Convergent synthesis of potent peptide inhibitors of the Grb2-SH2 domain by palladium catalyzed coupling of a terminal alkyne

Article abstract of DOI:10.1016/S0960-894X(01)00173-1

A new strategy was developed to prepare in a very efficient and convergent manner C-terminal modified tripeptides with high affinities for the Grb2-SH2 domain. Using Pd(PPh₃)₂Cl₂ as catalyst, selected naphthyl iodides and

Full text of DOI:10.1016/S0960-894X(01)00173-1

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1201–1203
Convergent Synthesis of Potent Peptide Inhibitors of the
Grb2-SH2 Domain by Palladium Catalyzed Coupling
of a Terminal Alkyne
Joseph Schoepfer,* Brigitte Gay, Nicole End, Evelyne Muller,
Gisela Scheffel, Giorgio Caravatti and Pascal Furet
Oncology Research, NOVARTIS Pharma AG, 4002 Basel, Switzerland
Received 22 December 2000; revised 27 February 2001; accepted 8 March 2001
Abstract—A new strategy was developed to prepare in a very efficient and convergent manner C-terminal modified tripeptides with
high affinities for the Grb2-SH2 domain. Using Pd(PPh3)2Cl2 as catalyst, selected naphthyl iodides and triflates were coupled to
Ac-Pmp(t-Bu)2-Ac6c-Asn-NH(prop-2-ynyl). The resulting alkyne derivatives were hydrogenated and deprotected to afford potent
Grb2-SH2 inhibitors. # 2001 Elsevier Science Ltd. All rights reserved.
The signal transduction pathways of tyrosine kinase
growth factor receptors offer different possibilities of
intervention in anticancer drug research. Besides target-
ing the kinase enzymatic activity of these receptors,1,2
inhibiting events downstream in the signalling cascade
constitutes another approach of potential interest in the
search for new antitumor agents. A possible strategy in
this direction is to block the interaction between the
phosphotyrosine-containing activated receptors and the
Src homology 2 (SH2) domain of the growth factor
receptor-bound protein 2. Recent papers reported sig-
nificant advances in the use of SH2 inhibitors in cancer
therapy3ꢀ5 and osteoporosis.6 Phosphonopeptide 1, the
corresponding prodrug,3,4 and the peptide 2 containing
a malonic acid isostere of pTyr5 showed encouraging
cellular activities (Fig. 1). Furthermore, combining Src-
SH2 inhibition and the bone targeting properties of
phosphonates, compound 3 displayed in vivo anti-
resorptive activity.6
Grb2-SH2 domain.8,9 This modification resulted in
phosphopeptides with affinity and selectivity.
Continuing along this line, we have designed, synthe-
sized, and tested C-terminal modified tripeptides
[general structure Ac-Pmp-Ac6c-Asn-NH-(3-naphtha-
lenyl-propyl)] 4. These compounds contain 4-(phos-
phonomethyl)phenylalanine (Pmp) as a stable pTyr
The X-ray structure of the Grb2-SH2 domain in com-
plex with a specific phosphopeptide ligand7 has revealed
an extended hydrophobic area adjacent to the primary
binding site. Therefore, hydrophobic residues were
added to the terminal Asn of the minimal peptide
sequence Ac-pTyr-X+1-Asn-NH2 recognised by the
*Corresponding author. Tel: +41-61-696-36-09; fax: +41-61-696-62-
46; e-mail: joseph.schoepfer@pharma.novartis.com
Figure 1. Examples of biologically profiled SH2 inhibitors.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00173-1

1202
J. Schoepfer et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1201–1203
replacement and maintain high affinities for the Grb2-
SH2 domain.
The terminal alkyne of 7 was then coupled with the dif-
ferent naphthyl iodides or triflate using Sonogashira14
reaction conditions: Pd(PPh3)2Cl2, copper iodide, and
triethylamine in DMF; in the presence of tetra-
butylammonium iodide15 (Scheme 3). The alkyne deri-
vative was then hydrogenated, deprotected and purified
with reverse phase medium pressure liquid chromato-
graphy.
Chemistry
A new strategy was developed to synthesize naphthal-
enyl peptides in a convergent and efficient way.
Starting from commercial BOC-Asn-OSu, the suitable
alkyne amino acid 5 was obtained by reaction with
propargylamine and subsequent TFA deprotection
(Scheme 1). From the chloromethyl derivative 6,10
reaction with deprotonated di-tert-butylphosphite and
coupling of the resulting free acid with 5 afforded the
common intermediate 7.
Results and Discussion
Earlier results have already shown that targeting the
hydrophobic patch was a successful strategy.8,10 Intro-
duction at the C-terminus of 3-naphthalenyl-propyl
substituents, whose size and shape allow to bury a sig-
nificant portion of the surface of the hydrophobic patch,
led to high affinity peptides (Table 1). The introduction
of hydroxyl and methoxy groups, in order to have elec-
tron-rich naphthols16 afforded in the case of 4f a com-
pound with a significantly lower IC50 value than 4a.
The iodonaphthols 8b,c were obtained by diazotation11
of the commercially available aminonaphthols. The tri-
flates 8d,e were obtained by aromatization of the
corresponding methoxy-tetralone with Pd(C) in
either triisopropylbenzenze12 or b-methylstyrene,13 fol-
lowed by treatment with triflic anhydride in pyridine
(Scheme 2).
The gain in activity obtained with 4f can be ascribed to
a favorable conformational restriction effect. Modeling
suggests the possibility of an intramolecular hydrogen
bond between the hydroxyl group of the naphthol and
the C-terminal carbonyl of the compound. This internal
hydrogen bond preorganizes the naphthol moiety in the
conformation giving the most extended surface of con-
tact with the hydrophobic patch, and thus burying the
largest amount of surface (Fig. 2).
Triflate 8f was prepared the same way from 8-benzyl-
oxy-naphthalen-2-ol, the major isomer obtained after
alkylation of naphthalene-1,7-diol and chromatographic
separation.
Conclusions
Structure base design in conjunction with a new syn-
thetic strategy to incorporate C-terminal groups has
allowed us to improve the Grb2-SH2 affinity of the Ac-
Pmp-Ac6c-Asn-NH-(3-naphthalenyl-propyl) compound
class. Peptide 7 with a terminal alkyne group can be
used for further exploration of the Grb2-SH2 hydro-
phobic patch.
Scheme 1. (i) Propargylamine, DMF, rt; (ii) TFA, 0 ꢁC; (iii) NaH,
HOP(Ot-Bu)2, DMF, rt; (iv) EDC, HOBT, NMM, 5, DMF, rt.
Scheme 2. (i) NaNO2, HCl, KI, H2SO4, THF/H2O; (ii) for precursor
of 8d: Pd(C), triisopropylbenzene, 240 ꢁC; for precursor of 8e: Pd(C),
b-methylstyrene, 250 ꢁC; (iii) benzylbromide, Cs2CO3, dioxan reflux;
(iv) Tf2O, pyridine, 0 ꢁC.
Scheme 3. (i) 8, Pd(PPh3)2Cl2, CuI, TBAI, NEt3, DMF, rt; (ii) Pd(C),
H2, MeOH; (iii) TFA, 0 ꢁC.

J. Schoepfer et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1201–1203
Table 1. Starting naphthalenes and IC50’s from Grb2-SH2 ELISA binding assay9
1203
X=I
8a
X=I
8b
X=I
8c
X=OTf
8d
X=OTf
8e
X=OTf, R=Bn
8f
Compound
X=Peptide
4a
42.9
R=H
4f
11.1
ꢂ0..4
Compound
IC50 [nM]
4b
4c
4d
4e
24.6
ꢂ2.9
42.7
ꢂ2.9
129.9
ꢂ5.9
84.4
ꢂ5.0
ꢂ2.0
4. Gay, B.; Suarez, S.; Weber, C.; Rahuel, J.; Fabbro, D.;
Furet, P.; Caravatti, G.; Schoepfer, J. J. Biol. Chem. 1999,
274, 23311.
5. Gao, Y.; Luo, J.; Yao, Z.-J.; Guo, R.; Zou, H.; Kelley, J.;
Voigt, J. H.; Yang, D.; Burke, T. R. J. J. Med. Chem. 2000,
43, 911.
6. Shakespeare, W.; Yang, M.; Bohacek, R.; Cerasoli, F.;
Stebbins, K.; Sundaramoorthi, R.; Azimioara, M.; Vu, C.;
Pradeepan, S.; Metcalf, C., III; Haraldson, C.; Merry, T.;
Dalgarno, D.; Narula, S.; Hatada, M.; Lu, X.; Van Schra-
vendijk, M. R.; Adams, S.; Violette, S.; Smith, J.; Guan, W.;
Bartlett, C.; Herson, J.; Iuliucci, J.; Weigele, M.; Sawyer, T.
Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 9373.
7. Rahuel, J.; Gay, B.; Erdmann, D.; Strauss, A.; Garcia-
Echeverria, C.; Furet, P.; Caravatti, G.; Fretz, H.; Schoepfer,
J.; Gruetter, M. G. Nat. Struct. Biol. 1996, 3, 586.
8. Furet, P.; Gay, B.; Caravatti, G.; Garcia-Echeverria, C.;
Rahuel, J.; Schoepfer, J.; Fretz, H. J. Med. Chem. 1998, 41,
3442.
Figure 2. Compound 4f minimized in the Grb2-SH2 binding site.
Residues of the hydrophobic patch: a=Phe bE3, b=Leu bD01, c=Lys
bD6.
9. Furet, P.; Gay, B.; Garcia-Echeverria, C.; Rahuel, J.; Fretz,
H.; Schoepfer, J.; Caravatti, G. J. Med. Chem. 1997, 40, 3551.
10. Schoepfer, J.; Fretz, H.; Gay, B.; Furet, P.; Garcia-Eche-
verria, C.; End, N.; Caravatti, G. Bioorg. Med. Chem. Lett.
1999, 9, 221.
Acknowledgements
11. Snyckers, F.; Zollinger, H. Helv. Chim. Acta 1970, 53,
1294.
12. Katritzky, A. R.; Balasubramanian, M.; Siskin, M. Org.
Prep. Proced. Int. 1993, 25, 585.
We gratefully acknowledge support for NMRspectro-
scopy from Dr. Joseph Schneider.
13. JP Patent 58065235, 1983; Chem. Abstr. 1983, 99,
158060.
14. Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron
Lett. 1975, 4467.
References and Notes
15. Powell, N. A.; Rychnovsky, S. D. Tetrahedron Lett. 1996,
37, 7901.
16. Previous work10 with indolyl analogues had suggested
that electronic enrichment of the ring interacting with the
hydrophobic patch was beneficial in terms of binding affi-
nity.
1. Garcia-Echeverria, C.; Traxler, P.; Evans, D. B. Med. Res.
Rev. 2000, 20, 28.
2. Sedlacek, H. H. Drugs 2000, 59, 435.
3. Gay, B.; Suarez, S.; Caravatti, G.; Furet, P.; Meyer, T.;
Schoepfer, J. Int. J. Cancer 1999, 83, 235.