Synthesis of 3-[(N-carboalkoxy)ethylamino]-indazole-dione derivatives and their biological activities on human liver carbonyl reductase

Article abstract of DOI:10.1016/j.bmc.2009.11.011

A series of indazole-dione derivatives were synthesized by the 1,3-dipolar cycloaddition reaction of appropriate substituted benzoquinones or naphthoquinones and N-carboalkoxyamino diazopropane derivatives. These compounds were evaluated for their effects on human carbonyl reductase. Several of the analogs were found to serve as substrates for carbonyl reductase with a wide range of catalytic efficiencies, while four analogs display inhibitory activities with IC₅₀ values ranging from 3-5 μM. Two of the inhibitors were studied in greater detail and were found to be noncompetitive inhibitors against both NADPH and menadione with K_I values ranging between 2 and 11 μM. Computational studies suggest that conformation of the compounds may determine whether the indazole-diones bind productively to yield product or nonproductively to inhibit the enzyme.

Full text of DOI:10.1016/j.bmc.2009.11.011

Bioorganic & Medicinal Chemistry 18 (2010) 134–141
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of 3-[(N-carboalkoxy)ethylamino]-indazole-dione derivatives
and their biological activities on human liver carbonyl reductase
Solomon Berhe a, Andrew Slupe b, Choice Luster b, Henry A. Charlier Jr. b, Don L. Warner b, Leon H. Zalkow c,
Edward M. Burgess c, Nkechi M. Enwerem a, Oladapo Bakare a,
*
a Department of Chemistry, Howard University, Washington, DC 20059, USA
b Department of Chemistry and Biochemistry, Boise State University, Boise, ID 83725, USA
c School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of indazole-dione derivatives were synthesized by the 1,3-dipolar cycloaddition reaction of
appropriate substituted benzoquinones or naphthoquinones and N-carboalkoxyamino diazopropane
derivatives. These compounds were evaluated for their effects on human carbonyl reductase. Several
of the analogs were found to serve as substrates for carbonyl reductase with a wide range of catalytic effi-
Received 5 September 2009
Revised 4 November 2009
Accepted 5 November 2009
Available online 10 November 2009
ciencies, while four analogs display inhibitory activities with IC50 values ranging from 3–5
inhibitors were studied in greater detail and were found to be noncompetitive inhibitors against both
NADPH and menadione with KI values ranging between 2 and 11 M. Computational studies suggest that
lM. Two of the
Keywords:
Carbonyl reductase
Indazole-dione
Pyrazoloquinone
Anthracycline therapy
l
conformation of the compounds may determine whether the indazole-diones bind productively to yield
product or nonproductively to inhibit the enzyme.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
observed cardiotoxicity.10–17 Enzymes with NADPH-dependent
carbonyl reductase activity are known to reduce the C13-carbonyl
Anthracyclines such as doxorubicin and daunorubicin (Fig. 1A)
are anti-neoplastic agents commonly used to treat a variety of can-
cers.1–5 However, the use of anthracyclines in treating cancer is sig-
nificantlylimited by a potentially lethal cardiotoxicity.3–5 Formation
of reactive oxygen species has been proposed as a major pathway for
the cause and development of anthracycline-induced cardiotoxic-
ity.6 As a result, extensive studies and tests have been conducted
to evaluate the cardio-protective effectiveness of antioxidants
against anthracycline-induced cardiotoxicity, but these efforts have
resulted in only limited success.7,8 So far, the antioxidant and iron
chelator dexrazoxane is the only drug approved by the FDA as an
adjuvant therapy for protection against anthracycline-induced car-
diotoxicity.6,9 However, this drug offers only partial cardio-protec-
tion, and its use is accompanied by dose-dependent leukopenia.7
Therefore, it is necessary to develop new and effective means of
stemming anthracycline-induced cardiotoxicity based on mecha-
nisms that are different from those involving the generation of reac-
tive oxygen species. One promising strategy involves inhibiting a
pathway by which anthracyclines are converted to alcohol
metabolites.
of anthracyclines to form the cardiotoxic hydroxyl metabolites.
Specifically, several studies have correlated the concentrations of
carbonyl reductase (CR; EC 1.1.1.184) with the risk of developing
anthracycline-induced cardiotoxicity.18,19 For example, decreased
levels of CR from heterozygous mice for a null allele of a CR gene
protected against the development of anthracycline cardiotoxity.19
However, overexpression of CR in transgenic mice greatly
increased susceptibility to anthracycline cardiotoxicity.18
It was also found that the parent anthracyclines, rather than the
alcohol metabolites, are largely responsible for the anti-neoplastic
B
A
O
OH
s
O
OH
O
OH
R
R
OH
OH
OMe O
OH
O
OMe O
OH
O
O
O
NH2
OH
NH2
OH
Several reports suggest that the C13-hydroxy metabolites of the
anthracyclines (Fig. 1) are the principal agents responsible for the
1A, R = OH
2A, R = H
1B, R = OH
2B, R = H
Figure 1. Structures of anthracyclines and C13-hydroxy metabolites. (A) Doxoru-
bicin, R = OH and daunorubicin, R = H. (B) Doxorubicinol, R = OH and daunorubi-
cinol, R = H.
* Corresponding author. Tel.: +1 202 806 6888; fax: +1 202 806 5442.
E-mail address: obakare@howard.edu (O. Bakare).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.011

S. Berhe et al. / Bioorg. Med. Chem. 18 (2010) 134–141
135
properties,11 while the cardiotoxic alcohol metabolites possess sig-
nificantly reduced anticancer properties. Thus, anthracycline
reduction has been postulated to be a mechanism for anthracycline
drug resistance.20 Since anthracycline reduction by CR leads to the
formation of alcohol metabolites that are more cardiotoxic and less
cytotoxic than the parent anthracyclines, it appears that anthracy-
cline reduction by CR contributes to the cardiotoxicity problem in
at least two ways: (1) It reduces the amount of anthracycline in cir-
culation thereby necessitating an increase in the dose needed for
effective treatment; (2) An increased anthracycline dosage results
in an increase in the amount of cardiotoxic metabolite formed.
Consequently, this increase in metabolite concentration increases
the risk of cardiotoxicity. Such observations suggest that CR could
be a pharmacological target for inhibitors that block its action in
order to reduce the risk of cardiotoxicity associated with anthracy-
cline therapy and improve anthracycline anticancer efficacy.
Numerous compounds have been found to inhibit CR,21,22 with
flavonoids such as rutin, quercetin, and quercitrin being among
the most potent inhibitors.21 Tanaka et al.23 have recently reported
the pyrazolopyrimidine 3-(1-tert-butyl-4-amino-1H-pyrazolo[3,4-
d]pyrimidin-3-yl)phenol (3) of the Src family kinase inhibitors as a
potent inhibitor of CR-catalyzed NADPH-dependent reduction of
menadione to menadiol (IC50 = 788 nM). In order to study the efficacy
of CR inhibition on anthracycline efficacy, they prepared an analog
which showedpoorinhibition against proteinkinasesbut maintained
potentinhibitionagainstCR(IC50 = 759 nM). Thispotentandselective
inhibitor of CR, 3-(7-isopropyl-4-(methylamino)-7H-pyrrolo[2,3-
d]pyrimidin-5yl)phenol (4), was able to effect a 25% enhancement
ofdaunorubicin-mediatedA549-cellkilling,consistentwithitsability
to inhibit CR-mediated metabolism of daunorubicin.23
but no purification was done to separate the two as only the mono-
substituted derivatives react in a 1,3-dipolar cycloaddition reaction
to give phenylamino indazole-diones.
In addition, the 1,3-dipolar cycloaddition of the diazopropane
intermediates with almost all the quinone dipolarophiles was
found to be regioselective and furnished only the 6-substituted
indazole-dione. However, the cycloaddition of 3-(N-carbometh-
oxyamino)-1-diazopropane to 2-chloro-1,4-benzoquinone pro-
duced
a regioisomeric mixture of 6- and 5-chloroindazol-4,
7-dione (19A and 19B, Scheme 3).
We have previously reported24 the synthesis of compounds 20
and 21 while all the other analogs in this study are new.
2.2. Biological activities on carbonyl reductase
Quinones are known to be good substrates of carbonyl reduc-
tase.21 Most of the indazole-dione derivatives in this study were also
found to be substrates of CR with different catalytic efficiencies.
Table 1 lists the different kinetic constants of CR for the different
indazole-dione substrates. The kcat/Km, which is a measure of an en-
zyme’s catalytic efficiency, varied by about 450-fold for CR and the
indazole-dione substrates in this work. The substrates with the
greatest catalytic efficiencies (ranging from 0.087 to 1.3 l )
Mꢀ1 sꢀ1
are indazole-diones 19A, 20 and 21, which have either a chlorine
or a methoxy substituent at position 6 of the indazole-dione. The
substrates with the lowest catalytic efficiencies (ranging from
0.0030 to 0.0051 l
Mꢀ1 sꢀ1) are indazole-diones 14–16, which have
a phenyl amino substituent at position 6 of the indazole-dione. By
way of comparison, menadione, an excellent CR substrate which is
often used as a model quinone substrate, has a kcat/Km value of
0.098 l . The differences in kcat/Km values between the best
Mꢀ1 sꢀ1 26
and worst substrates most likely results from a combination of elec-
tronic and steric factors. On one hand the phenyl amino substituents
are far bulkier than the substituents present at position 6 in com-
pounds 19A, 20, and 21 and, therefore, could interfere with binding
interactions that promoteefficientcatalysis. On theother hand, elec-
tronic factors seem to underlie the differences in the catalytic effi-
ciencies of compounds 19A and 21. Whereas compound 19A (the
best substrate) possesses an electron-withdrawing chlorine group
at position 6, compound21possesses an electron-donating methoxy
group at this position. The slight difference in the substrate efficien-
cies of compounds 20 and 21 further underscores the importance of
steric contributions to the activities of these compounds upon inter-
action with thebindingdomainof CR. While bothcompounds20 and
21 possess a methoxy substituent at position 6 of the indazole-dione
scaffold, compound 20 incorporates a bulkier benzyl group (com-
pared to the smaller methyl group of compound 21) on the carba-
mate appendage making it a less efficient substrate than 21.
OH
OH
NH2
N
NH
N
N
N
N
N
N
4
3
While investigating the anticancer properties of novel indazole-
dione derivatives, we were prompted to evaluate the biological
activities of these compounds on the CR pathway. Our screening
revealed this new class of indazole-diones to be inhibitors and sub-
strates of CR, and we herein report the synthesis of several 3-[(N-
carboalkoxy)ethylamino]indazole-dione derivatives and their
biological activities on human CR.
2. Results and discussion
2.1. Chemistry
Four indazole-dione derivatives (11, 12, 22, and 23) were found
to be inhibitors of CR with IC50 values of 3–5
lM against menadi-
one and 11–25 M against daunorubicin (Table 2). Further studies
l
with indazole-diones 11 and 12 revealed that they are noncompet-
itive inhibitors against both NADPH and menadione (Table 3). Sim-
ilar inhibition patterns were observed for the similarly structured
pyrazolopyrimidine inhibitors.23,27 Such a finding suggests that
these inhibitors bind to several forms of the enzyme, as indicated
in Figure 2.
The chemical structures of these inhibitors are only slightly dif-
ferent from those of the indazole-dione substrates discussed
above. For example, among the benzene-fused indazole-diones,
the substitution of hydrogen at position 8 of substrate 17 with a
hydroxyl group gives the indazole-dione 23, which is an inhibitor
of CR. The small structural changes on the benzene-fused inda-
zole-diones that give rise to the inhibitory activities may involve
interactions, perhaps through hydrogen bonding with amino acids
in the active site, that disrupt the proper positioning of the
The indazole-diones in this work were prepared as outlined
in Scheme 2. Nitrosation of tetrahydro-2-pyrimidone furnished
N-nitrosotetrahydro-2-pyrimidone which reacted with alcoholic
potassium alkoxides to generate 3-(N-carboalkoxyamino)-1-diazo-
propanes. Theinsitucycloadditionofthediazopropaneintermediates
with appropriate benzoquinone and naphthoquinone derivatives in a
mixture of THF/Et2O at room temperature furnished the correspond-
ing indazole-dione derivatives.24 Some of the quinones used as dipol-
arophiles in the cycloaddition reaction were obtained commercially,
but the phenylamino-1,4-benzoquinones were synthesized by add-
ing a solution of aniline derivatives (5) in aqueous acetic acid to an
aqueous solution of 1,4-benzoquinone (6) in an ice bath (Scheme 1)
according to the method of Barakat et al.25 This reaction is known to
usually give a mixture of mono- and di-anilino-1,4-benzoquinones,

136
S. Berhe et al. / Bioorg. Med. Chem. 18 (2010) 134–141
O
O
H2O, HOAc
+
Ar NHR
Ar
N
R
O
O
5a- f
7a- f
6
H
H
N
NH2
N
Et
Me
Ar NHR
5a
5b
5c
NH2
NH2
NH
Me
MeO
5d
5e
5f
Scheme 1.
ethyl substituent (Fig. 3). For example, the phenyl ring of the sub-
strate indazole-dione 15 is almost coplanar with the indazole-
dione ring. However, substituting the hydrogen on the 6-amino
group with an ethyl or methyl substituent (11 and 12, respectively)
presumably induces unfavorable eclipsing interactions that cause
the phenyl ring to twist perpendicular to the indazole-dione ring.
This apparent difference in orientation could result in different
binding modes for the substrate and inhibitor phenylamino inda-
zole-diones such that the catalytic residues and NADPH are posi-
tioned properly for catalysis when the phenyl ring is more in
plane with the indazole-dione ring. Further work is underway to
gain additional insight into the structural requirements for a sub-
strate versus an inhibitor of CR for this class of compounds.
O
O
Ice; Conc.H2SO4
, NaNO2
HN
NH
HN
N NO
8
9
KOH,ROH,Et2O
R = CH3, PhCH2
O
H
N
O
O
OR
H
R1
N
N
O
N2
RO
N
O
R1
O
H
THF
10
11-23
3. Conclusion
Scheme 2.
We have synthesized a small library of novel indazole-diones
and assessed their biological activities on human carbonyl reduc-
tase (CR). Most of the indazole-diones were found to be substrates,
while four analogs were found to be inhibitors of human CR with
substrate carbonyls for reduction by NADPH. In fact, X-ray struc-
ture analysis of CR with the pyrazolpyrimidine inhibitor 3 embed-
ded in the binding site showed the hydroxyl group on 3 to make
key H-bond interactions with some residues of the active site of
CR.23 A similar interaction with indazole-dione 23 may also be
responsible for the inhibitory activity.
For indazole-diones with a phenylamino substituent, replace-
ment of hydrogen attached to nitrogen of the phenylamino group
by a methyl or ethyl group converted the indazole-dione derivative
from a substrate to an inhibitor. In an attempt to explain why such
a substitution converts a phenylamino-substituted indazole-dione
from a substrate to an inhibitor, computational methods were em-
ployed. A comparison of the energy minimized structures for inda-
zole-diones 11, 12, and 15 shows that the orientation of the phenyl
ring with respect to the indazole-dione ring is markedly different
when the amino hydrogen is replaced with either a methyl or an
IC50 values of 3–5
lM. These inhibitors offer new leads and the po-
tential to improve anthracycline therapy by reducing the cardio-
toxicity associated with anthracyclines, while also increasing the
anticancer efficacy of anthracyclines. More importantly, this study
serves as a lead in a pursuit of the understanding of the structural
requirements for substrate versus inhibitory effects on CR.
4. Experimental section
4.1. Chemistry: general procedures.
All reactions were carried out using laboratory grade materials
and solvents. Purification by column chromatography was done
using 70–230 mesh silica gel. Melting points were determined in
H
O
H
H
N2
MeO
N
O
O
NCO2Me
NCO2Me
Cl
O
+
N
N
Cl
N
H
N
H
Cl
O
O
19A
O
19B
Scheme 3.

S. Berhe et al. / Bioorg. Med. Chem. 18 (2010) 134–141
137
Table 1
Values of Km, kcat, and kcat/Km for indazole-dione substrates of carbonyl reductase
Compound
Km
(
lM)
kcat (sꢀ1
)
kcat/Km (l )
Mꢀ1 sꢀ1
H
O
NCO2CH3
N
22 (4)
25 (5)
57 (4)
0.26 (0.02)
0.012 (0.001)
N
H
N
H
O
13
H
O
NCO2CH3
N
0.073 (0.005)
0.0030 (0.0005)
N
H
N
O
H
O
14
H
O
NCO2CH3
N
0.270 (0.009)
0.0048 (0.0001)
N
H
N
H
O
15
H
O
O
NCO2CH3
H3CO
N
27 (4)
0.14 (0.01)
0.37 (0.016)
0.72 (0.06)
0.0051 (0.0003)
0.0082 (0.0004)
0.0056 (0.0003)
N
H
N
H
16
H
O
O
NCO2CH3
N
45 (4)
N
H
17
H
O
NCO2CH3
N
130 (20)
N
H
O
18
H
O
NCO2CH3
N
0.76 (0.057)
16 (2.5)
5 (2)
1.0 (0.02)
1.4 (0.08)
0.91 (0.05)
1.3 (0.08)
N
Cl
H
O
19A
H
O
NCO2CH2Ph
N
N
0.087 (0.009)
O
O
H
O
O
20
H
NCO2CH3
N
N
0.17 (0.05)
H
O
21
open capillary tubes on a Mel-Temp melting point apparatus and
are uncorrected. The IR spectra were performed in KBr on a Nicolet
Magna 560 or a Nicolet Avatar 370 FTIR spectrometer. The 1H NMR
spectra were obtained on a Bruker Avance 400 MHz spectrometer
in deuterated dimethyl sulfoxide (DMSO-d6). Chemical shifts are
in d units (ppm) with (CH3)2SO (2.50 ppm) as the internal standard.

138
S. Berhe et al. / Bioorg. Med. Chem. 18 (2010) 134–141
NADP+ (Q)
Table 2
Menadione (B)
Menadiol (P)
NADPH (A)
IC50 values of indazole-dione inhibitors on carbonyl reductase
Compound
IC50 versus
menadione
IC50 versus
daunorubicin
E
EA
EAB EPQ
EQ
E
(l
M)
(lM)
H
O
NCO2CH3
EI
EAI
EPQI and/or EQI
N
N
Figure 2. Kinetic mechanism of human carbonyl reductase with inhibitor binding.
The kinetic mechanism for human carbonyl reductase is ordered bi–bi as indicated
above. E is the enzyme, A is NADPH, B is menadione, P is menadiol, and Q is NADP+. I
represents the inhibitor indazole-diones 11 and 12. Arrows pointing into the
horizontal line indicate binding to enzyme and arrows pointing away indicate
release from the enzyme.
4
4
5
3
13
11
25
20
N
H
N
N
O
11
H
O
NCO2CH3
was used as the solvent, and all the mass spectra showed signals
corresponding to the molecular ion plus sodium (Na). N-Nitroso-
tetrahydro-2(H)-pyrimidone was prepared as previously
reported.24
N
H
O
12
H
O
O
NCO2CH3
Cl
4.2. Synthesis of phenylamino-1,4-benzoquinones (7a–f) as
exemplified by the synthesis of 2-(N-ethylphenylamino)-1,4-
benzoquinone (7a)
N
N
Cl
O
H
O
22
Benzoquinone (6, 2.0884 g, 19.32 mmol) was dissolved in hot
distilled water (200 mL) and the solution was filtered. The filtrate
was cooled down to ꢀ8 °C, and a solution of N-ethylaniline (5a,
1.2 mL, 9.506 mmol) was added to it (the solution of N-ethylaniline
was prepared by adding N-ethylaniline (1.2 mL) into water (75 mL)
and then adding glacial acetic acid until all the N-ethylaniline dis-
solved). The resulting mixture was stirred at 0 °C for 15 min, and
filtered under suction to yield a dark purple solid (0.7320 g) which
was used without purification for the next experiment.
H
OH
O
NCO2CH3
N
N
H
O
23
Peaks due to methylene protons
a to the urethane group of the
indazole-diones in this study are buried or overlap significantly
with the water peak at 3.25–3.50 ppm. This was confirmed by
obtaining the 1H NMR spectrum of one indazole-dione (compound
11) in deuterated chloroform (CDCl3). The 1H NMR spectrum of 11
in CDCl3 displayed a 2H multiplet centered at 3.53 ppm for the
4.3. General procedure for the synthesis of indazole-diones with
phenylamino substituents as exemplified by the synthesis of 3-
(methyl-N-ethylcarbamate)-6-(N0-ethylphenylamino)indazol-
4,7-dione (11)
methylene protons
a
to the urethane group, whereas, the peak
N-Nitrosotetrahydro-2-pyrimidone (9, 0.7040 g, 5.4523 mmol)
for the same protons was buried in the water peak at 3.33 ppm
in the DMSO-d6 spectrum. For most of the compounds, mass spec-
tral measurements were done using a Finnigan Navigator mass
spectrometer; while for three compounds, high resolution mass
spectra (EI or FAB) were recorded on a VG Analytical 70-SE mass
spectrometer equipped with a 11–250 J data system. Methanol
was added in small portions to a cooled mixture of KOH
(1.0044 g) in methanol (10 mL) and diethyl ether (50 mL). The
resultant mixture was stirred at room temperature for 25 min
and washed with saturated NaCl solution (2 ꢁ 25 mL), 50% aque-
ous NaCl (2 ꢁ 25 mL), and saturated NaCl (2 ꢁ 25 mL). The ether
layer was added to
a solution of 2-(N-ethylphenylamino)-1,
Table 3
Inhibition constants for indazole-dione inhibitors 11 and 12
Inhibitor
Fixed substrate (concentration)
NADPH
Varied substrate (concentrations)
Menadione
Kinetic constants (lM)
KII = 3.2 (0.14)
KIS = 11.3 (3.4)
KII = 3.3 (0.15)
KIS = 8.0 (1.8)
KII = 3.4 (0.17)
KIS = 5.2 (1.0)
KII = 2.5 (0.08)
KIS = 16.2 (7.8)
KII = 2.0 (0.16)
KIS = 2.3 (0.63)
H
O
(10
NADPH
(50 M)
NADPH
(300 M)
Menadione
(50 M)
Menadione
(250 M)
NADPH
(10 M)
NADPH
(50 M)
NADPH
(300 M)
lM)
(10–200
Menadione
(10–250 M)
Menadione
lM)
NCO2CH3
l
l
N
N
H
N
l
(5–130
NADPH
lM)
O
11
l
(3–50
lM)
NADPH
l
(3–50 lM)
Menadione
(20–250 M)
Menadione
(14–250 M)
Menadione
(16–250 M)
KII = 2.9 (0.18)
KIS = 3.4 (0.83)
KII = 2.2 (0.21)
KIS = 7.3 (4.0)
KII = 4.6 (0.72)
KIS = 4.4 (4.2)
H
O
l
l
NCO2CH3
l
l
N
N
H
N
l
l
O
12

S. Berhe et al. / Bioorg. Med. Chem. 18 (2010) 134–141
139
Figure 3. Energy minimized structures of indazole-diones 11 and 15. (A) Indazole-dione 11 (inhibitor). (B) Indazole-dione 15 (substrate).
4-benzoquinone (7a, 0.6606 g, 2.9068 mmol) in THF (25 mL), and
the resultant mixture was stirred at room temperature for 3 days.
The mixture was concentrated in vacuo and diethyl ether (100 mL)
was added to it. The resulting mixture was placed in the freezer
overnight and filtered under suction to furnish a brick red solid
of 11 (282.7 mg, 26.40%). Mp 95–96 °C. IR (cmꢀ1) 3336, 3121,
1697, 1615, 1548. 1H NMR (DMSO-d6) d 1.10 (t, 3H, J = 6.5 Hz),
2.99 (t, 2H, J = 6.8 Hz), 3.51 (s, 3H), 3.78 (m, 2H), 5.84 (s, 1H),
7.13 (d, 2H, J = 7.5 Hz), 7.18–7.24 (m, 2H), 7.36 (t, 2H, J = 7.8 Hz),
14.00 (s, 1H). ESI MS m/z 391.0 ([M+Na]+ calcd 391.1). 1H NMR
(CDCl3) d 1.23 (t, 3H, J = 7.0 Hz), 3.17 (dist. t, 2H), 3.53 (m, 2H),
3.63 (s, 3H), 3.84 (q, 2H, J = 7.0 Hz), 5.52 (br s, 1H), 5.82 (s, 1H),
7.10 (d, 2H, J = 8.0 Hz), 7.25 (t, 1H, J = 7.4 Hz), 7.37 (t, 2H,
J = 7.73 Hz), 12.43 (br s, 1H).
4.4. General procedure for the synthesis of indazole-diones
without phenylamino substituents as exemplified by the
synthesis of 3-(methyl-N-ethylcarbamate)-benz[f]indazol-4,9-
dione (17)
N-Nitrosotetrahydro-2-pyrimidone (9, 0.6974 g, 5.4012 mmol)
was added in small portions to
a cooled mixture of KOH
(0.9962 g) in methanol (10 mL) and diethyl ether (50 mL). The
resultant mixture was stirred at room temperature for 25 min
and washed with saturated NaCl (2 ꢁ 25 mL), 50% aqueous NaCl
(2 ꢁ 25 mL), and saturated NaCl (2 ꢁ 25 mL). The ether layer was
added to
a
solution of 1,4-naphthoquinone (0.4852 g,
3.0680 mmol) in THF (20 mL). The resultant mixture was stirred
at room temperature for 1.5 h and filtered under suction to furnish
a pale yellowish solid of 17 (444.8 mg, 48.44%). Mp 249–252 °C. IR
(cmꢀ1) 3289, 3122, 1680, 1596. 1H NMR (DMSO-d6) d 3.13 (t, 2H,
J = 6.5 Hz), 3.51 (s, 3H), 7.25 (br t, 1H), 7.84–7.91 (m, 2H), 8.12–
8.16 (m, 2H), 14.40 (s, 1H). ESI MS m/z 322.1 ([M+Na]+ calcd 322.1).
4.3.1. 3-(Methyl-N-ethylcarbamate)-6-(N0-methylphenylamino)
indazol-4,7-dione (12)
Mp 194–196 °C. IR (cmꢀ1) 3354, 3125, 1696, 1603, 1554. 1H
NMR (DMSO-d6) d 3.02 (t, 2H, J = 6.8 Hz), 3.52 (s, 3H), 5.93 (s,
1H), 7.13 (d, 2H, J = 7.6 Hz), 7.18 (t, 1H, J = 7.3 Hz), 7.27 (br t, 1H,
J = 5.4 Hz), 7.34 (t, 2H, J = 7.8 Hz), 14.00 (s, 1H). ESI MS m/z 377.2
([M+Na]+ calcd 377.1).
4.4.1. 3-(Methyl-N-ethylcarbamate)-6-methylindazol-4,7-dione
(18)
Mp 204–206 °C. IR (cmꢀ1) 3305, 3183, 3117, 1685, 1642, 1560.
1H NMR (DMSO-d6) d 2.02 (d, 3H, J = 1.5 Hz), 3.00 (t, 2H, J = 6.9 Hz),
3.50 (s, 3H), 6.66 (d, 1H, J = 1.6 Hz), 7.25 (t, 1H, J = 5.7 Hz), 14.00 (s,
1H). ESI MS m/z 286.1 ([M+Na]+ calcd 286.1).
4.3.2. 3-(Methyl-N-ethylcarbamate)-6-(p-methylphenylamino)
indazol-4,7-dione (13)
Mp 212–214 °C. IR (cmꢀ1) 3335, 3250, 3118, 1696, 1624, 1591.
1H NMR (DMSO-d6) d 2.31 (s, 3H), 3.00 (t, 2H, J = 7.0 Hz), 3.51 (s,
3H), 5.74 (s, 1H), 7.22 (s, 5H), 8.87 (s, 1H), 14.10 (s, 1H). FAB HRMS
calcd for C18H19N4O4 m/z 355.1406 (M++1), found 355.1440.
4.4.2. 3-(Methyl-N-ethylcarbamate)-6-chloroindazol-4,7-dione
(19)
Mp 192–193 °C. IR (cmꢀ1) 3322, 3179, 3109, 1700, 1642, 1572.
1H NMR (DMSO-d6) d 3.03 (t, 2H, J = 6.8 Hz), 3.51 (s, 3H), 7.18–7.26
(m, 2H), 14.35 (s, 1H). EI HRMS calcd for C11H10N3O4Cl m/z
283.0360, found 283.0320.
4.3.3. 3-(Methyl-N-ethylcarbamate)-6-(o-methoxyphenylamino)
indazol-4,7-dione (14)
Mp 217–220 °C. IR (cmꢀ1) 3334, 3113, 1701, 1582. 1H NMR
(DMSO-d6) d 3.02 (t, 2H, J = 6.8 Hz), 3.51 (s, 3H), 3.85 (s, 3H), 5.54
(s, 1H), 7.03 (dt, 1H, J = 7.6, 1.3 Hz), 7.15 (dd, 1H, J = 8.3, 1.2 Hz),
7.20–7.28 (m, 2H), 7.33 (dd, 1H, J = 7.8, 1.5 Hz), 8.34 (s, 1H),
14.10 (s, 1H). EI HRMS calcd for C18H18N4O5 m/z 370.1277, found
370.1271.
4.4.3. 3-(Benzyl-N-ethylcarbamate)-6-methoxylindazol-4,7-
dione(20)
See Ref. 24.
4.4.4. 3-(Methyl-N-ethylcarbamate)-6-methoxylindazol-4,7-
dione (21)
See Ref. 24.
4.3.4. 3-(Methyl-N-ethylcarbamate)-6-phenylaminoindazol-
4,7-dione (15)
4.4.5. 3-(Methyl-N-ethylcarbamate)-6,7-dichloro-5,8-dimethoxybenz
[f]indazol-4,9-dione (22)
Mp 202–203 °C. IR (cmꢀ1) 3401, 3327, 3109, 1697, 1578. 1H
NMR (DMSO-d6) d 3.02 (t, 2H, J = 6.2 Hz), 3.51 (s, 3H), 5.83 (s,
1H), 7.17–7.21 (m, 1H), 7.24–7.27 (m, 1H), 7.35 (d, 2H,
J = 7.4 Hz), 7.39–7.44 (m, 2H), 8.95 (s, 1H), 14.10 (s, 1H). ESI MS
m/z 363.1 ([M+Na]+ calcd 363.1).
Mp 216–219 °C. IR (cmꢀ1) 3357, 3117, 1676, 1534. 1H NMR
(DMSO-d6) d 3.10 (br s, 2H), 3.51 (s, 3H), 3.86 (s, 3H), 3.87 (s,
3H), 7.25 (br s, 1H), 14.30 (s, 1H). ESI MS m/z 450.2 ([M+Na]+ calcd
450.0).
4.3.5. 3-(Methyl-N-ethylcarbamate)-6-(p-methoxyphenylamino)
indazol-4,7-dione (16)
4.4.6. 3-(Methyl-N-ethylcarbamate)-5-hydroxybenz[f]indazol-
4,9-dione (23)
Mp 191 °C (dec.). IR (cmꢀ1) 3324, 3222, 1696, 1613, 1571. 1H
NMR (DMSO-d6) d 3.00 (t, 2H, J = 6.9 Hz), 3.51 (s, 3H), 3.77 (s,
3H), 5.63 (s, 1H), 6.99 (d, 2H, J = 9.0 Hz), 7.22–7.31 (m, 3H), 8.86
(s, 1H), 14.10 (s, 1H). ESI MS m/z 393.2 ([M+Na]+ calcd 393.1).
Mp 205–207 °C. IR (cmꢀ1) 3342, 3251, 1662, 1646, 1628, 1600.
1H NMR (DMSO) d 3.14 (br s, 2H), 3.51 (s, 3H), 7.14–7.44 (m, 2H),
7.58–7.87 (m, 2H), 12.60 (d, 1H), 14.50 (s, 1H). ESI MS m/z 338.1
([M+Na]+ calcd 338.1).

140
S. Berhe et al. / Bioorg. Med. Chem. 18 (2010) 134–141
4.5. Expression and purification of recombinant human
carbonyl reductase
initial studies onto the quinone containing core. Another geometry
optimization was done with the side-chain intact using Hartree–
Fock 3-21G*. These structures were then imported into GAUSSIAN
0334 and subjected to a geometry optimization and frequency cal-
culation using Hartree–Fock 6-31G*. For each indazole-dione, the
lowest energy structures were analyzed for conformational and
electronic differences and similarities.
Recombinant human carbonyl reductase was expressed in Esch-
erichia coli, BL21 DE3 cells harboring pET-5a-HCBR.26 The cells were
grown in 6 L of LB-broth according to published methods.26 Re-
combinant human liver carbonyl reductase was purified to homo-
geneity following a previously described method.26 Throughout
the preparation, human carbonyl reductase was distinguished
from the bacterial enzyme by sensitivity to rutin inhibition.28 Pur-
ity was confirmed via SDS–PAGE and the resulting protein was
found to have kcat and Km values for menadione and 4-benzoylpyr-
idine consistent with previously reported values for human car-
bonyl reductase.21,29 Enzyme concentration was determined
Acknowledgments
The work described in this paper was supported in part by NIH
NCRR P20-RR16454 (H.A.C. and A.S.), NIH NCI 1 R15 CA102119-01
(H.A.C. and C.L.), Research Corporation Cottrell College Science
Award CC5404 (H.A.C.), Howard University New Faculty Grant
(O.B.) and Grant 2 G12 RR003048 from the RCMI Program, Division
of Research Infrastructure, National Center for Research Resources,
NIH.
spectrophotometrically (e
280 = 0.699 mgꢀ1ml) and protein concen-
trations were determined by the method of Bradford using bovine
serum albumin as the standard.30
4.6. Steady-state kinetics of carbonyl reductase
References and notes
Kinetic measurements were made for the indazole-dione sub-
strates with different substrate concentrations and 50 lM NADPH
1. Young, R. C.; Ozols, R. F.; Myers, C. E. N. Eng. J. Med. 1981, 305, 139–153.
2. Cortes, E. P.; Lutman, G.; Wanka, J.; Wang, J. J.; Pickren, J.; Wallace, J.; Holland, J.
F. Cancer Chemother. Rep. 1975, 6, 215–255.
3. Minow, R. B. R.; Gottleeb, J. Cancer Chemother. Rep. 1975, 6, 195–210.
4. von Hoff, D. D.; Layard, M. W.; Basa, P.; Davis, H. L., Jr.; von Hoff, A. L.;
Rozencweig, M.; Muggia, F. M. Ann. Int. Med. 1979, 91, 710–717.
5. Lipshultz, S. E.; Colan, S. D.; Gelber, R. D.; Perez-Atyde, A. R.; Sallan, S. E.;
Sanders, S. P. N. Eng. J. Med. 1991, 324, 808–815.
6. Horenstein, M. S.; Vander Heide, R. S.; L’Ecuyer, T. J. Mol. Genet. Metab. 2000, 71,
436–444.
7. Schimmel, K. J. M.; Richel, D. J.; van den Brink, R. B. A.; Guchelaar, H.-J. Cancer
Treat. Rev. 2004, 30, 181–191.
in 100 mM potassium phosphate. It was necessary to use up to 1%
DMSO or 2% methanol for several of the carbonyl compounds to
enhance their solubility. In control experiments using the standard
assay described below, the enzyme was unaffected by methanol
concentrations up to 2% and DMSO concentrations up to 1%. Car-
bonyl substrate concentrations were determined from mass or
spectrophotometrically (menadione,
norubicin,
486 = 12,100 Mꢀ1 cmꢀ1). Assays were measured by fol-
lowing NADPH oxidation to NADP+ spectrophotometrically
e
340 = 2500 Mꢀ1 cmꢀ1; dau-
e
8. Wojtacki, J.; Lewicka-Nowak, E.; Lesniewski-Kmak, K. Med. Sci. Monit. 2000, 6,
411–420.
(e
340 = 6220 Mꢀ1 cmꢀ1) as a function of time using a Varian Cary
9. Cusack, B. J.; Gambliel, H.; Musser, B.; Hadjokas, N.; Shadle, S. E.; Charlier, H.;
Olson, R. D. Cancer Chemother. Pharmacol. 2006, 58, 517–526.
10. Olson, R. D.; Mushlin, P. S.. Mechanism of Anthracycline Cardiotoxicity: Are
Metabolites Involved? In Cellular and Molecular Toxicology in In Vitro Toxicology;
CRC Press: Boca Raton, 1990.
11. Olson, R. D.; Mushlin, P. S.; Brenner, D. E.; Fleischer, S.; Cusack, B. J.; Chang, B.
K., ; Robert, R. J., Jr. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 3585–3589.
12. Boucek, R. J., Jr.; Olson, R. D.; Brenner, D. E.; Ogunbunmi, E. M.; Inui, M.;
Fleischer, S. J. Biol. Chem. 1987, 262, 15851–15856.
100 Bio.31 Initial slopes of the resulting progress curves were esti-
mated from fits of the curves to either a line or a parabola, and
these slopes were used to calculate the initial velocities. Steady-
state kinetic data were fit to the Michaelis–Menten equation using
HYPERW.32
4.7. Inhibition of carbonyl reductase
13. Cusack, B. J.; Mushlin, P. S.; Voulelis, L. D.; Li, X.; Boucek, R. J., Jr.; Olson, R. D.
Toxicol. Appl. Pharmocol. 1993, 118, 177–185.
14. Minotti, G.; Cavaliere, A. F.; Mordente, A.; Rossi, M.; Schiavello, R.; Zamparelli,
R.; Possati, G. J. Clin. Invest. 1995, 95, 1595–1605.
15. Minotti, G.; Recalcati, S.; Mordente, A.; Liberi, G.; Calafiore, A. M.; Mancuso, C.;
Preziosi, P.; Cairo, G. FASEB J. 1998, 12, 541–551.
16. Minotti, G.; Cairo, G.; Monti, E. FASEB J. 1999, 13, 199–211.
17. Olson, R. D.; Li, X.; Palade, P.; Shadle, S. E.; Mushlin, P. S.; Gambliel, H. A.; Fill,
M., ; Boucek, R. J., Jr.; Cusack, B. J. Toxicol. Appl. Pharmacol. 2000, 169, 168–176.
18. Forrest, G. L.; Gonzalez, B.; Tseng, W.; Li, X.; Mann, J. Cancer Res. 2000, 60,
5158–5164.
19. Olson, L. E.; Bedja, D.; Alvey, S. J.; Cardounel, A. J.; Gabrielson, K. L.; Reeves, R. H.
Cancer Res. 2003, 63, 6602–6606.
20. Ax, W.; Soldan, M.; Koch, L.; Maser, E. Biochem. Pharmacol. 2000, 59, 293–300.
21. Wermuth, B. J. Biol. Chem. 1981, 256, 1206–1213.
22. Iwata, N.; Inazu, N.; Satoh, T. J. Biochem. 1989, 105, 556–564.
23. Tanaka, M.; Bateman, R.; Rauh, D.; Vaisberg, E.; Ramachandani, S.; Zhang, C.;
Hansen, K. C.; Burlingame, A. L.; Trautman, J. K.; Shokat, K. M.; Adams, C. L. PLoS
Biol. 2005, 3, 764–776.
24. Bakare, O.; Zalkow, L. H.; Burgess, E. M. Synth. Commun. 1997, 27, 1569–
1576.
Inhibitors were evaluated by determining the IC50 values in the
following standard assay. Enzyme was added to different concen-
trations of inhibitor, 250 lM menadione, and 50 lM NADPH in
100 mM potassium phosphate, pH 7.0 at 25 °C. IC50 values were
extrapolated from the plot of % activity remaining versus inhibitor
concentration. For more complete inhibition studies, concentra-
tions of indazole-diones 11 and 12 were varied against varied men-
adione or NADPH concentrations. For experiments with varied
menadione concentrations the NADPH concentrations were fixed
at either 10
NADPH concentrations, the menadione concentrations were held
constant at either 50 M or 250 M. The resulting data were fit
lM, 50 lM, or 300 lM. For experiments with varied
l
l
to competitive, uncompetitive, and noncompetitive inhibition
equations using COMP, UNCOMP, and NONCOMP, respectively.32
25. Barakat, M. Z.; Shehab, S. K.; El-Sadr, M. M. J. Chem. Soc. 1958, 901–902.
26. Slupe, A.; Williams, B.; Larson, C.; Primbs, T.; Lee, L. M.; Breusch, A. J.;
Bjorklund, C.; Warner, D. L.; Peloquin, J.; Shadle, S. E.; Gambliel, H. A.; Cusack,
B. J.; Olson, R. D.; Charlier, H. A., Jr. Cardiovasc. Toxicol. 2005, 5, 365–376.
27. Tanaka, M.; Zhang, C.; Shokat, K. M.; Burlingame, A. L.; Hansen, K.; Bateman, R.
L.; Dimagno, S. G. Pyrazolo Pyrimidine Derivatives and Methods of Use Thereof.
In WHO: PCT, 2005.
28. Bohren, K. M.; Wermuth, B.; Harrison, D.; Dagmar, R.; Petsko, G. A.; Gabbay, K.
H. J. Mol. Biol. 1994, 244, 659–664.
29. Bohren, K. M.; von Wartburg, J. P.; Wermuth, B. Biochem. J. 1987, 244, 165–171.
30. Bradford, M. M. Anal. Biochem. 1976, 72, 248–254.
4.8. Computational chemistry for select indazole-diones
Indazole-diones 11, 12, 15, 17, 22, and 23 were subjected to a
conformation search using Hartree–Fock 3-21G* with SPARTAN
04.33 The two lowest energy conformers for each compound were
then geometry optimized in SPARTAN 04 using Hartree–Fock 6-31G*.
The remaining structures were subjected to a conformer search
excluding the carbamate side-chain using Hartree–Fock 3-21G*
with SPARTAN 04. For each indazole-dione, the lowest energy con-
formers within a calculated 2 kcal/mol range of each other were
carried forward by appending the carbamate side-chain from the
31. Wermuth, B.. Aldo–Keto Reductases. In Enzymology of Carbonyl Metabolism:
Aldehyde Dehydrogenase, Aldo/Keto Reductase, and Alcohol Dehydrogenase; Alan
R. Liss: New York, 1985.
32. Cleland, W. W. Methods Enzymol. 1979, 63, 103–138.

S. Berhe et al. / Bioorg. Med. Chem. 18 (2010) 134–141
141
33. SPARTAN ‘04 Windows; Version 1.0.1 ed.; Wavefunction: Irvine, CA, (2004).
34. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.;
Cheeseman, J. R.; Montgomery, Jr., J. A.; Vreven, T.; Kudin, K. N.; Burant, J. C.;
Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.;
Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.;
Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao,
O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken,
V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A.
J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G.
A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.;
Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.;
Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.;
Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.;
Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe,
M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A.
GAUSSIAN 03, Revision C.02; Gaussian: Wallingford, CT, 2004.