Scaffold oriented synthesis. Part 1: Design, preparation, and biological evaluation of thienopyrazoles as kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.09.042

We report the synthesis of kinase targeted libraries based on the thienopyrazole scaffold. Several thienopyrazole analogs have been identified as submicromolar inhibitors of KDR.

Full text of DOI:10.1016/j.bmcl.2005.09.042

Bioorganic & Medicinal Chemistry Letters 16 (2006) 96–99
Scaffold oriented synthesis. Part 1: Design, preparation,
and biological evaluation of thienopyrazoles as kinase inhibitors
Irini Akritopoulou-Zanze,* Daria Darczak, Kathy Sarris, Kathleen M. Phelan,
Jeffrey R. Huth, Danying Song, Eric F. Johnson, Yong Jia and Stevan W. Djuric
Abbott, 100 Abbott Park Road, Abbott Park, IL 60044, USA
Received 23 August 2005; revised 12 September 2005; accepted 16 September 2005
Available online 10 October 2005
Abstract—We report the synthesis of kinase targeted libraries based on the thienopyrazole scaffold. Several thienopyrazole analogs
have been identified as submicromolar inhibitors of KDR.
Ó 2005 Elsevier Ltd. All rights reserved.
As part of our groupꢀs ongoing effort to enhance the Ab-
bott compound collection with novel and/or rare chem-
otypes, we have initiated an effort to design new kinase
inhibitors for current and future kinase programs.
Kinases have emerged in recent years as targets in a
variety of therapeutic areas such as cancer,1 diabetes,2
inflammation,3 cardiovascular,4 and neurodegenerative5
diseases, and several kinase inhibitors have been
approved by the FDA for use in human.6
the amino groups of the pyrazole ring.11,12 Subsequent-
ly, we were planning to explore the surrounding space
by functionalizing the scaffold in different regions. A
survey of the literature revealed very few references for
their preparation, all of which were unsuitable for
obtaining versatile scaffolds. Therefore, we developed a
new route for the synthesis of thienopyrazoles and
chemistries that provided access to all sites of the
molecule.
Much work has been directed toward the design and
synthesis of kinase inhibitors,7 including computational8
and fragment-based9 approaches. De novo design of
new kinase inhibitors is greatly facilitated by the archi-
tecture of kinases having a mostly conserved catalytic
site that binds ATP. ATP forms key hydrogen bond
interactions in the hinge region of the catalytic site of
protein kinases. Therefore, small and relatively flat het-
erocyclic molecules containing hinge-binding elements
are expected to form weak interactions with the enzymes
that can be amplified by functionalization to access
additional pockets in the active site.
We have prepared three small libraries around thienopy-
razole scaffolds as well as various smaller groups of ana-
logs to probe the various sites. Emphasis was placed on
preparing a variety of structurally diverse analogs rather
than closely following SAR trends. Our ultimate goal
was to create a lead generation set that would provide
good starting points for medicinal chemistry lead opti-
mization exercises. The libraries were designed to have
lead-like physicochemical properties.13 In all, we have
prepared a collection of 95 compounds with an average
MW of 292, an average clogP of 2.5, and an average
polar surface area of 71.
In the above context, we have decided to investigate
the underexplored class of thienopyrazole structures as
kinase inhibitors.10 We expected to obtain the key
donor/acceptor hydrogen bond hinge interactions from
Scheme 1 illustrates the initial hurdles encountered
with the synthesis of the basic thienopyrazole core.
Reaction of the commercially available methyl dihy-
dro-pyrazolone 1 with POCl3 provided the intermedi-
ate 4-formyl-5-chloro Vilsmeier product, which was
protected with p-methoxybenzyl chloride to yield two
different regioisomers 2a and 3a in a two to one ratio,
respectively. The identity of each regioisomer was con-
firmed by NMR experiments. An NOE was observed
between the benzylic methylene protons and the meth-
Keywords: Thienopyrazoles; Kinase inhibitors; KDR inhibitors; Tar-
geted libraries; De novo design.
*
Corresponding author. Tel.: +1 847 937 5006; fax: +1 847 935
0310; e-mail: irini.zanze@abbott.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.09.042

I. Akritopoulou-Zanze et al. / Bioorg. Med. Chem. Lett. 16 (2006) 96–99
97
R2
O
O
R3
R1
N
N
H
H
R1
CO2Me
a, b
a, b, c
S
O
N
N
N
N
Cl
O
PG
Cl
S
N
N
N
N
N
H
PG
PG
H
1
29%
40%
PG = PMB
PG = BPMPM
2a
2b
3a 16%
R1 = Me PG = PMB
R1 = Ph PG = BPMPM
8a-e
9a-f
5%
3b
c
c
Scheme 2. Reagents and conditions: (a) 1:1 1 M NaOH/MeOH, 50 °C,
R1 = Me, 82%, R1 = Ph, 44%; (b) R1 = Me, 1:1 TFA/CH2Cl2, 120 °C,
20 min, microwave (CEM, 300 W, ramp time 1 min), 59%, R1 = Ph,
4 M HCl in dioxane, 99%; (c) PS-carbodiimide, HOBt, DIEA,
R2R3NH, DMA, microwave (Personal Chemistry, 300 W), 100 °C,
6 min, 2–12%.
d or e
CO2Me
CO2Me
No product
N
S
N
S
N
N
H
PG
60%
53%
PG = PMB
PG = BPMPM
4a
4b
5
OH
f, g
N
O
N
b
H
a
R1
N
Br
R2
N
N
N
h, i
Cl
Cl
Cl
CO2Me
N
N
N
CO2Me
BPMPM
BPMPM
BPMPM
N
S
S
N
N
N
2b
10
11
Boc
H
R
NH2
6
7a,b
NH2
O
NH
O
d, e
NH2
O
c
Scheme 1. Reagents and conditions: (a) POCl3, DMF, 0 °C then reflux
2 h, 32%; (b) PG = PMB, PMBCl, K2CO3, DMF, 120 °C, 1 h;
PG = BPMPM, BPMPMCl,14 Et3N, THF, rt, 1 h; (c) Mercapto-acetic
acid methyl ester, Na2CO3, MeOH, reflux; (d) PG = PMB, 1:1 TFA/
CH2Cl2, 120 °C, 20 min, microwave (CEM, 300 W, ramp time 1 min),
59%; (e) PG = BPMPM, 4 M HCl in dioxane, rt, 1 h, 60%; (f) Boc2O,
Et3N, DMAP, MeCN, 3 h, 66%; (g) NBS, (PhCO2)2, CCl4, reflux, 6 h,
24%; (h) R1R2NH, EtOH; (i) 1:1 TFA/CH2Cl2, 24–84% in two steps.
N
S
N
S
N
N
BPMPM
H
12
13a-h
Scheme 3. Reagents and conditions: (a) NH2OHÆHCl, satd NaHCO3,
EtOH/H2O, 7 h, 83%; (b) Ac2O, reflux, 1 h, 73%; (c) 2-mercapto-
acetamide, K2CO3, EtOH, reflux, 17 h, 77%; (d) RCOCl, Py, rt, 24 h;
(e) 4 M HCl, dioxane, 6 h, 4–20% in two steps.
yl group in 3a but not in 2a. Interestingly, only regio-
isomer 2a participated in the subsequent reaction with
mercapto-acetic acid methyl ester to provide the fused
thienopyrazole product, while 3a remained completely
unreactive.15 Deprotection of the p-methoxybenzyl
group was easily achieved by heating 4a in the micro-
wave with a one- to one-mixture of TFA/dichloro-
methane. Alternatively, the bulkier and more acid
labile bis(4-methoxyphenyl)methyl group provided a
better yield of the desired regioisomer 2b over 3b
and can be readily removed at room temperature with
4 M HCl in dioxane. Thus, bis(4-methoxyphenyl)meth-
yl became the protecting group of choice. Protection
of 5 (Scheme 1) with Boc, subsequent NBS bromina-
tion of the methyl group, and reaction of the bromo
intermediate 6 with amines provided final products 7
in good yields.
An interesting diversion from the above synthetic proto-
col was the formation of 4-amino substituted analogs 13
(Scheme 3). Aldehyde 2b was transformed in two steps
to the corresponding cyano product 11, which under-
went reaction with 2-mercapto-acetamide to provide 4-
amino thienopyrazole 12. The 4-amino group could be
further reacted with acyl chlorides in pyridine to yield
the acylated products 13.
All compounds were tested against a panel of 5 kinases
of interest.17 Although exploration of the 3-position
(Table 1) was limited to a few analogs, low micromolar
activity was already observed against some of the ki-
nases. Simple analog 5 was active against KDR and
CK2, while the benzylamine analog 7a exhibited activity
against Plk1, Pak4, CK2, and Akt. Piperidinyl analog 7b
was inactive indicating an initial preference of the site
for benzylamine analogs. It should be noted that mole-
cules such as 5, which exhibit high binding efficiency
(BEI = 24), i.e., strong binding in relation to their
molecular weight,18 represent excellent starting points
for followup libraries to improve upon activity and
selectivity.
Similar sequences were followed starting from phenyl
dihydro-pyrazolone16 to provide the 3-phenyl thieno-
pyrazole of Scheme 2. Both 3-methyl and 3-phenyl
thienopyrazoles in Scheme 2 were first saponified with
NaOH in MeOH to provide the carboxylic acids and
then the pyrazole ring was deprotected. We have ob-
served that this sequence was cleaner and higher yield-
ing than first deprotecting the pyrazole and then
saponifying. The final unprotected thienopyrazole
acids were used for the synthesis of small amide li-
braries 8 and 9.
Table 2 summarizes the activity of amides 8 and 9 against
an extended panel of 9 kinases.19 In general, amides 8
showed similar trends in activity as the ester 5 inhibiting

98
I. Akritopoulou-Zanze et al. / Bioorg. Med. Chem. Lett. 16 (2006) 96–99
Table 1. Kinase inhibitory activity of position 3 analogsa
Table 3. Kinase inhibitory activity of position 4-analogsa
O
R
R
NH
S
O
O
NH2
O
S
N
N
H
N
N
H
Compd
R
IC50 (lM)
Compd
R
IC50 (lM)
KDR
Plk1
Pak4
CK2
Akt1
KDR
Plk1
Pak4
CK2
Akt1
5
H
23b
>100
>100
>100
21
>100
>100
37
>100
38
5b
>100
16
>100
13a
13b
13c
13d
13e
13f
13g
13h
Me
19b
17
>20
8
>20
>100
>100
>100
>100
>100
>100
13b
9b
3c
>20
>100
>100
17
7a
7b
NHPh
Piperidinyl
CO2Me
CH2Oph
2-Thienyl
2-ClPh
3-ClPh
4-ClPh
NHPh
>100
11
6
>100
>100
23
6
a IC50 values are based on an 11 point curves at 10 lM ATP; >100
indicates less than 50% inhibition at that concentration or an
IC50 > 100 lM.
>100
9c
8
>100
>100
>100
9b
>100
>100
20
>100
>100
>20
>100
>100
11
b Average of two values.
KDR and CK2 with low micromolar potency. However,
replacement of the methyl group with a phenyl separated
the KDR and CK2 activity, and resulted in submicromo-
lar hits against KDR. Depending on the amide substitu-
tions some 3-phenyl analogs have also showed activity
against MK2 (9c and 9e) and CDK2 (9b).
a IC50 values are based on 11 point curves at 10 lM ATP; >100 indi-
cates less than 50% inhibition at that concentration or an
IC50 > 100 lM.
b Average of two IC50 values.
c Average of three IC50 values.
4-Amino analogs 13 (Table 3) also showed a preference
for KDR and CK2 kinases. However, depending on the
substitution on the 4-amino group, leads for Plk1 13b
and 13e, and Akt1 13d and 13h were obtained. Urea
analog 13h20 is an interesting case in that it inhibits most
of the kinases with low micromolar potency.
leads with low micromolar potency against a variety of
kinase targets. In addition, a subset of 3-phenyl substi-
tuted thienopyrazoles exhibited submicromolar potency
against KDR.
Acknowledgments
In conclusion, we have prepared a diverse set of thieno-
pyrazole-based lead-like molecules that were evaluated
in a panel of kinase assays. This lead generation exercise
was quite successful, as we have identified numerous
The authors thank Vincent Stoll, Cele Abad-Zapatero,
Kent Stewart, James Metz, and Phillip Hajduk for ideas
and suggestions regarding the thienopyrazole scaffold,
Table 2. Kinase inhibitory activity of analogs 8 and 9a
R2
R3
N
R1
O
S
N
N
H
Compound
R1
R2
R3
IC50 (lM)
KDR
Plk1
Pak4
CK2
Akt1
CDK2d,e
p-38d,e
MK2e
COTd,e
8a
8b
8c
8d
8e
9a
9b
9c
9d
9e
9f
Me
Me
Me
Me
Me
Ph
H
H
n-Bu
17b
16
>20
>20
>20
>20
16
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
4c
21
24b
>20
19b
5
>20
>20
>20
>20
>20
>20
>20
15
ND
ND
ND
ND
ND
>20
11
ND
ND
ND
ND
ND
>20
>20
ND
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
17
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
H
Me
H
n-Bu
>20
>20
12
Cyclohexyl
Bn
n-Bu
H
Me
H
0.35
>20
12b
ND
17
Ph
Ph
Cyclohexyl
Bn
0.49
0.5
2
>20
2
H
ND
>20
>20
>20
Ph
Ph
H
CH2Bn
p-MeOBn
-CH2CH2N(Me)CH2CH2-
>20
>20
>20
>20
>20
>20
>20
16
H
0.51
4
>20
>20
Ph
>20
a IC50 values are based on 11 point curves at 10 lM ATP; >20 indicates less than 50% inhibition at that concentration or an IC50 > 20 lM; ND, not
determined.
b Average of two IC50 values.
c Average of four IC50 values.
d 100 lM ATP.
e IC50 values are based on seven point curves.

I. Akritopoulou-Zanze et al. / Bioorg. Med. Chem. Lett. 16 (2006) 96–99
99
13. Hann, M. M.; Oprea, T. I. Curr. Opin. Chem. Biol. 2004,
8, 255.
14. Prepared by refluxing for 3 h 4,40-dimethoxybenzhydrol in
SOCl2 and then evaporating the reaction mixture to
dryness.
Jan Waters for the NOE experiments, and the Abbott
High Throughput Purification and Structural Chemistry
groups for their support with purification and analytical
data, respectively.
15. Also, no reaction was observed with the unprotected
4-formyl-5-chloro Vilsmeier intermediate derived from 1.
16. Prepared in one step by heating 3-oxo-3-phenyl-propionic
acid ethyl ester, hydrazine hydrate, and acetic acid in
ethanol at 90 °C for 2 h.
References and notes
1. (a) Medinger, M.; Drevs, J. Curr. Pharm. Des 2005, 11,
1139; (b) Mazitschek, R.; Giannis, A. Curr. Opin. Chem.
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36, 462.
2. (a) Bridges, A. J. Biochem. Soc. Trans. 2005, 33, 343; (b)
Cohen, P.; Goedert, M. Nat. Rev. Drug Discov. 2004, 3, 479.
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ii64; (b) Wong, W. S. F.; Leong, K. P. Recent Res. Dev.
Immun. 2003, 5, 57.
4. Force, T.; Kuida, K.; Namchuk, M.; Parang, K.; Kyriakis,
J. M. Circulation 2004, 109, 1196.
5. Resnick, L.; Fennell, M. Drug Discovery Today 2004, 9,
932.
6. For a list of approved drugs and late clinical development
candidates as well as their profiling in a panel of 119
kinase assays, see: Fabian, M. A.; Biggs, W. H.; Treiber,
D. K.; Atteridge, C. E.; Azimioara, M. D.; Benedetti, M.
G.; Carter, T. A.; Ciceri, P.; Edeen, P. T.; Floyd, M.;
Ford, J. M.; Galvin, M.; Gerlach, J. L.; Grotzfeld, R. M.;
Herrgard, S.; Insko, D. E.; Insko, M. A.; Lai, A. G.;
Lelias, J.-M.; Mehta, S. A.; Milanov, Z. V.; Velasco, A.
M.; Wodicka, L. M.; Patel, H. K.; Zarrinkar, P. P.;
Lockhart, D. J. Nat. Biotechnol. 2005, 23, 329.
7. (a) McInnes, C.; Fischer, P. M. Curr. Pharm. Des. 2005, 11,
1845; (b) Parang, K.; Sun, G. Curr. Opin. Drug Disc. Dev.
2004, 7, 617; (c) Prien, O. Chem. Biochem. 2005, 6, 500.
8. Woolfrey, J. R.; Weston, G. S. Curr. Pharm. Des. 2002, 8,
1527.
9. Gill, A. Mini-Rev. Med. Chem. 2004, 4, 301.
10. (a) Recently, two patent applications have been disclosed
describing thienopyrazoles as kinase inhibitors: Ohi, N.;
Sato, N.; Soejima, M.; Doko, T.; Terauchi, T.; Naoe, Y.;
Motoki, T. WO 2003101968, 2003; (b) Bigot, A.; Clerc, F.;
Doerflinger, G.; Mignani, S.; Minoux, H. U.S.
2005026984, 2005.
11. The bioisosteric indazoles have shown kinase inhibitory
activity against a number of kinases, for example Atsuya,
T. I.; Masayuki, O.; Yuji, K.; Takehisa, O. H.; Takahashi,
N.; Shindo, K.; Kimura, K.; Tagami, Y.; Miyake, M.;
Fukushima, K.; Inagaki, M.; Amano, M.; Kaibuchi, K.;
Iijima, H. Bioorg. Med. Chem. 2004, 12, 2115.
17. In vitro kinase assays. Recombinant CK2 (Calbiochem, San
Diego, California) was commercially obtained. His-tagged
AKT1[S378A, S381A, T450D, S473D] (139–480), His-
tagged KDR (789–1354), His-tagged PAK4 (290–581),
and GST-tagged PLK1 (1–331) were expressed using the
FastBac baculovirus expression system (GIBCO BRL,
Gaithersburg, MD) and purified using either nickel (His-
tag) or glutathione (GST) affinity chromatography. Peptide
substrates had the general structure biotin-Ahx-peptide
with sequences: AKT, EELSPFRGRSRSAPPNLWA
AQR; CK2, RRADDSDDDDD; KDR, AEEEYFFLFA-
amide; PAK4, KEVPRRKSLVGTPYWMAPE; PLK1,
AKMETTFYDDALNASFLPSEKKK-Amide. Inhibition
of kinase activity was assessed using a radioactive Flash-
Plate-based assay platform as previously described in Luo,
Y.; Smith, R. A.; Guan, R.; Liu, X.; Klinghofer, V.; Shen, J.;
Hutchins, C.; Richardson, P.; Holzman, T.; Rosenberg, S.
H.; Giranda, V. L. Biochemistry 2004, 43, 1254.
18. Abad-Zapatero, C.; Metz, J. T. Drug Discovery Today
2005, 10, 464.
19. Additional in vitro kinase assays. The kinase assays were
performed using the homogeneous time-resolved fluores-
cence (HTRF) method (G. Mathis, Clin. Chem. 1993, 39,
1953–1959). COT (made in-house) assay contained
13.7 nM COT, 0.5 lM biotin-MEK-peptide, 0.1 mM
ATP, and compound in a buffer containing 50 mM Tris–
HCl, pH 7.5, 10 mM MgCl2, 1 mM EGTA, 2 mM DTT,
0.01% Brij 35, 5 mM b-phosphoglycerol. MK2 (made in
house) assay contained 1.8 nM MK2, 1 lM biotin-cdc25-
peptide, 10 lM ATP and compound in the MK2 Buffer
(20 mM Mops, pH 7, 10 mM MgCl2, 5 mM EGTA, 5 mM
b-phosphoglycerol, 1 mM Na3VO4, 0.01% Triton X-100,
and 1 mM DTT). p38a and CDK2 (UBI) assays contained
either 7.8 nM p38a or 2.7 nM CDK2/cyclin A, and 0.5 lM
biotin-MBP-peptide, 0.1 mM ATP, and compound in the
MK2 Buffer. All assays were carried out at RT for 60 min
and stopped by addition of EDTA. The products were
detected by addition of revelation reagents containing
Europium labeled phospho-specific antibodies and SAXL.
The plates were incubated in dark at 4 °C overnight or RT
for 10 min (for MK2) and read in the HTRF reader
RUBYstar (BMG).
12. Further proof of the pyrazole amino groups interacting
with the hinge region provided the synthesis of N-
methylated thienopyrazoles following procedures similar
to the ones shown in Schemes 1 and 3, which were void of
any inhibitory activity against kinases.
20. Synthesized from 12 upon reaction with phenyl-isocyanate
in dichloromethane and subsequent deprotection with 4 M
HCl in dioxane.