N-Terminal carboxyl and tetrazole-containing amides as adjuvants to Grb2 SH2 domain ligand binding

Article abstract of DOI:10.1016/S0968-0896(01)00014-1

High affinity binding of peptides to Src homology 2 (SH2) domains, often requires the presence of phosphotyrosyl (pTyr) or pTyr-mimicking moieties in the N-terminal position of the binding ligand. Several reports have shown that N^α-acylation of the critical pTyr residue can result in increased SH2 domain binding potency. For Grb2 SH2 domains which recognize pTyr-Xxx-Asn-NH₂ motifs, significantly potency enhancement can be incurred by N^α-(3-amino)Z derivatization of tripeptides such as pTyr-Ile-Asn-NH₂. Using ligands based on the high affinity pY-Ac₆c-Asn-(naphthylpropylamide) motif, (where Ac₆c= 1-aminocyclohexanecarboxylic acid), additional reports have shown moderate potentiating effects of N^α-oxalyl derivatization. The current study examined variations of the N^α oxalyl theme in the context of a Xxx-Ac₆c-Asn-(naphthylpropylamide) paltform, where Xxx = the hydrolytically stable pTyr mimetics phosphonomethyl phenylalanine (Pmp) or carboxymethyl phenylalanine (Cmf). The effects of N^α(3-amino)Z derivatization were also investigated for this platform, to ascertain whether the large binding enhancement reported for tripeptides such as pTyr-Ile-Asn-NH₂ could be observed. In ELISA-based extracellular Grb2 SH2 domain binding assays, it was found for the pmp-based series, that extending the oxalyl carboxyl out by one methylene unit or replacing carboxyl functionality with a tetrazole isostere, resulted in binding potency greater than the parent N^α-acetyl-containing compound, with enhancement approximately that observed for the NN^α-oxalyl derivative. When Cmf was used as the pTyr mimetic, only modest differences in IC₅₀ approximating values were obsered for the series. Examination of the N^α-(3-amino)Z derivatized Pmp-Ac₆c-Asn-(naphthylpropylamide), showed that binding affinity was reduced relative to the parent N^α-acetyl analogue, in contrast to the reported significant enhancement of affinity observed with other peptide ligands. Treatment of MDA-453 tumor cells, which are mitogenically driven through erbB-2 tyrosine kinase-dependent pathways, with Pmp-containing inhibitors resulted in growth inhibition, with the N^αoxalyl and N^α-malonyl-containing compounds exhibiting IC₅₀ values (4.3 and 4.6 μM, respectively) approximately five-fold lower than the parent N^α-acetyl-containing compound. Tetrazole and N^α-(3-amino)Z-containing inhibitors were from two- to four-fold less potent than these latter analogues in the growth inhibition assays. Copyright

Full text of DOI:10.1016/S0968-0896(01)00014-1

Bioorganic & Medicinal Chemistry 9 (2001) 1439–1445
N-Terminal Carboxyl and Tetrazole-containing Amides as
Adjuvants to Grb2 SH2 Domain Ligand Binding
Terrence R. Burke Jr.,a,* Zhu-Jun Yao,a Yang Gao,a Jane X. Wu,b Xiaofeng Zhu,b
Juliet H. Luo,b Ribo Guob and Dajun Yangb,y
aLaboratory of Medicinal Chemistry, Division of Basic Sciences, National Cancer Institute, National Institutes of Health,
Building 376, FCRDC, Frederick, MD 21702-1201, USA
bDepartment of Oncology, Lombardi Cancer Center, Georgetown University, Washington, DC 20007, USA
Received 9 November 2000; accepted 11 January 2001
Abstract—High affinity binding of peptides to Src homology 2 (SH2) domains, often requires the presence of phosphotyrosyl (pTyr)
or pTyr-mimicking moieties in the N-terminal position of the binding ligand. Several reports have shown that Na-acylation of the
critical pTyr residue can result in increased SH2 domain binding potency. For Grb2 SH2 domains which recognize pTyr-Xxx-Asn-
NH2 motifs, significant potency enhancement can be incurred by Na-(3-amino)Z derivatization of tripeptides such as pTyr-Ile-Asn-
NH2. Using ligands based on the high affinity pY-Ac6c-Asn-(naphthylpropylamide) motif, (where Ac6c=1-aminocyclohex-
anecarboxylic acid), additional reports have shown moderate potentiating effects of Na-oxalyl derivatization. The current study
examined variations of the Na-oxalyl theme in the context of a Xxx-Ac6c-Asn-(naphthylpropylamide) platform, where Xxx=the
hydrolytically stable pTyr mimetics phosphonomethyl phenylalanine (Pmp) or carboxymethyl phenylalanine (Cmf). The effects of
Na-(3-amino)Z derivatization were also investigated for this platform, to ascertain whether the large binding enhancement reported
for tripeptides such as pTyr-Ile-Asn-NH2 could be observed. In ELISA-based extracellular Grb2 SH2 domain binding assays, it was
found for the Pmp-based series, that extending the oxalyl carboxyl out by one methylene unit or replacing carboxyl functionality
with a tetrazole isostere, resulted in binding potency greater than the parent Na-acetyl-containing compound, with enhancement
approximating that observed for the Na-oxalyl derivative. When Cmf was used as the pTyr mimetic, only modest differences in IC50
values were observed for the series. Examination of the Na-(3-amino)Z derivatized Pmp-Ac6c-Asn-(naphthylpropylamide), showed
that binding affinity was reduced relative to the parent Na-acetyl analogue, in contrast to the reported significant enhancement of
affinity observed with other peptide ligands. Treatment of MDA-453 tumor cells, which are mitogenically driven through erbB-2
tyrosine kinase-dependent pathways, with Pmp-containing inhibitors resulted in growth inhibition, with the Na-oxalyl and Na-
malonyl-containing compounds exhibiting IC50 values (4.3 and 4.6 mM, respectively) approximately five-fold lower than the parent
Na-acetyl-containing compound. Tetrazole and Na-(3-amino)Z-containing inhibitors were from two- to four-fold less potent than
these latter analogues in the growth inhibition assays. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
involved in the etiology of a variety of cancers,3 inhibi-
tors of SH2 domain binding may potentially afford
attractive new therapeutic approaches by disrupting the
continuity of signal transmission.4 Included among
important targets for development of antiproliferative
agents, are Grb2 SH2 domains, which have been asso-
ciated with breast cancer5 and MET-dependent can-
cers.6 For most SH2 domains, high affinity ligand
binding depends both on the combined interactions of
pTyr residues within well defined pTyr binding pockets,
as well as secondary interactions of amino acid residues
proximal to this pocket.7 For Grb2 SH2 domains, an
Asn residue in the pY+2 position, provides critical ele-
ments of this recognition. Major design considerations
in the development of most Grb2 SH2 domain inhibitors
Protein-tyrosine kinase (PTK)-dependent signal trans-
duction depends on phosphorylation of tyrosyl residues
in protein substrates, with subsequent recognition and
binding to these newly generated phosphotyrosyl
(pTyr)-containing sequences, by pTyr-binding modules
such as Src homology 2 (SH2) and phosphotyrosyl
binding (PTB) domains.1,2 Because PTK pathways are
*Corresponding Author. Tel.:+1-301 846-5906; fax:+1-301 846-6033;
e-mail: tburke@helix.nih.gov
ySupported in part by the Susan G. Komen Breast Cancer Founda-
tion.
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00014-1

1440
T. R. Burke, Jr. et al. / Bioorg. Med. Chem. 9 (2001) 1439–1445
to date, have been predicated on the binding of pTyr-
containing peptide leads, with interactions in the pTyr
binding pocket being an important component.8ꢀ10
Both X-ray11ꢀ13 and NMR solution structures14ꢀ16 of
ligated Grb2 SH2 domain protein have shown that pTyr
binding involves key hydrogen bonding between the
pTyr phosphoryl oxygens and bC and aA arginine
residues (Arg86 and Arg67, respectively).17 Develop-
ment of phenyl phosphate mimetics which can engage
these critical Arg residues in fashions similar to parent
pTyr residues, has been an integral component of efforts
to derive SH2 domain inhibitors which are stable to
cellular phosphatases.18ꢀ22 To date, a major focus of
these efforts has been on substituents at the 4-
position23ꢀ31 or 3,4-positions28,32 of phenylalanine resi-
dues. However, studies have also shown that bonding
interactions can be enhanced by substituents originating
from the tyrosyl a-position.29,33ꢀ37 For Grb2 SH2
domains, binding enhancement of certain Na-sub-
stituents has been attributed to interactions with the
Arg67 residue.12,28,38 Potency enhancement through Na-
auxiliary functionality is particularly attractive, since it
supplements and maintains interactions already pro-
vided by phosphate mimicking groups at the phenylala-
nine 4-position. The Na-oxalyl group is one example of
a substituent which both enhances binding potency and
potentiates cellular efficacy.28 To date however, investi-
gations of acidic Na-derivatives in Grb2 SH2 domain
inhibitors have not been extensively reported. There-
fore, the current study was undertaken to examine a
series of oxalyl-related analogues in a Grb2 SH2
domain-binding platform.
and 6) bearing phosphonomethyl40ꢀ42 or carbox-
ymethyl43,44 phosphate mimicking functionalities at the
phenylalanyl 4-position, were examined (Scheme 1).
Synthesis of both series of naphthyl-containing analo-
gues were accomplished by solution methods, starting
from previously reported tert-butyl-protected 128 or 2.30
Acylation of free a-amino groups of 1 or 2 as described
in the Experimental, provided protected intermediates
3a–d and 4a–c, respectively (Scheme 1), which after
acidic deprotection and purification by HPLC, gave
final products 5g–j and 6g–i. Na-acetyl analogues 5e and
6e and Na-oxalyl analogues 5f and 6f have been pre-
viously reported.28,30
Results and Discussion
Development of Grb2 SH2 domain inhibitors pre-
dicated on binding of pTyr-Xxx-Asn’’ peptides, is
heavily influenced both by interactions within the pTyr
binding pocket as well as by interactions afforded by the
Asn residue.21 Previously disclosed N-terminal 3-
(naphth-1-yl)propan-1-yl)-containing tripeptide 5e pro-
vides high affinity interactions outside the pTyr-binding
pocket.39 Using 5e as a model, in prior reports we have
investigated binding interactions within the pTyr-bind-
ing pocket, through a series of phenylalanine analogues
bearing various phosphate-mimicking functionality.
Among pTyr mimetics examined phosphonomethyl
phenylalanine (Pmp)-containing 5e28 and carbox-
ymethylphenylalanine (Cmf)-containing 6e30 were the
most potent phosphorus and monocarboxy-based inhi-
bitors, examined. A critical component of high affinity
binding of these analogues, the interaction of their
anionic phosphate mimicking functionality with Arg86
and Arg67 residues, can be augmented by appending
oxalyl species at the phenylalanyl Na-position.28,30 The
basis for binding enhancement has been inferred from
molecular modelling studies, to potentially reside in
Synthesis
Inhibitors were based on an N-terminal 3-(naphth-1-
yl)propan-1-yl)-containing tripeptide platform, origin-
ally disclosed as providing high affinity Grb2 SH2
domain inhibitors.39 Two parallel series of analogues (5
Scheme 1.

T. R. Burke, Jr. et al. / Bioorg. Med. Chem. 9 (2001) 1439–1445
1441
anionic interactions between the oxalyl b-carboxyl
group and the positively charged Arg67 guanidino
group.28,30
In order to examine structural parameters other than
chain length, the effect of replacing the oxalyl carboxyl
group by ‘‘carboxylic-mimicking’’ functionality was
investigated. Use of tetrazole groups as carboxylic iso-
steres has been well documented.45ꢀ47 and in the present
study, tetrazolyl analogues 5h (IC50=0.045 mM) and 6h
(IC50=6 mM) were prepared as counterparts of oxalyl-
containing 5f and 6f, respectively. For Pmp-based 5h, a
higher potency relative to the parent Na-acetyl analogue
was observed. Similar relative potency ratios were
observed for Cmf-based 6g. Just as in the oxalyl series,
where the effect of chain elongation was examined,
similar chain extension was undertaken in the tetrazole
series, with compounds 5i (IC50=0.034 mM) and 6i
(IC50=4 mM) being prepared as tetrazolyl congeners of
Na-malonyl analogues 5g and 6g respectively. These
were found to be approximately equipotent to their
malonyl counterparts.
Relative Grb2 SH2 domain binding affinities in
extracellular ELISA assays
In the current study, Na-oxalyl functionality enhanced
binding potency relative to the parent Na-acetyl con-
gener, by approximately three-fold in both the Pmp-
containing series (5e, IC50=0.065 mM versus 5f,
IC50=0.02 mM) and the Cmf-containing series (6e,
IC50=9 mM versus 6f, IC50=2.7 mM) (Table 1). Since
one objective of the current study was to explore
potential binding enhancement afforded by acidic Na-
substituents other than the oxalyl group, the Na-oxalyl
group was initially replaced by an Na-malonyl sub-
stituent, which effectively extended the oxalyl carboxyl
out from the Na-position by an additional methylene
unit. In the Pmp-series, this change resulted in a binding
potency (5g, IC50=0.03 mM) which was approximately
two-fold higher than the parent Na-acetyl analogue (5e),
and approximately equipotent to the Na-oxalyl com-
pound (5f). In the Cmf series, the Na-malonyl analogue
(6g, IC50=6 mM) was only slightly more potent than the
parent Na-acetyl compound (6e), and showed a two-fold
loss of potency relative to the Na-oxalyl analogue (6f).
It had been expected that enhancement of potency could
potentially be observed for tetrazolyl versus carboxyl
analogues in the latter series, due in part to possible
cation–p interactions between the Arg67 guanidino
group and the tetrazole rings. The experimental obser-
vation that tetrazolyl analogues did not exhibit
enhanced binding potency relative to their carboxyl
counterparts, indicated that cation–p interaction may
not have come into play. Nonetheless, it remained of
interest to examine the potential utility of cation–p
interactions with Arg67 in the context of the naphthyl-
containing Pmp-Ac6c-Asn-(naphthylpropylamide) plat-
form. In this regard, it had previously been reported
that the Grb2 SH2 domain binding potency of Xxx-
pTyr-Ile-Asn-NH2 could be enhanced over 100-fold
when Xxx=m-aminobenzyloxycarbonyl [(3-amino)Z]
as compared to Xxx=Acetyl.38 By analogy to X-ray
studies of a related N-terminal anthranilic acid-deriva-
tized inhibitor,12 the exceptional enhancement in affinity
afforded by the Na-(3-amino)Z group could be partially
attributed to p-stacking between the (3-amino)Z aryl
ring and the Arg67. Surprisingly, in the current study,
the (3-amino)Z-derivatized analogue 5j (IC50=0.078 mM)
exhibited an affinity lower than the parent Na-acetyl
analogue (5e), indicating the absence of beneficial bind-
ing effects.
Table 1. Inhibition of Grb2 SH2 domain bindinga
R
No
IC50ꢁs.d. (mM)
0.065ꢁ0.008
No
IC50 (mM)b
5e
6e
9
5f
5g
5h
5i
0.02ꢁ0.004
0.03ꢁ0.02
6f
6g
6h
6i
2.7
6
Inhibition of tumor MDA-453 cell growth
ELISA data presented in Table 1 and discussed above,
reflect the ability of inhibitors to block binding of a
short pTyr-containing peptide to isolated Grb2 SH2
domain protein. This kind of data is a useful indicator
of molecular interactions between the inhibitors and
Grb2 SH2 domains, and it provides a basis for com-
parison of effects on binding induced by structural
changes within a series of compounds. However, in
physiological contexts, Grb2 SH2 domains exist intra-
cellularly as subunits of larger proteins which bind to
pTyr residues that are contained within protein sequen-
ces, and which require cell membrane transport of inhi-
bitors before access to the Grb2 protein can be
achieved. Additionally, the ultimate aim of these studies
is to block Grb2 SH2 domain-dependent mitogenic
0.045ꢁ0.018
0.034ꢁ0.002
0.078ꢁ0.081
6
4
5j
aIC50 values were determined using Grb2 SH2 domain fusion protein
in an ELISA assay as previously described in ref 45.
bSingle determinations.

1442
T. R. Burke, Jr. et al. / Bioorg. Med. Chem. 9 (2001) 1439–1445
signalling. Therefore, in order to more faithfully exam-
ine the ability of compounds to inhibit interaction of
Conclusions
erbB-2
native Grb2 with cognate p185
in whole cells and
Previously it has been shown that for pTyr-containing
ligands, derivatization at the tyrosyl Na-position can
result in increased Grb2 SH2 domain binding potency,
including potentiating effects of Na-oxalyl functionality
when incorporated into the naphthylpropylamido tri-
peptide platform 5. The primary intent of the current
study was to examine variations of the Na-oxalyl theme
in the context of both Pmp-containing platform 5 as
well as Cmf-containing platform 6, and also to examine
Na-(3-amino)Z derivatization in platform 5. As with the
Na-oxalyl derivative 5f, it was found that extending the
oxalyl carboxyl out by one methylene unit or replacing
carboxyl functionality with a tetrazole isostere, resulted
in increased potency both in ELISA-based extracellular
Grb2 SH2 domain binding assays and erbB-2 tyrosine
kinase dependent cell growth assays. Interestingly, in
spite of the fact that Na-(3-amino)Z derivatization has
been reported to significantly enhance the binding
potency of pTyr-containing tripeptides, when applied to
the naphthyl-containing platform 5, this pronounced
enhancement was not observed.
to block mitogenic signalling, tests were conducted
using MDS-453 cells, which are derived from human
breast cancer where there is an amplification of erbB-2
gene. These growth inhibition assays (results shown in
Table 2) reflect the combined effects of cell membrane
transport as well as physiologically relevant inhibition
of Grb2 SH2 domain binding. Na-oxalyl 5f has been
reported previously to more potently inhibit intracellular
Grb2 SH2 domain binding than does the Na-acetyl parent
5e.28 In the current study, 5f (IC50=4.3 mM) was approxi-
mately five-fold more potent than 5e (IC50=23 mM) as an
inhibitor of erbB-2-dependent cell growth. Consistent
with extracellular ELISA binding results (Table 1), Na-
malonyl derivative 5g (IC50=4.6 mM) was equi-potent
to 5f in inhibiting cell growth. Tetrazole-containing
analogue 5h (IC50=9.6 mM) was approximately twice as
potent as Na-acetyl parent 5e, while inhibitors 5i
(IC50=19.7 mM) and 5j (IC50=17.0 mM) were minimally
more potent.
Of interest, was a comparison of the relative cellular
potencies of tetrazole derivatives (5h and 5i) relative to
their carboxyl counterparts (5f and 5g). While being
approximately as acidic as carboxyl groups at physi-
ological pH, tetrazole groups are almost 10 times more
lipophilic.48 In previous reports, replacement of car-
boxylate functionality with tetrazole groups can result
either in enhanced potency, as exemplified by CCK-B
ligands,49 or as in the case of phospholipase A(2)
antagonists, it can lead to both increased or decreased
affinity, depending on the display platform.50 Such
results are exemplary of the general observation, that
bioisoterism of the tetrazole moiety with carboxyl func-
tionality, is not uniformly consistent. This is further
seen with certain anti-allergic, anti-lipemic and anti-
inflammatory agents, where tetrazoles exhibit increased
potency relative to their respective carboxylate con-
geners, while for select oestrogenic acids and anti-
arrythmics, lowered potency is observed.51 The lack of a
clearly defined bioisosteric relationship between car-
boxyl and tetrazole functionality is reflected in the cur-
rent study, where cellular potency of tetrazole-
containing 5h and 5i was not enhanced relative to their
carboxylic counterparts (5f and 5g), in spite of the fact
that 5h and 5i would be expected to exhibit greater
lipophilicity.
Experimental
Cells and cell cultures
Cell lines were obtained from the American Type Cul-
ture Collection (Rockville, MD) and the Lombardi
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 ꢂC and 5% CO2.
ELISA assay of Grb2 binding inhibition
A biotinated Shc-derived phosphopeptide (20 ng/mL),
was bound to 96-well plate by reaction overnight. Non-
specific interactions were inhibited by addition of 5%
bovine serum albumin containing TBS. Recombinant
purified Grb2 SH2-GST fusion protein, incubated with
test compounds at a series of dilutions from 1 mM to
1 mM, were added to each well. Following extensive
washing with 0.1% bovine serum albumin in TBS, Grb2
SH2 domain binding was detected using anti-GST anti-
bodies and goat anti-mouse antibody conjugated to
alkaline phosphatase. Color reactions were developed
using para-nitrophenyl phosphate.
Table 2. Inhibition of MDA-453 tumor cell growth by treatment
with phosphonate-containing Grb2 SH2 domain antagonistsa
Cell growth inhibition
No
IC50ꢁs.d. (mM)
No
IC50ꢁs.d. (mM)
5e
5f
5g
23ꢁ2b
4.3ꢁ0.8
4.6ꢁ1.4
5h
5i
5j
9.6ꢁ6.4
19.7ꢁ3.2
17.0ꢁ1.4b
The effect of Grb2 inhibitors on cell proliferation was
determined by direct cell counting. Briefly, 25,000 cells
were plated into 24-well plates and Grb2 SH2 domain
inhibitors at appropriate concentrations were added and
cultured for 8 to 10 days. Cells were collected every
other day and counted using a Coulter counter.
aIC50 values were determined as previously descibed in reference 45,
and represent the average of three independent experiments, unless
otherwise indicated.
bAverage of two independent determinations.

T. R. Burke, Jr. et al. / Bioorg. Med. Chem. 9 (2001) 1439–1445
1443
General synthetic methods
7.15 (9H, m), 7.02–6.99 (2H, d, J=7.81 Hz), 6.90 (1H,
d, J=7.57 Hz), 6.69 (1H, s), 6.51 (1H, s), 5.65 (1H, s),
5.31 (1H, d, J=5.1 Hz), 5.07 (1H, d, J=12.0 Hz), 4.89
(1H, d, J=12.0 Hz), 7.74 (1H, m), 7.25 (1H, m), 3.37
(2H, m), 3.15–2.69 (8H, m), 2.01–1.65 (8H, m), 1.50
(9H, s), 1.40 (18H, m). FABMS (+VE, NBA) m/z
1027.5 [MH+].
Fast atom bombardment mass spectra (FABMS) were
acquired with a VG Analytical 7070E mass spectro-
meter under the control of a VG 2035 data system. H
1
NMR data were obtained on Bruker AC250 (250 MHz)
are reported in ppm relative to TMS and referenced to
the solvent in which they were run. Solvents were
removed by rotary evaporation under reduced pressure
and anhydrous solvents were obtained commercially
and used without further drying. Preparative high pres-
sure liquid chromatography (HPLC) was performed
using a Waters PrepLC 4000 system with photodiode
array detection and an Advantage C18 5m column
(20 mm diaꢃ250 mm) at a flow rate of 10 mL/min, using
a solvent system of A=0.1% aqueous TFA and
B=0.1% TFA in acetonitrile.
General procedure for formation of protected N-acyl
intermediates 4a–c14
Preparation of compound 4a. To a solution of amine 2
(0.1 mmol) in DMF (1 mL) was added an active ester
solution formed by reacting mono tert-butyl malonate
(17m, 0.11 mmol), HOBt (15 mg, 0.11 mmol) and
DIPCDI (17m, 0.11 mmol) in DMF (0.5 mL) (10 min).
The resulting solution was stirred (overnight) then taken
to dryness under high vacuum and purified by silica gel
chromatography (EtOAc then 5% MeOH in EtOAc) to
yield 4a as a syrup (60 mg, 83% yield). 1H NMR
(CDCl3) d 8.12 (d, 1H, J=6.8 Hz), 8.00–7.86 (m, 2H),
7.80–7.66 (m, 3H), 7.56–7.38 (m, 5H), 7.22 (s, 4H), 6.65
(brs, 1H), 5.74 (brs, 1H), 4.86–4.72 (m, 2H), 3.55 (s,
2H), 3.55–3.35 (m, 2H), 3.26 (s, 2H), 3.25–3.15 (m, 2H),
3.10–2.95 (m, 2H), 2.70 (dd, 1H, J=3 Hz & 15 Hz),
2.20–1.20 (m, 12H), 1.52 (s, 9H), 1.46 (s, 9H). FABMS
(+VE, NBA) m/z 828.6 (MH+).
Formation of protected N-acyl intermediates 3a,c and d
Preparation of compound 3a. To the solution of 128
(0.066 mmol) in anhydrous DMF (1 mL) was added an
activated ester solution formed by reacting mono tert-
butyl malonate (Aldrich) (11.2 mL 0.073 mmol), 1-
hydroxybenzotirazole
hydrate
(HOBt)
(9.9 mg,
0.073 mmol) and 1,3-diisopropylcarboddimide (DIPCDI)
(11.2mL, 0.073 mmol) in anhydrous DMF (1 mL) (10 min).
The resulting solution was stirred (overnight) then taken
to dryness under high vacuum and purified by silica gel
chromatography (CHCl3/EtOAc/MeOH) to provide 3a
Preparation of compound 4b. Acylation of 2 with 1-(4-
methoxybenzyl)tetrazole-5-carboxylic acid chloride53
according to the general procedure and purification by
silica gel chromatography (EtOAc with MeOH, from 0
1
as white foam (37 mg, 60% yield). H NMR (CDCl3) d
8.05 (1H, dd, J=1.95, 7.20 Hz), 7.82 (2H, m), 7.68 (1H,
m), 7.51–7.38 (4H, m), 7.36 (2H, d, J=4.6 Hz), 7.16–
7.04 (5H, m), 6.46 (1H, br), 5.37 (1H, br), 4.77 (2H, m),
3.42–3.34 (2H, m), 3.18 (2H, d, J=1.47 Hz), 3.16–3.10
(3H, m), 3.02 (1H, s), 2.97–2.87 (2H, m), 2.93 (1H, s),
2.53 (1H, dd, J=5.1, 15.3 Hz), 2.15 (1H, d, J=5.0 Hz),
2.05–1.62 (6H, m), 1.43 (9H, s), 1.42 (9H, s), 1.40 (9H,
s). (+VE, NBA) m/z 920.5 [MH+].
1
to 2.5%) provided 4b as a syrup (53% yield). H NMR
(CDCl3) d 8.44 (d, 1H, J=7.3 Hz), 8.25 (d, 1H,
J=7.3 Hz), 8.20–8.05 (m, 2H), 7.80–7.30 (m, 10H), 6.94
(d, 2H, J=8.5 Hz), 6.44 (s, 1H), 6.36 (s, 1H), 5.92 (s,
1H), 5.88 (d, 1H, J=13 Hz), 5.78 (d, 1H, J=13 Hz),
5.10–4.95 (m, 1H), 4.78–4.66 (m, 1H), 3.82 (s, 3H), 3.56
(s, 2H), 3.45 (m, 1H), 3.30–3.05 (m, 4H), 2.54 (dd, 1H,
J=9 Hz & 14 Hz), 2.15–0.90 (m, 12H), 1.51 (s, 9H).
FABMS (+VE, NBA) m/z 902.9 (MH+).
Preparation of compound 3c. Acylation of 1 with 2-(5-
(2-methoxybenzyl)tetrazolyl)acetyl chloride (prepared
from ethyl 2-(5-(2-methoxybenzyl)tetrazolyl)acetate52
by initial hydrolysis to the free acid (LiOH) followed by
reaction with oxalyl chloride) according to the general
procedure, and purification of crude product by silica
gel chromatography (CHCl3/MeOH, 20:1) provided 3c
Preparation of compound 4c. Acylation of 2 with 2-(5-
(2-methoxybenzyl)tetrazolyl)acetyl chloride (prepared
from ethyl 2-(5-(2-methoxybenzyl)tetrazolyl)acetate48
by initial hydrolysis to the free acid (LiOH) followed by
reaction with oxalyl chloride) according to the general
procedure, and purification of crude product by silica
gel chromatography (EtOAc with MeOH, from 0 to
20%) provided 4c as a foam (34% yield).1H NMR
(CDCl3) d 8.07 (d, 1H, J=7.3 Hz), 7.90–7.80 (m, 3H),
7.74 (d, 1H, J=6.4 Hz), 7.55–7.30 (m, 10H), 6.92 (d,
2H, J=8 Hz), 5.60 (2H), 4.90–4.68 (brm, 2H), 3.95–3.75
(m, 3H), 3.82 (s, 3H), 3.55 (s, 2H), 3.45–2.55 (m, 5H,
2.15–1.10 (m, 12H), 1.51 (s, 9H). FABMS (+VE, NBA)
m/z 917 (MH+).
1
as a foam (62% yield). H NMR (CDCl3) d 8.02 (m,
2H), 7.85 (m, 2H), 7.68 (m, 1H), 7.45 (m, 2H), 7.39–7.07
(m, 9H), 6.88 (s, 2H), 6.83 (s, 1H), 6.41 (brs, 1H), 5.56
(s, 2H), 5.36 (brs, 1H), 4.66 (m, 2H), 3.77 (s, 3H), 3.74
(s, 2H), 3.35 (m, 2H), 3.13–2.84 (m, 5H), 2.92 (d, 2H,
J=18 Hz), 2.47 (dd, 1H, J=4.9 Hz & 15 Hz), 2.05–1.13
(brm, 12 Hz), 1.41 (s, 9H), 1.41 (s, 9H). FABMS (+VE,
NBA) m/z 1008.6 (MH+).
Preparation of compound 3d. Acylation of 1 with 3-N-
((tert-butyloxy)carbonylamino)benzyl-4-nitrophenylcar-
bonate38 according to the general procedure, and pur-
ification of crude product by silica gel chromatography
(CHCl3/EtOAc/MeOH) provided 3d as a white solid
(47% yield). 1H NMR (CDCl3) d 8.04 (1H, dd, J=1.90,
7.08 Hz), 7.82 (2H, m), 7.67 (1H, m), 7.58 (1H, m), 7.48–
Global deprotection and formation of final products 5g–j
Preparation of compound 5g. A solution of 3a (30 mg,
0.033 mmol)
in
TFA/H2O/triethylsilane
(TES)
(1.9 mL:100 mL : 50 mL) was stirred at room temperature
(1 h), then solvent was removed under high vacuum and

1444
T. R. Burke, Jr. et al. / Bioorg. Med. Chem. 9 (2001) 1439–1445
residue was purified by preparative HPLC (linear gra-
dient 5 to 50% B over 10 mins, then 50 to 100% B over
15 min: retention time=16.2 min) to provide product 5g
by preparative HPLC (linear gradient 20 to 90% B over
20 min: retention time=14.7 min) to provide product 6g
as a white solid (25 mg; 46% yield). H NMR (DMSO-
d6) d 8.55 (d, 1H, J=6.4 Hz), 8.12 (s, 2H), 7.99–7.86 (m,
2H) 7.84–7.75 (m, 1H), 7.60–7.35 (m, 6H), 7.20 (s, 4H),
6.94 (brs, 1H), 4.70–4.60 (m, 2H, 4.50–4.35 (m, 2H),
3.55 (s, 2H), 3.30–3.05 (m, 6H), 2.98–2.84 (m, 1H),
2.08–1.20 (m, 12H). FABMS (ꢀVE, Gly) m/z 714
(MꢀH).
1
1
as a white solid (23 mg, 94% yield). H NMR (DMSO-
d6) d 8.50 (1H, d, J=7.08 Hz), 8.08 (1H, s), 7.92–7.72
(2H, m), 7.75 (1H, m), 7.49 (3H, m), 7.37 (3H, m), 7.29
(1H, s), 7.15–7.05 3H, s), 6.89 (1H, m), 4.58 (1H, m),
4.39 (1H, m), 3.25. FABMS (ꢀVE, Gly) m/z 714
(MꢀH).
Preparation of compound 5h. Acylation of 1 with 1-(4-
methoxybenzyl)tetrazole-5-carboxylic acid chloride49
according to the general procedure outlined above
and purification by silica gel chromatography
(CHCl3:MeOH, 20:1) provided intermediate 3b as a
glass (65% yield) which was directly treated with TFA/
H2O/TES (2 mL:100 mL:50 mL) (room temperature,
overnight) as described above and taken to dryness
under vacuum. Residue was purified by preparative
HPLC (linear gradient 5 to 60% B over 25 min:
retention time=24.8 min) to provide product 5h as a
Preparation of compound 6h. As described in the general
procedure for deprotection of 4a to 6g, treatment of 4b
(overnight) and purification by preparative HPLC (lin-
ear gradient 30 to 50% B over 20 min: retention
time=19.5 min) provided 6h as a white solid (40%
1
yield). H NMR (DMSO-d6) d 9.32 (d, 1H, J=7.3 Hz),
8,48 (s, 1H), 8.16–7.92 (m, 3H), 7.86–7.75 (m, 1H),
7.64–7.38 (m, 6H), 7.32 (d, 2H, J=7.7 Hz), 7.16 (d, 2H,
J=8.1 Hz), 7.04–6.95 (m, 1H), 5.05–4.90 (m, 1H), 4.50–
4.40 (m, 2H), 3.54 (s, 2H), 3.35–2.96 (m, 6H), 2.85–2.68
(m, 1H), 2.15–1.15 (m, 12H). FABMS (ꢀVE, Gly) m/z
724.5 (MꢀH).
1
white solid (6.8 mg, 21% yield). H NMR (D2O) d 8.43
(brs, 1H), 8.28–8.18 (m, 1H), 8.05–7.90 (m, 3H), 7.85–
7.75 (m, 2H), 7.67–7.40 (m, 4H), 7.35–7.20 (m, 6H),
4.76–4.65 (m, 1H), 4.60–4.50 (m, 1H), 3.45–2.8 (m,
10H), 2.1–1.0 (m, 12H). FABMS (ꢀVE, Gly) m/z 760
(MꢀH).
Preparation of compound 6i. As described in the general
procedure for deprotection of 4a to 6g, treatment of 4c
(1.5 h) and purification by preparative HPLC (linear
gradient 20 to 80% B over 20 min: retention time=14.5
min) provided 6i as a white solid (32% yield). 1H NMR
(DMSO-d6) d 8.20–8.10 (m, 1H), 8.05–7.95 (m, 3H),
7.60–7.45 (m, 8H), 7.32–7.16 (m, 4H), 7.04 (brs, 1H),
6.60 (brs, 1H), 4.80–4.70 (m, 1H), 4.65–4.55 (m, 1H),
4.48–4.38 (m, 1H), 3.94 (d, 1H, J=5.6 Hz), 3.55 (s, 2H),
3.35–2.96 (m, 6H), 2.15–1.15 (m, 12H). FABMS (ꢀVE,
Gly) m/z 738 (MꢀH).
Preparation of compound 5i. As described in the general
procedure for deprotection of 3a to 5g, treatment of 3c
(overnight) and purification by preparative HPLC (lin-
ear gradient 5 to 60% B over 25 min, then 60 to 100% B
over 5 min: retention time=23.8–27.2 min) provided 5i
1
as a white solid (43% yield). H NMR (DMSO-d6) d
8.68 (1, J=7.3), 8.21 (s, 1H), 8.12–8.06 (m, 1H), 7.95–
7.88 (m, 2H), 7.78–7.72 (m, 2H), 7.56–7.48 (m, 3H),
7.43–7.37 (m, 3H), 7.20–7.08 (m, 4H), 6.94 (brs, 1H),
4.74–4.63 (m, 1H), 4.45–4.35 (m, 1H), 3.94 (d, 1H,
J=16 Hz), 3.82 (d, 1H, J=16 Hz), 3.25–2.58 (m, 5H),
2.92 (d, 2H, J=21 Hz), 2.06–1.10 (m, 12 H). FABMS
(ꢀVE, Gly) m/z 774 (MꢀH).
Acknowledgements
The authors express their appreciation to Dr. James
Kelley and Ms. Lynne Anderson of the LMCH for mass
spectral analysis.
Preparation of compound 5j. As described in the general
procedure for deprotection of 3a to 5g, treatment of 3d
(1 h) and purification by preparative HPLC (linear gra-
dient from 5 to 50% B over 10 min: retention
time=16.8 min) provided 5j as a white solid (quantita-
tive). 1H NMR (DMSO-d6) d 8.29 (1H, s), 8.08 (1H, m),
7.96 (1H, d, J=8.05 Hz), 7.89 (1H, m), 7.74 (1H, t,
J=4.40 Hz), 7.61 (1H, d, J=8.05 Hz), 7.55–7.35 (7H,
m), 7.30–7.09 (6H, m), 7.05–6.88 (4H, m), 4.95 (1H, d,
J=12.94 Hz), 4.88 (1H, d, J=12.94 Hz), 4.40 (2H, m),
3.25–2.98 (5H, m), 2.92 (2H, d, J=21.24 Hz), 2.70–2.50
93H, m), 2.10–1.10 (12H, m). FABMS (ꢀVE, Gly) m/z
813.7.
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