N3-Arylmalonamides: A new series of thieno[3,2-b]pyridine based inhibitors of c-Met and VEGFR2 tyrosine kinases

Article abstract of DOI:10.1016/j.bmcl.2009.10.095

A family of thieno[3,2-b]pyridine based small molecule inhibitors of c-Met and VEGFR2 were designed based on lead structure 2. These compounds were shown to have IC₅₀ values in the low nanomolar range in vitro and were efficacious in human tumo

Full text of DOI:10.1016/j.bmcl.2009.10.095

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6836–6839
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N3-Arylmalonamides: A new series of thieno[3,2-b]pyridine based inhibitors
of c-Met and VEGFR2 tyrosine kinases
a
a
a
Oscar Saavedra a, Stephen Claridge a, , Lijie Zhan , Franck Raeppel , Marie-Claude Granger ,
Stéphane Raeppel a, Michael Mannion a, Frédéric Gaudette a, Nancy Zhou a, Ljubomir Isakovic a,
Naomy Bernstein a, Robert Déziel a, Hannah Nguyen b, Normand Beaulieu b, Carole Beaulieu b,
Isabelle Dupont b, James Wang c, A. Robert Macleod b, , Jeffrey M. Besterman b, Arkadii Vaisburg a
*
a Department of Medicinal Chemistry, 7220 Frederick-Banting, Montréal, QC, Canada H4S 2A1
b Department of Biology, 7220 Frederick-Banting, Montréal, QC, Canada H4S 2A1
c Department of PK/Analytical Chemistry, 7220 Frederick-Banting, Montréal, QC, Canada H4S 2A1
a r t i c l e i n f o
a b s t r a c t
Article history:
A family of thieno[3,2-b]pyridine based small molecule inhibitors of c-Met and VEGFR2 were designed
based on lead structure 2. These compounds were shown to have IC50 values in the low nanomolar range
in vitro and were efficacious in human tumor xenograft models in mice in vivo.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 22 September 2009
Revised 20 October 2009
Accepted 21 October 2009
Available online 25 October 2009
Keywords:
c-Met
VEGFR2
RTK inhibitors
Thieno[3,2-b]pyridine scaffold
Oncology
When overexpressed or mutated, protein tyrosine kinases (PTKs)
become potent oncoproteins that can cause deregulated cell growth,
angiogenesis and metastasis.1 Because of these characteristics, they
are key targets for small molecule inhibitors in the treatment of
cancer. Moreover, tumor survival could potentially be more effi-
cientlyreduced ifmultiplesignalingpathwaysare interruptedeither
by multi-targeted agents or by combination of single targeted
drugs.2 Several PTK inhibitors have been found to have effective
anti-tumor activity and some of them have been approved or are
In one of our previous Letters, the synthesis and biological evalu-
ation of a novel family of potent thieno[3,2-b]pyridine based inhib-
itors of c-Met and VEGFR2 bearing a carbamothioyl-arylacetamide
group were described, in which substitution at position 2 of the
thieno[3,2-b]pyridine system was explored.8a To further understand
the structure–activity relationship (SAR) of this novel series of
compounds, certain surrogates of the carbamothioyl-arylacetamide
fragment were investigated.9 Modifications to the carbamothioyl-
arylacetamide moiety had been reported previously by Kirin
Breweryfor a series of quinoline based inhibitors of c-Met10 in which
replacement of the sulfur atom with an oxygen atom of the
TM4
in clinical trials.3 Recent FDA approved drugs Sutent and Nexa-
varTM5 are two such examples of multi-targeted agents (Fig. 1).
In particular, the simultaneous inhibition of the vascular endo-
thelial growth factor (VEGF) receptors6 and c-Met7 is a promising
approach for the treatment of cancer.
c-Met and its ligand the Hepatocyte Growth Factor/Scatter
Factor (HGF/SF) are associated with the development of various
human malignancies and are overexpressed or mutated in a num-
ber of malignancies while the VEGFRs play key roles in tumor
angiogenesis.
H
H
O
N
N
N
NEt2
O
H
O
N
Cl
CF3
N
F
H
H
O
N
N
H
O
Sorafenib (NexavarTM
)
* Corresponding author. Tel.: +1 514 337 3333; fax: +1 514337 0550.
E-mail address: claridges@methylgene.com (S. Claridge).
Present address: Isis Pharmaceuticals, 1896 Rutherford Road, Carlsbad, CA 92008,
USA.
Sunitinib (SutentTM
(Pfizer/Sugen)
)
(Bayer/Onyx)
Figure 1.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.095

O. Saavedra et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6836–6839
6837
Table 1
H
H
In vitro13,14 profile of compounds 2, 5–11
F
N
N
R1
c-Met,
IC50 (nM)
VEGFR2,
IC50 (nM)
Phospho-TPR-
Met, IC50 (nM)
S
O
Compd
O
S
H
H
2
H–
270
200
4
nt
F
N
N
O
N
O
O
5
6
40
65
2 lM
O
N
1
S
O
2
N
89
560
N
N
Figure 2.
N
7
8
36
25
50
30
40
thiocarbonyl unit and switching the CH and the NH groups had affor-
ded N3-arylmalonamides, possessing similar potency compared to
the parent compounds. We were interested to see if such modifica-
tions were possible within our thieno[3,2-b]pyridine class of
molecules8a (Fig. 2).
The chemistry described in Scheme 1 shows the synthetic route
chosen to obtain 2 (R1 = H). Thus, reaction of methyl 3-chloro-3-
oxo-propionate with aniline afforded 38b which upon reaction with
48a using a BOP coupling procedure afforded 2. The benefit of this
protocol was the ease with which compound libraries could be made
simply by using different amines (aromatic or aliphatic) or appropri-
ately substituted amine 4. A comparison of the activities of 18a and 2
against c-Met (116 nM and 270 nM, respectively) showed that the
change was well tolerated. However, a similar comparison of activ-
N
N
N
340
N
9
19
5
180
190
N
N
N
10
11
N
20
36
6
N
O
19
>5 lM
HO
N
position of the phenylmalonamide ring were also investigated.
ortho-Methoxy substitution was well tolerated by both enzymes,
while substitution with a trifluoromethyl group caused a ꢀ5-fold
decrease in potency against c-Met (compound 9 vs compound
17) but retained good activity against VEGFR2. Interestingly the
2-hydroxy analogue 18, which is essentially a demethylated
version of compound 16, had almost identical potency enzymati-
cally against both enzymes but the cellular activity was signifi-
cantly less, again possibly due to decreased cellular penetration.
It has been shown previously that substitution at the C-2 position
or the N-terminal amide atom of the head group was tolerated by
c-Met or VEGFR2 in a series of quinoline based inhibitors10,11 and
the question was raised as to whether this would be tolerated within
the thieno[3,2-b]pyridine class of molecules as well. The chemistry
used to obtain these novel molecules is shown in Scheme 2. The
N-substituted analogues were synthesized using the chemistry
shown in Scheme 1 by using the appropriate N-substituted anilines.
N-Methylation of the malonamide was well tolerated, as
compound 22 was equipotent to compound 9 against both enzymes.
A further improvement in cell-based properties, presumably at the
level of cell permeability, was achieved with compound 21 from
the introduction of a cyclopropyl moiety at the methylene unit of
the malonamide fragment. It was found, however, that a combina-
tion of both the N-methyl group and a cyclopropyl group was detri-
mental to activity against both targets (data not shown) perhaps due
to the conformation restrictions placed on such a molecule.9
ities against VEGFR2 (1.1 lM and 200 nM, respectively) showed a
significant improvement of 2 over 1. With this information in hand,
a library of compounds was synthesized (Scheme 1) and the results
are given in Table 1. Previously we have shown8a that the preferred
substituent at the 2 position of thieno[3,2-b]pyridine was either a
cyclic amide or an imidazole, as these tended to give molecules of
higher potency against the target enzymes. Thus efforts were
focused on these groups over other moieties (Table 1). The results
show, as expected, both the amides and imidazole analogues were
highly potent against both enzymes. The difference, however, was
that inhibitors of the imidazole class were significantly more potent
in the cellular assay. The only exception to this was 11 in which it
appears the terminal carboxylic acid was detrimental to activity,
possibly due to poor cellular penetration.
Attention was turned next to investigate the SAR around the
malonamide unit. Using the same approach as in Scheme 1 except
replacing aniline for an appropriately substituted amine, a series of
compounds was made (Table 2). It was decided to make analogues
of 9 as it had shown good activity against both enzymes (enzymat-
ically and cellularly) and was easily synthesized in bulk. Replace-
ment of the phenyl group (9) by a cyclohexyl group (12) caused
a small decrease in c-Met and VEGFR2 enzymatic inhibitory activ-
ity as well as its cellular potency. Addition of fluorine to the malo-
namide unit in either the ortho (13), meta (14) or para (15)
positions essentially had no effect on the potency of the molecules
when compared with 9. However, compound 14, was less potent in
the c-Met cellular assay than either 13 or 15. Introduction of
electron donating and electron withdrawing groups at the ortho-
Certain compounds that had reasonable solubility (data not
shown) were further evaluated for their pharmacokinetic properties
H
H
F
N
N
H
N
ii.
i.
OH
Cl
OMe
O
O
O
F
NH2
O
O
O
O
S
O
3
R1
2, 5-11
S
48a
R1
N
N
Scheme 1. Reagents and conditions: (i) aniline, DCM, rt, 8 h, then LiOH, THF, water, rt, 1 h; (ii) BOP, DIPEA, DMF, rt, 2 h.

6838
O. Saavedra et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6836–6839
Table 2
Table 3
In vitro13,14 profile of compounds 12–18
In vitro13,14 profile of compounds 21–23
H
H
N
F
N
NHR2
F
R3
O
O
O
O
O
N
S
N
S
N
N
N
N
Compd R3
c-Met,
IC50 (nM)
VEGFR-2
IC50 (nM)
Phospho-TPR-
Met, IC50 (nM)
Compd R2
c-Met IC50
(nM)
VEGFR-2 IC50
(nM)
Phospho-TPR-Met,
IC50 (nM)
H
N
9
19
5
180
4
9
19
52
5
180
590
O
O
H
N
12
10
21
24
33
8
6
F
N
22
23
31
13
32
18
35
O
O
F
H
N
F
F
38
15
120
14
15
52
24
10
5
870
23
Table 4
Pharmacokinetic profile of selected compounds in ratsa
OMe
CF3
OH
Parameter
9
21
22
16
17
18
25
91
26
6
13
7
44
150
600
t1/2 (h), iv
CL (L/h/kg)
Vss (L/kg)
Tmax (h), po
2.6
3.1
0.99
2.2
2
0.10
0.62
33
0.91
0.52
0.33
0.5
0.29
2.28
60
0.33
0.82
5.7
0.18
0.77
12
Cmax
(l
M/(mg/kg)), po
AUC (lM h/(mg/kg)), po
%F
a
iv doses 2.17–2.86 mg/kg; po doses 4.28–4.98 mg/kg.
Table 5
Effect of compound 21 on c-Met cellular endpoints
in the rat (Table 4). Compounds 9, 21 and 22—all showed an appre-
ciable half-life, a reasonably low rate of clearance, a low steady-state
volume of distribution and good oral bioavailability.
Compd A549 wound healing, IC50 (nM) DU145 cell scattering, IC50 (nM)
21 220 40
Compound 21 was further evaluated in additional in vitro
assays as it had a reasonable overall PK profile and in addition
was the most potent of the three compounds in the cellular assay
(Table 3). Compound 21 potently inhibited HGF-induced epithelial
cell migration and scattering (Table 5), showing that it is able to
inhibit c-Met-dependent cell motility events.
Second, compound 21 was tested in a variety of VEGF-depen-
dent cellular assays (Table 6). It potently inhibited the VEGF-in-
duced phosphorylation of the downstream effector ERK, and also
impeded the VEGF-dependent proliferation of human umbilical
vein endothelial cells (HUVEC). In an in vitro angiogenesis assay,
which measures the formation of tubules (AngiokitTM; TCS Cell-
works), compound 21 significantly affected tubule growth and
was also able to almost completely inhibit tubule formation at
the 100 nM dose (Table 6). These results show that compound 21
has a significant effect on both c-Met and VEGF-dependent func-
tional activity in cells.
In addition, compound 21 was also profiled against a panel of
kinases and was found to inhibit other therapeutically significant
H
H
H
i
N
N
F
ii
N
OH
HO
OH
O
O
O
O
O
O
O
H2N
N
F
19
N
S
O
N
S
N
N
21
N
20
Scheme 2. Reagents and conditions: (i) SOCl2, THF, 0 °C, 30 min then aniline, triethylamine, 0 °C, 2 h; (ii) EDC, DMF, 3 h, rt.

O. Saavedra et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6836–6839
6839
Table 6
Siegmund, A.; Xi, N.; Kaplan-Lefko, P.; Rex, K.; Chen, A.; Lin, J.; Moriguchi, J.;
Berry, L.; Huang, L.; Teffera, Y.; Yang, Y.; Zhang, Y.; Bellon, S. F.; Lee, M.;
Shimanovich, R.; Bak, A.; Dominguez, C.; Norman, M. H.; Harmange, J.-C.;
Dussault, I.; Kim, T.-S. J. Med. Chem. 2008, 51, 3688; (j) Kim, K. S.; Zhang, L.;
Schmidt, R.; Cai, Z.-W.; Wei, D.; Williams, D. K.; Lombardo, L. J.; Trainor, G. L.;
Xie, D.; Zhang, Y.; An, Y.; Sack, J. S.; Tokarski, J. S.; Darienzo, C.; Kamath, A.;
Marathe, P.; Zhang, Y.; Lippy, J.; Jeyaseelan, R., Sr.; Wautlet, B.; Henley, B.;
Gullo-Brown, J.; Manne, V.; Hunt, J. T.; Fargnoli, J.; Borzilleri, R. M. J. Med. Chem.
2008, 51, 5330; (k) D’Angelo, N. D.; Bellon, S. F.; Booker, S. K.; Cheng, Y.; Coxon,
A.; Dominguez, C.; Fellows, I.; Hoffman, D.; Hungate, R.; Kaplan-Lefko, P.; Lee,
M. R.; Li, C.; Liu, L.; Rainbeau, E.; Reider, P. J.; Rex, K.; Siegmund, A.; Sun, Y.;
Tasker, A. S.; Xi, N.; Xu, S.; Yang, Y.; Zhang, Y.; Burgess, T. L.; Dussault, I.; Kim,
T.-S. J. Med. Chem. 2008, 51, 5766; (l) Toschi, L.; Jänne, P. A. Clin. Cancer Res.
2008, 14, 5941.
Effect of compound 21 on VEGFR2 cellular endpoints
Compd
ERK phosphorylation
IC50 (nM)
HUVEC proliferation
IC50 (nM)
Tubule length
IC50 (nM)
21
1
0.3
<3
Table 7
The effect of oral dosage of 21 on various human tumor models in vivo at a dosage of
40 mg/kg once daily
8. (a) Claridge, S.; Raeppel, F.; Granger, M.-C.; Bernstein, N.; Saavedra, O.; Zhan, L.;
Llewellyn, D.; Wahhab, A.; Deziel, R.; Rahil, J.; Beaulieu, N.; Nguyen, H.; Dupont,
I.; Barsalou, A.; Beaulieu, C.; Chute, I.; Gravel, S.; Robert, M.-F.; Lefebvre, S.;
Dubay, M.; Pascal, R.; Gillespie, J.; Jin, Z.; Wang, J.; Besterman, J.; Macleod, A. R.;
Vaisburg, A. Bioorg. Med. Chem. Lett. 2008, 18, 2793; (b) Saavedra, O.; Claridge,
S.; Zhan, L.; Raeppel, F.; Vaisburg, A.; Raeppel, S.; Deziel, R.; Mannion, M.; Zhou,
N.; Gaudette, F.; Isakovic, L.; Wahhab, A.; Granger, M.-C.; Bernstein, N. WO
2007/0004675 A1.
Tumor model
Experiment duration
(days)
% Tumor growth
inhibition
A549 (lung)
U87MG (glioblastoma)
MKN45 (gastric)
14
10
12
54
70
101
9. Raeppel, S.; Claridge, S.; Saavedra, O.; Gaudette, F.; Zhan, L.; Mannion, M.; Zhou,
N.; Raeppel, F.; Granger, M.-C.; Isakovic, L.; Déziel, R.; Nguyen, H.; Beaulieu, N.;
Beaulieu, C.; Dupont, I.; Robert, M.-F.; Lefebvre, S.; Dubay, M.; Rahil, J.; Wang, J.;
Ste-Croix, H.; Macleod, A. R.; Besterman, J.; Vaisburg, A. Bioorg. Med. Chem. Lett.
2009, 19, 1323.
kinases.12 Compound 21 showed good efficacy in vivo when eval-
uated in several human tumor xenograft models, with daily oral
administration of 40 mg/kg (Table 7).
In conclusion, novel dual c-Met/VEGF receptor tyrosine kinase
inhibitors based upon the thieno[3,2-b]pyridine scaffold were
designed and synthesized. These compounds exhibit potent activi-
ties against target enzymes and in cell-based assays. Lead molecules
possess favorable pharmacokinetic profiles and demonstrate signif-
icantoralanti-tumoractivityinvivo. ThisworkwaspartoftheMeth-
ylGene kinase inhibitor research program, which led to the
identification of the clinical candidate, MGCD265, currently in Phase
II clinical development.
10. (a) Fujiwara, A.; Senga, T.; Nishitoba, T.; Osawa, T.; Miwa, A.; Nakamura, K. PCT
Int. Patent Appl. WO 2003/000660; (b) Fujiwara, A.; Senga, T.; Nishitoba, T.;
Osawa, T.; Miwa, A.; Nakamura, K. U.S. Patent 2004/0242603.
11. Bannen, L. C.; Chan, D. S.; Chen, J.; Dalrymple, L. E.; Forsyth, T. P.; Huynh, T. P.;
Jammalamadaka, V.; Khoury, R. G.; Leahy, J. W.; Mac, M. B.; Mann, G.; Mann, L.
W.; Nuss, J. M.; Parks, J. J.; Takeuchi, C. S.; Wang, Y.; Xu, W. PCT Int. Patent Appl.
WO 2005/030140.
12. Compound 21 at a concentration of 100 nM inhibited the following enzymes:
Flt-3 (100%), Tie-2 (100%), PDGFR
a (99%), c-Kit (99%), VEGFR1 (98%), VEGFR3
(97%), Aurora A (96%), EphB4 (95%), c-SRC (93%). Compound 21 had <5%
inhibitory activity against the following enzymes CDK2/Cyclin E, EGFR, GSK3b,
IKKb, PKBa
. Compound 21 was evaluated using the KinaseprofilerTM Kinase
Selectivity Screening Service (radiometric protein kinase assays) by Millipore.
13. In vitro kinase assays (c-Met and VEGFR-2): Preparation of GST fusion proteins:
recombinant baculovirus containing the catalytic domain of c-Met and of the
VEGFR-2 receptor fused to glutathione S-transferase (GST) fusion genes were
used to infect High five (c-Met) or Sf9 (VEGFR-2) cells at a multiplicity of
infection of 1 or 0.1, respectively. Cell lysates were prepared after ꢀ72 h of
References and notes
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infection in 1% Triton X-100, 2 lg of leupeptin/mL, and 2 lg of aprotinin/mL
after ꢀ72 h of infection in phosphate-buffered saline, and the fusion proteins
were purified over glutathione agarose (Sigma) according to manufacturer’s
instructions. Biochemical kinase assays for IC50 determination and kinetic
studies: Inhibition of c-Met and VEGFR2 was measured in a DELFIATM assay
(Perkin–Elmer). The substrate poly(Glu4,Tyr) was immobilized onto black
high-binding polystyrene 96-well plates (Nunc Maxisorp). The c-Met kinase
reaction was conducted in 25 mM Hepes pH 7.5 containing 20 mM NaCl,
10 mM MgCl2, 5 mM b-mercaptoethanol, 0.1 mg/mL bovine serum albumin
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(BSA) and 20
60 mM Hepes pH 7.5 containing 3 mM MgCl2, 3 mM MnCl2, 1.2 mM b-
mercaptoethanol, 0.1 mg/mL BSA and 3 M vanadate. ATP concentrations in
the assay were 10 M for VEGFR-2/KDR (2Â
M for c-Met (5Â the Km) and 0.6
lM vanadate, while the VEGFR-2/KDR reaction was conducted in
l
l
l
the Km). Enzyme concentration was 25 nM (c-Met) or 5 nM (VEGFR-2). The
recombinant enzymes were pre-incubated with inhibitor and Mg-ATP on ice in
polypropylene 96-well plates for 4 min, and then transferred to the substrate
coated plates. The subsequent kinase reaction took place at 30 °C for 30 min (c-
Met) or 10 min (VEGFR2). After incubation, the kinase reactions were
quenched with EDTA and the plates were washed. Phosphorylated product
was detected by incubation with Europium-labeled anti-phosphotyrosine
MoAb. After washing the plates, bound MoAb was detected by time-resolved
fluorescence in a Gemini SpectraMax reader (Molecular Devices). Inhibitors
were tested at seven different concentrations each in triplicate. IC50s were
calculated in a four parameters equation curve plotting inhibition (%).
14. Cellular assay conditions: A cellular clone of 293T cells stably expressing TPR-
Met (Park, M.; Dean, M.; Cooper, C. S.; Schmidt, M.; O’Brien, S. J.; Blair, D. G.;
Vande Woude, G. F. Cell 1986, 45, 895), the activated mutated form of the
receptor Met, under a CMV promoter was derived. Cells were treated with
compounds dilutions for 150 min and lysate samples from treatment wells
were transferred to high-binding white polysterene 96-well plates (Corning).
TPR-Met autophosphorylated levels were detected by ELISA using the primary
antibodies anti-phospho-Tyrosine (Millipore, 4G10) and a reporter antibody,
anti-mouse-horseradish peroxidase (Sigma). Plates were washed on a plate
washer (SkanWasher, Molecular Devises) and subsequently incubated with
chemiluminescent substrate solution (ECL, Roche). Luminescence signal was
captured on a Polar Star Optima apparatus (BMG LabTech). Average values of
triplicate treatment points were used to prepare IC50 curves using
a 4-
parameter fit model. These curves were calculated using GRAFIT 5.0 software.