Synthesis of a 5-Methylindolyl-Containing Macrocycle That Displays Ultrapotent Grb2 SH2 Domain-Binding Affinity

Article abstract of DOI:10.1021/jm030440b

The growth factor receptor-bound protein 2 (Grb2) is an SH2 domain-containing docking module that represents an attractive target for anticancer therapeutic intervention. Here, a ring-closing metathesis approach is utilized to synthesize a 5-methylindolyl

Full text of DOI:10.1021/jm030440b

788
J . Med. Chem. 2004, 47, 788-791
Letters
Syn th esis of a
5-Meth ylin d olyl-Con ta in in g Ma cr ocycle
Th a t Disp la ys Ultr a p oten t Gr b2 SH2
Dom a in -Bin d in g Affin ity
Zhen-Dan Shi,† Kyeong Lee,† Chang-Qing Wei,†
Lindsey R. Roberts,‡ Karen M. Worthy,‡
Robert J . Fisher,‡ and Terrence R. Burke, J r.*,†
Laboratory of Medicinal Chemistry, CCR,
National Cancer Institute, National Institutes of Health,
Frederick, Maryland 21702, and Protein Chemistry
Laboratory, SAICsFrederick, Frederick, Maryland 21702
Received September 5, 2003
Abstr a ct: The growth factor receptor-bound protein 2 (Grb2)
is an SH2 domain-containing docking module that represents
an attractive target for anticancer therapeutic intervention.
Here, a ring-closing metathesis approach is utilized to syn-
thesize a 5-methylindolyl-containing tetrapeptide mimetic (6)
that exhibits unprecedented in vitro Grb2 SH2 domain-binding
affinity (Kd ) 93 pM). Key to the preparation of 6 is the
enantioselective synthesis of (2S)-2-(3-(5-methylindolyl)meth-
yl)pent-4-enylamine (12) as one of two ring-closing segments.
Induction of conformational constraint through mac-
rocyclization is a potentially useful technique for en-
hancement of biological potency.1,2 This is exemplified
by the recent macrocyclization of a Grb2 SH2 domain-
binding tetrapeptide mimetic using ring-closing me-
tathesis (RCM) on a simplified phosphatase-stable vari-
ant of the high-affinity Grb2 SH2 domain-binding
tetrapeptide mimetic 13 that lacked a substituent at the
pTyr mimetic R-position (2, Figure 1).4 Although ana-
logues such as 35 containing carboxylic acid functional-
ity at the pTyr mimetic R-position had been shown to
exhibit enhanced Grb2 SH2 domain-binding potency in
whole cell systems, for reasons of synthetic accessibility
the first RCM-derived macrocycle (4) lacked such im-
portant R-functionality. Even though it was structurally
simplified, 4 exhibited good Grb2 SH2 domain-binding
affinity in extracellular ELISA-based assays (IC50 ) 20
nM). However, disappointing potency was observed in
whole cells (IC50 g 10 µM).6 More recently, the fully ela-
borated macrocycle 5 bearing an R-carboxymethyl group
was prepared and found to have high Grb2 SH2 domain-
binding potency in both extracellular (IC50 ) 2 nM) and
whole cell assays (IC50 e 1 µM).7 This was significant,
since the issue of potency in whole cell systems is central
to SH2 domain-signaling inhibitor development because
of the unique requirement for high SH2 domain-binding
affinity of anionic phosphate-mimicking functionality8,9
and the limitations, which this typically presents for cell
membrane transit. Inhibitor 5 could be termed a “second
generation” analogue that results from modification of
F igu r e 1. Structures of synthetic Gr2 SH2 domain-binding
analogues.
the lower pTyr-mimicking portion of the ring-closing
olefin segment (Figure 1). To further extend this genre
of RCM macrocyclization, it was of interest to modify
functionality residing on the upper ring-closing olefin
segment. Accordingly, efforts were undertaken to re-
place the naphthyl ring of 5 with a 5-methylindolyl
group. As reported herein, the resulting macrocycle (6)
has been found to exhibit unprecedented Grb2 SH2
domain-binding potency.
Syn th esis. Key to the construction of title compound
6 was the enantioselective synthesis of methylindolyl-
containing pentenylamine 12 bearing the R-configura-
tion (Scheme 1).19 Stereochemistries at both upper and
lower macrocycle ring junctures were determined previ-
ously as being most appropriate for binding to the Grb2
SH2 domain.6,7 Starting from 3-(5-methylindolyl)pro-
panoic acid (7),11 chiral induction was achieved by coupl-
ing with commercially available (S)-4-phenyl-1,3-oxazo-
lidin-2-one12 using trimethylacetyl chloride mixed an-
hydride coupling in the presence of n-BuLi, (92% yield)
to yield oxazolidine 8. This was subjected to R-alkylation
using lithium hexamethyldisilylamide (LHMDS) and
3-iodopropene to provide 9 (69% de, 61% yield; Scheme
* To whom correspondence should be addressed. Phone: (301) 846-
5906. Fax: (301) 846-6033. E-mail: tburke@helix.nih.gov.
† National Cancer Institute.
‡ SAICsFrederick.
10.1021/jm030440b CCC: $27.50 © 2004 American Chemical Society
Published on Web 01/13/2004

Letters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 789
Sch em e 1a
a
Reagents and conditions: (i) Me3CCOCl, N-methylmorpholine, BuLi, THF, -78 °C, 2 h, (87% yield); (ii) LHMDS, THF, -78 °C, 2 h,
(69% de in 61% yield); (iii) LiAlH4, THF, -78 °C, 1 h, then 0 °C, 3 h, (92% yield); (iv) DIAD, Ph3P, THF, room temp, 12 h, (88% yield); (v)
N2H4‚H2O, EtOH, H2O, reflux, 3 h, (84% yield).
Sch em e 2a
a
Reagents and conditions: (i) (a) Boc-Asn-OH, DIPCDI, HOBt, DMF, room temp, 12 h; (b) HCl(aq) (2 N), CH3CN, room temp, 12 h (75%
yield); (ii) (a) N-Fmoc Ac6c, EDCI‚HCl, HOBt, DMF, room temp, 12 h (83% yield); (b) piperidine, CH3CN, room temp, 2 h (86% yield); (iii)
15,7 EDCI‚HCl, HOAt, DMF, 50 °C, 24 h (56% yield); (iv) 17, CH2Cl2, reflux, 48 h (70% yield); (v) TFA-HS(CH2)2SH-H2O, room temp,
1 h (35% yield); (vi) aqueous NaHCO3(aq) (quantitative).
1). The unexpectedly low chiral induction was attributed
to potential chelation interference by the indolyl nitro-
gen atom. The undesired (3S)-diastereomer could be
removed by careful column flash chromatography. This
approach was the inverse of that used to prepare the
corresponding naphthyl-containing analogue, where
alkylation of 4-pentenoyl-derivatized (R)-4-phenyl-1,3-
oxazolidin-2-one was achieved using a 1-(bromomethyl)-
naphthalene group.6 Lithium aluminum hydride reduc-
tion of 9 at -78 °C afforded a mixture of alcohol and
aldehyde. However, warming to 0 °C provided complete
reduction, yielding 10 as the sole product. The synthesis
of 12 was completed in a two-step process by Mitsunobu-
mediated phthalimide coupling with 10 that yielded 11,
which was subjected to hydrazine reflux (74% combined
yield).
diisopropylcarbodiimide (DIPCDI) in the presence of
1-hydroxybenzotriazole (HOBt) proceeded successfully.
Subsequent attempted Boc deprotection using TFA in
organic solvent resulted in the generation of highly
colored decomposition byproducts. Use of aqueous 2 N
HCl in acetonitrile cleanly provided the free amine 13
in 72% yield from 12. Coupling of 13 with commercially
available N-Fmoc 1-aminocyclohexanecarboxylic acid
(N-Fmoc Ac6c) followed by piperidine-mediated N-depro-
tection gave 14 in good yield. Condensation of 14 with
known 157 proved troublesome. This was attributed to
steric interaction of the Ac6c ring with the bulky R-(CH2-
t
CO2 Bu) group. However, use of active ester coupling
(HOAt and 1-[3-(dimethylamino)propyl]-3-ethylcarbo-
diimide hydrochloride (EDCI‚HCl)) gave satisfactory
yields of 16 (56%) if conducted at elevated temperatures
(50 °C). Finally, metathesis ring closure was achieved
using second generation Grubbs’ catalyst [((PCy3)(Im-
(Mes)2)RudCHPh) (17))]12,13 to yield protected macro-
cycle 18 (70% yield) exclusively as the trans isomer
(vinylic coupling constant J ) 15.0 Hz). Global removal
of tBu ester protecting groups using TFA in the presence
With the upper 5-methylindolyl-containing ring-clos-
ing segment (12) in hand, synthesis of metathesis
precursor 16 was accomplished next (Scheme 2). Un-
expectedly, coupling of 12 with N-Fmoc Asn-OH failed,
even with the use of 1-hydroxy-7-azabenzotriazole
(HOAt). However, coupling of N-Boc Asn-OH with

790 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Letters
F igu r e 3. SPR-derived steady-state binding of 6s to Grb2 SH2
domain protein immobilized by amine coupling to a carboxym-
ethylated dextran-coated sensor chip.
(SPR) performed on Biacore 2000 and Biacore 3000
instruments (Biacore, Inc., Piscataway, NJ ). Previously
reported SH2 domain SPR studies have provided indi-
rect determinations of Kd values by measuring the
ability of test ligands to compete with sensor chip-bound
reference pTyr-peptide for binding to Grb2 SH2 domain
protein in solution.16,17 The current protocol examined
direct binding of 6s to Grb2 SH2 domain protein
immobilized to the sensor chip. Binding affinity deter-
mined in this fashion has consistently provided Kd
values in the range ∼60-90 pM, with a typical value of
Kd ) 75 ( 16 pM (n ) 6) having been reported
recently.18 For this previously reported set of experi-
ments, the Kd value was calculated as the ratio of Koff/
Kon, where a value of Kon ) 4.7 × 108 s-1 appeared to
indicate that binding was diffusion-limited. In the
current study, the very high binding affinity was sup-
ported by steady-state analysis of the rate at which
binding saturation was achieved. This provided a Kd
value of 92.7 ( 11.9 pM (Figure 3). This is significantly
more potent than the naphthyl-containing congener 5
(Kd ) 0.91 nM18), and to our knowledge, it is the highest
affinity yet reported for a synthetic Grb2 SH2 domain-
binding ligand.
F igu r e 2. Molecular mechanics docking of macrocycles 6 (A)
and 5 (B) into the Grb2 SH2 domain highlighting potential
interactions of ligands (ball-and-stick rendering) with a hy-
drophobic patch of the protein (CPK rendering).
of 1,2-ethanedithiol gave HPLC-purified 6, which was
converted to its trisodium salt 6s.
Gr b2 SH2 Dom a in Bin d in g In ter a ction s. To ap-
proximate potential binding interactions of 6 with the
Grb2 SH2 domain, molecular modeling studies were
carried out using MacroModel 8.0 (Schroding, L.L.C.)
and Sybyl 6.9 (Tripos, Inc.) on a Silicon Graphics Octane
2 workstation based on the previously reported X-ray
structure of a phosphohexapeptide bound to the Grb2
SH2 domain (PDB entry 1TZE14). Low-energy binding
modes were compared for naphthyl-containing 5 and
5-methylindolyl-containing 6 (Figure 2). Both the indolyl
(6) and the naphthyl (5) rings are involved in hydro-
phobic interactions with the side chains of residues Lys
âD6 and Leu âD′1.3,15 However, the 5-methyl substitu-
ent of 6 participated in more extended contacts with the
hydrophobic area of the protein without observable
steric clashes. It has also been previously reported that
electron enrichment of aromatic ring systems by electron-
donating substituents, such as methyl or hydroxyl,
result in enhanced CH/π interactions.15 On the basis of
these modeling results, the indolyl analogue 6 was
expected to exhibit higher affinity than the correspond-
ing naphthyl analogue 5.
A characteristic of the previously reported naphthyl-
containing macrocycle 5 was its ability to exhibit high
potency in whole cell assays without the need for
prodrug protection or use of carrier peptide motifs.7 In
similar whole cell assays, 5-methylindolyl-containing 6
behaves in similar fashion, only with significantly
higher potency. Recent studies have shown that 6 is able
to block the association of Grb2 with cytoplasmic erbB-2
with an apparent IC50 value of e10 nM and to inhibit
the growth-factor-promoted growth of MDA-MB-453
breast cancer cells with an IC50 value of 0.6 µM.18
Con clu sion s. Using ring-closing metathesis, synthe-
sis of 5-methylindolyl-containing macrocyclic tetrapep-
tide mimetic 6 has been achieved. Macrocycle 6 exhibits
a low-picomolar Grb2 SH2 domain-binding affinity in
extracellular assays while exerting blockade of Grb2
association with cognate intracellular proteins in whole
cell assays at low nanomolar concentrations. This is
achieved without the use of prodrug protection or carrier
peptide motifs. The ability of 6 to elicit antimitogenic
effects in growth-factor-driven breast cancer cells at
noncytotoxic submicromolar concentrations may indi-
Affinity of macrocycle 6 for the Grb2 SH2 domain
protein was measured using surface plasmon resonance

Letters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 791
(8) Bradshaw, J . M.; Mitaxov, V.; Waksman, G. Investigation of
phosphotyrosine recognition by the SH2 domain of the Src
kinase. J . Mol. Biol. 1999, 293, 971-985.
cate the potential therapeutic utility of this class of
signal-transduction-altering agent.
(9) Bradshaw, J . M.; Waksman, G. Molecular recognition by SH2
domains. Adv. Protein Chem. 2003, 61, 161-210.
Ack n ow led gm en t. Appreciation is expressed to
Drs. Christopher Lai and J ames Kelley of the LMC for
mass spectral analysis.
(10) The change in designation of stereochemistry to (R) at the upper
ring juncture relative to previous reports of (S) stereochemistry
is due to nomenclature only.
(11) Basanagoudar, L. D.; Siddappa, S. Synthesis of 1-(3-aminopro-
pyl)indoles and 3-indol-1-ylpropionic acids. J . Chem. Soc. C 1967,
2599-2601.
(12) Commercially available from Sigma-Aldrich Chemical Corp.
(13) Scholl, M.; Ding, S.; Lee, C. W.; Grubbs, R. H. Synthesis and
activity of a new generation of ruthenium-based olefin metath-
esis catalysts coordinated with 1,3-dimesityl-4,5 dihydroimida-
zol-2-ylidene ligands. Org. Lett. 1999, 1, 953-956.
(14) Rahuel, J .; Gay, B.; Erdmann, D.; Strauss, A.; GarciaEcheverria,
C.; Furet, P.; Caravatti, G.; Fretz, H.; Schoepfer, J .; Grutter, M.
G. Structural basis for specificity of GRB2-SH2 revealed by a
novel ligand binding mode. Nat. Struct. Biol. 1996, 3, 586-589.
(15) Schoepfer, J .; Fretz, H.; Gay, B.; Furet, P.; GarciaEcheverria,
C.; End, N.; Caravatti, G. Highly potent inhibitors of the Grb2-
SH2 domain. Bioorg. Med. Chem. Lett. 1999, 9, 221-226.
(16) Morelock, M. M.; Ingraham, R. H.; Betageri, R.; J akes, S.
Determination of receptor-ligand kinetic and equilibrium bind-
ing constants using surface plasmon resonance: Application to
the lck SH2 domain and phosphotyrosyl peptides. J . Med. Chem.
1995, 38, 1309-1318.
(17) Yao, Z. J .; King, C. R.; Cao, T.; Kelley, J .; Milne, G. W. A.; Voigt,
J . H.; Burke, T. R. Potent inhibition of Grb2 SH2 domain binding
by non-phosphate-containing ligands. J . Med. Chem. 1999, 42,
25-35.
(18) Shi, Z.-D.; Lee, K.; Liu, H.; Zhang, M.; Roberts, L. R.; Worthy,
K. M.; Fivash, M. J .; Fisher, R. J .; Yang, D.; Burke, T. R., J r. A
novel macrocyclic tetrapeptide mimetic that exhibits low-pico-
molar Grb2 SH2 domain-binding affinity. Biochem. Biophys. Res.
Commun. 2003, 310, 378-383.
Su p p or tin g In for m a tion Ava ila ble: Synthetic proce-
dures and spectral characterization for compounds 6, 6s, 8-14,
16, and 18. This material is available free of charge via the
Internet at http://pubs.acs.org.
Refer en ces
(1) Fairlie, D. P.; Abbenante, G.; March, D. R. Macrocyclic
peptidomimeticssforcing peptides into bioactive conformations.
Curr. Med. Chem. 1995, 2, 654-686.
(2) McGeary, R. P.; Fairlie, D. P. Macrocyclic peptidomimetics:
potential for drug development. Curr. Opin. Drug Discovery Dev.
1998, 1, 208-217.
(3) Furet, P.; Gay, B.; Caravatti, G.; GarciaEcheverria, C.; Rahuel,
J .; Schoepfer, J .; Fretz, H. Structure-based design and synthesis
of high affinity tripeptide ligands of the Grb2-SH2 domain. J .
Med. Chem. 1998, 41, 3442-3449.
(4) Gao, Y.; Wei, C.-Q.; Burke, T. R., J r. Olefin metathesis in the
design and synthesis of a globally constrained Grb2 SH2 domain
inhibitor. Org. Lett. 2001, 3, 1617-1620.
(5) Wei, C.-Q.; Li, B.; Guo, R.; Yang, D.; Burke, T. R., J r. Develop-
ment of a phosphatase-stable phosphotyrosyl mimetic suitably
protected for the synthesis of high affinity Grb2 SH2 domain-
binding ligands. Bioorg. Med. Chem. Lett. 2002, 12, 2781-2784.
(6) Gao, Y.; Voigt, J .; Wu, J . X.; Yang, D.; Burke, T. R., J r.
Macrocyclization in the design of a conformationally constrained
Grb2 SH2 domain inhibitor. Bioorg. Med. Chem. Lett. 2001, 11,
1889-1892.
(7) Wei, C.-Q.; Gao, Y.; Lee, K.; Guo, R.; Li, B.; Zhang, M.; Yang,
D.; Burke, T. R., J r. Macrocyclization in the design of Grb2 SH2
domain-binding ligands exhibiting high potency in whole cell
systems. J . Med. Chem. 2003, 46, 244-254.
J M030440B