A novel chemotype of kinase inhibitors: Discovery of 3,4-ring fused 7-azaindoles and deazapurines as potent JAK2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.11.021

Pictet-Spengler condensation of aldehydes or alpha-keto-esters with 4-(2-anilinophenyl)-7-azaindole (11) or deazapurine (12) gave high yields of the 3,4-fused cyclic compounds. SAR studies, by varying the substituted benzaldehyde components, lead to the discovery of a series of potent JAK2 kinase inhibitors.

Full text of DOI:10.1016/j.bmcl.2009.11.021

Bioorganic & Medicinal Chemistry Letters 20 (2010) 153–156
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A novel chemotype of kinase inhibitors: Discovery of 3,4-ring fused
7-azaindoles and deazapurines as potent JAK2 inhibitors
*
*
Tiansheng Wang , Mark W. Ledeboer , John P. Duffy, Albert C. Pierce, Harmon J. Zuccola, Eric Block,
Dina Shlyakter, James K. Hogan, Youssef L. Bennani
Vertex Pharmaceuticals Inc., 130 Waverly St., Cambridge, MA 02139-4242, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Pictet–Spengler condensation of aldehydes or alpha-keto-esters with 4-(2-anilinophenyl)-7-azaindole
(11) or deazapurine (12) gave high yields of the 3,4-fused cyclic compounds. SAR studies, by varying
the substituted benzaldehyde components, lead to the discovery of a series of potent JAK2 kinase
inhibitors.
Received 18 September 2009
Revised 31 October 2009
Accepted 5 November 2009
Available online 12 November 2009
Ó 2009 Elsevier Ltd. All rights reserved.
T.W. would like to dedicate this Letter to his
PhD mentor, Professor Zhengming Li of
Nankai University, Tianjin, China, in honor
of his more than 50 years of teaching and
research in organic chemistry
Keywords:
Azaindole
Deazapurine
JAK2
Kinase
Inhibitor
The Janus kinases (JAK) are a family of intracellular non-recep-
tor tyrosine kinases that transduce cytokine-mediated signals via
the JAK-STAT pathway. There are four known mammalian kinases
in the JAK family: JAK1, JAK2, JAK3 and TYK2.1 Among the JAK pro-
teins, JAK3 has been a drug target for extensive study, cultimating
in the discovery of a small molecule JAK3 inhibitor, CP-690550 (1)
currently in advanced clinical trial for treatment of Rheumatoid
Arthritis.2
In 2005 it was independently reported by several research
teams that the occurrence of a single residue mutation (V617F)
in JAK2 was commonly found in patients diagnosed with myelo-
proliferative disorders such as polycythemia vera, essential throm-
bocythemia and chronic idiopathic myelofibrosis.3 The inhibition
of cascade events mediated by JAK2 (V617F) therefore became an
attractive approach for developing new targeted therapies against
myeloproliferative disorders.4 We became interested in the discov-
ery of compounds that block the kinase activity of JAK2 by target-
ing the ATP binding site.
One class of JAK inhibitors contains an azaindole (2a) or deazap-
urine (2b) hinge-binding motif, as represented by CP-690550 (1).
We started a program directed at the discovery of new JAK2 inhib-
itors where the 7-azaindole or deazapurine were of interest as
starting points for design (Fig. 1).
Although mono- or poly-substituted 2a and 2b are documented
in recent patent applications as novel kinase inhibitors, tricyclic,
tetracyclic and polycyclic azaindoles and deazapurines have not
been well represented.
Inspired by polycyclic Ergot indole alkaloid natural products,
where a bridged ring exists between the 3 and 4 positions of in-
dole,5 we realized that an additional ring can be formed between
3 and 4 positions of 7-azaindole and deazapurine. This would re-
N
N
N
CN
4
3
O
N
N
N
H
N
H
N
H
N
N
* Corresponding authors. Tel.: +1 617 444 6484; fax: +1 617 444 7824 (T.W.).
E-mail addresses: tiansheng_wang@vrtx.com (T. Wang), mark_ledeboer@vrtx.-
com (M.W. Ledeboer).
1 CP-690,550
2a
Figure 1.
2b
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.021

154
T. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 153–156
sult in a novel polycyclic chemotype, which should be rigid in nat-
ure and might have additional benefits of favorable physicochem-
ical properties. Furthermore, screening of our corporate deck
revealed that simple 3- and 4-substituted compounds such as 3
and 4 possessed moderate JAK2 activities. Overlaying the putative
hinge binding orientations of 3 and 4 suggested that a hybrid struc-
ture such as 5, with a bridge between positions 3 and 4 on the aza-
indole/deazapurine core, might provide a novel series of JAK2
inhibitors (Fig. 2).
With such a goal in mind, we investigated several possibilities to
form the ring between the 3 and 4 positions on azaindole and dea-
zapurine. After initial trial and error exploring several synthetic pos-
sibilities, we found a robust methodology based on Pictet–Spengler
condensation to form such ring-fused systems. Our approach al-
lowed rapid access to well over 150 examples of this chemotype
and led to the discovery of a series of potent JAK2 inhibitors.
Suzuki coupling of 2-acetamidophenylboronic acid (8) with 4-
bromo-7-azaindole (6) or 4-chlorodeazapurine (7) afforded inter-
mediate 4-(2-acetamidophenyl)-azaindole (9) or deazapurine
(10), which were treated in refluxing concentrated hydrochloric
acid to produce high yields of 11 and 12 (Scheme 1). Next we
examined the condensation between the anilines 11 and 12 with
an aldehyde. To our delight, the Pictet–Spengler reaction pro-
ceeded well. Thus, heating either 11 or 12 with aldehydes in meth-
anol and 4 N HCl-dioxane for 30 min, gave, after simple filtration
and washing with ether, the resulting ring fused compounds 13
and 14. Most workups were quite straightforward and consisted
of partial evaporation of methanol, addition of 4–5 volumes of
ethyl ether, filtration and washing with ether twice. For aldehydes
containing basic nitrogen, one equivalent of the aldehyde could be
used. For sterically demanding aldehydes such as t-pentaldehyde,
longer reaction times and large excesses of the aldehydes were
necessary. When the reaction was refluxed in methanol in a flask
open to air for an extended period of time (2 days), the imino prod-
ucts 15 could be isolated in good yields. Obviously, 15 were the re-
sult of oxidation of products of 13. This was further confirmed by
refluxing of 13y in methanol for two days to afford 15a as a clean
product.
The cyclo-condensation could also be readily carried out with
other aldehyde equivalents such as cyclized acetals 16 and 17.
Thus, heating 11 with either 2-ethoxytetrahydrofuran (16) or 2-
methoxytetrahydro-2H-pyran (17) under microwave conditions
(120 °C, 20 min; methanol, 4 N HCl-dioxane) afforded 13j or 13k,
which released terminal primary alcohols. Likewise, extended
heating of 17 with 11 (90 °C, overnight) yielded the imino product
18 (77%) (Scheme 2).
When esters of alpha-formal benzoate 19 were used in this
reaction, the hexacyclic products of type 20 were obtained in
almost quantitative yields (Scheme 3).
Figure 2.
Activated ketones such as 2-oxo-2-phenylacetic esters 21 could
also condense with 11 and 12 to form tetracycles 22 and 23 con-
taining a quaternary center (Scheme 4).6
The methodology described in Scheme 1 was further expanded
to include synthesis of an 8-membered 3,4-fused ring. Thus, Suzuki
coupling of 4-bromoazaindole (6) with 2-cyanophenylboronic acid
produced benzamide 24, which condensed with aldehydes to af-
ford lacams 25 and 26 (Scheme 5).
Table 1 summarizes JAK2 inhibition data for 3,4-ring-fused
compounds 13, 14, 15, 18, 20, 22, 23, 25 and 26.7 When R is ali-
phatic (13a to 13k), only micromolar JAK2 activity was observed.
In the deazapurine cases 14a and 14b, the observed Ki was greater
B(OH)2
O
NHCOCH3
Br
N
Cl
N
H
8
N
or
a
b
X
N
N
N
H
H
N
H
N
6
7
9
X = CH
10 X = N
NH
N
NH2
RCHO
c
R
R
than 5 lM. When R is aryl as in 13, 14 and 15, the enzyme inhib-
X
itory potency against JAK2 improved (13l to 13t). Introduction of
one or more fluorine atoms around the phenyl ring improved po-
tency. The most potent fluoro analog was 13r (2,3,6-trifluorophe-
nyl) with a Ki of 56 nM. It was found that when R is a para-
hydroxy phenyl group, very potent JAK2 activities were observed
(13u to 13w, 13y, 13z, 14e, 14f and 15a in Table 1). This was also
true in the case of eight-membered ring (25).
X
N
H
N
N
H
N
H
N
N
11
13
15
X = CH
12 X = N
X = CH
14 X = N
Scheme 1. Reagents and conditions: (a) Pd(PPh3)4, K2CO3, DME, 128 °C, overnight;
(b) concd HCl, reflux (99.9% for 11 and 84.1% for 12 each over two steps); (c) RCHO,
MeOH, 4 N HCl-dioxane, 100 °C, 30 min, (>90%).
OH
OH
NH
N
n
NH2
a
n
+
OR
O
N
H
N
H
N
N
N
N
H
11
16 n = 1, R = Et
17 n = 2, R = Me
13j
18
n = 1
13k n = 2
Scheme 2. Reagents and conditions: MeOH, 4 N HCl-dioxane, 120 °C, microwave, 20 min (30% for 13j and 25% for 13k).

T. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 153–156
155
Table 1
O
R
JAK2 Ki and cellular IC50 determinations
CHO
R1
N
TF1-GMCSF8 IC50
(lM)
R
CO2Me
Compds
R
JAK2 Ki (
l
M)
NH2
a
+
13a
13b
13c
13d
13e
13f
13g
13h
13i
Et
-iBu
-iPr
-t-Bu
CH2CO2Me
H
cyclohexyl
CH2OCH2Ph
CH2Ph
1.7
3.4
2
2.1
0.46
0.75
2.9
1.8
1
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
1.1
0.86
0.72
>20
5.6
5
ND
1.1
ND
ND
ND
ND
ND
ND
ND
ND
ND
0.20
0.14
0.16
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
R
X
X
N
H
N
H
N
N
19a R = H
19b R = OMe
20a X = CH, R = H
20b X = CH, R = OMe
20c
11
X = CH
12 X = N
X = N, R = H
Scheme 3. Reagents and conditions: (a) MeOH, 4 N HCl-dioxane, 100 °C, 30 min,
>90%.
13j
13k
13l
(CH2)4OH
(CH2)3OH
Ph
2-F-Ph
3-F-Ph
2.4
2.4
0.37
0.22
0.2
0.49
0.13
0.21
0.056
0.28
1.1
0.0016
0.0005
0.0006
0.013
0.004
0.002
0.042
0.003
0.071
2.1
3.1
4.2
1.3
>5
>5
3.8
1.5
0.001
0.001
0.0008
0.4
4.5
>5
13m
13n
13o
13p
13q
13r
13s
13t
13u
13v
13w
13x
13y
13z
13aa
13ab
13ac
13ad
13ae
13af
13ag
14a
14b
14c
14d
14e
14f
O
RO
O
O
NH
OR
R2
4-F-Ph
R2
R1
NH2
+
2,6-F2-Ph
2,4-F2-Ph
2,3,6-F3-Ph
2,3,4-F3-Ph
2-OH-Ph
4-OH-Ph
3-F-4-OH-Ph
2-F-4-OH-Ph
4-B(OH)2-Ph
2-Cl-4-OH-Ph
3-Br-4-OH-Ph
4-MeO-Ph
4-HOCH2Ph
4-NH2-Ph
4-MeSO2NHPh
4-CO2H-Ph
4-MeNHCOPh
4-Pyridyl
CF3
X
X
N
H
N
H
N
R1
N
R = Me, R1 = NO2, R2 = H 22a
R = Me, R1 = OH, R2 = F 22b
X = CH, R = Me, R = NO2, R2 = H
X = CH, R = Me, R1 = OH, R2 = F
22c X = CH, R = Me, R1 = OH, R2 = H
23 X = N, R = Et, R1 = OH, R2 = H
1
21a
21b
11 X = CH
12
X = N
21c R = Me, R1 = OH, R2 = H
21d R = Et, R1 = OH, R2 = H
Scheme 4. Reagents and conditions: (a) MeOH, 4 N HCl-dioxane 100 °C, 30 min,
>90%.
O
O
R1
NH
Br
NH2
b
R2
a
CH2OCH2Ph
Ph
-3-F-Ph
2-F-4-OH-Ph
3-F-4-OH-Ph
2-Cl-4-OH-Ph
N
N
N
N
N
H
N
H
H
1 = F, R2 = OH
26 R1 = H, R2 = OMe
24
25
R
15a
18
Scheme 5. Reagents and conditions: (a) 2-Cyanophenylboronic acid, Pd(PPh4)3,
Na2CO3, DME, 95 °C, 4 h (47% amide plus 48% nitrile); (b) aldehyde, MeOH, 4 N HCl-
dioxane, 90 °C, 1 h (13% for 25 and 26).
20a
20b
20c
22a
22b
22c
23
>5
0.21
0.029
0.21
0.63
0.034
3.8
Several other polar substitutions at the para-phenyl position
were studied (13x, 13aa to 13ag). The para-boronic acid analog
13x also had potent activity. Methoxy analog 13aa caused more
than 20-folds loss in potency compared to the phenol analog 13u
while the hydroxymethyl group maintained activity (13ab). The
4-amino analog had a Ki of 71 nM (13ac) but the corresponding
methylsulfonamide (13ad) caused a loss of potency (13ae). Car-
boxylic acid (13ae) and carboxamide (13af) groups did not help
with the binding affinity. Finally, 4-pyridyl analog 13af only had
25
26
a Ki of 1.3 lM.
The role of the para-phenol functionality in high affinity binding
to JAK2 was revealed by X-ray studies of a co-complex of 15a with
JAK2. As shown in Figure 3, compound 15a forms traditional hinge
hydrogen bonds to Leu 932 and Glu 930. In addition, its hydroxyl
group donates a hydrogen bond to Glu 898 and accepts a hydrogen
bond from the backbone NH of Phe 995. It is these latter two
hydrogen bonds, buried deep in a hydrophobic pocket, that ac-
count for the extraordinary potency of the para-phenol containing
analogues.
Since compounds 13 and 14 possess a chiral center, the effect of
the chirality on activity was also examined. By a chiral column sep-
aration, we isolated two enantiomers of 13y. The one enantiomer
of 13y had Ki of 2 nM while the other, 4 nM. When the chiral center
in 13y was eliminated by formation of imino 15a, the potency was
increased to a Ki of 0.8 nM.
Figure 3. Crystal structure of compound 15a bound to JAK2. Dashed lines represent
hydrogen bonds. Crystallographic data for the structures has been deposited with
the RCSB Protein Data Bank. Compound 15a, PDB accession code: 3KCK.

156
T. Wang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 153–156
Tefferi, A.; Lasho, T. L.; Schwager, S. M.; Steensma, D. P.; Mesa, R. A.; Li, C.-Y.;
The hexacyclic analogs 20 did not show JAK2 potency while the
Wadleigh, M.; Gilliland, D. G. Br. J. Haematol. 2005, 131, 320.
4. For recent review on Jak2 inhibitors in potential treatment of myeloproliferative
disorders, see: (a) Atallah, E.; Verstovsek, S. Exp. Rev. Anticancer Ther. 2009, 9,
663; (b) Takenaka, K. Ketsueki, Shuyoka 2009, 58, 70; (c) Pardanani, A. Leukemia
2008, 22, P23; (d) Skoda, R. C. Blood 2008, 111, 5419; (e) Morgan, K. J.; Gilliland,
D. G. Annu. Rev. Med. 2008, 59 213.
5. For a review of the Ergot alkaloids, see: Ninomiya, I.; Kiguchi, T.. In The Alkaloids;
Brossi, A., Ed.; Academic Press: San Diego, CA, 1990; Vol. 38, p 1.
6. For the preparation of 21c, see: Chen, Y. T.; Seto, C. T. J. Med. Chem. 2002, 45,
3946.
compounds 22 and 23, with a quaternary center, still possessed
reasonable potency against the JAK2 enzyme.
For potent JAK2 inhibitors 14e, 14f, 13v, 13w and 15a, the JAK2
mediated cellular inhibitory activity was measured in a TF-1 cell
line (an erythroleukemic cell line). In this assay, TF-1 cells were
stimulated with GM-CSF and FACS was used to measure the intra-
cellular concentration of pSTAT5.8 As shown in Table 1, some of the
most potent JAK2 inhibitors demonstrated sub-micromolar inhibi-
7. JAK2 Ki Determination: ATP and polyE4Y were obtained from Sigma Chemical
Co. (St. Louis, MO, USA). 33P- -ATP, GF/B filter plates, and Ultima GoldTM
c
tory IC50
.
scintillant were purchased from Perkin–Elmer Life Sciences (Boston, MA, USA).
JAK2 used in Vertex assays were expressed and purified by the Gene Expression
and Protein Biochemistry groups, respectively, at Vertex Pharmaceuticals
Incorporated using standard recombinant methods.a Methods: The inhibitory
activity of compounds in Table 1 against JAK2 was determined by following the
In summary, we have developed synthetic methodology based
on Pictet–Spengler like condensation that allows for quick synthe-
sis of the 3,4-ring fused azaindoles and deazapurines. Assessment
of these compounds for activity against the JAK2 enzyme identified
a new class of potent JAK2 inhibitors, several of which also exhib-
ited promising potencies in a GM-CSF stimulated TF-1 cell assay.
residual kinase activity of JAK2 using
a radiometric assay. The final
concentration of the components in the assay were as follows: 100 mM HEPES
(pH 7.5), 10 mM MgCl2, 1 mM DTT, 0.01% BSA, 0.6 nM JAK2, 0.5 mg/ml polyE4Y,
and 12
which additional dilutions were made in DMSO; a 1.5
inhibitor in DMSO was added to each well. 50
L of a 2Â substrate mixture
(100 mM HEPES, 10 mM MgCl2, 1.0 mg/mL polyE4Y, and 24 -ATP) was
M 33P-
added and mixed with the inhibitor/DMSO. The reaction was initiated by the
addition of 50
L of a 2Â enzyme mixture (100 mM HEPES (pH 7.5), 10 mM
MgCl2, 2 mM DTT, 0.02% BSA, 1.0 nM JAK2). After 15 min, the reaction was
quenched with 50 L of 20%TCA. The quenched reaction was transferred to the
GF/B filter plates and washed three times with 5% TCA. Following the addition of
Ultimate GoldTM scintillant (50
L), the samples were counted in a Packard
l
M 33P-
c
-ATP. A stock solution of inhibitor was made up in DMSO from
l
L aliquot of DMSO or
Acknowledgement
l
l
c
We would like to acknowledge our colleague Paul Charifson for
proof reading of the manuscript.
l
l
References and notes
l
TopCount. The radioactivity trapped is a measure of the residual JAK2 kinase
activity. From the activity versus inhibitor concentration, the Ki value was
determined by fitting the data to an equation for competitive tight binding
inhibition kinetics2 using Prism software, version 4.0, San Diego, CA, USA. (a)
Fox, T.; Coll, J. T.; Ford, P. J.; Germann, U. A.; Porter, M. D.; Pazhanisamy, S.;
Fleming, M. A.; Galullo, V.; Su, M.-S.; Wilson, K. P. Protein Sci. 1998, 7, 2249; (b)
Morrison, J. F.; Stone, S. R. Comments Mol. Cell Biophys. 1985, 2, 347.
1. For general review of JAK protein kinase inhibitors, see: Thompson, J. E. Drug
News Perspect. 2005, 18, 305.
2. Sorbera, L. A.; Serradell, N.; Bolos, J.; Rosa, E.; Bozzo, J. Drugs Future 2007, 32,
674.
3. (a) James, C.; Ugo, V.; Le Couedic, J.-P.; Staerk, J.; Delhommeau, F.; Lacout, C.;
Garcon, L.; Raslova, H.; Berger, R.; Bennaceur-Griscelli, A.; Villeval, Jean L.;
Constantinescu, S. N.; Casadevall, N.; Vainchenker, W. Nature 2005, 434, 1144;
(b) Levine, R. L.; Wadleigh, M.; Cools, J.; Ebert, B. L.; Wernig, G.; Huntly, B. J. P.;
Boggon, T. J.; Wlodarska, I.; Clark, J. J.; Moore, S.; Adelsberger, J.; Koo, S.; Lee, J.;
Gabriel, S.; Marynen, P.; Vandenberghe, P.; Mesa, R. A.; Tefferi, A.; Griffin, J. D.;
Eck, M. J.; Sellers, W. J.; Meyerson, M.; Golub, T. R.; Lee, S. J.; Gilliland, D. G.
Cancer Cell 2005, 7, 387; (c) Kralovics, R.; Passamonti, F.; Buser, A. S.; Teo, S-S.;
Tiedt, R.; Passweg, J. R.; Tichelli, A.; Cazzola, M.; Skoda, R. C. N. Eng. J. Med. 2005,
352, 1779; (d) Baxter, E. J.; Scott, L. M.; Campbell, P. J.; East, C.; Fourouclas, N.;
Swanton, S.; Vassiliou, G. S.; Bench, A. J.; Boyd, E. M.; Curtin, N.; Scott, M. A.;
Erber, W. N.; Green, A. R. Lancet 2005, 365, 1054; (e) Zhao, R.; Xing, S.; Li, Z.; Fu,
X.; Li, Q.; Krantz, S. B.; Zhao, Z. J. J. Biol. Chem. 2005, 280, 22788; (f) Tono, C.; Xu,
G.; Toki, T.; Takahashi, Y.; Sasaki, S.; Terui, K.; Ito, E. Leukemia 2005, 19, 1843; (g)
8. IC50 determinations: TF-1 cells were obtained from American Type Culture
Collection, Manassas, VA and cultured according to the provider’s instructions in
the presence of variable concentrations of compounds or DMSO. Method: JAK2-
STAT5 signaling was stimulated with the addition of 2 ng/mL granulocyte-
macrophage colony-stimulating factor (GM-CSF, R&D systems, Minneapolis,
MN) for fifteen minutes to TF-1 cells. Stimulated cells were fixed with the
addition of 4% formaldehyde and permeabilized with 90% methanol. Phospho-
STAT5 (pSTAT5) was quantified by flow cytometry in a Guava PCA-96 system
(Guava Technologies, Hayward, CA) using an anti-STAT5 monoclonal antibody
conjugated to phycoerythrin (BD Biosciences, San Jose, CA). IC50 values were
calculated with Softmax Pro (Molecular Devices, Sunnyvale CA).