (DMSO-d6) d 13.37 (br s, 1H), 11.74 (s, 1H), 7.84 (d, 1H,
J = 1.5 Hz), 7.81 (s, 1H), 7.56 (d, 1H, J = 8.4 Hz), 7.49 (t, 2H,
J = 7.5 Hz), 7.40 (d, 1H, J = 1.5 Hz), 7.37 (t, 1H, J = 7.5 Hz), 7.15 (s,
1H), 7.00 (d, 1H, J = 7.8 Hz), 6.85 (s, 1H), 3.42–3.40 (m, 2H), 2.95
(s, 3H), 2.00 (s, 1H), 0.91–0.78 (br, 6H); LC–HRMS (ESI+) m/z
373.2022 [M+H]+ (Calcd for C23H25N4O: 373.20229).
5.13. General procedure
5.13.1. 2-(5-Phenyl-1H-pyrazol-3-yl)-1H-indole-6-carboxylic
acid (2-methoxy-ethyl)-amide (18)
Prepared by the general procedure as in Section 5.8.
1H NMR (DMSO-d6) d 13.50 (s, 1H), 11.76 (s, 1H), 8.39 (s, 1H),
7.95 (s, 1H), 7.82 (d, 2H, J = 7.5 Hz), 7.60–7.48 (m, 4H), 7.41 (d,
1H, J = 7.8 Hz), 7.18 (s, 1H), 6.83 (s, 1H), 3.49–3.41 (m, 4H), 3.28
(s, 3H); LC–HRMS (ESI+) m/z 361.16578 [M+H]+ (Calcd for
C21H21N4O2: 361.1659).
5.14. 1-(Tetrahydro-pyran-2-yl)-1H-pyrazole (25)
A solution of pyrazole (12.0 g, 176.26 mmol) and trifluoroacetic
acid (2 ml, 3.06 g, 26.83 mmol) in toluene (35 ml) was heated to
80 °C and to it was added 3,4-dihydro-2H-pyran (15.7 ml,
15.57 g, 185.1 mmol). After stirring the reaction mixture at 80 °C
for 2 h, the toluene was evaporated. The resulting residue was di-
luted with water and extracted with EtOAc. The combined extracts
were washed with brine, dried over sodium sulfate and concen-
trated in vacuo. The residue was purified by column chromatogra-
phy with hexanes–EtOAc as eluent to afford the title compound
(24.1 g, 90%) as a pale yellow liquid. 1H NMR (DMSO-d6) d 7.86
(1H, d, J = 2.4 Hz), 7.47 (1H, d, J = 1.5 Hz), 6.29 (1H, d, J = 1.8 Hz),
5.39 (1H, dd, J = 10.2, 2.7 Hz), 3.94–3.87 (1H, m), 3.65–3.57 (1H,
m), 2.13–2.02 (1H, m), 1.96–1.84 (2H, m), 1.73–1.60 (1H, m),
1.56–1.47 (2H, m); GC–MS (EI) m/z 151.91 [M+].
5.13.2. 2-(5-Phenyl-1H-pyrazol-3-yl)-1H-indole-6-carboxylic
acid (6-trifluoromethyl-pyridin-3-ylmethyl)-amide (19)
Prepared by the general procedure as in Section 5.8.
1H NMR (DMSO-d6) d 13.51 (s, 1H), 11.83 (s, 1H), 9.11 (s, 1H),
8.76 (d, 1H, J = 1.8 Hz), 8.02 (dd, 1H, J = 8.1, 1.8 Hz), 7.99 (s, 1H),
7.89 (d, 1H, J= 8.1 Hz), 7.84 (s, 1H), 7.82 (s, 1H), 7.59 (s, 2H), 7.49
(t, 2H, J = 7.5 Hz), 7.38 (d, 1H, J = 6.3 Hz), 7.18 (s, 1H), 6.88 (s,
1H), 4.62 (d, 2H, J = 5.7 Hz); LC–HRMS (ESI+) m/z 462.1534
[M+H]+ (Calcd for C25H19F3N5O: 462.15362).
5.13.3. 2-(5-Phenyl-1H-pyrazol-3-yl)-1H-indole-6-carboxylic
acid (pyrazin-2-ylmethyl)-amide (20)
5.15. 5-Benzyl-1-(tetrahydro-pyran-2-yl)-1H-pyrazole (26)
Prepared by the general procedure as in Section 5.8.
1H NMR (DMSO-d6) d 13.51 (s, 1H), 11.80 (s, 1H), 9.08 (s, 1H),
8.65 (s, 1H), 8.60 (dd, 1H, J = 2.7, 1.5 Hz), 8.54 (d, 1H, J = 2.7 Hz),
8.00 (s, 1H), 7.82 (d, 2H, J = 7.5 Hz), 7.64 (s, 1H), 7.58 (s, 1H), 7.51
(t, 2H, J = 7.5 Hz), 7.39 (t, 1H, J = 7.5 Hz), 7.19 (s, 1H), 6.85 (s, 1H),
4.64 (d, 2H, J = 5.7 Hz), LC–HRMS (ESI+) m/z 395.16084 [M+H]+
(Calcd for C23H19N6O: 395.16149).
To a stirred solution of 25 (20 g, 131.4 mmol) in anhydrous THF
(700 ml) at ꢀ78 °C was added n-BuLi (83 ml, 131.8 mmol) and the
reaction mixture was stirred at ꢀ78 °C for 1 h. Benzyl bromide
(22.7 g, 132.2 mmol) was added dropwise at ꢀ78 °C and the reac-
tion mixture was warmed to room temperature over 3 h and stir-
red overnight. The reaction mixture was quenched at 0 °C by ice-
cold water and extracted with EtOAc. The combined extracts were
washed with brine, dried over sodium sulfate and concentrated in
vacuo. The residue was purified by column chromatography with
hexanes–EtOAc as eluent to yield the title compound (12.0 g,
38%). 1H NMR (DMSO-d6) d 7.37 (d, 1H, J = 1.8 Hz), 7.34–7.29 (m,
2H), 7.25–7.20 (m, 4H), 5.95 (d, 1H, J = 1.5 Hz), 5.36 (dd, 1H,
J = 9.9, 2.7 Hz), 4.06 (s, 2H), 3.87–3.80 (m, 1H), 3.61–3.53 (m, 1H),
2.31–2.18 (m, 1H), 1.80–1.72 (m, 1H), 1.62–1.45 (m, 4H); GC–MS
(EI) m/z 242.02 [M+].
5.13.4. 2-(5-Phenyl-1H-pyrazol-3-yl)-1H-indole-6-carboxylic
acid (3-methyl-3H-imidazol-4-ylmethyl)-amide (21)
Prepared by the general procedure as in Section 5.8.
1H NMR (DMSO-d6) d 13.33 (br s, 1H), 11.78 (s, 1H), 8.74 (s, 1H),
7.96 (s, 1H), 7.84 (d, 1H, J = 1.5 Hz), 7.81 (s, 1H), 7.55–7.52 (m, 3H),
7.49 (t, 2H, J = 7.5 Hz), 7.37 (t, 1H, J = 7.5 Hz), 7.77 (s, 1H), 6.86 (s,
1H), 6.83 (s, 1H), 4.48 (d, 2H, J = 4.5 Hz), 3.64 (s, 3H); LC–HRMS
(ESI+) m/z 397.1771 [M+H]+ (Calcd for C23H21N6O: 397.17714).
5.16. 3-Benzyl-1-(tetrahydro-pyran-2-yl)-1H-pyrazole (27)
5.13.5. 2-(5-Phenyl-1H-pyrazol-3-yl)-1H-indole-6-carboxylic
acid (2-methyl-2H-pyrazol-3-ylmethyl)-amide (22)
To a solution of 26 (0.6 g, 2.48 mmol) in MeOH (5 ml) at 0 °C
was added ethereal HCl (15 ml). The reaction mixture was then
stirred overnight at room temperature and then concentrated in
vacuo. The residue was neutralized with aqueous NaHCO3 solution
and extracted with EtOAc. The combined extracts were washed
with brine, dried over sodium sulfate and concentrated in vacuo.
To the resulting residue were added trifluoroacetic acid (0.01 ml,
0.015 g, 0.13 mmol) and toluene (10 ml). The mixture was heated
to 80 °C and to it was added 3,4-dihydro-2H-pyran (0.25 ml,
0.23 g, 2.74 mmol). After stirring the reaction mixture overnight
at 80 °C, the toluene was evaporated. The resulting residue was di-
luted with water and extracted with EtOAc. The combined extracts
were washed with brine, dried over sodium sulfate and concen-
trated in vacuo. The residue was purified by column chromatogra-
phy with hexanes–EtOAc (80:20 v/v) to afford the title compound
(0.5 g, 83%). 1H NMR (DMSO-d6) d 7.74 (d, 1H, J = 2.4 Hz), 7.30–7.17
(m, 5H), 6.06 (d, 1H, J = 2.4 Hz), 5.30 (dd, 1H, J = 10.5, 2.7 Hz), 3.93–
3.87 (m, 1H), 3.86 (s, 2H), 3.63–3.54 (m, 1H), 2.10–2.01 (m, 1H),
Prepared by the general procedure as in Section 5.8.
1H NMR (DMSO-d6) d 13.41 (br s, 1H), 11.87 (s, 1H), 8.89 (t, 1H,
J = 5.7 Hz), 7.97 (s, 1H), 7.84 (d, 1H, J = 3.0 Hz), 7.82 (s, 1H), 7.57–
7.56 (m, 2H), 7.49 (t, 2H, J = 7.5 Hz), 7.37 (t, 1H, J = 7.5 Hz), 7.31
(d, 1H, J = 2.1 Hz), 7.18 (s, 1H), 6.82 (s, 1H), 6.17 (d, 1H,
J = 1.8 Hz), 4.54 (d, 2H, J = 5.4 Hz), 3.85 (s, 3H); LC–HRMS (ESI+)
m/z 397.17645 [M+H]+ (Calcd for C23H21N6O: 397.17714).
5.13.6. 2-(5-Phenyl-1H-pyrazol-3-yl)-1H-indole-6-carboxylic
acid (5-methyl-isoxazol-4-ylmethyl)-amide (23)
Prepared by the general procedure as in Section 5.8.
1H NMR (DMSO-d6) d 13.51 (s, 1H), 11.78 (s, 1H), 8.78 (t, 1H,
J = 5.7 Hz), 8.41 (s, 1H), 7.94 (s, 1H), 7.83 (d, 1H, J = 1.5 Hz), 7.80
(s, 1H), 7.57 (s, 1H), 7.54 (s, 1H), 7.51 (t, 2H, J = 8.1 Hz), 7.45–
7.37 (m, 1H), 7.19 (d, 1H, J = 1.8 Hz), 6.82 (d, 1H, J = 1.8 Hz), 4.26
(d, 2H, J = 5.7 Hz), 2.45 (s, 3H); LC–HRMS (ESI+) m/z 398.16079
[M+H]+ (Calcd for C23H20N5O2: 398.16115).

4556
A. D. Velankar et al. / Bioorg. Med. Chem. 18 (2010) 4547–4559
1.95–1.82 (m, 2H), 1.71–1.57 (m, 1H), 1.55–1.46 (m, 2H); GC–MS
reaction mixture was refluxed overnight. The reaction mixture
was then concentrated in vacuo, acidified with 1 N HCl and the
resulting precipitate was filtered to afford the title compound
(0.51 g, 89%). 1H NMR (DMSO-d6) d 11.73 (s, 1H), 7.99 (d, 1H,
J = 0.9 Hz), 7.56 (d, 1H, J = 1.5 Hz),7.54 (s, 1H), 7.34–7.33 (m, 1H),
7.32–7.29 (m, 3H), 7.26–7.21 (m, 1H), 6.77 (d, 1H, J = 1.2 Hz),
6.49 (s, 1H), 4.01 (s, 2H); LC–ESMS m/z 318.13 [M+H]+.
(EI) m/z 242.02 [M+] (Calcd for C15H18N2O).
5.17. 3-Benzyl-5-iodo-1-(tetrahydro-pyran-2-yl)-1H-pyrazole (28)
To a stirred solution of 27 (4.2 g, 17.3 mmol) in anhydrous THF
(15 ml) at ꢀ78 °C was added n-BuLi (11 ml, 17.6 mmol) and the
reaction mixture was stirred at ꢀ78 °C for 30 min. A solution of
I2 (4.5 g, 17.7 mmol) in THF (10 ml) was added dropwise at
ꢀ78 °C, the reaction mixture was warmed to room temperature
over 2 h and stirred for 3 h. The reaction mixture was quenched
at 0 °C by a saturated aqueous solution of NaHSO3 and extracted
with EtOAc. The combined extracts were washed with brine, dried
over sodium sulfate and concentrated in vacuo. The residue was
purified by column chromatography with hexanes–EtOAc as eluent
to yield the title compound (2.6 g, 41%). 1H NMR (DMSO-d6) d
7.30–7.16 (m, 5H), 6.31 (s, 1H), 5.30 (dd, 1H, J = 10.2, 2.7 Hz),
3.93–3.87 (m, 1H), 3.86 (s, 2H), 3.62–3.53 (m, 1H), 2.35–2.22 (m,
1H), 2.01–1.93 (br m, 1H), 1.87–1.79 (m, 1H), 1.75–1.61 (m, 1H),
1.55–1.46 (m, 2H); GC–MS (EI) m/z 367.99 [M+].
5.21. General procedure
5.21.1. 2-(5-Benzyl-1H-pyrazol-3-yl)-1H-indole-6-carboxylic
acid (3-methyl-3H-imidazol-4-ylmethyl)-amide (32)
Prepared by the general procedure as in Section 5.8.
1H NMR (DMSO-d6) d 12.92 (s, 1H), 11.64 (s, 1H), 8.70 (s, 1H),
7.90 (s, 1H), 7.54 (s, 1H), 7.49 (s, 2H), 7.37–7.28 (m, 4H), 7.25–
7.21 (m, 1H), 6.82 (s, 1H), 6.71 (s, 1H), 6.48 (s, 1H), 4.47 (d, 2H,
J = 5.4 Hz), 4.02 (s, 2H), 3.63 (s, 3H); LC–HRMS (ESI+) m/z
411.19334 [M+H]+ (Calcd for C24H23N6O: 411.19279).
5.21.2. 2-(5-Benzyl-1H-pyrazol-3-yl)-1H-indole-6-carboxylic
acid (2-methyl-2H-pyrazol-3-ylmethyl)-amide (33)
5.18. 2-[5-Benzyl-2-(tetrahydro-pyran-2-yl)-2H-pyrazol-3-yl]-
indole-1,6-dicarboxylic acid 1-tert-butyl ester 6-methyl ester
(29)
Prepared by the general procedure as in Section 5.8.
1H NMR (DMSO-d6) d 12.93 (s, 1H), 11.66 (s, 1H), 8.84 (s, 1H),
7.90 (s, 1H), 7.50 (s, 2H), 7.34 (s, 1H), 7.32–7.30 (m, 4H), 7.29 (d,
1H, J = 1.8 Hz), 6.72 (s, 1H), 6.48 (s, 1H), 6.15 (d, 1H, J 1.5 Hz),
4.51 (d, 2H, J = 5.7 Hz), 4.02 (s, 2H), 3.83 (s, 3H); LC–HRMS (ESI+)
m/z 411.19234 [M+H]+ (Calcd for C24H23N6O: 411.19279).
A pressure bottle containing a solution of 28 (2.5 g, 6.79 mmol)
and 1-(t-butoxycarbonyl)-6-(methoxycarbonyl)-1H-indol-2-ylbo-
ronic acid (3.24 g, 10.15 mmol) in 1,4-dioxane (30 ml) was purged
with nitrogen for 3 min. To it was added in succession Cs2CO3
(6.62 g, 20.32 mmol) and [1,10-Bis(diphenylphosphino)ferro-
cene]dichloropalladium(II), complex with dichloromethane
(0.39 g, 0.48 mmol), the reaction mixture was purged with nitro-
gen again for 3 min and the reaction bottle was sealed. The reac-
tion mixture was heated at 80 °C for 6 h, cooled to room
temperature, diluted with water and extracted with EtOAc. The
combined extracts were washed with brine, dried over sodium sul-
fate and concentrated in vacuo. The residue was purified by col-
umn chromatography with hexanes–EtOAc as eluent to yield the
title compound (1.1 g, 31%). 1H NMR (DMSO-d6) d 8.80 (t, 1H,
J = 0.9 Hz), 7.88 (dd, 1H, J = 8.1, 1.5 Hz), 7.76 (d, 1H, J = 8.1 Hz),
7.34–7.26 (m, 4H), 7.23–7.16 (m, 1H), 6.92 (d, 1H, J = 0.6 Hz),
6.27 (s, 1H), 5.04 (dd, 1H, J = 9.9, 2.4 Hz), 3.92 (s, 2H), 3.89 (s,
3H), 3.87–3.79 (m, 2H), 2.26–2.15 (m, 1H), 1.90–1.82 (m, 1H),
1.80–1.70 (br, 1H), 1.60–1.35 (m, 3H), 1.19 (s, 9H); LC–ESMS m/z
432.40 [MꢀTHP+H]+.
5.21.3. 2-(5-Benzyl-1H-pyrazol-3-yl)-1H-indole-6-carboxylic
acid isobutyl-methyl-amide (34)
Prepared by the general procedure as in Section 5.8.
1H NMR (DMSO-d6) d 12.90 (s, 1H), 11.50 (s, 1H), 7.49 (d, 1H,
J = 8.4 Hz), 7.34–7.29 (m, 5H), 7.26–7.22 (m, 1H), 6.96 (d, 1H,
J = 8.4 Hz), 6.69 (s, 1H), 6.45 (s, 1H), 4.02 (s, 2H), 3.42–3.40 (m,
2H), 2.93 (s, 3H), 1.99 (s, 1H), 0.85 (br, 6H); LC–HRMS (ESI+) m/z
387.21812 [M+H]+ (Calcd for C24H27N4O: 387.21794).
5.21.4. 2-(5-Benzyl-1H-pyrazol-3-yl)-1H-indole-6-carboxylic
acid (pyrazin-2-ylmethyl)-amide (35)
Prepared by the general procedure as in Section 5.8.
1H NMR (CDCl3) d 10.51 (s, 1H), 8.65 (s, 1H), 8.44 (s, 2H), 8.06 (s,
1H), 7.60–7.53 (m, 2H), 7.45 (dd, 1H, J = 8.4, 1.5 Hz), 7.28–7.25 (m,
3H), 7.24–7.21 (m, 2H), 6.70 (s, 1H), 6.39 (s, 1H), 5.47 (br s, 1H),
4.79 (d, 2H, J = 5.1 Hz), 4.02 (s, 2H); LC–HRMS (ESI+) m/z
409.17741 [M+H]+ (Calcd for C24H21N6O: 409.17714).
5.19. 2-(5-Benzyl-2H-pyrazol-3-yl)-1H-indole-6-carboxylic acid
methyl ester (30)
5.21.5. 2-(5-Benzyl-1H-pyrazol-3-yl)-1H-indole-6-carboxylic
acid methylamide (36)
To 29 (1.1 g, 2.13 mmol) at 0 °C was added 4 M HCl in 1,4-diox-
ane (15 ml). The reaction mixture was stirred overnight and then
concentrated in vacuo. To the crude solid were added K2CO3
(0.67 g, 4.85 mmol) and MeOH (10 ml) and the reaction mixture
was stirred at room temperature for 5 h. The reaction mixture
was acidified at 0 °C with 1 N HCl to pH 3, the resulting precipitate
was filtered and dried to give pure the title compound (0.6 g, 85%).
1H NMR (DMSO-d6) d 11.84 (s, 1H), 8.01 (d, 1H, J = 0.9 Hz), 7.58 (d,
1H, J = 1.2 Hz), 7.57 (s, 1H), 7.36–7.29 (m, 4H), 7.26–7.20 (m, 2H),
6.80 (dd, 1H, J = 2.1, 0.9 Hz), 6.51 (s, 1H), 4.02 (s, 2H), 3.84 (s,
3H); LC–ESMS m/z 332.07 [M+H]+.
Prepared by the general procedure as in Section 5.8.
1H NMR (DMSO-d6) d 12.91 (s, 1H), 11.62 (s, 1H), 8.28 (s, 1H),
7.87 (s, 1H), 7.48 (s, 2H), 7.36–7.28 (m, 4H), 7.26–7.23 (m, 1H),
6.70 (s, 1H), 6.48 (s, 1H), 4.02 (s, 2H), 2.79 (d, 3H, J = 4.5 Hz); LC–
HRMS (ESI+) m/z 331.15508 [M+H]+ (Calcd for C20H19N4O:
331.15534).
5.21.6. 2-(5-Benzyl-1H-pyrazol-3-yl)-1H-indole-6-carboxylic
acid (5-methyl-isoxazol-4-ylmethyl)-amide (37)
Prepared by the general procedure as in Section 5.8.
1H NMR (DMSO-d6) d 12.92 (s, 1H), 11.64 (s, 1H), 8.74 (t, 1H,
J = 6.3 Hz), 8.40 (s, 1H), 7.88 (s, 1H), 7.48 (s, 2H), 7.36–7.22 (m,
5H), 6.71 (s, 1H), 6.48 (s, 1H), 4.25 (d, 2H, J = 5.7 Hz), 4.02 (s, 2H),
2.44 (s, 3H); LC–HRMS (ESI+) m/z 412.17724 [M+H]+ (Calcd for
C24H22N5O2: 412.1768).
5.20. 2-(5-Benzyl-2H-pyrazol-3-yl)-1H-indole-6-carboxylic acid
(31)
To a solution of 30 (0.6 g, 1.81 mmol) in EtOH (10 ml) was
added 5% aqueous KOH (0.203 g, 3.62 mmol in 4 ml H2O) and the

A. D. Velankar et al. / Bioorg. Med. Chem. 18 (2010) 4547–4559
4557
5.22. 2-(4-Phenyl-1H-pyrazol-3-yl)-1H-indole-6-carboxylic acid
methyl ester (38)
(s, 3H), 1.98 (m, 1H), 0.84 (br, 6H); LC–HRMS (ESI+) m/z
522.28607 [M+H]+ (Calcd for C32H36N5O2: 522.28635).
To a solution of 5 (0.5 g, 0.97 mmol) in MeOH (12 ml) was
added aqueous KOH (0.17 g, 3.03 mmol, in 2 ml H2O) and the reac-
tion mixture was at room temperature for 3 h. It was then concen-
trated, acidified with 1 N HCl and extracted with EtOAc. The
combined organic layers were dried over Na2SO4 and concentrated
to give the title compound (0.3 g, 97%). 1H NMR (DMSO-d6) d 13.36
(s, 1H), 11.79 (s, 1H), 8.06 (s, 1H), 7.99 (s, 1H), 7.56 (d, 1H,
J = 8.1 Hz), 7.50–7.38 (m, 5H), 7.34 (d, 1H, J = 6.9 Hz), 6.32 (s, 1H),
3.84 (s, 3H); LC–ESMS m/z 318.13 [M+H]+.
5.26.2. 3-[(3-Hydroxy-propylamino)-methyl]-2-(4-phenyl-1H-
pyrazol-3-yl)-1H-indole-6-carboxylic acid isobutyl-methyl-
amide (43)
Prepared by the procedure in Section 5.26.1 for compound 42.
1H NMR (CDCl3) d 8.49 (s, 1H), 7.68 (s, 1H), 7.59 (d, 1H,
J = 8.1 Hz), 7.45–7.40 (m, 4H), 7.39–7.35 (m, 1H), 7.24 (s, 1H),
7.14 (d, 1H, J = 8.4 Hz), 4.08 (s, 2H), 3.83 (t, 2H, J = 5.7 Hz), 3.38
(br, 2H), 3.17 (br, 3H), 2.95 (t, 3H, J = 6.0 Hz), 1.85 (t, 3H,
J = 6.3 Hz), 1.67 (s, 1H), 0.87 (d, 6H, J = 6.9 Hz); LC–HRMS (ESI+)
m/z 460.27014 [M+H]+ (Calcd for C27H34N5O2: 460.2707).
5.23. 3-Formyl-2-(4-phenyl-1H-pyrazol-3-yl)-1H-indole-6-
carboxylic acid methyl ester (39)
5.26.3. 3-Hydroxymethyl-2-(4-phenyl-1H-pyrazol-3-yl)-1H-
indole-6-carboxylic acid isobutyl-methyl-amide (44)
To a solution of 41 (15 mg, 0.037 mmol) in CH3OH at 0 °C was
added NaBH4 (3 mg, 0.08 mmol) and the reaction was stirred at
room temperature for 30 min. The reaction was quenched with
water (5 ml) and extracted with EtOAc. The combined extracts
were washed with brine, dried over sodium sulfate and concen-
trated in vacuo. The residue was purified by column chromatogra-
phy with CH3OH/CH2Cl2 (8:92) as eluent to yield the title
compound (13 mg, 85%). 1H NMR (DMSO-d6) d 7.98 (s, 1H), 7.81
(d, 1H, J = 8.1 Hz), 7.40 (s, 1H), 7.31–7.26 (m, 3H), 7.25 (s, 1H),
7.22–7.19 (m, 1H), 7.18–7.12 (m, 1H), 4.66 (s, 2H), 3.44–3.43 (m,
1H), 3.24–3.23 (m, 1H), 3.07 (s, 3H), 2.00–1.97 (m, 1H), 1.02 (d,
3H, J = 5.1 Hz), 0.75 (d, 3H, J = 6.0 Hz); LC–HRMS (ESI+) m/z
403.21255 [M+H]+ (Calcd for C24H27N4O2: 403.21285).
To DMF (1.1 g 15.1 mmol) at 0 °C was added phosphorous oxy-
chloride (2.32 g, 15.1 mmol). The reaction mixture was stirred at
room temperature for 1 h and to it was then slowly added a solu-
tion of 38 (0.48 g, 1.51 mmol) in DMF (2 ml). After stirring for 3 h at
70 °C, the reaction was quenched with H2O and the resulting solid
was filtered to afford the title compound (0.35 g, 67%). 1H NMR
(DMSO-d6) d 13.75 (s, 1H), 12.65 (s, 1H), 9.59 (s, 1H), 8.29 (s,
1H), 8.19 (d, 1H, J = 8.7 Hz), 8.07 (s, 1H), 7.84 (dd, 1H, J = 8.7,
1.5 Hz), 7.30 (d, 1H, J = 1.5 Hz), 7.30–7.26 (m, 3H), 7.25–7.23 (m,
1H), 3.87 (s, 3H); LC–ESMS m/z 346.13 [M+H]+.
5.24. 3-Formyl-2-(4-phenyl-1H-pyrazol-3-yl)-1H-indole-6-
carboxylic acid (40)
5.27. 2-(5-Benzyl-2H-pyrazol-3-yl)-3-formyl-1H-indole-6-
carboxylic acid methyl ester (45)
Prepared from 39 by the procedure in Section 5.7 for compound
6.
1H NMR (DMSO-d6) d 13.70 (s, 1H), 12.41 (s, 1H), 9.59 (s, 1H),
8.31 (d, 1H, J = 1.5 Hz), 8.12 (d, 1H, J = 7.8 Hz), 7.43 (s, 1H), 7.31
(s, 1H), 7.30–7.26 (m, 3H), 7.25–7.23 (m, 1H), 7.22–7.20 (m, 1H);
LC–ESMS m/z 332.13 [M+H]+.
To DMF (0.33 g, 4.52 mmol) at 0 °C was added phosphorous
oxychloride (0.69 g, 4.52 mmol). The reaction mixture was stirred
at room temperature for 1 h and to it was then slowly added a
solution of 30 (0.6 g, 1.81 mmol) in DMF (2 ml). After stirring for
4 h at room temperature, the reaction was quenched with H2O,
the resulting solid was filtered and dried to afford the title com-
pound (0.45 g, 69%). 1H NMR (DMSO-d6) d 12.65 (s, 1H), 10.56 (s,
1H), 8.47 (br s, 1H), 8.23 (d, 1H, J = 8.1 Hz), 8.06 (s, 1H), 7.80 (dd,
1H, J = 8.4, 1.5 Hz), 7.38–7.32 (m, 4H), 7.28–7.23 (m, 1H), 6.78 (s,
1H), 4.09 (s, 2H), 3.87 (s, 3H); LC–ESMS m/z 360.13 [M+H]+.
5.25. 3-Formyl-2-(4-phenyl-1H-pyrazol-3-yl)-1H-indole-6-
carboxylic acid isobutyl-methyl-amide (41)
Prepared by the general procedure as in Section 5.8.
1H NMR (DMSO-d6) d 13.69 (s, 1H), 12.41 (s, 1H), 9.58 (s, 1H),
8.30 (d, 1H, J = 1.5 Hz), 8.12 (d, 1H, J = 7.8 Hz), 7.94 (s, 1H), 7.42
(s, 1H), 7.30–7.26 (m, 5H), 2.95 (br, 2H), 2.93 (s, 3H), 1.98 (m,
1H), 0.83 (br, 6H); LC–ESMS m/z 401.20 [M+H]+.
5.28. 5.282-(5-Benzyl-2H-pyrazol-3-yl)-3-formyl-1H-indole-6-
carboxylic acid (46)
5.26. General procedure
To a solution of 45 (0.45 g, 1.25 mmol) in EtOH (10 ml) was
added 5% aqueous KOH (0.3 g, 5.34 mmol in 6 ml H2O) and the
reaction mixture was refluxed overnight. The reaction mixture
was then concentrated in vacuo, acidified with 1 N HCl and the
resulting precipitate was filtered to afford the title compound
(0.3 g, 69%). 1H NMR (DMSO-d6) d 12.59 (s, 1H), 10.56 (s, 1H),
8.21 (d, 1H, J = 8.1 Hz), 8.05 (s, 1H), 7.79 (dd, 1H, J = 8.1, 1.5 Hz),
7.35–7.32 (m, 4H), 7.28–7.23 (s, 1H), 6.77 (s, 1H), 4.09 (s, 2H);
LC–ESMS m/z 346.20 [M+H]+.
5.26.1. 3-[(4-Methoxy-benzylamino)-methyl]-2-(4-phenyl-1H-
pyrazol-3-yl)-1H-indole-6-carboxylic acid isobutyl-methyl-
amide (42)
To a solution of 41 (0.1 g, 0.25 mmol) in MeOH (8 ml) was
added 4-methoxybenzylamine (0.035 g, 0.25 mmol) followed by
catalytic amount of glacial acetic acid. The reaction was stirred at
room temperature for 12 h, cooled to 0 °C and treated with sodium
borohydride (0.014 g, 0.37 mmol). After stirring at 0 °C for 15 min,
the reaction was quenched with water and extracted with EtOAc.
The combined extracts were washed with brine, dried over sodium
sulfate and concentrated in vacuo. The residue was purified by col-
umn chromatography with CH3OH/CH2Cl2 (5:95) as eluent to yield
the title compound (0.022 g, 17%). 1H NMR (DMSO-d6) d 11.34 (s,
1H), 8.12 (s, 1H), 7.70 (d, 1H, J = 8.1 Hz), 7.32 (s, 1H), 7.29–7.25
(m, 4H), 7.18 (dd, 1H, J = 8.7, 4.5 Hz), 7.11 (d, 2H, J = 8.7 Hz), 7.04
(d, 1H, J = 7.5 Hz), 6.82 (d, 2H, J = 8.7 Hz), 3.72 (s, 3H), 3.71 (s,
2H), 3.54 (s, 2H), 3.42–3.40 (m, 2H, obscured by H2O signal), 2.94
5.29. General procedure
5.29.1. 2-(5-Benzyl-1H-pyrazol-3-yl)-3-formyl-1H-indole-6-
carboxylic acid (3-methyl-3H-imidazol-4-ylmethyl)-amide (47)
Prepared by the general procedure as in Section 5.8,
yield = 0.28 g, 69%.
1H NMR (DMSO-d6) d 13.44 (s, 1H), 12.48 (s, 1H), 10.55 (s, 1H),
8.86 (t, 1H, J = 5.4 Hz), 8.16 (d, 1H, J = 8.1 Hz), 7.95 (s, 1H), 7.70 (t,
1H, J = 8.4 Hz), 7.57 (s, 1H), 7.38–7.32 (m, 4H), 7.28–7.24 (m, 1H),

4558
A. D. Velankar et al. / Bioorg. Med. Chem. 18 (2010) 4547–4559
6.85 (s, 1H), 6.74 (s, 1H), 4.48 (d, 2H, J = 5.4 Hz), 4.08 (s, 2H), 3.64 (s,
3H); LC–ESMS m/z 439.27 [M+H]+.
1H), 1.50–1.43 (m, 4H); LC–HRMS (ESI+) m/z 432.23967 [M+H]+
(Calcd for C25H30N5O2: 432.2394).
5.29.2. 2-(5-Benzyl-1H-pyrazol-3-yl)-3-formyl-1H-indole-6-
carboxylic acid isobutyl-methyl-amide (48)
5.30.5. 2-(5-Benzyl-1H-pyrazol-3-yl)-3-hydroxymethyl-1H-
indole-6-carboxylic acid (3-methyl-3H-imidazol-4-ylmethyl)-
amide (54)
Prepared by the procedure in Section 5.26.3 for compound 44.
1H NMR (DMSO-d6) d 13.1 (br s, 1H), 11.52 (s, 1H), 8.73 (t, 1H,
J = 5.7 Hz), 7.89 (s, 1H), 7.64 (d, 1H, J = 8.4 Hz), 7.53 (dd, 1H,
J = 8.4, 1.5 Hz), 7.52 (s, 1H), 7.35–7.30 (m, 4H), 7.26–7.21 (m, 1H),
6.82 (d, 1H, J = 1.2 Hz), 6.54 (s, 1H), 4.93 (s, 1H), 4.81 (s, 2H), 4.46
(d, 2H, J = 5.1 Hz), 4.04 (s, 2H), 3.63 (s, 3H); LC–HRMS (ESI+) m/z
441.20352 (Calcd for C25H25N6O2: 441.20335).
Prepared by the general procedure as in Section 5.8,
yield = 0.51 g, 85%
1H NMR (DMSO-d6) d 13.43 (s, 1H), 12.37 (s, 1H), 10.56 (s, 1H),
8.16 (d, 1H, J = 8.1 Hz), 7.40 (s, 1H), 7.36 (d, 1H, J = 0.9 Hz), 7.35 (s,
1H), 7.33–7.32 (m, 2H), 7.28–7.22 (m, 1H), 7.18–7.14 (m, 1H), 6.72
(d, 1H, J = 2.1 Hz), 4.08 (s, 2H), 3.42–3.40 (m, 2H), 2.92 (s, 3H), 1.99
(s, 1H), 0.85 (br, 6H); LC–ESMS m/z 415.20 [M+H]+.
5.29.3. 2-(5-Benzyl-1H-pyrazol-3-yl)-3-formyl-1H-indole-6-
carboxylic acid methylamide (49)
5.31. Methods for biological assays
Prepared by the general procedure as in Section 5.8.
1H NMR (DMSO-d6) d 13.45 (s, 1H), 12.48 (s, 1H), 10.56 (s, 1H),
8.44 (d, 1H, J = 4.2 Hz), 8.16 (d, 1H, J = 8.1 Hz), 7.93 (s, 1H), 7.68 (dd,
1H, J = 8.4, 1.5 Hz), 7.39–7.32 (m, 4H), 7.28–7.22 (m, 1H), 6.75 (d,
1H, J = 2.1 Hz), 4.08 (s, 2H), 2.80 (d, 3H, J = 4.5 Hz); LC–ESMS m/z
359.20 [M+H]+.
5.31.1. ITK enzymatic activity (IMAP assay)
GST-ITK full length was from Invitrogen (PV3875). Substrate
was FAM-Blk peptide (5-FAM-EFPIYDFLPAKKK-NH2). Reaction
conditions were as following: 10 ng ITK/well in a final volume of
20
0.01% Tween; 5 mM DTT; 0.05% BSA) containing 500 nM substrate,
M ATP. The reaction time was 2 h and the binding time was 2 h.
The reaction was stopped by addition of 60 l binding solution
ll of final reaction buffer (20 mM Hepes pH 7.4; 2.5 mM MgCl2;
6
l
5.30. General procedure
l
(Progressive Binding IMAP system, 60% buffer A: 40% buffer B,
beads 1:1000, Molecular Devices). Signal was detected with an
envision reader (PE) by measuring fluorescence polarization at
485 nm/535 nm.
5.30.1. 2-(5-Benzyl-1H-pyrazol-3-yl)-3-morpholin-4-ylmethyl-
1H-indole-6-carboxylic acid (3-methyl-3H-imidazol-4-
ylmethyl)-amide (50)
Prepared by the procedure in Section 5.26.1 for compound 42.
1H NMR (DMSO-d6) d 13.01 (s, 1H), 11.55 (s, 1H), 8.71 (s, 1H),
7.88 (s, 1H), 7.67 (s, 1H), 7.64 (s, 1H), 7.54 (s, 1H), 7.34–7.31 (m,
4H), 7.26–7.22 (m, 1H), 6.82 (s, 1H), 6.51 (s, 1H), 4.47 (dd, 2H,
J = 4.8, 2.7 Hz), 4.40 (t, 1H, J = 5.7 Hz), 4.02 (s, 2H), 3.77 (s, 2H),
3.62 (s, 3H), 3.55–3.46 (m, 3H), 2.45–2.34 (m, 4H); LC–HRMS
(ESI+) m/z 510.26122 [M+H]+ (Calcd for C29H32N7O2: 510.2612).
5.31.2. Insulin receptor (InsR) enzymatic activity (IMAP assay)
The enzyme was a GST-fusion protein of the recombinant cata-
lytic domain (AA 987-1381) expressed in Sf21 cells infected with
recombinant Baculovirus. Substrate was FAM-peptide (5-FAM-
KKSRGDYMTMQIG-NH2). The reaction conditions were as follow-
ing: 25 ng InsR/well in a final volume of 20
fer (50 mM Tris pH 7.5; 10 mM MnCl2; 5 mM DTT; 0.01% BSA)
containing 100 nM substrate, 3 M ATP. The reaction time was
80 min and the binding time was 2 h. The reaction was stopped
by addition of 60 l binding solution (Progressive Binding IMAP
ll of final reaction buf-
5.30.2. 2-(5-Benzyl-1H-pyrazol-3-yl)-3-[(4-fluoro-
benzylamino)-methyl]-1H-indole-6-carboxylic acid isobutyl-
methyl-amide (51)
l
l
Prepared by the procedure in Section 5.26.1 for compound 42.
1H NMR (DMSO-d6) d 11.47 (s, 1H), 7.57 (d, 1H, J= 8.4 Hz), 7.40–
7.34 (m, 3H), 7.33–7.31 (m, 3H), 7.30–7.21 (m, 2H), 7.13 (t, 2H,
J = 8.4 Hz), 6.99 (dd, 1H, J = 8.4, 1.5 Hz), 6.42 (s, 1H), 4.04 (s, 2H),
4.01 (s, 2H), 3.79 (s, 2H), 3.42–3.40 (m, 2H, obscured by H2O sig-
nal), 2.93 (s, 3H), 1.98 (br, 1H), 0.85 (br, 6H); LC–HRMS (ESI+) m/z
524.28197 [M+H]+ (Calcd for C32H35FN5O: 524.28202).
system, 85% buffer A: 15% buffer B, beads 1:400, Molecular De-
vices). Signal was detected with an envision reader (PE) by measur-
ing fluorescence polarization at 485 nm/ 535 nm.
5.31.3. PKA enzymatic activity (IMAP assay)
His-PKA (catalytic domain) was from Panvera. Substrate was
FAM- peptide (5-FAM-GRTGRRNSI-NH2). The reaction conditions
were as following: 100 pg PKA/well in a final volume of 20
nal reaction buffer (10 mM Tris pH 7.2; 10 mM MgCl2; 0.05% NaN3;
1 mM DTT; 0.1% BSA) containing 100 nM substrate, 1 M ATP. The
reaction time was 40 min and binding time was 2 h. The reaction
was stopped by addition of 60 l binding solution (Progressive
Binding IMAP system, 100% buffer A, beads 1:400, Molecular De-
vices). Signal was detected with an envision reader (PE) by measur-
ing fluorescence polarization at 485 nm/ 535 nm.
ll of fi-
5.30.3. 2-(5-Benzyl-1H-pyrazol-3-yl)-3-{[(pyridin-4-ylmethyl)-
amino]-methyl}-1H-indole-6-carboxylic acid methylamide (52)
Prepared by the procedure in Section 5.26.1 for compound 42.
1H NMR (DMSO-d6) d 12.95 (br s, 1H), 11.51 (s, 1H), 8.47 (d, 2H,
J = 5.7 Hz), 8.31 (d, 1H, J = 4.5 Hz), 7.85 (s, 1H), 7.55 (d, 1H,
J = 8.4 Hz), 7.48 (dd, 1H, J = 8.1, 1.2 Hz), 7.34–7.33 (m, 3H), 7.32–
7.30 (m, 3H), 7.26–7.21 (m, 2H), 6.47 (s, 1H), 4.01 (overlapping s,
4H), 3.74 (s, 2H), 2.79 (d, 3H, J = 4.5 Hz); LC–HRMS (ESI+) m/z
451.22343 [M+H]+ (Calcd for C27H27N6O: 451.22409).
l
l
5.31.4. Akt1 enzymatic activity (IMAP assay)
His-Akt1 full length was from Upstate. Substrate was FAM- pep-
tide (5-FAM-GRTGRRNSI-NH2). The reaction conditions were as
5.30.4. 2-(5-Benzyl-1H-pyrazol-3-yl)-3-[(4-hydroxy-
butylamino)-methyl]-1H-indole-6-carboxylic acid methylamide
(53)
Prepared by the procedure in Section 5.26.1 for compound 42.
1H NMR (DMSO-d6) d 11.57 (s, 1H), 8.32 (d, 1H, J = 4.5 Hz), 7.84
(s, 1H), 7.63 (d, 1H, J = 8.4 Hz), 7.50 (dd, 1H, J = 8.7, 1.5 Hz), 7.34–
7.32 (m, 3H), 7.26–7.20 (m, 2H), 6.88 (s, 1H), 6.50 (s, 1H), 5.32
(br t, 1H), 4.02 (s, 2H), 2.79 (d, 3H, J = 4.5 Hz), 2.73–2.71 (m, 1H),
2.59 (t, 2H, J = 6.3 Hz), 2.53 (m, 2H), 2.27 (m, 1H), 2.03–1.97 (m,
following: 200 pg Akt1/well in a final volume of 20
tion buffer (10 mM Tris pH 7.2; 10 mM MgCl2; 0.05% NaN3; 1 mM
DTT; 0.1% BSA) containing 100 nM substrate, 20 M ATP. The reac-
tion time was 1 h and the binding time was 2 h. The reaction was
stopped by addition of 60 l binding solution (Progressive Binding
ll of final reac-
l
l
IMAP system, 100% buffer A, beads 1:400, Molecular Devices). Sig-
nal was detected with an envision reader (PE) by measuring fluo-
rescence polarization at 485 nm/ 535 nm.

A. D. Velankar et al. / Bioorg. Med. Chem. 18 (2010) 4547–4559
4559
5.31.5. CDK2 enzymatic activity (IMAP assay)
implemented in Macromodel v. 9.6. The gradient convergence
threshold was set to 0.001.
GST-CDK2/CyclinE full length was from ProQinase. Substrate
was FAM- peptide (5-FAM-GGGPATPKKAKKL-OH). The reaction
conditions were as following: 2 ng CDK2/well in a final volume
Partial atomic charges for compounds as well as the protein
were assigned according to the OPLS-AA force field. The standard
precision (SP) Glide docking method was used to dock all the com-
pounds into the catalytic site of ITK. Grids for Glide docking were
calculated using the bound inhibitor as the reference of the cata-
lytic site in ITK. Upon completion of each docking calculation, a
maximum of 10 poses per ligand were generated. The top-scored
pose was chosen using the Glidescore (Gscore) function.
of 20
0.05% NaN3; 1 mM DTT; 0.1% BSA) containing 100 nM substrate,
30 M ATP. The reaction time was 40 min and the binding time
was 1 h. The reaction was stopped by addition of 60 l binding
ll of final reaction buffer (10 mM Tris pH 7.2; 10 mM MgCl2;
l
l
solution (Progressive Binding IMAP system, 100% buffer A, beads
1:400, Molecular Devices). Signal was detected with an envision
reader (PE) by measuring fluorescence polarization at 485 nm/
535 nm.
Supplementary data
5.31.6. GSK3ß enzymatic activity (ATP consumption assay)
GST-GSK3ß full length was from ProQinase. Enzymatic activity
was measured using the Easylite–Kinase Detection Assay System
from PE. The substrate was the N-terminus biotinylated peptide
[YRRAAVPPSPSLSRHSSPHQ(pS)EDEEE] from Upstate. The reaction
conditions were as following: 100 ng GSK3/well in a final volume
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.04.056.
References and notes
1. Felices, M.; Falk, M.; Kosaka, Y.; Berg, L. J. Adv. Immunol. 2007, 93, 145.
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Immunol. 2006, 117, 780.
of 20 ll of final reaction buffer (50 mM Hepes pH 7.5; 3 mM MgCl2;
3 mM MnCl2; 0.05% BSA; 1 mM DTT) containing 500 nM substrate,
4. Forssell, J.; Sideras, P.; Eriksson, C.; Malm-Erjefält, M.; Rydell-Törmänen, K.;
Ericsson, P.-O.; Erjefält, J. S. Am. J. Respir.Cell Mol. Biol. 2005, 32, 511.
5. Readinger, J. A.; Schiralli, G. A.; Jiang, J.-K.; Thomas, C. J.; August, A.; Henderson,
A. J.; Schwartzberg, P. L. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 6684.
6. (a) Riether, D.; Zindell, R.; Kowalski, J. A.; Cook, B. N.; Bentzien, J.; De Lombaert,
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H.; O’Shea, K.; Woska, J. R.; Jeanfavre, D.; Sellati, R.; Ralph, K. L. M.; Ahlberg, J.;
Labissiere, G.; Kashem, M. A.; Pullen, S. S.; Takahashi, H. Bioorg. Med. Chem. Lett.
2009, 19, 1588; (b) Cook, B. N.; Bentzien, J.; White, A.; Nemoto, P. A.; Wang, J.;
Man, C. C.; Soleymanzadeh, F.; Khine, H. H.; Kashem, M. A.; Kugler, S. Z., Jr.;
Wolak, J. P.; Roth, G. P.; De Lombaert, S.; Pullen, S. S.; Takahashi, H. Bioorg. Med.
Chem. Lett. 2009, 19, 773; (c) Lo, H. Y.; Bentzien, J.; White, A.; Man, C. C.; Fleck,
R. W.; Pullen, S. S.; Khine, H. H.; King, J.; Woska, J. R., Jr.; Wolak, J. P.; Kashem,
M. A.; Roth, G. P.; Takahashi, H. Tetrahedron Lett. 2008, 49, 7337.
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W.; Shuster, D. J.; O’Day, K. D.; Penhallow, B.; Hung, C.-Y.; Doweyko, A. M.;
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O’Day, K. D.; Penhallow, B.; Hung, C.-Y.; Kanner, S. B.; Lin, T.-A.; Dodd, J. H.;
Barrish, J. C.; Wityak, J. Bioorg. Med. Chem. Lett. 2006, 16, 2411.
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Kleinheinz, T.; Krell, H.-W.; Li, J.; Liang, J.; Limberg, A.; McNutt, A.; Moffat, J.;
Phillips, G.; Ran, Y.; Safina, B.; Ultsch, M.; Walker, L.; Wiesmann, C.; Zhang, B.;
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200 nM ATP. The reaction time was 60 min at room temperature in
dark. The reaction was stopped by addition of 20 ll Easylite re-
agent. Luminescence was detected with an Envision reader (PE).
5.32. Molecular modeling
Molecular docking studies were performed using Maestro, ver-
sion 8.5, and Glide, version 5.0 (Schrodinger,L.L.C.). The crystal
structure of ITK complexed with thiophene-2-carboxylic acid [1-
(2-carbamoyl-ethyl)-5-(cyclohexanecarbonyl-methyl-amino)-1H-
benzoimidazol-2-yl]-amide inhibitor was used as a template for
the Glide docking. Among several ITK crystal structures bound
with inhibitors, this structure was selected because of the struc-
tural similarity of the bound thiazolyl benzimidazole ligand with
the pyrazolyl-indole derivatives under study.
The crystal structure of ITK was optimized for docking using the
Protein Preparation Wizard provided by Schrodinger. This involved
assignment of bond orders, addition of hydrogens and capping of
uncapped termini. The hydrogen-bonding network was optimized
by reorienting all the hydroxyl groups and the amide groups of Asn
and Gln. Appropriate states and orientations of the imidazole ring
in the His residues were selected. Finally the whole structure was
refined with a constrained minimization to 0.3 rmsd using the
Optimised Potentials for Liquid Simulations (OPLS) force field.
The pyrazolyl-indoles were constructed using the fragment dic-
tionary of Maestro 8.5 and geometry optimized under the OPLS
force field using the Polak-Ribiere Conjugate gradient protocol as
11. Noble, M. E. M.; Endicott, J. A.; Johnson, L. N. Science 2004, 303, 1800.
12. Vulpetti, A.; Boscotti, R. Il Farmaco 2004, 59, 759.