Macrocyclization in the design of Grb2 SH2 domain-binding ligands exhibiting high potency in whole-cell systems

Article abstract of DOI:10.1021/jm0203635

While most SH2 domains bind phosphotyrosyl (pTyr) containing peptides in extended fashion, the growth factor receptor-bound protein 2 (Grb2) SH2 domain preferentially binds ligands in bend conformations. Accordingly, incorporation of bend-inducing functionality into synthetic ligands could potentially enhance their affinity for this SH2 domain. A macrocyclic tripeptide mimetic that contains a simplified pTyr surrogate lacking an α-nitrogen has recently been shown to exhibit high Grb2 SH2 domain-binding affinity in extracellular ELISA-based assays. However, the same compound is largely ineffective in whole-cell assays. It is known that acidic functionality originating from the α-nitrogen of pTyr residues or from the α-position of P⁰ pTyr mimetics not only increases binding affinity of peptides to Grb2 SH2 domains in extracellular assays but also enhances potency in cell-based systems. Such functionality is absent from the previously reported macrocycle. Therefore, the current study was undertaken to examine the effects of introducing carboxylic functionality at the pTyr mimetic α-position of macrocyclic ligands. It was found that such a modification not only enhanced Grb2 SH2 domain binding in extracellular assays but also conferred high efficacy in whole-cell systems. The most potent compound of the current study exhibited an IC₅₀ value of 0.002 μM in an extracellular ELISA-based assay, and in MDA-MB-453 cells, it both inhibited the association of Grb2 with p185^erbB-2 and exhibited antimitogenic effects with submicromolar IC₅₀ values.

Full text of DOI:10.1021/jm0203635

244
J . Med. Chem. 2003, 46, 244-254
Ma cr ocycliza tion in th e Design of Gr b2 SH2 Dom a in -Bin d in g Liga n d s
Exh ibitin g High P oten cy in Wh ole-Cell System s
Chang-Qing Wei,† Yang Gao,† Kyeong Lee,† Ribo Guo,‡ Bihua Li,‡ Manchao Zhang,‡ Dajun Yang,‡ and
Terrence R. Burke, J r.*,†
Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer InstitutesFrederick, National Institutes of
Health, Frederick, Maryland 21702, and Department of Hematology/ Oncology, University of Michigan Medical School,
Ann Arbor, Michigan 48109
Received August 22, 2002
While most SH2 domains bind phosphotyrosyl (pTyr) containing peptides in extended fashion,
the growth factor receptor-bound protein 2 (Grb2) SH2 domain preferentially binds ligands in
bend conformations. Accordingly, incorporation of bend-inducing functionality into synthetic
ligands could potentially enhance their affinity for this SH2 domain. A macrocyclic tripeptide
mimetic that contains a simplified pTyr surrogate lacking an R-nitrogen has recently been
shown to exhibit high Grb2 SH2 domain-binding affinity in extracellular ELISA-based assays.
However, the same compound is largely ineffective in whole-cell assays. It is known that acidic
functionality originating from the R-nitrogen of pTyr residues or from the R-position of P0 pTyr
mimetics not only increases binding affinity of peptides to Grb2 SH2 domains in extracellular
assays but also enhances potency in cell-based systems. Such functionality is absent from the
previously reported macrocycle. Therefore, the current study was undertaken to examine the
effects of introducing carboxylic functionality at the pTyr mimetic R-position of macrocyclic
ligands. It was found that such a modification not only enhanced Grb2 SH2 domain binding in
extracellular assays but also conferred high efficacy in whole-cell systems. The most potent
compound of the current study exhibited an IC50 value of 0.002 µM in an extracellular ELISA-
based assay, and in MDA-MB-453 cells, it both inhibited the association of Grb2 with p185erbB-2
and exhibited antimitogenic effects with submicromolar IC50 values.
In tr od u ction
Overactivation of growth factor protein tyrosine ki-
nase (PTK) pathways can promote cellular proliferation
and metastasis and thereby contribute to the etiology
of human cancers.1 Down regulation of pathogenic
PTK signaling by kinase inhibitors affords a new and
potentially noncytotoxic approach toward anticancer
therapy.2-5 Src homology 2 (SH2) domains play impor-
tant roles in PTK signaling pathways, and SH2 domain-
binding antagonists potentially represent complemen-
F igu r e 1. Macrocyclization in the design of phosphatase-
tary alternatives to PTK inhibitors as new therapeutics
stable Grb2 SH2 domain-binding ligands.
for treatment of a variety of diseases.6-9 Of numerous
families of SH2 domains, the growth factor receptor-
bound protein 2 (Grb2) SH2 domain is included among
those considered to be attractive targets for antagonist
development.10,11 On the basis of the preferential bind-
ing of Grb2 SH2 domains to pY-X-N-containing se-
quences in type I â-bend conformations,12 scientists at
Novartis Corporation developed a variety of high-
affinity Grb2 SH2 domain-binding tripeptides that
incorporate a bend-inducing R-aminocyclohexylcarboxy-
lic acid residue (Ac6c) in the pTyr + 1 position.13 The
naphthylpropylamido-containing 1 is representative of
this class of compound (Figure 1).14 In a followup study,
replacement of pTyr with the hydrolytically stable
phosphonomethylphenylalanine (Pmp) residue bearing
an NR-oxalyl residue (compound 2) was reported as a
variant of 1 that displayed enhanced antagonism of
Grb2 SH2 domain binding in cellular assays.15 More
recently, the R-carboxymethyl analogue 3 has also been
shown to exhibit good cellular efficacy.16
In a separate study, macrocycle 4 was prepared in
an effort to reduce binding entropy penalties by con-
straining the peptide backbone of 2 to bend conforma-
tions.17 Although such a constraint would be in addition
to the bend-inducing effects already afforded by the Ac6c
residue,18,19 it was felt that ring closure achieved by
joining the â-position of the pTyr-mimicking residue to
the central methylene of the N-terminal naphthylpropyl
group could potentially achieve additional conforma-
tional benefit through side chain rotational constraint.
To reduce synthetic complexity, carboxylic acid contain-
ing -NHCOCO2H or -CH2CO2H functionalities at the
pTyr mimetic R-positions of open-chain 2 or 3 respec-
* To whom correspondence should be addressed. Address: Labora-
tory of Medicinal Chemistry, Center for Cancer Research, NCIs
Frederick, P.O. Box B, Building 376, Boyles Street, Frederick, MD
21702-1201. Phone: (301) 846-5906. Fax: (301) 846-6033. E-mail:
tburke@helix.nih.gov.
† National Institutes of Health.
‡ University of Michigan Medical School.
10.1021/jm0203635 CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/12/2002

Ligands in Whole-Cell Systems
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2 245
Sch em e 1a
Sch em e 2a
a
Reagents and conditions: (a) NaHMDS, trisyl azide, THF, -78
a
Reagents and conditions: (a) H2O2, LiOH, THF-H2O, 90%
°C, 73% yield; (b) H2O2, LiOH, THF-H2O, 92% yield; (c) HOBt,
DIPCDI, DMF, 70% yield; (d) Ph3P, THF-H2O, reflux, quantita-
tive; (e) Ac2O, Et3N, CH2Cl2, 80% yield; (f) (PCy3)2Cl2RudCHPh,
CH2Cl2, reflux; (g) (PCy3)(IMes)Cl2RudCHPh, toluene, 80 °C.
yield; (b) HOBt, EDCI, DMF, 79% yield; (c) (PCy3)2Cl2RudCHPh,
CH2Cl2, reflux, 77% yield; (d) TFA-SiEt3-H2O, quantitative.
tively, were deleted from 4.20 By use of an in vitro
enzyme-linked immunosorbent assay (ELISA) based
binding assay, it was observed that 4 exhibited an
approximate 100-fold increase in binding affinity rela-
tive to the open-chain congener.17 However, the ability
of 4 to block the association of activated erbB-2 PTK
with cognate Grb2 in whole cells was paradoxically less
than would be expected on the basis of in vitro binding
data. Additionally, attenuation of growth-factor-driven
mitogenesis of MDA-MB-453 cells was also significantly
reduced relative to what would have been expected.
in their synthesis.20 Finally, removal of phosphonate
tert-butyl protection provided the desired ring-expanded
12.
Syntheses of macrocycles bearing an R-amino sub-
stituent within the pTyr-mimicking residue were at-
tempted next. As outlined in Scheme 2, the initial
approach was similar to that used to prepare parent
macrocycle 420 except that the R-azido functionality was
employed as a protected, latent amine.25 Accordingly,
azidation of known 1320 using trisyl azide and sodium
hexamethyldisilylamide (NaHMDS) gave R-azido 14 in
good yield. This was then converted to the free acid 15
using standard hydrolysis conditions. Coupling of 15 to
dipepeptide 9 using HOBt-active ester conditions pro-
vided the corresponding azido-containing tripeptide 16
(70% yield), which was reduced to the R-amino-contain-
ing 17 using triphenylphosphine in refluxing aqueous
THF. Acetylation using acetic anhydride gave metath-
esis precursor 18. Unexpectedly, ring closure to 19 could
not be accomplished using either standard Grubbs
catalyst [(PCy3)2Cl2RudCHPh]26 or a more recently
reported second-generation catalyst 4,5-dihydroomida-
zol-2-ylidene-substituted ruthenium-based catalyst
[(PCy3)(IMes)Cl2RudCHPh)].27
Since failure of ring closure might be due to steric
crowding by the Ac6c residue, ring closure was at-
tempted with open-chain precursors in which the cy-
clohexane portion of the Ac6c residue had been replaced
by less bulky groups (29-31, Scheme 3). In initial
studies, these open-chain precursors lacked functional-
ity in the pTyr mimetic R-position. Synthesis of requisite
open-chain analogues 29-31, as well as Ac6c-containing
32, began by HOBT-active ester coupling of appropriate
commercially available N-Fmoc amino acids, Gly, Ile,
R-amino isobutyric acid and R-cyclohexane carboxylic
acid, with known asparagine amide 2020 to give the
corresponding dipeptides 21-24, respectively. Depro-
tection (piperidine in DMF) yielded the free amines 25-
27 and 9, which were coupled in good yield with known
It is known for open-chain ligands that the function-
ality arising from the pTyr R-position provides impor-
tant binding interactions with the RA2 Arg67 residue
of the Grb2 SH2 domain.12 The lack of equivalent
functionality in macrocycle 4 could potentially reduce
its in vivo efficacy. To examine the importance both in
vitro and in vivo of functionality arising from the pTyr
mimetic R-position and to enhance the efficacy of
macrocyclic ligands, the current study was undertaken
to prepare analogues of types 5 and 6, which represent
fully functionalized macrocyclized versions of the parent
open-chain compounds 2 and 3, respectively.
Syn th esis. The synthesis of macrocycle 12 (Scheme
1), which represents a one-carbon ring expansion of 4,
was analogous to that used to make 420 except that the
pTyr-mimicking residue (8) contained an allylic group
at the â-position rather than a vinyl group. Hydrolysis
of Evans chiral auxiliary functionality from starting 7
(previously prepared as an intermediate in the synthesis
of a pipecolic acid based pTyr mimetic21) provided 8,
which was coupled with known dipeptide 9.20 This gave
the metathesis precursor 10, which was ring-closed to
11 in good yield using the method of Grubbs.22-24 As
previously reported, for both 12 and all other macro-
cycles contained in the current paper, stereochemistries
at sites of ring juncture for both the naphthylpropyl and
pTyr mimetic portions were predicated on the absolute
configurations of the corresponding Evans reagents used

246 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2
Wei et al.
Sch em e 3a
a
Reagents and conditions: (a) for 21, Fmoc-Gly-OH, HOBt, DIPCDI, DMF, 89% yield; for 22, Fmoc-Ile-OH, HOBt, DIPCDI, DMF,
86% yield; for 23, Fmoc-Aib-OH, HOBt, DIPCDI, DMF, 88% yield; for 24, Fmoc-1-amino-cyclohexane carboxylic acid, HOBt, DIPCDI,
DMF, 82% yield; (b) piperidine, DMF, quantitative; (c) HOBt, DIPCDI, DMF, 82% yield (29); 81% yield (30); 78% yield (31); 76% yield
(32); (d) (PCy3)2Cl2RudCHPh, CH2Cl2, reflux, 63% yield (35); 67% yield (36); (e) TFA-SiEt3-H2O, quantitative.
â-vinyl-containing desamino analogue 2820 to provide
the desired olefin metathesis precursors 29-32. At-
tempted synthesis of macrocycles 33 and 34 by olefin
metathesis ring closure of 29 and 30, respectively, failed
despite repeated attempts. Alternatively, ring closure
of 31 proceeded in good yield to provide macrocycle 35,
as did ring closure of 32 to yield 36, consistent with the
previous synthesis of this latter compound.20 Deprotec-
tion of the acidic functionality in 35 and 36 gave the
desired final products 37 and 4, repectively.
One consequence of the above study using simplified
pTyr mimetics lacking R-substituents was that success-
ful ring closure appeared to require a proximal R,R-
disubstituted residue at the pTyr mimetic + 1 position.
Accordingly, the fully elaborated macrocycles 45 and 6
were next prepared bearing R-carboxymethyl substitu-
ents on their pTyr mimetics (Scheme 4). The requisite
tert-butyl-protected pTyr mimetic 39, containing R-car-
boxymethyl and â-vinyl substituents, was prepared from
precursor 13 by procedures similar to the synthesis of
the analogous compound lacking a â-vinyl group.16
Metathesis precursosrs 40, 41, and 42 were then
prepared by coupling of 39 with dipeptides 25, 27, and
9, respectively. Analogue 40, which lacked an R,R-
disubstituted residue at the pTyr mimetic + 1 position
was included, since it was uncertain whether introduc-
tion of the R-carboxymethyl substituents on the pTyr
mimetic might induce conformational factors favoring
ring closure even in the absence of the otherwise
required pTyr mimetic + 1 R,R-substituents. Intramo-
lecular ring closure of 41 and 42 yielded desired mac-
rocycles 43 and 44, respectively, in good yields. The
geometry of the ring-closing double bond in macrocycle
44, which contains a cyclohexyl-bearing pTyr mimetic
+ 1 residue, was established as E on the basis of NMR
coupling constants of the olefinic proton at δ 5.74 (dd,
J ) 10.1 and 14.1 Hz). This was in agreement with
similar results previously obtained with compound 36
(δ 5.66; dd, J ) 9.3 and 14.4 Hz). Compound 40 failed
to undergo olefin metathesis, further supporting the
requirement for ring closure of an R,R-disubstituted
residue at the pTyr mimetic + 1 position. Finally, global
hydrolysis of tert-butyl protection on 43 and 44 provided
target macrocycles 45 and 6, which were purified by
HPLC.
Resu lts a n d Discu ssion
Syn th etic Con sid er a tion s. It was unclear whether
the ring-closing three-carbon bridge of parent macro-
cycle 4 provided the optimum ring size and geometry
for bindng to the Grb2 SH2 domain. Therefore, prior to
undertaking introduction of the pTyr mimetic R-func-
tionality, which was the focus of the current study, we
first examined whether a larger ring size would exhibit
higher affinity. This resulted in the preparation of
expanded analogue 12, which contains one additional
methylene in its ring-closing segment. Unexpectedly,
such a seemingly minor modification resulted in a near-
total loss of binding affinity (12; IC50 > 10 µM; Table
1). Molecular modeling studies indicated that binding
interactions of 12 differ significantly from those of 4,
which contains a three-carbon ring-closing segment. Of
particular note are differences in positioning of the
critical Asn side chain carboxamido groups as well as
naphthyl rings, both of which are critical for tight Grb2
SH2 domain binding (Figure 2). This indicated the

Ligands in Whole-Cell Systems
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2 247
Sch em e 4a
a
Reagents and conditions: (a) NaHMDS, BrCH2CO2But, THF, -78 °C, 77% yield; (b) LiOH, H2O2, THF-H2O, 85% yield; (c) HOAt,
EDCI, DMF, 85% yield (40), 75% yield (41), 72% yield (42); (d) (PCy3)2Cl2RudCHPh, CH2Cl2, reflux, 83% yield (43), 87% yield (44); (e)
TFA-SiEt3-H2O, quantitative.
Ta ble 1. Inhibition Data Obtained in Extracellular
ELISA-Based Grb2 SH2 Domain-Binding Assays
F igu r e 2. Overlay of energy-minimized conformations of 4
(colored by atom) and 12 (orange). The ligands were extracted
from energy-minimized protein-ligand complexes.
observed in the synthesis of macrocycles 4 and 12, where
functionality at the pTyr mimetic R-position was absent,
the inability of 18 to undergo ring closure potentially
a
Values were obtained as described in the Experimental
Section.
indicated that steric or electronic considerations pre-
sented by the R-amino functionality were incompatible
with the olefin metathesis process.
importance of maintaining the original three-carbon
bridge in further analogues.
On the basis of the above finding, further analogues
in the study were prepared employing a three-carbon
ring-closing segment. Accordingly, preparation of
macrocycle 19, bearing the acetamido functionality in
the pTyr mimetic R-position, was undertaken. How-
ever, it was found that olefin-metathesis-induced ring
closure of the corresponding open-chain precursor 18
could not be achieved using standard Grubbs catalyst
[(PCy3)(IMes)Cl2RudCHPh] or a more recently reported
second-generation catalyst [((PCy3)(Im(Mes)2)Rud
CHPh)]. In light of the relatively facile ring closure
Several factors were considered that might hinder
olefin metathesis of 18, including steric crowding be-
tween the highly hindered Ac6c residue and the R-amino
group of the pTyr-mimicking residue. Original reasons
for using Ac6c in the pTyr + 1 position of open-chain
compounds such as 1 included its ability to induce bend
conformations.13 Since in the context of macrocyclized
ligands “bend conformations” are ensured without the
aid of the Ac6c residue, its importance as a bend inducer
might be reduced in such compounds. Accordingly,

248 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2
Wei et al.
macrocycles were designed in which Ac6c was replaced
with residues containing less bulky groups. The syn-
thesis of these compounds was first undertaken without
functionality in the pTyr mimetic R-position. Ring-
opened precursors 29-32 were prepared bearing R,R-
substituents consisting of the following: H, H (Gly); H,
sec-butyl (Ile); Me, Me (R-amino isobutyric acid); as well
as the previously reported R,R-cyclohexane carboxylic
acid.20
F igu r e 3. Association of Grb2 with p185erbB-2 in MDA-MB-
453 cells following treatment with indicated concentrations of
2 and 6 measured by immunoprecipitation with Grb2 antibod-
ies followed by Western blotting with pTyr antibodies as
described in the Experimental Section.
Attempted olefin metathesis ring closure of precursors
29 and 30, which bear a single substituent in this
position, failed despite repeated attempts. Alternatively,
ring closure of analogues containing two substituents
(31 and 32) proceeded in good yield to provide macro-
cycles 35 and 36. The synthesis of 36 was consistent
with the previous synthesis of this compound.20 These
results indicated that successful ring-closing metathesis
appeared to require an R,R-disubstituted residue proxi-
mal to the site of ring closure. It is known that such
R,R-disubstituted residues induce bend structures,19 and
our findings indicate that preexisting bend conforma-
tions favor olefin metathesis ring closure.
On the basis of the above study, fully elaborated
macrocycles 45 and 6 bearing pTyr mimetic R-car-
boxymethyl substituents in addition to R,R-disubstituted
residues at the pTyr mimetic + 1 position were pre-
pared. Olefin metathesis ring closure leading to these
analogues went smoothly in high yield. By comparison
of ring-closing yields of 43 and 44 with 35 and 36, it
was observed that an approximate 20% increase in yield
was obtained by introduction of a substituent at the
pTyr mimetic R-position. The inability to induce ring
closure of analogue 18, which has an acetamido group
at the pTyr mimetic R-position, indicated that the
nature of the substituent at this position also affects
ring closure. Finally, it was also observed that having
a cyclohexyl-bearing Ac6c residue at the pTyr mimetic
+ 1 position induced slightly more facile ring closure
than the corresponding residue with R,R-dimethyl sub-
stituents. This further supported the importance of
steric and conformational effects in ring-closing reac-
tions.
F igu r e 4. Molecular mechanics generated model of 6 (ren-
dered in ball-and-stick fashion) complexed to the Grb2 SH2
domain as determined in the Experimental Section: (A) overall
binding of 6 to the Grb2 SH2 domain; (B) highlight of ligand-
protein interactions in the pTyr-binding pocket.
In h ibition of Gr b2 SH2 Dom a in Bin d in g in Ex-
tr a cellu la r ELISA Assa ys. The current study was
undertaken with the intent of preparing macrocycles
that contain pTyr mimetic R-functionality. Additionally,
since, for macrocycles such as 4, the potential impor-
tance of a pTyr mimetic + 1 Ac6c residue was uncertain
because a ring structure is inherently constrained in
bend conformations, we also examined various replace-
ments for the Ac6c residue. Although attempts to replace
the Ac6c residue in 4 with either a Gly (compound 33)
or an Ile residue (compound 34) failed, ring closure of
open-chain R,R-dimethyl-substituted analogues (35 and
43) was readily achieved. Final products 37 (IC50 ) 0.7
µM) and 45 (IC50 ) 0.008 µM), which were obtained
following deprotection of 31 and 41, respectively, showed
reduced potency relative to their Ac6c-containing coun-
terparts 4 (IC50 ) 0.02 µM17) and 6 (IC50 ) 0.002 µM,
Table 1), respectively. As with open-chain tripeptides
in the original study by Novartis,13 this again shows the
preference for an Ac6c residue in the pTyr mimetic + 1
position. These results were supported by molecular
modeling studies, which showed that compared with
Ac6c side chain Cγ methylenes, R,R-dimethyl-substitut-
ed residues exhibit reduced van der Waals contacts with
the protein pY + 1 binding sites (Phe âD5 and Gln âD3).
In tr a cellu la r In h ibition of Gr b2 SH2 Dom a in
Bin d in g in Wh ole Cell Assa ys. The primary focus of
the current study was introduction into macrocycles of
carboxylic acid containing functionality at the pTyr
mimetic R-position in order to enhance binding interac-
tions with the Grb2 SH2 domain Arg67 residue. This
objective was achieved with compound 6, which exhib-
ited high binding potency in an extracellular ELISA-
based binding assay (Table 1). Of greater interest was
the ability of 6 to function in whole-cell assays. The
effective inhibition by synthetic ligands in whole-cell
environments differs from extracellular ELISA assays
in that ligands must cross cell membranes. Additionally,
in cellular contexts, full-length Grb2 to which synthetic
ligands must bind and cognate phosphorylated erbB-2
proteins with which the inhibitors must compete are
much larger and more complex than in extracellular

Ligands in Whole-Cell Systems
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2 249
F igu r e 5. Plot of cell survival versus dose following treatment of MDA-MB-453 (A) and 2-LMP (B) cells in culture with varying
concentrations of compounds as described in the Experimental Section.
ELISA assays (where competition is between the Grb2
SH2 domain fusion protein and a short pTyr-containing
peptide). As shown in Figure 3, when open-chain Ox-
oPmp (2) and functionalized macrocycle 6 were exam-
ined for their ability to inhibit the association of Grb2
with p185erbB-2, 6 was significantly more potent than
2. This is in contrast to macrocycle 4, which had
previously been shown to exhibit much reduced potency
relative to 2 in a similar assay.17 Shown in Figure 4
(panel A) is the binding of 6 to the Grb2 SH2 domain
as determined by molecular mechanics calculations,
which highlights potential interactions of R-carboxym-
ethyl functionality with the Arg67 residue (panel B).
This represents another example where inclusion of the
acidic functionality at the pTyr mimetic R-position
enhances cellular Grb2 SH2 domain-binding potency.15,16
E xa m in a t ion of An t ip r olifer a t ive E ffect s in
Br ea st Ca n cer Cell Cu ltu r es. Finally, it was of
interest to examine the ability of macrocycles prepared
in this study to inhibit the growth of breast cancer cells
that are mitogenically driven through erbB-2 pathways.
For these studies, MDA-MB-453/m1 cells (American
Type Culture Collection, Rockville, MD) were treated
with macrocycles 6, 12, 37, 45 and with control open-
chain 2 at varying concentrations for 8-10 days, and
resulting cell numbers were then determined by direct
counting. As shown in Figure 5A, similar to the previous
report,17 OxoPmp (2) was able to exert a concentration-
dependent antimitogenic effect, with an IC50 value of
approximately 5 µM. In contrast, macrocycles 12 and
37, which lack carboxylic acid containing functionality
at the pTyr mimetic R-position, failed to exhibit anti-
mitogenic effects up to 50 µM concentrations. This was
consistent with the previously reported poor antimito-
genic profile of macrocycle 4.17 Alternatively, macrocyles
6 and 45, which possess carboxymethyl functionality at
the pTyr mimetic R-position, did show good antimito-
genic potencies, with Ac6c-containing 6 exhibiting a
submicromolar IC50 value that was consistent with its
ability to block the association of Grb2 to p185erbB-2 in
the same cell line (Figure 3). When examined in the
LMP-2 breast cancer cell line, which is not dependent
on signaling through the erbB-2 system for growth and
survival, macrocycle 6 had no effect at similar concen-
trations (Figure 5B), indicating that activity observed
in MDA-MB-453 cells is most probably not associated
with unrelated cytotoxicity.
Con clu sion s
The current study was predicated on previous findings
that macrocyclization leading to compounds of type 4
could potentially enhance Grb2 SH2 domain-binding
potency. Structural aspects of such macrocycles that
might enhance Grb2 SH2 domain-binding potency and
cellular efficacy were therefore examined. These in-
cluded the role of ring size and the role of the pTyr
mimetic + 1 residue in binding affinity. Here, it was
found that enlarging the ring-closing bridge from three
to four carbons significantly reduced binding potency
and that the Ac6c residue plays an important role in
maintaining high inhibitory potency. However, the most
significant aspect of the current study is its examination
of the effects of carboxylic functionality at the pTyr
mimetic R-position. It was observed that macrocycles
such as 4, 12, and 37 that lack functionality at the pTyr
mimetic R-position exhibit high Grb2 SH2 domain-
binding potency in extracellular assays, yet they fail to
demonstrate high potency in whole-cell assays. Alter-
natively, introduction of the carboxymethyl functionality
at the pTyr mimetic R-position not only resulted in
enhanced extracellular Grb2 SH2 domain-binding af-
finity but also conferred high potency in whole-cell
systems. This latter fact may render such compounds
as leads for the development of therapeutically relevant
Grb2 SH2 domain signaling inhibitors.
Exp er im en ta l Section
Cells a n d Cell Cu ltu r e. Cell lines were obtained from the
American Type Culture Collection (Rockville, MD) and Lom-
bardi Cancer Center, Georgetown University Medical Center.
Cells were routinely maintained in improved minimal essential
medium (IMEM, Biofluids, Rockville, MD) with 10% fetal
bovine serum. Cultures were maintained in a humidified
incubator at 37 °C and 5% CO2.
In h ibition of Gr b2 SH2 Dom a in Bin d in g Usin g ELISA
Tech n iqu es. As previously reported,28 a biotinated phospho-
peptide encompassing a Grb2 SH2 domain-binding sequence
derived from SHC protein was bound at 20 ng/mL to 96-well

250 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2
Wei et al.
plates overnight. Nonspecific interactions were inhibited by
5% bovine serum albumin containing TBS. Samples of recom-
binant purified Grb2 SH2-GST fusion protein were preincu-
bated with serial dilutions of inhibitors and then added into
each well. After extensive washing with 0.1% bovine serum
albumin in TBS, bound Grb2 SH2 domain was detected using
anti-GST antibodies and goat antimouse antibody conjugated
to alkaline phosphatase. Quantitation of bound alkaline phos-
phatase was achieved by a colorimetric reaction employing
p-nitrophenyl phosphate as substrate. Each compound is
subjected to a minimum of three determinations. Assays are
conducted with a positive control of known potency, with
additional controls being performed without peptide or protein.
In h ib it ion of Gr b 2 SH2 Dom a in Bin d in g in Wh ole
Cells. As previously reported,28 erbB-2 overexpressing breast
cancer cells, MDA-MB-453, were treated with inhibitors (25
µM) for 3 h in serum-free IMEM medium (Gibco). Cells were
washed twice with PBS to remove inhibitor, and then cell
lysates were prepared using 1% Triton X-100 in PBS contain-
ing 0.2 mM NaVO4. Grb2 and associated Grb2-binding proteins
were immunoprecipitated from each lysate (500 µg) with anti-
Grb2 antibodies and collected using protein A Sepharose.
Immunoprecipitated proteins were separated by SDS-PAGE
on 8-16% gradient gels (Novex), and pTyr-containing proteins
were detected by Western blotting using anti-phosphotyrosine
antibodies (Upstate Biochemicals Inc.). Previous experiments
have shown that a major tyrosine phosphorylated protein in
these cells is the p185 erbB-2, which is overexpressed as a
consequence of gene amplification. Western blotting with Grb2
MAb was done as a control. At lease two determinations are
performed for each compound.
binary solvent systems as indicated, where A ) 0.1% aqueous
TFA and B ) 0.1% TFA in acetonitrile, and either Vydac C18
(10 µm) Peptide & Protein or Advantage C18 (5 µm) columns
(preparative size, 20 mm diameter × 250 mm long with a flow
rate of 10 mL/min; semipreparative size, 10 mm diameter ×
250 mm long, with a flow rate of 2 mL/min).
Gen er a l P r oced u r e for Oxa zolid in on e Hyd r olysis:
Syn th esis of Com p ou n d s 8, 15, a n d 39. To a solution of
oxazolidinone 7, 14, or 38 (2.17 mmol) in THF/H2O (3:1) (32
mL) at 0 °C was added H2O2 (30%, 1.107 mL, 10.85 mmol) via
a syringe over 1 min. This was followed by the addition of
LiOH (182.4 mg, 4.34 mmol) in H2O (4.4 mL). After being
stirred at 0 °C (1 h), the reaction mixture was raised to room
temperature, and stirring was continued for an additional 6
h. A solution of Na2SO3 (1.37 g, 10.85 mmol) in H2O (8.5 mL)
was added to quench unreacted peroxide, and THF was
evaporated at 30 °C. The resulting mixture was extracted with
dichloromethane to remove cleaved oxazolidinone, and then
the aqueous solution was acidified with ice-cooled 0.2 M
aqueous HCl (54 mL), extracted with EtOAc, washed with
brine, dried over Na2SO4, concentrated, and taken to dryness
under high vacuum to provide free acids 8, 15, and 39.
Gen er a l P r oced u r e for F m oc Dep r otection of Na p h -
th yl-Con ta in in g Dip ep tid es: Syn th esis of Com p ou n d s
9 a n d 25-27. To a solution of Fmoc-containing compounds
21-24 (1.0 mmol) in acetonitrile (5 mL) was added piperidine
(1.0 mL), and the resulting solution was stirred at room
temperature (2 h). Solvent was evaporated and residue was
purified by silica gel chromatography to provide the corre-
sponding free amines.
Gen er a l Syn th etic P r oced u r e for Cou p lin g of Am in o
Acid An a logu es: Syn th esis of Com p ou n d s 10, 16, 21-
24, 29-32, a n d 40-42. To a solution of amine (0.626 mmol),
amino acid (0.626 mmol), and HOBt or HOAt (0.626 mmol) in
anhydrous DMF (5 mL) was added EDCI (0.688 mmol) at 0
°C, and the resulting mixture was stirred at room temperature
(12-24 h). Then DMF was evaporated under high vacuum.
Residue was dissolved in ethyl acetate (50 mL) and then
washed sequentially with saturated aqueous NaHCO3, H2O,
and brine and dried over Na2SO4. Concentration and purifica-
tion by silica gel chromatography afforded the title compounds.
Gr ow th In h ibition of MDA-453 Br ea st Ca n cer Cells.
Growth inhibition assays were run using 96-well plates.
Individual wells were seeded with either MDA-453 cells (500
per well) or MDA-MB-231 (a subclone 2LMP; 2000 per well).
Inhibitors were diluted to desired concentrations and added
to plates, with fresh drug solutions being added every day for
5 days. Plates were incubated in a humidified incubator at 37
°C under 5% CO2. Cell growth was detected using MTT (Sigma
M-2128). Briefly, 20 µL of MTT (0.5 mg/mL) was added to each
well, followed by incubation under CO2 (4 h). The medium was
then removed, and cells dissolved were read at 570 nm using
a microplate reader (Molecular Device). Final results were
presented as a percent of control (no drug added).
Molecu la r Mod elin g. Molecular modeling of enzyme-
substrate complexes was carried out using Sybyl 6.8 (Tripos,
Inc., St. Louis, MO) on a Silicon Graphics Octane 2 worksta-
tion. Construction of the protein-ligand complex was based
on the X-ray structure of the Grb2 SH2 domain complexed with
a heptapeptide inhibitor (PDB entry 1TZE).12 A model of the
binding site was constructed consisting of residues between
His 58 and Glu 152. Water molecules were removed, and
backbone and side chain hydrogens were added using standard
average bond angles and lengths. Amber4.1 atom type was
given to the ligand, and Amber all-atom charges were applied
to the protein and the ligand using the biopolymer module in
Sybyl. Ligands were manually docked into the active site by
adjusting torsion angles to those found in the X-ray structure.
Protein-ligand complexes were minimized using a Powell
conjugate gradient minimizer according to a MAXIMIN pro-
cedure with the Amber4.1 force field.
Gen er a l Syn th etic Meth od s. Elemental analyses were
obtained from Atlantic Microlab Inc., Norcross, GA, and fast
atom bombardment mass spectra (FAB-MS) were acquired
with a VG Analytical 7070E mass spectrometer under the
control of a VG 2035 data system. Where indicated, FAB-MS
matrixes used were glycerol (Gly) or nitrobenzoic acid (NBA).
1H NMR data were obtained on a Varian 400 MHz instrument
and are reported in ppm relative to TMS and referenced to
the solvent in which they were run. Solvent was removed by
rotary evaporation under reduced pressure, and anhydrous
solvents were obtained commercially and used without further
drying. HPLC measurements were conducted using a Waters
Prep LC4000 system having photodiode array detection and
Gen er a l P r oced u r e for Rin g-Closin g Meta th esis: Syn -
th esis of Com p ou n d s 11, 35, 36, 43, a n d 44. To a solution
of either 10, 31, 32, 41, or 42 (0.518 mmol) in anhydrous
dichloromethane (150 mL) (deoxygenated by purging with
argon) was added, via syringe, ruthenium catalyst (180 mg,
0.218 mmol) in dichloromethane (7 mL), and the solution was
refluxed (60 °C) under argon with monitoring by TLC. Fol-
lowing reaction completion (approximate reaction times of 60
h were required), the solvent was evaporated and residue was
purified by silica gel chromatography to provide the title
compounds.
Gen er a l P r oced u r e for Rem ova l of ter t-Bu tyl P r otec-
tion : Syn th esis of F in a l P r od u cts 4, 6, 12, 37, a n d 45. The
tert-butyl-protected analogues 11, 35, 36, 43, and 44 were
treated with a solution of TFA/TES/H2O (3.7 mL:0.1 mL:0.2
mL) (room temperature, 1 h), and then solvents were evapo-
rated. To the residue was added ether, providing a precipitate,
which was collected by centrifugation. The solid was washed
with ether and separated again by centrifugation, and the
resulting material was dried under high vacuum to provide
the crude product, which was dissolved in acetonitrile/H2O (1:
1) (16 mL) and purified by reversed-phase HPLC to provide
the desired products as white solids.
(4S)-3-[(3R)-3-(4-{[Bis(ter t-bu toxy)p h osp h on o]m eth yl}-
p h en yl)h ex-5-en oyl]-4-p h en yl-1,3-oxa zolid in -2-on e (7).
Compound 7 was prepared as previously indicated.21
(3S)-3-(4-{[Bis(ter t-bu toxy)ph osph on o]m eth yl}ph en yl)-
h ex-5-en oic a cid (8). Mp 89-90 °C; 1H NMR (CDCl3) δ 7.15
(4H, m), 5.65 (1H, m), 4.97 (2H, m), 3.20 (1H, m), 2.93 (2H, d,
J ) 21.2 Hz), 2.72 (1H, dd, J ) 6.4, 15.4 Hz), 2.57 (1H, dd, J
) 8.8, 15.4 Hz), 2.38 (2H, t, J ) 7.1 Hz), 1.39 (9H, s), 1.38
(9H, s); FAB-MS (-VE) m/z 395 (M - H)-.

Ligands in Whole-Cell Systems
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2 251
(3S)-N-{[N-((1S)-1-{N-[(2S)-2-(Na p h th ylm eth yl)p en t-4-
e n yl]ca r b a m oyl}-2-ca r b a m oyle t h yl)ca r b a m oyl]cyclo-
h exyl}-3-(4-{[bis(ter t-bu toxy)ph osph on o]m eth yl}ph en yl)-
N-{[N-((1S)-1-{N-[(2S)-2-(Na p h th ylm eth yl)p en t-4-en yl]-
ca r b a m oyl}-2-ca r b a m oylet h yl)ca r b a m oyl]cycloh exyl}-
(2S,3R)-2-azido-3-(4-{[bis(ter t-bu toxy)ph osph on o]m eth yl}-
p h en yl)p en t-4-en a m id e (16). Mp 148-149 °C; 1H NMR
(CDCl3) δ 8.16 (1H, d, J ) 7.8 Hz), 8.01 (1H, dd, J ) 6.6, 14.8
Hz), 7.78 (1H, d, J ) 8.2 Hz), 7.65 (2H, m), 7.44 (1H, dt, J )
1.4, 6.8 Hz), 7.38 (1H, dd, J ) 1.2, 7.0 Hz), 7.33 (2H, m), 7.14
(2H, m), 7.04 (1H, s), 7.02 (1H,s), 6.97 (1H, s), 6.49 (1H, s),
6.10 (1H, m), 5.83 (1H, m), 5.10 (4H, m), 4.58 (1H, m), 4.17
(1H, d, J ) 4.3 Hz), 3.93 (1H, dd, 4.1, 8.8 Hz), 3.23 (2H, dd, J
) 5.6, 5.8 Hz), 3.02 (2H, m), 2.96 (2H, d, J ) 21.4 Hz), 2.39
(1H, dd, J ) 5.1, 15.0 Hz), 2.10 (3H, m), 1.84 (2H, m), 1.74
(1H, m), 1.59 (3H, m), 1.39 (9H, s), 1.37 (9H, s), 1.23 (4H, m),
0.80 (1H, m); FAB-MS (+VE) m/z 870 (MH+).
N-{[N-((1S)-1-{N-[(2S)-2-(Na p h th ylm eth yl)p en t-4-en yl]-
ca r b a m oyl}-2-ca r b a m oylet h yl)ca r b a m oyl]cycloh exyl}-
(2S ,3R )-2-a m in o -3-(4-{[b is (t er t -b u t o x y )p h o s p h o n o ]-
m eth yl}p h en yl)p en t-4-en a m id e (17). To a solution of 16
(363 mg, 0.418 mmol) in a mixture of THF/H2O (8 mL:0.5 mL)
was added triphenylphosphine (163 mg, 0.618 mmol, 1.5
equiv), and the resulting mixture was refluxed (12 h). Then
THF was removed by evaporation. The resulting aqueous
solution was extracted with ethyl acetate, and the organic
layers were washed with brine and dried over Na2SO4. After
removal of solvent, residue was purified by silica gel chroma-
tography (CHCl3/methanol, from 100:0 to 9:1) to provide 17
as a sticky oil (341 mg; 97% yield). 1H NMR (CDCl3) δ 8.12
(1H, m), 7.98 (2H, m), 7.77 (1H, d, J ) 7.8 Hz), 7.64 (1H, dd,
J ) 4.7, 5.1 Hz), 7.43 (1H, dt, J ) 1.2, 6.6 Hz), 7.36 (1H, m),
7.32 (2H, m), 7.12 (2H, m), 7.02 (1H, s), 7.00 (1H, s), 6.68 (1H,
s), 5.98 (1H, m), 5.79 (1H, m), 5.31 (1H, s), 5.08 (4H, m), 4.58
(1H, m), 3.87 (1H, m), 3.60 (1H, d, J ) 4.3 Hz), 3.21 (2H, t, J
) 5.5 Hz), 3.07 (1H, m), 2.97 (1H, m), 2.95 (2H, d, J ) 21.4
Hz), 2.38 (1H, dd, J ) 5.1, 14.8 Hz), 2.09 (3H, m), 1.87 (2H,
m), 1.74 (1H, m), 1.57 (3H, m), 1.38 (9H, s), 1.37 (9H, s), 1.23
(4H, m), 0.77 (1H, m); FAB-MS (+VE) m/z 844 (MH+).
1
h ex-5-en a m id e (10). Mp 82-83 °C; H NMR (CDCl3) δ 8.02
(1H, d, J ) 8.5 Hz), 7.78 (2H, t, J ) 7.9 Hz), 7.67 (1H, d, J )
2.4 Hz), 7.65 (1H, d, J ) 2.8 Hz), 7.42 (2H, m), 7.31 (2H, m),
7.09 (1H, d, J ) 2.5 Hz), 7.07 (1H, d, J ) 2.5 Hz), 6.93 (1H, s),
6.90 (1H, s), 6.64 (1H, s), 6.01 (1H, s), 5.80 (1H, m), 5.45 (1H,
m), 5.43 (1H, s), 5.03 (2H, m), 4.83 (2H, m), 4.63 (1H, m), 3.30
(1H, m), 3.09 (2H, m), 3.03 (1H, m), 2.98-2.84 (2H, m), 2.92
(2H, d, J ) 21.4 Hz), 2.48 (2H, m), 2.39 (1H, dd, J ) 4.8, 15.1
Hz), 2.20 (3H, m), 2.07 (2H, m), 1.95 (1H, d, J ) 14.2 Hz),
1.82 (1H, d, J ) 12.9 Hz), 1.69 (1H, dt, J ) 3.6, 13.6 Hz), 1.60
(1H, dt, J ) 3.2, 12.9 Hz), 1.46 (3H, m), 1.37 (9H, s), 1.35 (9H,
s), 1.23 (1H, m), 1.09 (1H, m), 0.94 (1H, m); FAB-MS (+VE)
m/z 843 (MH+).
2-[(9S,13S,18S)-8,11,21-Tr ia za -18-(4-{[bis(ter t-bu toxy)-
p h osp h on o]m et h yl}p h en yl)-13-(n a p h t h ylm et h yl)-7,10,-
20-tr ioxospir o[5.15]h en icos-15-en -9-yl]acetam ide (11). Mp
215 °C; 1H NMR (CDCl3) δ 8.01 (1H, d, J ) 8.2 Hz), 7.97 (1H,
s), 7.78 (1H, dd, J ) 1.3, 7.9 Hz), 7.64 (1H, d, J ) 8.1 Hz),
7.54 (1H, s), 7.42 (2H, m), 7.29 (1H, dd, J ) 7.0, 8.1 Hz), 7.22
(3H, m), 7.13 (1H, s), 7.11 (1H, s), 6.35 (1H, s), 6.23 (1H, s),
5.68 (1H, m), 5.53 (1H, s), 5.23 (1H, m), 4.77 (1H, s), 3.54 (1H,
dd, J ) 11.8, 22.7 Hz), 3.46 (1H, dd, J ) 3.2, 13.6 Hz), 3.15
(1H, d, J ) 13.2 Hz), 3.00 (1H, m), 2.97 (2H, d, J ) 21.4 Hz),
2.86 (1H, d, J ) 12.9 Hz), 2.62 (3H, m), 2.46-2.22 (4H, m),
1.88 (3H, m), 1.52 (5H, m), 1.36 (9H, s), 1.35 (9H, s), 1.29 (2H,
m), 1.20 (2H, m); FAB-MS (+VE) m/z 815 (MH+).
2-[(9S,13S,18S)-8,11,21-Tr ia za -13-(n a p h t h ylm et h yl)-
7,10,20-tr ioxo-18-[4-(ph osph on om eth yl)ph en yl]spir o[5.15]-
h en icos-15-en -9-yl]a ceta m id e (12). Mp 220 °C (dec); 1H
NMR (CD3OD) δ 8.06 (1H, d, J ) 8.1 Hz), 7.83 (1H, d, J ) 7.9
Hz), 7.69 (1H, d, J ) 6.0 Hz), 7.46 (2H, m), 7.34 (2H, m), 7.26
(2H, d, J ) 7.2 Hz), 7.13 (2H, d, J ) 7.5 Hz), 5.65 (1H, m),
5.21 (1H, m), 4.65 (1H, s), 3.51 (1H, m), 3.43 (1H, dd, J ) 3.2,
13.5 Hz), 3.08 (2H, d, J ) 21.2 Hz), 2.98 (1H, m), 2.90-2.50
(6H, m), 2.36 (1H, s), 2.25 (1H, d, J ) 5.4 Hz), 2.08 (1H, m),
1.90 (1H, m), 1.75 (2H, m), 1.50 (5H, m), 1.33 (2H, m), 1.21
(2H, m); FAB-MS (-VE) m/z 701 (M - H)-. HR-FAB-MS calcd
for C38H46N4O7P (M - H)-: 701.3104. Found: 701.314 43.
(4S )-3-[(2S ,3R )-2-Azid o-3-(4-{[b is(t er t -b u t oxy)p h os-
ph on o]m eth yl}ph en yl)pen t-4-en oyl]-4-ph en yl-1,3-oxazoli-
d in -2-on e (14). To a solution of sodium hexamethyldisilyla-
mide (1.0 M in THF, 3.33 mL, 3.33 mmol) at -78 °C was added
a precooled solution (-78 °C) of 1320 (1.596 g, 3.024 mmol) in
THF (10 mL). The resulting solution quickly became deep-
purple. After the mixture was stirred at -78 °C (30 min), a
precooled (-78 °C) solution of trisyl azide (1.122 g, 3.628 mmol)
was added, and the resulting solution was stirred at -78 °C
(5 min). The reaction was quenched by the addition of glacial
acetic acid (0.95 g, 16.6 mmol) followed by the addition of
potassium acetate (1.217 g, 12.41 mmol), and the mixture was
then stirred at 30 °C for an additional 1.5 h. To the mixture
was added saturated aqueous NaHCO3 solution (20 mL). Then
THF was removed by evaporation, the residue was extracted
with ethyl acetate, and the organic layers were washed with
brine and dried over Na2SO4. Concentration and purification
by silica gel chromatography (hexane/ethyl acetate, from 4:1
to 2:1) afforded 14 as an oil (1.26 g, 73% yield). 1H NMR
(CDCl3) δ 7.40-7.14 (9H, m), 6.15 (1H, m), 5.48 (1H, d, J )
10.2 Hz), 5.29 (2H, dd, J ) 10.2, 17. 0 Hz), 4.90 (1H, dd, J )
2.8, 8.3 Hz), 4.24 (1H, t, J ) 8.5 Hz), 4.04 (1H, dd, J ) 2.7, 8.8
Hz), 3.82 (1H, t, J ) 8.5 Hz), 3.01 (2H, d, J ) 21.2 Hz), 1.46
(9H, s), 1.45 (9H, s); FAB-MS (+VE) m/z 569 (MH+).
N-{[N-((1S)-1-{N-[(2S)-2-(Na p h th ylm eth yl)p en t-4-en yl]-
ca r b a m oyl}-2-ca r b a m oylet h yl)ca r b a m oyl]cycloh exyl}-
(2S,3R)-2-(acetylam in o)-3-(4-{[bis(ter t-bu toxy)ph osph on o]-
m eth yl}p h en yl)p en t-4-en a m id e (18). To a solution of 17 (84
mg, 0.1 mmol) in anhydrous dichloromethane (10 mL) was
added acetic anhydride (189 µL, 2.0 mmol, 20 equiv) and
triethylamine (355 µL, 2.4 mmol, 24 equiv), and the resulting
mixture was stirred at room temperature (12 h). The reaction
was quenched by addition of aqueous NaHCO3, and the
solution was extracted with dichloromethane and washed with
brine and dried over Na2SO4. Solvent was removed by evapo-
ration, and residue was purified by silica gel chromatography
(CHCl3/methanol, 9:1) to provide 18 as a foam (71 mg, 80%
1
yield). H NMR (CDCl3) δ 8.02 (1H, d, J ) 8.2 Hz), 7.80 (1H,
d, J ) 7.8 Hz), 7.68 (1H, d, J ) 7.4 Hz), 7.46 (3H, m), 7.34
(3H, m), 7.12 (2H, m), 7.02 (2H, m), 6.75 (1H, s), 6.67 (1H, s),
6.11 (1H, s), 5.80 (3H, m), 5.00 (4H, m), 4.64 (2H, m), 3.57
(1H, m), 3.35 (1H, m), 3.11 (1H, m), 3.04 (1H, m), 2.98 (1H,
m), 2.93 (2H, d, J ) 21.4 Hz), 2.74 (1H, m), 2.60 (1H, m), 2.10
(3H, m), 1.83 (3H, s), 1.80 (2H, m), 1.63 (2H, m), 1.40 (9H, s),
1.37 (9H, s), 1.28 (2H, m), 1.09 (2H, m), 0.81 (1H, m), 0.74
(1H, m); FAB-MS (+VE) m/z 886 (MH+).
(2S)-N-[(2S)-2-(Na p h th ylm eth yl)p en t-4-en yl]-2-(2-a m i-
n oa cetyla m in o)-3-ca r ba m oylp r op a n a m id e (25). Mp 182
1
°C; H NMR (CD3OD) δ 7.98 (1H, d, J ) 8.6 Hz), 7.78 (1H, d,
J ) 8.8 Hz), 7.66 (1H, d, J ) 7.8 Hz), 7.46 (1H, m), 7.39 (1H,
m), 7.29 (2H, m), 5.80 (1H, m), 5.01 (2H, m), 4.68 (1H, dd, J )
6.1, 6.6 Hz), 3.25 (2H, m), 3.05 (2H, m), 2.97 (2H, m), 2.63
(2H, d, J ) 6.3 Hz), 2.06 (3H, m); FAB-MS (+VE) m/z 397
(MH+).
(2S ,3R )-2-Azid o-3-(4-{[Bis(t er t -b u t oxy)p h osp h on o]-
m eth yl}p h en yl)p en t-4-en oic Acid (15). Mp 125 °C; 1H NMR
(CDCl3) δ 7.22 (2H, d, J ) 7.8 Hz), 7.14 (2H, dd, J ) 2.4, 7.8
Hz), 6.16 (1H, m), 5.25 (2H, dd, J ) 13.7, 16.6 Hz), 3.95 (1H,
d, J ) 9.3 Hz), 3.85 (1H, t, J ) 9.3 Hz), 2.78 (2H, dd, J ) 12.2,
21.2 Hz), 1.41 (9H, s), 1.35 (9H, s); FAB-MS (-VE) m/z 422
(M+ - H).
N-((1S)-1-{N-[(2S)-2-(Na p h th ylm eth yl)p en t-4-en yl]ca r -
b a m oyl}-2-ca r b a m oylet h yl)-(2S,3R)-2-a m in o-3-m et h yl-
p en ta n a m id e (26). Mp 152-153 °C; 1H NMR (CD3OD) δ 7.98
(1H, d, J ) 8.0 Hz), 7.80 (1H, d, J ) 8.4 Hz), 7.68 (1H, d, J )
7.8 Hz), 7.48 (1H, m), 7.40 (1H, m), 7.31 (2H, m), 5.80 (1H,
m), 5.02 (2H, m), 4.67 (1H, dd, J ) 6.4, 6.6 Hz), 3.25 (2H, m),
3.13 (2H, m), 2.98 (2H, m), 2.63 (2H, d, J ) 6.4 Hz), 2.07 (2H,

252 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2
Wei et al.
m), 1.66 (1H, m), 1.40 (1H, m), 1.08 (1H, m), 0.87 (3H, d, J )
6.8 Hz), 0.81 (3H, t, J ) 7.4 Hz); FAB-MS (+VE) m/z 453
(MH+).
2-[(1S ,5S ,9R )-3,12,15-Tr ia za -9-(4-{[b is(t er t -b u t oxy)-
p h osp h on o]m eth yl}p h en yl)-13,13-d im eth yl-5-(n a p h th yl-
m et h yl)-2,11,14-t r ioxocyclop en t a d ec-7-en yl]a cet a m id e
(35). Mp 218 °C; 1H NMR (CD3OD) δ 8.78 (1H, s), 8.48 (1H, d,
J ) 8.2 Hz), 8.14 (1H, d, J ) 8.5 Hz), 7.78 (1H, dd, J ) 1.2,
8.2 Hz), 7.74 (1H, m), 7.65 (1H, d, J ) 7.6 Hz), 7.48 (1H, m),
7.40 (1H, m), 7.30 (2H, m), 7.12 (2H, m), 5.61 (1H, m), 5.39
(1H, m), 4.50 (1H, m), 3.82 (1H, m), 3.72 (1H, dd, J ) 6.6, 13.0
Hz), 3.28 (1H, m), 2.98 (1H, m), 2.96 (2H, d, J ) 21.4 Hz),
2.78 (1H, m), 2.63 (2H, m), 2.46 (2H, m), 2.20 (1H, m), 1.98
(1H, m), 1.80 (1H, m), 1.45 (3H, s), 1.37 (3H, s), 1.35 (18H, s);
FAB-MS (+VE) m/z 761 (MH+).
N-((1S)-1-{N-[(2S)-2-(Na p h th ylm eth yl)p en t-4-en yl]ca r -
b a m oyl}-2-ca r b a m oylet h yl)-2-a m in o-2-m et h ylp r op a n -
1
a m id e (27). Mp 89 °C; H NMR (CDCl3) δ 8.80 (1H, d, J )
7.0 Hz), 7.98 (1H, d, J ) 8.2 Hz), 7.85 (1H, m), 7.72 (1H, d, J
) 8.2 Hz), 7.49 (2H, m), 7.38 (1H, m), 7.31 (1H, d, J ) 7.0 Hz),
7.21 (1H, m), 6.29 (1H, s), 5.81 (1H, m), 5.54 (1H, s), 5.09 (2H,
m), 4.61 (1H, m), 3.25 (2H, m), 3.06 (1H, dd, J ) 6.6, 14.0 Hz),
2.96 (1H, dd, J ) 6.6, 14.0 Hz), 2.80 (1H, dd, J ) 4.3, 15.2
Hz), 2.54 (1H, dd, J ) 7.0, 15.2 Hz), 2.11 (3H, d, J ) 6.2 Hz),
1.36 (3H, s), 1.34 (3H,s); FAB-MS (+VE) m/z 425 (MH+).
2-[(9S,13S,17R)-8,11,20-Tr ia za -17-(4-{[bis(ter t-bu toxy)-
p h osp h on o]m et h yl}p h en yl)-13-(n a p h t h ylm et h yl)-7,10,-
19-tr ioxosp ir o[5.14]icos-15-en -9-yl]a ceta m id e (36). Mp
(2S)-N-[(2S)-2-(Na p h t h ylm et h yl)p en t -4-en yl]-2-[(a m i-
n o c y c lo h e x y l)c a r b o n y la m in o ]-3-c a r b a m o y lp r o p a n -
1
1
210 °C; H NMR (CDCl3) δ 8.30-8.00 (2H, m), 7.83 (1H, m),
a m id e (9). H NMR (CDCl3): δ 8.89 (1H, d, J ) 6.8 Hz), 7.98
7.69 (1H, d, J ) 7.8 Hz), 7.59 (1H, m), 7.55-7.30 (4H, m),
7.25-7.05 (4H, m), 6.35 (1H, s, br), 6.28 (1H, s, br), 5.66 (1H,
dd, J ) 9.3, 14.4 Hz), 5.31 (1H, m), 5.08 (1H, s, br), 4.66 (1H,
m), 3.90-3.70 (2H, m), 3.62 (1H, m), 3.33-3.08 (2H, m), 3.06-
2.88 (4H, m), 2.85-2.50 (4H, m), 2.37-1.15 (11H, m), 1.42
(18H, s); FAB-MS (+VE) m/z 801 (MH+).
2-{(1S,5S,9R)-3,12,15-Tr ia za -13,13-d im eth yl-5-(n a p h th -
ylm eth yl)-2,11,14-tr ioxo-9-[4-(p h osp h on om eth yl)p h en yl]-
cyclop en ta d ec-7-en yl]a ceta m id e (37). Mp 230 °C (dec); 1H
NMR (CD3OD) δ 8.79 (1H, s), 8.48 (1H, d, J ) 8.0 Hz), 8.14
(1H, d, J ) 8.4 Hz), 7.78 (1H, d, J ) 7.8 Hz), 7.74 (1H, m),
7.64 (1H, d, J ) 7.4 Hz), 7.48 (1H, t, J ) 7.0 Hz), 7.40 (1H, t,
J ) 7.4 Hz), 7.28 (2H, m), 7.14 (4H, m), 5.60 (1H, m), 5.37
(1H, m), 4.50 (1H, m), 3.80 (1H, dd, J ) 10.1, 10.7 Hz), 3.72
(1H, dd, J ) 6.3, 13.7 Hz), 3.31 (1H, m), 3.02 (2H, d, J ) 21.3
Hz), 2.97 (1H, m), 2.78 (1H, m), 2.65 (1H, dd, J ) 10.1, 13.5
Hz), 2.58 (1H, dd, J ) 2.3, 12.5 Hz), 2.49 (1H, dd, J ) 4.7,
15.2 Hz), 2.42 (1H, dd, J ) 12.3, 12.5 Hz), 2.19 (1H, m), 1.95
(1H, m), 1.78 (1H, m), 1.46 (3H, s), 1.36 (3H, s); FAB-MS (-VE)
m/z 647 (MH+). HR-FAB-MS calcd for C34H40N4O7P (M - H)-:
647.2635. Found: 647.2667.
(1H, m), 7.85 (1H, m), 7.72 (1H, d, J ) 7.8 Hz), 7.55-7.26 (7H,
m), 6.31 (1H, s, br), 5.91-5.75 (1H, m), 5.54 (1H, s, br), 5.13-
5.07 (2H, m), 4.68 (1H, dd, J ) 6.1, 10.7 Hz), 3.35-3.21 (2H,
m), 3.08 (1H, dd, J ) 6.6, 14.2 Hz), 2.97 (1H, dd, J ) 6.4, 13.9
Hz), 2.83 (1H, dd, J ) 4.4, 15.1 Hz), 2.56 (1H, dd, J ) 6.5,
14.6 Hz), 2.17 (2H, d, J ) 5.9 Hz), 2.05-1.23 (11H, m). FAB-
MS (+VE) m/z: 465 (MH+).
(3R)-N-{[N-((1S)-1-{N-[(2S)-2-(Na p h th ylm eth yl)p en t-4-
en yl]ca r ba m oyl}-2-ca r ba m oyleth yl)ca r ba m oyl]m eth yl}-
3-(4-{[bis(ter t-bu toxy)p h osp h on o]m eth yl}p h en yl)p en t-4-
1
en a m id e (29). Mp 120 °C; H NMR (CDCl3) δ 7.95 (1H, d, J
) 8.4 Hz), 7.78 (1H, dd, J ) 1.3, 8.1 Hz), 7.66 (1H, d, J ) 7.9
Hz), 7.61 (1H, d, J ) 7.9 Hz), 7.43 (2H, m), 7.29 (2H, m), 7.12
(1H, d, J ) 2.4 Hz), 7.09 (1H, d, J ) 2.4 Hz), 7.01 (1H, s), 6.98
(1H, s), 6.90 (1H, m), 6.78 (1H, s), 5.81 (2H, m), 5.71 (1H, s),
5.03 (2H, m), 4.93 (2H, m), 4.68 (1H, dd, J ) 7.1, 12.4 Hz),
3.75 (1H, m), 3.65 (2H, m), 3.28 (1H, m), 3.07 (1H, m), 2.97
(2H, m), 2.93 (2H, d, J ) 21.4 Hz), 2.70 (1H, dd, J ) 4.9, 15.9
Hz), 2.52 (3H, m), 2.06 (3H, m), 1.37 (9H, s), 1.35 (9H, s); FAB-
MS (+VE) m/z 761 (MH+).
(3R)-N-{[N-((1S)-1-{N-[(2S)-2-(Na p h th ylm eth yl)p en t-4-
en yl]ca r ba m oyl}-2-ca r ba m oyleth yl)ca r ba m oyl]-(1S,2R)-
2-m eth ylbu tyl}-3-(4-{[bis(ter t-bu toxy)ph osph on o]m eth yl}-
p h en yl)p en t-4-en a m id e (30). Mp 175-176 °C; 1H NMR
(CD3OD) δ 7.98 (1H, d, J ) 8.2 Hz), 7.79 (1H, dd, J ) 1.2, 8.6
Hz), 7.65 (1H, dd, J ) 2.3, 7.4 Hz), 7.43 (1H, m), 7.38 (1H, m),
7.29 (2H, m), 7.10 (1H, d, J ) 2.3 Hz), 7.08 (1H, d, J ) 2.3
Hz), 7.00 (1H, s), 6.97 (1H, s), 5.82 (2H, m), 5.00 (2H, m), 4.89
(2H, m), 4.55 (1H, t, J ) 6.2 Hz), 3.98 (1H, d, J ) 7.0 Hz),
3.66 (1H, dd, J ) 7.4, 15.6 Hz), 3.19 (1H, dd, J ) 6.6, 13.6
Hz), 3.02 (1H, m), 2.97 (1H, m), 2.95 (2H, d, J ) 21.4 Hz),
2.63 (3H, m), 2.50 (1H, dd, J ) 6.2, 14.0 Hz), 2.10 (2H, m),
1.99 (1H, m), 1.74 (1H, m), 1.42 (1H, m), 1.38 (9H, s), 1.34
(9H, s), 1.13 (1H, m), 0.81 (6H, m), 0.69 (1H, m); FAB-MS
(+VE) m/z 817 (MH+).
(3R)-N-{[N-((1S)-1-{N-[(2S)-2-(Na p h th ylm eth yl)p en t-4-
en yl]car bam oyl}-2-car bam oyleth yl)car bam oyl]isopr opyl}-
3-(4-{[bis(ter t-bu toxy)p h osp h on o]m eth yl}p h en yl)p en t-4-
en a m id e (31). Mp 108-110 °C; 1H NMR (CDCl3) δ 8.04 (1H,
d, J ) 8.2 Hz), 7.80 (2H, m), 7.69 (2H, m), 7.44 (2H, m), 7.34
(2H, m), 7.13 (1H, d, J ) 2.7 Hz), 7.11 (1H, d, J ) 2.7 Hz),
6.96 (1H, s), 6.94 (1H, s), 6.79 (1H, s), 6.51 (1H, s), 5.84 (2H,
m), 5.58 (1H, s), 5.05 (2H, m), 4.95 (2H, m), 4.62 (1H, m), 3.71
(1H, dd, J ) 7.0, 14.0 Hz), 3.32 (1H, m), 3.10 (2H, m), 3.00
(1H, m), 2.96 (2H, d, J ) 21.4 Hz), 2.90 (1H, m), 2.54 (2H, m),
2.45 (1H, dd, J ) 5.1, 15.2 Hz), 2.23 (1H, m), 2.10 (2H, m),
1.41 (9H, s), 1.39 (9H, s), 1.36 (3H, s), 1.32 (3H, s); FAB-MS
(+VE) m/z 789 (MH+).
2-[(9S,13S,17R)-8,11,20-Tr ia za -13-(n a p h th ylm eth yl)-7,-
10,19-tr ioxo-17-[4-(p h osp h on om eth yl)p h en yl]sp ir o[5.14]-
icos-15-en -9-yl]a ceta m id e (4). Mp 225 °C (dec); 1H NMR
(400 MHz, d6-DMSO) δ 8.40 (1H, s), 8.23 (1H, d, J ) 7.8 Hz),
8.12 (1H, d, J ) 8.1 Hz), 7.89 (1H, d, J ) 7.6 Hz), 7.75 (1H,
m), 7.56-7.38 (6H, m), 7.15-7.02 (5H, m), 5.55 (1H, dd, J )
9.5, 14.2 Hz), 5.37 (1H, m), 4.27 (1H, m), 3.86 (1H, m), 3.59
(1H, dd, J ) 5.6, 11.7 Hz), 3.16 (1H, dd, J ) 5.7, 14.4 Hz),
2.95-2.30 (8H, m), 2.25-1.10 (13H, m); FAB-MS (-VE) m/z
687.6 (M - H)-. HR-FAB-MS calcd for C37H44N4O7P (M - H)-:
687.2948. Found: 687.2958.
ter t-Bu tyl 3-[((4S)-2-Oxo-4-p h en yl(1,3-oxa zolid in -3-yl))-
ca r b on yl]-(3S ,4S )-4-(4-{[b is(t er t -b u t oxy)p h osp h on o]-
m eth yl}p h en yl)h ex-5-en oa te (38). To a solution of sodium
hexamethyldisilylamide (1.0 M in THF, 1.2 mL, 1.2 mmol) at
-78 °C was added a solution of 13 (527 mg, 1.0 mmol) in THF
(2 mL). The solution quickly became deep-purple. After the
mixture was stirred at -78 °C (30 min), a precooled (-78 °C)
solution of tert-butyl bromoacetate (234 mg, 1.2 mmol) was
added, and the resulting solution was stirred at -78 °C (2 h).
Then the mixture was brought to room temperature and
stirred overnight. The reaction was quenched by addition of
saturated aqueous NH4Cl (20 mL), and then THF was evapo-
rated and the resulting liquid was extracted with ethyl acetate,
washed with brine, and dried over Na2SO4. Concentration and
purification by silica gel chromatography (hexanes/ethyl ac-
etate, 1:1.25) afforded 38 as an oil (495 mg, 77% yield). 1H
NMR (CDCl3) δ 7.30-7.15 (9H, m), 6.08 (1H, m), 5.18 (2H,
m), 4.80 (2H, m), 4.04 (1H, dd, J ) 8.1, 8.6 Hz), 3.95 (1H, dd,
J ) 2.4, 8.6 Hz), 3.35 (1H, t, J ) 9.8 Hz), 3.02 (2H, d, J ) 21.5
Hz), 2.77 (1H, dd, J ) 11.0, 17.1 Hz), 2.61 (1H, dd, J ) 3.9,
17.1 Hz), 1.49 (18H, s), 1.31 (9H, s); FAB-MS (+VE) m/z 642
(MH+).
(3R)-N-{[N-((1S)-1-{N-[(2S)-2-(Na p h th ylm eth yl)p en t-4-
e n yl]ca r b a m oyl}-2-ca r b a m oyle t h yl)ca r b a m oyl]cyclo-
h exyl}-3-(4-{[bis(ter t-bu toxy)ph osph on o]m eth yl}ph en yl)-
p en t-4-en a m id e (32). Mp 87-88 °C; 1H NMR (CDCl3) δ 8.05
(1H, d, J ) 8.1 Hz), 7.85-7.64 (4H, m), 7.55-7.38 (4H, m),
7.15 (2H, dd, J ) 2.4, 7.8 Hz), 6.99 (2H, d, J ) 8.1 Hz), 6.28
(1H, s, br), 5.96-5.77 (3H, m), 5.41 (1H, s, br), 5.13-4.90 (4H,
m), 4.72 (1H, m), 3.73 (1H, m), 3.37 (1H, m), 3.22-2.91 (6H,
m), 2.65-2.45 (2H, m), 2.30-2.01 (3H, m), 1.95-1.09 (11H,
m), 1.43 (9H, s), 1.41 (9H, s); FAB-MS (+VE) m/z 828 (MH+).
(2S ,3S )-3-(4-{[Bis(t er t -b u t oxy)p h osp h on o]m e t h yl}-
p h en yl)-2-{[(ter t-bu tyl)oxyca r bon yl]m eth yl}p en t-4-en o-
1
ic Acid (39). Mp 155 °C; H NMR (CDCl3): δ 7.16 (4H, m),
5.97 (1H, m), 5.15 (2H, m), 3.50 (1H, t, J ) 9.3 Hz), 3.15 (1H,

Ligands in Whole-Cell Systems
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2 253
dt, J ) 4.4, 9.8 Hz), 2.83 (2H, dd, J ) 3.4, 21.5 Hz), 2.60 (2H,
m), 1.43 (9H, s), 1.41 (9H, s), 1.37 (9H, s); FAB-MS (-VE) m/z
495 (M - H)-.
1.70 (6H, m), 1.52 (3H, m), 1.35 (9H, s), 1.34 (9H, s), 1.30 (9H,
s); FAB-MS (+VE) m/z 915 (MH+).
2-{(1S,7S,11S,15S)-3,6,9-Tr ia za -7-(ca r b a m oylm et h yl)-
4,4-d im e t h yl-11-(n a p h t h ylm e t h yl)-2,5,8-t r ioxo-15-[4-
(p h osp h on om et h yl)p h en yl]cyclop en t a d ec-13-en yl]a ce-
tic Acid (45). Mp 240 °C (dec); 1H NMR (CD3OD) δ 8.86 (1H,
s), 8.48 (1H, d, 8.2 Hz), 8.18 (1H, d, J ) 8.6 Hz), 7.78 (1H, d,
J ) 7.6 Hz), 7.68 (2H, m), 7.48 (1H, dd, J ) 6.8, 7.4 Hz), 7.40
(1H, t, J ) 7.6 Hz), 7.32 (2H, m), 7.20 (3H, s), 5.85 (1H, m),
5.44 (1H, m), 4.53 (1H, m), 3.97 (1H, d, J ) 9.4 Hz), 3.72 (1H,
dd, J ) 7.2, 13.3 Hz), 3.34 (1H, m), 3.10 (1H, m), 3.02 (2H, d,
J ) 21.3 Hz), 2.97-2.77 (3H, m), 2.68 (1H, m), 2.48 (1H, dd,
J ) 4.7, 15.0 Hz), 2.22 (1H, m), 2.15 (1H, d, J ) 15.4 Hz), 2.02
(1H, m), 1.94 (1H, m), 1.414 (3H, s), 1.407 (3H, s). FAB-MS
(-VE) m/z 705 (M - H)-. HR-FAB-MS calcd for C39H42N4O9P
(M - H)-: 705.268 94. Found: 705.268 96.
ter t-Bu tyl 3-(N-{[N-((1S)-1-{N-[(2S)-2-(Na p h th ylm eth -
yl)p en t -4-en yl]ca r ba m oyl}-2-ca r b a m oylet h yl)ca r ba m o-
yl]m e t h yl}ca r b a m oyl)-(3S ,4S )-4-(4-{[b is(t er t -b u t oxy)-
p h osp h on o]m eth yl}p h en yl)h ex-5-en oa te (40). Mp 104-
105 °C; 1H NMR (CDCl3): δ 7.97 (1H, d, J ) 8.5 Hz), 7.80
(1H, dd, J ) 1.4, 8.2 Hz), 7.68 (1H, d, J ) 7.9 Hz), 7.45 (2H,
m), 7.34 (3H, m), 7.12 (3H, m), 7.05 (1H, s), 7.03 (1H, s), 6.40
(1H, s), 5.79 (2H, m), 5.60 (1H, s), 5.04 (4H, m), 4.61 (1H, dd,
J ) 6.4, 13.0 Hz), 3.59 (1H, dd, J ) 5.4, 16.7 Hz), 3.33 (3H,
m), 3.13 (1H, m), 3.00 (2H, d, J ) 6.7 Hz), 2.92 (2H, d, J )
21.5 Hz), 2.86 (1H, dt, J ) 3.7, 10.1 Hz), 2.70 (2H, m), 2.58
(1H, m), 2.49 (1H, dd, J ) 3.8, 17.1 Hz), 2.12 (3H, m), 1.37
(9H, s), 1.35 (9H, s), 1.34 (9H, s); FAB-MS (+VE) m/z 875
(MH+).
2-[(9S ,10S ,14S ,18S )-7,16,19-T r ia za -18-(c a r b a m o y l-
m eth yl)-14-(n a p h th ylm eth yl)-8,17,20-tr ioxo-10-[4-(p h os-
p h on om et h yl)p h en yl]sp ir o[5.14]icos-11-en -9-yl]a cet ic
ter t-Bu tyl 3-(N-{1-[N-((1S)-1-{N-[(2S)-2-(Na p h th ylm e-
thyl)pent-4-enyl]carbamoyl}-2-carbamoylethyl)carbamoyl]-
isop r op yl}ca r b a m oyl)-(3S ,4S )-4-(4-{[b is(t er t -b u t oxy)-
p h osp h on o]m eth yl}p h en yl)h ex-5-en oa te (41). Mp 92 °C;
1H NMR (CDCl3) δ 8.04 (1H, d, J ) 8.5 Hz), 7.80 (1H, dd, J )
1.3, 8.5 Hz), 7.68 (1H, m), 7.48 (1H, m), 7.42 (1H, m), 7.35
(3H, m), 7.10 (2H, m), 7.00 (1H, m), 6.91 (1H, s), 6.89 (1H, s),
6.63 (1H, s), 6.00 (1H, m), 5.84 (1H, m), 5.66 (1H, m), 5.38
(1H, s), 5.05 (2H, m), 4.92 (2H, m), 4.45 (1H, dd, J ) 6.2, 13.0
Hz), 3.29 (1H, m), 3.11 (2H, m), 3.04 (2H, m), 2.92 (2H, d, J )
21.4 Hz), 2.78 (2H, m), 2.66 (1H, dd, J ) 7.5, 15.2 Hz), 2.48
(2H, d, J ) 7.0 Hz), 2.16 (2H, m), 2.06 (1H, m), 1.39 (9H, s),
1.37 (9H, s), 1.35 (9H, s), 1.14 (3H, s), 1.04 (3H, s); FAB-MS
(+VE) m/z 903 (MH+).
1
Acid (6). Mp 260 °C (dec); H NMR (CD3OD) δ 8.64 (1H, s),
8.48 (1H, d, 7.9 Hz), 8.16 (1H, d, J ) 8.3 Hz), 7.78 (1H, d, J )
7.9 Hz), 7.66 (1H, d, J ) 9.1 Hz), 7.60 (1H, t, J ) 6.3 Hz), 7.48
(1H, t, J ) 7.0 Hz), 7.40 (1H, dd, J ) 6.9, 7.8 Hz), 7.32 (2H,
m), 7.22 (3H, m), 5.84 (1H, dd, J ) 11.1, 13.7 Hz), 5.40 (1H,
m), 4.49 (1H, m), 4.04 (1H, d, J ) 9.4 Hz), 3.72 (1H, dd, J )
6.7, 13.9 Hz), 3.37 (2H, m), 3.02 (2H, d, J ) 21.7 Hz), 2.96
(1H, dd, J ) 4.5, 15.2 Hz), 2.88 (1H, m), 2.82 (1H, m), 2.68
(1H, dd, J ) 10.1, 13.8 Hz), 2.45 (1H, dd, J ) 4.8, 15.2 Hz),
2.28-1.90 (6H, m), 1.80 (2H, m), 1.58 (5H, m), 1.26 (1H, m);
FAB-MS (-VE) m/z 745 (M - H)-; HR-FAB-MS calcd for
C
39H46N4O9P (M - H)-: 745.300 24. Found: 745.298 77.
Ack n ow led gm en t. This work was supported in part
by a grant from the Susan G. Komen Breast Cancer
Foundation to D.Y.
ter t-Bu tyl 3-(N-{[N-((1S)-1-{N-[(2S)-2-(Na p h th ylm eth -
yl)p en t -4-en yl]ca r ba m oyl}-2-ca r b a m oylet h yl)ca r ba m o-
y l]c y c lo h e x y l }c a r b a m o y l)-(3 S ,4S )-4-(4-{[b i s (t e r t -
bu toxy)p h osp h on o]m eth yl}p h en yl)h ex-5-en oa te (42). Mp
Refer en ces
1
85 °C; H NMR (CDCl3) δ 8.05 (1H, d, J ) 8.4 Hz), 7.81 (1H,
(1) Renhowe, P. A. Growth factor receptor kinases in cancer. Annual
Reports in Medicinal Chemistry; Academic Press: London, 2001;
pp 109-118.
(2) Wilkinson, S. E.; Harris, W. Selective tyrosine kinase inhibitors.
Emerging Drugs 2000, 5, 287-297.
d, J ) 8.5 Hz), 7.68 (1H, dd, J ) 2.6, 6.6 Hz), 7.45 (2H, m),
7.36 (3H, m), 7.09 (2H, m), 6.90 (1H, s), 6.88 (1H, s), 6.74 (1H,
s), 6.06 (1H, s), 5.84 (1H, m), 5.61 (1H, m), 5.38 (1H, s), 5.07
(2H, m), 4.90 (2H, m), 4.57 (1H, dd, J ) 7.4, 12.5 Hz), 3.32
(1H, m), 3.20 (1H, dd, J ) 10.0, 10.4 Hz), 3.04 (2H, m), 2.93
(4H, m), 2.74 (1H, m), 2.62 (1H, m), 2.46 (2H, m), 2.18 (2H,
m), 2.08 (1H, m), 1.70 (6H, m), 1.40 (9H, s), 1.38 (9H, s), 1.36
(9H, s), 1.05 (3H, m), 0.78 (1H, m); FAB-MS (+VE) m/z 943
(MH+).
(3) Mendel, D. B.; Laird, A. D.; Smolich, B. D.; Blake, R. A.; Liang,
C.; Hannah, A. L.; Shaheen, R. M.; Ellis, L. M.; Weitman, S.;
Shawver, L. K.; Cherrington, J . M. Development of SU5416, a
selective small molecule inhibitor of VEGF receptor tyrosine
kinase activity, as an anti-angiogenesis agent. Anti-Cancer Drug
Des. 2000, 15, 29-41.
(4) Noonberg, S. B.; Benz, C. C. Tyrosine kinase inhibitors targeted
to the epidermal growth factor receptor subfamily: Role as
anticancer agents. Drugs 2000, 59, 753-767.
ter t-Bu t yl 2-[(1S,7S,11S,15S)-3,6,9-Tr ia za -15-(4-{[b is-
(ter t-bu toxy)p h osp h on o]m eth yl}p h en yl)-7-(ca r ba m oyl-
m et h yl)-4,4-d im et h yl-11-(n a p h t h ylm et h yl)-2,5,8-t r ioxo-
cyclop en ta d ec-13-en yl]a ceta te (43). Mp 229-230 °C; 1H
NMR (CD3OD) δ 8.88 (1H, s), 8.50 (1H, d, J ) 8.5 Hz), 8.18
(1H, d, J ) 8.5 Hz), 7.78 (1H, dd, J ) 1.2, 8.2 Hz), 7.66 (2H,
m), 7.48 (1H, m), 7.40 (1H, m), 7.32 (2H, m), 7.18 (3H, m),
5.84 (1H, ddd, J ) 2.0, 10.4, 12.3 Hz), 5.45 (1H, ddd, J ) 3.1,
10.5, 14.2 Hz), 4.53 (1H, m), 3.98 (1H, d, J ) 10.3 Hz), 3.72
(1H, dd, J ) 6.9, 13.2 Hz), 3.34 (1H, dd, J ) 4.4, 13.3 Hz),
3.05 (1H, dt, J ) 12.4, 2.3 Hz), 3.00 (2H, d, J ) 21.4 Hz), 2.98
(1H, m), 2.80 (2H, m), 2.67 (1H, dd, J ) 10.2, 13.6 Hz), 2.47
(1H, dd, J ) 4.8, 15.2 Hz), 2.22 (1H, m), 2.03 (2H, m), 1.94
(1H, m), 1.418 (3H, s), 1.412 (3H, s), 1.352 (9H, s), 1.348 (9H,
s), 1.32 (9H, s).
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ter t-Bu tyl
2-[(9S,10S,14S,18S)-7,16,19-Tr ia za -10-(4-
{[b is(ter t-b u t oxy)p h osp h on o]m et h yl}p h en yl)-18-(ca r -
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7.74 (1H, d, J ) 8.4 Hz), 7.64 (1H, d, J ) 7.9 Hz), 7.49 (1H,
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6.36 (1H, s), 6.05 (1H, s), 5.74 (1H, dd, J ) 10.1, 14.1 Hz),
5.26 (1H, m), 4.98 (1H, s), 4.63 (1H, m), 3.92 (1H, d, J ) 9.4
Hz), 3.74 (1H, dd, J ) 6.1, 13.8 Hz), 3.27 (1H, dd, J ) 5.0,
13.8 Hz), 3.09 (1H, m), 3.00 (1H, m), 2.95 (2H, d, J ) 21.4
Hz), 2.81 (1H, dd, J ) 11.8, 17.3 Hz), 2.72 (1H, m), 2.64 (1H,
dd, J ) 9.7, 13.8 Hz), 2.25 (2H, m), 2.10 (3H, m), 1.88 (1H, m),
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