Structure-based design, synthesis, and X-ray crystallography of a high- affinity antagonist of the Grb2-SH2 domain containing an asparagine mimetic

Article abstract of DOI:10.1021/jm991013u

Previous efforts in the search for molecules capable of blocking the associations between the activated tyrosine kinase growth factor receptors and the SH2 domain of Grb2 had resulted in the identification of 3-amino-Z- pTyr-Ac₆c-Asn-NH2, a hig

Full text of DOI:10.1021/jm991013u

2358
J . Med. Chem. 1999, 42, 2358-2363
Str u ctu r e-Ba sed Design , Syn th esis, a n d X-r a y Cr ysta llogr a p h y of a
High -Affin ity An ta gon ist of th e Gr b2-SH2 Dom a in Con ta in in g a n Asp a r a gin e
Mim etic
Pascal Furet,*,† Carlos Garc´ıa-Echeverr´ıa,† Brigitte Gay,† J oseph Schoepfer,† Martin Zeller,‡ and J oseph Rahuel§
Oncology Research Department, Novartis Pharma Inc., CH-4002 Basel, Switzerland
Received February 2, 1999
Previous efforts in the search for molecules capable of blocking the associations between the
activated tyrosine kinase growth factor receptors and the SH2 domain of Grb2 had resulted in
the identification of 3-amino-Z-pTyr-Ac6c-Asn-NH2, a high-affinity and selective antagonist of
this SH2 domain. In the present paper, we report the successful replacement of asparagine in
this compound by a â-amino acid mimetic, which brings us closer to our objective of identifying
a Grb2-SH2 antagonist suitable for pharmacological investigations.
In tr od u ction
The adapter protein Grb2 is an essential component
of the molecular machinery used by tyrosine kinase
growth factor receptors to transmit mitogenic signals
inside the cell.1-3 Grb2 contains a Src homology 2 (SH2)
domain whose function is to specifically associate with
phosphotyrosine (pTyr)-containing motifs present in the
intracellular part of the activated receptors. Interrup-
tion of the transmission of mitogenic signals in deregu-
lated cancerous cells by chemical blockade of these
F igu r e 1. The asparagine of the ligand makes two hydrogen
associations is regarded as a possible strategy to dis-
cover new antitumor agents.4-6 Using the minimal
peptide sequence pTyr-Ile-Asn retaining micromolar
affinity for the Grb2-SH2 domain as an initial lead
structure, we have engaged in a medicinal chemistry
effort based on this concept. We recently reported the
structure-based design of several modifications impart-
ing high affinity to the minimal sequence.7-9 In par-
ticular, attachment of a 3-aminobenzyloxycarbonyl (Z)
N-terminal group combined with replacement of the
central isoleucine residue by 1-aminocyclohexane car-
boxylic acid (Ac6c) resulted in 3-amino-Z-pTyr-Ac6c-Asn-
NH2 (1), a potent (IC50 ) 1 nM in an ELISA assay) and
selective antagonist of the Gbr2-SH2 domain. The high
potency of this compound prompted us to undertake
additional efforts directed at reducing its peptidic
character. Along this line, we present here the design,
synthesis, and confirmation of the binding mode by
X-ray crystallography of an analogue of 1 in which the
Asn residue has been replaced by a nonproteinogenic
cyclic â-amino acid mimetic without loss of activity.
bonds in a bidentate manner with the backbone NH and
carbonyl of Lys âD6 and one hydrogen bond with the backbone
carbonyl of Leu âE4 (dashed lines). The â-turn intramolecular
hydrogen bond between the NH of residue pTyr + 3 and the
backbone carbonyl of pTyr is also represented. For clarity, the
residues of the ligand N-terminal to pTyr are not shown.
F igu r e 2. Substructure query used in the database search.
Rn means bond belonging to a ring.
asparagine residue in position pTyr + 211 of the se-
quence of phosphopeptide ligands.12,13 In the X-ray
structure, this asparagine occupies position i + 2 of a
type I â-turn allowing the carboxamide group of its side
chain to make three specific hydrogen bonds with the
peptide backbone of the SH2 domain at residues Lys
âD6 and Leu âE414 as depicted in Figure 1. Using this
structural information, we looked for possible mimetics
of asparagine in 1.
Design
The X-ray crystal structure of the Grb2-SH2 domain
complexed with the phosphotyrosyl peptide H-Lys-Pro-
Phe-pTyr-Val-Asn-Val-NH2 (the 174-180 sequence of
the BCR-abl protein) has been determined in our
group.10 This has revealed with full atomic details the
structural basis of ligand recognition by the Grb2-SH2
domain. One of the key elements for recognition is the
The C-terminal carboxamide group of 1 corresponds
to the peptide bond connecting residues Asn (pTyr + 2)
and Val (pTyr + 3) in the phosphopeptide of the X-ray
structure. Inspection of the latter shows that this amide
bond has no interactions with the SH2 domain. How-
ever, its NH group is involved in the intramolecular
hydrogen bond with the backbone carbonyl of the
phosphotyrosine residue that stabilizes the â-turn con-
formation. Deletion of this carboxamide group in 1 by
* Corresponding author. E-mail: pascal.furet@pharma.novartis.com.
The authors contributed equally to the reported work.
† Oncology Research.
‡ Novartis CropProtection
§ Core Technology Area.
10.1021/jm991013u CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/09/1999

High-Affinity Antagonists of the Grb2-SH2 Domain
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13 2359
F igu r e 3. Designed cyclic â-amino acid mimetic (black) of the asparagine moiety (gray). Hydrogen bonds are shown as thick
lines (stereoview).
Sch em e 1a
replacement of Asn by â-alanine, leading to a molecule
which retains the capacity to make the same hydrogen
bonds as with the side chain of asparagine but lacking
the C-terminal carboxamide group, resulted in a dra-
matic loss of affinity for the Grb2-SH2 domain.15 This
result suggested the importance of introducing confor-
mational restriction in potential mimetics of the aspar-
agine moiety.
Interactive modeling based on the X-ray structure
showed that it was possible to construct a ring connect-
ing the R and â carbons of the phosphopeptide aspar-
agine while preserving the orientation of the side chain
carboxamide necessary to form the three hydrogen
bonds. In addition, the ring did not clash with the SH2
domain if it was built in the direction opposite to residue
Trp EF1 of the protein. This led to the idea of replacing
asparagine by a cyclic â-amino acid, providing rigidity.
Following this direction, a substructure search in the
chemical archives of the company based on the query
shown in Figure 2 was performed. The search returned
cis-2-amino-cyclohex-3-ene carboxylic acid (cis-Achec) as
an available six-membered ring â-amino acid synthe-
sized in an unrelated project. Molecular modeling
confirmed the suitability of this amino acid as a
substitute for asparagine. As shown in Figure 3, cis-
Achec-NH2 constructed as the (1S,2R) enantiomer in a
half-chair conformation with equatorial and axial posi-
tions for the amino and carboxy groups, respectively,
can adequately mimic the asparagine of the ligand in
its interactions with the SH2 domain. Thus, Asn in 1
was replaced by cis-Achec resulting in compounds 2a
and 2b. Although still able to form the three hydrogen
bonds with Lys âD6 and Leu âE4, the compound
containing the (1R,2S) enantiomer of cis-Achec was
expected to be less potent due to steric clash of its
cyclohexene ring with the SH2 domain residue Trp EF1.
a
Conditions: (a) ClSO2NCO, DCM; (b) KCO3-Na2SO3, 35%; (c)
Boc2O, TEA, DMAP, DCM, 82%; (d) LiOH‚H2O, THF/H2O, 91%;
(e) 4 N HCl in dioxane; (f) FmocOSu, TEA, MeCN:H2O (1:1, v/v),
80%.
F igu r e 4. Chromatograms for the separation and purification
of compounds 2a and 2b.
Compound 2 (3-amino-Z-pTyr-Ac6c-(cis)-Achec-NH2)
was synthesized manually on a Rink amide MBHA
resin,18 employing the Fmoc/tert-butyl strategy.19 The
protected cyclic â-amino acid 3 and the required NR-
Fmoc-amino acids were incorporated to the solid support
using standard coupling protocols. After completion of
the synthesis, the peptide resin was simultaneously
cleaved and deprotected with 95% TFA-water (19:1,
v/v). The HPLC trace of the crude phosphopeptide
displayed two main products with identical masses
(Figure 4). As expected from the use of the racemic
â-amino acid 3, the two main compounds (2a and 2b)
correspond to the two diastereomers of phosphopeptide
2, which were purified to homogeneity by preparative
reversed-phase MPLC (Figure 4).
Ch em istr y a n d Biology
The cyclic â-amino acid 3 was prepared as depicted
in Scheme 1. rac,cis-7-Azabyciclo[4.2.0]-oct-4-en-8-one
(4) was synthesized by known procedures16 using so-
dium sulfite for the reduction of the N-chlorosulfonyl
â-lactam.17 The cis orientation assigned to the fused
â-lactam is that expected for a dipolar addition of
chlorosulfonyl isocyanate with olefinic substrates.17
After protection of the amino functionality with the tert-
butoxycarbonyl group, the â-lactam was hydrolyzed with
lithium hydroxide to afford the Nâ-Boc protected cyclic
â-amino acid 6. For synthetic reasons, the Boc group
was removed under acidic conditions and replaced with
the Fmoc group.

2360 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13
Furet et al.
Ta ble 1. Inhibitory Activity of Compounds 1, 2a , and 2b in
EGFR Assaya
entry
compound
IC50 (nM)
1
2a
2b
3-amino-Z-pTyr-Ac6c-Asn-NH2
3-amino-Z-pTyr-Ac6c-(1R,2S)-Achec-NH2
3-amino-Z-pTyr-Ac6c-(1S,2R)-Achec-NH2
1.0 ( 0.2
>5000
1.6 ( 0.1
b
a
IC50 concentration to inhibit the binding of the phosphorylated
C-terminal intracellular domain of EGFR to the Grb2-SH2 domain.
Dose-response relationships were constructed by nonlinear re-
gression of the competition curves with GraFit 3.0 (Erithacus
Software Limited, London, U.K.). The errors quoted correspond
b
to the standard error in the fits of the data. Stereochemistry
assignment based on X-ray structure.
The Grb2-SH2 inhibitory activity of compounds 2a
and 2b was determined in our EGFR assay (Table 1).
This assay measures the potency of a compound to
inhibit the binding of the phosphorylated intracellular
domain of the epidermal growth factor receptor (EGFR)
to the Grb2-SH2 domain. The most potent diastereomer
is 2b which, remarkably, has a binding affinity almost
identical to that observed for our reference compound,
the asparagine-containing phosphopeptide 1 (IC50 ) 1.6
nM versus 1.0 nM). In addition, we found that 2b binds
selectively to the SH2 domain of Grb2 as testified by
the lack of affinity of the compound for the SH2 domain
of p56lck, a SH2 domain having a different specificity
than that of Grb2 (no inhibition at a concentration of
10 µM in a phosphopeptide competition assay previously
described9).
F igu r e 5. X-ray crystal structure of the Grb2-SH2 domain
in complex with 2b (yellow). Only the amino acid residues of
the SH2 domain within a distance of 4 Å of the ligand are
shown.
As shown by X-ray crystallography, the â-amino acid
(amino protected), cis-2-amino-cyclohex-3-ene carboxylic
acid, mimics the hydrogen bond interactions of aspar-
agine with the SH2 domain in full agreement with our
design. Thus with 2b, we have obtained a high-affinity
ligand of low molecular weight in which two of the three
initial natural amino acids of the minimal sequence
have been replaced by nonproteinogenic amino acids.
Furthermore, our asparagine mimetic allows the elimi-
nation of one peptide bond (the C-terminal carboxamide
group involved in the â-turn intramolecular hydrogen
bond) compared to previously described compound 1.
Therefore, the work reported here constitutes a progress
in our effort to identify an antagonist of the Grb2-SH2
domain suitable for pharmacological investigations.
The X-ray structure of the Grb2-SH2 domain com-
plexed with compound 2b allowed the determination of
the configuration of the cylic â-amino acid in the highly
potent diastereomer.
X-r a y Cr ysta llogr a p h y. The Grb2-SH2 domain was
cocrystallized with compound 2b and the X-ray struc-
ture of the complex solved at 3.0 Å resolution. The
structure revealed the expected (1S,2R) configuration
of cis-2-amino-cyclohex-3-ene carboxylic acid in 2b. The
main interactions of the ligand with the SH2 domain
are shown in Figure 5. As can be seen, the asparagine
mimetic functions as designed. Its carboxamide moiety
forms two hydrogen bonds in a bidentate manner with
the backbone carbonyl and NH of Lys âD6 and an
additional one with the backbone carbonyl of Leu âE4.
Moreover, the structure confirms the validity of earlier
design. As predicted, one observes a stacking interaction
of the phenyl ring of the 3-amino-Z N-terminal group
with the guanidinium moiety of Arg RA2,7 and Ac6c
adopts a right-handed 310 helical conformation with its
side chain making hydrophobic van der Waals contacts
with Phe âD5 and Gln âD3.8 Other important interac-
tions are identical to those seen in the X-ray structure
of the Grb2-SH2 domain complexed with the phospho-
peptide H-Lys-Pro-Phe-pTyr-Val-Asn-Val-NH2.10 In par-
ticular, the phosphotyrosine binds exactly the same way,
its phosphate group forming hydrogen bonds with the
side chains of Arg âB5 and Arg RA2. The backbone NH
of residue pTyr + 1 is also involved in a hydrogen bond
with the backbone carbonyl of His âD4.
Exp er im en ta l Section
Gen er a l. 4-(2′,4′-Dimethoxyphenyl-Fmoc-aminomethyl)-
phenoxy-polystyrene resin (1% DVB cross-linked, 0.4 mequiv/
g, 100-200 mesh) was purchased from NovaBiochem (La¨ufelfin-
gen, Switzerland). TPTU reagent was from Senn Chemicals
AG (Dielsdorf, Switzerland). HATU reagent was from PerSep-
tive Biosystems (Hamburg, Germany). The required Fmoc
derivatives were obtained using standard protocols. Na-Fmoc-
Tyr(PO3H2)-OH was synthesized as described previously.20
Trifluoroacetic acid, diisopropylethylamine, and piperidine
were from Fluka (Buchs, Switzerland). N,N-Dimethylaceta-
mide was from Merck (Hohenbrunn, Germany) and N-meth-
ylpyrrolidin-2-one was from SDS (Peypin, France). All com-
merical chemicals used were of the highest quality available
(AR grade or higher). TLC was performed on precoted silica
gel plates (60F-254, 0.2 mm thick, Merck); visualization was
obtained by dipping the plates in a stainning solution (2.5 g
of phosphormolybdic acid and 0.6 g of cerium(IV) sulfate in 1
L of 1 M H2SO4) following by heating the plates with an air
gun. The melting points were determined on a Bu¨chi 510
melting point instrument and are uncorrected. Silica gel
column chromatography was carried out with Merck silica gel
60 (particle size 0.040-0.063 mm) at 0.5 bar. Peptide retention
times (tR) were measured by RP-HPLC on a Spectra-Physics
Con clu sion
In phosphotyrosyl peptides, specificity for binding to
the SH2 domain of Grb2 is determined by the presence
of asparagine in position pTyr + 2 of the sequence. We
have identified a suitable replacement for this residue.

High-Affinity Antagonists of the Grb2-SH2 Domain
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13 2361
liquid chromatography system. Preparative purification was
carried out on a Bu¨chi system (Bu¨chi 687 gradient former and
Bu¨chi 688 chromatography pump) using a C18-column (Merck
LICHROPREP RP-18, 15-25 µm bead diameter, reversed-
phase HPLC column material based on C18-derivatized silica
gel, Merck, Darmstadt, FRG; column length 46 cm, diameter
3.6 cm; flow rate 53.3 mL/min; detection at 215 nm) eluted
with an acetonitrile-water gradient containing 0.1% of TFA.
NMR spectra of the compounds were recorded on a Varian
Gemini-300, Bruker 250, or Bruker Avance-500 spectrometers.
All 1H NMR spectra are reported in δ units, ppm downfield
from tetramethylsilane using the residual solvent signal as
an internal standard. Coupling constants (J ) are reported in
hertz (Hz). Electrospray ionization mass spectra (ESI MS) were
obtained with a Fisons Instruments VG Platform II. High-
resolution mass spectra (HRMS) were recorded on a Micromass
Quattro II instrument.
r a c,cis-7-Aza bicyclo[4.2.0]-oct-4-en -8-on e (4). A solution
of cyclohexa-1,3-diene (27.5 mL, 0.29 mol) in dichloromethane
(200 mL) was added to a solution of chlorosulfonyl isocyanate
(25 mL, 0.29 mol) in dichloromethane (200 mL) over 10 min.
After 30 min at room temperature, the reaction mixture was
added to a stirred solution of potassium carbonate (84 g, 0.86
mol) and sodium sulfite (3.6 g 0.03 mol) in water (600 mL).
Additional water (500 mL) was added to the mixture. The
organic layer was separated, and the aqueous layer was
extracted with ether (2 × 800 mL). The combined organic layer
and extracts were washed with water (2 × 200 mL), dried over
anhydrous magnesium sulfate, and evaporated to dryness
under reduced pressure. Recrystallization from diisopropyl
ether/n-hexane afforded pure compound 4 (12.2 g, 35%) as
colorless crystals: mp 74-76 °C (lit.16mp 70.5-71.5 °C); 1H
NMR (250 MHz, CDCl3) δ 5.9-6.2 (m, 3H), 4.05 (t, J ) 4.8
Hz, 1H), 3.51 (m, 1H), 2.12 (m, 3H), 1.5-1.8 (m, 1H); ESI MS
(positive ion mode) 124 [M + H]+. Anal. (C7H9NO) C, H, N.
nylamino)-cyclohex-3-ene carboxylic acid (2 g, 8.3 mmol) was
added to a stirred solution of 4 N HCl in dioxane (20 mL).
After 50 min at room temperature, the solution was concen-
trated to dryness and the residue was evaporated two times
from petrolether to remove traces of HCl. The residue was
triturated with cold ether, filtered, and dried overnight in a
desiccator over NaOH pellets. The compound (0.6 g, 3.6 mmol)
was dissolved in MeCN:H2O (1:1, v/v; 12 mL) and stirred
vigorously. To this stirred solution were added triethylamine
(1.64 mL, 11.76 mmol) and fluorenylmethylsuccinimidyl car-
bonate (1.7 g, 5.03 mmol). After the mixture was stirred for 3
h, the acetonitrile was evaporated, water (20 mL) was added,
and the suspension was extracted with ether (3 × 15 mL). The
aqueous layer was acidified with 1 N HCl to pH 2-3, and the
suspension was extracted with ethyl acetate (3 × 25 mL). The
combined ethyl acetate extracts were washed with brine, dried
over anhydrous magnesium sulfate, and evaporated to dryness
under reduced pressure. The crude compound was purified by
flash chromatogray on silica gel using dichloromethane/
methanol to obtain the title compound (979 mg, 80%) as an
oil. A sample was crystallized from chloroform/diisopropyl
1
ether/petroleum ether: mp 128-130 °C; H NMR (500 MHz,
DMSO-d6) δ 12.07 (bs, 1H, COOH), 7.88 (d, J ) 7.6 Hz, 2H),
7.74 (d, J ) 7.5 Hz, 1H), 7.69 (d, J ) 7.5 Hz, 2H), 7.41 (dd, J
) 7.5 Hz, 2H), 7.36 (d, J ) 9.9 Hz, 1H, NH), 7.33 (m, 2H),
5.79 (m, 1H, H-olefin), 5.61 (m, 1H, H-olefin), 4.45 (m, 1H,
CH(â)), 4.19 (m, 3H), 2.59 (m, 1H, CH(a)), 1.70-2.06 (m, 4H);
ESI MS (positive ion mode) 386.2 [M + Na]+. Anal. (C22H21N1O4)
C, H, N.
P h osp h op ep tid e Syn th esis. The synthesis of peptide 1
was published previously.8 Phosphopeptide 2 was synthesized
manually on a 4-(2′,4′-dimethoxyphenyl-aminomethyl)-phe-
noxy resin, employing the fluorenylmethoxycarbonyl strategy.
Fmoc removal was with piperidine/DMA (1:4, v/v; 6 × 2 min),
followed by washing with MeOH (3 × 1 min), NMP (2 × 1 min),
MeOH (3 × 1 min), and NMP (3 × 2 min). Coupling was
achieved by first dissolving the Fmoc-amino acid (2 equiv),
DIEA (5 equiv), and HATU or TPTU (2 equiv) in NMP, waiting
3 min for preactivation, adding the mixture to the resin, and
finally shaking for at least 2 h. The incorporation of 1 and
Fmoc-1-aminocyclohexyl carboxylic acid was accomplished
with HATU (first coupling) and TPTU (second coupling) as
described above. Na-Fmoc-Tyr(PO3H2)-OH was coupled with
TPTU (first coupling) and HATU (second coupling) as de-
scribed above. 3-N-tert-Butoxycarbonyl-aminobenzyl-4-nitro-
phenyl-carbonate (3 equiv8) was coupled to the N-terminal
residue of the peptide resin in the presence of an equimolar
amount of DIEA in NMP during 17 h at room temperature.
The complete peptide resin obtained after the last coupling
step was simultaneosly deprotected and cleaved by treatment
with 95% TFA/5% H2O for 3 h at room temperature. The
filtrate from the cleavage reaction was precipitated in diiso-
propyl ether/petroleum ether (1:1, v/v, 0 °C), and the precipi-
tate collected by filtration. The crude peptide was purified by
medium-pressure liquid chromatography using a C18-column
eluted with H2O/0.1% TFA (buffer A) and MeCN/0.09% TFA
(buffer B): 7 min with 100% buffer A, 7 min linear gradient
from 0% to 10% buffer B, 30 min from 10% to 16% buffer B,
15 min from 16% to 18% buffer B, and 7 min from 18% to 19%
buffer B; detection at 215 nm. A second purification was
carried out to obtain pure peptide 2b: 7 min with 100% buffer
A, 10 min linear gradient from 0% to 13% buffer B, 30 min
from 13% to 17% buffer B, 15 min from 17% to 18% buffer B,
and 10 min from 18% to 19% buffer B; detection at 215 nm.
Fractions shown by analytical HPLC to be >95% pure were
pooled and lyophilized to provide the title compounds as white
powder TFA salts: HRMS obs. 680.2452 (2a ) [M + Na]+, calcd
for C31H39N5O9P1Na1 680.2463; HRMS obs. 680.2456 (2b) [M
+ Na]+, calcd for C31H39N5O9P1Na1 680.2463. The purity of the
peptides was verified by reversed-phase analytical HPLC on
a Nucleosil C18-column (250 × 4 mm, 5 µm, 100 Å): linear
gradient over 10 min of MeCN/0.09% TFA and H2O/0.1% TFA
from 1:49 to 3:2, flow rate 2.0 mL/min, detection at 215 nm;
r a c,cis-N-ter t-Bu toxyca r bon yl-7-a za bicyclo[4.2.0]-oct-
4-en -8-on e (5). Triethylamine (6.8 mL, 49 mmol), di-tert-butyl
dicarbonate (21.3 g, 97 mmol), and 4-(dimethylamino)pyridine
(6.0 g, 49 mmol) were added to a solution of rac,cis-7-
azabicyclo[4.2.0]-oct-4-en-8-one (6.0 g, 49 mmol) in dichlo-
romethane (100 mL) under
a nitrogen atmosphere. The
reaction mixture was stirred for 6 h at room temperature and
then poured into 2 M HCl (150 mL). The organic layer was
separated, and the aqueous layer was extracted with dichlo-
romethane (2 × 150 mL). The combined organic layer and
extracts were washed with water (100 mL), dried over anhy-
drous magnesium sulfate, and evaporated to dryness under
reduced pressure. The residue was purified by flash column
chromatography on silica gel using ethyl acetate/n-hexane (1:
4) to obtain 5 (8.9 g, 82%) as a yellow oil: 1H NMR (250 MHz,
CDCl3) δ 6.15 (m, 2H), 4.31 (dd, J ) 4.3 Hz, J ) 4.2 Hz, 1H),
3.49 (m, 1H), 2.04-2.14 (m, 3H), 1.66-1.77 (m, 1H), 1.52 (s,
9H); ESI MS (positive ion mode) 224 [M + H]+. Anal. (C12H17
NO3) calcd C, H, N.
-
r a c,ci s-2-(t er t -B u t o x y c a r b o n yla m in o )-c y c loh e x-3-
en e Ca r boxylic Acid (6). A solution of lithium hydroxide
monohydrate (4.1 g 97 mmol) in water (100 mL) was added to
a stirred solution of rac,cis-N-tert-butoxycarbonyl-7-azabicyclo-
[4.2.0]-oct-4-en-8-one (7.0 g; 31 mmol) in THF (140 mL). The
reaction mixture was stirred for 7 h at room temperature. After
this time, the THF was removed by evaporation, and the
solution was acidified to pH 4 by adding 10% acetic acid. The
mixture was extracted with ether (2 × 400 mL), dried over
anhydrous magnesium sulfate, and evaporated to dryness
under reduced pressure. The residue was recrystallized from
ethyl acetate/n-hexane to afford pure 6 (6.9 g, 91%) as colorless
crystals: mp 122-123 °C (lit.21 mp 110-118 °C); 1H NMR (250
MHz, DMSO-d6) δ 11.72 (bs, 1H, COOH), 6.11 (bd, 1H, NH),
5.72 (m, 1H, H-olefin), 5.60 (m, 1H, H-olefin), 4.36 (m, 1H,
CH(â)) 2.61 (m, 1H, CH(a)), 2.06-1.70 (m, 4H), 1.85-2.0, 1.38
(s, 9H, t-Bu); ESI MS (positive ion mode) 242 [M + H]+. Anal.
(C12H19NO4) C, H, N.
r a c,cis-2-(9-F lu or en ylm eth oxyca r bon yla m in o)-cyclo-
h ex-3-en e Ca r boxylic Acid (3). rac,cis-2-(tert-Butoxycarbo-
1
single peak at tR) 6.18 min (2a ); tR) 6.32 min (2b); H NMR

2362 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13
Furet et al.
nm on a plate reader by adding 100 µL/well of a solution of
tetramethylbenzidine as substrate.
Da ta An a lysis. Compound inhibition was calculated as a
percentage of the reduction in absorbance in the presence of
each inhibitor concentration compared to the absorbance
obtained with GST/SH2 in the absence of inhibitor. Dose-
response relationships were constructed by nonlinear regres-
sion of the competition curves with Grafit (Erithacus Software,
London, U.K.). Fifty percent inhibitory concentrations (IC50
were calculated from the regression lines (Figure 6).
)
X-r a y Cr ysta llogr a p h y. Cloning and expression of the
Grb2 SH2 domain have been described earlier.10 A solution of
pure protein (Grb2 55-153) was concentrated with a Centricon
(Amicon Grace Company) to 10-10.4 mg/mL and mixed with
a 5-fold molar excess of 2b. Small crystals of the complex with
2b were obtained after a few days in 0.1 M HEPES, pH 7.5,
and 1.4 M sodium acetate by the hanging drop vapor diffusion
method. The crystals belong to the orthorhombic space group
P212121 with lattice constants 73.8 × 93.3 × 232.8 Å and
diffract to 3.0 Å resolution.
F igu r e 6. Inhibition curve of compound 2b in the EGFR assay
([) and that of the Ac-EpYINQ-NH2 phosphopeptide, IC50
5.8 ( 0.5 as control (2). The standard deviation represents
the mean of triplicates.
)
One of these crystals (0.1 × 0.1 × 0.05 mm3) was cryocooled
at 100 K, and data were collected at this temperature on a
MAR area detector mounted on an FR591 rotating anode X-ray
generator (Enraf-Nonius) operating at 4.1 kW and producing
graphite monochromated Cu KR radiation. Frames (expo-
sure: 25 min, crystal rotation: 0.5°) were processed using the
MARXDS and the MARSCALE software programs.22 The
Rmerge for all data between 30.0 and 3.0 Å is 20% with a 3.9-
fold redundancy. The overall completeness of the data is 96%,
and the same for data in the shell between 3.5 and 3.0 Å.
The large crystal unit cell (1.6 × 106 Å3) indicates the
presence of many molecules of the complex in the asymmetric
unit (Vm ) 2.9 Å3/Da of protein for 12 protein molecules). A
model comprising a dimer of the protein, which has been
observed in the five different crystal forms obtained so far in
our laboratory, was used as a probe for molecular replacement
with the program AMORE.23 The highest 10 peaks obtained
in the rotation function were applied in the translation
function. Due to the weakness of the data it was not possible
to define unambiguously to which orthorhombic space group
the crystal belongs to. The translation function had to be
repeated in the four possible space groups (P222, P2221,
P21212, and P212121) plus the four axis permutations in space
groups P2221 and P21212. Only the space group P212121 gave
acceptable results in the calculation of the translation function,
giving a strong indication that it was the correct space group.
Nevertheless, the peak obtained for the first dimer was not
(500 MHz, 300 K, DMSO-d6) of 2b δ 7.82 (s, 1H, NH), 7.44 (d,
J ) 8.3 Hz, 1H, NH-urethane), 7.27 (d, J ) 8.6 Hz, 2H,
H-phenyl, pTyr), 7.23 (s, 1H, NH-carboxamide), 7.15 (d, J )
8.8 Hz, 1H, NaH), 7.05 (d, J ) 8.2 Hz, 2H, H-phenyl, pTyr),
7.01 (t, J ) 7.5 Hz, 1H, H-benzyloxy), 6.77 (s, 1H, NH-
carboxamide), 6.55 (m, 3H, H-benzyloxy), 5.68 (m, 1H, H-
olefin), 5.49 (m, 1H, H-olefin), 4.81 (s, 2H, CH2-benzyloxy) 4.49
(m, 1H, H-C(â)), 4.28 (m, 1, H-C(a), pTyr), 3.01 (dd, J ) 13.5
Hz, J ) 3.5 Hz, H-C(â), pTyr), 2.68 (dd, J ) 13.5 Hz, J ) 11.5
Hz, H-C(â), pTyr), 2.53 (m, 1H, H-C(a)), 1.12-2.09 (m, 14H).
Abbr evia tion s. The abbreviations for amino acids and the
nomenclature of peptide structures follow the recommenda-
tions of the IUPAC-IUB Commission on Biochemical Nomen-
clature (Eur. J . Biochem. 1984, 138, 9). Other abbreviations
are as follows: Ac6c, 1-aminocyclohexylcarboxylic acid; Boc,
tert-butyloxycarbonyl; DCM, dichloromethane; DIEA, diiso-
propylethylamine; DMA, N,N-dimethylacetamide; DMAP,
4-(dimethylamino)pyridine; EGFR, epidermal growth factor
receptor; ESI MS, electrospray ionization mass spectra; Fmoc,
9-fluorenylmethyloxycarbonyl; Grb2, growth factor receptor-
bound protein 2; HATU, N-[(dimethylamino)-1H-1,2,3-triazolo-
[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexaflu-
orophosphate N-oxide;HPLC,high-performance liquid chromato-
graphy; HRMS, high-resolution mass spectra; MBHA, meth-
oxybenzhydrylamine; MPLC, medium-pressure liquid chro-
matography; NMR, nuclear magnetic resonance; NMP, N-me-
thylpyrrolidin-2-one; SH2, Src homology 2; TEA, triethylamine;
TFA, trifluoroacetic acid; THF, tetrahydrofuran; TLC, thin-
layer chromatography; TPTU, 2-(2-oxo-1(2H)-pyridyl)-1,1,3,3-
tetramethyluronium tetrafluoroborate; tR, retention time.
EGF R Assa y. The EGFR assay has already been described
in detail previously.10 Briefly, phosphorylated MBP-EGFR
immobilized on a solid phase (polystyrene microtiter plates,
NUNC MAXYSORB) was incubated with a GST/Grb2-SH2
fusion protein capable of binding to it, in the presence of
phosphopeptide in buffer or buffer alone. Bound SH2 was
detected with polyclonal rabbit anti-GST antibody. Following
washing, horseradish peroxidase-conjugated mouse anti-rabbit
antibody was added. Peroxidase activity is monitored at 655
completely convincing (correlation factor ) 0.249, Rfactor
)
0.493). In the course of iteration of the translation function
with additional dimers, the correlation factor increased con-
stantly while the Rfactor decreased considerably. After the
introduction of six dimers in the model the correlation factor
was 0.642 and the Rfactor 0.369. Visual inspection confirmed
that the resulting packing in the crystal cell unit was reason-
able.
The refinement was performed with the program XPLOR,
24
and manual rebuilding of the models was performed with
the program O.25 All data between 30.0 and 3.0 Å were used,
and a bulk solvent correction was applied. A first cycle of rigid
body refinement was performed to position the 12 molecules
F igu r e 7. Stereoview of an Fobs-Fcalc electron density map in the binding site of Grb2-SH2 after refinement. The inhibitor which
has been omitted from Fcalc is represented. The electron density is contoured at 3 σ above the mean density.

High-Affinity Antagonists of the Grb2-SH2 Domain
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13 2363
(10) Rahuel, J .; Gay, B.; Erdmann, D.; Strauss, A.; Garcia-Echeverria,
C.; Furet, P.; Caravatti, G.; Fretz, H.; Schoepfer, J .; Gruetter,
M. Structural Basis for Specificity of Grb2-SH2 Revealed by a
Novel Ligand Binding Mode. Nature Struct. Biol. 1996, 3, 586-
589.
(11) Ligand residues are numbered relative to the position of the
phosphotyrosine which is denoted pTyr 0.
(12) Songyang, Z.; Shoelson, S. E.; McGlade, J .; Olivier, P.; Pawson,
T.; Bustelo, X. R.; Barbacid, M.; Sabe, H.; Hanafusa, H.; Yi. T.;
Ren, R.; Baltimore, D.; Ratnofsky, S.; Feldman, R. A.; Cantley,
L. C. Specific Motifs Recognized by the SH2 Domains of Csk,
3BP2, fps/fes, GRB-2, HCP, SHC, Syk and Vav. Mol. Cell. Biol.
1994, 14, 2777-2785.
(13) Songyang, Z.; Shoelson, S. E.; Chaudhuri, M.; Gish, G.; Pawson,
T.; Hase, W. G.; King, F.; Roberts, T.; Ratnofsky, S.; Lechleider,
R. J .; Neel, B. G.; Birge, R. B.; Fajardo, J . E.; Chou, M. M.;
Hanafusa, H.; Schaffhausen, B.; Cantley, L. C. SH2 Domains
Recognize Specific Phosphopeptide Sequences. Cell 1993, 72,
767-778.
(14) For the nomenclature of the SH2 domain residues see: Lee, C.
H.; Kominos, D.; J acques, S.; Margolis, B.; Schlessinger, J .;
Shoelson, S. E.; Kuriyan, J . Crystal Structures of Peptide
Complexes of the Amino-Terminal SH2 Domain of the Syp
Tyrosine Phosphatase. Structure 1994, 2, 423-438.
(15) 3-amino-Z-pTyr-Ac6c-âAla-NH2 has an activity in the micromolar
range in the EGFR assay (Garcia-Echeverria, C.; et al. Novartis
Pharma Inc., Oncology Research Department, unpublished
results).
(16) Malpass, J . R.; Tweddle, N. J . Reaction of Chlorosulphonyl
Isocyanate with 1,3-Dienes. Control of 1,2- and 1,4-Addition
Pathways and the Synthesis of Aza- and Oxa-bicyclic Systems.
J . Chem. Soc., Perkin Trans. I 1977, 874-884.
(17) Durst, T.; O’Sullivan, M. J . Reduction of N-Chlorosulfonyl
â-Lactams to â-Lactams with Sodium Sulfite. J . Org. Chem.
1970, 35, 2043-2045.
(18) Rink, H. Solid-phase Synthesis of Protected Peptide Fragments
Using a Trialkoxy-Diphenyl-Methyl Ester Resin. Tetrahedron
Lett. 1987, 28, 3787-3790.
more precisely. The second step consisted of refinement of the
protein alone using strict noncrystallographic symmetry.
Visual inspection of an electron density map confirmed the
presence of the ligand in the active site and allowed its
complete insertion into the model. Further refinement was
performed, followed by minor modifications of the protein and
addition of two solvent molecules per protein monomer. The
final model consists of 96 protein residues, 53 inhibitor atoms,
and 4 water molecules. The Rfactor (Rfree) dropped to 0.305
(0.318) with acceptable deviations of ideal geometry (rms
deviation 0.027 Å and 2.536° for bonds and bond angles,
respectively). Analysis of main chain torsion angles using
PROCHECK26 showed that 86% of the residues are located in
the most favored regions, the remaining 14% occurring in the
additional allowed regions of the Ramachandran plot. All the
atoms of the inhibitor have been located within the electron
density (Figure 7).
The coordinates of the X-ray structure have been deposited
in the Brookhaven Protein Data Bank with accession code
1CJ 1 for immediate availability.
Molecu la r Mod elin g. The modeling work was performed
in MacroModel v.4.027 (“in house” version enhanced for graph-
ics by A. Dietrich, unpublished results). Energy minimizations
were performed using the AMBER force field28 in conjunction
with the GB/SA water solvation model.29 The conformation of
cis-Achec necessary to mimic the asparagine of 1 (shown in
Figure 3) is of low energy (within 1 kcal/mol of the calculated
global minimum). The two-dimensional query shown in Figure
2 and the subsequent substructure search were made using
the ISIS DRAW-ISIS BASE software, version 2.1.3d (MDL).
The full Novartis corporate database of compounds was
searched. The search gave 10 hits from which cis-Achec was
selected as an available and rigid building block.
(19) Atherton, E.; Sheppard, R. C. In Solid-Phase Peptide Synthesis
- A Practical Approach; Richwood, D., Hames, B. D., Eds.; IRL
Press at Oxford University Press: Oxford, 1989.
Ack n ow led gm en t. We thank R. Wille, D. Arz, V.
von Arx, and C. Stamm for their technical assistance.
(20) Ottinger, E. A.; Shekels, L. L.; Bernlohr, D. A.; Barany, G.
Synthesis of Phosphotyrosine-Containing Peptides as Their Use
as Substrates for Protein Tyrosine Phosphatases. Biochemistry
1993, 32, 4354-4361.
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J M991013U