Synthesis of casimiroin and optimization of its quinone reductase 2 and aromatase inhibitory activities

Article abstract of DOI:10.1021/jm801335z

An efficient method has been developed to synthesize casimiroin (1), a component of the edible fruit of Casimiroa edulis, on a multigram scale in good overall yield. The route was versatile enough to provide an array of compound 1 analogues that were evaluated as QR2 and aromatase inhibitors. In addition, X-ray crystallography studies of QR2 in complex with compound 1 and one of its more potent analogues has provided insight into the mechanism of action of this new series of QR2 inhibitors. The initial biological investigations suggest that compound 1 and its analogues merit further investigation as potential chemopreventive or chemotherapeutic agents.

Full text of DOI:10.1021/jm801335z

J. Med. Chem. 2009, 52, 1873–1884
1873
Synthesis of Casimiroin and Optimization of Its Quinone Reductase 2 and Aromatase
Inhibitory Activities
Arup Maiti,† P. V. Narasimha Reddy,† Megan Sturdy,‡ Laura Marler,§ Scott D. Pegan,‡ Andrew D. Mesecar,‡ John M. Pezzuto,§
and Mark Cushman*,†
Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmaceutical Sciences, and the Purdue Cancer
Center, Purdue UniVersity, West Lafayette, Indiana 47907, Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry
and Pharmacognosy, College of Pharmacy, The UniVersity of Illinois at Chicago, Chicago, Illinois 6060, College of Pharmacy, UniVersity of
Hawaii at Hilo, Hilo, Hawaii 96720
ReceiVed October 22, 2008
An efficient method has been developed to synthesize casimiroin (1), a component of the edible fruit of
Casimiroa edulis, on a multigram scale in good overall yield. The route was versatile enough to provide an
array of compound 1 analogues that were evaluated as QR2 and aromatase inhibitors. In addition, X-ray
crystallography studies of QR2 in complex with compound 1 and one of its more potent analogues has
provided insight into the mechanism of action of this new series of QR2 inhibitors. The initial biological
investigations suggest that compound 1 and its analogues merit further investigation as potential
chemopreventive or chemotherapeutic agents.
Introduction
49% sequence identity with the NAD(P)H-dependent quinone
reductase 1 (QR1), but does not recognize NADH or NAD-
(P)Ha as cosubstrates. Instead, it catalyzes the oxidation of
reduced N-alkyl- and N-ribosylnicotinamides by menadione and
other quinones. Because of its high similarity in both primary
amino acid sequence and tertiary structure to QR1, it is believed
that QR2 might also serve as a detoxification enzyme that can
produce hydroquinones that are less toxic to cells compared to
the parent quinones. However, recent studies have indicated that
QR2 may actually transform certain quinone substrates into more
highly reactive species that are capable of causing more cellular
damage.9-11 Therefore, it is hypothesized that inhibition of QR2
in certain cases may lead to protection of cells against these
reactive species. Melatonin, a known antioxidant, inhibits QR2
and its X-ray structure in complex with QR2 has been
determined.6 Coenzyme Q0, another antioxidant, has been
shown to be an efficient substrate of the QR2-catalyzed
reaction.12 These observations suggest a mysterious link between
QR2 and cellular antioxidant status. Several QR2 inhibitors have
been reported in the literature.13 QR2 is inhibited by a variety
of natural polyphenols,12 and the X-ray crystal structure of QR2
in complex with the putative chemopreventive agent resveratrol,
a potent inhibitor of the enzyme, has been determined.6,14 These
latter observations motivated the present study.
The medicinal values of zapote blanco, an edible fruit of
Casimiroa edulis Llave et Lex (Rutaceae), have been proposed
for many years. Our continuous efforts to discover chemopre-
ventive agents from natural products led us to perform a study
on casimiroin (1), a key constituent of the seeds of zapote
blanco, that showed the ability to inhibit mutagenicity induced
by 7,12-dimethylbenz[a]anthracene (DMBA) in Salmonella
typhimurium strain TM677, as well as inhibition of the formation
of DMBA-induced preneoplastic lesions in mouse mammary
gland organ culture (MMOC).1 Compound 1, therefore, has the
potential to inhibit carcinogenesis. The goals of the present
investigation were to devise and execute a practical synthesis
of compound 1 that would afford larger quantities for advanced
biological testing and also to optimize the potential of compound
1 analogues as inhibitors of QR2 and aromatase (CYP19A1).
Quinone reductase 2 (QR2), a cytosolic FAD-dependent
flavoenzyme that catalyzes metabolic reduction of quinones with
unexpected cosubstrate specificity, is expressed in various organs
including heart, liver, skeletal muscle, and kidney.2 It has been
described as an enzyme of surprises and mysteries,3 and recent
studies have revealed that genetic polymorphisms of QR2 are
associated with a number of neurological diseases such as
Parkinson’s, schizophrenia, and others.4-6 QR2 also has a
unique relationship with quinoline-containing antimalarial
drugs,7 and it binds melatonin, which has led to its classification
as the third melatonin receptor binding site or MT3.8 QR2 shares
Aromatase, a key cytochrome P450 enzyme, catalyzes the
rate-limiting aromatization step in the conversion of androgens
(testosterone and androstenedione) to estrogens (estradiol and
estrone). Aromatase inhibitors decrease bioavailable estrogen
and have been shown to have considerable antitumor activity
in certain breast cancers.15 Such compounds ultimately reduce
estradiol receptor stimulation and reduce formation of genotoxic
estrogen metabolites.16 Certain nonsteroidal aromatase inhibitors
a Abbreviations: DPPA, diphenylphosphoryl azide; TMS, trimethylsilyl;
FAD, flavin adenine dinucleotide; HRMS, high-resolution mass spectrom-
etry; EIMS, electron-impact mass spectrometry; DMF, dimethylformamide;
ESIMS, electrospray-ionization mass spectrometry; MTT, (3-(4,5-dimeth-
ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; BSA, bovine serum albu-
min; DMSO, dimethylsulfoxide; SER-CAT, Southeast Regional Collabo-
rative Access Team; CCD, charge-coupled device; CCP4, collaborative
computational project number 4; NADPH, the reduced form of nicotinamide
adenine dinucleotide phosphate (NADP+).
* To whom correspondence should be addressed. Phone: 765-494-1465.
Fax: 765-494-6790. E-mail: cushman@pharmacy.purdue.edu.
†
Purdue University.
University of Illinois at Chicago.
University of Hawaii at Hilo.
‡
§
10.1021/jm801335z CCC: $40.75
2009 American Chemical Society
Published on Web 03/06/2009

1874 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Scheme 1. Retrosynthetic Analysis of Compound 1
Maiti et al.
Scheme 2a
are already in clinical use for the treatment of breast cancer,
and several naturally occurring nonsteroidal aromatase inhibitors
have shown promising chemopreventive activity.17
In our group, QR2 and aromatase have been targeted for
identification of naturally occurring chemopreventive agents.6
Our preliminary observation of the antimutagenic activity of
compound 1,1 along with the limited quantities of compound 1
from natural sources, led us to synthesize compound 1 and its
analogues for further biological testing. This report discloses a
new and improved total synthesis of compound 1, the discovery
of its QR2 and aromatase inhibitory activities, and the optimiza-
tion of these activities resulting in a new series of potent
inhibitors of both enzymes. In addition, X-ray crystallography
studies of QR2 in complex with compound 1 and a more potent
analogue are described. These studies have provided insight into
the mechanism of QR2 inhibition by compound 1 and related
compounds.
a Reagents and conditions: (i) (PhO)2PON3, Et3N, PhCH2OH, PhH, 3 h;
(ii) NaH, MeI, rt, 2 h; (iii) Pd/C, H2, EtOAc, 30 psi, 1 h; (iv) diethyl
malonate, 100-110°C, 3 h; (v) (a) NaOH, 60°C, 2 h, (b) HCl; (vi) PPA,
95-100 °C 3 h; (vii) TMSCH2N2, MeOH-Et2O, rt, 24 h.
Results and Discussion
biological assays to determine its chemopreventive activity. The
encouraging biological results motivated the synthesis of
additional compound 1 congeners in order to gain insight into
the structure-activity relationships and mechanism of action.
Compound 1 analogues 1a-q (Scheme 3) were synthesized
by treating the corresponding amines 3a-q with diketene in
refluxing benzene to yield ketoamides 10a-q, followed by a
polyphosphoric acid (PPA)-mediated cyclization that afforded
very high yields, except for compounds 1j and 1p. PPA-
mediated cyclization of compounds 10j and 10p produced
quinolinones 1j and 1p, respectively, as minor products. The
desired products 1j and 1p were therefore prepared in very high
yield by methylating compounds 1i and 1o, respectively, using
NaH and MeI in DMF, as shown in Scheme 4. Quinolinone 1r
was also prepared from 1q in a similar way. Compound 1a was
synthesized in 82% yield by treating ketoamide compound 10a
with trifluoroacetic acid at room temperature.
Most of the free amines or monomethylated amines are
commercially available. However, some of them had to be
synthesized from the corresponding acids via Curtius rearrange-
ment as shown in Scheme 5. Amines 3l and 3q were synthesized
in good yields following literature procedures.20,21 Quinolinone
1l was used to synthesize quinone 12 following a previously
reported literature procedure as outlined in Scheme 6.21
Chemistry. Although a synthesis of compound 1 has been
reported in the literature, it has a month-long critical cyclization
step that provides the desired intermediate as a minor product
in only 10% yield, resulting in a very poor overall yield (0.67%)
over 11 steps.18 The final step involves several recrystallizations
followed by sublimation to remove contaminating byproduct.
A retrosynthetic analysis of compound 1 is shown in Scheme
1. The key step is the synthesis of intermediate 3b, a degradation
product of compound 1, by a Curtius rearrangement. The final
product 1 was planned to be made by acid-mediated intramo-
lecular Friedel-Crafts acylation followed by a methylation of
an enol.
In practice, the present synthesis of compound 1 was executed
in a straightforward manner, without any unexpected difficulties,
as outlined in Scheme 2. The degradation product 3b of
compound 1 was easily prepared in six steps from commercially
available 2,3-dihydroxy benzoic acid (4) using a modified
literature procedure19 in 27% overall yield. Curtius rearrange-
ment of dioxole benzoic acid 5a using DPPA and Et3N in
refluxing benzene, followed by in situ quenching with benzyl
alcohol, produced compound 6a. N-Methylation of 6a using
NaH and MeI in DMF, followed by hydrogenolysis of the benzyl
group and decarboxylation, yielded N-methylated amine 3b.
Acylation of the secondary amine 3b with diethyl malonate
afforded intermediate 8, which was saponified using sodium
hydroxide to provide the acid 2. Polyphosphoric acid-mediated
cyclization led to intermediate 9. Methylation of 9 with TMS-
diazomethane afforded compound 1 as a white powder in ∼15%
overall yield from commercially available acid 4. The 1H NMR,
13C NMR, IR, mass spectral, and elemental analysis data of the
synthetic compound 1 were consistent with those reported for
the natural product. Synthetic compound 1 was used for various
Quinone Reductase 2 Inhibitory Activity. QR2 inhibition
assays were performed on synthetic compound 1 and its various
analogues, as described in the Experimental Section. The IC50
values are reported in Table 1. Synthetic compound 1 was
determined to have an IC50 value of 54.1 ( 6.7 µM. Most of
the compound 1 analogues displayed superior inhibitory potency
compared with the natural product and were active in the lower
micromolar range. In general, N-methylated analogues showed

Casimiroin Synthesis and Inhibitory ActiVities
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1875
Scheme 5a
Scheme 3a
a Reagents and conditions: (i) (PhO)2PON3, Et3N, PhCH2OH, PhH, 3 h;
(ii) NaH, MeI, DMF, rt, 2 h; (iii) Pd/C, H2, EtOAc, 30 psi, 1 h.
Scheme 6a
a Reagents and conditions: (i) diketene, PhH, 95-100 °C, 3 h, or
CF3CO2H, rt; (ii) PPA, 95-100 °C, 3 h.
Scheme 4a
a Reagents and conditions: (i) HBr, heat, 48 h; (ii) K2Cr2O7, H2SO4,
acetone, 0 °C, 10 min.
another methoxy group at the 7 position in 8-methoxylated
quinolines (1e,1d) decreased the inhibitory potency (1g, IC50
29.3 ( 3.4 µM, 1h, IC50 9.3 ( 2.3 µM). In contrast,
N-methylated 6,8-dimethoxy derivative 1j (IC50 1.9 ( 0.2 µM)
and the non-N-methylated 6,8-dimethoxy compound 1i (IC50
8.8 ( 1.1 µM) have superior potency over other disubstituted
analogues. Dimethoxy derivatives 1k (IC50 10.8 ( 1.5 µM), 1l
(IC50 4.1 ( 0.6 µM), and trimethoxy derivative 1m (IC50 10.0
( 1.4 µM) and 1n (IC50 7.0 ( 1.1 µM) revealed a similar pattern
but were less potent than the 6,8-dimethoxy derivatives.
Surprisingly, a change in pattern was observed for other
trimethoxylated series. Non-N-methylated 1o (IC50 6.0 ( 0.6
µM) had better potency than N-methylated 1p (IC50 10.8 ( 1.3
µM). A decrease in inhibitory potency for N-methylated 6,7,8-
trimethoxy derivatives relative to 5,6,8-trimethoxy analogues
and completely inactive tetramethoxy analogues 1q and 1r
suggests that the steric bulk of the consecutive methoxy groups
may force them to be pointed out of the planar ring system,
making the π-π stacking interaction with FAD unfavorable.
X-Ray Structure Analysis. To gain insight into the binding
mode of this new series of QR2 inhibitors, the X-ray crystal
structures of compound 1 and one of the most potent analogues,
1l, from the series in complex with human QR2 were deter-
mined. Complete X-ray data sets were collected and processed
a Reagents and conditions: (i) NaH, MeI, DMF, 80 °C, 12 h.
better potency than non-N-methylated quinolinones in inhibiting
QR2. Compound 1j, with IC50 1.9 ( 0.2 µM, is comparable in
potency with most of the QR2 inhibitors reported in the
literature. The replacement of the 6-methoxy group of compound
1 by a methyl group increases the inhibitory activity more than
8-fold (1b, IC50 ) 6.2 ( 0.8 µM). Non-N-methylated quinoli-
none 1a (IC50 ) 10.8 ( 2.1 µM) showed a decrease in activity
relative to 1b but still has better potency than the natural product.
Complete removal of the dioxole ring from 1a made the
resulting compound 1c inactive; however, N-methylated com-
pound 1d showed an IC50 value of 18.2 ( 3.1 µM, an
approximately 3-fold improvement of activity relative to
compound 1. The presence of an 8-methoxy group (1e,1f)
improved the activity relative to the unsubstituted derivatives
(1c,1d). Quinolinone 1f had an IC50 value 5.8 ( 0.9 µM, which
is an approximately 10-fold increase in potency compared with
the natural product.
To analyze the effect of methoxy substitutions on the benzene
ring, several dimethoxylated (1g-l), trimethoxylated (1m-p),
and tetramethoxylated (1q-r) quinolinone derivatives were
synthesized and evaluated. It was found that introduction of

1876 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Maiti et al.
Table 1. Inhibition of QR2 and Aromatase by Compound 1 and Its Analogues
cytotoxicity IC50 (µM)
quinolinones
QR2 IC50 (µM)
aromatase IC50 (µM)
Hepa 1c1c7
MCF7
LNCaP
LU
1
54.1 ( 6.7
10.8 ( 2.1
6.2 ( 0.8
>500
3.92 ( 0.67
>98.49
1.25 ( 0.03
>125.7
>115.6
>105.8
2.02 ( 0.1
31.5 ( 3.1
5.76 ( 0.07
>91.29
0.96 ( 0.02
26.8 ( 13.77
0.96 ( 0.01
18.47 ( 3.7
2.72 ( 0.02
14.8 ( 0.65
0.10 ( 0.01
5.68 ( 0.24
0.76 ( 0.02
>98.4
>85.81
>98.49
>92.14
>125.7
>115.6
>105.8
>98.48
>91.29
>85.80
>91.29
>85.80
>91.22
>85.74
>80.29
>76.01
34.52
>85.81
>98.49
>92.14
>125.7
>115.6
>105.8
>98.48
>91.29
>85.80
>91.29
>85.80
>91.22
>85.74
>80.29
>76.01
42.95
>85.81
>98.49
>92.14
>125.7
>115.6
>105.8
>98.48
>91.29
>85.80
>91.29
>85.80
>91.22
>85.74
>80.29
>76.01
45.76
>85.81
>98.49
>92.14
>125.7
>115.6
>105.8
>98.48
>91.29
>85.80
>91.29
>85.80
>91.22
>85.74
>80.29
>76.01
35.28
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
18.2 ( 3.1
24.1 ( 3.4
5.8 ( 0.9
29.3 ( 3.4
9.3 ( 2.3
8.8 ( 1.1
1.9 ( 0.2
10.8 ( 1.5
4.1 ( 0.6
10.0 ( 1.4
7.0 ( 1.1
6.0 ( 0.6
10.8 ( 1.3
>500
1o
1p
1q
1r
70.73
>76.01
>71.66
>68.23
60.53
NT
NT
>76.01
>71.66
>68.23
58.07
NT
NT
68.47
>71.66
>68.23
54.14
NT
NT
>71.66
>68.23
49.22
NT
NT
>500
>500
0.96 ( 0.13
11.3 ( 2.1
12
resveratrol6
melatonin6
aminoglutethimide
NT
NT
0.27
NT
NT
NT
NT
a Not tested.
Table 2. Data Collection and Refinement Statistics for QR2-Inhibitor
Complexes
(Figure 1A, color code yellow), the methoxy group of compound
1 points toward Phe178, whereas in the second orientation, it
orients toward solvent-exposed surface (Figure 1A, color code
green). Despite this difference, the interactions of compound 1
within the QR2 active sites are similar and involve mainly a
hydrogen bond between the carbonyl oxygen of compound 1
and the side chain amide nitrogen of Asn161 and a series of
hydrophobic contacts with surrounding side chains and the flavin
ring of FAD. These interactions are maintained for both
compound 1 and compound 1l.
QR2-inhibitor complex
1
1l
data collection
space group
unit cell dimensions
a, b, c (Å)
P212121
56.73, 84.23, 106.31
P212121
56.34, 83.88, 106.69
resolution (Å)
no. reflections observed
no. unique reflections
Rmerge (%)
66.08-1.85(1.92-1.85) 65.49-1.98(2.04-1.98)
241450
197663
44812
35682
9.3(62.9)a
17.8(2.0)a
99.9(99.4)a
8.4(29.1)a
21.1(4.5)a
99.3(95.2)a
I/σI
% completeness
The single orientation of 1l is nearly identical between sites
1 and 2 of the dimer (Figure 1B). A superposition of site 1
from the QR2-compound 1 structure with site 1 of the QR2-1l
structure reveals that one of the compound 1 orientations is
nearly identical to that of 1l with respect to the quinolinone
ring (Figure 2). In general, a careful analysis of the two crystal
structures reveal that these two inhibitors, 1 and 1l, bind within
the active site and sit parallel to the plane of the isoalloxazine
ring of the bound cofactor FAD, occupying very similar
positions as menadione in the QR2-menadione complex,22
resveratrol in the QR2-resveratrol complex,13 CB1954 in the
QR2-CB1954 complex,23 and melatonin in the QR2-melatonin
complex.6 All of these molecules bind deeply within the
hydrophobic cleft of QR2, which is surrounded by side chains
of Tyr132′, Phe178′, Phe126′, Met164′, and Cys121′ from one
side of a QR2 monomer, and Tyr155, Phe106, and the
isoalloxazine ring of FAD from the other monomer. The side
chain of Trp105 forms the bottom of the hydrophobic cleft, and
on one end of the cleft reside the side chains of Asn161 and
the hydroxyl groups of Tyr155 and Tyr132. These latter two
amino acid side chains form a relatively hydrophilic surface at
one end of the cleft and also the hydroxyl group of Thr71′ and
main chain carbonyls from Gly68 and Asp117 all point toward
the cavity on the other end. The positions of these amino acid
residues indicate that both ends have numerous functional groups
accessible for hydrogen bonding. The fourth side of this unique
17 Å long and 7 Å wide cavity is accessible to the solvent.
Because of the narrow width of this cavity, the enzyme can
typically only accommodate molecules that can adopt nearly
flat conformations. The crystal structures indicate that the
refinement
resolution range
no. reflections in working set 42486
no. reflections in test set
Rwork (%)
66.08-1.84
65.49-1.98
33839
2256
1788
18.0 (28.5)a
23.1 (35.7)a
22.2
18.1 (22.2)a
22.7 (33.0)a
26.2
Rfree (%)
average B-factor (Å2)
a Highest resolution data shell is given in parentheses.
and the final structural models were refined to 1.84 Å and 1.98
Å for the compound 1 and compound 1l complexes, respec-
tively. The final data collection and refinement statistics are
summarized in Table 2. QR2 is a dimer in solution, and it
crystallizes as a dimer in the asymmetric unit with two nearly
equivalent catalytic sites defined as site 1 for chain A and site
2 for chain B.6 Crystals of QR2 in complex with compound 1
produced strong electron density in Fo - Fc difference maps
consistent with the natural product being bound in two different
orientations within each active site of the dimer (Figure 1A).
In contrast, QR2 crystals complexed with compound 1l produced
strong electron density for only one orientation of the inhibitor
within each active site of the dimer (Figure 1B).
Two molecules of compound 1 were manually built within
the electron density of the QR2-compound 1 active sites, and
the model was refined with the occupancy of each molecule to
50%. The two orientations of compound 1 within the active
site of a single monomer (site 1) are nearly identical to
the orientations within the active site of the second monomer
(site 2). The two orientations of compound 1 within a single
site are related to each other by a 180° flip. In the first orientation

Casimiroin Synthesis and Inhibitory ActiVities
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1877
Figure 2. Stereoview of a superposition of compound 1 and compound
1l bound within the active sites of human QR2. The two orientations
of compound 1 bound within the active sites of chain A (blue) and
chain B (magenta) are shown with compound 1l (yellow).
orientations, whereas their higher affinity analogues, such as
iodomelatonin, tend to bind in only one orientation.6 In that
study, we found that melatonin, with an IC50 of 11.3 µM, binds
in at least three orientations depending on which of three crystal
structures of this complex are analyzed, whereas iodomelatonin,
with an IC50 of 1.1 µM, binds in only one orientation in the
active sites of two independently determined X-ray structures.
Our observation that inhibitor binding to QR2 can be a stochastic
process dependent on the strength of the interaction has recently
been supported by X-ray structures of QR2 in complex with
dopamine and andrenochrome, where dopamine was found to
bind in two orientations.24
Aromatase Inhibitory Activity. An aromatase inhibition
assay was performed on synthetic compound 1 and its analogues,
as described in the Experimental Section. The IC50 values of
the compounds are reported in Table 1. Synthetic compound 1
was determined to have an IC50 value of 3.9 ( 0.67 µM. The
analogues showed good inhibitory potency against aromatase
except for a few N-nonmethylated analogues (1a, 1c, 1e, 1i)
and two N-methylated analogues (1d and 12). The replacement
of the 6-methoxy group of compound 1 by a methyl group
increased the inhibitory activity (1b, IC50 ) 1.2 ( 0.03 µM).
However, removal of the dioxole ring made the resulting
compounds 1c and 1d inactive. Introduction of an 8-methoxy
restored the inhibitory potency of 1f (IC50 2.0 ( 0.1 µM),
however, analogue 1e was not active. Interestingly, 7,8-
dimethoxy analogue 1g (IC50 31.5 ( 3.1 µM) showed moderate
inhibitory activity, but in comparison with the 8-methoxy
N-methylated analogue 1f, the activity of 1h was reduced (IC50
5.8 ( 0.07 µM). The 6,8-dimethoxy derivative 1i was found to
be inactive, but its congener 1j (IC50 0.96 ( 0.02 µM) showed
very potent inhibitory activity in the aromatase assay. All other
N-nonmethylated analogues (1k, 1m, 1o, 1q) and N-methylated
analogues (1l, 1n, 1p, 1r) showed a similar trend as we have
seen for 7,8-dimethoxy analogues (1g, 1h), with the N-
methylated inhibitors being more potent. Inhibitory activity for
1j, 1l, and 1r are almost the same and the IC50 values are in the
submicromolar range. 5,6,8-Trimethoxy-N-methylated analogue
1p displayed the best inhibitory potency, with an IC50 value of
0.1 ( 0.01 µM, which compares favorably with the clinically
useful aromatase inhibitor aminoglutethimide. These observa-
tions show that N-methylated analogues are better aromatase
inhibitors and methoxy substitution on the ring makes a
significant contribution to the potency.
Figure 1. X-ray structure and electron density surrounding inhibitors
in the active site of human QR2. In each stereoview, QR2 inhibitor
complexes depict all FAD and enzyme carbons in gray and beige,
oxygen atoms are red, and all nitrogen atoms are blue. Hydrogen bonds
between Asn161 and the inhibitors are represented as blue dashed lines.
(A) QR2 in complex with compound 1. The 2Fo - Fc electron density
surrounding the two orientations of compound 1 is contoured at 1σ in
black. Site 1, compound 1 binds in two orientations in both site 1 (green
and yellow) of chain A (top panel) and site 2 (blue and magenta) of
chain B (lower panel). The two binding orientations are rotated by
∼180° degrees from each other. Each pose was refined at 50%
occupancy. The different orientations in the two separate sites are
depicted in different colors for clarity. Ile128 and Met154 have been
removed for clarity. (B) QR2 in complex with compound 1l binds in
one orientation in both site 1 (blue) of chain A (top panel) and site 2
(yellow) of chain B (lower panel). The 2Fo - Fc electron density
surrounding compound 1l is contoured at 1σ in black.
benzene rings of these quinolinones stack with the C ring of
FAD, similar to the benzene ring of CB1954. Hydrophobic
interactions with Trp105, Gly68, Phe126, and Phe178 help to
stabilize inhibitor binding via interactions with the quinolinone
ring and carbonyl group. These two inhibitors occupy about
two-thirds of the active site.
Cytotoxicity Studies. Ideally, potential chemotherapeutic
agents should not be highly cytotoxic. To evaluate this aspect,
the cytotoxicities of the compounds were evaluated in four
different cell lines, and the data are presented in Table 1. The
results indicate that the present series of quinolinones are in
The observation that the weaker inhibitor, compound 1, binds
in two orientations within the active site of QR2, whereas the
10-fold more potent inhibitor 1l binds in only one orientation
is reminiscent of our seminal observation that weak binding
inhibitors of QR2, such as melatonin, tend to bind in multiple

1878 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Maiti et al.
phoric acid had dissolved, the solution was neutralized with 4 N
NaOH solution. The deposited solid was collected by filtration or
extracted with chloroform and evaporated to dryness. Solid was
purified by column chromatography on silica gel, eluting with ethyl
ether or ethyl acetate-hexane mixture to yield the pure product in
higher yield than obtained with method A.
general not significantly cytotoxic. Those expressing a low
degree of cytotoxicity were compounds 1o, 1p, and 12.
Conclusion
An efficient total synthesis of compound 1 and a series of
compound 1-based QR2 and aromatase inhibitors have been
successfully carried out. Structural modification of the parent
natural product resulted in the most active compound 1j vs QR2
and 1p vs aromatase. The crystal structures of compound 1 and
one of the potent analogues 1l in complex with QR2 provide a
detailed description of the enzyme active site that can be utilized
in the further design of new QR2 inhibitors.
The present series of compounds inhibits both aromatase and
QR2, two distinct targets for the development of chemopre-
ventive agents. As is the case with combination drug therapy,
dual targeting through a pleiotropic mechanism offers the
potential to enhance efficacy if the two drug targets can function
in a synergistic fashion.
Method C: Using Trifluoroacetic Acid. A solution of the
acetoacetamide (2 mmol) in trifluoroacetic acid (5 mL) was stirred
at room temperature for 24 h. The reaction mixture was evaporated
to dryness under reduced pressure. The residue was purified by
column chromatography on silica gel, eluting with ethyl ether or
ethyl acetate-hexane mixture to afford the pure product in good
yield. This method was used when isolation of the product was
very difficult with methods A and B because of solubility issues.
6-Methyl-[1,3]dioxolo[4,5-h]quinolin-8(9H)-one (1a). This com-
pound was prepared in 82% yield by following method C. Colorless
solid: mp 285 °C (dec). IR (KBr) 1655, 1645, 1573, 1477, 1435,
1405, 1395, 1292, 1242 cm-1. 1H NMR (CDCl3, 300 MHz) δ 7.31
(d, J ) 8.7 Hz, 1 H), 6.94 (dd, J ) 8.4, 0.9 Hz, 1 H), 6.49 (s, 1 H),
6.19 (d, J ) 0.9 Hz, 2 H), 2.51 (s, 3 H). 13C NMR (CDCl3, 75
MHz) δ 163.8, 152.8, 149.6, 134.2, 121.7, 119.1, 117.6, 116.1,
106.5, 103.3, 19.7. EIMS (m/z, relative intensity) 203 (M+, 100),
174 (17), 161 (6), 146 (35), 117 (27), 90 (11), 69 (23), 57 (27).
HRMS calcd for C11H9NO3 203.0582, found 203.0584. Anal.
(C11H9NO3) C, H, N.
6,9-Dimethyl-[1,3]dioxolo[4,5-h]quinolin-8(9H)-one (1b). This
compound was prepared in 73% yield by following method B.
Colorless solid: 158-159 °C. IR (KBr) 1652, 1590, 1506, 1416,
1382, 1328, 1261, 1124, 1093, 1050, 1004, 939, 753 cm-1. 1H NMR
(CDCl3, 300 MHz) δ 7.19 (d, J ) 8.4 Hz, 1 H), 6.77 (d, J ) 8.4
Hz, 1 H), 6.39 (s, 1 H), 6.01 (s, 2 H), 3.84 (s, 3 H), 2.35 (s, 3 H).
13C NMR (CDCl3, 75 MHz) δ 162.0, 149.0, 146.3, 133.5, 126.2,
119.5, 118.6, 117.9, 104.1, 100.8, 32.0, 19.4; EIMS (m/z, relative
intensity) 217 (M+, 100), 202 (6), 188 (41), 174 (42), 160 (20),
146 (10), 130 (25), 116 (8), 89 (14), 63 (11). HRMS calcd for
C12H11NO3 217.0739, found 217.0738. Anal. (C12H11NO3) C,
H, N.
4-Methylquinolin-2(1H)-one (1c). This compound was prepared
in 62% yield as a colorless solid by following method B: mp
214-216 °C (lit.25 mp 220-222 °C). IR (KBr) 3300, 2800, 1658,
1635, 1557, 1508, 1492, 1435, 1371, 1261 cm-1. 1H NMR (DMSO-
d6, 300 MHz) δ 7.69 (d, J ) 8.1 Hz, 1 H), 7.49 (m, 1 H), 7.29 (d,
J ) 8.1 Hz, 1 H), 7.18 (m, 1 H), 6.39 (s, 1 H), 2.41 (s, 3 H). 13C
NMR (DMSO-d6, 75 MHz) δ 161.6, 147.9, 138.6, 130.2, 124.7,
121.6, 120.8, 119.5, 115.4, 18.4. EIMS (m/z, relative intensity) 159
(M+, 44), 130 (100), 103 (9), 89 (5), 77 (11), 63 (6), 51 (7). HRMS
calcd for C10H9NO 159.0684, found 159.0683. Anal. (C10H9NO)
C, H, N.
1,4-Dimethylquinolin-2(1H)-one (1d). This compound was
prepared in 82% yield as a colorless solid by following method B:
mp 132-134 °C (lit.26 mp 130-132 °C). IR (KBr) 1652, 1615,
1592, 1562, 1454, 1370, 1321 cm-1. 1H NMR (CDCl3, 300 MHz)
δ 7.68 (dd, J ) 1.2, 7.8 Hz, 1 H), 7.54 (dd, J ) 1.2, 8.4, Hz, 1 H),
7.34 (d, J ) 8.4 Hz, 1 H), 7.23 (dd, J ) 1.2, 7.8 Hz, 1 H), 6.57 (s,
1 H), 3.67 (s, 3 H), 2.43 (s, 3 H). 13C NMR (CDCl3, 75 MHz) δ
162.0, 146.3, 139.7, 130.4, 125.1, 121.8, 121.3, 121.0, 114.3 29.1,
18.9. EIMS (m/z, relative intensity) 173 (M+, 82), 144 (100), 130
(71), 115 (15), 103 (12), 89 (7), 77 (17), 63 (8), 51 (9). HRMS
calcd for C11H11NO 173.0841, found 173.0841. Anal. (C11H11NO)
C, H, N.
Experimental Section
NMR spectra were obtained at 300 MHz (1H) and 75 MHz (13C)
in CDCl3 using CHCl3 as internal standard. Flash chromatography
was performed with 230-400 mesh silica gel. TLC was carried
out using commercially available precoated glass silica gel plates
of 2.5 mm thickness. Melting points are uncorrected. Microanalyses
were performed at the Purdue Microanalysis Laboratory. All yields
refer to yields of isolated compounds. Unless otherwise stated,
chemicals and solvents were of reagent grade and used as obtained
from commercial sources without further purification. Acetone was
distilled from potassium carbonate, dichloromethane from CaH2,
benzene from P2O5, and triethylamine from KOH prior to use.
6-Methoxy-9-methyl-[1,3]dioxolo[4,5-h]quinolin-8(9H)-one (1).
A suspension of hydroxyquinolone 9 (300 mg, 1.3 mmol) in
ether (1 mL) at 0 °C was treated with excess TMS-diazomethane
(2 M in hexanes, 10 mL), and the mixture was stirred for 20
min. Absolute methanol (10 mL) was added dropwise, and the
mixture was stirred for 6 h. An additional amount of TMS-
diazomethane (2 M in hexanes, 5 mL) and absolute methanol
(5 mL) were added successively to the mixture, and the mixture
was stirred for another 12 h. After completion of the reaction,
the mixture was treated with a solution of dilute acetic acid (0.5
mL) to decompose the excess diazomethane. Evaporation of the
solution and purification by silica gel column chromatography,
eluting with an acetone-hexane (1:1) mixture, yielded compound
1 (240 mg, 78%) as a colorless solid: mp 199-201 °C (lit.18
mp 202-203 °C). IR (KBr) 3319, 1735, 1645, 1592, 1444, 1465,
1331, 1265, 1232, 1159, 1057, 940 cm-1. 1H NMR (CDCl3, 300
MHz) δ 7.51 (d, J ) 8.4 Hz, 1 H), 6.74 (d, J ) 8.4 Hz, 1 H),
6.01 (s, 2 H), 5.86 (s, 1 H), 3.87 (s, 3 H) 3.80 (s, 3 H). 13C
NMR (CDCl3, 75 MHz) δ 164.0, 162.6, 149.8, 133.5, 126.5,
118.0, 112.9, 104.2, 100.2, 94.5, 55.8, 31.9. EIMS (m/z, relative
intensity) 233 (M+, 100), 218 (26), 190 (31), 176 (24), 160 (10),
146 (9), 132 (8), 117 (7), 69 (17). HRMS calcd for C12H11NO4
233.0688, found 233.0692. Anal. (C12H11NO4) C, H, N.
General Procedures for Cyclization, Method A: Using
H2SO4. A solution of the acetoacetamide (2 mmol) in concd sulfuric
acid (5 mL) was stirred at room temperature for 30-60 min. The
reaction mixture was poured on ice (1 g) and neutralized or slightly
basified to pH ∼8 with 28-30% aqueous ammonia (5-10 mL).
The aqueous solution was extracted with chloroform (3 × 50 mL)
and the organic extract was dried and evaporated to dryness under
reduced pressure. The residue was purified by column chromatog-
raphy on silica gel, eluting with ethyl ether or ethyl acetate-hexane
mixture to produce the pure product in moderate yield.
Method B: Using Polyphosphoric Acid (PPA). The acetoac-
etamide (2 mmol) was added to polyphosphoric acid (5 g) at
95-100 °C and the mixture was allowed to stir for 2.5 h at the
same temperature and then poured into ice-water. After polyphos-
8-Methoxy-4-methylquinolin-2(1H)-one (1e). This compound
was prepared in 90% yield as a colorless solid by following method
B: mp 188-190 °C (lit.27 187-189 °C). IR (KBr) 3400, 2800,
1
1649, 1605, 1473, 1463, 1390, 1266 cm-1. H NMR (CDCl3, 300
MHz) δ 9.33 (brs, 1 H), 7.25 (d, J ) 8.1 Hz, 1 H), 6.79 (m, 1 H),
6.97 (d, J ) 8.1 Hz, 1 H), 6.53 (s, 1 H), 3.96 (s, 3 H), 2.45 (s, 3
H). 13C NMR (CDCl3, 75 MHz) δ 161.6, 148.7, 145.5, 128.1, 121.7,
121.2, 120.5, 116.1, 109.7, 55.9, 19.1. EIMS (m/z, relative intensity)
189 (M+, 100), 174 (17), 160 (58), 146 (32), 130 (27), 118 (31),
91 (21), 65 (20), 51 (14). HRMS calcd for C11H11NO2 189.0790,
found 189.0787. Anal. (C11H11NO2) C, H, N.

Casimiroin Synthesis and Inhibitory ActiVities
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1879
8-Methoxy-1,4-dimethylquinolin-2(1H)-one (1f). This com-
pound was prepared in 72% yield as a yellowish solid by following
method B: mp 72-74 °C (lit.28 mp 69-70.5 °C). IR (KBr) 1657,
1611, 1595, 1459, 1385, 1260 cm-1. 1H NMR (CDCl3, 300 MHz)
δ 7.36 (d, J ) 8.1 Hz, 1 H), 7.32 (m, 1 H), 7.11 (d, J ) 8.1 Hz,
1 H), 6.64 (s, 1 H), 3.97 (s, 3 H), 3.95 (s, 3 H), 2.47 (s, 3 H). 13C
NMR (CDCl3, 75 MHz) δ 163.2, 148.6, 145.9, 131.1, 123.4, 122.3,
121.2, 117.4, 113.5, 56.3, 35.2, 19.4. EIMS (m/z, relative intensity)
203 (M+, 80), 188 (100), 173 (27), 160 (51), 144 (43), 130 (45),
103 (7), 69 (13), 55 (12). HRMS calcd for C12H13NO2 203.0946,
found 203.0946. Anal. (C12H13NO2) C, H, N.
7,8-Dimethoxy-4-methylquinolin-2(1H)-one (1g). This com-
pound was prepared in 55% yield as a colorless solid by following
method A: mp 187-189 °C. IR (KBr) 1651, 1620, 1600, 1515,
1469, 1396, 1284, 1104 cm-1. 1H NMR (CDCl3, 300 MHz) δ 9.26
(brs, 1 H), 7.28 (d, J ) 8.1 Hz, 1 H), 6.77 (d, J ) 8.1, Hz, 1 H),
6.61 (s, 1 H), 3.88 (s, 3 H), 3.87 (s, 3 H), 2.35 (s, 3 H). 13C NMR
(CDCl3, 75 MHz) δ 162.3, 152.5, 148.6, 133.6, 132.3, 119.9, 118.6,
115.3, 107.2, 60.8, 56.0, 18.9. EIMS (m/z, relative intensity) 219
(M+, 100), 204 (43), 190 (19), 130 (12), 69 (17), 43 (16). HRMS
calcd for C12H13NO3 219.0895, found 219.0895. Anal. (C12H13NO3)
C, H, N.
7,8-Dimethoxy-1,4-dimethylquinolin-2(1H)-one (1h). This com-
pound was prepared in 78% yield as a colorless solid by following
method B: mp 118-120 °C. IR (KBr) 1655, 1589, 1557, 1454,
1387, 1324, 1268, 1057, 795 cm-1. 1H NMR (CDCl3, 300 MHz) δ
7.35 (d, J ) 9.0 Hz, 1 H), 6.85 (d, J ) 9.0, Hz, 1 H), 6.40 (s, 1 H),
3.91 (s, 3 H), 3.87 (s, 3 H), 3.72 (s, 3 H), 2.34 (s, 3 H). 13C NMR
(CDCl3, 75 MHz) δ 163.8, 154.9, 145.9, 137.0, 135.0, 120.9, 118.9,
117.6, 107.0, 61.6, 56.1, 33.8, 19.2. EIMS (m/z, relative intensity)
233 (M+, 61), 218 (93), 202 (35), 175 (100), 130 (20), 118 (18),
77 (22), 51 (17). HRMS calcd for C13H15NO3 233.1052, found
233.1057. Anal. (C13H15NO3) C, H, N.
(s, 1 H), 3.80 (s, 3 H), 3.79 (s, 3 H), 3.77 (s, 3 H), 2.55 (s, 3 H).
13C NMR (CDCl3, 75 MHz) δ 163.8, 152.7, 147.4, 143.2, 133.9,
121.9, 115.4, 104.1, 57.8, 55.8, 36.0, 25.1. EIMS (m/z, relative
intensity) 233 (M+, 52), 218 (100), 203 (38), 190 (34), 175 (68),
147 (14), 132 (10), 118 (12), 91 (8), 77 (7). HRMS calcd for
C13H15NO3 233.1052, found 233.1056. Anal. (C13H15NO3) C,
H, N.
6,7,8-Trimethoxy-4-methylquinolin-2(1H)-one (1m). This com-
pound was prepared in 70% yield as a colorless solid by following
method B: mp 218-220 °C. IR (KBr) 1647, 1598, 1495, 1461,
1406, 1388, 1378, 1276, 1195, 1120, 1015, 981, 795 cm-1. 1H NMR
(CDCl3, 300 MHz) δ 9.28 (brs, 1 H), 6.76 (s, 1 H), 6.44 (s, 1 H),
3.97 (s, 3 H), 3.91 (s, 3 H), 3.88 (s, 3 H), 2.39 (s, 3 H). 13C NMR
(CDCl3, 75 MHz) δ 161.8, 149.0, 147.9, 143.7, 139.1, 127.0, 120.5,
115.9, 100.9, 61.3, 61.1, 56.2, 19.2. EIMS (m/z, relative intensity)
249 (M+, 100), 234 (88), 220 (24), 203 (16), 191 (50), 176 (7),
163 (23), 148 (19), 134 (4), 120 (16), 92 (6), 65 (7). HRMS calcd
for C13H15NO4 249.1001, found 249.1005. Anal. (C13H15NO4) C,
H, N.
6,7,8-Trimethoxy-1,4-dimethylquinolin-2(1H)-one (1n). This
compound was prepared by following method B in 85% yield as a
colorless solid: mp 144-146 °C. IR (KBr) 1655, 1611, 1596, 1554,
1
1421, 1452, 1392, 1280, 1114, 1045, 915, 795 cm-1. H NMR
(CDCl3, 300 MHz) δ 6.73 (s, 1 H), 6.38 (s, 1 H), 3.84 (s, 3 H),
3.80 (s, 3 H), 3.75 (s, 3 H), 3.70 (s, 3 H), 2.26 (s, 3 H). 13C NMR
(CDCl3, 75 MHz) δ 162.8, 148.3, 145.5, 145.2, 142.5, 129.4, 120.1,
118.2, 102.0, 61.6, 60.8, 55.8, 33.5, 19.2. EIMS (m/z, relative
intensity) 263 (M+, 100), 248 (86), 205 (70), 162 (34), 133 (29),
124 (15), 93 (26), 69 (46). HRMS calcd for C14H17NO4 263.1058,
found 263.1160. Anal. (C14H17NO4) C, H, N.
5,6,8-Trimethoxy-4-methylquinolin-2(1H)-one (1o). This com-
pound was prepared as a colorless solid in 74% yield by following
method B: mp 149-151 °C (lit.31 mp 150-152 °C). IR (KBr) 1649,
1605, 1567, 1472, 1381, 1363, 1245, 1203, 1165, 1077, 1050, 1020,
6,8-Dimethoxy-4-methylquinolin-2(1H)-one (1i). This com-
pound was prepared in 67% yield as a light-brown solid by
following method B: mp 232-234 °C (lit.29 mp 231-233 °C). IR
1
971, 795 cm-1. H NMR (CDCl3, 300 MHz) δ 9.09 (brs, 1 H),
6.64 (s, 1 H), 6.31 (s, 1 H), 3.83 (s, 3 H), 3.80 (s, 3 H), 3.75 (s, 3
H), 2.53 (s, 3 H). 13C NMR (CDCl3, 75 MHz) δ 160.7, 148.7, 147.1,
141.4, 140.2, 123.1, 115.0, 99.7, 61.0, 57.0, 56.1, 23.1. EIMS (m/
z, relative intensity) 249 (M+, 76), 234 (100), 206 (56), 191 (25),
176 (5), 163 (10), 134 (7), 77 (6), 51 (4). HRMS calcd for
C13H15NO4 249.1001, found 249.1004. Anal. (C13H15NO4) C,
H, N.
1
(KBr) 1653, 1619, 1605, 1462, 1399, 1380, 1277, 1217 cm-1. H
NMR (DMSO-d6, 300 MHz) δ 10.5 (brs, 1 H), 6.80 (s, 1 H), 6.72
(s, 1 H), 6.41 (s, 1 H), 3.87 (s, 3 H), 3.81 (s, 3 H), 2.40 (s, 3 H).
EIMS (m/z, relative intensity) 219 (M+, 100), 203 (15), 190 (5),
176 (63), 161 (10), 133 (9), 105 (4), 69 (14), 55 (14). HRMS calcd
for C12H13NO3 219.0895, found 219.0894. Anal. (C12H13NO3) C,
H, N.
6,8-Dimethoxy-1,4-dimethylquinolin-2(1H)-one (1j). This com-
pound was prepared in 17% yield as a colorless solid by following
method B: mp 157-159 °C. IR (KBr) 1656, 1619, 1595, 1577,
1456, 1394, 1317, 1276, 1166 cm-1. 1H NMR (CDCl3, 300 MHz)
δ 6.50 (m, 2 H), 6.43 (s, 1 H), 3.74 (s, 3 H), 3.73 (s, 3 H), 3.72 (s,
3 H), 2.23 (s, 3 H). 13C NMR (CDCl3, 75 MHz) δ 162.5, 154.5,
149.5, 145.2, 125.7, 123.4, 121.5, 102.6, 98.3, 56.0, 55.2, 34.8,
19.4. EIMS (m/z, relative intensity) 233 (M+, 100), 218 (70), 188
(68), 175 (35), 160 (23), 147 (26), 118 (7), 97 (10), 69 (36). HRMS
calcd for C12H13NO3 233.1052, found 233.1048. Anal. (C12H13NO3)
C, H, N.
5,6,8-Trimethoxy-1,4-dimethylquinolin-2(1H)-one (1p). So-
dium hydride (60% oil dispersion, 10 mg, 0.25 mmol) was added
to a solution of compound 1o (50 mg, 0.2 mmol) in dry DMF (1
mL), and the mixture was stirred at 75-85 °C for 2 h. The reaction
mixture was cooled to room temperature, and methyl iodide (0.05
mL, 0.76 mmol) was added. After being stirred at 80 °C for 6 h,
the reaction mixture was quenched with ice water and extracted
with ether. The organic layer was dried and evaporated to dryness
under reduced pressure. The crude product was purified by silica
gel column chromatography, eluting with ethyl acetate-hexanes
(1:1), to yield compound 1p (40 mg, 74%) and 2,5,6,8-tet-
ramethoxy-4-methylquinoline (5 mg, 10%); yellowish solid, mp
128-130 °C (lit.31 mp 130-131 °C). IR (KBr) 1657, 1606, 1463,
5,8-Dimethoxy-4-methylquinolin-2(1H)-one (1k). This com-
pound was prepared in 84% yield as a light-brown solid by
following method A: mp 187-189 °C (lit.30 mp 188-189 °C). IR
1
1362, 1260 cm-1. H NMR (CDCl3, 300 MHz) δ 6.78 (s, 1 H),
6.45 (s, 1 H), 3.88 (s, 3 H), 3.82 (s, 3 H), 3.77 (s, 3 H), 3.76 (s, 3
H), 2.57 (s, 3 H). 13C NMR (CDCl3, 75 MHz) δ 162.9, 147.7, 146.1,
144.9, 141.1, 127.2, 123.6, 118.4, 103.0, 61.3, 57.6, 56.8, 35.8,
23.7. EIMS (m/z, relative intensity) 263 (M+, 100), 248 (43), 233
(26), 220 (47), 205 (40), 190 (12), 176 (11), 162 (10), 134 (8), 91
(7), 69 (15). HRMS calcd for C14H17NO4 263.1158, found 263.1160.
Anal. (C14H17NO4) C, H, N.
(KBr) 3400, 2800, 1651, 1613, 1489, 1463, 1441, 1369, 1266 cm-1
.
1H NMR (CDCl3, 300 MHz) δ 9.09 (brs, 1 H), 6.79 (d, J ) 8.7
Hz, 1 H), 6.44 (d, J ) 8.7 Hz, 1 H), 6.31 (s, 1 H), 3.84 (s, 3 H),
3.77 (s, 3 H), 2.56 (s, 3 H). 13C NMR (CDCl3, 75 MHz) δ 161.2,
151.9, 150.3, 139.6, 129.8, 121.4, 111.4, 109.9, 102.1, 56.1, 55.5,
24.5. EIMS (m/z, relative intensity) 219 (M+, 41), 204 (100), 190
(14), 176 (42), 161 (32), 148 (12), 133 (23), 120 (5), 104 (9), 89
(6), 77 (8). HRMS calcd for C12H13NO3 219.0895, found 219.0896.
Anal. (C12H13NO3) C, H, N.
5,6,7,8-Tetramethoxy-4-methylquinolin-2(1H)-one (1q). This
compound was prepared as a brown solid in 70% yield by following
method B: mp 158-160 °C. IR (KBr) 1653, 1597, 1556, 1483,
1
5,8-Dimethoxy-1,4-dimethylquinolin-2(1H)-one (1l). This com-
pound was prepared in 80% yield as a colorless solid by following
method B: mp 122-124 °C (lit.20 mp 122-123 °C). IR (KBr) 1644,
1393, 1378, 1259, 1170, 1099, 1073, 1012, 952 cm-1. H NMR
(CDCl3, 300 MHz) δ 9.65 (brs, 1 H), 6.27 (s, 1 H), 3.93 (s, 3 H),
3.85 (s, 3 H), 3.81 (s, 3 H), 3.80 (s, 3 H), 2.52 (s, 3 H). 13C NMR
(CDCl3, 75 MHz) δ 161.6, 148.9, 147.8, 147.6, 141.8, 134.6, 129.2,
121.3, 110.8, 61.2, 23.1. EIMS (m/z, relative intensity) 279 (M+,
1
1593, 1478, 1459, 1437, 1398, 1263 cm-1. H NMR (CDCl3, 300
MHz) δ 6.98 (d, J ) 8.7 Hz, 1 H), 6.58 (d, J ) 8.7 Hz, 1 H), 6.41

1880 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Maiti et al.
88), 264 (100), 249 (6), 236 (45), 221 (37), 206 (20), 178 (29),
135 (6), 79 (4). HRMS calcd for C14H17NO5 279.1107, found
279.1108. Anal. (C14H17NO5) C, H, N.
(13), 152 (32), 137 (8). HRMS calcd for C9H13NO2 + H+ 168.1024,
found 168.1025.
2,4-Dimethoxyaniline (3i). Yield 94%. Colorless solid: mp
5,6,7,8-Tetramethoxy-1,4-dimethylquinolin-2(1H)-one (1r). Yel-
34-36 °C (lit.32 33-35 °C). IR (neat) 3436, 2939, 2833, 1597,
1
lowish solid, mp 66-68 °C. IR (KBr) 1655, 1578, 1551, 1458,
1515, 1465, 1285, 1240, 1206, 1154, 1035, 919 cm -1. H NMR
1
1416, 1400, 1387, 1372, 1260, 1089, 1043, 1021 cm-1. H NMR
(CDC13, 300 MHz) δ 6.80 (d, J ) 8.1 Hz, 1 H), 6.63 (d, J ) 2.7
Hz, 1 H), 6.52 (dd, J ) 8.1, 2.7 Hz, 1 H), 3.96 (s, 3 H), 3.91 (s, 3
H). 13C NMR (CDCl3, 75 MHz) δ 152.6, 147.9, 129.5, 114.8, 103.8,
98.9, 55.3, 55.0. EIMS (m/z, relative intensity) 153 (90), 138 (100),
110 (51), 95 (41), 79 (8), 67 (18), 52 (26). HRMS calcd for
C8H11NO2 153.0790, found 153.0788.
(CDCl3, 300 MHz) δ 6.35 (s, 1 H), 3.99 (s, 3 H), 3.85 (s, 3 H),
3.83 (s, 3 H), 3.76 (s, 3 H), 3.68 (s, 3 H), 2.53 (s, 3 H). 13C NMR
(CDCl3, 75 MHz) δ 163.1, 149.8, 148.0, 146.3, 142.0, 138.5, 132.5,
121.6, 113.6, 61.8, 61.4, 61.3, 61.1, 35.2, 23.7. EIMS (m/z, relative
intensity) 293 (M+, 100), 278 (90), 263 (21), 248 (40), 220 (38),
192 (28), 164 (7), 131 (4), 123 (11), 93 (10), 84 (15). HRMS calcd
for C15H19NO5 293.1263, found 293.1265. Anal. (C14H17NO4) C,
H, N.
3-(Benzo[d][1,3]dioxol-4-yl(methyl)amino)-3-oxopropanoic Acid
(2). A mixture of diketo ester 8 (2.0 g, 7.5 mmol) and 5% aqueous
sodium hydroxide solution (15 mL) was heated at 60 °C for 2 h.
The solution was concentrated to about one-quarter of the original
volume and cooled in an ice bath. The mixture was acidified to pH
3.5 by the addition of 2 N HCl. The mixture was extracted with
chloroform, dried over Na2SO4, and evaporated to dryness under
vacuum to give acid 2 (1.5 g, 85%) as colorless solid: mp 115-117
°C. IR (KBr) 2914, 1737, 1663, 1614, 1486, 1459, 1386, 1362,
1253, 1196, 1054, 1028, 794 cm-1. 1H NMR (CDCl3, 300 MHz) δ
6.83 (m, 2 H), 6.64 (dd, J ) 7.8, 1.8 Hz, 1 H), 6.00 (s, 2 H), 3.26
(s, 3 H), 3.21 (s, 2 H). 13C NMR (CDCl3, 75 MHz) δ 170.0, 167.8,
149.1, 142.4, 123.1, 123.0, 119.7, 109.2, 101.9, 36.3, 36.2. EIMS
(m/z, relative intensity) 237 (M+, 35), 220 (3), 193 (10), 151 (100),
122 (23), 93 (49), 69 (17), 55 (26). HRMS calcd for C11H11NO5
237.0637, found 237.0644.
General Procedure for CBz Group Deprotection. A mixture
of CBz-protected amine (5.0 mmol) and 10% Pd-C catalyst (300
mg) in EtOAc (30 mL) was hydrogenated at 20 psi for 6 h. The
suspension was filtered, and the filtrate was evaporated. The residue
was dissolved in CHCl3 (30 mL), and the solution was extracted
with dilute HCl (3 × 50 mL). The combined aqueous extracts were
then made basic with concd NH4OH, and the aqueous mixture was
again extracted with CHCl3. The combined extracts were dried
(Na2SO4) and evaporated to provide amines in high yield, which
were pure enough to use for the next step without further
purification. A small portion of each compound was taken out and
purified by silica gel column chromatography for analysis.
Benzo[d][1,3]dioxol-4-amine (3a). Yield 92%. Colorless oil. IR
(KBr) 3360, 2887, 1654, 1499, 1467, 1358, 1255, 1212, 1212, 1050,
925 cm-1. 1H NMR (CDCl3, 300 MHz) δ 6.70 (m, 1 H), 6.37 (m,
2 H), 5.9 (s, 2 H), 3.37 (brs, 2 H). EIMS (m/z, relative intensity)
137 (M+, 84), 108 (8), 91 (8), 79 (80), 52 (100). HRMS calcd for
C7H7NO2 137.0477, found 137.0478.
2,4-Dimethoxy-N-methylaniline (3j). Colorless oil. IR (neat)
3419, 2935, 2831, 1596, 1520, 1454, 1438, 1286, 1259, 1206, 1153,
1
1034, 918 cm -1. H NMR (300 MHz, CDCl3) δ 6.50 (m, 3 H),
3.80 (s, 3 H), 3.76 (s, 3 H), 2.83 (s, 3 H). 13C NMR (75 MHz,
CDCl3) δ 151.4, 147.6, 133.5, 109.0, 103.3, 98.3, 55.2, 54.9, 30.6.
EIMS (m/z, relative intensity) 167 (75), 152 (100), 124 (27), 109
(28), 93 (7), 80 (6), 66 (7), 52 (9). HRMS calcd for C9H13NO2
167.0946, found 167.0947.
2,3,4-Trimethoxyaniline (3m). Yield 90%. Colorless oil. IR
(neat) 3442, 2937, 1496, 1478, 1424, 1266, 1204, 1128, 1085, 1041,
1012 cm-1. 1H NMR (CDC13, 300 MHz) δ 6.33 (d, J ) 8.1 Hz, 1
H), 6.35 (d, J ) 8.1 Hz, 1 H), 3.79 (s, 3 H), 3.78 (s, 3 H), 3.69 (s,
3 H). 13C NMR (CDCl3, 75 MHz) δ 145.7, 142.8, 141.1, 134.2,
109.3, 108.1, 60.6, 60.1, 56.5. EIMS (m/z, relative intensity) 183
(M+, 100), 168 (87), 153 (15), 140 (12), 125 (43), 110 (17), 108
(12), 82 (16), 54 (31). HRMS calcd for C9H13NO3 183.0895, found
183.0897.
2,3,4-Trimethoxy-N-methylaniline (3n). Colorless oil. IR (neat)
3411, 2936, 2830, 1611, 1506, 1486, 1268, 1228, 1203, 1158, 1094,
1
1026, 937 cm -1. H NMR (300 MHz, CDCl3) δ 6.55 (d, J ) 8.7
Hz, 1 H), 6.24 (d, J ) 8.7 Hz, 1 H), 3.84 (s, 3 H), 3.82 (s, 3 H),
3.75 (s, 3 H), 2.76 (s, 3 H). 13C NMR (75 MHz, CDCl3) δ 144.8,
142.5, 140.6, 137.8, 108.0, 104.0, 60.6, 60.1, 56.4, 30.7. EIMS (m/
z, relative intensity) 197 (91), 182 (100), 167 (29), 152 (10), 139
(20), 136 (6), 124 (16), 110 (5), 96 (15), 68 (22), 53 (8). HRMS
calcd for C10H15NO3 197.1052, found 197.1050.
2,4,5-Trimethoxyaniline (3o). Yield 94%. Colorless solid: mp
84-86 °C. IR (KBr) 3392, 1527, 1468, 1413, 1233, 1204, 1188,
1
1044, 1027, 824 cm-1. H NMR (CDC13, 300 MHz) δ 6.49 (s, 1
H), 6.35 (s, 1 H), 3.78 (s, 3 H), 3.77 (s, 3 H), 3.75 (s, 3 H). 13C
NMR (CDCl3, 75 MHz) δ 143.7, 141.4, 140.7, 128.4, 101.8, 100.0,
57.2, 56.5. EIMS (m/z, relative intensity) 183 (M+, 74), 168 (100),
140 (39), 125 (34), 108 (16), 94 (9), 80 (5), 44 (16). HRMS calcd
for C9H13NO3 183.0895, found 183.0894.
2,4,5-Trimethoxy-N-methylaniline (3p). Yield 91%. Colorless
solid: mp 58-60 °C. IR (KBr) 3435, 2942, 1529, 1457, 1449, 1397,
1
1223, 1210, 1199, 1035, 824 cm-1. H NMR (CDC13, 300 MHz)
δ 6.51 (s, 1 H), 6.27 (s, 1 H), 3.82 (s, 3 H), 3.78 (s, 3 H), 3.76 (s,
3 H), 2.82 (s, 3 H). 13C NMR (CDCl3, 75 MHz) δ 144.0, 140.5,
139.7, 133.9, 99.9, 97.4, 57.6, 56.8, 56.2, 30.9. EIMS (m/z, relative
intensity) 197 (M+, 74), 182 (100), 166 (8), 154 (31), 139 (35),
123 (31), 109 (14), 82 (12), 69 (11), 53 (12). HRMS calcd for
C10H15NO3 197.1052, found 197.1051.
2,3,4,5-Tetramethoxyaniline (3q). Colorless solid: mp 98-100
°C. (lit.20 99-100 °C). IR (KBr) 3360, 2936, 1617, 1499, 1469,
1227, 1124, 1079, 1044, 1007 cm-1. 1H NMR (CDC13, 300 MHz)
δ 6.05 (s, 1 H), 3.90 (s, 3 H), 3.76 (s, 3 H), 3.75 (s, 3 H), 3.73 (s,
3 H). 13C NMR (CDCl3, 75 MHz) δ 149.7, 147.3, 135.7, 134.6,
94.9, 61.3, 61.0, 60.4, 56.0. EIMS (m/z, relative intensity) 213 (60),
198 (100), 183 (13), 168 (10), 155 (17), 140 (22), 112 (11), 84
(10), 68 (5), 53 (6). HRMS calcd for C10H15NO4 213.1001, found
213.0994.
General Procedure to Synthesize Protected Amines from
Acids. A mixture of diphenylphosphoryl azide (12 mmol), the
required acid (11 mmol), and triethylamine (12 mmol) in benzene
(20 mL) was heated under reflux for 2-3 h. After the evolution of
N2 had ceased, benzyl alcohol (11 mmol) was added and the mixture
was heated at reflux for another 2-5 h. The reaction was monitored
by TLC. After cooling, the mixture was washed successively with
cold 1 N HC1, aq NaHCO3 solution, and brine, and then dried
N-Methylbenzo[d][1,3]dioxol-4-amine (3b). Yield 95%. Color-
less oil. IR (neat) 3401, 2972, 1646, 1474, 1454, 1353, 1275, 1258,
1092, 1052, 926 cm-1. 1H NMR (CDC13, 300 MHz) δ 6.73 (t, J )
8.1 Hz, 1 H), 6.29 (m, 2 H), 5.87 (s, 2 H), 3.58 (brs, 1 H), 2.86 (s,
3 H). 13C NMR (CDCl3, 75 MHz) δ 146.9, 134.0, 133.6, 122.3,
105.7, 100.4, 98.9, 30.6. Positive ion ESIMS (m/z, relative intensity)
152 (MH+, 100), 136 (35), 133 (8), 105 (4). HRMS calcd for
C8H9NO2 152.0711, found 152.0712.
2,3-Dimethoxyaniline (3g). Yield 96%. Colorless oil. IR (neat)
3367, 2938, 1613, 1499, 1478, 1321, 1264, 1228, 1132, 1087, 1052,
1
1003, 775 cm-1. H NMR (CDC13, 300 MHz) δ 6.81 (t, J ) 8.4
Hz, 1 H), 6.25 (m, 2 H), 3.84 (m, 2 H), 3.80 (s, 3 H), 3.79 (s, 3 H).
13C NMR (CDCl3, 75 MHz) δ 152.8, 140.5, 135.7, 124.0, 108.6,
102.0, 59.5, 55.5. Positive ion ESIMS (m/z, relative intensity) 153
(M+, 100), 138 (100), 110 (6), 95 (25), 67 (10). HRMS calcd for
C8H11NO2 153.0790, found 153.0786.
2,3-Dimethoxy-N-methylaniline (3h). Yield 93%. Colorless oil.
IR (neat) 3417, 1935, 1602, 1514, 1482, 1304, 1263, 1218, 1161,
1110, 1079, 1050, 1003 cm-1. 1H NMR (CDC13, 300 MHz) δ 6.93
(t, J ) 8.1 Hz, 1 H), 6.31 (m, 2 H), 4.30 (brs, 1 H), 3.84 (s, 3 H),
3.77 (s, 3 H), 2.82 (s, 3 H). 13C NMR (CDCl3, 75 MHz) δ 152.2,
143.4, 135.1, 124.3, 124.1, 108.7, 103.7, 101.0, 59.7, 55.6, 30.4.
Positive ion ESIMS (m/z, relative intensity) 168 (MH+, 100), 166

Casimiroin Synthesis and Inhibitory ActiVities
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1881
(Na2SO4). The organic extract was evaporated under reduced
pressure and the residue was chromatographed on silica gel, eluting
with ethyl ether-hexane (1:1).
285 (100), 242 (68). HRMS calcd for C16H15NO4 + Na+ 308.0899,
found 308.0906.
Benzyl 2,3-Dimethoxyphenyl(methyl)carbamate (7g). Yield
89%. Colorless solid: mp 92-94 °C (lit.33 mp 92-93 °C). IR (KBr)
Benzyl Benzo[d][1,3]dioxol-4-ylcarbamate (6a). Yield 75%.
Colorless oil. IR (KBr) 3400, 2900, 1735, 1616, 1537, 1450, 1374,
1707, 1587, 1488, 1479, 1389, 1316, 1265, 1157, 1075, 794 cm-1
.
1H NMR (CDCl3, 300 MHz) δ 7.22 (m, 5 H), 6.99 (t, J ) 8.4 Hz,
1 H), 6.81 (m, 2 H), 5.12 (s, 2 H), 3.83 (s, 3 H), 3.73 (s, 3 H), 3.20
(s, 3 H). 13C NMR (CDCl3, 75 MHz) δ 155.7, 153.3, 145.2, 136.7,
128.2, 127.6, 127.5, 123.5, 120.6, 111.4, 67.0, 60.6, 55.8, 37.5.
Positive ion ESIMS (m/z, relative intensity) 324 (MNa+, 49), 283
(18), 279 (35), 278 (8), 277 (60), 244 (7), 243 (10). HRMS calcd
for C17H19NO4 + Na+ 324.1212, found 324.1208.
1
1220, 1101, 1013, 795 cm-1. H NMR (CDCl3, 300 MHz) δ 7.33
(m, 7 H), 7.17 (m, 1 H), 6.79 (m, 1 H), 6.57 (d, J ) 8.4 Hz, 1 H),
5.90 (s, 2 H), 5.19 (s, 2 H). 13C NMR (CDCl3, 75 MHz) δ 153.0,
147.3, 135.8, 129.6, 128.5, 128.3, 128.2, 128.0, 125.2, 121.9, 121.5,
120.0, 113.3, 104.1, 101.1, 67.1. Positive ion ESIMS (m/z, relative
intensity) 272 (MH+, 20), 264 (6), 102 (100). HRMS calcd for
C15H14NO54 272.0923, found 272.0924.
Benzyl 2,4-Dimethoxyphenyl(methyl)carbamate (7i). Yield
90%. Colorless oil. IR (KBr) 1705, 1609, 1587, 1513, 1455, 1389,
1352, 1209, 1159, 1033 cm-1. 1H NMR (CDCl3, 300 MHz) δ 7.22
(m, 6 H), 6.40 (m, 2 H), 5.11 (s, 2 H), 3.77 (s, 3 H), 3.66 (s, 3 H),
3.14 (s, 3 H). 13C NMR (CDCl3, 75 MHz) δ 159.7, 155.8, 155.7,
137.2, 128.9, 128.1, 127.9, 127.4, 127.2, 125.0, 103.9, 99.2, 66.7,
55.4, 55.3, 37.5. EIMS (m/z, relative intensity) 301 (M+, 22), 166
(33), 151 (42), 91 (100), 65 (11). HRMS calcd for C16H19NO4
301.1314, found 301.1315.
Benzyl Methyl(2,3,4-trimethoxyphenyl)carbamate (7m). Yield
92%. Colorless oil. IR (KBr) 1708, 1598, 1497, 1471, 1418, 1390,
1352, 1212, 1155, 1094 cm-1. 1H NMR (CDCl3, 300 MHz) δ 7.35
(m, 5 H), 6.86 (d, J ) 8.7 Hz, 1 H), 6.61 (d, J ) 8.7 Hz, 1 H),
5.11 (s, 2 H), 3.84 (s, 6 H), 3.79 (s, 3 H), 3.21 (s, 3 H). 13C NMR
(CDCl3, 75 MHz) δ 155.9, 152.8, 149.7, 142.4, 136.6, 129.4, 128.0,
127.3, 122.7, 106.3, 66.8, 60.7, 55.8, 37.9. EIMS (m/z, relative
intensity) 331 (M+, 9), 196 (100), 181 (45), 166 (10), 147 (12), 91
(82), 77 (2), 65 (13). HRMS calcd for C18H21NO5 331.1420, found
331.1424.
Benzyl 2,3-Dimethoxyphenylcarbamate (6g). Yield 92%. Col-
orless oil. IR (KBr) 3422, 2940, 1736, 1607, 1531, 1481, 1459,
1
1421, 1299, 1232, 1203, 1102, 1043 cm-1. H NMR (CDCl3, 300
MHz) δ 7.75 (brs, 1 H), 7.33 (m, 5 H), 6.93 (t, J ) 8.1 Hz, 1 H),
6.53 (m, 1 H), 5.11 (s, 2 H), 3.73 (s, 6 H). 13C NMR (CDCl3, 75
MHz) δ 152.9, 151.8, 136.7, 135.8, 131.8, 128.3, 128.1, 123.9,
110.6, 106.4, 66.7, 60.3, 55.4. EIMS (m/z, relative intensity) 287
(M+, 19), 243 (19), 228 (52), 213 (3), 179 (11), 164 (7), 136 (8),
91 (100), 77 (8), 65 (21). HRMS calcd for C16H17NO4 287.1158,
found 287.1160.
Benzyl 2,4-Dimethoxyphenylcarbamate (6i). Yield 95%. Col-
orless semisolid. IR (KBr) 3400, 2900, 1728, 1618, 1531, 1492,
1417, 1208, 1158, 1037 cm-1. 1H NMR (CDCl3, 300 MHz) δ 7.98
(brs, 1 H), 7.35 (m, 5 H), 7.06 (s, 1 H), 6.46 (m, 2 H), 5.17 (s, 2
H), 3.78 (s, 3 H), 3.76 (s, 3 H). 13C NMR (CDCl3, 75 MHz) δ
155.8, 153.3, 148.9, 136.2, 128.5, 128.2, 120.9, 119.8, 119.0, 103.7,
98.6, 66.7, 55.5, 55.4. EIMS (m/z, relative intensity) 287 (M+, 19),
243 (9), 179 (30), 152 (100), 136 (22), 124 (57), 109 (17), 91 (88),
79 (16), 65 (23). HRMS calcd for C16H17NO4 287.1158, found
287.1156.
Benzyl 2,3,4-Trimethoxyphenylcarbamate (6m). Yield 73%.
Colorless solid: mp 53-55 °C. IR (KBr) 3400, 2900, 1731, 1599,
1525, 1495, 1459, 1412, 1211, 1102, 1023 cm-1. 1H NMR (CDCl3,
300 MHz) δ 7.75 (d, J ) 9.0 Hz, 1 H), 7.35 (m, 5 H), 6.61 (d, J
) 9.0 Hz, 1 H), 5.17 (s, 2 H), 3.87 (s, 3 H), 3.83 (s, 3 H), 3.80 (s,
3 H). 13C NMR (CDCl3, 75 MHz) δ 153.3, 149.0, 142.4, 141.7,
136.0, 128.5, 128.3, 125.1, 112.9, 107.0, 66.8, 61.0, 60.8, 56.1.
EIMS (m/z, relative intensity) 317 (M+, 20), 273 (8), 258 (10),
182 (65), 167 (29), 123 (27), 91 (100), 77 (2), 65 (19). HRMS
calcd for C17H19NO5 317.1263, found 317.1261.
Benzyl 2,4,5-Trimethoxyphenylcarbamate (6o). Yield 90%.
Colorless solid: mp 99-100 °C. IR (KBr) 3400, 2900, 1728, 1614,
1531, 1457, 1456, 1407, 1202, 1137, 1036 cm-1. 1H NMR (CDCl3,
300 MHz) δ 7.81 (brs, 1 H), 7.35 (m, 5 H), 7.11 (s, 1 H), 6.50 (s,
1 H), 5.16 (s, 2 H), 3.82 (s, 6 H), 3.78 (s, 3 H). 13C NMR (CDCl3,
75 MHz) δ 153.3, 144.5, 143.0, 141.5, 136.1, 128.5, 128.28, 120.5,
103.9, 97.8, 66.8, 56.59, 56.50, 56.3. EIMS (m/z, relative intensity)
317 (M+, 44), 258 (6), 209 (8), 182 (22), 123 (33), 91 (100), 65
(14). HRMS calcd for C17H19NO5 317.1263, found 317.1265.
General Procedure for N-Methylation. A mixture of NaH (12
mmol, 60% dispersion in oil, washed twice with n-hexane) and
monoprotected amine (8 mmol) in DMF (10 mL) was stirred at
room temperature for 30 min. A solution of CH3I (24 mmol) was
added, and the resulting mixture was stirred at room temperature
for 2 h. Ice water (20 g) was added to this mixture, and the resulting
mixture was extracted with CHC13 (3 × 100 mL). The combined
extracts were washed with brine (50 mL), dried (Na2SO4), and
evaporated. The residue was purified by column chromatography
on silica gel, eluting with a 9:1 mixture of petroleum ether and
ethyl acetate. Evaporation of the solvents gave methylated carbam-
ate in very high yield.
Benzyl Methyl(2,4,5-trimethoxyphenyl)carbamate (7o). Yield
85%. Colorless solid: mp 98-99 °C. IR (KBr) 1704, 1608, 1515,
1
1455, 1386, 1330, 1206, 1148, 1036 cm-1. H NMR (CDCl3, 300
MHz) δ 7.76 (m, 5 H), 7.14 (s, 1 H), 6.98 (s, 1 H), 5.56 (s, 2 H),
4.33 (s, 3 H), 4.23 (s, 3 H), 4.16 (s, 3 H), 3.64 (s, 3 H). 13C NMR
(CDCl3, 75 MHz) δ 155.9, 148.9, 148.4, 142.3, 136.8, 128.1, 127.9,
127.6, 127.3, 127.0, 123.1, 112.4, 97.6, 66.5, 56.1, 55.9, 37.2. EIMS
(m/z, relative intensity) 331 (M+, 73), 196 (45), 181 (42), 166 (22),
137 (10), 91 (100), 65 (15). HRMS calcd for C18H21NO5 331.1420,
found 331.1421.
Ethyl 3-(Benzo[d][1,3]dioxol-4-yl(methyl)amino)-3-oxopro-
panoate (8). A mixture of amine 3b (1.51 g, 10.0 mmol) and excess
diethyl malonate (15 mL) was heated at 110 °C for 3 h while argon
gas was bubbled through the solution. The excess diethyl malonate
and ethanol produced during the reaction were removed under
reduced pressure. The crude product was purified by silica gel
column chromatography, eluting with 30% ethyl acetate in hexanes,
to afford carbamate 8 (2.1 g, 82%) as a colorless oil. IR (neat)
3459, 2971, 1741, 1672, 1631, 1460, 1375, 1253, 1096, 1055, 928,
795 cm-1. 1H NMR (CDCl3, 300 MHz) δ 6.75 (m, 2 H), 6.62 (dd,
J ) 7.8, 1.8 Hz, 1 H), 5.92 (s, 2 H), 4.00 (q, J ) 7.2 Hz, 2 H),
3.19 (s, 2 H), 3.15 (s, 3 H), 1.11 (t, J ) 8.4 Hz, 3 H). 13C NMR
(CDCl3, 75 MHz) δ 167.2, 165.9, 148.6, 142.5, 125.0, 122.2, 120.6,
108.2, 101.4, 60.9, 41.1, 35.8, 13.8. EIMS (m/z, relative intensity)
265 (M+, 35), 220 (10), 178 (9), 151 (100), 120 (5), 93 (12), 77
(3), 65 (20). HRMS calcd for C13H15NO5 + Na+ 288.0848, found
288.0847.
6-Hydroxy-9-methyl-[1,3]dioxolo[4,5-h]quinolin-8(9H)-one (9).
The acid 2 (500 mg, 2.1 mmol) was added to polyphosphoric acid
(5 g) at 95-100 °C, and the mixture was allowed to stir for 3 h at
the same temperature and then poured into ice-water. After
polyphosphoric acid had dissolved, a white solid product appeared
in the flask. The solid hydroxyquinolone 9 (400 mg, 88%) was
collected by filtration, washed with ether, and dried under vacuum.
The solid was used for the next step without further purification:
mp 310 °C (dec, lit.18 mp 310-315 °C). 1H NMR (CF3CO2D, 300
MHz) δ 8.06 (d, J ) 8.4 Hz, 1 H), 7.29 (d, J ) 8.4 Hz, 1 H), 6.72
(s, 1 H), 6.27 (s, 2 H), 4.28 (s, 3 H). EIMS (m/z, relative intensity)
219 (M+, 100), 190 (27), 149 (22), 120 (15), 93 (29), 69 (27), 46
(8). HRMS calcd for C11H9NO4 219.0532, found 219.0538.
Benzyl Benzo[d][1,3]dioxol-4-yl(methyl)carbamate (7a). Yield
94%. Colorless oil. IR (KBr) 1712, 1633, 1492, 1459, 1388, 1345,
1
1301, 1252, 1152, 1102, 1059, 795 cm-1. H NMR (CDC13, 300
MHz) δ 7.26 (m, 5 H), 6.75 (m, 3 H), 5.86 (brs, 2 H), 5.16 (brs,
2 H), 3.26 (s, 3 H). 13C NMR (CDCl3, 75 MHz) δ 155.0, 148.4,
141.9, 136.4, 128.2, 127.7, 125.3, 121.3, 120.1, 106.8, 101.0, 67.2,
36.7. Positive ion ESIMS (m/z, relative intensity) 308 (MH+, 35),

1882 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
Maiti et al.
General Procedure for the Synthesis of Amides (10a-g).
Diketene (10.0 mmol) was added to a magnetically stirred solution
of the substituted anilines (3a-g, 11.0 mmol) in benzene (10 mL),
and the mixture was heated at 100 °C for 90-120 min. The reaction
mixtures were monitored by TLC. After completion of the reactions,
the solutions were evaporated under reduced pressure and the
residues were chromatographed on silica gel, eluting with ethyl
ether-hexane (1:1).
50.3, 31.0. EIMS (m/z, relative intensity) 237 (M+, 68), 206 (8),
153 (51), 138 (100), 95 (18), 43 (99). HRMS calcd for C12H15NO4
237.1001, found 237.1009.
N-(2,3-Dimethoxyphenyl)-N-methyl-3-oxobutanamide (10h).
Yield 70%, colorless oil. IR (neat) 1721, 1656, 1587, 1478, 1429,
1378, 1356, 1317, 1267, 1234, 1148, 1096, 1051, 1003, 794 cm-1
.
1H NMR (CDCl3, 300 MHz) δ 6.98 (m, 1 H), 6.85 (m, 1 H), 6.63
(d, J ) 8.1 Hz, 1 H), 3.79 (s, 3 H), 3.74 (s, 5 H), 3.15 (s, 3 H),
2.20 (s, 3 H). 13C NMR (CDCl3, 75 MHz) δ 202.2, 167.1, 153.3,
136.6, 128.0, 124.1, 120.6, 112.2, 60.7, 55.7, 49.1, 36.5, 29.8.
Positive ion ESIMS (m/z, relative intensity) 274 (MNa+, 68), 252
(MH+), 180 (100). HRMS calcd for C13H17NO4 + Na+ 274.1055,
found 274.1054.
N-(Benzo[d][1,3]dioxol-4-yl)-3-oxobutanamide (10a). Yield
68%. Colorless solid: mp 64-66 °C. IR (KBr) 3300, 2800, 1716,
1668, 1636, 1542, 1419, 1451, 1358, 1254, 1178, 1158, 1068, 1022,
1
934, 794 cm-1. H NMR (CDCl3, 300 MHz) δ 9.10 (brs, 1 H),
7.54 (d, J ) 8.4 Hz, 1 H), 6.77 (m, 1 H), 6.59 (d, J ) 7.8 Hz, 1
H), 5.93 (s, 2 H), 3.57 (s, 2 H), 2.29 (s, 3 H). 13C NMR (CDCl3,
75 MHz) δ 205.1, 163.3, 147.5, 137.3, 121.8, 121.0, 114.9, 105.1,
101.3, 49.2, 31.2. EIMS (m/z, relative intensity) 221 (M+, 14), 163
(33), 137 (100), 109 (8), 79 (17), 52 (13). HRMS calcd for
C11H11NO4 221.0688, found 221.0690.
N-(2,4-Dimethoxyphenyl)-3-oxobutanamide (10i). Yield 80%,
colorless solid: mp 92-94 °C. IR (KBr) 3300, 1713, 1667, 1615,
1603, 1535, 1459, 1416, 1302, 1262, 1282, 1208, 1158, 1128, 1030,
750 cm-1. 1H NMR (CDCl3, 300 MHz) δ 9.00 (brs, 1 H), 8.15 (d,
J ) 8.7 Hz, 1 H), 6.44 (d, J ) 8.7 Hz, 1 H), 6.40 (brs, 1 H), 3.84
(s, 3 H), 3.75 (s, 3 H), 3.54 (s, 3 H), 2.29 (s, 3 H). 13C NMR (CDCl3,
75 MHz) δ 204.6, 162.8, 156.6, 149.7, 120.9, 120.8, 103.5, 98.6,
55.8, 55.5, 50.6, 31.0. EIMS (m/z, relative intensity) 237 (M+, 67),
179 (55), 153 (40), 138 (100), 110 (12), 93 (5), 52 (4). HRMS
calcd for C12H15NO4 237.1001, found 237.1001.
N-(Benzo[d][1,3]dioxol-4-yl)-N-methyl-3-oxobutanamide (10b).
Yield 70%. Colorless oil. IR (neat) 1721, 1662, 1630, 1604, 1487,
1459, 1425, 1377, 1358, 1253, 1194, 1090, 1055, 1032, 927 cm-1
.
1H NMR (CDCl3, 300 MHz) δ 6.78 (m, 2 H), 6.63 (m, 1 H), 5.96
(s, 2 H), 3.30 (s, 2 H), 3.21 (s, 3 H), 2.11 (s, 3 H). 13C NMR (CDCl3,
75 MHz) δ 202.1, 166.9, 148.8, 142.6, 125.3, 122.4, 120.6, 108.5,
101.5, 49.6, 35.9, 30.0. EIMS (m/z, relative intensity) 235 (M+,
31), 178 (4), 151 (100), 122 (12), 93 (23), 65 (10). HRMS calcd
for C12H13NO4 235.0845, found 235.0846.
N-(2,4-Dimethoxyphenyl)-N-methyl-3-oxobutanamide (10j).
Yield 80%, colorless oil. IR (neat) 1720, 1655, 1609, 1586, 1513,
1462, 1421, 1379, 1317, 1283, 1210, 1162, 1113, 1031, 795 cm-1
.
1H NMR (CDCl3, 300 MHz) δ 6.99 (d, J ) 1.5 Hz, 1 H), 6.42 (m,
2 H), 3.74 (s, 3 H), 3.72 (s, 3 H), 3.09 (s, 5 H), 2.05 (s, 3 H). 13C
NMR (CDCl3, 75 MHz) δ 202.4, 167.8, 160.5, 155.5, 129.1, 124.7,
104.4, 99.3, 55.4, 55.2, 49.6, 36.1, 29.7. EIMS (m/z, relative
intensity) 251 (M+, 5), 220 (3), 167 (65), 152 (100), 136 (7), 124
(23), 109 (12), 98 (22), 56 (9). HRMS calcd for C13H17NO4
251.1158, found 251.1161.
3-oxo-N-Phenylbutanamide (10c). Yield 80%, colorless solid:
mp 82-84 °C (lit.34 mp 84-86 °C). IR (KBr) 3300, 1716, 1661,
1
1599, 1544, 1444, 1410, 1314, 1231, 1157, 1029, 755 cm-1. H
NMR (CDCl3, 300 MHz) δ 9.16 (brs, 1 H), 7.50 (m, 2 H), 7.26
(m, 2 H), 7.06 (m, 1 H), 3.52 (s, 2 H), 2.25 (s, 3 H). 13C NMR
(CDCl3, 75 MHz) δ 204.9, 163.7, 137.4, 128.8, 124.4, 120.0, 49.8,
31.0. EIMS (m/z, relative intensity) 177 (M+, 8), 119 (18), 93 (100),
66 (28), 58 (12), 43 (11). HRMS calcd for C10H11NO2 177.0790,
found 177.0794.
N-(2,5-Dimethoxyphenyl)-3-oxobutanamide (10k). Yield 94%,
colorless solid: mp 70-72 °C (lit.36 mp 67-68 °C). IR (KBr) 3300,
2800, 1715, 1676, 1600, 1537, 1486, 1465, 1430, 1361, 1279, 1221,
1159, 1046, 795 cm-1. 1H NMR (CDCl3, 300 MHz) δ 9.20 (brs, 1
H), 7.97 (d, J ) 3.0 Hz, 1 H), 6.72 (d, J ) 9.0 Hz, 1 H), 6.50 (dd,
J ) 9.0, 3.0 Hz, 1 H), 3.76 (s, 3 H), 3.68 (s, 3 H), 3.51 (s, 2 H),
2.22 (s, 3 H). 13C NMR (CDCl3, 75 MHz) δ 204.2, 163.3, 153.4,
142.3, 127.8, 110.7, 108.4, 106.3, 56.1, 55.5, 50.4, 30.7. Positive
ion ESIMS (m/z, relative intensity) 260 (MNa+, 100). HRMS calcd
for C12H15NO4 + Na+ 260.0899, found 260.0900.
N-Methyl-3-oxo-N-phenylbutanamide (10d). Yield 85%, color-
less oil. IR (neat) 1721, 1655, 1595, 1496, 1427, 1379, 1358, 1343,
1
1302, 1162, 1123 776 cm-1. H NMR (CDCl3, 300 MHz) δ 7.37
(m, 3 H), 7.13 (m, 2 H), 3.24 (s, 5 H), 2.04 (s, 3 H). 13C NMR
(CDCl3, 75 MHz) δ 202.2, 166.5, 143.4, 129.8, 129.5, 128.1, 127.1,
49.8, 37.2, 30.2. Positive ion ESIMS (m/z, relative intensity) 382
(2M+, 100), 192 (MH+, 63), 122 (30), 93 (100), 66 (28), 58 (12),
43 (11). HRMS calcd for C11H13NO2 + H+ 192.1024, found
192.1023.
N-(2,5-Dimethoxyphenyl)-N-methyl-3-oxobutanamide (10l).
Yield 80%, colorless solid: mp 69-71 °C (lit.20 mp 68.5-69 °C).
IR (KBr) 1720, 1658, 1611, 1508, 1464, 1424, 1376, 1310, 1274,
1219, 1181, 1107, 1041, 728 cm-1. 1H NMR (CDCl3, 300 MHz) δ
6.81 (m, 2 H), 6.67 (m, 1 H), 3.70 (s, 3 H), 3.69 (s, 3 H), 3.12 (m,
5 H), 2.08 (s, 3 H). 13C NMR (CDCl3, 75 MHz) δ 202.3, 167.3,
153.6, 148.7, 132.0, 114.6, 114.2, 112.5, 55.6, 49.5, 35.9, 29.7.
Positive ion ESIMS (m/z, relative intensity) 274 (MNa+, 100).
HRMS calcd for C13H17NO4 + Na+ 274.1055, found 274.1056.
3-oxo-N-(2,3,4-Trimethoxyphenyl)butanamide (10m). Yield
85%, colorless solid: mp 86-88 °C. IR (KBr) 3300, 2800, 1711,
1674, 1601, 1535, 1494, 1462, 1412, 1360, 1272, 1164, 1095, 1060,
N-(2-Methoxyphenyl)-3-oxobutanamide (10e). Yield 70%,
yellow solid: mp 82-84 °C (lit.35 83-84 °C). IR (KBr) 3300, 1714,
1677, 1600, 1537, 1488, 1461, 1435, 1336, 1253, 1159, 750 cm-1
.
1H NMR (CDCl3, 300 MHz) δ 9.20 (brs, 1 H), 8.27 (d, J ) 8.1
Hz, 1 H), 7.01 (m, 1 H), 6.96 (m, 1 H), 6.84 (m, 1 H), 3.83 (s, 3
H), 3.54 (s, 2 H), 2.25 (s, 3 H). 13C NMR (CDCl3, 75 MHz) δ
204.3, 163.2, 148.1, 127.1, 123.9, 120.7, 119.9, 109.9, 55.6, 50.6,
30.8. EIMS (m/z, relative intensity) 207 (M+, 47), 149 (41), 108
(100), 92 (7), 80 (35), 43 (52). HRMS calcd for C12H13NO3
207.0895, found 207.0891.
1
1010, 795 cm-1. H NMR (CDCl3, 300 MHz) δ 9.28 (brs, 1 H),
N-(2-Methoxyphenyl)-N-methyl-3-oxobutanamide (10f). Yield
75%, colorless oil. IR (neat) 1720, 1655, 1510, 1433, 1464, 1357,
1379, 1279, 1240, 1108, 795 cm-1. 1H NMR (CDCl3, 300 MHz) δ
7.42 (m, 1 H), 7.26 (m, 1 H), 7.02 (m, 2 H), 3.93 (s, 3 H), 3.31 (s,
3 H), 3.29 (s, 2 H), 2.42 (s, 3 H). 13C NMR (CDCl3, 75 MHz) δ
202.3, 167.4, 154.6, 131.5, 129.7, 128.8, 121.0, 111.8, 55.2, 49.6,
36.0, 29.7. EIMS (m/z, relative intensity) 221 (M+, 10), 190 (21),
122 (100), 94 (27), 65 (15), 43 (46). HRMS calcd for C12H15NO3
221.1052, found 221.1050.
7.86 (d, J ) 9.0 Hz, 1 H), 6.53 (d, J ) 9.0 Hz, 1 H), 3.86 (s, 3 H),
3.80 (s, 3 H), 3.77 (s, 3 H), 3.51 (s, 2 H), 2.21 (s, 3 H). 13C NMR
(CDCl3, 75 MHz) δ 204.5, 163.1, 149.6, 143.1, 141.5, 125.0, 114.7,
106.6, 60.9, 60.6, 55.8, 49.8, 30.8. EIMS (m/z, relative intensity)
267 (M+, 20), 209 (100), 183 (83), 168 (49), 153 (7), 140 (11),
123 (19), 108 (12), 95 (4), 80 (12), 69 (8). HRMS calcd for
C13H17NO5 267.1107, found 267.1111.
N-Methyl-3-oxo-N-(2,3,4-trimethoxyphenyl)butanamide (10n).
Yield 78%, colorless oil. IR (neat) 1712, 1633, 1492, 1459, 1388,
1345, 1301, 1252, 1152, 1102, 1059, 795 cm-1. 1H NMR (CDCl3,
300 MHz) δ 6.69 (d, J ) 9.0 Hz, 1 H), 6.51 (d, J ) 9.0 Hz, 1 H),
3.73 (s, 3 H), 3.70 (s, 3 H), 3.69 (s, 3 H), 3.04 (m, 5 H), 1.96 (s,
3 H). 13C NMR (CDCl3, 75 MHz) δ 202.0, 167.3, 153.6, 149.0,
142.2, 129.2, 122.6, 106.6, 60.8, 60.5, 55.6, 49.1, 36.5, 29.6.
Positive ion ESIMS (m/z, relative intensity) 282 (MH+, 100), 251
N-(2,3-Dimethoxyphenyl)-3-oxobutanamide (10g). Yield 67%,
colorless oil. IR (neat) 3300, 1712, 1681, 1603, 1539, 1478, 1463,
1
1422, 1297, 1260, 1090, 1002, 794 cm-1. H NMR (CDCl3, 300
MHz) δ 9.47 (brs, 1 H), 7.92 (d, J ) 8.1 Hz, 1 H), 6.99 (dd, J )
8.1, 16.5 Hz, 1 H), 6.63 (d, J ) 8.1 Hz, 1 H), 3.88 (s, 3 H), 3.83
(s, 3 H), 3.59 (s, 2 H), 2.29 (s, 3 H). 13C NMR (CDCl3, 75 MHz)
δ 204.4, 163.4, 152.0, 137.8, 131.9, 124.0, 112.6, 107.7, 60.7, 55.7,

Casimiroin Synthesis and Inhibitory ActiVities
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7 1883
(3), 198 (21), 183 (2). HRMS calcd for C14H19NO5 + H 282.1343,
found 282.1341.
QR2 Crystallization and X-ray Structure Determination.
Crystals of the QR2 apoenzyme were grown according to our
previously reported protocols.6 The resulting crystals were soaked
overnight with inhibitor in a solution that consisted of 1 µL of a
10 mM inhibitor solution (dissolved in 100% DMSO) that was
mixed into 9 µL of the mother liquor solution that contained 1.339
M ammonium sulfate, 0.1 M bis-Tris, pH 6.7, 0.1 M NaCl, 5 mM
DTT, and 12 µM FAD. Crystals were then retrieved with a nylon
loop, which was then used to swipe the crystals through the well
solution supplemented with 30% glycerol. The crystals were
immediately flash-frozen by plunging into liquid nitrogen. Crystals
were stored in liquid nitrogen in Dewar flasks until X-ray data
collection.
3-oxo-N-(2,4,5-Trimethoxyphenyl)butanamide (10o). Yield
67%. Colorless solid: mp 90-92 °C (lit.37 mp 92-93 °C). IR (KBr)
3300, 2800, 1714, 1668, 1534, 1465, 1405, 1342, 1219, 1203, 1031,
750 cm-1. 1H NMR (CDCl3, 300 MHz) δ 9.02 (brs, 1 H), 7.97 (s,
1 H), 6.46 (s, 1 H), 3.78 (s, 3 H), 3.76 (s, 3 H), 3.73 (s, 3 H), 3.49
(s, 2 H), 2.23 (s, 3 H). 13C NMR (CDCl3, 75 MHz) δ 204.2, 162.9,
145.0, 142.3, 120.2, 105.4, 97.3, 56.5, 56.2, 50.3, 30.8. EIMS (m/
z, relative intensity) 267 (M+, 64), 252 (5), 209 (44), 194 (32),
168 (100), 140 (28), 108 (13), 69 (15). HRMS calcd for C13H17NO5
267.1107, found 267.1106.
N-Methyl-3-oxo-N-(2,4,5-trimethoxyphenyl)butanamide (10p).
X-Ray diffraction data were collected at the SER-CAT beamline
22-BM located at the Advanced Photon Source (APS) at Argonne
National Laboratories. Flash-frozen crystals were mounted on a
goniostat under a stream of dry N2 at 100 K. X-ray data were
collected on either a MAR 225 mm or MAR315 CCD detector.
Individual data sets were processed and scaled in the P212121 space
group using HKL2000. Initial phases for the QR2-compound
complexes were determined via molecular replacement using the
program Phaser in the CCP4 suite. Model building and structural
refinement were performed using the programs WinCoot and
Refmac from the CCP4 suite. The atomic coordinates and structure
factors have been deposited in the Protein Data Bank (www.pd-
b.org)38 under PDB codes 3G5M for the QR2-compound 1 complex
and 3GAM for the 1l complex.
Aromatase Assay. Aromatase activity was assayed as previously
reported, with the necessary modifications to assay in a 384-well
plate. Briefly, the test compound (3.5 µL) was preincubated with
30 µL of NADPH regenerating system (2.6 mM NADP+, 7.6 mM
glucose 6-phosphate, 0.8 U/mL glucose-6-phosphate dehydrogenase,
13.9 mM MgCl2, and 1 mg/mL albumin in 50 mM potassium
phosphate buffer, pH 7.4) for 10 min at 37 °C. The enzyme and
substrate mixture (33 µL of 1 µM CYP19 enzyme, BD Biosciences,
0.4 µM dibenzylfluorescein, 4 mg/mL albumin in 50 mM potassium
phosphate, pH 7.4) was added, and the plate was incubated for 30
min at 37 °C before quenching with 25 µL of 2 N NaOH. After
termination of the reaction and shaking for 5 min, the plate was
further incubated for 2 h at 37 °C. This enhances the ratio of signal
to background. Fluorescence was measured at 485 nm (excitation)
and 530 nm (emission). IC50 values and dose-response curves were
based on three independent experiments performed in duplicate
using five concentrations of test substance (Table 1).
Cytotoxicity Testing. LNCaP (prostate), MCF7 (breast), and
Hepa 1c1c7 (mouse liver) cell lines were acquired from ATCC
(Manassas, VA). The LU lung cancer cell line was provided by
the Department of Surgical Oncology, University of Illinois
Chicago, Chicago, IL. Evaluation of cytotoxic potential was
performed as described previously.39-41 In brief, using 96-well
plates, cells were seeded at a density of 5.0 × 104 cells/mL, and
treated with a 10 µL solution of each compound in 10% DMSO to
yield final concentrations ranging from 0-20 µg/mL. A zero day
control plate was incubated for 30 min at 37 °C. All other plates
were incubated for 72 h. After incubation, cells were fixed by the
addition of 50 µL of cold 50% aqueous trichloroacetic acid,
incubated at 4 °C for 30 min, washed with tap water, and air-dried.
Cells were then stained with 100 µL of 0.4% sulforhodamine B in
1% acetic acid for 30 min at 23 °C. After washing with 1% acetic
acid (4×) to remove excess dye and air-drying, bound dye was
solubilized by the addition of 200 µL 10 mM Tris base (pH 10).
Finally, the plates were shaken for 5 min, and absorption at 515
nm was determined with a Beckman Coulter ELx800 plate reader.
All determinations were performed in triplicate, and the 50%
inhibitory (IC50) values of active samples were assessed by linear
regression analyses of dose-response data.
Yield 81%. Colorless oil. IR (neat) 1731, 1599, 1525, 1495, 1459,
1
1412, 1270, 1240, 1211, 1102, 1089, 1023, 795 cm-1. H NMR
(CDCl3, 300 MHz) δ 6.61 (s, 1 H), 6.46 (s, 1 H), 3.82 (s, 3 H),
3.71 (s, 6 H), 3.13 (s, 2 H), 3.08 (s, 3 H), 2.04 (s, 3 H). 13C NMR
(CDCl3, 75 MHz) δ 202.5, 167.7, 149.5, 148.8, 142.8, 123.0, 112.2,
97.3, 56.4, 56.0, 55.8, 49.5, 36.0, 29.8. EIMS (m/z, relative intensity)
281 (M+, 37), 250 (10), 209 (12), 197 (33), 182 (100), 166 (12),
154 (17), 123 (10), 98 (11), 69 (26). HRMS calcd for C14H19NO5
281.1263, found 281.1261.
3-oxo-N-(2,3,4,5-Tetramethoxyphenyl)butanamide (10q). Yield
77%. Colorless oil. IR (neat) 3500, 2800, 1710, 1682, 1604, 1532,
1
1486, 1464, 1410, 1364, 1219, 1129, 1098, 1006 cm-1. H NMR
(CDCl3, 300 MHz) δ 9.4 (brs, 1 H), 7.73 (s, 1 H), 3.86 (s, 3 H),
3.85 (s, 6 H), 3.82 (s, 3 H), 3.77 (s, 3 H), 3.54 (s, 2 H), 2.04 (s, 3
H). 13C NMR (CDCl3, 75 MHz) δ 204.5, 163.2, 149.0, 146.3, 138.6,
136.1, 126.9, 99.2, 61.2, 61.1, 61.0, 51.0, 49.9, 31.0. EIMS (m/z,
relative intensity) 297 (M+, 47), 282 (10), 239 (22), 198 (100),
183 (21), 155 (27), 140 (17), 127 (5), 69 (16). HRMS calcd for
C14H19NO6 297.1212, found 297.1210.
Expression and Purification of Human QR2 Gene. Human
QR2 was purified from 3 L of E. coli BL21(DE3) grown in
Luria-Bertani medium supplement with 100 µg/mL ampicillin
(Fisher Scientific, Pittsburgh, PA) following our previously reported
procedure.6 Freshly prepared and nonfrozen QR2 was used to set
up crystallization experiments. For kinetic assays and inhibitor
studies, QR2, previously stored at -80 °C, was thawed on ice and
then adjusted to the desired concentration by diluting in storage
buffer without glycerol.
Steady-State Kinetic Assays and IC50 Value Determination.
The activity of QR2 under steady-state conditions was evaluated
on SpectraMax Plus 384 UV/vis spectrophotometer thermostatted
at 23 °C by monitoring the increase in absorbance of the product
formazan at 610 nm which is produced from MTT. The assays
were run in 96-well plates at a final assay volume of 200 µL. Each
assay mixture contained 12 nM QR2, 17.5 µM NMeH, and 200
µM MTT in a reaction buffer containing 100 mM NaCl, 0.1%
Triton-X100 (Fisher Biotech 93004) and 0.6 mg/mL BSA (Sigma
A3059). The reactions were initiated by the addition of QR2. The
initial slopes of the reaction (∆Abs/∆time) were used to calculate
the initial rates using a molar extinction coefficient of 11300 mM-1
cm-1 for MTT.
IC50 values were determined at 23 °C in 96-well plates using an
assay volume of 200 µL and the assay conditions described above.
Inhibitor concentrations ranged from 0.4 to 500 µM, and 11 different
inhibitor concentrations were chosen for each IC50 value determi-
nation. Assays at each inhibitor concentration were performed in
triplicate, and the average rate values and standard deviation at each
rate were used to determine the IC50 values. The resulting data were
used to calculate the percent inhibition values (%I) versus the
noninhibited control, which consisted of reaction mixture with no
inhibitor. The percent inhibition data were then plotted as a function
of inhibitor concentration ([I]) and the data were fit via nonlinear
regression to the following equation: %I ) %Imax/ (1 + (IC50/[I]))
describing a simple binding isotherm. Data were fit using the
Enzyme Kinetics Module of program SigmaPlot from SPSS
Scientific. IC50 values are reported along with standard errors.
Acknowledgment. This work was supported by program
project grant P01 CA48112 awarded by the National Cancer
Institute. Some of this research was conducted in a facility
constructed with the financial support of a Research facilities

1884 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 7
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Supporting Information Available: Elemental analyses, 1H
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3g-h, 3i-j, 3m-q, 6a, 6g, 6i, 6m, 6o, 7a, 7g, 7i, 7m, 7o, 8, and
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