Imidazole-based ligands of the Src SH2 protein

Article abstract of DOI:10.1016/S0960-894X(02)00138-5

Starting from known Src SH2 inhibitors incorporating five-membered heterocycles or benzamide scaffolds, we prepared tetrasubstituted imidazole compounds able to interact with the pY, pY+1 and pY+3 binding sites of the Src SH2 protein. The synthesis and bi

Full text of DOI:10.1016/S0960-894X(02)00138-5

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1287–1289
Imidazole-based Ligands of the Src SH2 Protein
Pierre Deprez,* Eliane Mandine, Annie Vermond and Dominique Lesuisse
Aventis Pharma, Paris Research Center, Medicinal Chemistry, 102 route de Noisy, 93235 Romainville Cedex, France
Received 23 October 2001; accepted 19 February 2002
Abstract—Starting from known Src SH2 inhibitors incorporating five-membered heterocycles or benzamide scaffolds, we prepared
tetrasubstituted imidazole compounds able to interact with the pY, pY+1 and pY+3 binding sites of the Src SH2 protein. The
synthesis and biological data are presented. # 2002 Elsevier Science Ltd. All rights reserved.
The pp60c-src (Src) is a nonreceptor tyrosine kinase
involved in intracellular signal transduction.1 The
observation that osteopetrosis is the major phenotype in
In this paper, we would like to disclose the synthesis and
biological evaluation of Src SH2 inhibitors based on the
imidazole scaffold, incorporating a carboxamide moiety
susceptible to displace the water molecule. This
should enhance the entropic contribution to binding
when compared to the five-membered rings already
described.10,11
2
SrcÀ/Àmice
incited us to develop a program for
the inhibition of the Src protein via its SH2 domain for
the treatment of osteoporosis. Src SH2 domain3 con-
tains approximately 100 amino acids and binds to
phosphotyrosine (pTyr) containing peptides.
The target compound identified for synthesis is the
tetrasubstituted imidazole 15b. The cyclohexyl moiety
was introduced to interact with the hydrophobic pocket
while the 5-CONH2 was intended to displace the struc-
tural water molecule. Moreover, the substitution at the
2-position of the imidazole was designed to pick up the
pY as well as the pY+1 interactions (originally pY-E
on the tetrapeptide pYEEI). However, Buchanan et al.10
at Ariad showed that simple alkyl side chains can be
acceptable replacements for the (pY+1) Glu residue
within the five-membered ring oxadiazole series. This
was also the case in the benzamide series developed by
Parke Davies.8 Thus, aware of the importance of redu-
cing the overall charge of the molecule but also for
synthetic feasibility, we decided to replace the original
Glu residue by a methyl group.
Extensive crystallographic studies4,5 between the pY-E-
E-I tetrapeptide and the Src SH2 protein have revealed
the presence of two major binding pockets, one inter-
acting with the pTyr and the other with the pY+3 Ileu
residue. From rational drug design, new potent and less
peptidic inhibitors6À11 have been found, based on vari-
ous scaffolds which are able to interact positively with
the protein and also to deliver their side chains into the
respective pY and pY+3 pockets. Among these scaf-
folds, benzamide8 is potent, the phenyl group being in
contact with the hydrophobic surface generated by the
Tyr bD5 and the carboxamide function taking the place
of a structural water molecule and thus interacting
directly with Lys 60. Five-membered ring scaffolds
(thiazole, oxadiazole) have also been disclosed
recently10,11 and proved to efficiently deliver the side
chains to their respective pockets. However, these het-
erocycles are lacking the interaction generated by the
carboxamide of the benzamide scaffold, and thus the
inhibitors designed from them appeared to be a slightly
less active than pYEEI and those derived from the
benzamide scaffold.
Compound 7 was the key intermediate for the synthesis
of 15b and was prepared via a multigram four-step
sequence with an adaptation of the pathway developed
for the synthesis of angiotensin II antagonists.12,13
Compound 7 proved to be a versatile scaffold which
allowed easy functionalization of positions 2, 4 and 5 of
the imidazole ring. Thus several compounds could be
derived from imidazole 7 via chemical modifications of
the 2-carboxaldehyde, the 4-masqued thiol and 5-car-
boxyester, as shown with the synthesis of the target
compounds 15a–c (Scheme 1).
*Corresponding author. Tel.: +33-1-4991-3069; fax: +33-1-4991-
3089; e-mail: pierre.deprez@aventis.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00138-5

1288
P.Deprez et al./ Bioorg.Med.Chem.Lett.12 (2002) 1287–1289
Scheme 1. Synthesis of the tetrasubstituted imidazole scaffold 7. Reagents and conditions: (a) pyridine, THF, 2 h, 62%; (b) MeSH excess in EtOH,
TEA, overnight 5 ꢀC, 84%; (c) propane phosphonic anhydride, AcOEt, 30mn rt then reflux 2 h, 79%; (d) K CO3, DMF, 1.1 equiv MeI 3 h, 90%;
(e) 50equiv of HCOCOOH, H 2O in CH3COOH HClconc (50equiv), 30h, rt, 89%.
2
Condensation of dithioethylacetylchloride14 1 with ethyl
aminocyanoacetate 2 led to intermediate 3. Addition of
methylthiol onto the nitrile gave rise to the amino com-
pound 4 which could easily undergo an intramolecular
cyclization using propane phosphonic anhydride in 75%
yield. Alkylation of the resulting imidazole 5 with
methyl iodide afforded N–Me imidazole 6 as the major
regioisomer (90% yield). Final deprotection of the
dithioacetal proved to be rather difficult and attempts
using HgCl2–HgO in CH2Cl2, HgCl2 with CaCO3 in
CH3CN, HCl in MeOH or SOCl2 with SiO2 in CH2Cl2
were unsuccessful. Finally, the use of glyoxylic acid
which gave smooth displacement of the dithioethyl
group was the method of choice15 and led to the desired
2-carboxaldehyde in 89% yield (Scheme 2).
desired fragment from the aldehyde and generation of
the thiol from the thiomethyl group.
Modification of the aldehyde was first achieved by
introduction of the pY+1 side chain as a methyl group
by addition of methyl Grignard. Then, in order to pre-
pare the required amine, the resulting alcohol 8 was
converted into azido derivative 9 using DPPA in toluene
with a base. This reaction was more efficient with DBU
as a base than using TEA (85% yield vs 20%).
The azido group of compound 9 was then used as an
amino protecting group during the functionalization of
the 4-thiomethyl moiety. Conversion of thiomethyl to
another thioalkyl (methylcyclohexyl in this paper) was
performed according to a straightforward two-step
procedure developed recently in our team.16 The first
step consists of oxidation of the sulfur atom with
mCPBA at 0 ꢀC. The resulting sulfoxide 10 was then
submitted to Pummerer reaction with trifluoroacetic
With this scaffold 7 in hand, we could functionalize the
2- and 4-positions of the imidazole ring in order to
deliver the pY and pY+3 side chains into their respec-
tive pockets. This was done by construction of the
Scheme 2. Synthesis of imidazole based Src SH2 ligands 15a–c. Reagents and conditions: (a) MeMgBr, THF, 0 ꢀC 15 min, 55%; (b) DPPA, toluene,
DBU 24 h, 85%; (c) mCPBA, CH2Cl2 0 ꢀC; 15 min, 85%; (d) (i) TFAA, CH2Cl2 15 min, then evaporation; (ii) degassed MeOH, TEA 5 min,
followed by addition of ICH2cHexyl, overnight at rt, 81%; (e) (i) 5 equiv of 2 N NaOH, EtOH, overnight (91%); (ii) EDC, HOBT, NMM in THF
1 h, then NH4OH 30% 3 h (63%); (f) H2, Pd/C AcOEt (100%); (g) EDC, HOBt, RCOOH, 3 h, 88%; (h) H2, Pd/C MeOH, 70% [for PO(OBn)2 or
TMSBr in CH2Cl2], 3 h at 0 ꢀC [for PO(OEt)2].

P.Deprez et al./ Bioorg.Med.Chem.Lett.12 (2002) 1287–1289
1289
Table 1. Src SH2 binding affinities
interacting by displacement of the water molecule as
expected.
References and Notes
1. (a) Brown, M. T.; Cooper, J. A. Biochim.Biophys.Acta
1996, 1287, 121. (b) Liu, D.; Wang, L.-H. J.Biomed.Sci. 1994,
1, 65. (c) Thomas, S. M.; Brugge, J. S. Annu.Rev.Cell Dev.
Biol. 1997, 13513.
2. Soriano, P.; Montgomery, C.; Geske, R.; Bradley, A. Cell
1991, 64, 693.
Compd
R1
R2
IC50 (mM)18
3. (a) Sawyer, T. K. Biopolymers 1998, 47, 243. (b) Pawson, T.
Nature 1994, 373, 573.
4. Waksman, G.; Shoelson, S. E.; Pant, N.; Cowburn, D.;
Kuriyan, J. Cell 1993, 72, 779.
15a
15b
15c
COOEt
CONH2
CONH2
OPO3H2
OPO3H2
CF2PO3H2
6.9
8.6
5.3
5. (a) Eck, M. J.; Shoelson, S. E.; Harrison, S. C. Nature 1993,
362, 87. (b) Eck, M. J.; Atwell, S. K.; Shoelson, S. E.; Harri-
son, S. C. Nature 1994, 368, 764.
6. Charifson, P. S.; Shewchuk, L. M.; Rocque, W.; Hummel,
C. W.; Jordan, S. R.; Mohr, C.; Pacofsky, G. J.; Peel, M. R.;
Rodriguez, M.; Sternbach, D. D.; Consler, T. G. Biochemistry
1997, 36, 6283.
7. Lunney, E. A.; Para, K. S.; Rubin, J. R.; Humblet, C.;
Fergus, J. H.; Marks, J. S.; Sawyer, T. K. J.Am.Chem.Soc.
1997, 119, 12471.
anhydride to generate the intermediate protected thiol
SCH2OCOCF3. Basic treatment with TEA in MeOH
liberated in situ the thiol which was immediately con-
verted one pot in the desired thiomethylcyclohexyl 11 by
addition of the corresponding iodide (81% yield for the
two steps from sulfoxide).
Final functional group modifications led to the desired
compound. Thus, introduction of the carboxamide
group was achieved via saponification of the ester group
followed by a EDC coupling of the resulting acid with
ammonia. Functionalization of the 2-position of the
imidazole was achieved after conversion of the azido to
the amino group by hydrogenation (13) and EDC cou-
pling with dibenzyl protected phosphotyrosine (or
F2PMP tyrosine17). Final deprotection (hydrogenolysis
in EtOH for debenzylation of the dibenzylphosphate or
TMSBr in CH2Cl2 for removal of the ethyl groups from
the CF2PO(OEt)2 fragment afforded the final desired
compounds 15b–c. To evaluate the influence of the
substitution in the position 5 of the imidazole (ester vs
carboxamide), the intermediate 5-ethyl carboxylate ester
imidazole 11 was also coupled to phosphotyrosine after
hydrogenation of the azido group to finally give 15a.
8. (a) Plummer, M. S.; Holland, D. R.; Shahripour, A.; Lun-
ney, E. A.; Fergus, J. H.; Marks, J. S.; McConnel, P.; Mueller,
W. T.; Sawyer, T. K. J.Med.Chem. 1997, 40, 3719. (b) Lun-
ney, E. A.; Para, K. S.; Rubin, J. R.; Humblet, C.; Fergus,
J. H.; Marks, J. S.; Sawyer, T. K. J.Am.Chem.Soc.
119, 12471.
1997,
9. Beaulieu, P. L.; Cameron, D. R.; Ferland, J.-M.; Gauthier,
J.; Ghiro, E.; Gillard, J.; Gorys, V.; Poirier, M.; Rancourt, J.;
Wernic, D.; Llinas-Brunet, M. J.Med.Chem. 1999, 42, 1757.
10. Buchanan, J. L.; Vu, C. B.; Merry, T. J.; Corpuz, E. G.;
Pradeepan, S. G.; Mani, U. N.; Yang, M.; Plake, H. R.; Var-
khedkar, V. M.; Lynch, B. A.; MacNeil, I. A.; Loiacono,
K. A.; Tiong, C. L.; Holt, D. A. Bioorg.Med.Chem.Lett.
1999, 9, 2359.
11. Buchanan, J. L.; Bohacek, R. S.; Luke, G. P.; Hatada, M.;
Lu, X.; Dalgarno, D. S.; Narula, S. S.; Yuan, R. Y.; Holt,
D. A. Bioorg.Med.Chem.Lett. 1999, 9, 2353.
12. Caille, J. C.; Didierlaurent, S.; Lefrancois, D.; Lelievre,
M. H.; Surry, C.; Aszodi, J. Synthesis 1995, 6, 635.
13. Deprez, P.; Guillaume, J.; Becker, R.; Corbier, A.;
Didierlaurent, S.; Fortin, M.; Frechet, D.; Hamon, G.; Heck-
mann, H. H.; Kleemann, H.-W.; Vevert, J.-P.; Vincent, J.-C.;
Wagner, A.; Zhang, J. J.Med.Chem. 1995, 38, 2357.
These three compounds 15a–c have been evaluated
using a competition assay (Scintillation Proximity assay,
SPA)18 and the biological results are indicated in Table
1. The mM level of activity indicates that some positive
interactions between the protein and these ligands exist.
However, these results are not as promising as expected
because the prepared compounds are around 30times
less active than the reference peptide pYEEI in our
assay (150nM). Moreover, there is no difference in
binding between compound 15a and 15b (5-COOEt vs
5-CONH2). By comparison with the benzamide scaf-
fold,8 the carboxamide imidazole scaffold appears to be
less attractive. This suggests that our ligand are not
14. Bates, S. J.Chem.Soc,. Chem.Commun.
1979, 161.
15. Muxfeldt, H.; Unterweger, W. D.; Helmchen, G. Synthesis
1976, 694.
16. Deprez, P.; Vevert, J. P. J.Fluorine Chem. 1996, 80, 159.
17. Burke, T. R.; Smyth, M. S.; Otaka, A.; Roller, P. R.
Tetrahedron Lett. 1993, 34, 4125.
18. (a) Mandine, E.; Gofflo, D.; Jean-Baptiste, V.; Gaillard-
Kelly, M.; Zoller, M.; Baron, R.; Lesuisse, D. JMBM 1998,
23, S221. (b) Mandine, E.; Gofflo, D.; Jean-Baptiste, V.;
Sarubbi, E.; Touyer, G.; Deprez, P.; Lesuisse, D. J.Mol.
Recog. 2001, 14, 254.