Design, synthesis, and evaluation of potent and selective benzoyleneurea-based inhibitors of protein geranylgeranyltransferase-I

Article abstract of DOI:10.1016/j.bmc.2004.10.053

A series of novel protein geranylgeranyltransferase-I (PGGTase-I) inhibitors based on a benzoyleneurea scaffold has been synthesized. Using a benzoyleneurea scaffold as a mimetic for the central dipeptide (AA), we have developed CAAX peptidomimetic inhibi

Full text of DOI:10.1016/j.bmc.2004.10.053

Bioorganic & Medicinal Chemistry 13 (2005) 677–688
Design, synthesis, and evaluation of potent and selective
benzoyleneurea-based inhibitors of protein
geranylgeranyltransferase-I
Dora Carrico,a Michelle A. Blaskovich,b Cynthia J. Bucher,b
Sa¨ıd M. Sebtib and Andrew D. Hamiltona,*
aDepartment of Chemistry, Yale University, PO Box 208107, New Haven, CT 06520, USA
bDrug Discovery Program, H. Lee Moffitt Cancer Center and Research Institute, Department of Oncology and Department
of Biochemistry and Molecular Biology, University of South Florida, Tampa, FL 33612, USA
Received 21 September 2004; revised 18 October 2004; accepted 25 October 2004
Available online 10 December 2004
Abstract—A series of novel protein geranylgeranyltransferase-I (PGGTase-I) inhibitors based on a benzoyleneurea scaffold has been
synthesized. Using a benzoyleneurea scaffold as a mimetic for the central dipeptide (AA), we have developed CAAX peptidomimetic
inhibitors that selectively block the activity of PGGTase-I over the closely related enzyme protein farnesyltransferase. In this new
class of PGGTase-I inhibitors, compound (6c) with X = L-phenylalanine displayed the highest inhibition activity against PGGTase-
I with an IC50 value of 170nM. The inhibitors described in this study represent novel and promising leads for the development of
potent and selective inhibitors of mammalian PGGTase-I for potential application as antitumor agents.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
PFTase) or a geranylgeranyl group (20 carbon by
PGGTase-I and-II) to cysteine residues near the C-
terminus of certain proteins.1 Protein prenyltransferase
activity plays an important role in mediating protein–
membrane and protein–protein interactions that affect
a variety of cellular processes associated with, for exam-
ple, cell cycle progression, cytoskeleton remodeling,
apoptosis, signal transduction, and intracellular traffick-
ing pathways.2–6
Inhibition of protein prenyltransferases has received
considerable interest in recent years as a promising strat-
egy for the development of chemotherapeutic agents for
a variety of diseases. The prenyltransferase family of
lipid-modifying enzymes consists of protein farnesyl-
transferase (PFTase), geranylgeranyltransferase type I
(PGGTase-I), and geranylgeranyltransferase type II
(PGGTase-II). To date, most work has focused on
developing PFTase inhibitors as anticancer agents be-
cause malignant Ras activity was found to be dependent
on post-translational modification by PFTase. However,
recent findings linking protein prenylation of PGGTase-
I substrates to tumorigenesis and metastasis have
highlighted PGGTase-I as a new potential target in
anticancer drug research.
The protein prenyltransferases are usually classified into
two categories: (1) the CAAX prenyltransferases, which
include PFTase and PGGTase-I and (2) PGGTase-II.
The CAAX prenyltransferases modify eukaryotic pro-
tein substrates that contain a C-terminal CAAX
sequence. In the CAAX motif, C represents a cysteine
amino acid residue, A represents any aliphatic amino
acid and X is the determining residue that specifies
PFTase or PGGTase-I activity. PFTase modifies sub-
strates that contain a CAAX motif where X is pre-
dominantly methionine and serine, but other amino
acid residues such as alanine or glutamine are common
as well.1,7 Protein substrates terminating with CAAX
sequences where X is leucine, isoleucine or phenylalanine
are usually modified by PGGTase-I.1,7 The non-
CAAX prenyltransferase PGGTase-II catalyzes the
The protein prenyltransferase enzymes catalyze the
covalent transfer of a farnesyl group (15 carbon by
Keywords: Geranylgeranyltransferase-I; Peptidomimetics; Farnesyl-
transferase; Antitumor agents.
*
Corresponding author. Tel.: +1 203 432 5570; fax: +1 203 432
3221; e-mail: andrew.hamilton@yale.edu
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.10.053

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D. Carrico et al. / Bioorg. Med. Chem. 13 (2005) 677–688
modification of protein substrates terminating in a CC
or a CXC motif.
opens into the extensive subunit interface and extends
into the hydrophobic funnel-shaped cavity of the b-sub-
unit. This funnel-shaped cavity hosts a single zinc ion,
which marks the location of the active site of PGG-
Tase-I. Since an X-ray crystal structure of PGGTase-I
was not available until very recently, the work described
herein was based on a rational approach in which design
improvements were guided by: (1) comparison between
the active sites of PFTase, (from published PFTase
structural information), and PGGTase-I (from sequence
alignment)26 and (2) extensive structure activity relation-
ship studies on previous PGGTase-I inhibitors.
Nearly 30% of all human cancers contain oncogenic
forms of Ras, with higher prevalence in pancreatic
(90%), colorectal (50%), and lung (40%). Since Ras
malignant activity was found to be dependent on post-
translational modification by PFTase, significant efforts
have been directed toward the development of PFTase
inhibitors for application as antitumor agents.8–10 How-
ever, recent studies have indicated that PGGTase-I is a
new potential target for the development of anticancer
therapeutics. These studies have shown that several
PGGTase-I substrates such as RhoC, RhoA, Rac-1,
Cdc42, and R-Ras play a critical role in tumorigenesis
and metastasis.11–15 In addition, the most frequently
mutated form of Ras in human cancers, Ki-Ras4B,
was found to be geranylgeranylated when PFTase is
inhibited.16,17 Thus, inhibition of both PFTase and
PGGTase-I is required to inhibit prenylation of Ki-
Ras4B in vivo.18,19 Finally, the development of highly
potent and selective PGGTase-I inhibitors (GGTIs) is
needed to determine the effect of PGGTase-I in normal
and oncogenic cell growth. Based on these recent find-
ings, we have directed our efforts toward the develop-
ment of novel selective inhibitors of PGGTase-I for
further development of useful chemotherapeutic agents
with in vivo antitumor activity.
Our design for peptidomimetic inhibitors of PGGTase-I
was based on the C-terminal CAAX motif (X = L, F)
sequence found in many geranylgeranylated proteins.
The first generation of PGGTase-I inhibitors were ob-
tained by replacing the central dipeptide (AA) portion
with a rigid spacer, where X was chosen to be leucine.
By employing a 2-aryl-4-aminobenzoic acid scaffold as
a mimetic for the dipeptide (AA), we developed a series
of PGGTase-I inhibitors from which the most potent
compound was found to be GGTI-2154, exhibiting an
IC50 for PGGTase-I of 21nM and of 5,600nM for
PFTase (Fig. 1). In an effort to improve the potency,
selectivity and in vivo antitumor activity of our
PGGTase-I inhibitors, we sought new inhibitors based
on novel scaffolds that could be readily constructed
and modified to provide useful structure activity profiles.
Similar to PFTase, PGGTase-I is a heterodimeric zinc
metalloenzyme, consisting of a 48kDa a-subunit and a
43kDa b-subunit.20,21 Both PFTase and PGGTase-I
share an identical a-subunit and the b-subunit is known
to contain approximately 35% of amino acid sequence
identity.22–24 The recently published X-ray crystal struc-
ture of protein PGGTase-I shows that the overall
structural arrangement is mainly helical in nature.25
The a-subunit of PGGTase-I is folded in a crescent-
shaped domain composed of a-helices that are arranged
in a-helical hairpins. The b-subunit also contains mostly
helical domains, which form a compact a–a barrel struc-
ture with a central cavity. The substrate binding site
A variety of potentially suitable scaffolds were consid-
ered in terms of: (a) synthetic versatility and (b) struc-
tural features common to our existing two most potent
PGGTase-I inhibitors based on a 2-aryl-4-aminobenzoic
acid (GGTI-2154)27 and piperazine (GGTI-2418) scaf-
folds.28 Simple energy-minimizing molecular modeling
studies revealed that a benzoyleneurea unit as in com-
pound 6g, would be an attractive scaffold for the devel-
opment of a new series of GGTIs (Fig. 2). This scaffold
shared structural features common to both GGTI-2154
and GGTI-2418, and suggested that inhibitors designed
around it might bind effectively to the active site of
Figure 1. Evolution of CAAX peptidomimetic PGGTase-I inhibitor design.

D. Carrico et al. / Bioorg. Med. Chem. 13 (2005) 677–688
679
Figure 2. Comparison of proposed novel PGGTase-I inhibitor 6g (atom color) with (a) GGTI-2418 (orange) and (b) GGTI-2154 (purple). Inhibitors
were built and minimized in the Insight II (2000) suite of programs builder module.
PGGTase-I. In addition, this scaffold has the advantage
of being commercially available and ease of substitution
would allow potential access to a wide range of novel
GGTI leads.
azane and ammonium sulfate gave the monosilylated
product 1, which was then alkylated with benzyl chloro-
acetate followed by desilylation with ethanol to afford
intermediate 2. The regiochemistry of alkylation in 2
was initially established as the N1-position by 2DNMR
where an NOE signal was observed between protons Ha
and Hb (Scheme 1) and later confirmed by X-ray crystallo-
graphy (Fig. 3).
2. Results and discussion
2.1. Chemistry
Treatment of compound 2 with 1-t-butoxycarbonyl-4-
chloromethylimidazole and potassium carbonate gave
the dialkylated compound 3. Benzyl group removal
was accomplished by hydrogenation with 10%
The general synthetic route utilized to prepare these
compounds is outlined in Scheme 1. Silylation of com-
mercially available benzoyleneurea with hexamethyldisil-
Scheme 1. Reagents and conditions: (a) (CH3)3SiNHSi(CH3)3, (NH4)2SO4, toluene; (b) benzyl chloroacetate, KI, toluene; (c) EtOH, 90% for three
steps; (d) 1-t-butoxycarbonyl-4-chloromethylimidazole, K2CO3, acetone, 89%; (e) H2, Pd/C, MeOH, CH2Cl2, 91%; (f) H2NR, HOBT, EDCI, Et3N,
DMF, 25–100%; (g) TFA, 82–100% or NaOH, 81–100%.

680
D. Carrico et al. / Bioorg. Med. Chem. 13 (2005) 677–688
amino acid methyl esters or amines using EDCI/HOBT
coupling conditions to give the respective amides 5.
Standard Boc deprotection and, if necessary, saponifica-
tion conditions using TFA and NaOH, respectively,
were then employed to give the final deprotected com-
pounds 6 (Fig. 4).
2.2. Biological activity
The enzyme inhibition activity displayed by the inhibi-
tors shown in Figure 4 is reported in Table 1 as IC50 val-
ues (the concentration at which PFTase or PGGTase-I
activity was inhibited by 50%). The ability of the benzo-
yleneurea GGTI compounds to inhibit PFTase and
PGGTase-I in vitro was investigated by using partially
purified PFTase and PGGTase-I from human Burkitt
Lymphoma (Daudi) cells as described earlier.29–31 Inhi-
bition activity was determined by the ability of the com-
pounds to inhibit the transfer of [3H]farnesyl and
[3H]geranylgeranyl from [3H]farnesyl PPi and [3H]geran-
ylgeranyl PPi to H-Ras-CVLS and H-Ras-CVLL,
respectively.
Figure 3. X-ray crystal structure of compound 2.
palladium on activated charcoal. The resulting carboxy-
lic acid derivative 4 was then coupled with a variety of
A comparison between the active sites of PFTase and
PGGTase-I suggested that the central dipeptide played
Figure 4. Benzoyleneurea-based PGGTase-I inhibitors.

D. Carrico et al. / Bioorg. Med. Chem. 13 (2005) 677–688
Table 1. PFTase and PGGTase-I activity for compounds 6a–p
681
Though these compounds were designed before a crystal
structure of PGGTase-I was available, the obtained
structure-activity relationships can be rationalized by
docking studies based on the recently published X-ray
crystal structure of mammalian protein PGGTase-I
(PDB code: 1N4Q).25 Docking studies were performed
with the genetic algorithm included in Gold v. 2.1 with
10 independent runs per ligand.34 Crystallographic
water molecules were removed and the GGPP analog
used in the crystal structure was retained for the mole-
cular modeling studies. The parent compound of this
series, 6g, was docked into mammalian PGGTase-I,
and the single low energy enzyme-bound conformation
is shown in Figure 5.
Inhibitor
PGGTase-I
IC50 (nM)
PFTase
IC50 (nM)
PFTase/
PGGTase-I
6a
6b
6c
6d
6e
6f
6300
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>1.5
>10,000
170
4500
>58
>2
2700
>10,000
580
>3.5
6g
6h
6i
>17
>10,000
6425
>1.5
>2.5
6j
>10,000
3350
6k
6l
>10,000
>10,000
>10,000
>10,000
>10,000
6m
6n
6o
6p
Docking studies show that compound 6g binds to
PGGTase-I in the CVIL binding site, suggesting that
its activity may arise from a peptide-competitive mecha-
nism. As with CVIL, compound 6g adopts an extended
conformation, with the imidazole nitrogen coordinating
to the catalytic zinc ion. The benzoyleneurea scaffold
makes extensive hydrophobic contacts with the hydro-
phobic pocket formed by the geranylgeranylpyrophos-
phate analog. The C-terminus carboxylate group of 6g
interacts strongly with Arg 173b and the leucine side
chain fits tightly to the hydrophobic cleft formed by
the GGPP analog, Phe 53b and Leu 320b.
a simple hydrophobic role in binding to PGGTase-I,
and could thus be substituted by other scaffolds that
would allow for better occupation of the PGGTase-I ac-
tive site and consequently lead to an improvement in
binding affinity. Previous studies have indicated that
the cysteine sulfur of CAAL substrates coordinates to
the zinc ion of PGGTase-I and constitutes an important
step in PGGTase-I catalytic activity.32 Replacement of
the cysteine thiol by an imidazole metal-coordinating
group has been previously explored and shown to in-
crease the metabolic stability and also the selectivity
for PGGTase-I.33 Based on the CAAL tetrapeptide
motif, we replaced the central dipeptide AA portion
by a benzoyleneurea scaffold and substituted the
cysteine residue with an imidazole and the X residue
with several groups.
In this series, compounds 3 (Scheme 1) and 5 (Fig. 4),
containing a Boc-substituted imidazole, showed no
activity against PGGTase-I. Interestingly, previous
studies with our PFTase inhibitors, have shown that
substitution on the imidazole ring leads to an increase
in activity and selectivity against PFTase. The lack of
activity exhibited by 3 and 5 was presumed to be due
to an unfavorable conformational change or steric inter-
action between the inhibitor and the enzyme imposed by
Figure 5. Comparison of the GOLD-predicted docked conformation of 6g (atom color) in the active site of PGGTase-I (pdb 1N4Q, colored by
hydrophobicity, red hydrophobic to blue hydrophilic) with the enzyme bound conformation of the native substrate peptide CVIL (cyan blue).

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D. Carrico et al. / Bioorg. Med. Chem. 13 (2005) 677–688
the presence of the t-butyl group on the imidazole.
This possible explanation was further supported by
X-ray crystal structures, which show that the zinc ion
binding pocket in PGGTase-I is more congested than
in PFTase, suggesting a strict size limitation for the pres-
ence of any substitution on either imidazole nitrogen.
Thus, increased size of the imidazole substitution cannot
be tolerated by the tight PGGTase-I zinc binding pock-
et, which likely leads to the observed loss in activity.
Furthermore, the electron-withdrawing Boc-group may
decrease the basicity of the imidazole and weaken its
coordination to zinc in PGGTase-I, which may also con-
tribute to a decrease in binding of the inhibitor to the
enzyme.
6c and 6g showed potent activities against the enzyme
and that most of the methyl ester compounds, with the
exception of 6d, showed no inhibition against PGG-
Tase-I. These results are clearly explained by docking
studies, which show that compounds lacking the carb-
oxylate can no longer form a hydrogen bond with Arg
173b.
2.3. Conclusions
In conclusion, a series of novel benzoyleneurea-based
inhibitors for PGGTase-I were successfully synthe-
sized. These derivatives exploit a benzoyleneurea scaf-
fold as a replacement for the AA portion of the
CAAX tetrapeptide, and led to a highly potent and
selective inhibitor series for protein PGGTase-I. Only
compounds containing a free carboxyl group exhibi-
ted potent activity against PGGTase-I and phenyl-
alanine seems to be the preferred amino acid residue
to bind in the hydrophobic X specificity pocket of the
enzyme. In addition, the L-configuration of the amino
acid side chain is of great importance for PGGTase-I
inhibition activity, as significant loss of activity was
observed for compounds containing D-configured
residues.
In order to probe the binding environment of the specifi-
city pocket of PGGTase-I, compounds containing a
variety of side chains at the X-position of the CAAX
motif were prepared and evaluated. The glycine-contain-
ing compounds, 6m and 6n showed no activity against
PGGTase-I, which suggested that hydrophobic interac-
tions between the side chain group and the enzyme are
important for activity. This observation was further
confirmed by the high activity exhibited by L-leucine
derivative 6g and L-phenylalanine derivative 6c, with
IC50 values of 580 and 170nM, respectively. These
results suggest that a favorable interaction is gained
through the hydrophobic side-chain of the phenyl-
alanine and leucine residues. The potency of 6g and
6c is also consistent with the known preference of
PGGTase-I for proteins that contain leucine or phenyl-
alanine on their X terminal residue. Docking studies
with 6g show that the hydrophobic leucine side chain
can indeed occupy the hydrophobic specificity pocket
of PGGTase-I. The docking studies with 6g also point
to a strict size limitation for substitution on the C-termi-
nal position and suggest that only small aliphatic groups
will be tolerated in the PGGTase specificity pocket.
3. Experimental
3.1. General methods
Melting points were determined with an electrothermal
capillary melting point apparatus and are uncorrected.
1H and 13C NMR spectra were recorded on a Bruker
AM-500 and 400 spectrometer. Chemical shifts were
reported in d (ppm) relative to tetramethylsilane. All
coupling constants were described in Hz. Flash column
chromatography was performed on silica gel (40–63lm)
under a pressure of about 4psi. Solvents were obtained
from commercial suppliers and purified as follows: tetra-
hydrofuran and ether were distilled from sodium benzo-
phenone ketyl; dichloromethane was distilled over
calcium hydride. Synthesized final compounds were
checked for purity by analytical HPLC, which was
performed using a Rainin HP controller and a Rainin
UV-C detector with a Rainin 250mm · 4.6mm, 5lm
Microsorb C-18 column, eluted with gradient 10–90%
of CH3CN in 0.1% TFA in H2O in 30min. High-
resolution mass spectra (HRMS) and low-resolution
mass spectra (LRMS) were performed on a Varian
MAT-CH-5 (HRMS) or VG 707 (LRMS) mass
spectrometer.
In order to determine if the L-configuration is required
to observe PGGTase-I inhibition in this series, we pre-
pared inhibitors containing both D-phenylalanine (6a,
6b), and D-leucine (6e, 6f) residues as well as an achiral
cyclohexyl side chain (6i, 6j). Compounds 6b and 6f
showed no inhibition, and 6a and 6e showed negligible
activity against PGGTase-I. Cyclohexyl achiral peptido-
mimetics 6i and 6j also showed no or poor activity
against the enzyme. The importance of the L-configura-
tion of the side chain for PGGTase-I inhibition in this
series is most probably due to the strict size limitation
of the specificity pocket in the PGGTase-I active site
as shown in Figure 5. A change in configuration of the
side chain is not well tolerated by the tight specificity
pocket of PGGTase-I, forcing the inhibitor to bind in
an alternative unfavorable orientation leading to the
observed loss in activity.
3.1.1. 3-Trimethylsilanyl-1H-quinazoline-2,4-dione (1). A
suspension
of
1H-quinazoline-2,4-dione
(1.62g,
10mmol), 1,1,1,3,3,3-hexamethyldisilazane (5.05mL,
24mmol), ammonium sulfate (0.20g, 1.5mmol) in anhy-
drous toluene was refluxed under N2 overnight. The
reaction mixture was allowed to reach room tempera-
ture and the solvent was removed under reduced pres-
sure to afford a light yellow residue that was used for
the next reaction without further purifications.
Finally, peptidomimetics 6o and 6p lacking a free carb-
oxyl group did not show any activity against PGGTase-
I, which suggested the presence of a critical interaction
between the carboxyl group and a positively charged
residue in the PGGTase-I active site. This was further
supported by the fact that only the free acid compounds

D. Carrico et al. / Bioorg. Med. Chem. 13 (2005) 677–688
683
3.1.2. (2,4-Dioxo-3,4-dihydro-2H-quinazolin-1-yl)-acetic
acid benzyl ester (2). A mixture of 1, potassium iodide
(1.66g, 10mmol), and benzyl chloroacetate (6mL,
39.5mmol) was stirred at 85ꢁC under N2 overnight.
To the reaction mixture, ethanol (20mL) was added
and the reaction was refluxed for 2h. The reaction mix-
ture was allowed to reach room temperature and the sol-
vent was removed under reduced pressure. The resulting
residue was rinsed first with acetone (2 · 25mL), H2O
(2 · 20mL), and with additional acetone (2 · 20mL).
The solid was dried in a vacuum oven overnight to af-
ford the product as a white powder (2.81g, 90%). Mp
114.63, 85.75, 45.15, 38.95, 27.72; LRMS (FAB) m/z
calcd for C19H20N4O6H+ 401, found 401; HRMS
(FAB) m/z calcd for C19H20N4O6H+ 401.1461, found
401.1460.
3.1.5. 4-{1-[(1-Methoxycarbonyl-3-methyl-butylcarba-
moyl)-methyl]-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl-
methyl}-imidazole-1-carboxylic acid tert-butyl ester (5h).
A stirred solution containing acid 4 (68mg, 0.17mmol),
L-leucine methyl ester hydrochloride (34mg, 0.19mmol),
HOBt (25mg, 0.19mmol), Et3N (26lL, 0.19mmol) in
anhydrous DMF (4mL) was cooled to ꢀ10ꢁC. To the
cooled reaction mixture was added EDCI (33mg,
0.17mmol) and the reaction was stirred at room temper-
ature overnight. The solvent was removed under re-
duced pressure and the white residue was diluted with
EtOAc (60mL) and washed with saturated K2CO3
(10mL). The aqueous phase was washed with EtOAc
(10mL) and the organic phases were combined. The or-
ganic phase was extracted with saturated K2CO3
(10mL) and saturated NaCl (2 · 10mL). Combined or-
ganic phases were dried over Na2SO4, filtered, and con-
1
236–238ꢁC; H NMR (DMSO-d6) d 4.99 (s, 2H), 5.19
(s, 2H), 7.29–7.38 (m, 7H), 7.71 (t, J = 7.0Hz, 1H),
8.02 (dd, J = 1.5 and 7.8Hz, 1H), 11.78 (s, 1H); 13C
NMR (DMSO-d6) d 168.56, 162.04, 150.67, 141.22,
135.92, 135.75, 128.80, 128.55, 128.27, 127.99, 123.34,
115.77, 114.97, 66.89, 44.12; LRMS (FAB) m/z
calcd for C17H14N2O4H+ 311, found 311; HRMS
(FAB) m/z calcd for C17H14N2O4H+ 311.1032, found
311.1033.
3.1.3. 4-(1-Benzyloxycarbonylmethyl-2,4-dioxo-1,4-dihy-
dro-2H-quinazolin-3-ylmethyl)-imidazole-1-carboxylic acid
tert-butyl ester (3). A solution of 2 (116mg, 0.37mmol),
4-chloromethyl-imidazole-1-carboxylic acid tert-butyl
ester35 (105mg, 0.48mmol) and K2CO3 (62mg,
0.45mmol) in dry acetone (2mL) was heated at 60ꢁC
under N2 overnight. The reaction mixture was cooled
to room temperature and concentrated under reduced
pressure. The resulting white residue was diluted with
EtOAc (50mL) and washed with H2O (2 · 25mL).
Combined organic phases were dried over Na2SO4, fil-
tered, and concentrated under reduced pressure. Flash
chromatography (2:1:0.01 CHCl3/ethyl acetate/NH4OH
eluent) furnished dialkylated product 3 (161mg, 89%)
as a clear, colorless oil. HPLC Rt 19.21; 1H NMR
(CDCl3) d 8.26 (dd, J = 1.5, 7.9Hz, 1H), 7.97 (d,
J = 1.2Hz, 1H), 7.58 (ddd, J = 1.6, 7.4, 8.7Hz, 1H),
7.24–7.36 (m, 7H), 6.89 (d, J = 8.4Hz, 1H), 5.25 (s,
2H), 5.21 (s, 2H), 4.97 (s, 2H), 1.58 (s, 9H); 13C NMR
(CDCl3) d 168.05, 161.67, 151.25, 147.28, 140.01,
139.14, 137.20, 135.63, 135.32, 129.79, 129.01,
128.95, 128.69, 123.73, 116.01, 115.74, 113.42,
85.91, 67.93, 45.30, 39.36, 28.24; LRMS (FAB) m/z
calcd for C26H26N4O6H+ 491, found 491; HRMS
(FAB) m/z calcd for C26H26N4O6H+ 491.1931, found
491.1932.
centrated
under
reduced
pressure.
Flash
chromatography (2:1:0.01 CHCl3/ethyl acetate/Et3N
eluent) furnished L-leucine derivative product 5h
(38mg, 42%) as a clear, colorless oil. 1H NMR
(CD3OD) d 8.06 (dd, J = 1.3, 7.9Hz, 1H), 7.98 (d,
J = 1.0Hz, 1H), 7.63 (t, J = 7.1Hz, 1H), 7.34 (s, 1H),
7.21 (t, J = 7.5Hz, 1H), 7.09 (d, J = 8.5Hz, 1H), 5.10
(s, 2H), 4.88 (d, J = 17.3Hz, 1H), 4.77 (d, J = 17.1Hz,
1H), 4.38–4.42 (m, 1H), 3.58 (s, 3H), 1.48–1.57 (m,
12H), 0.79–0.86 (m, 6H); 13C NMR (CD3OD) d
174.70, 170.33, 163.54, 152.88, 148.46, 141.98, 140.23,
138.49, 136.93, 129.98, 124.83, 117.24, 116.99, 115.73,
87.65, 53.14, 52.67, 47.53, 41.54, 40.12, 28.37, 26.35,
23.75, 22.05; LRMS (ESI) m/z calcd for C26H33N5O7H+
528, found 528; HRMS (ESI) m/z calcd for
C26H33N5O7H+ 528.2458, found 528.2463.
3.1.6. 2-{2-[3-(1H-Imidazol-4-ylmethyl)-2,4-dioxo-3,4-di-
hydro-2H-quinazolin-1-yl]-acetylamino}-4-methyl-penta-
noic acid (6g). Compound 5h (8.2mg, 0.015mmol) was
dissolved in MeOH (1.0mL) and 1N NaOH (0.1mL)
was added. The reaction mixture was stirred at room
temperature for 1h and 1N HCl (0.15mL) was then
added to the solution. The solvent was removed under
reduced pressure and the resulting white residue was
further purified by gel permeation on Sephadex LH-20
with MeOH to afford to the final compound (6.5mg,
96%) as a white foam. HPLC Rt 10.79; 1H NMR
(CD3OD) d 8.72 (s, 1H), 8.10 (dd, J = 1.2, 7.8Hz, 1H),
7.64 (t, J = 7.2Hz, 1H), 7.45 (s, 1H), 7.24
(t, J = 7.5Hz, 1H), 7.15 (d, J = 8.2Hz, 1H), 5.24 (s,
2H), 4.96 (d, J = 17.2Hz, 1H), 4.81 (d, J = 17.5Hz,
1H), 4.37 (t, J = 7.4Hz, 1H), 1.54–1.67 (m, 3H), 0.86
(dd, J = 6.1, 22.5Hz, 6H); 13C NMR (CD3OD) d
174.76, 170.37, 163.57, 153.05, 141.90, 137.01, 134.93,
132.84, 129.95, 124.88, 117.07, 115.89, 115.74, 52.80,
47.46, 41.52, 26.38, 23.73, 22.12; LRMS (FAB) m/z
calcd for C20H23N5O5H+ 414, found 414; HRMS
(FAB) m/z calcd for C20H23N5O5H+ 414.1777, found
414.1778.
3.1.4. 4-(1-Carboxymethyl-2,4-dioxo-1,4-dihydro-2H-quin-
azolin-3-ylmethyl)-imidazole-1-carboxylic acid tert-butyl
ester (4). A flask containing 3 (2.07g, 4.21mmol) in
MeOH (15mL) and CH2Cl2 (10mL) was evacuated 3
times and filled with N2. To the flask 10% Pd–C
(0.18g) was added and the reaction mixture was hydro-
genated at atmospheric pressure for 2.5h. The catalyst
was filtered and the filtrate concentrated under reduced
pressure to afford the carboxylic acid 4 (1.54g, 91%) as a
1
clear, colorless oil. H NMR (DMSO-d6) d 8.08–8.11
(m, 2H), 7.58 (t, J = 7.0Hz, 1H), 7.30–7.43 (m, 3H),
5.06 (s, 2H), 4.89 (s, 2H), 1.55 (s, 9H); 13C NMR
(DMSO-d6) d 169.78, 161.07, 150.61, 146.91, 140.23,
139.14, 137.02, 135.88, 128.41, 123.48, 114.99, 114.92,

684
D. Carrico et al. / Bioorg. Med. Chem. 13 (2005) 677–688
3.1.7. 2-{2-[3-(1H-Imidazol-4-ylmethyl)-2,4-dioxo-3,4-di-
hydro-2H-quinazolin-1-yl] acetylamino}-4-methyl-penta-
noic acid methyl ester (6h). A solution of 5h (18mg,
0.034mmol) in CH2Cl2 (1mL) was cooled to 0ꢁC and
TFA was added (0.17mL). The reaction mixture was
allowed to reach room temperature and was stirred for
an additional 35min. The mixture was concentrated
and the residue was rinsed with Et2O (2 · 5mL) to give
6h as a light yellow beige foamy residue (19mg, 100%).
HPLC Rt 11.75; 1H NMR (CD3OD) d 8.70 (s, 1H),
8.09 (dd, J = 1.4, 7.9Hz, 1H), 7.65 (t, J = 7.1Hz, 1H),
7.47 (s, 1H), 7.25 (t, J = 7.3Hz, 1H), 7.12 (d,
J = 8.4Hz, 1H), 5.24 (s, 2H), 4.94 (d, J = 17.3Hz, 1H),
4.80 (d, J = 17.0Hz, 1H), 4.39 (t, J = 6.7Hz, 1H), 3.60
(s, 3H), 1.52–1.60 (m, 3H), 0.84 (dd, J = 6.2, 21.3Hz,
6H); 13C NMR (CD3OD) d 173.08, 168.68, 161.81,
151.27, 140.21, 135.20, 134.93, 132.84, 128.29, 123.13,
118.80, 115.53, 114.01, 51.57, 51.11, 45.86, 39.95,
37.55, 24.74, 22.13, 20.52; LRMS (FAB) m/z calcd for
C21H25N5O5H+ 428, found 428; HRMS (FAB) m/z
calcd for C21H25N5O5H+ 428.1934, found 428.1932.
with 5b to afford the final compound as a clear colorless
oil (96%). HPLC Rt 11.10; 1H NMR (CD3OD) d 8.69 (d,
J = 0.9Hz, 1H), 8.04 (dd, J = 1.3, 7.9Hz, 1H), 7.54 (t,
J = 7.2Hz, 1H), 7.44 (s, 1H), 7.05–7.24 (m, 6H), 6.79
(d, J = 8.4Hz, 1H), 5.20 (s, 2H), 4.71–4.90 (m, 2H),
4.62–4.66 (m, 1H), 3.61 (s, 3H), 3.11 (dd, J = 5.11,
14.0Hz, 1H), 2.87 (dd, J = 9.6, 13.9Hz, 1H); 13C
NMR (CD3OD) d 173.50, 169.88, 163.33, 152.76,
141.74, 138.51, 137.34, 135.41, 131.01, 130.68, 130.03,
129.96, 128.42, 125.00, 120.05, 116.88, 115.68, 55.69,
53.31, 47.14, 38.69, 36.46; LRMS (ESI) m/z calcd for
C24H23N5O5H+ 462, found 462; HRMS (ESI) m/z calcd
for C24H23N5O5H+ 462.1777, found 462.1776.
3.1.11. {1-[(1-Methoxycarbonyl-2-phenyl-ethylcarbamo-
yl)-methyl]-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl}
methyl-imidazole-1-carboxylic acid tert-butyl ester (5d).
This compound was prepared in a similar manner to
that described for compound 5h with 4 and L-phenylala-
nine methyl ester hydrochloride to afford a white oily
25
residue (100%). HPLC Rt 17.63; ½aꢁ þ 0:7 (c 0.01,
D
MeOH); 1H NMR (CD3OD) d 8.19 (dd, J = 1.5,
7.8Hz, 1H), 8.11 (d, J = 1.3Hz, 1H), 7.68 (t,
J = 7.3Hz, 1H), 7.47 (d, J = 1.2Hz, 1H), 7.18–7.36 (m,
6H), 6.97 (d, J = 8.6Hz, 1H), 5.22 (s, 2H), 4.93 (d,
J = 17.2Hz, 1H), 4.85 (d, J = 17.0Hz, 1H), 4.74–4.80
(m, 1H), 3.73 (s, 3H), 3.23 (dd, J = 5.4, 14.1Hz, 1H),
3.00 (dd, J = 9.3, 14.0Hz, 1H), 1.63 (s, 9H); 13C NMR
(CD3OD) d 173.51, 169.86, 163.41, 152.77, 148.44,
141.78, 140.22, 138.54, 138.50, 137.02, 130.70, 129.99,
129.92, 128.35, 124.76, 117.15, 117.05, 115.63, 87.65,
55.62, 53.31, 47.41, 40.14, 38.66, 28.43; LRMS (FAB)
m/z calcd for C29H31N5O7H+ 562, found 562; HRMS
(FAB) m/z calcd for C29H31N5O7H+ 562.2302, found
562.2302.
3.1.8. 4-{1-[(1-Methoxycarbonyl-2-phenyl-ethylcarbamo-
yl)-methyl]-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-ylmeth-
yl}-imidazole-1-carboxylic acid tert-butyl ester (5b). This
compound was prepared in a similar manner to that
described for compound 5h with 4 and D-phenylalanine
methyl ester hydrochloride to afford a white oily residue
1
(84%). HPLC Rt 16.54; H NMR (CD3OD) d 8.09 (dd,
J = 1.5, 7.9Hz, 1H), 8.01 (d, J = 1.3Hz, 1H), 7.57 (t,
J = 7.4Hz, 1H), 7.43 (d, J = 1.0Hz, 1H), 7.12–7.27 (m,
6H), 6.89 (d, J = 8.5Hz, 1H), 5.15 (s, 2H), 4.86 (d,
J = 17.2Hz, 1H), 4.73–4.77 (m, 2H), 3.68 (s, 3H), 3.19
(dd, J = 5.2, 13.9Hz, 1H), 2.96 (dd, J = 9.3, 13.8Hz,
1H), 1.57 (s, 9H); 13C NMR (CD3OD) d 173.51,
169.76, 163.32, 152.71, 148.45, 141.78, 140.29, 138.54,
138.50, 136.99, 130.74, 130.01, 129.94, 128.37, 124.75,
117.09, 117.04, 115.65, 87.62, 55.61, 53.39, 47.49,
40.25, 38.77, 28.52; LRMS (ESI) m/z calcd for
C29H31N5O7H+ 562, found 562; HRMS (ESI) m/z calcd
for C29H31N5O7H+ 562.2302, found 562.2292.
3.1.12. 2-{2-[3-(1H-Imidazol-4-ylmethyl)-2,4-dioxo-3,4-di-
hydro-2H-quinazolin-1-yl]-acetylamino}-3-phenyl-propi-
onic acid (6c). This compound was prepared in a similar
manner to that described for compound 6g with 5d to
afford the final compound as a clear colorless oil
25
(87%). HPLC Rt 11.80; ½aꢁ þ 14:7 (c 0.01, MeOH);
D
3.1.9. 2-{2-[3-(1H-Imidazol-4-ylmethyl)-2,4-dioxo-3,4-di-
hydro-2H-quinazolin-1-yl]-acetylamino}-3-phenyl-propionic
acid (6a). This compound was prepared in a similar
manner to that described for compound 6g with 5b to
afford the final compound as a clear colorless oil
1H NMR (CD3OD) d 8.64 (br s, 1H), 8.06 (s, 1H),
7.53 (t, J = 7.1Hz, 1H), 7.08–7.26 (m, 7H), 6.81 (d,
J = 7.6Hz, 1H), 5.21 (br s, 2H), 4.59–4.91 (m, 3H),
3.17 (t, J = 10.9Hz, 1H), 2.89 (t, J = 10.9Hz, 1H); 13C
NMR (CD3OD) d 175.92, 169.77, 163.33, 152.78,
141.77, 138.93, 137.34, 130.74, 129.94, 128.27, 124.97,
116.91, 115.77, 60.17, 47.25, 38.78, 36.59; LRMS
(FAB) m/z calcd for C23H21N5O5H+ 448, found 448;
HRMS (FAB) m/z calcd for C23H21N5O5H+ 448.1621,
found 448.1622.
1
(100%). HPLC Rt 11.25; H NMR (CD3OD) d 8.81 (s,
1H), 8.15 (d, J = 7.5Hz, 1H), 7.64 (t, J = 7.9Hz, 1H),
7.17–7.62 (m, 7H), 6.91 (d, J = 8.4Hz, 1H), 5.31 (br s,
2H), 4.70–5.22 (m, 3H), 3.29 (dd, J = 4.5, 13.9Hz,
1H), 3.00 (dd, J = 9.7, 13.8Hz, 1H); 13C NMR
(CD3OD) d 173.05, 168.14, 161.66, 151.10, 140.07,
137.16, 135.69, 133.81, 129.32, 129.06, 128.31, 128.24,
126.64, 123.31, 118.41, 115.19, 114.10, 53.93, 45.55,
37.08, 34.88; LRMS (ESI) m/z calcd for C23H21N5O5H+
448, found 448; HRMS (ESI) m/z calcd for
C23H21N5O5H+ 448.1621, found 448.1621.
3.1.13. 2-{2-[3-(1H-Imidazol-4-ylmethyl)-2,4-dioxo-3,4-di-
hydro-2H-quinazolin-1-yl]-acetylamino}-3-phenyl-propi-
onic acid methyl ester (6d). This compound was prepared
in a similar manner to that described for compound 6h
with 5d to afford the final compound as a clear colorless
25
D
oil (82%). HPLC Rt 13.17; ½aꢁ þ 0:9 (c 0.012,
1
3.1.10. 2-{2-[3-(1H-Imidazol-4-ylmethyl)-2,4-dioxo-3,4-di-
hydro-2H-quinazolin-1-yl]-acetylamino}-3-phenyl-propi-
onic acid methyl ester (6b). This compound was prepared
in a similar manner to that described for compound 6h
MeOH); H NMR (CD3OD) d 8.68 (d, J = 1.3Hz, 1H),
8.05 (dd, J = 1.5, 7.9Hz, 1H), 7.54 (t, J = 7.4Hz, 1H),
7.44 (s, 1H), 7.08–7.21 (m, 6H), 6.80 (d, J = 8.4Hz,
1H), 5.20 (s, 2H), 4.82 (d, J = 17.3Hz, 1H), 4.69 (d,

D. Carrico et al. / Bioorg. Med. Chem. 13 (2005) 677–688
685
J = 17.3Hz, 1H), 4.62–4.67 (m, 1H), 3.60 (s, 3H), 3.12
(dd, J = 5.2, 13.9Hz, 1H), 2.87 (dd, J = 9.6, 13.9Hz,
1H); 13C NMR (CD3OD) d 173.49, 169.85, 163.34,
152.77, 141.75, 138.48, 137.33, 135.38, 131.04, 130.65,
130.02, 129.96, 128.40, 124.99, 120.04, 116.90, 115.67,
55.67, 53.28, 47.14, 38.69, 36.45; LRMS (FAB) m/z calcd
for C24H23N5O5H+ 462, found 462; HRMS (FAB) m/z
calcd for C24H23N5O5H+ 462.1777, found 462.1776.
3.1.17. 4-{1-[(1-Methoxycarbonyl-cyclohexylcarbamoyl)-
methyl]-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-ylmeth-
yl}-imidazole-1-carboxylic acid tert-butyl ester (5j). Prep-
aration of 1-amino-cyclohexanecarboxylic acid methyl
ester: A suspension of 1-amino-cyclohexanecarboxylic
acid (2.0g, 14mmol) in anhydrous MeOH (10mL) was
cooled to ꢀ10ꢁC. To the suspension was added cold
SOCl2 (2.81mL, 35.9mmol) dropwise. The reaction
was allowed to reach room temperature and was ref-
luxed for 4h. The solvent was removed under reduced
pressure and the residue was diluted with AcOEt
(50mL) and extracted with NaHCO3 solution
(3 · 25mL). The combined organic layers were washed
with saturated NaCl solution (25mL) and dried with
Na2SO4. The dried organic layer was filtered and con-
centrated to afford 1-amino-cyclohexanecarboxylic acid
methyl ester as a light yellow oil, that was used in the
synthesis of 5j without further purification (460mg,
21%). 1H NMR (CD3OD) d 3.61 (s, 3H), 1.87 (t,
J = 8.9Hz, 2H), 1.51–1.58 (m, 2H), 1.34–1.36 (m, 6H);
13C NMR (CD3OD) d 178.58, 58.84, 52.99, 36.78,
26.85, 23.55.
3.1.14. 4-{1-[(1-Methoxycarbonyl-3-methyl-butylcarbamo-
yl)-methyl]-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-yl-
methyl}-imidazole-1-carboxylic acid tert-butyl ester (5f).
This compound was prepared in a similar manner to
that described for compound 5h with 4 and D-leucine
methyl ester hydrochloride to afford a white oily residue
1
(84%). HPLC Rt 16.29; H NMR (CD3OD) d 8.01 (dd,
J = 1.5, 7.9Hz, 1H), 7.96 (d, J = 1.3Hz, 1H), 7.58 (t,
J = 7.3Hz, 1H), 7.32 (d, J = 1.2Hz, 1H), 7.17 (t,
J = 7.3Hz, 1H), 7.07 (d, J = 8.4Hz, 1H), 5.07 (s, 2H),
4.86 (d, J = 17.3Hz, 1H), 4.74 (d, J = 17.2Hz, 1H),
4.37–4.41 (m, 1H), 3.57 (s, 3H), 1.45–1.55 (m, 12H),
0.80 (dd, J = 6.2, 14.1Hz, 6H); 13C NMR (CD3OD) d
174.70, 170.26, 163.48, 152.86, 148.48, 141.94, 140.27,
138.51, 136.93, 129.99, 124.83, 117.21, 117.05, 115.74,
87.67, 53.21, 52.72, 47.58, 41.62, 40.18, 28.46, 26.39,
23.81, 22.18; LRMS (ESI) m/z calcd for C26H33N5O7H+
528, found 528; HRMS (ESI) m/z calcd for
C26H33N5O7H+ 528.2458, found 528.2448.
Compound 5j was prepared in a similar manner to that
described for compound 5h with 4 and 1-amino-cyclo-
hexanecarboxylic acid methyl ester to afford the desired
final compound as a white clear oil (100%). HPLC Rt
1
17.14; H NMR (CD3OD) d 8.01 (dd, J = 1.3, 7.9Hz,
1H), 7.97 (s, 1H), 7.59 (t, J = 7.0Hz, 1H), 7.31 (s, 1H),
7.16 (t, J = 7.4Hz, 1H), 7.06 (d, J = 8.4Hz, 1H), 5.06
(s, 2H), 4.81 (s, 2H), 3.47 (s, 3H), 1.90–1.98 (m, 2H),
1.64–1.73 (m, 2H), 1.44–1.48 (m, 15H); 13C NMR
(CD3OD) d 176.44, 169.62, 163.47, 152.86, 148.48,
142.01, 140.33, 138.47, 136.87, 129.96, 124.77, 117.13,
117.06, 115.78, 87.65, 61.05, 53.11, 47.22, 40.19, 33.76,
28.40, 26.75, 22.90; LRMS (FAB) m/z calcd for
C27H33N5O7H+ 540, found 540; HRMS (FAB) m/z
calcd for C27H33N5O7H+ 540.2458, found 540.2460.
3.1.15. 2-{2-[3-(1H-Imidazol-4-ylmethyl)-2,4-dioxo-3,4-di-
hydro-2H-quinazolin-1-yl]-acetylamino}-4-methyl-penta-
noic acid (6e). This compound was prepared in a similar
manner to that described for compound 6g with 5f to af-
ford the final compound as a clear colorless oil (100%).
HPLC Rt 10.46; 1H NMR (CD3OD) d 8.70 (s, 1H), 8.07
(d, J = 7.7Hz, 1H), 7.64 (t, J = 8.0Hz, 1H), 7.46 (s, 1H),
7.23 (t, J = 7.5Hz, 1H), 7.14 (d, J = 8.4Hz, 1H), 5.23 (s,
2H), 4.96 (d, J = 17.3Hz, 1H), 4.81 (d, J = 17.3Hz, 1H),
4.36 (t, J = 6.2Hz, 1H), 1.56–1.68 (m, 3H), 0.84 (dd,
J = 6.1, 22.9Hz, 6H); 13C NMR (CD3OD) d 176.08,
170.23, 163.42, 152.85, 141.89, 137.28, 135.48, 131.11,
129.97, 125.05, 120.02, 116.97, 115.94, 52.73, 47.32,
41.83, 36.78, 26.49, 23.85, 22.08; LRMS (ESI) m/z calcd
for C20 H23N5O5H+ 414, found 414; HRMS (ESI) m/z
calcd for C20H23N5O5H+ 414.1777, found 414.1778.
3.1.18. 1-{2-[3-(1H-Imidazol-4-ylmethyl)-2,4-dioxo-3,4-di-
hydro-2H-quinazolin-1-yl]-acetylamino}-cyclohexanecarb-
oxylic acid (6i). This compound was prepared in a sim-
ilar manner to that described for compound 6g with 5j
to afford the final compound as a clear colorless oil
1
(81%). HPLC Rt 10.87; H NMR (CD3OD) d 8.77 (s,
1H), 8.12 (d, J = 6.9Hz, 1H), 7.70 (t, J = 6.8Hz, 1H),
7.53 (s, 1H), 7.16–7.37 (m, 2H), 5.29 (s, 2H), 4.97 (s,
2H), 1.96–2.21 (m, 2H), 1.72–1.91 (m, 2H), 1.22–1.71
(m, 6H); 13C NMR (CD3OD) d 177.78, 169.70, 163.35,
152.80, 141.84, 137.22, 135.57, 131.04, 129.83, 124.98,
120.01, 116.88, 116.13, 61.04, 47.19, 36.57, 33.62,
26.79, 22.94; LRMS (FAB) m/z calcd for
C21H23N5O5H+ 426, found 426; HRMS (FAB) m/z
calcd for C21H23N5O5H+ 426.1777, found 426.1778.
3.1.16. 2-{2-[3-(1H-Imidazol-4-ylmethyl)-2,4-dioxo-3,4-di-
hydro-2H-quinazolin-1-yl]-acetylamino}-4-methyl-penta-
noic acid methyl ester (6f). This compound was prepared
in a similar manner to that described for compound 6h
with 5f to afford the final compound as a clear colorless
oil (98%). HPLC Rt 11.90; 1H NMR (CD3OD) d 8.63 (s,
1H), 8.07 (dd, J = 1.4, 7.8Hz, 1H), 7.64 (t, J = 7.1Hz,
1H), 7.44 (s, 1H), 7.23 (t, J = 7.5Hz, 1H), 7.11 (d,
J = 8.5Hz, 1H), 5.22 (s, 2H), 4.93 (d, J = 17.3Hz, 1H),
4.79 (d, J = 17.3Hz, 1H), 4.36–4.40 (m, 1H), 3.59 (s,
3H), 1.51–1.62 (m, 3H), 0.83 (dd, J = 6.2, 26.1Hz,
6H); 13C NMR (CD3OD) d 174.68, 170.24, 163.42,
152.85, 141.89, 137.24, 135.47, 131.29, 130.02, 125.06,
120.07, 117.01, 115.80, 53.18, 52.81, 47.26, 41.67,
36.65, 26.43, 23.68, 22.11; LRMS (ESI) m/z calcd for
C21H25N5O5H+ 428, found 428; HRMS (ESI) m/z calcd
for C21H25N5O5H+ 428.1934, found 428.1935.
3.1.19. 1-{2-[3-(1H-Imidazol-4-ylmethyl)-2,4-dioxo-3,4-di-
hydro-2H-quinazolin-1-yl]-acetylamino}-cyclohexanecarb-
oxylic acid methyl ester (6j). This compound was pre-
pared in a similar manner to that described for
compound 6h with 5j to afford the final compound as
1
a clear colorless oil (96%). HPLC Rt 12.34; H NMR
(CD3OD) d 8.69 (d, J = 1.4Hz, 1H), 8.08 (dd, J = 1.5,
7.9Hz, 1H), 7.67 (t, J = 7.3Hz, 1H), 7.46 (d,

686
D. Carrico et al. / Bioorg. Med. Chem. 13 (2005) 677–688
J = 1.2Hz, 1H), 7.23 (t, J = 7.3Hz, 1H), 7.12 (d,
J = 8.4Hz, 1H), 5.23 (s, 2H), 4.88 (s, 2H), 3.47 (s, 3H),
1.89–2.00 (m, 2H), 1.71–1.79 (m, 2H), 1.17–1.61 (m,
6H); 13C NMR (CD3OD) d 176.38, 169.76, 163.39,
152.84, 141.89, 137.22, 135.39, 131.11, 129.99, 125.05,
119.99, 116.97, 115.83, 61.14, 53.08, 46.98, 36.49,
33.74, 26.71, 22.89; LRMS (FAB) m/z calcd for
C22H25N5O5H+ 440, found 440; HRMS (FAB) m/z
calcd for C22H25N5O5H+ 440.1934, found 440.1936.
33.20, 32.09, 17.27; LRMS (FAB) m/z calcd for
C20H23N5O5SH+ 446, found 446; HRMS (FAB)
m/z calcd for C20H23N5O5SH+ 446.1498, found
446.1497.
3.1.23. 4-{1-[(Methoxycarbonylmethyl-carbamoyl)-meth-
yl]-2,4-dioxo-1,4-dihydro-2H-quinazolin-3-ylmethyl}-imi-
dazole-1-carboxylic acid tert-butyl ester (5n). This com-
pound was prepared in a similar manner to that
described for compound 5h with 4 and glycine methyl
ester hydrochloride to afford the desired final compound
3.1.20. 4-{1-[(1-Methoxycarbonyl-3-methylsulfanyl-propyl-
carbamoyl)-methyl]-2,4-dioxo-1,4-dihydro-2H-quinazo-
lin-3-ylmethyl}-imidazole-1-carboxylic acid tert-butyl
ester (5l). This compound was prepared in a similar
manner to that described for compound 5h with 4 and
L-methionine methyl ester hydrochloride to afford the
1
as a white clear oil (100%). HPLC Rt 12.31; H NMR
(CD3OD) d 8.01 (dd, J = 1.5, 7.9Hz, 1H), 7.96 (d,
J = 1.4Hz, 1H), 7.59 (t, J = 7.3Hz, 1H), 7.32 (d,
J = 1.2Hz, 1H), 7.16 (t, J = 7.3Hz, 1H), 7.12 (d, J =
8.5Hz, 1H), 5.05 (s, 2H), 4.80 (s, 2H), 3.85 (s, 2H),
3.57 (s, 3H), 1.47 (s, 9H); 13C NMR (CD3OD) d
169.41, 168.35, 161.08, 150.45, 146.02, 139.47, 137.75,
136.07, 134.56, 130.38, 127.47, 122.42, 114.77, 113.47,
85.21, 50.61, 45.29, 39.83, 37.69, 25.92; LRMS (FAB)
m/z calcd for C22H25N5O7H+ 472, found 472; HRMS
(FAB) m/z calcd for C22H25N5O7H+ 472.1832, found
472.1834.
1
desired final compound as a white clear oil (38%). H
NMR (CD3OD) d 8.04 (dd, J = 1.6, 7.9Hz, 1H), 7.98
(d, J = 1.4Hz, 1H), 7.61 (t, J = 7.0Hz, 1H), 7.33 (d,
J = 1.2Hz, 1H), 7.19 (t, J = 7.3Hz, 1H), 7.11 (d,
J = 8.5Hz, 1H), 5.08 (s, 2H), 4.86 (d, J = 17.3Hz, 1H),
4.77 (d, J = 17.2Hz, 1H), 4.50–4.54 (m, 1H), 3.59 (s,
3H), 2.31–2.48 (m, 2H), 1.97–2.06 (m, 1H), 1.93 (s,
3H), 1.82–1.92 (m, 1H), 1.49 (s, 9H); 13C NMR
(CD3OD) d 173.90, 170.34, 163.50, 152.87, 148.48,
141.97, 140.24, 138.51, 136.97, 129.99, 124.84, 117.23,
116.96, 115.77, 87.67, 54.47, 53.29, 47.70, 40.15,
32.11, 31.53, 28.42, 15.67; LRMS (FAB) m/z calcd
for C25H31N5O7SH+ 546, found 546; HRMS (FAB)
m/z calcd for C25H31N5O7SH+ 546.2022, found
546.2020.
3.1.24. {2-[3-(1H-Imidazol-4-ylmethyl)-2,4-dioxo-3,4-di-
hydro-2H-quinazolin-1-yl]-acetylamino}-acetic acid (6m).
This compound was prepared in a similar manner to
that described for compound 6g with 5n to afford the
final compound as a clear colorless oil (100%). 1H
NMR (CD3OD) d 8.44 (s, 1H), 8.01 (d, J = 6.3Hz,
1H), 7.65 (t, J = 7.6Hz, 1H), 7.37 (s, 1H), 7.18–7.27
(m, 2H), 5.22 (s, 2H), 4.88 (s, 2H), 3.84 (s, 2H); 13C
NMR (CD3OD) d 172.66, 170.61, 163.45, 152.96,
141.93, 137.35, 137.19, 130.28, 129.95, 125.08, 117.02,
116.06, 47.42, 42.29, 31.19; LRMS (FAB) m/z calcd
for C16H15N5O5H+ 358, found 358; HRMS (FAB) m/z
calcd for C16H15N5O5H+ 358.1151, found 358.1151.
3.1.21. 2-{2-[3-(1H-Imidazol-4-ylmethyl)-2,4-dioxo-3,4-di-
hydro-2H-quinazolin-1-yl]-acetylamino}-4-methylsulfan-
yl-butyric acid (6k). This compound was prepared in a
similar manner to that described for compound 6g with
5l to afford the final compound as a clear colorless oil
1
(97%). HPLC Rt 10.49; H NMR (CD3OD) d 8.73 (d,
J = 1.5Hz, 1H), 8.08 (dd, J = 1.5, 7.9Hz, 1H), 7.64 (t,
J = 7.0Hz, 1H), 7.48 (d, J = 1.2Hz, 1H), 7.24 (t,
J = 7.2Hz, 1H), 7.18 (d, J = 8.4Hz, 1H), 5.24 (s, 2H),
4.97 (d, J = 17.3Hz, 1H), 4.83 (d, J = 17.3Hz, 1H),
4.49–4.52 (m, 1H), 2.39–2.54 (m, 2H), 2.03–2.11 (m,
1H), 1.98 (s, 3H), 1.86–1.96 (m, 1H); LRMS (FAB)
m/z calcd for C19H21N5O5SH+ 432, found 432; HRMS
(FAB) m/z calcd for C19H21N5O5SH+ 432.1342, found
432.1344.
3.1.25. {2-[3-(1H-Imidazol-4-ylmethyl)-2,4-dioxo-3,4-di-
hydro-2H-quinazolin-1-yl]-acetylamino}-acetic acid methyl
ester (6n). This compound was prepared in a similar
manner to that described for compound 6h with 5n to
afford the final compound as a clear colorless oil
1
(89%). HPLC Rt 8.00; H NMR (CD3OD) d 8.69 (s,
1H), 8.07 (dd, J = 1.3, 7.8Hz, 1H), 7.65 (t, J = 7.2Hz,
1H), 7.47 (s, 1H), 7.23 (t, J = 7.5Hz, 1H), 7.17 (d,
J = 8.5Hz, 1H), 5.23 (s, 2H), 4.87 (s, 2H), 3.89 (s, 2H),
3.60 (s, 3H); 13C NMR (CD3OD) d 171.89, 170.70,
163.42, 152.85, 141.87, 137.33, 135.42, 131.08, 129.99,
125.09, 120.10, 117.00, 115.99, 53.09, 47.45, 42.31,
36.49; LRMS (FAB) m/z calcd for C17H17N5O5H+
372, found 372; HRMS (FAB) m/z calcd for
C17H17N5O5H+ 372.1308, found 372.1308.
3.1.22. 2-{2-[3-(1H-Imidazol-4-ylmethyl)-2,4-dioxo-3,4-di-
hydro-2H-quinazolin-1-yl]-acetylamino}-4-methylsulfan-
yl-butyric acid methyl ester (6l). This compound was pre-
pared in a similar manner to that described for
compound 6h with 5l to afford the final compound as
1
a clear colorless oil (82%). HPLC Rt 10.72; H NMR
(CD3OD) d 8.70 (d, J = 1.4Hz, 1H), 8.09 (dd, J = 1.5,
7.9Hz, 1H), 7.66 (t, J = 7.0Hz, 1H), 7.47 (d,
J = 1.1Hz, 1H), 7.24 (t, J = 7.2Hz, 1H), 7.15 (d,
J = 8.5Hz, 1H), 5.24 (s, 2H), 4.94 (d, J = 17.3Hz, 1H),
4.81 (d, J = 17.3Hz, 1H), 4.52–4.55 (m, 1H), 3.62 (s,
3H), 2.37–2.57 (m, 2H), 2.02–2.09 (m, 1H), 1.97 (s,
3H), 1.85–1.93 (m, 1H); 13C NMR (CD3OD) d 175.64,
172.09, 165.15, 154.54, 143.63, 138.99, 131.76, 126.80,
122.32, 118.77, 117.57, 55.03, 54.92, 49.07, 33.77,
3.1.26. [3-(1H-Imidazol-4-ylmethyl)-2,4-dioxo-3,4-dihydro-
2H-quinazolin-1-yl]-acetic acid benzyl ester (6o). This
compound was prepared in a similar manner to that de-
scribed for compound 6h with 3 to afford the final com-
pound as a clear colorless oil (90%). HPLC Rt 12.97; 1H
NMR (CDCl3) d 8.70 (d, J = 1.3Hz, 1H), 8.12 (dd,
J = 1.5, 7.9Hz, 1H), 7.63 (t, J = 7.3Hz, 1H), 7.44 (s,
1H), 7.21–7.28 (m, 6H), 7.17 (d, J = 8.5Hz, 1H), 5.23
(s, 2H), 5.13 (s, 2H), 4.98 (s, 2H); 13C NMR (CDCl3)

D. Carrico et al. / Bioorg. Med. Chem. 13 (2005) 677–688
687
d 170.05, 163.25, 152.65, 141.67, 137.42, 137.23, 135.49,
4. Supplementary data
131.08, 130.14, 130.02, 129.93, 129.71, 125.17, 120.18,
116.84, 115.71, 68.94, 46.41, 36.39; LRMS (FAB) m/z
calcd for C21H18N4O4H+ 391, found 391; HRMS
(FAB) m/z calcd for C21H18N4O4H+ 391.1406, found
391.1406.
Crystallographic data (excluding structure factors) for
structure 2 have been deposited with the Cambridge
Crystallographic Data Centre as supplementary publica-
tion no. CCDC 250739. Copies of the data can be ob-
tained, free of charge, on application to CCDC, 12
Union Road, Cambridge, CB2 1EZ, UK, (fax: +44-
(0)1223-336033 or e-mail: deposit@ccdc.cam.ac.uk).
3.1.27. 2-[3-(1H-Imidazol-4-ylmethyl)-2,4-dioxo-3,4-di-
hydro-2H-quinazolin-1-yl]-N-(3-methyl-butyl)-acetamide
(6p). This compound was prepared in a similar manner
to that described for compound 5h with 4 and isoamyl-
amine to afford the desired final compound as a white
Acknowledgements
1
clear oil (25%). HPLC Rt 12.39; H NMR (CD3OD) d
8.05 (dd, J = 1.4, 7.9Hz, 1H), 7.59 (t, J = 7.0Hz, 1H),
7.49 (s, 1H), 7.18 (t, J = 7.7Hz, 1H), 7.05 (d,
J = 8.5Hz, 1H), 6.95 (s, 1H), 5.12 (s, 2H), 4.74 (s, 2H),
3.13 (t, J = 7.1Hz, 2H), 1.44–1.51 (m, 1H), 1.24–1.34
(m, 2H), 0.79 (d, J = 6.7Hz, 6H); 13C NMR (CD3OD)
d 169.85, 163.56, 152.97, 141.98, 136.90, 136.45,
134.36, 129.99, 124.80, 120.16, 117.30, 115.51, 47.82,
39.66, 39.24, 39.03, 27.20, 23.18; LRMS (FAB) m/z
calcd for C19H23N5O3H+ 370, found 370; HRMS
(FAB) m/z calcd for C19H23N5O3H+ 370.1879, found
370.1880.
The authors wish to thank the National Cancer Institute
(CA 67771) for support, Dr. Christopher D. Incarvito
for the X-ray crystal structure of compound 2, and
Dr. Matthew Glenn for assistance with the docking
studies.
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