Design, synthesis, and biological evaluation of potent quinoline and pyrroloquinoline ammosamide analogues as inhibitors of quinone reductase 2

Article abstract of DOI:10.1021/jm201251c

A variety of ammosamide B analogues have been synthesized and evaluated as inhibitors of quinone reductase 2 (QR2). The potencies of the resulting series of QR2 inhibitors range from 4.1 to 25,200 nM. The data provide insight into the structural parameters necessary for QR2 inhibitory activity. The natural product ammosamide B proved to be a potent QR2 inhibitor, and the potencies of the analogues generally decreased as their structures became more distinct from that of ammosamide B. Methylation of the 8-amino group of ammosamide B was an exception, resulting in an increase in quinone reductase 2 inhibitory activity from an IC₅₀ of 61 nM to IC₅₀ 4.1 nM.

Full text of DOI:10.1021/jm201251c

Article
pubs.acs.org/jmc
Design, Synthesis, and Biological Evaluation of Potent Quinoline and
Pyrroloquinoline Ammosamide Analogues as Inhibitors of Quinone
Reductase 2
P. V. Narasimha Reddy,† Katherine C. Jensen,‡ Andrew D. Mesecar,‡ Phillip E. Fanwick,§
and Mark Cushman*,†
†Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, and The Purdue University Center for
Cancer Research, Purdue University, West Lafayette, Indiana 47907, United States
‡Departments of Biological Sciences and Chemistry, and The Purdue University Center for Cancer Research, Purdue University,
West Lafayette, Indiana 47907, United States
§Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, United States
S
* Supporting Information
ABSTRACT: A variety of ammosamide B analogues have been synthesized and
evaluated as inhibitors of quinone reductase 2 (QR2). The potencies of the resulting
series of QR2 inhibitors range from 4.1 to 25,200 nM. The data provide insight into
the structural parameters necessary for QR2 inhibitory activity. The natural product
ammosamide B proved to be a potent QR2 inhibitor, and the potencies of the
analogues generally decreased as their structures became more distinct from that of
ammosamide B. Methylation of the 8-amino group of ammosamide B was an
exception, resulting in an increase in quinone reductase 2 inhibitory activity from an
IC50 of 61 nM to IC50 4.1 nM.
Scheme 1. Approach to the Synthesis of the Ammosamides
INTRODUCTION
■
Ammosamides A−C are metabolites isolated from the marine
Streptomyces strain CNR-698.1−3 All three natural products are
thought to modulate tubulin and actin dynamics through
myosin binding.2,4 The administration of a fluorescent
ammosamide B conjugate to HCT-116 cells results in the
depolymerization of microtubules and an increase in actin
filaments, and histological staining is consistent with the
binding of the conjugate to several myosin families.4
2 (QR2) inhibitory activity.6 The FAD-dependent flavoenzyme
QR2 catalyzes the reduction of quinones by reduced N-alkyl-
and N-ribosylnicotinamides.7 QR2 is capable of transforming
some quinone substrates into highly reactive species that
damage cells.7−9 Inhibition of QR2 could therefore conceivably
protect cells from chemical damage.10 A number of QR2
inhibitors have been reported,11−20 and the structures of QR2
in complex with a number of inhibitors have been determined
Two conceptually distinct syntheses of ammosamide B have
recently been reported.3,5 Our synthesis relies on the
condensation of the diprotected 1,3,4,6-tetraaminobenzene
derivative 4 with the di(methylester) of 2-ketoglutaconic acid
(5) to produce the ammosamide framework 6 as the key step
(Scheme 1).5 As reported in the present communication, this
synthesis has proven to be quite short and flexible, allowing the
production of a focused library of ammosamide congeners that
have been evaluated as inhibitors of quinone reductase 2.
X-ray crystallographic-assisted dereplication methods have
revealed that the ammosamides have potent quinone reductase
Received: September 19, 2011
Published: December 29, 2011
© 2011 American Chemical Society
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Article
a
Scheme 2. Synthesis of Ammosamide Analogues 9−16
a
Reagents and conditions: (a) benzyl chloroformate, DIPEA, CH2Cl2 room temperature (24 h); (b) compound 5, PTSA, CH2Cl2, 40 °C (24 h); (c)
H2, Pd/C, CH3OH, room temperature (1 h); (d) NCS, CH3CN, 65 °C (2 h); (e) NH4OH, THF, room temperature (24 h); (f) Ac2O, DMAP,
CH2Cl2, room temperature (2 h); (g) NaH, CH3I, DMF, room temperature (24 h).
a
by X-ray crystallography.11,21−26 The present study was
motivated by the idea that novel and potent QR2 inhibitors
could be generated based on the structures of the
ammosamides and that these inhibitors could be possibly be
of value as cancer chemopreventive agents.
Scheme 3. Synthesis of Ammosamide Analogues 18−20
RESULTS AND DISCUSSION
■
Synthesis of Ammosamide Analogues. Our synthesis of
ammosamide B provided sufficient material for evaluation of its
biological properties, and it also enabled the synthesis of an
array of structural analogues of value in the investigation of
structure−activity relationships.27 As outlined in Scheme 2, the
substituted quinoline 9 was obtained by monoprotection of 7
using benzyl chloroformate followed by condensation of the
product 8 with compound 5. Quinoline 9 proved to be a
versatile intermediate that could be converted to a number of
derivatives. Hydrogenation of quinoline 9 at 30 psi for 1 h
afforded the free amine 10, which reacted with N-
chlorosuccinimide (NCS) to afford the dichloroquinoline 11.
Treatment of 10 with 30% ammonium hydroxide in THF at
room temperature afforded C-2 amide 12, while acetylation of
10 provided 13, which could be converted to the corresponding
C-2 amide 14 in good yield. Quinoline 10 on reaction with MeI
and NaH in DMF yielded the corresponding N,N-dimethylated
quinoline 15 in 80% yield, which on treatment with 30% aq
ammonia afforded the corresponding C2 amide 16 in 95%
yield. As outlined in Scheme 3, reaction of the starting material
1728 with compound 5 resulted in the cyclized intermediate 18,
which was converted to 19 and 20 with ammonium hydroxide
in THF. Quinoline 22, obtained by condensation of 21 with 5,
reacted with aq 30% NH3 in THF for 24 h to afford the
corresponding C2 amide 23 (Scheme 4).
a
Reagents and conditions: (a) (1) compound 5, CH2Cl2, room
temperature (30 min), (2) PTSA, Cu (OAc)2, reflux (24 h); (b)
NH4OH, THF, 70 °C (24 h).
acid (PTSA) and cupric acetate in methylene chloride afforded
compound 26, which on treatment with NaH in THF at room
temperature produced 33. However, reaction of 26 with SOCl2
provided the expected quinoline 27 in very high yield.
Quinoline 27 on treatment with Et3N in CH2Cl2 resulted in
the formation of corresponding pyrroloquinoline 28, which on
methylation with MeI, using NaH in DMF as the base, afforded
the N-methylpyrroloquinoline compound 29 in 92% yield.
Compound 29 on treatment with 30% aq ammonia in THF at
room temperature for 24 h resulted in corresponding amide
compound 30. Deprotonation of amide 30 with n-butyllithium
in THF, followed by reaction with benzyl bromide, afforded
benzylamide 31. However, deprotonation of 30 with sodium
hydride in DMF, followed by alkylation with methyl iodide,
yielded the corresponding N,N-dimethylamide 32. Surprisingly,
attempted nitration of the pyrroloquinoline 29 with HNO3 and
Intermediate 255 was obtained by reduction of the starting
material 24 with iron and ammonium chloride in aq DMF at
100 °C (Scheme 5). Reaction of 25 with (E)-dimethyl 4-
oxopent-2-enedioate (5) in the presence of p-toluenesulfonic
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a
a
Scheme 4. Synthesis of Ammosamide Analogue 23
Scheme 6. Synthesis of Ammosamide Analogue 38
a
Reagents and conditions: (a) (1) compound 5, CH2Cl2, room
temperature (30 min), (2) PTSA, Cu(OAc)2, reflux (24 h); (b)
NH4OH, THF, room temperature (24 h).
a
Reagents and conditions: (a) NaH, CH3I, DMF, 23 °C, 1 h; (b) 30%
aq NH4OH, THF 23 °C, 24 h.
H2SO4 afforded ortho-quinone 34 instead, as confirmed by X-
ray crystallography (see Supporting Information).
15% yield. The methyl ester 37 was then converted to amide 38
by treatment with ammonia in THF.
Inhibition of Quinone Reductase 2 by Tricyclic
Ammosamide Analogues. The QR2 inhibitory activities of
the tricyclic ammosamide analogues are summarized in Table 1,
while the inhibitory activities of a series of bicyclic compounds
The synthesis of the ammosamide analogue 38 is outlined in
Scheme 6. Deprotonation of compound 355 with sodium
hydride in DMF, followed by alkylation with methyl iodide,
afforded a mixture of the products 365 in 70% yield and 37 in
a
Scheme 5. Synthesis of Ammosamide Analogues 27−34
a
Reagents and conditions: (a) Fe, NH4Cl, DMF/H2O, 100 °C; (b) (1) compound 5, CH2Cl2, room temperature (30 min), (2) PTSA, Cu(OAc)2,
40 °C, 24 h, 65%; (c) SOCl2, 90 °C (3 h); (d) Et3N, CH2Cl2, room temperature (24 h); (e) NaH, CH3I, DMF, 90 °C (1 h); (f) 30% NH4OH, THF,
room temperature (24 h); (g) BuLi, BnBr, THF, −78 °C (3 h); (h) CH3I, NaH, DMF, room temperature (1 h); (i) NaH, THF, room temperature
(15 min); (j) HNO3, H2SO4, room temperature (1 h).
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Table 1. QR2 Inhibitory Activities of Ammosamide B and Tricyclic Analogues 30−32
compd
-R1
-R2
-R3
-R4
-R5
IC50 (μM)
% max inhibition
2 (ammosamide B)
-CH3
-CH3
-CH3
-CH3
-CH3
-NH2
-Cl
-Cl
-H
-H
-H
-Cl
-NH2
-Cl
-C(O)NH2
0.061 0.005
5.8 1.2
90.3 1.3
46.3 2.4
44.8 2.0
31.5 4.6
101.9 1.1
30
31
32
38
-C(O)NH2
-Cl
-Cl
-C(O)NH−CH2−Ph
-C(O)N(CH3)2
-C(O)NH2
7.8 1.2
-Cl
-Cl
25.2 9.4
-NH2
-NHCH3
0.0041 0.0002
are summarized in Table 2. The lead compound 2, or
ammosamide B, was previously shown be a potent QR2
inhibitor with an IC50 value of 61 nM.6 Removal of the R3
chlorine atom and substitution of the R2 and R4 amines with
chlorines in analogue 30 decreased the inhibitory potency 95-
fold. Modification of the R5 group of 30 further reduced the
inhibitory potency as observed with analogues 31 and 32.
Solubility issues with the dichloro analogues (27, and 30−32)
confounded the kinetic studies and are likely the reason
complete inhibition (% max inhibition <50%) of QR2 could
not be obtained.
Additional compounds, including 26 and 33, were tested, but
none showed inhibitory activity against QR2. The lack of
inhibition by these compounds may be due to their absence of
structural planarity. The active site of QR2 in general prefers
planar, rigid ligands that are capable of stacking with the planar
flavin ring system of the FAD cofactor. Therefore, removal of
structural planarity in inhibitor analogues tends to destroy this
interaction.23 Orthoquinone 34 was found to be too reactive
with the N-methyldihydronicotinamide (NMeH) cofactor in
the absence of enzyme and therefore could not be tested as an
inhibitor with the QR2 system. The only tricyclic ammosamide
analogue that showed improved inhibitory potency compared
to that of ammosamide B was 38. Methylation of the amine
group at R4 increased the potency toward QR2 around 15-fold
(4.1 nM versus 61 nM).
X-ray Structures of QR2 in Complex with 2 and 38. In
an attempt to gain structural insight into the potent inhibition
of QR2 by compounds 2 and 38, the X-ray crystal structures of
these complexes were determined. QR2 crystallized in space
group P212121 and contained one dimer per asymmetric unit.
Complete X-ray data sets were collected and refined to 1.53 Å
and 1.50 Å for the complexes containing compounds 2 and 38,
respectively. Strong electron density is observed for compounds
2 and 38 as well as for a number of water molecules associated
with the inhibitors (Figure 1a and b). Both compounds bind to
QR2 in identical orientations sitting directly above and
interacting with the FAD cofactor. Two direct hydrogen
bonds are observed between the primary amide group of the
inhibitors (R5 position) and the amide −NH2 group of Asn161.
In addition, both compounds form water-mediated hydrogen
bonds to the side chain −OH group of Thr71 via their −NH2
groups at the R2 position. Interestingly, the amide group at the
R5 position, the amine group at the R4 position, and the
nitrogen in the ring of the inhibitor all form hydrogen bonds
with an ordered active site water molecule that is hydrogen
bonded to the backbone carbonyl oxygen of Gly174 (not
shown). This water molecule is present in the active site of
Figure 1. X-ray crystal structures of ammosamide B (2) and
compound 38 in complex with QR2. Ligands are shown in ball and
stick representation and are colored according to atom type. Water
molecules are shown as solid spheres, and hydrogen bonds are shown
in gray dashes (2.8−3.4 Å). Electron density maps (2Fo − Fc) are
contoured to 1.0 σ and shown in gray mesh. Electron density
difference maps (Fo − Fc) are contoured to 3.0 σ and are shown in red
mesh. The binding orientation of the ligands are the same in both
active sites of the dimer, and therefore for simplicity, only the
inhibitors in the A-chain active site are shown. (A) X-ray structure of
QR2 in complex with compound 2. (B) X-ray structure of QR2 in
complex with compound 38. (C) Superposition of the X-ray structures
shown in A and B with the colors of inhibitors conserved. Active site
water molecules correspond to compound coloring. Fo − Fc electron
density omit maps for ammosamide B and compound 38 are provided
in Supporting Information, Figure S3.
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Table 2. QR2 Inhibitory Activities of Bicyclic Ammosamide Analogues
compd
-R2
-R4
-R5
-Cl
-R6
-H
-R7
R8
IC50 (μM)
% max inhibition
10
11
12
13
14
15
16
19
20
22
23
27
-COOMe
-COOMe
-COOMe
-COOMe
-COOMe
-COOMe
-COOMe
-COOMe
-C(O)NH2
-COOMe
-COOMe
-COOMe
-COOMe
-NH2
-NH2
-NH2
-H
-Cl
-H
-H
-H
-H
-H
-H
-H
-H
-H
-Cl
3.3 0.14
5.6 0.4
1.1 0.2
>100
87.9 0.9
91.1 1.9
75.5 2.8
-COOMe
-Cl
-Cl
-Cl
-Cl
-Cl
-Cl
-H
-H
-C(O)NH2
-H
-COOMe
-H
-NH(CO)CH3
-NH(CO)CH3
-N(CH3)2
-N(CH3)2
-NH2
-C(O)NH2
-COOMe
-H
9.0 0.7
4.0 0.3
1.6 0.1
1.5 0.2
1.8 0.11
0.24 20
0.15 0.02
22.1 5.2
96.0 2.1
92.2 1.5
80.5 1.5
64.0 2.0
83.9 1.0
85.7 1.2
89.7 1.4
36.6 3.2
-H
-C(O)NH2
-C(O)NH2
-C(O)NH2
-COOMe
-H
-I
-H
-I
-NH2
-Cl
-Cl
-NH2
-NH2
-NH2
-Cl
-NH2
-C(O)NH2
-NH2
-COOMe
-H
Table 3. Cytotoxicities of Quinoline and Pyrroloquinoline Ammosamide Analogues in Cancer Cell Lines at 10 μM
Concentration
a
growth percent
lung HOP-
62
colon HCT-
116
CNS SF-
539
melanoma UACC-
62
renal
SN12C
prostate DU-
145
breast
MCF7
b
compd
mean
ovarian OVCAR-3
2
101.07
105.27
104.88
104.38
109.97
103.06
104.52
103.35
104.17
108.57
105.39
96.95
101.05
108.20
100.36
98.84
88.22
100.09
101.70
97.93
115.24
106.32
109.55
110.07
124.69
120.20
115.27
108.21
103.52
126.09
123.43
98.89
114.59
108.11
108.85
105.75
112.18
124.60
138.91
97.82
98.04
105.75
100.27
97.23
100.11
89.58
78.61
96.11
95.79
122.71
94.99
10
11
12
13
14
15
30
31
32
34
108.99
104.53
109.87
112.21
103.41
114.23
105.32
113.20
118.26
110.60
117.31
99.06
102.78
99.49
104.82
109.23
107.48
100.61
104.34
97.49
102.96
112.51
108.54
109.82
90.84
102.63
109.64
109.00
113.39
101.49
107.48
106.07
102.66
103.44
104.18
104.18
107.00
103.06
102.47
104.56
101.93
110.63
96.96
104.81
127.34
112.19
101.43
106.99
a
b
Growth percent relative to control (untreated cell cultures). Mean growth percent in the NCI panel of 60 human cancer cell lines.
unliganded QR2 and is typically displaced by the binding of
inhibitors.22 The utilization of this water molecule for the
formation of a hydrogen bond with QR2 may contribute to the
nanomolar potency.
entropic gain for the QR2−compound 38 complex, thereby
improving its potency. The importance of active site water
molecules in QR2 inhibitor binding has recently been notes for
a series of imidazoacridin-6-ones.20
The X-ray structures of QR2 in complex with compounds 2
and 38 are superimposed in Figure 1c to gain structural insight
into the improved potency of compound 38 over 2. The
binding orientations of the two inhibitors within the QR2 active
site are identical within the coordinate error of the X-ray
structures. As a result, we cannot explain structurally why the
additional methyl group at the R1 position of compound 38
leads to the greater than 10-fold increase in potency compared
to that of compound 2. However, there is one observable
difference between the active sites of the two structures. There
are 7 highly ordered water molecules observed in the active site
of the QR2−compound 2 complex, whereas there are only 5
water molecules observed in the QR2−compound 38 complex.
We independently determined three X-ray structures of the
QR2−compound 38 complex from different crystals, and all
show the same number of water molecules suggesting that the
disappearance of the two water molecules for compound 38 is
likely not an artifact of soaking or flash-freezing the crystals.
Therefore, the loss of the 2 water molecules may provide some
Inhibition of Quinone Reductase 2 by Bicyclic
Ammosamide Analogues. To further explore the structural
space surrounding the ammosamides, we designed and
synthesized a series of bicyclic ammosamide derivatives and
tested their inhibitory potency against human QR2. The
structures of the analogues and their associated inhibitory
potencies are summarized in Table 2. A comparison of the
activities of a number of the bicyclic compounds documents a
regular increase in biological activity when the C-2 methyl ester
is converted into a primary amide, and the rest of the structure
is constant. Examples of this effect include 10 vs 12 (IC50 3.3 vs
1.1 μM), 15 vs 16 (IC50 4.0 vs 1.6 μM), 13 vs 14 (>100 vs 9.0
μM), and 22 vs 23 (IC50 0.24 vs 0.15 μM). Similarly, the
conversion of a hydrogen of 12 into a primary amine at the R6
position of 23 results in a 10-fold increase in activity (IC50 1.1
vs 0.15 μM) and the most potent bicyclic compound. The
primary amide substituent of 23 parallels the substitution of the
two most potent tricyclic compounds, 2 and 38. The
conversion of the 7-amino group in 10 to a dimethylamino
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group in 15 is well tolerated, as observed by their comparable
IC50 values (IC50 3.3 vs 4.0 μM). The addition of a second
chlorine at the 8-position, 11, as compared to 10, results in only
a slight decrease in potency (IC50 3.3 vs 5.6 μM). The effect of
acetylating the 7-amino group decreases activity as documented
by a comparison of 12 vs 14 (IC50 1.1 vs 9.0 μM). Additionally,
acetylation at R7 in compound 13 drastically decreases activity
as compared to compound 10 (IC50 >100 μM vs 3.3 μM).
Conversion of the methyl ester at C-4 in 19 to the amide in 20
causes only a slight increase in activity (IC50 1.8 vs 1.5 μM).
QR2 inhibitors are in general planar aromatic compounds
that can stack with the planar flavin moiety of the cofactor in
the active site.11−20 These structural requirements are similar to
those involved in DNA intercalation, and indeed, some QR2
inhibitors have been found to be cytotoxic due to a DNA
intercalation mechanism.20 In addition, the ammosamides were
originally isolated by cytotoxicity-guided (HCT-116) fractio-
nation, further suggesting that the present series of compounds
could be cytotoxic.2 The compounds were therefore submitted
to the NCI panel of 60 human cancer cell lines for cytotoxicity
evaluation.29,30 As documented in Table 3, all of the
compounds were surprisingly noncytotoxic at a concentration
of 10 μM. These data are in agreement with results indicating
that QR2 inhibitors that do not have off-target effects should
generally not be cytotoxic.20
The expression levels of QR2 have been documented in both
normal and transformed prostate cells.32,33 While QR2 is
expressed at levels that are below the limits of detection in
normal prostate epithelial cells (PrECs), it had robust levels of
expression in normal prostate stromal cells (PrSCs).32
Furthermore, PrSCs were subject to dose-dependent inhibition
of cellular proliferation by the QR2 inhibitor resveratrol.11,18,34
However, resveratrol had no effect on PrEC growth. This has
led to the hypothesis that QR2 has a role in the control of
cellular proliferation by resveratrol in PrSCs.32 According to
this idea, the lack of toxicity of the ammosamide analogues
shown in Table 3 might therefore have something to do with
low levels of QR2 expression. This unlikely possibility can be
eliminated in the case of DU-145 prostate cancer cells because
QR2 has been detected in significant levels in DU-145 cells, as
well as in LNCaP, CWR22Rv1, PC-3, and JCA1 prostate cancer
cells.33 Furthermore, significant expression levels of QR2 have
been documented in all of the other cell lines listed in Table 3
(see Supporting Information, Figure S2). The lowest QR2
expression rate is in HOP-62 cells, while the highest rate is in
HCT-116 cells.
chloroquine, quinacrine, and mefloquine, which are also
quinoline derivatives.12,19
EXPERIMENTAL SECTION
■
General Procedures. Melting points were determined in capillary
tubes using a Mel-Temp apparatus and are not corrected. Infrared
spectra were obtained as films on KBr salt plates except where
otherwise specified, using a Perkin-Elmer Spectrum One FT-IR
spectrometer, and are baseline corrected. 1H NMR spectra were
obtained with CDCl3 at 300 or 500 MHz, using Bruker ARX300 or
Bruker Avance 500 (TXI 5 mm probe) spectrometers (residual
chloroform referenced to 7.25 ppm) or DMSO-d6 (residual DMSO
referenced to 2.49 ppm and residual water in DMSO-d6 appearing at
3.33 ppm). 13C NMR spectra were recorded with CDCl3 at 75 or 125
MHz, using Bruker ARX300 or Bruker Avance 500 (TXI 5 mm probe)
spectrometers (residual chloroform referenced to 77.0 ppm) or
DMSO-d6 (residual DMSO referenced to 39.5 ppm). Mass spectral
analyses were performed at the Purdue University Campus-Wide Mass
Spectrometry Center. ESIMS was performed using a FinniganMAT
LCQ Classic mass spectrometer system. EI/CIMS was performed
using a Hewlett-Packard Engine or GCQ FinniganMAT mass
spectrometer system. Analytical thin-layer chromatography was carried
out on Baker-flex silica gel IB2-F plastic-backed TLC plates.
Preparative thin-layer chromatography was performed on Analtech
silica gel 1500 μm glass plates. Compounds were visualized with both
short- and long-wavelength UV light. Silica gel flash chromatography
was accomplished using 230−400 mesh silica gel. All yields reported
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. The intensity of
the major peak in the analytical HPLC trace of each target compound
was ≥95% that of the combined intensities of all of the peaks detected
at 254 nm on a reversed-phase C18 HPLC column.
General Procedure for Quinoline Formation. A solution of
(E)-dimethyl 4-oxopent-2-enedioate (5) (1.2 equiv) in dichloro-
methane (20 mL) was added to a solution of amine in dichloro-
methane (10 mL/0.1 mmol) and the reaction mixture stirred for 30
min. A catalytic amount of PTSA (0.1 equiv) was added, and the
solution was heated at reflux for 24 h. The reaction mixture was
washed 3 times with NaHCO3 (15 mL). The organic layer was
separated and dried over Na2SO4 and purified by silica gel column
chromatography.
General Procedure for Carboxamide Formation. Diester was
dissolved in THF (20 mL) and a 30% NH4OH solution (2 mL) was
added. The reaction mixture was stirred at room temperature for 24 h,
by which time all of the ester had been converted to amide. THF was
removed on a rotary evaporator, CHCl3 (15 mL) was added, and the
mixture was washed with water (2 × 10 mL). The combined organic
layer was dried over Na2SO4 and then concentrated to get the amide in
quantitative yield.
Benzyl 3-Amino-5-chlorophenylcarbamate (8). DIPEA (0.54 g,
4.1 mmol) was added to a stirred solution of diamine 7 (0.2 g, 1.40
mmol) in CH2Cl2 (10 mL) at room temperature and the reaction
mixture stirred at room temperature for 30 min. Benzyl chloroformate
(0.216 mL, 1.54 mmol) was added, and the reaction mixture was
stirred for 24 h. The reaction mixture was extracted with CH2Cl2 (2 ×
20 mL) and washed with aq NH4Cl. The organic layer was
concentrated and purified by silica gel column chromatography
using hexane−EtOAc 1:1 to afford the product 8 as a white solid
(0.230 g) in 60% yield: mp 156−157 °C 1H NMR (CDCl3, 300 MHz)
δ 7.36−7.32 (m, 5 H), 6.75 (s, 1 H), 6.65 (s, 2 H), 6.34 (s, 1 H), 5.15
(s, 2 H), 3.72 (br s, 1 H).
Dimethyl 7-(Benzyloxycarbonylamino)-5-chloroquinoline-2,4-di-
carboxylate (9). This compound was prepared using the general
procedure for carboxamide formation detailed above: mp 123−125 °C.
IR (KBr) 3366, 2978, 1712, 1702, 1238, 1221, 783, 729, 656 cm−1; 1H
NMR (CDCl3, 300 MHz) δ 8.23 (s, 1 H), 8.05−8.03 (m, 2 H), 7.40−
7.37 (m, 5 H), 7.10 (s, 1 H), 5.22 (s, 2 H), 4.04 (s, 3 H), 4.00 (s, 3 H);
13C NMR (CDCl3, 75 MHz) δ 165.7, 165.2, 153.4, 147.9, 141.3, 136.4,
In conclusion, a series of ammosamide B analogues were
designed, synthesized, and tested for their inhibitory potency
against human quinone reductase 2, but only one analogue was
found to have improved potency over the natural product. The
simple methylation of the amine at the R4 position of 2
producing compound 38 improved potency over 10-fold. X-ray
structural analysis of the QR2 bound with these inhibitors
suggests that since no differences are observed in the binding
orientations of the inhibitors, that the differences in potencies
may potentially be attributed to a difference in entropy
produced by binding of fewer water molecules in the active site
of the QR2-38 complex. However, further thermodynamic
measurements would need to be made in order to quantify any
differences.
The present series of QR2 inhibitors are structurally
reminiscent of the known QR2 inhibitors primaquine,
372
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Journal of Medicinal Chemistry
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132.6, 130.0, 128.5, 120.0, 127.8, 125.5, 125.0, 118.2, 114.5, 111.8,
66.2, 51.5; ESIMS (m/z, relative intensity) 429 (MH+, 100), 431
(MH+, 35, chlorine isotope), 490 (64); HRMS calcd for
C21H17ClN2O6 428.0775; found, 428.0779.
relative intensity) 322 (MH+, 100), 324 (MH+, 31, chlorine isotope);
HRMS calcd for C14H12ClN3O4 322.0594; found, 322.0596.
Dimethyl 5-Chloro-7-(dimethylamino)quinoline-2,4-dicarboxy-
late (15). Amine 10 (0.050 g, 0.170 mmol) was dissolved in
anhydrous DMF (4 mL), and then NaH (0.014 g, 0.510 mmol)
followed by MeI (0.160 g, 1.10 mmol) were added at room
temperature and the reaction mixture stirred at the same temperature
for 1 h. The reaction mixture was quenched with saturated aq NH4Cl,
ethyl acetate (30 mL) was added, and the solution was washed with
water (2 × 30 mL). The combined organic layer was dried over
Na2SO4 and then concentrated and purified by column chromatog-
raphy (EtOAc−hexane 1:1) to get the product 15 as yellowish solid
(0.040 g) in 80% yield: mp 155−157 °C. IR (KBr) 2864, 1734, 1704,
1023, 844, 734, 678, 546 cm−1; 1H NMR (CDCl3, 300 MHz) δ 7.84 (s,
1 H), 7.33 (d, J = 2.7 Hz, 1 H), 7.26 (d, J = 2.7 Hz, 1 H), 4.03(s, 3 H),
3.98 (s, 3 H), 3.09 (s, 6 H); 13C NMR (CDCl3, 75 MHz) δ 163.4,
162.8, 145.8, 145.7, 140.3, 131.8, 129.9, 117.4, 116.6, 111.8, 52.6, 41.8;
ESIMS (m/z, relative intensity) 323 (MH+, 100), 325 (MH+, 31,
chlorine isotope), 263 (19), 324 (16); HRMS calcd for C15H15ClN2O4
323.0799; found, 323.0803.
Methyl 2-Carbamoyl-5-chloro-7-(dimethylamino)quinoline-4-
carboxylate (16). Compound 15 was dissolved in THF (6 mL),
and a 30% NH4OH solution (2 mL) was added. The reaction mixture
was stirred at room temperature for 24 h, by which time all of the ester
had been converted to amide. THF was removed on a rotary
evaporator, CHCl3 (20 mL) was added, and the mixture was washed
with water (2 × 15 mL). The combined organic layer was dried over
Na2SO4 and then concentrated to get amide 16 (0.020 g) in
quantitative yield as yellowish solid: mp 260−262 °C. IR (KBr) 3416,
3186, 1737, 1687, 1612, 1212, 1116, 761, 658 cm−1; 1H NMR (CDCl3,
300 MHz) δ 7.94 (s, 1 H), 7.92 (brs, 1 H), 7.31 (d, J = 2.7 Hz, 1 H),
7.07 (d, J = 2.7 Hz, 1 H), 5.48 (brs, 1 H), 3.98 (s, 3 H), 3.11 (s, 6 H);
13C NMR (CD3OD, 125 MHz) δ 170.7, 163.3, 152.4, 151.2, 141.1,
Dimethyl 7-Amino-5-chloroquinoline-2,4-dicarboxylate (10). A
mixture of CBz-protected amine 9 (0.05 g, 0.17 mmol) and 10% Pd−
C catalyst (20 mg) in EtOAc−MeOH (1:1) (4 mL) was hydrogenated
at 30 psi for 1 h. The suspension was filtered, and the filtrate was
evaporated and then purified by silica gel column chromatography,
with hexane−EtOAc, 6:4 to get the amine 10 (0.030 g) in 95% yield as
yellowish solid: mp 153−155 °C. IR (KBr) 3372, 2953, 1724, 1708,
1612, 1259, 1232, 789, 726, 643 cm−1; 1H NMR (CDCl3, 300 MHz) δ
7.88 (s, 1 H), 7.33 (d, J = 2.1 Hz, 1 H), 7.19 (d, J = 2.1 Hz, 1 H), 4.25
(br s, 2 H), 4.04 (s, 3 H), 3.98 (s, 3 H); 13C NMR (CDCl3, 75 MHz)
δ 166.3, 165.6, 150.6, 148.3, 147.6, 135.4, 126.4, 122.4, 120.1, 118.7,
109.9, 53.1, 52.7; ESIMS (m/z, relative intensity) 295 (MH+, 100)],
297 (MH+, 33, chlorine isotope), 233 (62), 260 (51), 282 (49);
HRMS calcd for C13H11ClN2O4 295.0486; found, 295.0480.
Dimethyl 7-Amino-5,8-dichloroquinoline-2,4-dicarboxylate (11).
Compound 10 (0.05 g, 0.116 mmol) was taken in CH3CN (4 mL),
and NCS (0.020 g, 0.140 mmol) was added at room temperature and
the reaction mixture heated at 65 °C for 2 h. Ethyl acetate (10 mL)
was added to the mixture, and the solution was washed with aq
NaHCO3 (5 mL). The water layer was extracted again with EtOAc (10
mL), and the solution was washed with the aq NaHCO3 (5 mL). The
solvent was evaporated from the combined EtOAc layer, and the
residue was purified by column chromatography, eluting with hexane−
EtOAc 8:2 to get the dichloro compound 11 (0.040 g) in 74% yield:
mp 195−197 °C. IR (KBr) 3376, 2944, 1728, 1718, 1612, 1246, 1230,
790, 733 cm−1; 1H NMR (CDCl3, 300 MHz) δ 8.91 (s, 1 H), 8.13 (s, 1
H), 7.71 (s, 1 H), 7.42−7.37 (m, 5 H), 5.26 (s, 2 H), 4.05 (s, 3 H),
4.01 (s, 3 H); 13C NMR (CDCl3, 75 MHz) δ 165.9, 165.3, 148.5,
147.6, 140.1, 130.2, 130.0, 117.4, 116.3, 113.5, 51.5; ESIMS (m/z,
relative intensity) 463 (MH+, 100), 465 (MH+, 65, chlorine isotope);
HRMS calcd for C13H10Cl2N2O4 329.0096; found, 329.0093.
130.9, 120.9, 114.7, 107.6, 105.9, 53.6, 40.2; ESIMS (m/z, relative
intensity) 308 (MH+, 100), 310 (MH+, 27, chlorine isotope), 248
(22), 303 (29); HRMS calcd for C14H14ClN3O3 308.0802; found,
308.0803.
Methyl 7-amino-2-carbamoyl-5-chloroquinoline-4-carboxylate
(12). The general procedure above for carboxamide synthesis was
followed: mp 225−227 °C. IR (KBr) 3356, 2961, 1726, 1713, 1633,
Dimethyl 7-Amino-6-iodoquinoline-2,4-dicarboxylate (18). A
solution of (E)-dimethyl 4-oxopent-2-enedioate (0.80 g, 4.8 mmol)
in dichloromethane (30 mL) was added to a solution of 4,6-
diiodobenzene-1,3-diamine 17 (1.0 g, 2.7 mmol) in dichloromethane
(10 mL) and the reaction mixture stirred for 30 min. A catalytic
amount of PTSA (0.180 g, 0.949 mmol) and Cu(OAc)2 (0.116 g,
0.632 mmol) was added, and the solution was heated at reflux for 24 h.
The reaction mixture was washed 3 times with NaHCO3 (15 mL).
The organic layer was separated and dried over Na2SO4 and purified
by silica gel column chromatography using hexane−EtOAc 7:3 to get
product 18 (0.30 mg) in 30% yield: mp 133−135 °C. IR (KBr) 3364,
1
1247, 1242, 756, 725, 631 cm−1; H NMR (CDCl3, 300 MHz) δ 7.95
(s, 1 H), 7.23 (d, J = 2.1 Hz, 1 H), 7.18 (d, J = 2.1 Hz, 1 H), 4.30 (br s,
2 H), 3.94 (s, 3 H); 13C NMR (DMSO-d6, 125 MHz) δ 168.5, 165.3,
150.7, 150.4, 149.8, 139.0, 128.5, 121.9, 113.3, 113.0, 106.8, 79.1, 59.7,
53.0; ESIMS (m/z, relative intensity) 280 (MH+, 35), 282[(MH+, 14,
chlorine isotope)], 414 (33), 602 (100); HRMS calcd for
C12H10ClN3O3 279.0411; found, 279.0421.
Dimethyl 7-Acetamido-5-chloroquinoline-2,4-dicarboxylate (13).
Amine 10 (0.050 g, 0.17 mmol) was dissolved in anhydrous CH2Cl2 (4
mL), and then DMAP (0.062 g, 0.51 mmol) followed by Ac2O (0.034
g, 0.34 mmol) were added at room temperature and the reaction
mixture stirred at the same temperature for 2 h. The mixture was
neutralized with saturated aq NH4Cl, dichloromethane (30 mL) was
added, and the solution was washed with water (2 × 30 mL). The
combined organic layer was dried over Na2SO4 and then concentrated
and purified by column chromatography (EtOAc−hexane 7:3) to get
product 13 as white solid (0.045 g) in 90% yield: mp 233−235 °C. IR
1
2943, 1728, 1712, 1643, 1255, 1221, 791, 723, 631 cm−1; H NMR
(CDCl3, 300 MHz) δ 9.30 (s, 1 H), 8.37 (s, 1 H), 7.46 (s, 1 H), 4.04
(s, 3 H), 4.01 (s, 3 H); 13C NMR (CDCl3, 75 MHz) δ 166.2, 165.8,
149.2, 148.3, 147.9, 134.0, 133.1, 115.1, 87.1, 50.8; ESIMS (m/z,
relative intensity) 387 (MH+, 100); HRMS calcd for C14H11IN2O4
386.1405; found, 386.1409.
7-Amino-6-iodoquinoline-2,4-dicarboxamide (19) and 7-Amino-
6-iodo-4-methoxycarbonylquinoline-2-carboxamide (20). Com-
pound 18 was dissolved in THF (6 mL), and a 30% NH4OH solution
(2 mL) was added. The reaction mixture was stirred at 70 °C for 24 h
in a sealed tube. THF was removed on a rotary evaporator, CHCl3 (20
mL) was added, and the mixture was washed with water (2 × 15 mL).
The combined organic layer was dried over Na2SO4 and then
concentrated and purified by silica gel column chromatography using
hexane−EtOAc 4:6 to get the diamide product 19 (0.020 g) in 20%
yield along with monoamide 20 (0.040) in 40% yield: 19 mp 195−197
°C. IR (KBr) 3416, 3186, 1737, 1687, 1612, 1212, 1116, 761, 658
1
(KBr) 2867, 1746, 1723, 1678, 1106, 845, 716, 548 cm−1; H NMR
(CDCl3, 300 MHz) δ 8.31 (s, 1 H), 8.11 (s, 1 H), 8.07 (s, 1 H) 4.04
(s, 3 H), 4.01 (s, 3 H), 2.19 (s, 3 H); 13C NMR (CDCl3, 75 MHz) δ
168.2, 164.9, 164.5, 146.9, 146.2, 140.9, 132.3, 130.7, 118.3, 117.8,
111.4, 52.1, 24.8; ESIMS (m/z, relative intensity) 337 (MH+, 54), 339
(MH+, 18, chlorine isotope), 295 (16), 376 (21); HRMS calcd for
C15H13ClN2O5 336.0513; found, 336.0511.
Methyl 7-Acetamido-2-carbamoyl-5-chloroquinoline-4-carboxy-
late (14). The general procedure above for carboxamide synthesis was
followed: mp 240−242 °C. IR (KBr) 2956, 1745, 1729, 1646, 1243,
843, 789, 569 cm−1; 1H NMR (CDCl3, 300 MHz) δ 9.85 (s, 1 H), 8.32
(s, 1 H), 7.90 (s, 1 H), 7.79 (br s, 1 H), 6.65 (br s, 1 H), 3.78 (s, 3 H),
2.02 (s, 3 H); 13C NMR (CD3OD, 125 MHz) δ 170.6, 168.5, 166.3,
150.2, 148.7, 140.3, 139.7, 129.1, 118.7, 52.3, 22.5; ESIMS (m/z,
1
cm−1; H NMR (DMSO-d6, 500 MHz) δ 8.61 (s, 1 H), 8.28 (br s, 1
H), 8.19 (br s, 1 H), 7.84 (br s, 1 H), 7.76 (s, 1 H), 7.73 (br s, 1 H),
7.26 (s, 1 H), 6.03 (s, 2 H); 13C NMR (DMSO-d6, 125 MHz) δ 168.4,
166.0, 150.5, 149.9, 148.4, 141.4, 135.9, 118.5, 112.2, 107.2, 92.4;
ESIMS (m/z, relative intensity) 357 (MH+, 100); HRESIMS calcd for
373
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C11H9IN4O2 355.9770; found, 355.9774. 20: mp 164−166 °C. IR
(KBr) 3583, 3318, 1724, 1673, 1612, 1490, 1313, 1231, 1196, 1174,
726 cm−1; 1H NMR (CDCl3, 300 MHz) δ 9.31 (s, 1 H), 8.49 (s, 1 H),
7.30 (s, 2 H), 4.63 (br s, 2 H), 3.99 (s, 3 H); 13C NMR (DMSO-d6,
125 MHz) 165.7, 165.6, 150.4, 149.9, 148.8, 135.6, 133.6, 118.4, 115.2,
107.2, 93.5, 52.8; ESIMS (m/z, relative intensity) 372 (MH+, 100);
HRMS calcd for C12H10IN3O3 371.9845; found, 371.9851.
for the next reaction: mp >300 °C. IR (KBr) 3177, 2348, 1717, 1635,
1449, 1349, 1279, 1227, 1163, 1123, 1073, 1051, 740, 670, 524 cm−1;
1H NMR (DMSO-d6, 300 MHz) δ 11.84 (s, 1 H), 8.44 (s, 1 H), 7.99
(s, 1 H), 4.00 (s, 3 H); 13C NMR (DMSO-d6, 125 MHz) 168.4, 164.7,
158.8, 158.5, 158.2, 157.9, 118.5, 116.2, 113.9, 11.6; EIMS (m/z,
relative intensity) 296 (MH+, 100); HRMS calcd for C12H6Cl2N2O3
295.9755; found, 295.9759.
Dimethyl 6,7-Diamino-5-chloroquinoline-2,4-dicarboxylate (22).
A solution of (E)-dimethyl 4-oxopent-2-enedioate (1.30 g, 7.59 mmol)
in dichloromethane (30 mL) was added to a solution of triamine 21
(1.0 g, 4.1 mmol) in dichloromethane (10 mL) and the reaction
mixture stirred for 30 min. A catalytic amount of PTSA (0.180 g, 0.949
mmol) and Cu(OAc)2 (0.116 g, 0.632 mmol) were added, and the
solution was heated at reflux for 24 h. The reaction mixture was
washed 3 times with NaHCO3 (15 mL). The organic layer was
separated and dried over Na2SO4 and purified by silica gel column
chromatography using hexane−EtOAc 1:9 to get product 22 (0.5 mg)
in 25% yield: mp 203−205 °C. 1H NMR (CDCl3, 300 MHz) δ 7.96 (s,
1 H), 7.44 (s, 1 H), 4.60 (br. s 1 H), 4.00 (s, 3 H), 3.98 (s, 3 H) 13C
NMR (CDCl3, 75 MHz) δ 169.2, 165.0, 144.3, 142.2, 139.4, 147.6,
118.6, 112.6, 55.0; ESIMS (m/z, relative intensity) 310 (MH+, 100)],
312 (MH+, 35, chlorine isotope); HRMS calcd for C13H12ClN3O4
310.0598; found, 310.0598.
Methyl 6,8-Dichloro-1-methyl-2-oxo-1,2-dihydropyrrolo[4,3,2-
d,e]quinoline-4-carboxylate (29). A solution of 28 (0.621 g, 2.09
mmol) in DMF (20 mL) was heated to 90 °C, and then NaH (0.160 g,
4.1 mmol) followed CH3I (2.8 g, 20.9 mmol) were added. After the
addition of CH3I, a solid came out from solution. Reaction was
continued for 1 h, and then the solid was filtered off and washed with
water and dried to get compound 29 (0.600 g) in 92% yield as a
yellowish solid: mp 278−280 °C. IR (KBr) 2924, 2854, 1718, 1448,
1205, 1025, 736, 574 cm−1; 1H NMR (CDCl3, 300 MHz) δ 8.71 (s, 1
H), 7.66 (s, 1 H), 4.09 (s, 3 H), 3.68 (s, 3 H); EIMS(m/z, relative
intensity) 311 (MH+, 100), 313 (MH+, 59, chlorine isotope)]; HRMS
calcd for C13H8Cl2N2O3 309.9824; found, 309.9909.
6,8-Dichloro-1-methyl-2-oxo-1,2-dihydropyrrolo[4,3,2-d,e]-
quinoline-4-carboxamide (30). Compound 29 was dissolved in THF
(250 mL), and a 30% NH4OH solution (20 mL) was added. The
reaction mixture stirred at room temperature for 24 h, by which time
all of the ester had been converted to amide. THF was removed on a
rotary evaporator, CHCl3 (150 mL) was added, and the mixture was
washed with water (2 × 40 mL). The combined organic layer was
dried over Na2SO4 and then concentrated to get amide 30 (0.580 g) in
quantitative yield as yellowish solid: mp 288−290 °C. IR (KBr) 3452,
3184, 1716, 1245, 1024, 810, 651, 574 cm−1; 1H NMR (DMSO-d6, 300
MHz) δ 8.48 (s, 1 H), 8.17 (s, 1 H), 8.09 (s, 1 H), 7.98 (s, 1 H), 3.55
(s, 3 H); EI-CIMS (m/z, relative intensity) 296 (MH+, 100), 298
(MH+, 62, chlorine isotope); HRMS calcd for C12H7Cl2N3O2
294.9915; found, 294.9919.
Methyl 6,7-Diamino-2-carbamoyl-5-chloroquinoline-4-carboxy-
late (23). The general procedure above for carboxamide synthesis
1
was followed: mp 280−282 °C. H NMR (CDCl3, 300 MHz) δ 8.08
(s, 1 H), 7.88 (br. s, 1 H), 7.26 (s, 1 H), 5.66 (br. s, 1 H), 4.53 (br s, 4
H), 4.00 (s, 3 H); 13C NMR (DMSO-d6, 125 MHz) δ 169.6, 165.9,
144.6, 142.9, 141.1, 137.0, 134.7, 116.2, 113.6, 106.5, 104.7, 52.6;
ESIMS (m/z, relative intensity) 295 (MH+, 100), 297 (MH+, 30,
chlorine isotope), 317 [(MNa+, 17)], 319 (MNa+, 5, chlorine isotope);
HRMS calcd for C12H11ClN4O3Na 317.0417; found, 317.0420.
Dimethyl 5-Amino-6,8-dichloro-4-hydroxy-1,2,3,4-tetrahydroqui-
noline-2,4-dicarboxylate (26). A solution of (E)-dimethyl 4-oxopent-
2-enedioate (5) (1.70 g, 6.25 mmol) in dichloromethane (30 mL) was
added to a solution of diamine (1.0 g, 5.26 mmol) in dichloromethane
(10 mL) and the reaction mixture stirred for 30 min. A catalytic
amount of PTSA (0.180 g, 0.66 mmol) and 0.2 equiv of Cu(OAc)2
were added, and the solution was heated at reflux for 24 h. The
reaction mixture was washed 3 times with NaHCO3 (15 mL). The
organic layer was separated and dried over Na2SO4 and purified by
silica gel column chromatography using hexane−EtOAc 9:1 to get
product 26 (0.650 mg) in 65% yield: mp 170−172 °C. IR (KBr) 3476,
2952, 1743, 1022, 879, 743, 546 cm−1; 1H NMR (CDCl3, 300 MHz) δ
7.18 (s, 1 H), 5.23 (brs, 1 H), 4.21 (brs, 2 H), 4.00 (d, J = 12 Hz, 1 H),
3.81 (s, 3 H), 3.71 (s, 3 H), 2.30 (d, J = 10 Hz, 1 H), 2.13 (dd, J = 4.5,
10 Hz, 1 H); 13C NMR (DMSO-d6, 125 MHz) δ 175.2, 171.6, 142.3,
139.4, 128.0, 109.5, 106.9, 106.6, 79.1, 71.6, 52.5, 52.2, 49.7; EIMS(m/
z, relative intensity) 348 (MH+, 100); HRMS calcd for C13H14Cl2N2O5
348.0280; found, 348.0283.
N-Benzyl-6,8-dichloro-1-methyl-2-oxo-1,2-dihydropyrrolo[4,3,2-
d,e]quinoline-4-carboxamide (31). Compound 30 (0.050 g, 0.168
mmol) was dissolved in anhydrous THF (4 mL), and then BuLi (1.0
mL, 0.337 mmol) followed by BnBr (0.034 g, 0.202 mmol) were
added at −78 °C and the reaction mixture stirred at the same
temperature for 3 h. The reaction was quenched with saturated aq
NH4Cl, ethyl acetate (30 mL) was added, and the solution was washed
with water (2 × 30 mL). The combined organic layer was dried over
Na2SO4 and then concentrated and purified by column chromatog-
raphy (EtOAc−hexane 5:5) to afford product 31 as a yellowish solid
(0.030 g) in 65% yield: mp 236−238 °C. IR (KBr) 3397, 1718, 1678,
1
1529, 1519, 1244, 1023, 575 cm−1; H NMR (CDCl3, 300 MHz) δ
8.88 (s, 1 H), 8.62 (br s, 1 H), 7.69 (s, 1 H), 7.61−7.28 (m, 5 H) 4.75
(d, J = 6.3 Hz, 2 H), 3.68 (s, 3 H); 13C NMR (CDCl3, 125 MHz) δ
165.8, 162.9, 153.5, 139.4, 137.8, 135.5, 134.8, 134.4, 128.7, 128.5,
128.0, 127.7, 127.6, 127.3, 122.7, 117.8, 113.6, 43.7, 28.5; ESIMS (m/
z, relative intensity) 386 (MH+, 100), 388 (MH+, 60, chlorine
isotope); HRMS calcd for C19H13Cl2N3O2 386.0463; found, 386.0470.
6,8-Dichloro-N,N,1-trimethyl-2-oxo-1,2-dihydropyrrolo[4,3,2-
d,e]quinoline-4-carboxamide (32). Compound 30 (0.050 g, 0.168
mmol) was dissolved in anhydrous DMF (4 mL), and then NaH
(0.016 g, 0.675 mmol) followed by MeI (0.190 g, 1.34 mmol) were
added at room temperature and the reaction mixture stirred at the
same temperature for 1 h. The reaction was quenched with saturated
aq NH4Cl, ethyl acetate (30 mL) was added, and the solution was
washed with water (2 × 30 mL). The combined organic layer was
dried over Na2SO4 and then concentrated and purified by column
chromatography (EtOAc−hexane 6:4) to get product 32 as a yellowish
solid (0.040 g) in 85% yield: mp 236−238 °C. IR (KBr) 2840, 1734,
Dimethyl 5-Amino-6,8-dichloroquinoline-2,4-dicarboxylate (27).
Compound 26 (0.9 g, 2.5 mmol) was dissolved in thionyl chloride (8
mL) and then the reaction mixture was heated at 90 °C for 3 h, after
which thionyl chloride was removed on a rotary evaporator, ethyl
acetate (60 mL) was added, and the solution washed with saturated aq.
NaHCO3 (2 × 30 mL). The combined organic layer was dried over
Na2SO4 and then concentrated to get product 27 as reddish solid
(0.810 g) in 95% yield: mp 313−315 °C. IR (KBr) 3420, 2924, 1740,
1
1204, 845, 622 cm−1; H NMR (CDCl3, 300 MHz) δ 8.19 (s, 1 H),
7.85 (s, 1 H), 4.03 (s, 3 H), 4.02 (s, 3 H); ESIMS (m/z, relative
intensity) 351 (MNa+, 100), 353 (MNa+, 59, chlorine isotope), 253
(73), 255 (53); HRMS calcd for C13H10Cl2N2O4Na 350.9915; found,
350.9917.
1
1276, 1023, 865, 726, 547 cm−1; H NMR (CDCl3, 300 MHz) δ 8.30
Methyl 6,8-Dichloro-2-oxo-1,2-dihydropyrrolo[4,3,2-d,e]-
quinoline-4-carboxylate (28). Compound 27 (0.810 g, 2.39 mmol)
was dissolved in dichloromethane (25 mL), and excess Et3N (2 mL)
was added. The reaction mixture was kept aside for 24 h at room
temperature, by which time an undissolved solid was formed. The
solid was filtered off and dried to get product 28 as a dark yellowish
solid (0.621 g) in 85% yield, and the crude product was used as such
(s, 1 H), 7.62 (s, 1 H), 3.67 (s, 3 H), 3.21 (s, 6 H); ESIMS (m/z,
relative intensity) 324 (MH+, 60), 326 (MH+, 38, chlorine isotope),
346 (MNa+, 13); HRMS calcd for C14H11Cl2N3O2 324.0307; found,
324.0304.
Methyl 6,8-Dichloro-2a-hydroxy-2-oxo-1,2,2a,3,4,5-
hexahydropyrrolo[4,3,2-d,e]quinoline-4-carboxylate (33). To a
stirred solution of compound 26 (0.5 g, 1.43 mmol) in anhydrous
374
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Journal of Medicinal Chemistry
Article
THF (10 mL) was added NaH (0.1 g, 2.15 mmol) at room
temperature and the reaction mixture stirred for another 15 min and
then quenched with aq NH4Cl (10 mL) and then extracted with
EtOAc (2 × 20 mL). The combined ethyl acetate solution was washed
with water (15 mL) and brine (15 mL), and the ethyl acetate was
evaporated in the rotavapor to get product 33 as a white solid (0.390
average and standard deviations in the rate values were used to
determine the IC50 value by calculating the % inhibition at each
inhibitor concentration versus the negative control with zero inhibitor.
These data were plotted as the percent inhibition versus inhibitor
concentration. All data were fit to the equation: % I = (% Imax[1 + [I]/I
IC50)] using nonlinear regression in the Enzyme Kinetics Module of
the program SigmPlot. IC50 and % maximal inhibition values are
reported along with their standard error in the fitted parameter.
Crystallization and X-ray Structure Determination of QR2
Inhibitor Complexes. QR2 was crystallized using our previously
described methods with some modification.10,21,22,35 Briefly, the
hanging-drop, vapor-diffusion method was used by setting up drops
of 1 μL of purified QR2 (4 mg/mL) and then adding a series of 1 μL
aliquots of reservoir solution that contained between 1.3 to 1.7 M
ammonium sulfate in 0.1 M Bis-Tris buffer between pH 6.0 to 7.0,
with 0.1 M NaCl, 5 mM DTT, and 12 μM FAD. Diffraction quality,
rod-shaped crystals grew within 1 week with dimensions of
approximately 0.1 mm × 0.2 mm. Crystals were transferred from
hanging-drops to a 10 μL drop of an artificial mother-liquor solution
prepared with 9 μL reservoir solution and 1 μL stock solution of
inhibitor (10 mM in 100% DMSO). Crystals were allowed to soak for
3 to 24 h. Crystals were retrieved with a nylon loop, which was then
swiped through the same artificial mother-liquor solution supple-
mented with 20% glycerol. The crystals were then flash-frozen by
plunging into liquid nitrogen.
X-ray diffraction data were collected at beamline 21-ID-G at the Life
Sciences Collaborative Access Team (LS-CAT) at the Advanced
Photon Source (APS), Argonne National Laboratories. X-ray data sets
were collected on a MarMosaic 300 mm CCD detector. QR2−
inhibitor complexes of compound 2 and compound 38 were processed
and scaled using the program HKL2000.36 The crystals belonged to
the primitive orthorhombic space group P212121. Complete X-ray data
sets were obtained for 3 individual crystals of QR2 in complex with
compound 38. X-ray data on compound 2 were collected to 1.53 Å
(1.53−1.56 Å) where the data in parentheses represent the highest
resolution shell for the QR2−compound 2 complex. The overall
completeness was 97.0% (79.2%), the average I/σI was 31.7 (2.8), and
the average mosaicity was 0.17°. Data on the QR2−38 complex were
collected to 1.50 Å (1.50−1.55 Å) resolution. The overall
completeness was 99.6% (99.3%), the average I/σI was 37.4 (2.7),
and the average mosaicity was 0.61°.
Intensities were converted to structure−factor amplitudes by the
French and Wilson method using TRUNCATE in the CCP4 program
suite.37 The initial phases for the model were determined by molecular
replacement using the program PHASER in CCP4 using PDB 1SG0 as
the search model.11 The final structure contained a dimer per
asymmetric unit. Molecular library files and coordinates for the
inhibitors were built using Sketcher in CCP4. Fourier maps were
calculated and visualized using the program Coot,31 and the structures
were refined using the program Refmac. Water molecules were added
manually to 2Fo − Fc density peaks that were greater than 1.0 σ.
Iterative rounds of refinement using Refmac were continued until Rwork
and Rfree values reached their lowest values. At this point, TLS
refinement was employed by first submitting the coordinates to the
TLS (translation/libration/screw) server38 to generate a multigroup
TLS model. The resulting TLS groups were visualized using the
molecular viewer on the TLS Web site,39 and 13 TLS groups were
chosen. Two rounds of TLS and restrained refinement40 were
performed in REFMAC to arrive at the final models that were then
validated using MolProbity.41 The final model for the QR2−
compound 2 complex was refined to a value of 16.2% for Rwork and
18.4% for Rfree. The overall average B-factor was 14.1 Å2 for the protein
and 19.8 Å2 for the inhibitor. The X-ray structure for the QR2−
compound 38 complex was refined to 17.9% for Rwork and 19.7% for
Rfree. The overall average B-factor was 15.8 Å2 for the protein and 25.8
Å2 for the inhibitor. Electron density maps presented in the figures
were calculated using CCP4, and the figures were generated using the
program PYMOL.
1
g) in 85% yield: mp 241−243 °C. H NMR (DMSO-d6, 300 MHz) δ
10.59 (s, 1 H), 7.20 (s, 1 H), 6.48 (s, 1 H), 5.94 (s, 1 H), 4.37 (dd, J =
3.9, 12.9 Hz, 1 H), 3.71 (s, 3 H), 2.32 (dd, J = 3.9, 12.9 Hz, 1 H), 1.14
(t, J = 12.9 Hz, 1 H); ESIMS (m/z, relative intensity) 339 (MNa+,
100), 341 (MNa+, 62, chlorine isotope); HRMS calcd for
C12H10Cl2N2O4Na 338.9915; found, 338.9912.
Methyl 6,8-Dichloro-1-methyl-7-nitro-2-oxo-1,2-dihydropyrrolo-
[4,3,2-d,e]quinoline-4-carboxylate (34). Compound 29 (0.1 g,
0.322 mmol) was dissolved in a mixture of HNO3 and H2SO4 (3
mL, 2:1), and then the reaction mixture was stirred at room
temperature for 2 h. The mixture of acids was neutralized with
saturated aq NaHCO3, chloroform (40 mL) was added, and the
solution was washed with water (2 × 30 mL). The combined organic
layer was dried over Na2SO4 and then concentrated to get product 34
(structure confirmed by X-ray crystallography) as a yellowish solid (0.1
g) in 87% yield: mp 223−225 °C. IR (KBr) 2955, 2925, 1727, 1440,
1262, 1217, 1027, 740, 575 cm−1; 1H NMR (CDCl3, 300 MHz) δ 8.77
(s, 1 H), 4.09 (s, 3 H), 3.85 (s, 3 H); 13C NMR (CDCl3, 75 MHz) δ
173.7, 173.0, 164.2, 163.7, 155.1, 142.5, 142.0, 135.4, 134.4, 134.1,
123.4, 53.8, 29.6; ESIMS (m/z, relative intensity) 307 (MH+, 100),
309 (MH+, 65, chlorine isotope), 329 (MNa+, 45), 331 (MNa+, 30);
HRMS calcd for C13H7ClN2O5Na 328.9941; found, 328.9944.
8-Amino-7-chloro-1-methyl-6-(methylamino)-2-oxo-1,2-
dihydropyrrolo[4,3,2-d,e]quinoline-4-carboxamide (38). NaH (15
mg, 0.308 mmol) followed by CH3I (45 mg, 0.308 mmol) were added
to a stirred solution of 355 (0.075 g, 0.256 mmol) in DMF (3 mL).
The mixture was stirred at room temperature for 1 h, quenched with
saturated aq NH4Cl, and extracted with EtOAc (4 × 30 mL). The
combined organic layer was dried over Na2SO4, concentrated, and
purified by silica gel column chromatography using CHCl3−MeOH
(9.4:0.6) to furnish major product 365 (0.056 g, 70%) and minor
product 37 (0.012 g, 15%) as purple solids. Compound 37 (0.012 g,
0.037 mmol) was dissolved in THF (8 mL), and a 30% NH4OH
solution (0.5 mL) was added. The reaction mixture was stirred at
room temperature for 24 h. The THF was removed on a rotary
evaporator, and the product was purified by silica gel column
chromatography using CHCl3−MeOH (9.0:1.0) to yield product 38
(0.008 g, 80%) as a purple solid: mp >300 °C. IR (KBr) 3357, 3168,
1
2941, 2835, 1671, 1378, 1154, 595, 473 cm−1; H NMR (CDCl3, 300
MHz) δ 8.68 (s, 1 H), 8.10 (br s, 1 H), 5.97 (br s 1 H), 5.23 (br s 2
H), 3.54 (s, 3 H), 2.85 (s, 3 H); 13C NMR (DMSO-d6, 125 MHz) δ
165.9, 165.5, 146.5, 140.1, 135.1, 132.0, 131.9, 120.1, 115.0, 112.6,
108.6, 37.0, 30.3; ESIMS (m/z, relative intensity) 306 (MH+, 100),
308 (MH+, 31, chlorine isotope); HRESIMS calcd for C13H12ClN5O2
306.0758; found, 306.0755.
Steady-State Kinetic Assays and QR2 IC50 Value Determi-
nation. The enzymatic activity of QR2 was determined using MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and
NMeH as substrates as previously described.10,35 Briefly, assays were
run in a 96-well plate with a final assay volume of 200 μL, and the
appearance of the reduced form of the MTT substrate, formazan, was
monitored at 612 nm. The assay was performed at 23 °C using a
BioTek Synergy H1 Hybride multimode microplate reader. Each assay
mixture contained 12 nM QR2, 25 μM NMeH, and 200 μM MTT in a
reaction buffer containing 100 mM NaCl, 50 mM Tris, pH 7.5, and
0.1% TritonX-100. All reactions were initiated by the addition of QR2.
Initial slopes of the reactions (ΔOD@612 nm/min) were measured
and were used to calculate the initial rates of the reaction using a value
of 11,300 M−1cm−1 for the molar extinction coefficient of MTT.
IC50 values were determined using the same assay as described
above with the addition of inhibitor at varying concentrations. The
concentration ranges of inhibitor utilized to derive the final IC50 values
depended on the final potency of the inhibitor. Assays at each
concentration of inhibitor were performed in triplicate, and the
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Journal of Medicinal Chemistry
Article
Quinones and Anti-tumor Drugs. Biochem. Pharmacol. 2006, 72,
366−376.
(8) Jamieson, D.; Tung, A. T.; Knox, R. J.; Boddy, A. V. Reduction of
Mitomycin C Is Catalysed by Human Recombinant NRH:Quinone
Oxidoreductase 2 Using Reduced Nicotinamide Adenine Dinucleotide
As an Electron Donating Co-factor. Br. J. Cancer 2006, 95, 1229−
1233.
(9) Knox, R. J.; Jenkins, T. C.; Hobbs, S. M.; Chen, S.; Melton, R. G.;
Burke, P. J. Bioactivation of 5-(Aziridin-1-yl)-2,4-dinitrobenzamide
(CB 1954) by Human NAD(P)H Quinone Oxidoreductase 2: a Novel
Co-Substrate-Mediated Antitumor Prodrug Therapy. Cancer Res.
2000, 60, 4179−4186.
ASSOCIATED CONTENT
■
S
* Supporting Information
Crystallographic data for compound 34, gene expression profile
for QR2 in the NCI-60 cell lines (Figure S2), and Fo − Fc
electron density omit maps for ammosamide B and compound
38 (Figure S3). This material is available free of charge via the
Internet at http://pubs.acs.org.
Accession Codes
PDB code for ammosamide B (2) with QR2, QUXH; PDB
code for 38 with QR2, 3UXE.
(10) Maiti, A.; Sturdy, M.; Marler, L.; Pegan, S. D.; Mesecar, A. D.;
Pezzuto, J. M.; Cushman, M. Synthesis of Casimiroin and
Optimization of Its Quinone Reductase 2 and Aromatase Inhibitory
Activity. J. Med. Chem. 2009, 52, 1873−1884.
(11) Buryanovskyy, L.; Fu, Y.; Boyd, M.; Ma, Y. L.; Hsieh, T. C.; Wu,
J. M.; Zhang, Z. T. Crystal Structure of Quinone Reductase 2 in
Complex with Resveratrol. Biochemistry 2004, 43, 11417−11426.
(12) Kwiek, J. J.; Haystead, T. A. J.; Rudolph, J. Kinetic Mechanism
of Quinone Oxidoreductase 2 and Its Inhibition by the Antimalarial
Quinolines. Biochemistry 2004, 43, 4538−4547.
(13) Mailliet, F.; Ferry, G.; Vella, F.; Berger, S.; Coge, F.; Chomarat,
P.; Mallet, C.; Guenin, S. P.; Guillaumet, G.; Viaud-Massuard, M. C.;
Yous, S.; Delagrange, P.; Boutin, J. A. Characterization of the
Melatoninergic MT3 Binding Site on the NRH: Quinone Oxidor-
eductase 2 Enzyme. Biochem. Pharmacol. 2005, 71, 74−88.
(14) Rix, U.; Hantschel, O.; Duernberger, G.; Rix, L. L. R.;
Planyavsky, M.; Fernbach, N. V.; Kaupe, I.; Bennett, K. L.; Valent, P.;
Colinge, J.; Kocher, T.; Superti-Furga, G. Chemical Proteomic Profiles
of the BCR-ABL Inhibitors Imatinib, Nilotinib, and Dasatinib, Reveal
Novel Kinase and Nonkinase Targets. Blood 2007, 110, 4055−4063.
(15) Nolan, K. A.; Humphries, M. P.; Barnes, J.; Doncaster, J. R.;
Caraher, M. C.; Tirelli, N.; Bryce, R. A.; Whitehead, R. C.; Stratford, I.
J. Triazoloacridin-6-ones as Novel Inhibitors of the Quinone
Oxidoreductases NQO1 and NQO2. Bioorg. Med. Chem. 2010, 18,
696−706.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 765-494-1465. Fax: 765-494-6790. E-mail: cushman@
purdue.edu.
ACKNOWLEDGMENTS
■
This work was facilitated by the National Institutes of Health
(NIH) through support with Research Grant P01 CA48112.
We thank Dr. Karl Wood, Dr. Huaping Mo, and Dr. Daniel Lee
for consultation and valuable discussions. We also thank the
Life-Sciences CAT staff for their help at beamline 21-ID G,
which was used for X-ray data collection. Use of the Advanced
Photon Source was supported by the U.S. Department of
Energy, Office of Science, Office of Basic Energy Sciences,
under Contract No. DE-AC02-06CH11357. Use of the LS-
CAT Sector 21 was supported by the Michigan Economic
Development Corporation and the Michigan Technology Tri-
Corridor for the support of this research program (Grant
085P1000817).
ABBREVIATIONS USED
■
CIMS, chemical ionization mass spectrometry; DIPEA, N,N-
diisopropylethylamine; DMF, N,N-dimethylformamide;
DMSO, dimethyl sulfoxide; EIMS, electron impact mass
spectrometry; ESIMS, electrospray ionization mass spectrom-
etry; FAD, flavin adenine dinucleotide; HRMS, high resolution
mass spectrometry; NCS, N-chlorosuccinimide; NMeH, N-
methyldihydronicotinamide; PTSA, p-toluenesulfonic acid;
QR2, quinone reductase 2; THF, tetrahydrofuran
(16) Boyer, J. H.; Morgan, L. R. Conversion of 3-Aminocatechols to
6-Hydroxypicolinic Acids. J. Am. Chem. Soc. 1960, 82, 4748−4749.
(17) Sadighi, J. P.; Harris, M. C.; Buchwald, S. L. A Highly Active
Palladium Catalyst for the Arylation of Anilines. Tetrahderon Lett.
1998, 39, 5327−5330.
(18) Ferry, G.; Hecht, S.; Berger, S.; Moulharat, N.; Coge, F.;
Guillaumet, G.; Leclerc, V.; Yous, S.; Delagrange, P.; Boutin, J. A. Old
and New Inhibitors of Quinone Reductase 2. Chem.-Biol. Interact.
2010, 186, 103−109.
(19) Graves, P. R.; Kwiek, J. J.; Fadden, P.; Ray, R.; Hardeman, K.;
Coley, A. M.; Foley, M.; Haystead, T. A. J. Discovery of Novel Targets
of Quinoline Drugs in the Human Purine Binding Proteome. Mol.
Pharmacol. 2002, 62, 1364−1372.
REFERENCES
■
(1) Guaden
̂
cio, S. P.; MacMillan, J. B.; Jensen, P. R.; Fenical, W.
Ammosamides A and B New Cytotoxic Alkaloids Isolated from Marine
(20) Dunstan, M. S.; Barnes, J.; Humphries, M.; Whitehead, R. C.;
Bryce, R. A.; Leys, D.; Stratford, I. J.; Nolan, K. A. Novel Inhibitors of
NRH:Quinone Oxidoreductase 2 (NQO2): Crystal Structures,
Biochemical Activity, and Intracellular Effects of Inidazoaciidin-6-
ones. J. Med. Chem. 2011, 54, 6597−6601.
(21) Calamini, B.; Santarsiero, B. D.; Boutin, J. A.; Mesecar, A. D.
Kinetic, Thermodynamic and X-Ray Structural Insights into the
Interaction of Melatonin and Analogues with Quinone Reductase 2.
Biochem. J. 2008, 413, 81−91.
(22) Pegan, S. D.; Sturdy, M.; Ferry, G.; Delagrange, P.; Boutin, J. A.;
Mesecar, A. D. X-Ray Structural Studies of Quinone Reductase 2
Nanomolar Range Inhibitors. Protein Sci. 2011, 20, 1182−1195.
(23) Winger, J. A.; Hantschel, O.; Superti-Furga, G.; Kuriyan, J. The
Structure of the Leukemia Drug Imatinib Bound to Human Quinone
Reductase 2 (NQO2). BMT Struct. Biol. 2009, 9.
(24) AbuKhader, M.; Heap, J.; De Matteis, C.; Kellam, B.; Doughty,
S. W.; Minton, N.; Paoli, M. Binding of the Anticancer Prodrug
CB1954 to the Activating Enzyme NQO2 Revealed by the Crystal
Structure of Their Complex. J. Med. Chem. 2005, 48, 7714−7719.
Streptomyces sp. Planta Med. 2008, 74, 1083.
(2) Hughes, C. C.; MacMillan, J. B.; Gaudencio, S. R.; Jensen, P. R.;
Fenical, W. The Ammosamides: Structures of Cell Cycle Modulators
from a Marine-Derived Streptomyces Species. Angew. Chem. Int. Ed.
2009, 48, 725−727.
(3) Hughes, C. C.; Fenical, W. Total Synthesis of the Ammosamides.
J. Am. Chem. Soc. 2010, 132, 2528−2529.
(4) Hughes, C. C.; MacMillan, J. B.; Gaudencio, S. P.; Fenical, W.; La
Clair, J. J. Ammosamides A and B Target Myosin. Angew. Chem. Int.
Ed. Engl. 2009, 48, 728−732.
(5) Reddy, P. V. N.; Banerjee, B.; Cushman, M. Efficient Total
Synthesis of Ammosamide B. Org. Lett. 2010, 12, 3112−3114.
(6) Sturdy, M.; Pegan, S.; Gaudancio, S. P.; Mo, S.; Chlipala, G.;
̃
Orjala, J.; Fenical, W.; Mesecar, A. D. Discovery of the Ammosamides
as Quinone Reductase-2 Inhibitors Through Crystallographic
Methods. In American Society of Pharmacognosy (ASP) Meeting,
Honolulu, HI, 2009.
(7) Celli, C. M.; Tran, N.; Knox, R.; Jaiswal, A. K. NRH:Quinone
Oxidoreductase 2 (NQO2) Catalyzes Metabolic Activation of
376
dx.doi.org/10.1021/jm201251c | J. Med. Chem. 2012, 55, 367−377

Journal of Medicinal Chemistry
Article
(25) Fu, Y.; Buryanovskyy, L.; Zhang, Z. T. Crystal Structure of
Quinone Reductase 2 in Complex with Cancer Prodrug CB1954.
Biochem. Biophys. Res. Commun. 2004, 336, 332−338.
(26) Fu, Y.; Buryanovskyy, L.; Zhang, Z. T. Quinone Reductase 2 Is a
Catechol Quinone Reductase. J. Biol. Chem. 2008, 283, 23829−23835.
(27) Dietz, B. M.; Kang, Y. H.; Liu, G. W.; Eggler, A. L.; Yao, P.;
Chadwick, L. R.; Pauli, G. F.; Farnsworth, N. R.; Mesecar, A. D.; van
Breemen, R. B.; Bolton, J. L. Xanthohumol Isolated from Humulus
lupulus Inhibits Menadione-Induced DNA Damage through Induction
of Quinone Reductase. Chem. Res. Toxicol. 2005, 18, 1296−1305.
(28) Iskra, J.; Stavber, S.; Zupan, M. Nonmetal-catalyzed Iodination
of Arenes with Iodide and Hydrogen Peroxide. Synthesis 2004, 1869−
1873.
(29) Boyd, M. R. Status of the NCI Preclinical Antitumor Drug
Discovery Screen. Principles Pract. Oncol. 1989, 3, 1−12.
(30) Boyd, M. R.; Paull, K. D. Some Practical Considerations and
Applications of the National Cancer Institute In Vitro Anticancer Drug
Discovery Screen. Drug Dev. Res. 1995, 34, 91−109.
(31) Emsley, P.; Cowtan, K. Coot: Model-Building Tools for
Molecular Graphics. Acta Crystallogr., Sect. D 2004, 60, 2126−2132.
(32) Hsieh, T. C. Uptake of Resveratrol and Role of Resveratrol-
targeting Protein, Quinone Reductase 2, in Normally Cultured Human
Prostate Cells. Asian J. Androl. 2009, 11, 653−661.
(33) Wang, Z.; Hsieh, T.; Zhang, Z.; Ma, Y.; Wu, J. M. Identification
and Purification of Resveratrol Targeting Proteins Using Immobilized
Resveratrol Affinity Chromatography. Biochem. Biophys. Res. Commun.
2004, 323, 743−749.
(34) Sun, B.; Hoshino, J.; Jermihov, K.; Marler, L.; Pezzuto, J. M.;
Mesecar, A. D.; Cushman, M. Design, Synthesis, and Biological
Evaluation of Resveratrol Analogues as Aromatase and Quinone
Reductase 2 Inhibitors for Chemoprevention of Cancer. Bioorg. Med.
Chem. 2010, 18, 5352−5366.
(35) Conda-Sheridan, M.; Marler, L.; Park, E. J.; Kondratyuk, T. P.;
Jermihov, K.; Mesecar, A. D.; Pezzuto, J. M.; Asolkar, R. N.; Fenical,
W.; Cushman, M. Potential Chemopreventive Agents Based on the
Structure of the Lead Compound 2-Bromo-1-hydroxyphenazine,
Isolated from Streptomyces Species, Strain CNS284. J. Med. Chem.
2010, 53, 8688−8699.
(36) Otwinowski, Z.; Minor, Z. Processing of X-Ray Diffraction Data
Collected in Oscillation Mode. Methods Enzymol. 1997, 276, 307−326.
(37) Collaborative Computational Project, N. The CCP4 Suite:
Programs for Protein Crystallography. Acta Crystallogr., Sect D 1994,
50, 760−763.
(38) Painter, J.; Merritt, E. A. TLSMD Web Server for the
Generation of Multi-group TLS Models. J. Appl. Crystallogr. 2006,
39, 109−111.
(39) Painter, J.; Merritt, E. A. A Molecular Viewer for the Analysis of
TLS Rigid-body Motion in Macromolecules. Acta Crystallogr., Sect. D
2005, 61, 465−471.
(40) Winn, M. D.; Isupov, M. N.; Murshudov, G. N. Use of TLS
Parameters to Model Anisotropic Displacements in Macromolecular
Refinement. Acta Crystallogr., Sect. D 2001, 57, 122−133.
(41) Lovell, S. C.; Davis, I. W.; Adrendall, W. B.; de Bakker, P. I. W.;
Word, J. M.; Prisant, M. G.; Richardson, J. S.; Richardson, D. C.
Structure Validation by C Alpha Geometry: Phi,Psi and C Beta
Deviation. Proteins: Struct., Funct., Genet. 2003, 50, 437−450.
377
dx.doi.org/10.1021/jm201251c | J. Med. Chem. 2012, 55, 367−377