Asymmetric synthesis of 2,3-dihydro-2-arylquinazolin-4-ones: Methodology and application to a potent fluorescent tubulin inhibitor with anticancer activity

Article abstract of DOI:10.1021/jm800271c

For several decades the 2,3-dihydroquinazolinone (DHQZ) heterocycle has been known to possess a variety of important biological and medicinal properties. Despite the many interesting facets of these molecules, synthetic access to nonracemic DHQZ analogues has remained elusive. Herein, we disclose a synthetic route that allows access to either enantiomer of a variety of DHQZ derivatives. We illustrate the utility of this chemistry with the asymmetric preparation and biological evaluation of a new chiral fluorescent tubulin binding agent with extremely potent antiproliferative properties against human cancer cells. A computational rationale for the increased potency of the (S)-enantiomer over the (R)-enantiomer is given, based on the crystal structure of αβ-tubulin complexed with colchicine. Taking advantage of the inherent fluorescence of these molecules, confocal images of GMC-5-193 (compound 7) in the cytoplasm of human melanoma cells (MDA-MB-435) cells are presented.

Full text of DOI:10.1021/jm800271c

4620
J. Med. Chem. 2008, 51, 4620–4631
Asymmetric Synthesis of 2,3-Dihydro-2-arylquinazolin-4-ones: Methodology and Application to
a Potent Fluorescent Tubulin Inhibitor with Anticancer Activity
Gary M. Chinigo,† Mikell Paige,‡ Scott Grindrod,†,‡ Ernest Hamel,§ Sivanesan Dakshanamurthy,‡ Maksymilian Chruszcz,|
Wladek Minor,| and Milton L. Brown*,‡
Department of Chemistry, UniVersity of Virginia, CharlottesVille, Virginia 22904, Toxicology and Pharmacology Branch, DeVelopmental
Therapeutics Program, DiVision of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick, National Institutes of Health,
Frederick, Maryland 21702, Department of Molecular Physiology and Biological Physics, UniVersity of Virginia, CharlottesVille, Virginia
22908, and Department of Oncology, Drug DiscoVery Program, Georgetown UniVersity Medical Center, Washington, D.C. 20057
ReceiVed March 12, 2008
For several decades the 2,3-dihydroquinazolinone (DHQZ) heterocycle has been known to possess a variety
of important biological and medicinal properties. Despite the many interesting facets of these molecules,
synthetic access to nonracemic DHQZ analogues has remained elusive. Herein, we disclose a synthetic
route that allows access to either enantiomer of a variety of DHQZ derivatives. We illustrate the utility of
this chemistry with the asymmetric preparation and biological evaluation of a new chiral fluorescent tubulin
binding agent with extremely potent antiproliferative properties against human cancer cells. A computational
rationale for the increased potency of the (S)-enantiomer over the (R)-enantiomer is given, based on the
crystal structure of R,ꢀ-tubulin complexed with colchicine. Taking advantage of the inherent fluorescence
of these molecules, confocal images of GMC-5-193 (compound 7) in the cytoplasm of human melanoma
cells (MDA-MB-435) cells are presented.
Introduction
et al.,8followed by auxiliary removal from the amide moiety.
Initially, we envisioned two routes, as shown in Scheme 1. The
first route was semisuccessful in that, after separation of the
diastereomers, hydroxyl-iodine exchange of the serine-based
auxiliary followed by reaction with magnesium successfully
cleaved the auxiliary from the amide. However, when the
enantiomeric purity was assessed by polarimetry and chiral
HPLC, complete racemization of the desired stereocenter was
observed. We then tried a similar strategy that employed milder
conditions. The free acid of a phenylalanine-based chiral
auxiliary was treated with diphenylphosphorylazide (DPPA) and
heated to 60 °C under modified Curtius conditions.8 The
isocyanate product was quenched with water in situ, resulting
in decarboxylation and collapse of the unstable free aminal.
While the auxiliary was successfully cleaved, to our dismay
the desired stereocenter had once again racemized.
The 2,3-dihydroquinazolinone (DHQZa) family of compounds
has a rich pharmacology with reported antibiotic,1 antidefibril-
latory,2 antispermatogenic,3 vasodilatory,4 and analgesic5 ef-
ficacy. We have been interested in 2-biphenyl substituted DHQZ
analogues as potent tubulin inhibitors with impressive antipro-
liferative activity against a number of human cancer cell lines
(Figure 1). DHQZ analogues that are substituted at the 2-position
contain a stereocenter at the aminal carbon. Through compu-
tational docking experiments and molecular dynamics (Figure
2), we rationalized that the (S)-enantiomer should bind to tubulin
better than the (R)-enantiomer. To test this hypothesis, we set
out to synthesize enantiopure 2-aryl substituted DHQZ analogues.
Racemic synthesis of 2-aryl DHQZ analogues involves
condensation of an arylaldehyde with anthraquinoline. Despite
efforts by Lee,6 Priego,7 and possibly others, the enantiomeric
synthesis or chiral separation of racemic DHQZ compounds has
yet to be reported. To the best of our knowledge, there are no
general synthetic methods for obtaining enantiopure 2-aryl
DHQZ analogues.
We rationalized that the aminal stereocenter is sensitive to
racemization when a negative charge or even a transient negative
charge is placed on the amide nitrogen atom. With this in mind,
we hypothesized that the undesired racemization was occurring
through a mechanism similar to that illustrated in Figure 3. To
circumvent the racemization of our stereocenter, we sought a
similar approach in which the chiral auxiliary is not directly
bound to the heterocycle. We envisioned that a chiral auxiliary
strategically grafted on the adjacent aromatic ring would provide
a substrate that is not directly attached to the sensitive
heterocycle but retains close proximity to the new stereocenter
(Scheme 2).
Our efforts began with the synthesis of similar amino acid
based DHQZ diastereomers according to the methods of Priego
* To whom correspondence should be addressed. Phone: 202-687-8603.
Fax: 202-687-7659. E-mail: mb544@georgetown.edu.
†
Department of Chemistry, University of Virginia.
Georgetown University Medical Center.
National Cancer Institute at Frederick.
Department of Molecular Physiology and Biological Physics, University
‡
§
|
of Virginia.
a Abbreviations: CDCl3, deuterated chloroform; DCC, dicyclohexylcar-
bodiimide; DPPA, diphenylphosphorylazide; DHQZ, 2,3-dihydroquinazoli-
none; DIC, differential interference contrast; DMSO, dimethyl sulfoxide;
ESI, electrospray ionization; EtOAc, ethyl acetate; Et3N, triethylamine;
GAFF, general Amber force field; HCT-116, human colon cancer cells;
MDA-MB-435, human melanoma cells; MeCN, acetonitrile; MeMgBr,
methylmagnesium bromide; MgSO4, magnesium sulfate; MHz, megahertz;
NaOH, sodium hydroxide; NH4Cl, ammonium chloride; NMR, nuclear
magnetic resonance; RESP, restrained electrostatic potential; rmsd, root-
mean square displacement; THF, tetrahydrofuran.
Figure 1. Structure of 2-biphenyl-2,3-dihydroguinazoline.
10.1021/jm800271c CCC: $40.75
2008 American Chemical Society
Published on Web 07/09/2008

2,3-Dihydro-2-arylquinazolin-4-ones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4621
Figure 2. Binding model of (S)-(+)-7 (A) and (R)-(-)-7 (B) with the R-tubulin and ꢀ-tubulin polypeptide backbones. Residue side chains at the
colchicine site (Cys ꢀ241, Lys ꢀ254, and Asn R101) and 7 are shown in stick with the carbon atoms of colchicine colored green and the carbon
atoms of 7 colored white. Yellow dashed lines indicate potential intermolecular hydrogen bonds.
Scheme 1. Synthesis of Compound 6 Resulting in Racemization
Scheme 2. Synthesis of Compounds 3 and 4
Scheme 3. Synthesis of Nonracemic Compound 6
Removal of the auxiliary without affecting the sensitive
aminal stereocenter as shown in Scheme 3 would then give a
general method to nonracemic analogues of our target com-
pounds. Furthermore, we would be able to verify that the (S)-
enantiomer of our target compound binds more tightly than the
(R)-enantiomer to tubulin as predicted by our computational
simulations.
results for the screen including the yields of the major
diastereomers formed and the diastereomeric ratios are tabulated
in Table 1.
The phenylglycine-derived auxiliary gave moderate selectivity
but diminished yield (see Table 1). Increasing the steric bulk
of the auxiliary from methyl to isopropyl to tert-butyl signifi-
cantly enhanced the diastereoselectivity and the yield of the
reaction. Fortuitously, the diastereomers were separable both
by chromatography and by solubility differences. During most
of these cyclizations, the major diastereomer precipitated from
solution as a single pure isomer. In all cases examined thus far,
the major diastereomer exists as a crystalline solid while the
minor diastereomers appear to be an amorphous foam or a
viscous oil after purification. These dramatic solubility differ-
Results and Conclusions
A series of chiral salicylaldehyde-derived esters were pre-
pared, condensed with anthranilamide under acid-catalyzed
conditions, and screened for their ability to give separable
diastereomers. We found that the Boc-protected amino acid
derived auxiliaries furnished the best results in this regard. The

4622 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Chinigo et al.
Table 1. Yield and Diastereomeric Ratios for Selected Chiral
ences obviate the need for the tedious purifications and/or
multiple columns often required for purifying diastereomers with
similar Rf values. These conditions may be ideally suited for
preparative synthesis on a larger scale.
Auxiliaries
In order to probe the scope and limitations of this key
stereocenter formation, we condensed nonracemic aldehyde 1
in Scheme 2 with a variety of anthranilamide analogues that
encompass electron-donating, electron-withdrawing, and bulky
functional groups. The results of this screen are tabulated in
Table 2. We found, as expected, that the enantiomer of 1 gave
the opposite configuration at the aminal stereocenter (entry a).
We also noticed that the most deleterious effects on yield and
selectivity occurred with an electron-donating substituent (entry
d) at R5. A similar phenomenon was also observed by Lee and
co-workers.7 Examination of the electron-withdrawing nitro
groups (Table 2, entries e and f) show similar diastereoselectivity
but with a reduced yield. It should be noted that the nitro
containing molecules required reaction conditions 40-50 °C
higher than that of the other compounds listed.
Having successfully isolated several pure diastereomers, we
were now well positioned to determine the absolute configu-
ration of the stereocenter. X-ray crystallographic analysis of
compound 3 (see Supporting Information) shows that the S
configuration of the chiral auxiliary resulted in a heterocycle
with an R configuration at the aminal carbon.
Table 2. Yield and Diastereomeric Ratios for Compounds 3 and 4
entry R3
R4
R5
R6 auxilliary config % yield
dr
To remove the auxiliary and isolate the target DHQZ
enantiomers, we sought conditions that would cleave the ester
but not racemize the sensitive aminal stereocenter. Treatment
of the DHQZ analogues with several hydride sources resulted
in a yellow color, presumably from the DHQZ anion formed
by deprotonation of the amide nitrogen. These reagents proved
to be incompatible with the DHQZ diastereomers, resulting in
complete racemization of the aminal stereocenter. To our delight,
we found that reaction with hydrazine did not produce a yellow
color and resulted in cleavage of the ester at ambient temperature
to give the phenol with no detectable racemization.
The resulting phenol derived from ester 4 (see Scheme 3)
was treated with N-phenyl-bis-triflimide in the presence of
Hunig’s base to selectively give the aryl triflate 5.9 The
nonracemic triflate was then reduced with ammonium formate
and Pd(dppf)Cl2 to give the desired enantiomerically pure
DHQZ 6, as indicated by chiral HPLC. To test whether this
compound was sensitive to base-catalyzed racemization as we
hypothesized, we treated a 5.4 mM solution of the nonracemic
aminal (-)-2,3-dihydro-2-phenyl-4(1H)-quinazolinone in THF
([R]2D3 ) -160) with 50 µL of 6 M NaOH solution. Indeed, the
measured optical rotation was zero within 10 min, signifying
complete racemization of the aminal stereocenter.
a
b
c
d
e
f
g
h
i
H
H
H
H
H
H
Me
H
H
H
H
OMe
H
NO2
H
H
H
H
H
H
H
OMe
H
NO2
H
H
H
H
H
H
H
H
H
Me
H
R
S
S
S
S
S
S
S
S
71
85
78
36
66
58
62
34
59
11 89
91
9
85 15
75 25
85 15
89 11
79 21
83 17
83 17
Cl
Suzuki-Miyaura coupling10 of o-tolylboronic acid with
3-bromoacetoxybenzene gave phenol 8, which was converted
to aldehyde 9 via regioselective formylation using the method
of Wang and co-workers.11 Coupling N-Boc-D-tert-leucine or
N-Boc-L-tert-leucine to the phenol gave aldehyde 10a or 10b,
respectively. Condensation of aldehyde 10 with 5-nitroanthra-
nilamide gave the diastereomeric DHQZ analogues 11. The
three-step sequence for removal of the auxiliary was performed
as follows: treatment with hydrazine, followed by triflation, and
palladium-mediated reduction of the resulting triflate to give
enantiomers (-)-7 or (+)-7, depending on the configuration of
the N-Boc-leucine employed. The absolute configuration was
assigned by X-ray crystallography with the S configuration
belonging to the (+)-7 enantiomer.
Studies in our laboratory showed that racemic compound 7
had remarkably potent antiproliferative activity toward a number
of human cancer cell lines (see Table 3). Since the isolation or
resolution of the enantiomers of DHQZ 7 or other DHQZ
analogues has not been reported, the biological effects of each
enantiomer had not been previously assessed. To obtain this
information, we applied this newly developed synthetic meth-
odology to prepare both stereocenters of compound 7 as outlined
in Scheme 4.
The evaluation of racemic 7, (S)-(+)-7, and (R)-(-)-7 for
binding to the colchicine site on tubulin was performed. The
results revealed, as predicted, that the S enantiomer of 7 was
more effective than both the R enantiomer and the racemic
mixture of 7 as an inhibitor of the binding of colchicine to
tubulin (Table 3).
Inhibitory Effects of Enantiomers on Cell Growth and
on Tubulin Polymerization and the Binding of
[3H]Colchicine to Tubulin. Racemic 7 and each enantiomer,
(S)-(+)-7 and (R)-(-)-7, were separately assayed against two
different human cancer cell lines for their antiproliferative
effects. As Table 3 shows, both cell lines appear to be
significantly more sensitive to (S)-(+)-7 when compared to the
racemate. Interestingly, the “less active” (R)-(-)-7 still retains
significant cytotoxicity, though much less so than either (S)-
(+)-7 or the racemate. Particularly noteworthy is the picomolar
Figure 3. Proposed mechanism of racemization for compound 6.

2,3-Dihydro-2-arylquinazolin-4-ones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4623
Table 3. Biological Evaluation of Racemic-7, (R)-(–)-7, and, (S)-(+)-7a
% inhibition of 3H-colchicine binding
compd
MDA-MB-435 GI50, nM
HCT-116 GI50, nM
inhibition of tubulin assembly, IC50, µM
5 µM compd
50 µM compd
racemic-7
(R)-(-)-7
(S)-(+)-7
CA-4
100
1100
0.1
300
930
100
1.7 ( 0.3
16 ( 4
1.1 ( 0.2
1.3 ( 0.04
17 ( 2
2.1 ( 10
28 ( 1
51 ( 0.6
5.5 ( 0.9
66 ( 1
99 ( 0.9
not done
a In the colchicine binding experiment, the tubulin concentration was 1 µM and the [3H]colchicine was 5 µM. Paclitaxel GI50 for MDA-MB-435 is 4 nM
(see ref 12).
Scheme 4. Syntheses of Compound (S)-(+)-7
level growth inhibition induced by (S)-(+)-7 on MDA-MB-435
cells, approximately 10 times more active than paclitaxel.12
Similarly, (S)-(+)-7 was significantly more potent than (R)-
(-)-7 as an inhibitor of tubulin assembly and as an inhibitor of
colchicine binding to tubulin (Table 3). Although we found no
significant effect of (R)-(-)-7 on colchicine binding, this
enantiomer did have a reproducible, weak inhibitory effect on
tubulin assembly. It should be noted that (S)-(+)-7 was as potent
as combretastatin A-4 (CA-4) as an inhibitor of tubulin assembly
but much less active as an inhibitor of colchicine binding to
tubulin.
The effects of (S)-(+)-7 on the MDA-MB-435 and HCT-
116 cell proliferation revealed a 1000-fold difference compared
to (R)-(-)-7 (Table 3). This might be explained by a selectivity
of a multidrug resistance (MDR) mechanism including p-
glycoprotein toward (R)-(-)-7 over (S)-(+)-7 in regard to
substrate selectivity. Detailed studies addressing the chiral
interactions of (R)-(-)-7 and (S)-(+)-7 with p-glycoprotein will
provide more insight into this important resistance mechanism.
Molecular Modeling Studies. Our studies support that the
(S)-(+)-7 enantiomer was more effective at inhibiting tubulin
polymerization and the binding of [3H]colchicine to tubulin, as
well as human cancer cell proliferation than (R)-(-)-7. These
findings could be rationalized by molecular docking simulations.
Initially the docking procedure was validated by comparing the
binding mode obtained by docking the known tubulin depo-
lymerization agents colchicine, podophyllotoxin, and combre-
tastatin A-4. Automatic docking simulations of the R and S
configuration of compound 7 with R,ꢀ-tubulin were conducted
and produced several potential binding conformations. Since
inhibition of [3H]colchicine binding, as shown in Table 3,
provides strong evidence that compound 7 and its S enantiomer
bind to the colchicine site, we selected one conformer for the
S, as well as one conformer for the R, that closely resembled
the bound conformation of colchicine. Independent use of two
programs, Autodock13 and FlexX,14 also provided the same
docking pose for the 5-nitroanthranilamide interacting in similar
conformational space on the R,ꢀ-tubulin interface as our
simulated docking results.
Docking inhibitors into a protein binding site derived from a
crystal structure of the protein complexed with another ligand
(in this case, colchicine) may not have the correct binding mode
because the potential ligand may induce conformational changes
on the protein side chains. Thus, we further refined the binding
modes of compound 7 (R and S) using molecular dynamics
(MD) simulations. Initially, the tubulin-compound 7 (R and
S) complexes were subjected to energy minimization to relieve
any unfavorable interactions on the model structure. The
minimized complex structure was then subjected to MD
simulation of 300 ps, performed in the NVE ensemble. Since
the tubulin system is large, the residues within 15 Å of the ligand
were allowed to relax while fixing all other residues during the
simulation. Low energy minimum structures were collected
every 10 ps giving a total of 30 structures which were energy
minimized and further analyzed. To evaluate the readjustment
of the active site residues for the calculated tubulin-bound
compound 7 (R and S), the root-mean square displacement
(rmsd) between the atoms of the initial tubulin structure and
the corresponding atoms of each tubulin-compound complex
was evaluated. In the case of backbone atoms superimposition,
the rmsd was lower than 0.37 Å, while for side chains the
maximum rmsd found was about 1.38 Å. These data suggest
that ligand-induced conformational changes are more pro-
nounced for side chains compared with the polypeptide back-

4624 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Chinigo et al.
bone functional groups. Finally, the global energy minimum of
the 30 collected structures was selected as the final binding
model.
Upon analysis of 30 low energy minima structural models
for the (R)-(-)-7 and (S)-(+)-7 conformations, most of the
ligand-interacting residues were reoriented. In particular, for both
low energy conformations of (R)-(-)-7 and (S)-(+)-7, we
observed significant conformational reorientation of the Q-R11,
N-R101, and K-ꢀ254 side chains and the formation of hydrogen
bonds (H-bonds) with the nitro and NH groups. These residues
were not within the H-bond distance in the starting docked
model. The side chain reorientation of these residues is not
surprising because these residues are exposed and not interacting
with other active site residues. However, this suggests that
ligand-induced conformational changes may be critical in the
protein active site, and side chains may undergo rearrangement
during ligand binding. In the final model shown in Figure 2A
for the S conformation, we noticed a network of seven H-bonds
between compound 7 and Q-R11, N-R101, S-R178, T-R179,
N-ꢀ249, K-ꢀ254, Y-ꢀ224, and N-ꢀ258. We also noticed that
the biphenyl rings were buried inside the ꢀ-site of the
hydrophobic pocket containing ꢀ-tubulin residues L255, L248,
A250, A316, V318, K352, A354, and I378. This hydrophobic
region of compound 7 for both the R and S enantiomers overlaps
with the colchicine trimethoxyphenyl group. However, signifi-
cant differences in the interaction of the 5-nitroanthranilamide
moiety with the tubulin active site residues for the R and S
enantiomers were observed. The 5-nitroanthranilamide moiety
of the S enantiomer also appears to be more planar than the R
enantiomer in the final model with increased H-bonds for (S)-
(+)-7 with tubulin (seven H-bonds compared to four possible
H-bonds for (R)-(-)-7) (Figure 2A and Figure 2B). Specifically,
(R)-(-)-7 H-bond interactions with Y-ꢀ224, S-R178, and
T-R179 appear to be absent. These differential binding interac-
tions provide a rationale for the greater inhibitory activity of
(S)-(+)-7 compared with (R)-(-)-7 in inhibiting clochicine
binding to tubulin and tubulin assembly.
Intracellular Localization of Compound 7. A notable
property of compound 7 is its inherent fluorescence, which
allowed us to examine its intracellular localization in human
cancer cells. Treatment of the MDA-MB-435 human melanoma
cells with a 5 µM solution of compound 7 for 6 h was followed
by fixation of the cells, which were then subjected to photo-
excitation and visualization by confocal microscopy using a two-
photon laser. Propidium iodide staining (in red) was used to
visualize the nucleus (Figure 4A), and differential interference
contrast (DIC) was used to reveal the morphology of the cells.
Cytoplasmic images clearly show that compound 7 (in green)
localized in the cytoplasm (Figure 4C and Figure 4D), as would
be expected for an antitubulin agent.
In this study, we developed methodology to synthetically
resolve and determine the stereochemistry of each enantiomer
of 2-substitutued DHQZ compounds. Using molecular modeling,
we rationalized important interactions of each enantiomer with
the colchicine binding site. Evaluation of compound 7 for
inhibition of colchicine binding, tubulin assembly, and cancer
cell proliferation demonstrated that the (S)-(+)-7 enantiomer is
the more active stereoisomer. Finally, we provided evidence
using the inherent fluorescent property of compound 7, that it
localizes to the cytoplasm of human melanoma cancer cells.
Figure 4. Confocal image of fluorescent racemic 7 in human melanoma
cells (MDA-MB- 435): (A) propidium iodine staining of the nucleus;
(B) DIC image with 0.5 million MDA-MB-435 cells; (C) cytoplasmic
fluorescence of racemic 7; (D) merged DIC, propidium iodide, and
fluorescent 7 image. (E) MDA-MB-435 cells were treated for 1 h with
5 M racemic 7. Excitation was at 715 nm, and absorbance was read at
bandpath filter of 480-520 nm. Images were taken with a two photon
confocal system at 60× magnification.
not dried prior to use. Melting points were determined in open
capillary tubes on an Electrothermal melting point apparatus and
1
are uncorrected. H (300 or 500 MHz) and 13C NMR (75 or 125
MHz) spectra were measured at 25 °C on compounds in solution
in CDCl3 or DMSO-d6 on a Varian 300 or 500 MHz spectrometer.
Chemical shifts (δ) are given in ppm downfield from tetramethyl-
silane, an internal standard. Mass spectra were obtained on a
Finnagan LcQ Classic. Elemental analyses were performed by
Atlantic Microlabs, and microanalytical data were within (0.4%
of the calculated values. Reactions were monitored by TLC using
Merck 60 F254 silica gel aluminum sheets (20 cm × 20 cm).
N-Boc-L-tert-leucine(salicylaldehyde) Ester (1a). L-tert-Leucine
(5.0 g, 38.12 mmol) was added to a solution of 38.1 mL (8.1 mmol)
of 1 N NaOH in 50 mL of dioxane. Di-tert-butyldicarbonate (8.32
g, 38.12 mmol) was added, and the mixture was stirred at room
temperature for 14 h. The reaction mixture was concentrated and
partitioned between 100 mL of EtOAc and 100 mL of water. The
aqueous phase was acidified to a pH of ∼2 with concentrated HCl.
The resulting cloudy, aqueous solution was extracted with three
100 mL portions of EtOAc, and the combined organic extract was
dried over MgSO4. Filtration and removal of the solvent under
reduced pressure gave 6.7 g (29.0 mmol) of the Boc-protected
amino acid as a viscous clear oil, which was immediately taken up
in 50 mL of DMF. To this solution was added 6.6 g (31.9 mmol)
of DCC. After 30 min, 4.31 g (31.9 mmol) of HOBt was added.
After an additional 30 min, 4.0 mL (37.7 mmol) of salicylaldehyde
was added and the reaction mixture was stirred for 12 h and then
quenched with 100 mL of water. The reaction mixture was extracted
with EtOAc (3 × 100 mL), and the combined organic phase was
dried over MgSO4. Filtration and removal of the solvent gave the
crude product. Purification by flash chromatography eluting with
7:1 hexanes-EtOAc gave the aldehyde product. Further purification
by recrystallization from hexanes gave 4.7 g (48% over two steps)
of the product as colorless crystals. [R]D -2.1 (c 1.0, CHCl3); mp
104-106 °C; 1H NMR (300 MHz, CDCl3) δ 10.22 (s, 1H),
7.92-7.95 (dd, J ) 7.5 Hz, 1.5 Hz, 1H), 7.60-7.65 (dt, J ) 7.5,
1.8 Hz, 1H), 7.36-7.41 (t, J ) 7.8 Hz, 1H), 7.19-7.26 (d, J ) 8.1
Hz, 1H), 5.12-5.15 (d, J ) 0.9 Hz, 1H), 4.34-4.38 (d, J ) 9.3
Hz, 1H), 1.47 (s, 9H), 1.14 (s, 9H); 13C (75 MHz, CDCl3) δ 188.7,
Experimental Section
Chemistry: General Methods. Reagents were purchased from
Aldrich or Lancaster and were used as received. The solvents were

2,3-Dihydro-2-arylquinazolin-4-ones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4625
171.0, 156.0, 152.4, 135.4, 129.6, 128.5, 126.8, 123.1, 80.6, 62.6,
34.6, 28.5, 26.9; ESI m/z (rel intensity) 335.2 (100).
2R-(2′-N-Boc-D-tert-leucine phenyl ester)-7-methoxy-4-quinazoli-
none (3c). The title compound was prepared according to general
procedure A from 4-methoxyanthranilamide (0.20 g, 1.19 mmol)
and aldehyde 1 (0.40 g, 1.19 mmol) to give 0.45 g (78%) of the
resulting dihydroquinazolinone as colorless crystals. [R]D -105 (c
N-Boc-D-tert-leucine(salicylaldehyde) Ester (1b). D-tert-Leucine
(1.0 g, 7.6 mmol) was added to a solution of 7.6 mL (7.6 mmol)
of 1 N NaOH in 20 mL of dioxane. Di-tert-butyldicarbonate (1.8
g, 8.0 mmol) was added, and the reaction mixture was stirred at
room temperature for 14 h. The reaction mixture was concentrated
and partitioned between 100 mL of EtOAc and 100 mL of water.
The aqueous phase was acidified to a pH of ∼2 with concentrated
HCl. The resulting cloudy, aqueous solution was extracted with
three 50 mL portions of EtOAc, and the combined organic extract
was dried over MgSO4. Filtration and removal of the solvent under
reduced pressure gave 0.75 g (3.5 mmol) of the Boc-protected
amino acid as a viscous clear oil, which was immediately taken up
in 25 mL of DMF. To this solution was added 0.75 g (3.6 mmol)
of DCC. After 30 min, 0.49 g (3.6 mmol) of HOBt was added.
After an additional 30 min, 0.48 mL (4.5 mmol) of salicylaldehyde
was added and the reaction mixture was stirred for 12 h and then
quenched with 100 mL of water. The reaction mixture was extracted
with EtOAc (3 × 50 mL), and the combined organic phase was
dried over MgSO4. Filtration and removal of the solvent under
reduced pressure gave the crude product. Purification by flash
chromatography eluting with a 7:1 solution of hexanes-EtOAc gave
the aldehyde product. Further purification by recrystallization from
hexanes gave 0.58 g (53% over two steps) of the purified product
as colorless crystals. [R]D +2.1 (c 1.0, CHCl3); mp 104-106 °C;
1H NMR (300 MHz, CDCl3) δ 10.22 (s, 1H), 7.91-7.95 (dd, J )
10.8 Hz, 1.5 Hz, 1H), 7.60-7.65 (dt, J ) 8.1, 1.8 Hz, 1H),
7.36-7.41 (t, J ) 7.8 Hz, 1H), 7.19-7.22 (d, J ) 8.1 Hz, 1H),
5.13-5.16 (d, J ) 0.9 Hz, 1H), 4.34-4.37 (d, J ) 9.0 Hz, 1H),
1.47 (s, 9H), 1.14 (s, 9H); 13C (75 MHz, CDCl3) δ 188.8, 171.0,
156.0, 152.4, 135.5, 129.7, 128.5, 126.8, 123.2, 80.6, 62.6, 34.7,
28.6, 27.0; ESI m/z (rel intensity) 335.2 (100).
1
0.1, THF); mp 228-230 °C; H NMR (300 MHz, DMSO-d6) δ
7.79 (s, 1H), 7.50-7.54 (m, 3H), 7.36-7.41 (t, J ) 7.5 Hz, 1H),
7.24-7.29 (t, J ) 7.5 Hz, 1H), 7.11-7.13 (d, J ) 8.1 Hz, 1H),
6.67 (s, 1H), 6.22-6.27 (m, 2H), 5.92 (s, 1H), 4.09-4.11 (d, 1H),
3.67 (s, 3H), 1.35 (s, 9H), 1.07 (s, 9H); 13C (75 MHz, DMSO-d6)
δ 171.2, 164.3, 164.2, 157.1, 149.8, 148.3, 134.1, 130.2, 130.0,
128.3, 126.7, 123.0, 108.9, 105.7, 98.5, 79.7, 64.0, 61.7, 55.8, 34.0,
28.8, 27.4; ESI m/z (rel intensity) 483.2 (100). Anal. (C26H33N3O6)
C, H, N. C: calcd, 64.58; found, 64.58. H: calcd, 6.88; found, 6.99.
N: calcd, 8.69; found, 8.93.
2R-(2′-N-Boc-D-tert-leucine phenyl ester)-6-methoxy-4-quinazoli-
none (3d). To a 0 °C solution of the 5-methoxyanthranilamide (0.25
g, 1.51 mmol) and aldehyde 1 (0.51 g, 1.51 mmol) in 8 mL of
MeCN was added 1-2 drops of trifluoroacetic acid. After 1.5 h,
the solvent was removed under reduced pressure and the resulting
residue purified by flash chromatography eluting with 2:1
hexanes-EtOAc to give 0.26 g (36%) of the resulting dihydro-
quinazolinone as a light-green solid. [R]D -104 (c 0.1, THF); mp
1
192-194 °C; H NMR (300 MHz, DMSO-d6) δ 7.73-7.75 (d, J
) 7.5 Hz, 1H), 7.40-7.41 (d, J ) 3.3 Hz, 1H), 7.34-7.37 (dd, J
) 7.8, 1.5 Hz, 1H), 7.24-7.29 (t, J ) 7.5 Hz, 1H), 7.11-7.14 (d,
J ) 7.8 Hz, 1H), 6.87-6.911 (dd, J ) 8.7, 4.8 Hz, 1H), 6.70 (s,
1H), 6.62-6.65 (d, 1H), 6.15 (s, 1H), 5.26-5.29 (d, J ) 7.5 Hz,
1H), 5.04 (s, 1H), 4.21-4.23 (d, J ) 7.8 Hz, 1H), 3.78 (s, 3H),
1.39 (s, 9H), 1.15 (s, 9H); 13C (75 MHz, DMSO-d6) δ 171.6, 165.5,
156.3, 153.2, 148.0, 141.4, 132.6, 130.2, 128.3, 126.9, 123.0, 122.4,
117.0, 116.1, 110.4, 80.8, 63.0, 62.6, 56.0, 34.1, 28.5, 27.1; ESI
m/z (rel intensity) 483.2 (100). Anal. (C26H33N3O6) C, H, N. C:
calcd, 64.58; found, 64.53. H: calcd, 6.88; found, 6.71. N: calcd,
8.69; found, 8.75.
General Procedure A. Synthesis of Dihydroquinazolinone
Diastereomers. To a solution of the anthranilamide (1 equiv) and
the aldehyde (1 equiv) in 8 mL/mmol of MeCN at 0 °C was added
1-2 drops of trifluoroacetic acid. After 1.5 h, the precipitate was
collected by filtration and recrystallized from THF-hexanes.
2R-(2′-N-Boc-D-tert-leucine phenyl ester)-7-nitro-4-quinazoli-
none (3e). To a room temperature solution of 4-nitroanthranilamide
(0.22 g, 1.19 mmol) and chiral aldehyde 1 (0.40 g, 1.19 mmol) in
8 mL of MeCN was added 1-2 drops of trifluoroacetic acid. After
24 h, the precipitate was collected and recrystallized from
THF-hexanes to give 0.39 g (66%) of the resulting dihydro-
quinazolinone as a yellow solid. [R]D -167 (c 0.1, THF); mp
240-241 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.47 (s, 1H),
7.84-7.86 (d, J ) 8.4 Hz, 1H), 7.58-7.61 (m, 2H), 7.42-7.50
(m, 3H), 7.30-7.35 (t, J ) 6.6 Hz, 2H), 7.15-7.17 (d, J ) 7.8
Hz, 1H), 6.13 (s, 1H), 4.08-4.10 (d, J ) 6.6 Hz, 1H), 1.28 (s,
9H), 1.07 (s, 9H); 13C (75 MHz, DMSO-d6) δ 171.2, 162.7, 157.0,
151.4, 148.9, 148.4, 132.8, 130.7, 129.9, 128.6, 127.0, 123.1, 120.0,
111.9, 109.7, 79.6, 63.9, 61.6, 33.9, 28.7, 27.3; ESI m/z (rel
intensity) 498.2 (100). Anal. (C25H30N4O7 ·0.1H2O) C, H, N. C:
calcd, 60.02; found, 59.70. H: calcd, 6.08; found, 6.04. N: calcd,
11.24; found, 10.97.
2S-(2′-N-Boc-D-tert-leucine phenyl ester)-4-quinazolinone (4a).
The title compound was prepared according to general procedure
A from anthranilamide (0.13 g, 0.93 mmol) and aldehyde 1a (0.30
g, 0.93 mmol) to give 0.29 g (71%) of the resulting dihydro-
quinazolinone as colorless crystals. [R]D +120 (c 0.1, THF); mp
236-237 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.00 (s, 1H),
7.49-7.61 (m, 3H), 7.36-7.42 (t, J ) 7.7 Hz, 1H), 7.25-7.30 (t,
J ) 7.2 Hz, 1H), 7.18-7.23 (t, J ) 7.8 Hz, 1H), 7.10-7.13 (d,
J)7.8 Hz, 1H), 6.64-6.73 (m, 3H), 5.95 (s, 1H), 4.08-4.11 (d,
J)9.9 Hz, 1H), 1.33 (s, 9H), 1.06, (s, 9H); 13C (75 MHz, DMSO-
d6) δ 171.3, 164.3, 157.09, 148.4, 148.3, 134.1, 133.8, 130.3, 128.5,
128.1, 126.8, 123.1, 118.0, 115.5, 115.1, 79.7, 64.0, 61.7, 34.0,
28.8, 27.4; ESI m/z (rel intensity) 453.2 (100). Anal.
(C25H31N3O5 ·0.1H2O) C, H, N. C: calcd, 65.95; found, 65.80. H:
calcd, 6.91; found, 6.86. N: calcd, 9.23; found, 9.19.
2R-(2′-N-Boc-D-tert-leucine phenyl ester)-6-nitro-4-quinazoli-
none (3f). To a 50 °C solution of 5-nitroanthranilamide (0.27 g,
1.49 mmol) and aldehyde 1 (0.50 g, 1.49 mmol) in 8 mL of MeCN
was added 1-2 drops of trifluoroacetic acid. After 24 h, the
precipitate was collected and recrystallized from THF-hexanes to
give 0.43 g (58%) of the resulting dihydroquinazolinone as a yellow
2R-(2′-N-Boc-L-tert-leucine phenyl ester)-4-quinazolinone
(3b). The title compound was prepared according to general
procedure A from anthranilamide (0.20 g, 1.49 mmol) and aldehyde
1 (0.50 g, 1.49 mmol) to give 0.57 g (85%) of the resulting
dihydroquinazolinone as colorless crystals. [R]D -126 (c 0.1, THF);
mp 236-237 °C; 1H NMR (300 MHz, DMSO-d6) δ 7.98 (s, 1H),
7.59-7.62 (dd, J ) 7.8, 1.5 Hz, 1H), 7.54-7.57 (dd, J ) 4.5, 1.2
Hz, 1H), 7.48-7.50 (d, J ) 6.6 Hz, 1H), 7.36-7.42 (t, J ) 7.8
Hz, 1H), 7.18-7.30 (m, 2H), 7.11-7.13 (d, J ) 7.8 Hz, 1H),
6.64-6.73 (m, 3H), 5.95 (s, 1H), 4.09-4.11 (d, J ) 6.9 Hz, 1H),
1.33 (s, 9H), 1.07, (s, 9H); 13C (75 MHz, DMSO-d6) δ 171.2, 164.2,
157.0, 148.4, 148.2, 134.0, 133.7, 130.3, 128.5, 128.1, 126.7, 123.0,
118.0, 115.5, 115.1, 79.6, 64.0, 61.6, 34.0, 28.8, 27.3; ESI m/z (rel
intensity) 453.2 (100). Anal. (C25H31N3O5) C, H, N. C: calcd, 66.21;
found, 66.12. H: calcd, 6.89; found, 6.92. N: calcd, 9.27; found,
9.14.
1
solid. [R]D -182 (c 0.1, THF); mp 211-213 °C; H NMR (300
MHz, DMSO-d6) δ 8.49 (s, 1H), 8.42 (d, J ) 2.7 Hz, 1H), 8.15 (s,
1H), 8.07-8.11 (dd, J ) 9.0, 2.7 Hz, 1H), 7.58-7.61 (d, J ) 7.5
Hz, 1H), 7.43-7.47 (t, J ) 6.3 Hz, 2H), 7.31-7.37 (t, J ) 7.2 Hz,
1H), 7.14-7.16 (d, J ) 8.1 Hz, 1H), 6.83-6.86 (d, J ) 9.3 Hz,
1H), 6.21 (s, 1H), 4.06-4.08 (d, J ) 6.9 Hz, 1H), 1.28 (s, 9H),
1.06 (s, 9H); 13C NMR (75 MHz, DMSO-d6) δ 171.1, 162.0, 156.8,
153.0, 148.2, 137.8, 132.4, 130.7, 129.3, 128.5, 127.0, 124.6, 122.9,
114.9, 113.3, 79.4, 63.6, 61.3, 33.7, 28.5, 27.1; ESI m/z (rel
intensity) 498.2 (100). Anal. (C25H30N4O7) C, H, N. C: calcd, 60.23;
found, 60.35. H: calcd, 6.07; found, 6.25. N: calcd, 11.24; found,
11.19.

4626 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Chinigo et al.
2R-(2′-N-Boc-D-tert-leucine phenyl ester)-8-methyl-4-quinazoli-
none (3g). To a 0 °C solution of 3-methylanthranilamide (0.05 g,
0.30 mmol) and aldehyde 1 (0.10 g, 0.30 mmol) in 8 mL of MeCN
was added 1-2 drops of trifluoroacetic acid. After 2 h the solvent
was removed under reduced pressure, and the resulting residue was
purified by flash chromatography eluting with 2:1 hexanes-EtOAc,
followed by recrystallization from THF-hexanes to give 0.090 g
(62%) of the resulting dihydroquinazolinone as a colorless solid.
(3-12 h), the reaction mixture was concentrated and partitioned
between 50 mL of EtOAc and 50 mL of water. The aqueous phase
was extracted with EtOAc, and the combined phase was dried over
MgSO4. Filtration, removal of the solvent under reduced pressure,
and purification of the resulting residue by flash chromatography
eluting with 1:1 hexanes-EtOAc gave the product as a viscous
oil.
2R-(2′-Trifluoromethane sulfonyl phenyl ester)-4-quinazoli-
none (5a). The title compound was synthesized according to general
procedure B from 4a (0.40 g, 0.88 mmol) to give 0.20 g (96%) of
the phenol. [R]D -128 (c 0.1, THF); 1H NMR (300 MHz, DMSO-
d6) δ 9.84 (s, 1H), 7.92 (s, 1H), 7.58-7.61 (d, J ) 7.8 Hz, 1H),
7.30-7.33 (d, J ) 7.5 Hz, 1H), 7.17-7.22 (dt, J ) 7.2, 1.2 Hz,
1H), 7.10-7.15 (t, J ) 6.6 Hz, 1H), 6.82-6.85 (d, J ) 8.1 Hz,
1H), 6.73-6.80 (m, 3H), 6.61-6.66 (t, J ) 7.5 Hz, 1H), 5.98 (s,
1H). Subsequently, 0.22 g (94%) of the triflate was isolated as a
1
[R]D -109 (c 0.1, THF); mp 177-179 °C; H NMR (300 MHz,
DMSO-d6) δ 7.76-7.82 (m, 2H), 7.37-7.43 (dt, J ) 7.8, 1.5 Hz,
1H), 7.29-7.34 (t, J ) 7.5 Hz, 1H), 7.13-7.16 (d, J ) 7.2
Hz, 1H), 7.05-7.07 (d, J ) 7.2 Hz, 1H), 6.71-6.76 (t, J ) 7.5
Hz, 1H), 6.14-6.17 (d, J ) 8.4 Hz, 2H), 5.10-5.12 (d, J ) 8.7
Hz, 1H), 4.63 (s, 1H), 4.22-4.25 (d, J ) 8.1 Hz, 1H), 2.08 (s,
3H), 1.30 (s, 9H), 1.12 (s, 9H); 13C (75 MHz, DMSO-d6) δ 172.3,
165.5, 156.0, 148.2, 145.5, 135.1, 132.9, 130.8, 128.6, 127.5, 126.6,
122.5, 122.3, 118.8, 115.1, 81.1, 62.7, 62.4, 34.3, 28.4, 27.0, 16.8;
ESI m/z (rel intensity) 467.2 (100). Anal. (C26H33N3O5) C, H, N.
C: calcd, 66.79; found, 66.42. H: calcd, 7.11; found, 7.23. N: calcd,
8.99; found, 8.62.
1
clear viscous oil. [R]D -123 (c 0.1, THF); H NMR (300 MHz,
CDCl3) δ 7.93 (dd, J ) 7.5, 2.1 Hz, 1H), 7.83-7.86 (dd, J ) 7.8,
1.5 Hz, 1H), 7.42-7.50 (m, 2H), 7.26-7.34 (m, 2H), 6.84-6.89
(t, J ) 7.8 Hz, 1H), 6.66-6.68 (d, J ) 8.1 Hz, 1H), 6.63 (s, 1H),
6.24 (s, 1H), 4.78 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 165.1,
146.8, 146.5, 134.6, 132.9, 131.7, 130.1, 129.6, 128.7, 122.0, 120.0,
115.3, 115.0, 62.4 (CF3 signal not detected); ESI m/z (rel intensity)
372.0 (100).
2R-(2′-N-Boc-D-tert-leucine phenyl ester)-5-methyl-4-quinazoli-
none (3h). To a 0 °C solution of 6-methylanthranilamide (0.18 g,
1.19 mmol) and aldehyde 1 (0.40 g, 1.19 mmol) in 8 mL of MeCN
was added 1-2 drops of trifluoroacetic acid. After 2 h the solvent
was removed under reduced pressure, and the resulting residue was
purified by flash chromatography eluting with 2:1 hexanes-EtOAc,
2:1, followed by recrystallization from THF-hexanes to give 0.19 g
(34%) of the resulting dihydroquinazolinone as a colorless solid.
2S-(2′-Trifluoromethane sulfonyl phenyl ester)-4-quinazoli-
none (5b). The title compound was synthesized according to general
procedure B from 3b (0.15 g, 0.34 mmol) to give 0.076 g (94%)
of the phenol. [R]D +122 (c 0.1, THF); 1H NMR (300 MHz,
DMSO-d6) δ 9.92 (s, 1H), 8.01 (s, 1H), 7.67-7.69 (d, J ) 7.8 Hz,
1H), 7.39-7.41 (d, J ) 7.5 Hz, 1H), 7.18-7.30 (m, 2H), 6.91-6.93
(d, J ) 8.1 Hz, 1H), 6.81-6.88 (m, 3H), 6.70-6.75 (t, J ) 7.5
Hz, 1H), 6.06 (s, 1H).
Subsequently, 0.10 g (84%) of the triflate was isolated as a clear
viscous oil. [R]D +120 (c 0.1, THF); 1H NMR (300 MHz, CDCl3)
δ 7.96-7.98 (d, J ) 7.5 Hz, 1H), 7.89-7.92 (d, J ) 7.2 Hz, 1H),
7.50-7.54 (m, 2H), 7.35-7.40 (m, 2H), 6.90-6.95 (t, J ) 7.5 Hz,
1H), 6.70-6.73 (d, J ) 8.1 Hz, 1H), 6.56 (s, 1H), 6.29 (s, 1H),
4.75 (bs, 1H); 13C NMR (75 MHz, CDCl3) δ 165.11, 146.7, 146.5,
134.6, 132.9, 131.7, 130.1, 129.6, 128.7, 122.1, 120.1, 115.3, 115.1,
62.5 (CF3 signal not detected); ESI m/z (rel intensity) 372.0 (100).
1
[R]D -143 (c 0.1, THF); mp 148-150 °C; H NMR (300 MHz,
DMSO-d6) δ 7.73-7.74 (d, J ) 4.5 Hz, 1H), 7.34-7.37 (t, J )
4.5 Hz, 1H), 7.25-7.27 (t, J ) 3.3 Hz, 1H), 7.08-7.09 (d, J ) 4.8
Hz, 1H), 7.03-7.07 (t, J ) 4.5 Hz, 1H), 6.56-6.57 (d, J ) 4.5
Hz, 1H), 6.49-6.50 (d, J ) 4.8 Hz, 1H), 6.34 (s, 1H), 6.05 (s,
1H), 5.16-5.18 (d, J ) 4.5 Hz, 1H), 5.07 (s, 1H), 4.18-4.20 (d,
J ) 4.8 Hz, 1H), 2.63 (s, 3H), 1.35 (s, 9H), 1.11 (s, 9H); 13C (75
MHz, DMSO-d6) δ 171.4, 165.6, 156.0, 148.3, 147.8, 142.1, 132.7,
132.2, 130.0, 128.2, 126.7, 122.7, 122.0, 113.6, 113.2, 80.5, 62.6,
61.6, 33.8, 28.2, 26.8, 22.3; ESI m/z (rel intensity) 467.2 (100).
Anal. (C26H33N3O5) C, H, N. C: calcd, 66.79; found, 66.85. H: calcd,
7.11; found, 7.22. N: calcd, 8.99; found, 8.86.
2R-(2′-N-Boc-D-tert-leucine phenyl ester)-6-chloro-4-quinazoli-
none (3i). The title compound was prepared according to general
procedure A from 5-chloroanthranilamide (0.05 g, 0.30 mmol) and
aldehyde 1 (0.10 g, 0.30 mmol) to give 0.11 g (77%) of the resulting
dihydroquinazolinone as colorless crystals. [R]D -123 (c 0.1, THF);
mp 207-209 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.21 (s, 1H),
7.59-7.60 (d, J ) 4.8 Hz, 1H), 7.54 (d, J ) 1.2 Hz, 1H), 7.48-7.49
(d, J ) 3.9 Hz, 1H), 7.41-7.44 (t, J ) 4.5 Hz, 1H), 7.30-7.33 (t,
J ) 4.5 Hz, 1H), 7.26-7.28 (d, J ) 5.1 Hz, 1H), 7.12-7.14 (d, J
) 4.8 Hz, 1H), 6.93 (s, 1H), 6.76-6.78 (d, J ) 4.8 Hz, 1H), 5.99
(s, 1H), 4.08-4.10 (d, J ) 4.2 Hz, 1H), 1.31 (s, 9H), 1.07 (s, 9H);
13C NMR (75 MHz, DMSO-d6) δ 171.3, 163.2, 157.0, 148.5, 147.3,
133.7, 133.1, 130.6, 128.7, 127.1, 126.9, 123.0, 121.7, 117.2, 116.8,
79.6, 63.9, 61.7, 34.0, 28.8, 27.4; ESI m/z (rel intensity) 487.2 (100).
Anal. (C25H30ClN3O5) C, H, N. C: calcd, 61.53; found, 61.43. H:
calcd, 6.20; found, 6.13. N: calcd, 8.61; found, 8.52.
2S-(2′-Trifluoromethane sulfonyl phenyl ester)-7-methoxy-
4-quinazolinone (5c). The title compound was synthesized ac-
cording to general procedure B from 3c (0.30 g, 0.62 mmol) to
give 0.15 g (89%) of the phenol. [R]D -93 (c 0.1, THF); 1H NMR
(300 MHz, DMSO-d6) δ 9.85 (bs, 1H), 8.30 (s, 1H), 7.71 (s, 1H),
7.50-7.53 (d, J ) 8.7 Hz, 1H), 7.27-7.30 (d, J ) 7.8 Hz, 1H),
7.10-7.25 (t, J ) 6.6 Hz, 1H), 6.72-6.85 (m, 2H), 6.20-6.30 (m,
2H), 5.94 (s, 1H), 3.68 (s, 3H). Subsequently, 0.14 g (77%) of the
triflate was isolated as a clear viscous oil. [R]D -92 (c 0.1, THF);
1H NMR (300 MHz, CDCl3) δ 7.93-7.94 (dd, J ) 7.2, 2.4 Hz,
1H), 7.77-7.80 (d, J ) 8.7 Hz, 1H), 7.43-7.53 (m, 2H), 7.30-7.33
(dd, J ) 7.5, 1.8 Hz, 1H), 6.52 (s, 1H), 6.41-6.45 (dd, J ) 8.7,
2.1 Hz, 1H), 6.22 (s, 1H), 6.16-6.17 (d, J ) 2.1 Hz, 1H), 4.87
(bs, 1H), 3.79 (s, 3H); 13C (75 MHz, CDCl3) δ 165.0, 164.9, 148.5,
146.4, 133.2, 131.6, 130.6, 130.0, 129.6, 122.0, 108.7, 107.2, 98.9,
62.5, 55.6 (CF3 signal not detected); ESI m/z (rel intensity) 402.1
(100).
General Procedure B. Ester Cleavage and Phenol Triflation.
To a solution of the ester (1 equiv) in 30 mL/mmol of THF was
added hydrazine monohydrate (3 equiv). The mixture was stirred
at room temperature until ester cleavage was complete as visualized
by TLC (3-12 h). The solvent was removed under reduced
pressure, and the residue was taken up in 15 mL of chloroform.
The solution was brought to 0 °C for 2 h, and the resulting
precipitate was collected by filtration. The mother liquor was
concentrated and taken up in 5-10 mL of chloroform and brought
to 0 °C for 2 h and then filtered.
2S-(2′-Trifluoromethane sulfonyl phenyl ester)-6-methoxy-
4-quinazolinone (5d). The title compound was synthesized ac-
cording to general procedure B from 3d (0.16 g, 0.32 mmol) to
1
give 0.080 g (88%) of the phenol. [R]D -165 (c 0.1, THF); H
NMR (300 MHz, DMSO-d6) δ 9.86 (s, 1H), 7.99 (s, 1H), 7.37-7.39
(d, 7.8 Hz, 1H), 7.15-7.20 (m, 2H), 6.77-6.96 (m, 4H), 6.41 (s,
1H), 5.97 (s, 1H), 3.72 (s, 3H). Subsequently, 0.10 g (100%) of
the triflate was isolated as a clear viscous oil. [R]D -78 (c 0.1,
THF); 1H NMR (300 MHz, CDCl3) δ 7.94-7.97 (dd, J ) 6.9, 2.4
Hz, 1H), 7.50-7.55 (dt, J ) 6.6, 1.5 Hz, 2H), 7.45-7.46 (d, 2.7
Hz, 1H), 7.38-7.40 (m, 2H), 7.01-7.04 (dd, J ) 8.7, 2.7 Hz, 1H),
6.70-6.73 (d, J ) 9 Hz, 1H), 6.59 (bs, 1H), 6.24 (s, 1H), 3.82 (s,
3H); 13C NMR (75 MHz, CDCl3) δ 165.6, 154.0, 146.6, 140.9,
1
The combined precipitates were confirmed by H NMR to be
the corresponding phenol. The phenol (1 equiv) was then taken up
in 10 mL of THF and reacted with N-phenyl-bis(trifluorosulfon-
imide) (2 equiv) in the presence of diisopropylethylamine (10
equiv). After triflation was complete, as judged by TLC visualization

2,3-Dihydro-2-arylquinazolin-4-ones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4627
132.5, 131.8, 129.9, 129.6, 127.1, 123.5, 122.2, 117.2, 110.8, 62.8,
56.0 (CF3 signal not detected); ESI m/z (rel intensity) 402.1 (100).
2S-(2′-Trifluoromethane sulfonyl phenyl ester)-7-nitro-4-
quinazolinone (5e). The title compound was synthesized according
to general procedure B from 3e (0.17 g, 0.34 mmol) to give 0.090
J ) 7.2 Hz, 1H), 6.92 (s, 1H), 6.68-6.78 (m, 3H), 5.92 (s, 1H).
Subsequently, 0.11 g (100%) of the triflate was isolated as a clear
1
viscous oil. [R]D -97 (c 0.1, THF); H NMR (300 MHz, CDCl3)
δ 9.37 (s, 1H), 7.86-7.89 (dd, J ) 7.2, 3.2 Hz, 1H), 7.83 (d, J )
2.4 Hz, 1H), 7.46-7.55 (m, 2H), 7.24-7.24 (m, 1H), 6.63-6.69
(m, 2H), 6.24 (s, 1H), 4.79 (s, 1H); 13C NMR (75 MHz, CDCl3) δ
164.5, 146.4, 145.2, 134.8, 132.0, 129.8, 129.7, 128.3, 127.1, 125.3,
123.2, 122.3, 116.7, 62.5 (CF3 signal not detected); ESI m/z (rel
intensity) 406.0 (100).
1
g (91%) of the phenol. [R]D -233 (c 0.1, THF); H NMR (300
MHz, DMSO-d6) δ 9.96 (s, 1H), 8.41 (s, 1H), 7.81-7.84 (d, J )
8.4 Hz, 1H), 7.60 (d, J ) 1.8 Hz, 1H), 7.46 (s, 1H), 7.37-7.40
(dd, J ) 8.7, 2.4 Hz, 1H), 7.25-7.28 (d, J ) 8.4 Hz, 1H),
7.13-7.18 (t, J ) 8.1 Hz, 1H), 6.76-6.87 (m, 2H), 6.08 (s, 1H).
Subsequently, 0.12 g (100%) of the triflate was isolated as a clear
viscous oil. [R]D -132 (c 0.1, THF); 1H NMR (300 MHz, CDCl3)
δ 8.84 (s, 1H), 8.00-8.03 (d, J ) 8.4 Hz, 1H), 7.86-7.89 (d, J )
7.5 Hz, 1H), 7.62-7.64 (d, J ) 8.4 Hz, 1H), 7.48-7.58 (m, 3H),
6.76 (s, 1H), 6.33 (s, 1H), 5.17 (s, 1H); 13C NMR (75 MHz, CDCl3)
δ 158.7, 147.2, 142.3, 141.6, 127.5, 127.2, 125.7, 125.0, 122.5,
118.6, 117.7, 109.4, 105.3, 57.8 (CF3 signal not detected); ESI m/z
(rel intensity) 417.0 (100).
General Procedure C. Reduction of Aryl Triflate. Triethy-
lamine (15 equiv) and Pd(dppf)Cl2 (0.02 equiv) were added to a
solution of formic acid (10 equiv) in DMF (75 mL/mmol). After
the resulting red solution was thoroughly degassed, the correspond-
ing aryl triflate (1 equiv) was added (the formic acid must be
completely neutralized before adding the triflate, otherwise race-
mization may occur), and the reaction mixture was heated at
90-100 °C until the starting material was completely consumed
as judged by TLC (1.5 h). The dark solution was added to 100 mL
of H2O and extracted with EtOAc. The combined organic extracts
were dried over MgSO4 and filtered, and the solvent was removed
under reduced pressure. The resulting residue was purified by flash
chromatography eluting with 1:1 hexanes-EtOAc and further
purified by recrystallization from THF-hexanes.
General Procedure D. Reduction of Aryl Triflate for Nitro-
Containing Compounds. Triethylamine (15 equiv) and Pd(dppf)Cl2
(0.02 equiv) were added to a solution of formic acid (1.0 equiv) in
75 mL/mmol of DMF (excess formic acid results in the reduction
of the nitro functional group). After the resulting red solution was
thoroughly degassed, the corresponding aryl triflate (1 equiv) was
added (the formic acid must be completely neutralized before adding
the triflate, otherwise racemization may occur), and the reaction
mixture was heated at 90-100 °C until the starting material was
completely consumed as judged by TLC (1.5 h). The dark solution
was added to 100 mL of H2O and extracted with EtOAc. The
combined organic extracts were dried over MgSO4 and filtered,
and the solvent was removed under reduced pressure. The resulting
residue was purified by flash chromatography eluting with 1:1
hexanes-EtOAc and further purified by recrystallization from
THF-hexanes.
2R-(Phenyl)-4-quinazolinone (6a). The title compound was
synthesized according to general procedure C from triflate 5a (0.075
g, 0.20 mmol) to give 0.044 g (98%) of the resulting dihydro-
quinazolinone as a colorless solid. [R]D -158 (c 0.1, THF); mp
211-213 °C; 1H NMR (500 MHz, DMSO-d6) δ 8.33 (s, -
1H), 7.63-7.64 (d, J ) 3.0 Hz, 1H), 7.50-7.52 (d, J ) 7.5 Hz,
2H), 7.33-7.40 (m, 3H), 7.23-7.26 (t, J ) 7.0 Hz, 1H), 7.14 (s,
1H), 6.76-6.78 (d, J ) 8.0 Hz, 1H), 6.66-6.69 (t, J ) 8.0 Hz,
1H), 5.77 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 164.1, 148.4,
142.1, 133.8, 128.9, 128.8, 127.8, 127.3, 117.6, 115.4, 114.9, 67.1;
ESI m/z (rel intensity) 224.1 (100). Anal. (C14H12N2O) C, H, N. C:
calcd, 74.98; found, 74.86. H: calcd, 5.39; found, 5.25. N: calcd,
12.49; found, 12.50. The enantiopurity was 100% as confirmed by
chiral HPLC (ChiralPak AS column with 100% MeCN; flow of
0.2 mL/min; retention time (6b), 32.2 min; retention times of the
components of the racemate, 29.9 and 32.3 min).
2S-(Phenyl)-4-quinazolinone (6b). The title compound was
synthesized according to general procedure C from triflate 5b (0.070
g, 0.19 mmol) to give 0.038 g (90%) of the resulting dihydro-
quinazolinone as a colorless solid. [R]D +160 (c 0.1, THF); mp
211-213 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.32 (s, 1H), 7.60,
7.63 (d, J ) 7.8 Hz, 1H), 7.47-7.50 (d, J ) 6.6 Hz, 2H), 7.31-7.40
(m, 3H), 7.20-7.25 (t, J ) 7.2 Hz, 1H), 7.12 (s, 1H), 6.73-6.76
(d, J ) 8.1 Hz, 1H), 6.64-6.68 (t, J ) 7.2 Hz, 1H), 5.75 (s, 1H);
13C NMR (125 MHz, DMSO-d6) δ 164.4, 148.5, 142.3, 134.1,
129.2, 129.0, 128.1, 127.5, 117.8, 115.6, 115.1, 67.2; ESI m/z (rel
intensity) 224.1 (100). Anal. (C14H12N2O) C, H, N. C: calcd, 74.98;
found, 74.84. H: calcd, 5.39; found, 5.35. N: calcd, 12.49; found,
12.46. The enantiopurity was 100% as confirmed by chiral HPLC
(ChiralPak AS column with MeCN 100%; flow of 0.2 mL/min;
retention time (6a), 29.5 min; retention times of the components
of the racemate, 29.9 and 32.3 min).
2S-(2′-Trifluoromethane sulfonyl phenyl ester)-6-nitro-4-
quinazolinone (5f). The title compound was synthesized according
to general procedure B from 3f (0.46 g, 0.95 mmol) to give 0.23 g
(85%) of the phenol. [R]D -292 (c 0.1, THF); 1H NMR (300 MHz,
DMSO-d6) δ 9.98 (s, 1H), 8.43-8.45 (m, 2H), 8.36 (s, 1H),
8.04-8.08 (dd, J ) 9.0, 2.7 Hz, 1H), 7.24-7.27 (d, J ) 7.5 Hz,
1H), 7.15-7.21 (t, J ) 7.5, 1.5 Hz, 1H), 6.80-6.88 (m, 3H), 6.18
(s, 1H). Subsequently, 0.34 g (100%) of the triflate was isolated as
a yellow viscous oil. [R]D -194 (c 0.1, THF); 1H NMR (300 MHz,
CDCl3) δ 9.77 (s, 1H), 8.77 (s, 1H), 8.20-8.23 (d, J ) 9.0 Hz,
1H), 7.88-7.90 (d, J ) 6.6 Hz, 1H), 7.47-7.60 (m, 2H), 7.03 (s,
1H), 6.76-6.80 (dd, J ) 9.0, 2.7 Hz, 1H), 6.43 (s, 1H), 6.08 (s,
1H); 13C NMR (75 MHz, CDCl3) δ 162.7, 151.0, 146.0, 139.8,
134.9, 131.9, 129.7, 129.4, 126.6, 125.5, 122.8, 122.1, 114.6, 62.2
(CF3 signal not detected); ESI m/z (rel intensity) 417.0 (100).
2S-(2′-Trifluoromethane sulfonyl phenyl ester)-8-methyl-4-
quinazolinone (5g). The title compound was synthesized according
to general procedure B from 3g (0.50 g, 1.09 mmol) to give 0.20 g
(73%) of the phenol. [R]D -97 (c 0.1, THF); 1H NMR (300 MHz,
DMSO-d6) δ 9.97 (s, 1H), 7.98 (s, 1H), 7.48-7.50 (d, J ) 7.5 Hz,
1H), 7.16-7.19 (dd, J ) 7.2, 1.2 Hz, 1H), 7.08-7.13 (t, J ) 7.5
Hz, 2H), 6.82-6.84 (d, J ) 8.1 Hz, 1H), 6.71-6.76 (t, J ) 7.5
Hz, 1H), 6.57-6.62 (t, 7.5 Hz, 1H), 6.07 (s, 1H), 5.93 (s, 1H),
2.07 (s, 3H). Subsequently, 0.18 g (100%) of the triflate was isolated
as a clear viscous oil. [R]D -88 (c 0.1, THF); 1H NMR (300 MHz,
CDCl3) δ 7.95-7.98 (dd, J ) 7.5, 2.1 Hz, 1H), 7.74-7.76 (d, J )
7.8 Hz, 1H), 7.50-7.55 (m, 2H), 7.22-7.39 (m, 3H), 7.17 (s, 1H),
6.79-6.84 (t, J ) 8.1 Hz, 1H), 6.29 (s, 1H), 2.15 (s, 3H); 13C
NMR (75 MHz, CDCl3) δ 165.8, 146.5, 145.1, 135.4, 133.2, 131.6,
130.1, 129.6, 126.5, 122.8, 122.0, 119.4, 115.2, 62.3, 16.7 (CF3
signal not detected); ESI m/z (rel intensity) 386.1 (100).
2S-(2′-Trifluoromethane sulfonyl phenyl ester)-5-methyl-4-
quinazolinone (5h). The title compound was synthesized according
to general procedure B from 3h (0.22 g, 0.47 mmol) to give 0.10 g
(81%) of the phenol. [R]D -114 (c 0.1, THF); 1H NMR (300 MHz,
DMSO-d6) δ 9.83 (s, 1H), 7.81 (s, 1H), 7.38-7.41 (dd, J ) 7.5,
1.2 Hz, 1H), 7.15-7.21 (dt, J ) 8.1, 1.5 Hz, 1H), 7.06-7.11 (t, J
) 7.8 Hz, 1H), 6.81-6.90 (m, 2H), 6.64-6.69 (m, 2H), 6.48-6.51
(d, J ) 7.5 Hz, 1H), 5.91 (s, 1H), 2.57 (s, 3H). Subsequently, 0.10 g
(100%) of the triflate was isolated as a clear viscous oil. [R]D -116
1
(c 0.1, THF); H NMR (300 MHz, CDCl3) δ 7.96-7.99 (dd, J )
6.9, 2.7 Hz, 1H), 7.47-7.52 (m, 2H), 7.31-7.35 (m, 2H),
7.16-7.21 (t, J ) 7.5 Hz, 1H), 6.67-6.71 (m, 2H), 6.56-6.58 (d,
J ) 8.1 Hz, 1H), 6.16 (s, 1H), 2.64 (s, 3H); 13C NMR (75 MHz,
CDCl3) δ 165.7, 148.4, 146.7, 142.7, 133.4, 132.7, 131.6, 130.3,
129.5, 126.5, 123.8, 121.9, 113.4, 61.9, 22.5 (CF3 signal not
detected); ESI m/z (rel intensity) 386.1 (100).
2S-(2′-Trifluoromethane sulfonyl phenyl ester)-6-chloro-4-
quinazolinone (5i). The title compound was synthesized according
to general procedure B from 3i (0.24 g, 0.51 mmol) to give 0.11 g
(78%) of the phenol. [R]D -172 (c 0.1, THF); 1H NMR (300 MHz,
DMSO-d6) δ 9.82 (s, 1H), 8.07 (s, 1H), 7.45 (s, 1H), 7.20-7.22
(d, J ) 6.6 Hz, 1H), 7.14-7.17 (d, J ) 7.2 Hz, 1H), 7.04-7.08 (t,

4628 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Chinigo et al.
2R-(Phenyl)-7-methoxy-4-quinazolinone (6c). The title com-
pound was synthesized according to general procedure C from
triflate 5c (0.12 g, 0.30 mmol) to give 0.049 g (79%) of the resulting
dihydroquinazolinone as a colorless solid. [R]D -135 (c 0.1, THF);
mp 180-181 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.10 (s, 1H),
7.52-7.53 (d, J ) 5.7 Hz, 1H), 7.46-7.48 (d, J ) 4.5 Hz, 2H),
7.32-7.39 (m, 3H), 7.11 (s, 1H), 6.25-6.26 (m, 2H), 5.7 (s, 1H),
3.69 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ 164.0, 149.9, 142.3,
129.6, 128.8, 128.7, 127.4, 109.0, 105.5, 98.4, 67.2, 55.7; ESI m/z
(rel intensity) 254.1 (100). Anal. (C15H14N2O2) C, H, N. C: calcd,
70.85; found, 70.49. H: calcd, 5.55; found, 5.49. N: calcd, 11.02;
found, 10.87. The racematic mixture of 6c could not be resolved
into its separate diastereomeric components.
2R-(Phenyl)-5-methyl-4-quinazolinone (6h). The title com-
pound was synthesized according to general procedure C from
triflate 5h (0.09 g, 0.23 mmol) to give 0.035 g (78%) of the resulting
dihydroquinazolinone as a colorless solid. [R]D -172 (c 0.1, THF);
mp 134-135 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.22 (s, 1H),
7.53-7.56 (d, J ) 6.6 Hz, 2H), 7.40-7.42 (d, J ) 7.2 Hz, 3H),
7.09-7.13 (t, J ) 7.5 Hz, 1H), 7.05 (s, 1H), 6.68-6.71 (d, J )
7.5 Hz, 1H), 6.50-6.52 (d, J ) 6.9 Hz, 1H), 5.67 (s, 1H), 3.70 (s,
3H); 13C NMR (75 MHz, DMSO-d6) δ 165.2, 150.1, 142.1, 141.2,
132.9, 129.1, 129.0, 127.7, 121.7, 114.3, 113.7, 67.1, 22.8; ESI
m/z (rel intensity) 238.1 (100). Anal. (C15H14N2O) C, H, N. C: calcd,
75.61; found, 75.63. H: calcd, 5.92; found, 5.94. N: calcd, 11.76;
found, 11.74. The components of the racemate of 6h were not
separable under any conditions we examined.
2R-(Phenyl)-6-chloro-4-quinazolinone (6i). The title compound
was synthesized according to general procedure C from triflate 5i
(0.11 g, 0.27 mmol) to give 0.045 g (59%) of the resulting
dihydroquinazolinone as a colorless solid. [R]D -111 (c 0.1, THF);
mp 243-245 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.53 (s, 1H),
7.59 (d, J ) 2.1 Hz, 1H), 7.51-7.54 (d, J ) 6.6 Hz, 2H), 7.38-7.45
(m, 4H), 7.30-7.33 (dd, J ) 8.7, 2.4 Hz, 1H), 6.81-6.84 (d, J )
8.7 Hz, 1H), 5.83 (s, 1H); 13C NMR (75 MHz, DMSO-d6) δ 163.2,
147.3, 142.0, 133.8, 129.3, 129.1, 127.6, 127.2, 121.5, 117.1, 116.8,
67.2; ESI m/z (rel intensity) 258.1 (100). Anal. (C14H11ClN2O) C,
H, N. C: calcd, 65.00; found, 64.91. H: calcd, 4.29; found, 4.19.
N: calcd, 10.83; found, 10.81. The components of the racemate of
6i were not separable under any conditions we examined.
5-(2′-Tolyl)-2-formylphenol (9). A solution of 3.95 g (18.4
mmol) of 3-bromophenylacetate, 2.50 g (18.4 mmol) of 2-tolyl-
boronic acid, 8.97 g (27.6 mmol) of Cs2CO3, and 0.42 g (0.37
mmol) of Pd(PPh3)4 in 50 mL of dioxane was thoroughly degassed
and brought to reflux until the starting materials were consumed
(12 h) as judged by TLC. EtOH (10 mL) was added, and the
reaction mixture was stirred for 1 h. The reaction mixture was
concentrated under reduced pressure and partitioned between 100
mL of saturated NH4Cl and 100 mL of EtOAc. The aqueous phase
was extracted with EtOAc. The combined organic extracts were
dried over MgSO4 and filtered, and the solvent was removed under
reduced pressure. Purification by flash chromatography eluting with
7:1 hexanes-EtOAc gave 3.17 g (94%) of the phenol 8 as a brown
oil.
To a solution of 3.17 g (17.2 mmol) of phenol 8 in 25 mL of
THF at 0 °C was added 5.7 mL (3 M solution in Et2O, 17.2 mmol)
of MeMgBr. After 30 min, a precipitate formed, and all the THF
was removed under reduced pressure. Benzene (25 mL) was added
and removed under reduced pressure. Then another 25 mL portion
of benzene was added. Then 6.61 g (73.4 mmol) of paraformal-
dehyde and 6.1 mL (44.1 mmol) of Et3N were added, and the
mixture was brought to reflux for 24 h. The reaction was quenched
by addition of 150 mL of saturated NH4Cl and extracted with three
75 mL portions of EtOAc. The combined organic extracts were
dried over MgSO4 and filtered, and the solvent was removed under
reduced pressure. Purification by distillation (220 °C, 1 mmHg)
gave 2.4 g (66%) of the aldehyde as a viscous pale-yellow oil. 1H
NMR (300 MHz, CDCl3) δ 11.22 (s, 1H), 9.97 (s, 1H), 7.62-7.65
(d, J ) 7.8 Hz, 1H), 7.27-7.37 (m, 4H), 7.03-7.07 (m, 2H), 2.36
(s, 3H); 13C NMR (75 MHz, CDCl3) δ 196.5, 161.7, 151.5, 140.6,
135.3, 133.7, 130.9, 129.5, 128.5, 126.3, 121.6, 119.6, 118.5, 20.7;
ESI m/z (rel intensity) 212.1 (100).
N-Boc-D-tert-leucine[5-(2′-tolyl)-2-formyl]phenyl Ester (10).
To a room temperature solution of 1.07 g (5.0 mmol) of Boc-(L)-
tert-leucine was added 1.02 g (5.0 mmol) of DCC. After 45 min,
0.67 g (5.0 mmol) of HOBt was added. After an additional 45 min,
1.00 g (4.7 mmol) of aldehyde 9 was added and the mixture was
stirred overnight. Water (150 mL) was added, and the mixture was
extracted with three 75 mL portions of EtOAc. The combined
organic extracts were dried over MgSO4 and filtered, and the solvent
was removed under reduced pressure. The residue was purified by
flash chromatography eluting with 6:1 hexanes-EtOAc, and 1.20 g
(59%) of the product was obtained as a viscous oil. 1H NMR (300
MHz,CDCl3) δ 10.29 (s, 1H), 8.00-8.03 (d, J ) 8.1 Hz, 1H),
2R-(Phenyl)-6-methoxy-4-quinazolinone (6d). The title com-
pound was synthesized according to general procedure C from
triflate 5d (0.090 g, 0.22 mmol) to give 0.042 g (91%) of the
resulting dihydroquinazolinone as a colorless solid. [R]D -200 (c
1
0.1, THF); mp 206-208 °C; H NMR (300 MHz, DMSO-d6) δ
8.35 (s, 1H), 7.51-7.53 (d, J ) 7.5 Hz, 2H), 7.36-7.43 (m, 3H),
7.17 (s, 1H), 6.93-6.97 (dd, J ) 8.7, 3.0 Hz, 1H), 6.74-6.78 (m,
2H), 5.71 (s, 1H), 3.70 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ
164.4, 152.1, 143.0, 142.2, 129.1, 129.0, 127.7, 122.2, 116.9, 116.4,
110.5, 67.6, 56.0; ESI m/z (rel intensity) 254.1 (100). Anal.
(C15H14N2O2) C, H, N. C: calcd, 70.85; found, 70.85. H: calcd,
5.55; found, 5.49. N: calcd, 11.02; found, 10.91. The components
of the racemate of 6d were not separable under any conditions we
examined.
2R-(Phenyl)-7-nitro-4-quinazolinone (6e). The title compound
was synthesized according to general procedure D from triflate 5e
(0.03 g, 0.07 mmol) to give 0.015 g (78%) of the resulting
dihydroquinazolinone as a yellow solid. [R]D -279 (c 0.1, THF);
mp 219-221 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.73 (s, 1H),
7.82-7.84 (d, J ) 5.1 Hz, 1H), 7.76 (s, 1H), 7.57 (d, J ) 1.5 Hz,
1H), 7.48-7.49 (d, J ) 4.2 Hz, 2H), 7.36-7.43 (m, 4H), 5.91 (s,
1H); 13C NMR (75 MHz, DMSO-d6) δ 162.3, 151.2, 148.6, 141.5,
129.6, 129.2, 129.0, 127.2, 119.7, 111.4, 109.3, 66.8; ESI m/z (rel
intensity) 269.1 (100). Anal. (C14H11N3O3 ·0.2H2O) C, H, N. C:
calcd, 61.63; found, 61.59. H: calcd, 4.21; found, 4.00. N: calcd,
15.40; found, 15.29. The enantiopurity was 100% as confirmed by
chiral HPLC (ChiralPak AS column with MeCN 100%; flow of
0.4 mL/min; retention time (6e), 12.0 min; retention times of the
components of the racemate, 12.0 and 16.0 min).
2R-(Phenyl)-6-nitro-4-quinazolinone (6f). The title compound
was synthesized according to general procedure D from triflate 5f
(0.20 g, 0.48 mmol) to give 0.044 g (58%) of the resulting
dihydroquinazolinone as a yellow solid. [R]D -240 (c 0.1, THF);
mp 238-240 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.75 (s, 1H),
8.57 (s, 1H), 8.42 (d, J ) 1.5 Hz, 1H), 8.08-8.10 (d, J ) 5.4 Hz,
1H), 7.36-7.47 (m, 5H), 6.81-6.83 (d, J ) 5.7 Hz, 1H), 6.01 (s,
1H); 13C NMR (75 MHz, DMSO-d6) δ 161.8, 152.6, 141.5, 137.5,
129.5, 129.4, 129.1, 127.0, 124.6, 114.7, 113.1, 66.7; ESI m/z (rel
intensity) 269.1 (100). Anal. (C14H11N3O3 ·0.2H2O) C, H, N. C:
calcd, 61.63; found, 61.57. H: calcd, 4.21; found, 4.02. N: calcd,
15.61; found, 15.24. The enantiopurity was 100% as confirmed by
chiral HPLC (ChiralPak AS column with MeCN 100%; flow of
0.4 mL/min; retention time (6f), 12.6 min; retention times of the
components of the racemate, 12.5 and 15.0 min).
2R-(Phenyl)-8-methyl-4-quinazolinone (6g). The title com-
pound was synthesized according to general procedure C from
triflate 5g (0.18 g, 0.47 mmol) to give 0.069 g (77%) of the resulting
dihydroquinazolinone as a colorless solid. [R]D -150 (c 0.1, THF);
mp 176-178 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.40 (s, 1H),
7.48-7.50 (d, J ) 4.5 Hz, 1H), 7.44-7.46 (d, J ) 4.5 Hz, 2H),
7.32-7.35 (t, J ) 4.5 Hz, 2H), 7.27-7.29 (m, 1H), 7.12-7.13 (d,
J ) 4.2 Hz, 1H), 6.56-6.61 (t, J ) 4.5 Hz, 2H), 5.72 (s, 1H), 2.12
(s, 3H); 13C NMR (75 MHz, DMSO-d6) δ 164.1, 145.9, 143.0,
134.5, 128.7, 128.5, 126.9, 125.7, 122.8, 117.2, 115.6, 65.9, 17.4;
ESI m/z (rel intensity) 238.1 (100). Anal. (C15H14N2O) C, H, N. C:
calcd, 75.61; found, 75.55. H: calcd, 5.92; found, 5.87. N: calcd,
11.76; found, 11.68. The components of the racemate of 6g were
not separable under any conditions we examined.

2,3-Dihydro-2-arylquinazolin-4-ones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4629
7.28-7.40 (m, 5H), 7.21 (s, 1H), 5.19-5.22 (d, J ) 8.7 Hz, 1H),
4.40-4.43 (d, J ) 8.4 Hz, 1H), 2.32 (s, 3H), 1.51 (s, 9H), 1.18 (s,
9H); ESI m/z (rel intensity) 425.2 (100).
2R-[4′-(2′′-tolyl)-2′-N-Boc-L-tert-leucine phenyl ester)-6-nitro-
4-quinazolinone (14). The title compound was prepared according
to general procedure A from 5-nitroanthranilamide (0.28 g, 1.55
mmol) and aldehyde 10 (0.70 g, 1.55 mmol) to give 0.44 g (50%)
of the product as a yellow foam. [R]D +213 (c 0.1, THF); 1H NMR
(300 MHz, DMSO-d6) δ 8.78-8.79 (d, J ) 2.4 Hz, 1H), 8.05-8.09
(dd, J ) 9.0, 2.4 Hz, 1H), 7.61-7.63 (m, 2H), 7.16-7.27 (m, 6H),
6.84 (s, 1H), 6.74 -6.77 (d, J ) 9.3 Hz, 1H), 6.48 (s, 1H),
5.18-5.20 (d, J ) 5.7 Hz, 1H), 4.10-4.13 (dd, J ) 7.2, 2.4 Hz,
1H), 2.25 (s, 3H), 1.44 (s, 9H), 1.11 (s, 9H); 13C NMR (75 MHz,
DMSO-d6) δ 171.0, 163.9, 156.8, 151.1, 145.9, 144.3, 140.0, 139.2,
135.6, 130.9, 130.8, 129.9, 129.5, 128.2, 127.6, 127.0, 126.2, 125.8,
123.4, 115.0, 113.0, 81.3, 63.3, 61.8, 34.1, 28.6, 27.1, 20.6; ESI
m/z (rel intensity) 588.3 (100).
2R-[4′-(2′′-Tolyl)phenyl]-6-nitro-4-quinazolinone ((R)-(-)-7).
The title compound was prepared by making the triflate according
to general procedure C, followed by reduction of the triflate
according to general procedure D. Reaction of 0.22 g (0.38 mmol)
of ester 11 with 0.056 mL (1.15 mmol) of hydrazine gave 0.14 g
(95%) of the corresponding phenol as a yellow solid. [R]D +293
(c 0.1, THF); 1H NMR (300 MHz, DMSO-d6) δ 10.13 (s, 1H),
8.45-8.52 (m, 3H), 8.08-8.12 (dd, J ) 9.0, 2.7 Hz, 1H), 7.22-7.36
(m, 5H), 7.13-7.16 (m, 1H), 6.81-6.87 (m, 3H), 6.26 (s, 1H),
2.23 (s, 3H).
To 0.11 g (0.28 mmol) of the phenol was added 0.20 g (0.56
mmol) of N-phenyl-bis(trifluoromethanesulfonimide) and 0.49 mL
(2.8 mmol) of diisopropylethylamine to give 0.14 g (100%) of the
triflate as a viscous yellow oil. [R]D +155 (c 0.1, THF); 1H NMR
(300 MHz, CDCl3) δ 8.77-9.78 (d, J ) 2.7 Hz, 1H), 8.17-8.21
(dd, J ) 9.0, 2.7 Hz, 1H), 7.87-7.89 (d, J ) 8.1 Hz, 1H),
7.45-7.48 (dd, J ) 8.1, 1.5 Hz, 1H), 7.25-7.38 (m, 4H), 7.16-7.19
(d, J ) 7.2 Hz, 1H), 7.07 (s, 1H), 6.73-6.6.76 (d, J ) 9.0 Hz,
1H), 6.44 (s, 1H), 5.65 (s, 1H), 2.26 (s, 3H); 13C NMR (75
MHz,CDCl3) δ 163.0, 150.9, 146.7, 145.7, 140.4, 138.8, 135.4,
131.1, 130.7, 130.3, 130.0, 129.8, 129.3, 128.9, 127.3, 126.5, 125.8,
123.3, 123.1, 114.9, 62.3, 20.5 (CF3 signal not detected).
2S-[4′-(2′′-Tolyl)-2′-N-Boc-D-tert-leucine phenyl ester)-6-nitro-
4-quinazolinone (11). The title compound was prepared according
to general procedure A from 5-nitroanthranilamide (0.27 g, 1.50
mmol) and aldehyde 10 (0.64 g, 1.50 mmol) to give 0.33 g (37%)
of the resulting product as a yellow foam. [R]D -210 (c 0.1, THF);
1H NMR (300 MHz, DMSO-d6) δ 8.79-8.80 (d, J ) 2.7 Hz, 1H),
8.06-8.10 (dd, J ) 9.0, 2.7 Hz, 1H), 7.62-7.64 (m, 2H), 7.16-7.28
(m, 6H), 6.83 (s, 1H), 6.74-6.77 (d, J ) 9.0 Hz, 1H), 6.48 (s,
1H), 5.18-5.20 (d, J ) 6.0 Hz, 1H), 4.11-4.14 (m, 1H), 2.90 (s,
3H), 1.44 (s, 9H), 1.17 (s, 9H); 13C NMR (75 MHz, DMSO-d6) δ
171.0, 164.2, 156.8, 151.3, 147.0, 144.2, 140.0, 139.1, 135.5, 131.0,
130.8, 129.9, 129.5, 128.2, 127.5, 127.0, 126.2, 125.7, 123.4, 115.0,
113.0, 81.2, 63.4, 61.8, 33.7, 28.6, 27.1, 20.6; ESI m/z (rel intensity)
588.3 (100).
2S-[4′-(2′′-tolyl)Phenyl]-6-nitro-4-quinazolinone ((S)-(+)-7).
The title compound was prepared by making the triflate according
to general procedure C, followed by reduction of the triflate
according to general procedure D. Reaction of ester 11 (0.33 g,
0.56 mmol) with hydrazine (0.82 mL, 1.68 mmol) gave 0.17 g
(80%) of the corresponding phenol as a yellow solid. [R]D -288
(c 0.1, THF); 1H NMR (300 MHz, DMSO-d6) δ 10.17 (s, 1H),
8.49-8.57 (m, 2H), 8.12-8.17 (dd, J ) 9.0, 2.7 Hz, 1H), 7.28-7.40
(m, 5H), 7.18-7.20 (m, 1H), 6.85-6.92 (m, 3H), 6.30 (s, 1H),
2.27 (s, 3H).
To 0.080 g (0.22 mmol) of the phenol was added 0.16 g (0.44
mmol) of N-phenyl-bis(trifluoromethanesulfonimide) and 0.38 mL
(2.2 mmol) of diisopropylethylamine to give 0.11 g (100%) of the
triflate as a viscous yellow oil. [R]D -156 (c 0.1, THF); 1H NMR
(300 MHz, CDCl3) δ 8.87 (s, 1H), 8.29-8.31 (d, J ) 6.6 Hz, 1H),
7.98-8.01 (d, J ) 7.8 Hz, 1H), 7.56-7.59 (d, J ) 8.1 Hz, 1H),
7.36-7.51 (m, 4H), 7.23-7.28 (m, 2H), 6.85-6.88 (d, J ) 9.0
Hz, 1H), 6.56 (s, 1H), 5.79 (s, 1H), 2.38 (s, 3H); 13C NMR (75
MHz, CDCl3) δ 163.1, 151.0, 146.8, 145.8, 140.5, 138.9, 135.5,
131.2, 130.8, 130.5, 130.2, 129.9, 129.4, 129.0, 127.3, 126.6, 125.9,
123.4, 123.2, 115.0, 62.5, 20.6 (CF3 signal not detected).
The triflate was reduced according to general procedure D using
formic acid (0.013 mL, 0.35 mmol), triethylamine (0.17 mL, 1.20
mmol), and Pd(dppf)Cl2 (0.004 g, 0.005 mmol) to give 0.058 g
(67%) of dihydroquinazolinone 7 as yellow crystals. [R]D -213 (c
The triflate was reduced according to general procedure D with
formic acid (0.010 mL, 0.27 mmol), triethylamine (0.36 mL, 2.56
mmol), and Pd(dppf)Cl2 (0.004 g, 0.005 mmol) to give 0.050 g
(54%) of 7 as yellow crystals. [R]D +209 (c 0.1, THF); mp
258-260 °C; 1H NMR (300 MHz, DMSO-d6) δ 8.82 (s, 1H), 8.66
(s, 1H), 8.49 (s, 1H), 8.13-8.17 (dd, J ) 9.3, 2.7 Hz, 1H),
7.55-7.58 (d, J ) 7.8 Hz, 2H), 7.42-7.45 (d, J ) 8.1 Hz, 2H),
7.27-7.31 (m, 3H), 7.17-7.20 (m, 1H), 6.87-6.90 (d, J ) 9.0
Hz, 1H), 6.11 (s, 1H), 2.25 (s, 3H); 13C NMR (75 MHz, DMSO-
d6) δ 162.0, 152.8, 142.6, 141.3, 140.4, 137.8, 135.3, 131.1, 130.2,
130.1, 130.0, 128.2, 127.4, 126.7, 124.9, 115.0, 113.3, 66.9, 20.9;
ESI m/z (rel intensity) 359.1 (100). Anal. (C21H17N3O3 ·0.1H2O)
C, H, N. C: calcd, 69.83; found, 69.75. H: calcd, 4.80; found, 4.65.
N: calcd, 11.63; found, 11.67. The enantiopurity was 100% as
confirmed by chiral HPLC (ChiralPak AS column with MeCN
100%; flow of 0.2 mL/min; retention time (15), 27.2 min; retention
times of the components of the racemate, 24.3 and 27.0 min).
X-ray Crystallography. A crystal of (-)-3 suitable for X-ray
diffraction was obtained by slow evaporation of a solution contain-
ing 3 and a 1:1 mixture of isopropanol and 1-butanol. The crystal
(dimensions 0.37 mm × 0.09 mm × 0.07 mm) was mounted on
the Rigaku RAPID diffractometer, and data collection was con-
trolled by the HKL-2000 package.15 The diffraction experiment
was carried out at 103 K using Cu KR X-ray radiation. During
data collection ω scans with ꢁ offsets were used. The diffraction
images were recorded with ∆ω ) 5°. Indexing, integration, and
scaling were performed with HKL2000. The reflections were scaled
in space group P43212 with Friedel pairs separated. The collected
reflection composed a complete data set up to a resolution 0.81 Å.
The data collection and processing statistics are summarized in
Table 3. The structure solution and determination was performed
with HKL-3000SM.16 The absolute configuration of the aminal
carbon of (-)-3 was assigned as the R configuration.
1
0.1, THF); mp 258-260 °C; H NMR (300 MHz, DMSO-d6) δ
8.86 (s, 1H), 8.69 (s, 1H), 8.51 (s, 1H), 8.15-8.19 (dd, J ) 9.0,
2.4 Hz, 1H), 7.58-7.61 (d, J ) 8.4 Hz, 2H), 7.44-7.47 (d, J )
8.1 Hz, 2H), 7.29-7.32 (m, 3H), 7.21-7.22 (m, 1H), 6.90-6.93
(d, J ) 9.0 Hz, 1H), 6.14 (s, 1H), 2.27 (s, 3H); 13C NMR (75
MHz, DMSO-d6) δ 162.0, 152.9, 142.7, 141.4, 140.5, 137.9, 135.4,
131.1, 130.2, 130.1, 129.8, 128.2, 127.3, 126.7, 125.0, 115.0, 113.3,
67.0, 20.9; ESI m/z (rel intensity) 359.1 (100). Anal. (C21H17N3O3)
C, H, N. C: calcd, 70.18; found, 70.05. H: calcd, 4.77; found, 4.65.
N: calcd, 11.69; found, 11.71. The enantiopurity was 100% as
confirmed by chiral HPLC (ChiralPak AS column with MeCN
100%; flow of 0.2 mL/min; retention time ((-)-7), 24.1 min;
retention times of the components of the racemate, 24.3 and 27.0
min).
N-Boc-L-tert-leucine [5-(2′-tolyl)-2-formyl]phenyl Ester (13).
To a room temperature solution of 1.50 g (6.9 mmol) of Boc-D-
tert-leucine (1.50 g, 6.91 mmol) was added 1.57 g (7.6 mmol) of
DCC. After 45 min, 1.03 g (7.6 mmol) of HOBt was added. After
an additional 45 min, 1.10 g (5.2 mmol) of aldehyde 9 was added,
and the reaction mixture was stirred overnight. Water (150 mL)
was added, and the mixture was extracted with EtOAc. The
combined organic extracts were dried over MgSO4 and filtered,
and the solvent was removed under reduced pressure. Purification
by flash chromatography eluting with 6:1 hexanes-EtOAc gave
0.80 g (38%) of the product as a viscous oil. 1H NMR (300
MHz,CDCl3) δ 10.25 (s, 1H), 7.96-7.99 (d, J ) 8.1 Hz, 1H),
7.17-7.36 (m, 5H), 7.16 (s, 1H), 5.14-5.17 (d, J ) 8.1 Hz, 1H),
4.35-4.38 (d, J ) 8.4 Hz, 1H), 2.29 (s, 3H), 1.47 (s, 9H), 1.13 (s,
9H); ESI m/z (rel intensity) 425.2 (100).
Molecular Modeling. The X-ray crystal structure of R,ꢀ-tubulin
complexed with colchicine (PDB code 1SA0) was used for the

4630 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Chinigo et al.
automated molecular docking of compounds (S)-7 and (R)-7.
Inconsistencies between the PDB format and the tubulin residues
library translation to atomic potential types were corrected manually.
The tubulin was minimized using the DISCOVER (Accelrys, San
Diego, CA) program’s cff91 force field17 with distance-dependent
dielectrics.
evaluated for a dose response curve. The EC50 for each cell line
(the amount of compound needed for 50% inhibition of cell
proliferation) was calculated using GraphPad Prism 4 software.
Confocal Microscopy. Human melanoma (MDA-MB-435)
samples were plated on sterilized microscope slides and incubated
for 1 h with 5 µM racemic 7. Excitation at 715 nm and absorbance
were read with a band path filter of 480-520 nm. Images were
taken with a two-photon confocal system at 60× magnification.
The nuclei were stained with propidium iodide, and the differential
interference contrast (DIC) image was taken with 0.5 million MDA-
MB-435 cells.
Initially, the DHQZ ligand was constructed using Sybyl7.3
(Tripos Inc.) and optimized with the MP2/6-31G** basis set using
the Gaussian quantum mechanical program.18 Docking experiments
were performed with the AutoDock 3.0.512 and FlexX programs.19
The standard procedures were used for docking simulations using
AutoDock and FlexX. In the case of AutoDock, for the protein
and ligand torsion angles were identified for 10 independent runs
per ligand. A grid of 60 × 60 × 60 points in the x, y, and z
directions was built, centered on the center of the mass of the N
atom in compound 7 that is in the tubulin active site. A grid spacing
of 0.4 Å and a distance-dependent function of the dielectric constant
were used for the calculation of the energetic map. The default
settings were used for all other parameters. At the end of the docking
simulation, ligands with the most favorable free energy of binding
were selected. In the case of FlexX, all the parameters were used
with the default settings, except the number of solution conforma-
tions was set to 90.
The best docked geometry was minimized using the DISCOVER
module of Insight II (Accelrys Inc.) with the procedure described
below. A consistent valence force field (cff91) was used. The cutoff
for nonbonded interaction energies was set to ∞ (i.e., no cutoff)
with the dielectric constant set at 4 to account for the dielectric
shielding found in proteins. Each minimization was carried out in
two steps, first using steepest descent minimization for 200 cycles
and then using conjugate gradient minimization, until the average
gradient fell below 0.01 kcal/mol. All atoms within 6.0 Å of the
inhibitor were allowed to relax during the minimization, whereas
those atoms beyond 6.0 Å were held rigid.
The minimized complex was subjected to molecular dynamics
simulations using the DISCOVER module of Insight II (Accelrys
Inc.). Molecular dynamics simulations performed in the NVE
ensemble consisted of an initial equilibration of 25 ps (ps), followed
by a production run of 300 ps dynamics at 300 K. The final complex
structure at the end of the molecular dynamics simulations was
subjected to 2000 steps of steepest descent energy minimization,
followed by conjugate gradient energy minimization. A distance-
dependent dielectric constant and nonbonded distance cutoff of 12
Å were used. Molecular dynamics simulations were performed using
the AMBER8 package,20 with the general Amber force field
(GAFF)21 and RESP charge models.22
Acknowledgment. The authors gratefully acknowledge
Tamara Stoops and the MAPS Core facility at the University
of Virginia for their assistance with the cytological assays and
partial financial support from the Georgetown University Drug
Discovery Program. The authors also thank Professor Erick
Carreira for suggesting the racemization studies.
Supporting Information Available: Results from elemental
1
analysis of key compounds, X-ray crystal structure of 3b, and H
and 13C NMR spectra of 1a, 1b, 4a, 3c, 3d, 3e, 3f, 3g, 3h, 3i, 6a,
6b, 6c, 6d, 6e, 6f, 6g, 6h, 6i, (R)-11, (S)-(+)-7, and (R)-(-)-7.
This material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) Russel, H. E.; Alaimo, R. J. Antibacterial 2,3-dihydro-2-(5-nitro-2-
thienyl)quinazolin-4-(1H)-ones. J. Med. Chem. 1972, 15, 335–336.
(2) Bonola, G.; Da Re, P.; Magistretti, M. J.; Massarani, E.; Setnikar, I.
1-Aminoacyl-2,3-dihydro-4(1H)-quinazolinone derivatives with choler-
etic and antifibrillatory activity. J. Med. Chem. 1968, 11, 1136–1139.
(3) Neil, G. L.; Li, L. H.; Buskirk, H. H.; Moxley, T. E. Antitumor effects
of the antispermatogenic agent, 2,3-dihydro-2-(1-naphthyl)-4(1H)-
quinazolinone. Cancer Chemother. 1972, 56, 163–173.
(4) Levin, J. I.; Chan, P. S.; Bailey, T.; Katocs, A. S.; Venkatesan, A. M.
The synthesis of 2,3-dihydro-4(1H)-quinazolinone angiotensin II
receptor antagonists. Bioorg. Med. Chem. Lett. 1994, 4, 1141–1146.
(5) Okumura, K.; Oine, T.; Yamada, Y.; Hayashi, G.; Nakama, M. 4-Oxo-
1,2,3,4-tetrahydroquinazolines. I. Syntheses and pharmacological
properties of 2-methyl-3-aryl-4-oxo-1,2,3,4-tetrahydroquinazolines and
their 1-acyl derivatives. J. Med. Chem. 1968, 11, 348–352.
(6) Hour, M. J.; Huang, L. J.; Kuo, S. C.; Xia, Y.; Bastow, K.; Nakanishi,
Y.; Hamel, E.; Lee, K. H. 6-Alkylamino- and 2,3-dihydro-3′-methoxy-
2-phenyl-4-quinazolinones and related compounds: their synthesis,
cytotoxicity, and inhibition of tubulin polymerization. J. Med. Chem.
2000, 43, 4479–4487.
(7) Escalante, J.; Flores, P.; Priego, J. M. Synthesis of 2,3-dihydro-4(1H)-
quinazolinones. Heterocycles 2004, 63, 2019–2032.
(8) Shioiri, T.; Ninomiya, K.; Yamada, S. Diphenylphosphoryl azide. New
convenient reagent for a modified Curtius reaction and for peptide
synthesis. J. Am. Chem. Soc. 1972, 94, 6203–6205.
(9) Hendrickson, J. B.; Bergeron, R. Triflamides: new acylating and
triflating reagents. Tetrahedron Lett. 1973, 14, 4607–4610.
(10) Miyaura, N.; Yamada, K.; Suzuki, A. A new stereospecific cross-
coupling by the palladium-catalyzed reaction of 1-alkenylboranes with
1-alkenyl or 1-alkynyl halides. Tetrahedron Lett. 1979, 36, 3437–
3440.
(11) Wang, R. X.; You, X. Z.; Meng, Q. J.; Mintz, E. A.; Bu, X. R. A
modified synthesis of O-hydroxyaryl aldehydes. Synth. Commun. 1994,
24, 1757–1760.
(12) National Cancer Institute, Developmental Therapeutics Program. http://
d t p . n c i . n i h . g o v / d t p s t a n d a r d / s e r v l e t /
MeanGraph?searchtype)NSC&searchlist)125973&outputformat)HTML
&outputmedium)page&chemnameboolean)AND&debugswitch)false&
assaytype)&testshortname)NCI+Cancer+Screen+Current+Data&
dataarraylength)86&endpt)GI50&button)Mean+Graph&highconc)-
6.0 (accessed Apr 2008).
(13) Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.;
Belew, R. K.; Olson, A. J. Automated docking using a Lamarckian
genetic algorithm and an empirical binding free energy function.
J. Comput. Chem. 1998, 19, 1639–1662.
(14) Sybyl7.0; Tripos Inc.: St. Louis, MO, 2003; http://www.tripos.com.
(15) Otwinowski, Z.; Minor, W. Processing of X-ray diffraction data
collected in oscillation mode. Methods Enzymol. 1997, 276, 307–326.
(16) Minor, W.; Cymborowski, M.; Otwinowski, Z.; Chruszcz, M. HKL-
3000: the integration of data reduction and structure solution. From
[3H]Colchicine Binding Assays. The binding of [3H]colchicine
to tubulin was measured by the DEAE-cellulose filter method, as
described in detail previously.23 The tubulin and [3H]colchicine
concentrations were 1.0 µM (0.1 mg/mL) and 5.0 µM, respectively.
Tubulin Polymerization. Tubulin polymerization was followed
turbidimetrically at 350 nm in Beckman model DU-7400 and
Du7500 spectrophotometers equipped with electronic temperature
controllers, as described in detail previously.24 The tubulin con-
centration was 10 µM (1.0 mg/mL).
Cell Proliferation Assay. HCT-116 colon cancer cells (ATCC)
were maintained in McCoy’s 5a medium with 1.5 mM L-glutamine
(Invitrogen) supplemented with 5% fetal bovine serum (Atlanta
Biologicals). MDA-MB-435 cancer cells (ATCC) were maintained
in D-MEM with 1.5 mM L-glutamine supplemented with 5% fetal
bovine serum (Optima). Cells were grown at 37 °C in an incubator
with a humidified, 5% CO2 atmosphere.
Cells grown to confluency were trypinized, counted, and diluted
to 6400 cells per 100 µL, and 3200 cells/well were added to a clear
96-well tissue culture plate (Falcon). Cells were allowed to attach,
and 50 µL of media with vehicle or concentrations of drug at 10
nM, 30 nM, 100 nM, 300 nM, 1 µM, or 3 µM was added to each
well (n ) 8). The cells were incubated for 72 h. The CellTiter 96
AQueous nonradioactive cell proliferation assay (Promega) was used
to measure the number of living cells in each well. The absorbance
was read at 490 nm on an ELISA plate reader, and the data were

2,3-Dihydro-2-arylquinazolin-4-ones
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4631
diffraction images to an initial model in minutes. Acta Crystallogr.,
Sect. D 2006, 62, 859–866.
(20) Case, D. A.; Darden, T. A.; Cheatham, T. E., III; Simmerling, C. L.;
Wang, J.; Duke, R. E.; Luo, R.; Merz, K. M.; Wang, B.; Pearlman,
D. A.; Crowley, M.; Brozell, S.; Tsui, V.; Gohlke, H.; Mongan, J.;
Hornak, V.; Cui, G.; Beroza, P.; Schafmeister, C.; Caldwell, J. W.;
Ross, W. S.; Kollman, P. A. AMBER 8; University of California: San
Francisco, CA, 2004.
(21) Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D. A.
Development and testing of a general amber force field. J. Comput.
Chem. 2004, 25, 1157–1174.
(22) Bayly, C. I.; Cieplak, P.; Cornell, W. D.; Kollman, P. A. A well-
behaved electrostatic potential based method using charge restraints
for deriving atomic charges: the RESP model. J. Phys. Chem. 1993,
97, 10269–10280.
(23) Verdier-Pinard, P.; Lai, J. Y.; Yoo, H. D.; Yu, J. R.; Marquez, B.;
Nagle, D. G.; Nambu, M.; White, J. D.; Falck, J. R.; Gerwick, W. H.;
Day, B. W.; Hamel, E. Structure-activity analysis of the interaction
of curacin A, the potent colchicine site anti-mitotic agent, with tubulin
and effects of analogs on the growth of MCF-7 breast cancer cells.
Mol. Pharmacol. 1998, 53, 62–76.
(17) Maple, J. R.; Hwang, M.-J.; Stockfisch, T. P.; Dinur, U.; Waldman,
M.; Ewig, C. S.; Hagler, A. T. Derivation of class II force fields. 1.
Methodology and quantum force field for the alkyl functional group
and alkane molecules. J. Comput. Chem. 1994, 15, 162–182.
(18) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb,
M. A.; Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery, J. A., Jr.;
Stratmann, R. E.; Burant, J. C.; Dapprich, S.; Millam, J. M.; Daniels,
A. D.; Kudin, K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.; Barone,
V.; Cossi, M.; Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo, C.;
Clifford, S.; Ochterski, J.; Petersson, G. A.; Ayala, P. Y.; Cui, Q.;
Morokuma, K.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.;
Foresman, J. B.; Cioslowski, J.; Ortiz, J. V.; Stefanov, B. B.; Liu, G.;
Liashenko, A.; Piskorz, P.; Komaromi, I.; Gomperts, R.; Martin, R. L.;
Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara,
A.; Gonzalez, C.; Challacombe, M.; Gill, P. M. W.; Johnson, B. G.;
Chen, W.; Wong, M. W.; Andres, J. L.; Head-Gordon, M.; Replogle,
E. S.; Pople, J. A. Gaussian 98; Gaussian, Inc.: Pittsburgh, PA, 1998.
(24) Hamel, E. Evaluation of antimitotic agents by quantitative comparisons
of their effects on the polymerization of purified tubulin. Cell Biochem.
Biophys. 2003, 38, 1–21.
(19) Rarey, M.; Kramer, B.; Lengauer, T.; Klebe, G. Predicting receptor-
ligand interactions by an incremental construction algorithm. J. Mol.
Biol. 1996, 261, 470–489.
JM800271C