Identification of 1,6-dihydropyrazolo[4,3-c]carbazoles and 3,6-dihydropyrazolo[3,4-c]carbazoles as new Pim kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.05.011

New 1,6-dihydropyrazolo[4,3-c]carbazoles and 3,6-dihydropyrazolo[3,4-c] carbazoles were prepared and evaluated for their Pim kinase inhibitory potencies as well as their antiproliferative activities toward two prostatic cancer cell lines. Pyrazolocarbazole 15a was found to be a potent Pim kinase modulator with inhibitory potency toward the three isoforms. Compound 6c strongly inhibited Pim-3 with weaker effect toward Pim-1 and Pim-2, and thus could be used as an interesting molecular tool to study Pim-3 biological functions.

Full text of DOI:10.1016/j.bmc.2013.05.011

Bioorganic & Medicinal Chemistry 21 (2013) 4102–4111
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identification of 1,6-dihydropyrazolo[4,3-c]carbazoles
and 3,6-dihydropyrazolo[3,4-c]carbazoles as new Pim kinase
inhibitors
⇑
Virginie Suchaud, Laurent Gavara, Emmanuelle Saugues, Lionel Nauton, Vincent Théry, Fabrice Anizon ,
Pascale Moreau
⇑
Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand, BP 10448, 63000 Clermont-Ferrand, France
CNRS, UMR 6296, ICCF, BP 80026, 63171 Aubière, France
a r t i c l e i n f o
a b s t r a c t
Article history:
New 1,6-dihydropyrazolo[4,3-c]carbazoles and 3,6-dihydropyrazolo[3,4-c]carbazoles were prepared and
evaluated for their Pim kinase inhibitory potencies as well as their antiproliferative activities toward two
prostatic cancer cell lines. Pyrazolocarbazole 15a was found to be a potent Pim kinase modulator with
inhibitory potency toward the three isoforms. Compound 6c strongly inhibited Pim-3 with weaker effect
toward Pim-1 and Pim-2, and thus could be used as an interesting molecular tool to study Pim-3 biolog-
ical functions.
Received 10 April 2013
Revised 6 May 2013
Accepted 8 May 2013
Available online 16 May 2013
Keywords:
Ó 2013 Elsevier Ltd. All rights reserved.
Pyrazolocarbazoles
Kinase inhibitors
Pim kinases
In vitro antiproliferative activities
1. Introduction
Thus, as part of our ongoing program targeting the identification
of biologically active compounds and more particularly protein ki-
Due to the essential biological role of Pim kinases in carcinogen-
esis, the identification of new Pim inhibitors appeared as a major
concern during the last ten years. This led to the discovery of a large
variety of chemical scaffolds that demonstrated various inhibitory
potencies and selectivity toward Pim in regard to other protein ki-
nases. Accordingly, a number of recent reviews reported the Pim ki-
nase inhibitors published in the patent or nonpatent literature.1–5
Early reported inhibitors were only active toward one or two but
not against all Pim isoforms (Pim-1, Pim-2, Pim-3). Nowadays, sev-
eral pan-Pim inhibitors have been identified and studied to evaluate
their therapeutic efficiency.6–8 Only two Pim kinase inhibitors have
been submitted to clinical trials. Supergen discontinued the phase I
clinical development of SGI-1776 due to cardiac toxicity linked to
hERG inhibition. Astrazeneca is currently recruiting patients for a
phase I clinical trial studying the pharmacokinetics and efficacy in
acute myelogenous leukemia of pan-Pim inhibitor AZD1208.9–12
Actually, despite intensive work in this field and the major interest
of Pim kinases as biological targets in the development of new anti-
cancer agents, no Pim inhibitor has reached the market to date.
nase modulators, we focused on the identification of new series of
Pim inhibitors. Nitrogen-containing aromatic heterocyclic systems
are of interest for the development of protein kinase inhibitors.
For example, we showed the great potential of indole and indazole
nuclei and we recently described the synthesis and Pim inhibitory
potencies of diversely substituted pyrazolo[3,4-g]quinoxalines,
pyrrolo[2,3-a]carbazoles and pyrrolo[2,3-g]indazoles13–19 (Fig. 1).
In this paper, we report the synthesis and Pim kinase inhibitory po-
tency of new compounds showing fused indolic and indazolic moi-
eties (Fig. 1). Moreover, as over-expression of Pim kinases has
already been observed in prostatic tumors, the in vitro antiprolifer-
ative activity of the synthesized compounds was studied toward
two prostatic cancer cell lines: PC3 (androgen independent cells)
and LnCAP (androgen dependent cells).
2. Results and discussion
2.1. Chemistry
The synthesis of 1,6-dihydropyrazolo[4,3-c]carbazoles was per-
formed from indazole derivative 2 which was previously described
by our group (Scheme 1).20 For the construction of the pyrazol-
⇑
Corresponding authors. Tel.: +33 (0) 473405364; fax: +33 (0) 473407717 (F.A.);
tel.: +33 (0) 473407963; fax: +33 (0) 473407717 (P.M.).
E-mail addresses: Fabrice.ANIZON@univ-bpclermont.fr (F. Anizon), Pascale.
MOREAU@univ-bpclermont.fr (P. Moreau).
o[4,3-c]carbazole system, we first envisaged
a Suzuki cross-
coupling between 7-iodoindazole 2 and 2-halophenylboronic acid
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.05.011

V. Suchaud et al. / Bioorg. Med. Chem. 21 (2013) 4102–4111
4103
R1
R2
CHO
R1
R2
N
N
H
N
HN
R2
N
N
N
H
N
H
R3
N
R1
Pyrazolo[3,4-g]quinoxalines
Pyrrolo[2,3-g]indazoles
Pyrrolo[2,3-a]carbazole-3-carbaldehydes
N
N
HN
R2
R2
NH
N
H
N
H
R1
R1
Present work:
Pyrazolo[4,3-c]carbazoles and pyrazolo[3,4-c]carbazoles
Figure 1. Structure of various Pim-kinase inhibitor scaffolds previously/presently described by our group.
R
R
I
THP
THP
THP
B(OH)2
3a 3c
H2N
O2N
O2N
O2N
H2N
O2N
N
N
N
2 steps, 57% [20]
N
N
N
Pd(PPh3)4, Na2CO3
toluene/EtOH/H2O
1
4a 78%
4b 84%
4c 86%
2
3a 7a R = H
3b 7b R = OCH3
3c 7c R = Cl
Pd(OAc)2, PIDA
toluene
R
R
R
H2, PtO2
EtOAc
D
c. HCl
THP
H
N
A N
H
N
C
HN
HN
HN
N
B
N
N
H2N
O2N
O2N
7a 81%
7b 49%
7c 59%
6a 62%
6b 62%
6c 69%
5a 39%
5b 24%
5c 12%
Scheme 1. Synthesis of 1,6-dihydropyrazolo[4,3-c]carbazoles 5, 6 and 7.
derivatives, followed by an Ullmann-type copper-mediated cycli-
zation to form the carbazole 5-membered ring. However, none of
our attempts using 2-bromophenylboronic and 2-chlorophenylbo-
ronic acids led to the desired compounds due to the formation of
de-iodinated indazole product. Nevertheless, the Suzuki cross-cou-
pling between 2 and arylboronic acids 3a–3c successfully afforded
aryl-substituted products 4a–4c in 78–86% yield. Interestingly, the
1H and 13C NMR spectra of compounds 4b and 4c in DMSO-d6
clearly showed the presence of two detectable conformers, proba-
bly due to the restricted rotation around the newly formed C–C
bond. We next successfully performed the cyclization of 4a–4c
by using palladium acetate and PIDA21 to obtain carbazole deriva-
tives 5a–5c in modest yields. Finally, THP protecting group was re-
moved in refluxing concentrated hydrochloric acid, leading to
compounds 6a–6c, and a final hydrogenation of the nitro group
provided compounds 7a–7c.
The construction of isomeric pyrazolo[3,4-c]carbazole system
was performed from 4-iodoindazole 11 (Scheme 2). Compound
11 was obtained in low yield during the preparation of compound
2 from 1.20 However, in order to facilitate the preparation of
compound 11, we developed another synthetic pathway to
this compound. Therefore, compound 11 was prepared from
5-bromo-6-nitroindazole 8 which was already reported in the
literature.22 After protection of N1 with a THP group in the pres-
ence of dihydropyrane and PTSA,23 Buchwald–Hartwig coupling
at the five-position with benzophenone imine and subsequent
hydrolysis,24 10 was obtained in 86% yield. Finally, iodination in
the presence of I2 in DMSO afforded 11 in 71% yield.
Indazole 11 was then engaged in a Suzuki cross-coupling with
phenylboronic acids (Scheme 3). In this case, we were able to per-
form the reaction with 2-halophenylboronic acids 12a–12c.
Whereas 12a is commercially available, compound 12b was

4104
V. Suchaud et al. / Bioorg. Med. Chem. 21 (2013) 4102–4111
NH
DHP, PTSA
CH2Cl2
THP
THP
H
N
O2N
Br
O2N
Br
O2N
H2N
N
N
N
N
N
78%
Pd2(dba)3.CHCl3, BINAP
NaOtBu, toluene
86%
8
9
10
I2, DMSO
71%
THP
O2N
H2N
N
N
I
11
Scheme 2. Optimized synthesis of indazole 11.
R
THP
O2N
HN
N
B(OH)2
THP
THP
CuI, Cu
K2CO3, Bu2O
N
O2N
H2N
O2N
12a 12c
N
N
X
N
N
Pd(PPh3)4, Na2CO3
toluene/EtOH/H2O
H2N
X
I
R
11
R
13a, X = Br 78%
13b X = Br 76%
13c X = I, not isolated
14a 67%
14b 59%
14c 47% (2 steps)
12a 16a R = H
12b 16b R = OCH3
12c 16c R = Cl
c. HCl
H
H
H2N
HN
O2N
N
N
B
A
N
N
H2, PtO2
EtOAc
HN
C
D
R
R
16a 91%
16b 83%
16c 69%
15a 82%
15b 50%
15c 79%
H2N
H2N
Cl
I2, Ag2SO4
EtOH
43% [25]
NaNO2, HCl
CuCl
61%
B(OH)2
B(OH)2
B(OH)2
I
I
17
18
12c
Scheme 3. Synthesis of 3,6-dihydropyrazolo[3,4-c]carbazoles 14–16 and arylboronic acid 12c.
prepared according to literature procedure25 and 12c was prepared
from commercially available 3-aminophenylboronic acid 17 after
iodination25 and Sandmeyer reaction of 18 in the presence of CuCl.
Compounds 13a and 13b were isolated in good yields. In the case

V. Suchaud et al. / Bioorg. Med. Chem. 21 (2013) 4102–4111
4105
of 13c, the product was engaged directly in the next step due to
purification problems. As it was previously observed for com-
pounds 4b and 4c, the 1H and 13C NMR spectra of 13a and 13b in
DMSO-d6 showed signal pairs indicating the presence of two con-
formers. Next, cyclization of compounds 13a–13c was performed
in the presence of CuI/Cu to give pyrazolocarbazoles 14a–14c,
which were subsequently deprotected as described above. Finally,
hydrogenation of the nitro group afforded compounds 16a–16c in
good yields.
compound could be a useful tool to study Pim-3 biological functions
in regards to the ones of Pim-1 and Pim-2.
2.3. In vitro antiproliferative activities
Finally, in vitro antiproliferative activities of compounds 6a–c,
7a–c, 15a–c and 16a–c were evaluated toward two prostatic can-
cer cell lines (PC3 and LnCAP) using the colorimetric MTS assay
by the ICSN cellular target screening platform (Gif-sur-Yvette,
France).27 All the compounds were firstly tested at 10
l
M concen-
tration. IC50 values were determined when the growth inhibition
was found to be more than 70% with compounds tested at 10
2.2. Pim kinase inhibitory potencies
lM
The potency of compounds 6a–c, 7a–c, 15a–c and 16a–c to in-
hibit Pim kinases (Pim-1, Pim-2, Pim-3) was evaluated at 10 lM
(Table 1). As shown in Table 1, most of the compounds tested have
been more active toward androgen independent PC3 cells. All
derivatives demonstrated 40–60% of PC3 cell proliferation inhibi-
tion, except compounds 6b, 7a and 7b. The percentage of growth
inhibition was found to be more than 70% only for two compounds
and 1 lM concentrations in duplicate assays by the International
Centre for Kinase Profiling (Dundee, UK) as previously described.26
The percentages of residual kinase activities are reported in Table 1.
IC50 values were only determined when the remaining kinase
activity was less than 45% when the compounds were tested at
tested at 10
itor of this series, inhibited the growth of PC3 cells with an IC50 va-
lue of 3 M while 15b demonstrated an IC50 value of 2.30 M.
lM toward PC3 cells: 15a, that is the best Pim-1 inhib-
1
l
M.
l
l
As shown in Table 1, in both series nitro derivatives are more ac-
tive than their amino counterparts, except for the chlorinated 3,6-
dihydropyrazolo[3,4-c]carbazoles for which the amino analog 16c
exhibited a better activity toward Pim-3 than 15c bearing a nitro
group. Even if 15a, the unsubstituted nitro 3,6-dihydropyrazolo
[3,4-c]carbazole was found to be the most active derivative tested,
the two regioisomer series showed similar inhibition potential,
with Pim-1 and Pim-3 being the most inhibited isoforms. The Pim
kinase inhibitory profile of the tested compounds is not much influ-
enced by the nature of the substituent present at the position nine
of the pyrazolocarbazole scaffold. Except 7a, 7c and 16a, all the
compounds tested efficiently inhibited either Pim-1 and/or Pim-3
2.4. Molecular modeling experiments
We next carried out molecular modeling experiments in order
to get an insight into the possible binding mode of these com-
pounds in the ATP-binding pocket of Pim-1 and Pim-3 kinases.
Therefore, we decided to perform the docking study using 6b and
15a, the best Pim-1/Pim-3 inhibitors for each pyrazolocarbazole
regioisomeric series.
For these docking experiments, Pim-1 model was generated
from 3JPV X-ray crystal structure available in the Protein Data
Bank (PDB),13 and Pim-3 model was constructed by homology to
1XWS Pim-1 crystal structure, with the same method we reported
previouly,19 using Modeller9V11, UCSF Chimera,28–31 and Syb-
ylx2.0.32 The docking experiments were then performed with Syb-
ylx2.0 for compounds 6b and 15a in either Pim-1 or Pim-3 models,
and the best solution for each complex was minimized. The dock-
ing solutions found are depicted in Figure 2.
at more than 50% when tested at 1 lM. Two of them are particularly
interesting. First of all, 15a was found to be a potent Pim kinase
modulator that strongly inhibited Pim-1 and Pim-3 with IC50 values
of 0.04
lM and 0.10
lM, respectively. Next, 6c strongly inhibited
Pim-3 with an IC50 value of 0.09
lM and led to more than 50% of
Pim-1 and Pim-2 residual activities when tested at 10 lM. This
Table 1
Biological activities for compounds 6a–c, 7a–c, 15a–c, 16a–c: % of residual kinase activity at 10
lM and 1
lM, and antiproliferative activities as inhibition % of cellular
proliferation at 10
lM (IC50
(l
M) in brackets when determined)
Kinase inhibitory potencies
Pim-2
Compd
Antiproliferative activities
Pim-1
Pim-3
PC3
LNCaP
10
26
l
M
1
l
M
10
46
l
M
1
l
M
10
l
M
1
1 lM
6a
6b
6c
1
2
8
44
4
4
76
77
90
2
16
23
38
22
(0.21 0.08)
28
(0.11 0.01)
50
(0.09 0.05)
5
59
29
51
4
4
1
45
19
28
4
1
3
9
(0.4 0.1)
39
60
41
9
1
72
0
6
8
0
1
0
9
(0.22 0.05)
69
93 16
7a
7b
54
43
2
6
98
93
5
6
78 12
70 12
116 13
120
20
14
3
2
63 18
46 17
31
28
3
1
28
8
0
8
(0.7 0.6)
7c
15a
29
15
5
1
85
14
2
2
79
38
1
3
115
49
6
14
17
2
3
55
28
1
5
43
85
2
1
35
38
1
3
2
1
3
2
(0.04 0.03)
29 19
(0.15 0.02)
48
(0.10 0.06)
21
(0.11 0.00)
26
(0.23 0.03)
66
(3 1)
74
(2.30 0.01)
15b
15c
13
39
3
2
35
54
3
6
54
57
96
11
19
1
4
1
2
3
27
0
1
4
50
3
1
(0.2 0.1)
16a
16b
16
23
5
3
61
70
2
5
71
54
7
6
20
16
4
7
3
6
56
46
3
1
56
39
2
1
123 30
51
(0.6 0.5)
16c
8
1
45
3
34 10
107
1
7
3
28
0
45
2
27
4
(1.1 0.1)
(0.11 0.06)

4106
V. Suchaud et al. / Bioorg. Med. Chem. 21 (2013) 4102–4111
Figure 2. Docking models of (A) 15a bound to Pim-1 ATP binding site, (B) 15a bound to Pim-3 ATP binding site, (C) 6b bound to Pim-1 ATP binding site, (D) 6b bound to Pim-3
ATP binding site. Hydrogen bonds are indicated as dashed lines. Molecular graphics images were produced using UCSF Chimera.28
Regarding 15a, the best inhibitor of both pyrazolocarbazole ser-
ies, we found a similar binding mode with Pim-1 and Pim-3
(Fig. 2A and B). The indazole ring system of 15a is inserted in the
ATP binding cleft, with the nitro group oriented toward the hydro-
philic Lys67/Lys69 region, while the pyrazole ring is placed near
the hinge region, establishing a hydrogen bond between the N-3
hydrogen atom and the backbone carbonyl oxygen of Glu121 in
Pim-1 (2.18 Å) or Glu124 in Pim-3 (2.09 Å). A similar orientation
of the pyrazolocarbazole ring system was found in the case of com-
pound 6b (Fig. 2C and D). Nevertheless, as these two series are
regioisomers, in the case of 6b, the pyrazole NH is not well posi-
tioned to establish a hydrogen bond with Glu121/Glu124 hinge
residues. The only polar interaction that was observed was found
between the nitro group and the Lys67/Lys69 region, as it was al-
ready observed for 15a.
series showed similar profiles toward the three Pim isoforms,
Pim-1/Pim-3 being the most inhibited kinases. The most potent
compound of the series 15a also showed interesting antiprolifera-
tive activities toward PC3 cells in the micromolar range. Finally,
compound 6c, that strongly inhibited Pim-3 with lower effect to-
ward Pim-1 and Pim-2, could be used as an interesting molecular
tool to study Pim-3 biological functions. Due to the potential inter-
est of these compounds as Pim inhibitors, the structure–activity
relationship studies performed on this series are currently wid-
ened in our group.
4. Experimental section
4.1. Chemistry
For both regioisomeric series, the D ring of the pyrazolocarbaz-
ole ring system (Schemes 1 and 3) is oriented toward the outside of
the ATP-binding pocket. These results might explain why, as indi-
cated above, the Pim kinase inhibitory profile of these two dihyd-
ropyrazolocarbazole series is not very dependent on the nature of
the substituent present at the position nine of the heteroaromatic
scaffold.
4.1.1. General
Starting materials were obtained from commercial suppliers
and used without further purification. IR spectra were recorded
on Shimadzu FTIR-8400S or Perkin–Elmer Spectrum 65 FT-IR spec-
ꢀ
trometers (
m
in cmÀ1). NMR spectra, performed on a Bruker
AVANCE 400 spectrometer (1H: 400 MHz, 13C: 100 MHz), or a Bru-
ker AVANCE 500 spectrometer (1H: 500 MHz, 13C: 126 MHz), are
reported in ppm using the solvent residual peak as an internal
standard; the following abbreviations are used: singlet (s), doublet
(d), triplet (t), doublet of doublet (dd), doublet of doublet of dou-
blet (ddd), doublet of triplet (dt), multiplet (m), broad signal (br
s); High resolution mass spectra were determined on a high-reso-
lution Micro Q-Tof apparatus (CRMP, Université Blaise Pascal, Cler-
mont–Ferrand, France) or on a Waters Q-Tof 2 apparatus (CRMPO,
Université de Rennes, France). Chromatographic purifications were
3. Conclusions
In conclusion, 1,6-dihydropyrazolo[4,3-c]carbazoles and 3,6-
dihydropyrazolo[3,4-c]carbazoles were synthesized and identified
as potent Pim kinase inhibitors. In both series, except for chlori-
nated analogue 16c, nitro derivatives were more potent Pim inhib-
itors compared to their amino counterparts. Both regioisomeric

V. Suchaud et al. / Bioorg. Med. Chem. 21 (2013) 4102–4111
4107
performed by column chromatography using 40–63
l
m silica gel.
131.0, 131.3, 131.5, 136.94, 136.95 (CHarom), 106.31, 106.34,
116.98, 116.99, 130.96, 130.97, 133.9, 134.2, 134.8, 134.9, 140.50,
140.53, 141.51, 141.52 (Carom); HRMS (ESI+) calcd for
Reactions were monitored by TLC using fluorescent silica gel plates
(60 F254 from Merck). Melting points were measured on a Reichert
microscope or a Stuart SMP3 apparatus and are uncorrected.
C
18H1835ClN4O3 (M+H)+ 373.1067, found 373.1056.
4.1.2. General procedure for preparation of compounds 4a–4c
A mixture of indazole 2 in a 1:1:1 toluene/ethanol/H2O mixture
(0.043 mmol/mL), Pd(PPh3)4 (10 mol%), Na2CO3 (2.5 equiv) and
boronic acid 3 (1.5–2 equiv) was refluxed for 2–4 h. Water was
added and the mixture was extracted with EtOAc. The combined
organic fractions were dried over MgSO4 and evaporated. Column
chromatography (cyclohexane/EtOAc) provided the corresponding
coupling product.
4.1.6. General procedure for preparation of compounds 5a–5c
mixture of indazole in toluene (0.05 mmol/mL) and
A
4
Pd(OAc)2 (10 mol%) was stirred at room temperature. Phenyliodo-
nium diacetate (1.5 equiv) was then slowly added and the reaction
mixture was stirred at room temperature for 4 h. The solvent was
removed under reduced pressure and the residue was purified by
column chromatography.
4.1.7. 5-Nitro-1-(tetrahydro-2H-pyran-2-yl)-1,6-
4.1.3. 5-Nitro-7-phenyl-1-(tetrahydro-2H-pyran-2-yl)-1H-
dihydropyrazolo[4,3-c]carbazole (5a)
indazol-6-amine (4a)
From 4a (30.0 mg, 0.089 mmol), column chromatography
(cyclohexane/EtOAc, 9:1 then 8:2) provided 5a (11.6 mg,
0.034 mmol, 39%) as a yellow powder. Mp 240–243 °C; IR (ATR):
From
2 (200 mg, 0.52 mmol) and phenylboronic acid 3a
(2 equiv) (reflux, 4 h), column chromatography (cyclohexane/
EtOAc, 8:2) provided 4a (136 mg, 0.40 mmol, 78%) as an orange
powder. Mp 207–211 °C; IR (ATR): 3486, 3355, 1618, 1491, 1414,
3403, 1604, 1488, 1304 cmÀ1 1H NMR (400 MHz, DMSO-d6):
;
1.61–1.75 (2H, m), 1.78–1.90 (1H, m), 2.07–2.25 (2H, m), 2.53–
2.65 (1H, m), 3.90–3.97 (2H, m), 6.54 (1H, dd, J1= 8 Hz, J2 = 3 Hz),
7.41 (1H, ddd, J1 = 8 Hz, J2 = 7 Hz, J3 = 1 Hz), 7.53 (1H, ddd,
J1 = 8 Hz, J2 = 7 Hz, J3 = 1 Hz), 7.93 (1H, d, J = 8 Hz), 8.33 (1H, d,
J = 8 Hz), 8.53 (1H, s), 8.94 (1H, s), 12.47 (1H, br s); 13C NMR
(100 MHz, DMSO-d6): 21.7, 24.6, 29.3, 65.5 (CH2), 84.2 (CH),
113.1, 118.2, 120.9, 122.1, 125.7, 138.0 (CHarom), 106.8, 118.2,
119.7, 129.7, 131.9, 138.1, 139.2 (Carom); HRMS (ESI+) calcd for
1296, 1039, 988 cmÀ1 1H NMR (400 MHz, DMSO-d6): 0.92–1.05
;
(1H, m), 1.20–1.36 (2H, m), 1.61–1.68 (1H, m), 1.75–1.83 (1H,
m), 2.16–2.28 (1H, m), 2.39–2.52 (1H, m), 3.58–3.64 (1H, m),
4.23 (1H, dd, J1 = 10.5 Hz, J2 = 2 Hz), 6.03 (2H, br s), 7.34–7.38
(1H, m), 7.40–7.44 (1H, m), 7.59–7.69 (3H, m), 8.21 (1H, s), 8.71
(1H, s); 13C NMR (100 MHz, DMSO-d6): 22.9, 24.3, 28.8, 66.7
(CH2), 83.5 (CH), 120.6, 129.0, 129.6, 129.8, 130.8, 131.1, 137.0
(CHarom), 107.8, 117.0, 131.0, 132.6, 140.7, 141.3 (Carom); HRMS
(ESI+) calcd for C18H19N4O3 (M+H)+ 339.1457, found 339.1456.
C
18H17N4O3 (M+H)+ 337.1301, found 337.1319.
4.1.8. 9-Methoxy-5-nitro-1-(tetrahydro-2H-pyran-2-yl)-1,6-
dihydropyrazolo[4,3-c]carbazole (5b)
4.1.4. 7-(3-Methoxyphenyl)-5-nitro-1-(tetrahydro-2H-pyran-2-
yl)-1H-indazol-6-amine (4b)
From 4b (140 mg, 0.38 mmol), column chromatography (cyclo-
hexane/EtOAc, 9:1 then 8:2) provided 5b (32.8 mg, 0.090 mmol,
24%) as an orange powder. Mp 246–249 °C; IR (ATR): 3430, 1610,
From 2 (50 mg, 0.13 mmol) and 3-methoxyboronic acid 3b
(1.5 equiv) (reflux, 2 h), column chromatography (cyclohexane/
EtOAc, 8:2) provided 4b (40 mg, 0.11 mmol, 84%) as a yellow pow-
der. Mp 173–174 °C; IR (ATR): 3497, 3376, 1618, 1490, 1297,
1488, 1393, 1326, 1301 cmÀ1 1H NMR (400 MHz, DMSO-d6):
;
1.59–1.89 (3H, m), 2.06–2.15 (1H, m), 2.17–2.27 (1H, m), 2.50–
2.64 (1H, m), 3.87–3.97 (2H, m), 3.92 (3H, s), 6.55 (1H, dd,
J1 = 7.5 Hz, J2 = 3 Hz), 7.23 (1H, dd, J1 = 9 Hz, J2 = 2 Hz), 7.82–7.86
(2H, m), 8.52 (1H, s), 8.91 (1H, s), 12.32 (1H, br s); 13C NMR
(100 MHz, DMSO-d6): 56.0 (CH3), 21.6, 24.6, 29.3, 65.5 (CH2),
84.0 (CH), 105.6, 113.6, 114.5, 118.2, 138.1 (CHarom), 106.6, 117.8,
120.2, 129.6, 132.3, 134.2, 138.2, 154.3 (Carom); HRMS (ESI+) calcd
for C19H19N4O4 (M+H)+ 367.1406, found 367.1399.
1038 cmÀ1 1H NMR (400 MHz, DMSO-d6): 0.94–1.09 (1H, m),
;
1.21–1.40 (2H, m), 1.59–1.70 (1H, m), 1.75–1.85 (1H, m), 2.15–
2.30 (1H, m), 2.45–2.59 (1H, m), 3.60–3.70 (1H, m), 3.80/3.81
(3H, 2s); 4.26/4.32 (1H, 2dd, J1 = 10.5 Hz, J2 = 1.5 Hz), 6.02–6.15
(2H, m), 6.88–6.99 (2H, m), 7.15–7.21 (1H, m), 7.51–7.60 (1H,
m), 8.21 (1H, s), 8.70 (1H, s); 13C NMR (100 MHz, DMSO-d6):
55.2, 55.4 (CH3), 22.9, 23.0, 24.31, 24.33, 28.80, 28.85, 66.7, 66.8
(CH2), 83.62, 83.63 (CH), 115.0, 115.2, 115.5, 116.0, 120.6, 122.76,
122.83, 130.6, 130.9, 136.9, 137.0 (CHarom), 107.7, 116.96, 116.99,
131.0, 133.89, 133.90, 140.59, 140.62, 141.2, 141.3, 160.2, 160.3
(Carom); HRMS (ESI+) calcd for C19H20N4NaO4 (M+Na)+ 391.1382,
found 391.1400.
4.1.9. 9-Chloro-5-nitro-1-(tetrahydro-2H-pyran-2-yl)-1,6-
dihydropyrazolo[4,3-c]carbazole (5c)
From 4c (159 mg, 0.426 mmol), column chromatography
(cyclohexane/EtOAc, 8:2) provided 5c (18.8 mg, 0.051 mmol,
12%) as an orange powder. Mp >250 °C; IR (ATR): 3416, 1606,
4.1.5. 7-(3-Chlorophenyl)-5-nitro-1-(tetrahydro-2H-pyran-2-
yl)-1H-indazol-6-amine (4c)
1486, 1393, 1326, 1297, 1079, 1038, 994 cmÀ1 1H NMR
;
(400 MHz, DMSO-d6): 1.61–1.89 (3H, m), 2.04–2.14 (1H, m),
2.14–2.24 (1H, m), 2.54–2.65 (1H, m), 3.89–4.04 (2H, m), 6.46
(1H, dd, J1 = 8 Hz, J2 = 3 Hz), 7.57 (1H, dd, J1 = 8.5 Hz, J2 = 2 Hz),
7.93 (1H, d, J = 8.5 Hz), 8.34 (1H, d, J = 2 Hz), 8.55 (1H, s), 8.99
(1H, s), 12.62 (1H, br s); 13C NMR (100 MHz, DMSO-d6): 21.7,
24.6, 29.2, 65.7 (CH2), 84.2 (CH), 114.5, 119.1, 121.3, 125.6,
137.9 (CHarom), 106.0, 118.3, 120.8, 125.1, 129.7, 132.5, 137.6,
137.8 (Carom); HRMS (ESI+) calcd for C18H1635ClN4O3 (M+H)+
371.0911, found 371.0915.
From 2 (105 mg, 0.27 mmol) and 3-chlorophenylboronic acid 3c
(2 equiv) (reflux, 4 h), column chromatography (cyclohexane/
EtOAc, 8:2) provided 4c (87 mg, 0.23 mmol, 86%) as a yellow pow-
der. Mp 225–227 °C; IR (ATR): 3492, 3367, 1617, 1489, 1411, 1292,
1079, 1037, 991 cmÀ1 1H NMR (400 MHz, DMSO-d6), amajor iso-
;
mer, bminor isomer: 0.98–1.11 (1Ha+1Hb, m), 1.21–1.40 (2Ha+2Hb,
m), 1.64–1.74 (1Ha+1Hb, m), 1.76–1.86 (1Ha+1Hb, m), 2.17–2.32
(1Ha+1Hb, m), 2.40–2.56 (1Ha+1Hb, m), 3.59–3.65 (1Hb, m), 3.67–
3.74 (1Ha, m), 4.23 (1Ha, dd, J1 = 10.5 Hz, J2 = 1.5 Hz), 4.28 (1Hb,
dd, J1 = 10.5 Hz, J2 = 1.5 Hz), 6.19 (2Hb, br s), 6.20 (2Ha, br s), 7.31
(1Hb, dt, J1 = 6.5 Hz, J2 = 2 Hz), 7.38 (1Ha, t, J = 1.5 Hz), 7.41 (1Ha,
dt, J1 = 7 Hz, J2 = 1.5 Hz), 7.54–7.56 (1Hb, m), 7.63–7.72 (2Ha+2Hb,
m), 8.21 (1Ha+1Hb, s), 8.73 (1Ha+1Hb, s); 13C NMR (100 MHz,
DMSO-d6): 22.8, 22.9, 24.25, 24.30, 28.7, 28.8, 66.6, 66.7 (CH2),
83.7 (CH), 121.05, 121.08, 129.00, 129.05, 129.8, 130.1, 130.9,
4.1.10. General procedure for preparation of compounds 6a–6c
A mixture of compound 5 in concentrated hydrochloric acid
(0.1 mmol/mL) was refluxed for 3 h. An aqueous saturated NaHCO3
solution was added and the product was extracted with EtOAc. The
combined organic fractions were dried over MgSO4 and evapo-
rated. The residue was purified by column chromatography.

4108
V. Suchaud et al. / Bioorg. Med. Chem. 21 (2013) 4102–4111
4.1.11. 5-Nitro-1,6-dihydropyrazolo[4,3-c]carbazole (6a)
From 5a (38.0 mg, 0.113 mmol), column chromatography
(cyclohexane/EtOAc, 7:3) provided 6a (17.7 mg, 0.070 mmol, 62%)
as an orange powder. Mp >250 °C; IR (ATR): 3500–3000, 1646,
1309, 1216, 1146, 1030 cmÀ1 1H NMR (400 MHz, DMSO-d6): 3.90
;
(3H, s), 4.93 (2H, br s), 6.82 (1H, s), 6.99 (1H, dd, J1 = 8.5 Hz,
J2 = 2.5 Hz), 7.48 (1H, d, J = 8.5 Hz), 7.86 (1H, s), 8.07 (1H, s),
11.11 (1H, s), 13.25 (1H, br s); 13C NMR (125 MHz, DMSO-d6):
55.8 (CH3), 97.7, 103.5, 111.8, 113.4, 132.4a (CHarom), 104.6b,
117.3, 122.0, 129.5b, 129.7, 131.3, 132.5, 153.6 (Carom), achemical
shift measured from a HSQC 1H–13C experiment, bchemical shift
measured from a HMBC 1H–13C experiment; HRMS (ESI+) calcd
for C14H13N4O (M+H)+ 253.1089, found 253.1083.
1524, 1473, 1337, 1297, 1243, 1192, 1157 cmÀ1
;
1H NMR
(400 MHz, DMSO-d6): 7.39 (1H, ddd, J1 = 8 Hz, J2 = 7 Hz, J3 = 1 Hz),
7.52 (ddd, J1 = 8 Hz, J2 = 7 Hz, J3 = 1 Hz), 7.87 (1H, d, J = 8 Hz), 8.51
(1H, br s), 8.64 (1H, d, J = 8 Hz), 8.94 (1H, s), 12.32 (1H, br s); 13C
NMR (100 MHz, DMSO-d6): 112.8, 117.9, 120.7, 121.1, 125.6,
137.8 (CHarom), 105.9, 116.9, 120.0, 129.5, 130.8, 136.6, 138.9 (Car-
om); HRMS (ESI+) calcd for C13H9N4O2 (M+H)+ 253.0726, found
253.0736.
4.1.17. 9-Chloro-1,6-dihydropyrazolo[4,3-c]carbazol-5-amine
(7c)
From 6c (19.3 mg, 0.067 mmol), column chromatography
(cyclohexane/EtOAc, 2:8) provided 7c (10.2 mg, 0.040 mmol, 59%)
as a pinkish powder. Mp >250 °C; IR (ATR): 3500–2700, 1519,
4.1.12. 9-Methoxy-5-nitro-1,6-dihydropyrazolo[4,3-c]carbazole
(6b)
From 5b (41.0 mg, 0.112 mmol), column chromatography
(cyclohexane/EtOAc, 7:3 to 5:5) provided 6b (19.5 mg,
0.069 mmol, 62%) as an orange powder. Mp >250 °C; IR (ATR):
1462, 1289, 1159, 1062 cmÀ1 1H NMR (400 MHz, DMSO-d6):
;
5.00 (2H, br s), 6.90 (1H, s), 7.37 (1H, dd, J1 = 8.5 Hz, J2 = 2 Hz),
7.61 (1H, d, J = 8.5 Hz), 7.89 (1H, s), 8.62 (1H, s), 11.50 (1H, s),
13.27 (1H, br s); 13C NMR (100 MHz, DMSO-d6): 98.8, 112.7,
120.3, 123.6, 132.6a (CH), 103.9b, 117.8, 122.7, 123.6, 129.1b,
3500–3000, 1649, 1470, 1290, 1215, 1168 cmÀ1
;
1H NMR
(400 MHz, DMSO-d6): 3.93 (3H, s), 7.14 (1H, dd, J1 = 9 Hz,
J2 = 2.5 Hz), 7.75 (1H, d, J = 9 Hz), 8.19 (1H, d, J = 2 Hz), 8.49 (1H,
s), 8.91 (1H, s), 12.16 (1H, br s); 13C NMR (100 MHz, DMSO-d6):
55.9 (CH3), 103.1, 113.6, 115.5, 117.8, 137.8 (CHarom), 106.0,
116.5, 120.4, 129.5, 131.0, 133.6, 136.6, 154.6 (Carom); HRMS
(ESI+) calcd for C14H11N4O3 (M+H)+ 283.0831, found 283.0816.
a
129.7, 131.6, 136.1 (Carom), chemical shift measured from a HSQC
1H–13C experiment, bchemical shift measured from a HMBC 1H–13
C
experiment; HRMS (ESI+) calcd for C13H1035ClN4 (M+H)+ 257.0594,
found 257.0591.
4.1.18. 5-Bromo-6-nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-
indazole (9)
4.1.13. 9-Chloro-5-nitro-1,6-dihydropyrazolo[4,3-c]carbazole
(6c)
To a mixture of 5-bromo-6-nitroindazole 8 (1.30 g, 5.40 mmol)
in anhydrous CH2Cl2 (40 mL) were added 3,4-dihydro-2H-pyrane
(1.46 mL, 1.35 g, 16 mmol) and PTSA (monohydrate) (93 mg,
0.49 mmol). The mixture was stirred for 1 h at room temperature
and then was washed with a saturated aqueous NaHCO3 solution.
The assembled aqueous fractions were extracted with CH2Cl2.
The organic fractions were dried over MgSO4, evaporated and the
residue was purified by column chromatography (cyclohexane/
EtOAc, 95:5). Pentane was added to the residue, the solid was fil-
tered off and then was washed with a minimum of pentane/MeOH
9:1 mixture. Compound 9 (1.37 g, 4.2 mmol, 78%) was obtained as
a yellow-orange solid. Mp 96–98 °C; IR (ATR): 1574, 1528, 1468,
From 5c (50 mg, 0.135 mmol), column chromatography (cyclo-
hexane/EtOAc, 8:2 then 7:3) provided 6c (26.5 mg, 0.092 mmol,
69%) as a yellow powder. Mp >250 °C; IR (ATR): 3500–3000,
1463, 1319, 1291 cmÀ1 1H NMR (400 MHz, DMSO-d6): 7.54 (1H,
;
dd, J1 = 8.5 Hz, J2 = 2 Hz), 7.86 (1H, d, J = 8.5 Hz), 8.51 (1H, d,
J = 1.5 Hz), 8.80 (1H, d, J = 2 Hz), 8.98 (1H, s), 12.46 (1H, br s); 13C
NMR (100 MHz, DMSO-d6): 114.3, 118.9, 120.4, 125.5, 137.9 (CHar-
om), 105.1, 116.9, 121.2, 125.2, 129.5, 131.5, 136.4, 137.3 (Carom);
HRMS (ESI+) calcd for C13H835ClN4O2 (M+H)+ 287.0336, found
287.0344.
4.1.14. General procedure for preparation of compounds 7a–7c
To a solution of indazole 6 in ethyl acetate (0.01 mmol/mL) was
added platinum oxide (20 mol%). The mixture was stirred for 36 h
under hydrogen atmosphere (balloon), and then was evaporated
under reduced pressure. The residue was purified by column
chromatography.
1439, 1416 cmÀ1 1H NMR (400 MHz, CDCl3): 1.66–1.84 (3H, m),
;
2.09–2.18 (2H, m), 2.41–2.51 (1H, m), 3.73–3.80 (1H, m), 3.96–
4.02 (1H, m), 5.75 (1H, dd, J1 = 9.0 Hz, J2 = 2.5 Hz), 8.05 (1H, d,
J = 1.0 Hz), 8.07 (1H, s), 8.15 (1H, s); 13C NMR (100 MHz, CDCl3):
22.0, 25.0, 29.5, 67.4 (CH2), 86.5 (CH), 108.7, 126.9, 133.1 (CHarom),
105.1, 127.1, 136.8, 148.0⁄ (Carom), ⁄chemical shift measured from a
HMBC
C
1H–13
C
experiment;
HRMS
(ES+)
calcd
for
4.1.15. 1,6-Dihydropyrazolo[4,3-c]carbazol-5-amine (7a)
From 6a (36.0 mg, 0.143 mmol), column chromatography
(cyclohexane/EtOAc, 2:8) provided 7a (25.6 mg, 0.115 mmol,
81%) as a pinkish powder. Mp >250 °C; IR (ATR): 3500–2700,
12H1279BrN3NaO3 (M+Na)+ 347.9960, found 347.9960.
4.1.19. 6-Nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-5-
amine (10)
1517, 1461, 1401, 1350, 1319, 1242, 1160 cmÀ1
;
1H NMR
A mixture of Pd2(dba)3ÁCHCl3 (5.6 mg, 5.4
lmol) and BINAP
(400 MHz, DMSO-d6): 4.98 (2H, br s), 6.86 (1H, s), 7.24 (1H, ddd,
J1 = 8 Hz, J2 = 7 Hz, J3 = 1 Hz), 7.37 (1H, ddd, J1 = 8 Hz, J2 = 7 Hz,
J3 = 1 Hz), 7.59 (1H, d, J = 8 Hz), 7.89 (1H, s), 8.48 (1H, d, J = 8 Hz),
11.31 (1H, s), 13.23 (1H, br s); 13C NMR (100 MHz, DMSO-d6):
98.0, 111.2, 119.1, 121.1, 123.8, 132.4a (CHarom), 104.7b, 117.7,
121.6, 129.6b, 129.7, 130.6, 137.6 (Carom), achemical shift measured
from a HSQC 1H–13C experiment, bchemical shift measured from a
HMBC 1H–13C experiment; HRMS (ESI+) calcd for C13H11N4 (M+H)+
223.0984, found 223.0977.
(11.4 mg, 0.018 mmol) in toluene (1 mL) degassed with argon
was stirred at room temperature for 15 min, and then indazole 9
(50 mg, 0.153 mmol) and NaOtBu (20.6 mg, 0.21 mmol) were
added. To this dark-red solution, benzophenone imine (31 lL,
0.185 mmol) was added and the reaction mixture was stirred at
80 °C for 5 h. After cooling, EtOAc was added and the suspension
was filtered through a pad of Celite and the filtrate was concen-
trated under reduced pressure. The residue was then dissolved in
THF (1 mL) and a 3 M aqueous HCl solution (0.3 mL) was added.
The reaction mixture was stirred at room temperature for
30 min, and then was extracted with EtOAc. The assembled organic
fractions were dried over MgSO4, filtered, and concentrated under
reduced pressure. The residue was purified by column chromatog-
raphy (cyclohexane/EtOAc, 8:2) to give 10 (34.6 mg, 0.13 mmol,
86%) as a red solid. For spectral data, see reference.20
4.1.16. 9-Methoxy-1,6-dihydropyrazolo[4,3-c]carbazol-5-amine
(7b)
From 6b (47.7 mg, 0.169 mmol), column chromatography
(cyclohexane/EtOAc, 5:5 to 2:8) provided 7b (21.0 mg, 0.083 mmol,
49%) as a pinkish powder. Mp >250 °C; IR (ATR): 3500–2700, 1481,

V. Suchaud et al. / Bioorg. Med. Chem. 21 (2013) 4102–4111
4109
4.1.20. 5-Chloro-2-iodophenylboronic acid (12c)
(81 mg, 0.24 mmol, 67%) as
a
red powder. Mp >250 °C; IR
5-Amino-2-iodophenylboronic acid 18 (1.18 g, 4.5 mmol) was
dissolved at 5 °C in concentrated HCl (2.8 mL). A solution of sodium
nitrite (310 mg, 4.5 mmol) in a minimum of water was slowly
added and the reaction was stirred at 5 °C for 30 min. A cold solu-
tion of CuCl (1.78 g, 18 mmol) in concentrated HCl (1 mL) was then
added dropwise and the mixture was stirred at room temperature
for 2 h. Water was added and the mixture was extracted with
EtOAc. The combined organic fractions were dried over MgSO4
and evaporated. Column chromatography (cyclohexane/EtOAc,
7:3) provided 12c (771 mg, 2.73 mmol, 61%) as a pink powder.
(ATR): 3419, 1320, 1260, 1080, 1036, 1018 cmÀ1 1H NMR
;
(400 MHz, DMSO-d6): 1.60–1.69 (2H, m), 1.76–1.89 (1H, m),
2.04–2.15 (2H, m), 2.44–2.57 (1H under solvent signal), 3.83–
3.95 (2H, m), 6.16 (1H, d, J = 9 Hz), 7.33 (1H, t, J = 7.5 Hz), 7.50
(1H, t, J = 7.5 Hz), 7.84 (1H, d, J = 8 Hz), 8.45 (1H, d, J = 8 Hz),
8.80 (1H, s), 8.90 (1H, s), 12.12 (1H, s); 13C NMR (100 MHz,
DMSO-d6): 21.9, 24.8, 28.9, 66.4 (CH2), 84.3 (CH), 105.3, 112.7,
120.1, 121.3, 126.1, 131.4 (CHarom), 115.3, 120.7, 121.2, 127.0,
132.7, 133.2, 139.9 (Carom); HRMS (ESI+) calcd for C18H16N4NaO3
(M+Na)+ 359.1120, found 359.1121.
Mp 179–181 °C; IR (ATR): 3280, 1375, 1328, 1098, 1001 cmÀ1 1H
;
NMR (400 MHz, DMSO-d6): 7.13 (1H, dd, J1 = 8.5 Hz, J2 = 2.5 Hz),
7.22 (1H, d, J = 2.5 Hz), 7.74 (1H, d, J = 8.5 Hz); 13C NMR
(100 MHz, DMSO-d6): 129.8, 132.6, 139.5 (CHarom), 96.7, 132.6,
4.1.24. 9-Methoxy-5-nitro-3-(tetrahydro-2H-pyran-2-yl)-3,6-
dihydropyrazolo[3,4-c]carbazole (14b)
Same procedure as for the preparation of compound 14a. From
13b (123 mg, 0.27 mmol), column chromatography (cyclohexane/
EtOAc, 8:2–5:5) provided 14b (59 mg, 0.16 mmol, 59%) as a red
powder. Mp >250 °C; IR (ATR): 3419, 1516, 1445, 1436, 1313,
148.7⁄ (Carom), ⁄chemical shift measured from a HMBC 1H–13
C
experiment; HRMS (ESI–) calcd for C6H435Cl11BIO2 (MÀH)À
280.9038, found 280.9036.
1285, 1239, 1221, 1207, 1153, 1037 cmÀ1 1H NMR (400 MHz,
;
4.1.21. 4-(2-Bromophenyl)-6-nitro-1-(tetrahydro-2H-pyran-2-
yl)-1H-indazol-5-amine (13a)
DMSO-d6): 1.58–1.68 (2H, m), 1.75–1.88 (1H, m), 2.03–2.13 (2H,
m), 2.43–2.57 (1H under solvent signal), 3.86–3.93 (2H, m), 3.95
(3H, s), 6.19 (1H, dd, J1 = 9.5 Hz, J2 = 2 Hz), 7.18 (1H, dd,
J1 = 9 Hz, J2 = 2.5 Hz), 7.74 (1H, d, J = 9 Hz), 7.93 (1H, d,
J = 2.5 Hz), 8.81 (1H, d, J = 1 Hz), 9.06 (1H, d, J = 1 Hz), 12.03
(1H, br s); 13C NMR (100 MHz, DMSO-d6): 55.9 (CH3), 22.0, 24.8,
29.0, 66.5 (CH2), 84.2 (CH), 103.3, 105.4, 113.7, 116.5, 131.8 (CHar-
om), 115.2, 121.0, 121.2, 127.5, 132.8, 133.1, 134.9, 154.2 (Carom);
Same procedure as for the preparation of compounds 4a and 4b.
From 11 (200 mg, 0.52 mmol) and 2-bromophenylboronic acid 12a
(1.5 equiv) (reflux, 2 h), column chromatography (cyclohexane/
EtOAc, 8:2) provided 13a (168 mg, 0.40 mmol, 78%) as a red oil.
IR (ATR): 3496, 3386, 1526, 1287, 1246, 1163, 1080, 1042 cmÀ1
;
1H NMR (400 MHz, DMSO-d6): 1.49–1.64 (2H, m), 1.67–1.81 (1H,
m), 1.93–2.06 (2H, m), 2.28–2.41 (1H, m), 3.75–3.93 (2H, m),
5.77 (2H, br s), 5.96 (1H, d, J = 10 Hz), 7.37–7.51 (3H, m), 7.54–
7.64 (1H, m), 7.86/7.88 (1H, 2s), 8.59/8.60 (1H, 2s); 13C NMR
(100 MHz, DMSO-d6): 22.0, 22.1, 24.8, 28.7, 28.8, 66.3, 66.5 (CH2),
83.8, 83.9 (CH), 107.9, 108.0, 128.80, 128.81, 130.8, 131.30,
131.33, 132.20, 132.24, 133.4 (CHarom), 117.43, 117.45, 123.88,
123.91, 128.86, 128.91, 131.0, 134.96, 134.99, 135.7 (Carom); HRMS
HRMS (ESI+) calcd for
367.1424.
C
19H19N4O4 (M+H)+ 367.1406, found
4.1.25. 9-Chloro-5-nitro-3-(tetrahydro-2H-pyran-2-yl)-3,6-
dihydropyrazolo[3,4-c]carbazole (14c)
Step A: same procedure as for the preparation of compounds
4a–4c. From 11 (200 mg, 0.52 mmol) and 5-chloro-2-
iodophenylboronic acid 12c (2 equiv) (reflux, 4 h), 13c was pre-
pared and used for the next step without chromatographic
purification.
(ESI+) calcd for
439.0402.
C
18H1779BrN4NaO3 (M+Na)+ 439.0382, found
4.1.22. 4-(2-Bromo-5-methoxyphenyl)-6-nitro-1-(tetrahydro-
2H-pyran-2-yl)-1H-indazol-5-amine (13b)
Step B: same procedure as for the preparation of compound
14a. From the coupling product obtained step A, column chroma-
tography (cyclohexane/EtOAc, 95:5–9:1) provided 14c (89 mg,
0.24 mmol, 47%) as an orange powder. Mp 227–228 °C; IR (ATR):
Same procedure as for the preparation of compounds 4a–4c.
From 11 (150 mg, 0.39 mmol) and 2-bromo-5-methoxyphenylbo-
ronic acid 12b (2 equiv) (reflux, 4 h), column chromatography
(cyclohexane/EtOAc, 9:1) provided 13b (132 mg, 0.30 mmol, 76%)
as a red powder. Mp 85–95 °C; IR (ATR): 3490, 3382, 1524, 1282,
3417, 1520, 1445, 1315, 1034 cmÀ1 1H NMR (400 MHz, DMSO-
;
d6): 1.59–1.67 (2H, m), 1.74–1.88 (1H, m), 2.04–2.12 (2H, m),
2.43–2.57 (1H under the solvent signal), 3.86–3.92 (2H, m), 6.22
(1H, dd, J1 = 9.5 Hz, J2 = 2 Hz), 7.55 (1H, dd, J1 = 8.5 Hz, J2 = 2 Hz),
7.85 (1H, d, J = 8.5 Hz), 8.63 (1H, d, J = 2 Hz), 8.90 (1H, d, J = 1 Hz),
9.10 (1H, d, J = 1 Hz), 12.33 (1H, br s); 13C NMR (100 MHz,
DMSO-d6): 22.0, 24.8, 29.0, 66.5 (CH2), 84.1 (CH), 106.6, 114.4,
120.7, 126.3, 131.9 (CHarom), 114.3, 121.0, 121.8, 124.8, 127.8,
132.9, 133.4, 138.3 (Carom); HRMS (ESI+) calcd for
1241, 1159, 1079, 1039 cmÀ1 1H NMR (400 MHz, DMSO-d6):
;
1.51–1.63 (2H, m), 1.68–1.81 (1H, m), 1.93–2.07 (2H, m), 2.29–
2.41 (1H, m), 3.75–3.92 (2H, m), 3.777/3.783 (3H, 2s), 5.81 (2H,
s), 5.94–5.99 (1H, m), 6.96/6.99 (1H, 2d, J = 3 Hz), 7.06 (1H, dd,
J1 = 9 Hz, J2 = 3 Hz), 7.48 (1H, m), 7.740/7.744 (1H, 2d, J = 9 Hz),
8.58/8.60 (1H, 2d, J = 1 Hz); 13C NMR (100 MHz, DMSO-d6): 55.5,
55.6 (CH3), 22.0, 22.1, 24.8, 28.7, 28.9, 66.4, 66.5 (CH2), 83.8, 83.9
(CH), 107.9, 108.0, 116.9, 117.19, 117.23, 131.4, 131.5, 134.2 (CHar-
om), 113.90, 113.93, 117.42, 117.44, 128.8, 128.9, 131.0, 134.91,
134.94, 135.71, 135.72, 135.81, 135.83, 159.30, 159.31 (Carom);
HRMS (ESI+) calcd for C19H2079BrN4O4 (M+H)+ 447.0668, found
447.0658.
C
18H1535ClN4NaO3 (M+Na)+ 393.0731, found 393.0730.
4.1.26. 5-Nitro-3,6-dihydropyrazolo[3,4-c]carbazole (15a)
Same procedure as for the preparation of compounds 6a–6c.
From 14a (70 mg, 0.21 mmol) in concentrated hydrochloric acid
(5 mL), column chromatography (cyclohexane/EtOAc) provided
compound 15a (43 mg, 0.17 mmol, 82%) as a red powder. Mp
>250 °C; IR (ATR): 3438, 3224, 1343, 1322 cmÀ1
;
1H NMR
4.1.23. 5-Nitro-3-(tetrahydro-2H-pyran-2-yl)-3,6-
dihydropyrazolo[3,4-c]carbazole (14a)
(500 MHz, DMSO-d6): 7.35 (1H, t, J = 7.5 Hz), 7.52 (1H, t,
J = 7.5 Hz), 7.84 (1H, d, J = 8 Hz), 8.48 (1H, d, J = 8 Hz), 8.58 (1H,
s), 8.92 (1H, s), 12.11 (1H, s), 13.80 (1H, br s); 13C NMR
(126 MHz, DMSO-d6): 105.5, 112.8, 120.1, 121.4, 126.1, 131.8
(CHarom), 114.9, 120.1, 120.9, 126.5, 132.8, 134.0, 139.7 (Carom);
HRMS (ESI+) calcd for C13H8N4NaO2 (M+Na)+ 275.0545, found
275.0546.
A mixture of compound 13a (150 mg, 0.36 mmol), Cu (68 mg),
CuI (14 mg, 0.074 mmol) and K2CO3 (99 mg, 0.72 mmol) in dibu-
tyl ether (5 mL) was refluxed for 24 h. Water (10 mL) was added
and the mixture was extracted with EtOAc (2 Â 30 mL). The com-
bined organic fractions were dried over MgSO4 and evaporated.
Column chromatography (cyclohexane/EtOAc) provided 14a

4110
V. Suchaud et al. / Bioorg. Med. Chem. 21 (2013) 4102–4111
4.1.27. 9-Methoxy-5-nitro-3,6-dihydropyrazolo[3,4-c]carbazole
(15b)
beige powder. Mp >250 °C; IR (ATR): 3450–2800, 1467, 1291,
1066, 935 cmÀ1 1H NMR (400 MHz, DMSO-d6): 5.50 (2H, br s),
;
Same procedure as for the preparation of compounds 6a–6c.
From 14b (59 mg, 0.161 mmol), column chromatography (cyclo-
hexane/EtOAc, 5:5) provided 15b (22.6 mg, 0.080 mmol, 50%) as
a red powder. Mp >250 °C; IR (ATR): 3436, 3241, 1472, 1445,
6.74 (1H, d, J = 1 Hz), 7.34 (1H, dd, J1 = 8.5 Hz, J2 = 2 Hz), 7.59 (1H,
d, J = 8.5 Hz), 8.28 (1H, d, J = 2 Hz), 8.42 (1H, s), 11.28 (1H, s),
12.57 (1H, br s); 13C NMR (100 MHz, DMSO-d6): 89.8, 112.8,
120.1, 123.7 130.9a (CHarom), 108.8, 110.8, 123.0, 123.7, 127.2,
134.8, 136.3, 138.2b (Carom), achemical shift measured from a HSQC
1307, 1288, 1208 cmÀ1 1H NMR (400 MHz, DMSO-d6): 3.95 (3H,
;
s), 7.16 (1H, dd, J1 = 9 Hz, J2 = 2 Hz), 7.73 (1H, d, J = 9 Hz), 7.91
(1H, d, J = 1.5 Hz), 8.56 (1H, s), 9.00 (1H, s), 11.95 (1H, s), 13.77
(1H, br s); 13C NMR (100 MHz, DMSO-d6): 55.9 (CH3), 103.1,
105.7a, 113.6, 116.1, 131.7a (CHarom), 114.8, 120.0, 121.3, 126.9,
1H–13C experiment, bchemical shift measured from a HMBC 1H–13
C
experiment; HRMS (ESI+) calcd for C13H1035ClN4 (M+H)+ 257.0605,
found 257.0594.
a
132.8, 133.8b, 134.6, 154.1 (Carom), chemical shift measured from
4.2. In vitro kinase inhibition assays
a HSQC 1H–13C experiment, bchemical shift measured from a
HMBC 1H–13C experiment; HRMS (ESI+) calcd for C14H11N4O3
(M+H)+ 283.0831, found 283.0826.
The procedures for the in vitro protein kinase assays and for the
expression and activation of the protein kinases have been de-
scribed previously.26
4.1.28. 9-Chloro-5-nitro-3,6-dihydropyrazolo[3,4-c]carbazole
(15c)
Source and purification of kinases: All protein kinases were of
human origin and encoded full-length proteins. All proteins were
either expressed as GST (glutathione transferase) fusion proteins
in Escherichia coli or as hexahistidine (His6)-tagged proteins in
Sf21 (Spodoptera frugiperda 21) insect cells. GST fusion proteins
were purified by affinity chromatography on glutathione–Sephar-
ose, and His6-tagged proteins on nickel/nitrilotriacetate–agarose.
Same procedure as for the preparation of compounds 6a–6c.
From 14c (82 mg, 0.221 mmol), column chromatography (cyclo-
hexane/EtOAc, 5:5) provided 15c (50 mg, 0.174 mmol, 79%) as a
red powder. Mp >250 °C; IR (ATR): 3441, 3246, 1445, 1315, 1282,
;
1181 cmÀ1 1H NMR (400 MHz, DMSO-d6): 7.53 (1H, dd,
J1 = 8.5 Hz, J2 = 2 Hz), 7.84 (1H, d, J = 8.5 Hz), 8.60 (1H, d, J = 2 Hz),
8.63 (1H, d, J = 1 Hz), 9.04 (1H, t, J = 1 Hz), 12.27 (1H, s, NH),
13.87 (1H, br s); 13C NMR (100 MHz, DMSO-d6): 106.6, 114.3,
120.6, 126.0, 132.1 (CHarom), 114.0, 119.9, 122.0, 124.6, 127.3,
132.8, 134.0, 138.1 (Carom); HRMS (ESI/ASAP) calcd for
Protein kinase assays: All assays (25.5 lL volume) were carried
out robotically at room temperature (21 °C) and were linear with
respect to time and enzyme concentration under the conditions
used. Assays were performed for 30 min using Multidrop Micro re-
agent dispensers (Thermo Electron Corporation, Waltham, MA,
U.S.A.) in a 96-well format. The concentration of magnesium ace-
C
13H835ClN4O2 (M+H)+ 287.0336, found 287.0336.
tate in the assays was 10 mM and [
was used at 5 M for Pim-2 and 20 M for Pim-1 and Pim-3, in or-
c-
33P]ATP (800 cpm/pmol)
4.1.29. 3,6-Dihydropyrazolo[3,4-c]carbazole-5-amine (16a)
Same procedure as for the preparation of compounds 7a–7c.
From 15a (10 mg, 0.040 mmol) in EtOAc (3 mL) (24 h), column chro-
matography (cyclohexane/EtOAc) provided 16a (8 mg, 0,036 mmol,
91%) as a brown powder. Mp >250 °C; IR (ATR): 3500–2500, 1345,
l
l
der to be at or below the Km for ATP for each enzyme.
The assays were initiated with MgATP, stopped by the addition
of 5
plates using
U.S.A.). Kinase substrate was RSRHSSYPAGT (300 l
l
L of 0.5 M orthophosphoric acid and spotted on to P81 filter
unifilter harvester (PerkinElmer, Boston, MA,
M) for Pim-1,
a
1319, 1248, 1179, 1083, 948 cmÀ1
;
1H NMR (500 MHz, DMSO-d6):
5.47 (2H, br s), 6.71 (1H, s), 7.19 (1H, t, J = 7.5 Hz), 7.34 (1H, t,
J = 7.5 Hz), 7.57 (1H, d, J = 8 Hz), 8.21 (1H, d, J = 8 Hz), 8.33 (1H, s),
11.07 (1H, s), 12.52 (1H, br s); 13C NMR (126 MHz, DMSO-d6):
89.0, 111.3, 118.6, 120.9, 123.8, 130.7a (CHarom), 109.1, 111.5,
122.7, 126.1, 134.8, 137.9, 138.1b (Carom), achemical shift measured
from a HSQC 1H–13C experiment, bchemical shift measured from a
Pim-2 and Pim-3. The enzymes were diluted in a buffer consisting
of 50 mM Tris/HCl, pH 7.5, 0.1 mM EGTA, 1 mg/mL BSA and 0.1% 2-
mercaptoethanol and assayed in a buffer comprising 50 mM Tris/
HCl, pH 7.5, 0.1 mM EGTA and 0.1% 2-mercaptoethanol.
The inhibition profile of the tested compounds was expressed as
the percentage of the residual kinase activity for an inhibitor con-
HMBC 1H–13
C
experiment; HRMS (ESI+) calcd for C13H11N4
centration of 10 or 1 lM. The IC50 values of inhibitors were deter-
mined after carrying out assays at 10 different concentrations of
(M+H)+ 223.0984, found 223.0993.
each compound.
4.1.30. 9-Methoxy-3,6-dihydropyrazolo[3,4-c]carbazol-5-amine
(16b)
4.3. In vitro antiproliferative activity assays
Same procedure as for the preparation of compounds 7a–7c.
From 15b (25.0 mg, 0.089 mmol), column chromatography (cyclo-
hexane/EtOAc, 5:5–0:10) provided 16b (18.6 mg, 0.074 mmol, 83%)
as a pinkish powder. Mp 254–255 °C; IR (ATR): 3500–2700, 1299,
Prostate adenocarcinoma PC3 or LNCap cells were grown in
RPMI 1640 +
fetal calf serum, 100 UI penicillin, 100
1.5 g/mL fungizone and kept under 5% CO2 atmosphere at 37 °C.
96 well plates were seeded with 2500 PC3 cells or 5000 LNCap
cells per well in 200 L medium.
L-Glutamine medium supplemented with 10% (v/v)
l
g/mL streptomycin and
1214, 1144, 1032, 934 cmÀ1
;
1H NMR (400 MHz, DMSO-d6): 3.90
l
(3H, s), 5.43 (2H, br s), 6.68 (1H, d, J = 1 Hz), 6.98 (1H, dd,
J1 = 9 Hz, J2 = 2.5 Hz), 7.47 (1H, d, J = 9 Hz), 7.67 (1H, d, J = 2.5 Hz),
8.38 (1H, d, J = 0.5 Hz), 10.87 (1H, s), 12.48 (1H, br s); 13C NMR
(125 MHz, DMSO-d6): 55.7 (CH3), 88.9, 103.1, 112.0, 113.6, 130.8a
(CHarom), 109.0, 111.5, 122.9, 126.9, 133.0, 134.8, 137.9b, 153.1 (Car-
om), achemical shift measured from a HSQC 1H–13C experiment,
bchemical shift measured from a HMBC 1H–13C experiment; HRMS
(ESI+) calcd for C14H13N4O (M+H)+ 253.1089, found 253.1096.
l
Twenty four hours later, tested drugs dissolved in DMSO were
added for 72 h at a final concentration in a fixed volume of DMSO
(1% final concentration). Controls received an equal volume of
DMSO. The number of viable cells was measured at 490 nm with
the MTS reagent (Promega, Madison, WI) and IC50 was calculated
as the concentration of compound eliciting a 50% inhibition of cell
proliferation.
4.1.31. 9-Chloro-3,6-dihydropyrazolo[3,4-c]carbazol-5-amine
(16c)
4.4. Molecular modeling experiments
Same procedure as for the preparation of compounds 7a–7c.
From 15c (60 mg, 0.209 mmol), column chromatography (cyclo-
hexane/EtOAc, 1:9) provided 16c (37.0 mg, 0.144 mmol, 69%) as a
Pim-1 3JPV crystal structure from the Protein Data Bank (PDB)
was used to generate Pim-1 model. After removing 1DR ligand,

V. Suchaud et al. / Bioorg. Med. Chem. 21 (2013) 4102–4111
4111
Laird, A. D.; Lin, R.; Mock, L.; Ngan, I.; Pack, M.; Stott, G.; Stout, T. J.; Yu, P.;
Zaharia, C.; Zhang, W.; Zhou, P.; Nuss, J. M.; Kearney, P. C.; Xu, W. Bioorg. Med.
Chem. Lett. 2012, 22, 3732.
protein structure was prepared and all hydrogen atoms were
added using Sybylx32 with its biopolymer module.
Regarding Pim-3, Modeller9V11 software28 from the module in-
cluded in UCSF Chimera molecular modeling system29–31 was used
to generate the 3D model from Pim-1 1XWS crystal structure. The
Pim-3 model was generated taking into account water molecules
and hydrogen bonds existing in the model structure. The Pim-3
model having the best RMSD in comparison with 1XWS was pro-
cessed with Sybylx2.0. Protein structure was prepared and all
hydrogen atoms were added using Sybylx2.0 with its biopolymer
module.
7. Dakin, L. A.; Block, M. H.; Chen, H.; Code, E.; Dowling, J. E.; Feng, X.; Ferguson, A.
D.; Green, I.; Hird, A. W.; Howard, T.; Keeton, E. K.; Lamb, M. L.; Lyne, P. D.;
Pollard, H.; Read, J.; Wu, A. J.; Zhang, T.; Zheng, X. Bioorg. Med. Chem. Lett. 2012,
22, 4599.
8. Haddach, M.; Michaux, J.; Schwaebe, M. K.; Pierre, F.; O’Brien, S. E.; Borsan, C.;
Tran, J.; Raffaele, N.; Ravula, S.; Drygin, D.; Siddiqui-Jain, A.; Darjania, L.;
Stansfield, R.; Proffitt, C.; Macalino, D.; Streiner, N.; Bliesath, J.; Omori, M.;
Whitten, J. P.; Anderes, K.; Rice, W. G.; Ryckman, D. M. ACS Med. Chem. Lett.
2012, 3, 135.
9. Drygin, D.; Haddach, M.; Pierre, F.; Ryckman, D. M. J. Med. Chem. 2012, 55, 8199.
10. Chen, L. S.; Redkar, S.; Bearss, D.; Wierda, W. G.; Gandhi, V. Blood 2009, 114,
4150.
11. http://clinicaltrials.gov/ct2/show/NCT01239108
12. http://clinicaltrials.gov/ct2/show/NCT01489722
13. Akué-Gédu, R.; Rossignol, E.; Azzaro, S.; Knapp, S.; Filippakopoulos, P.; Bullock,
A. N.; Bain, J.; Cohen, P.; Prudhomme, M.; Anizon, F.; Moreau, P. J. Med. Chem.
2009, 52, 6369.
14. Akué-Gédu, R.; Nauton, L.; Théry, V.; Bain, J.; Cohen, P.; Anizon, F.; Moreau, P.
Bioorg. Med. Chem. 2010, 18, 6865.
15. Gavara, L.; Saugues, E.; Alves, G.; Debiton, E.; Anizon, F.; Moreau, P. Eur. J. Med.
Chem. 2010, 45, 5520.
16. Letribot, B.; Akué-Gédu, R.; Santio, N. M.; El-Ghozzi, M.; Avignant, D.; Cisnetti,
F.; Koskinen, P. J.; Gautier, A.; Anizon, F.; Moreau, P. Eur. J. Med. Chem. 2012, 50,
304.
17. Akué-Gédu, R.; Letribot, B.; Saugues, E.; Debiton, E.; Anizon, F.; Moreau, P.
Bioorg. Med. Chem. Lett. 2012, 22, 3807.
18. Giraud, F.; Akué-Gédu, R.; Nauton, L.; Candelon, N.; Debiton, E.; Théry, V.;
Anizon, F.; Moreau, P. Eur. J. Med. Chem. 2012, 56, 225.
19. Gavara, L.; Suchaud, V.; Nauton, L.; Théry, V.; Anizon, F.; Moreau, P. Bioorg. Med.
Chem. Lett. 2013, 23, 2298.
20. Gavara, L.; Anizon, F.; Moreau, P. Tetrahedron 2011, 67, 7330.
21. Jordan-Hore, J. A.; Johansson, C. C. C.; Gulias, M.; Beck, E. M.; Gaunt, M. J. J. Am.
Chem. Soc. 2008, 130, 16184.
22. Zhu, G.-D.; Gong, J.; Gandhi, V. B.; Woods, K.; Luo, Y.; Liu, X.; Guan, R.;
Klinghofer, V.; Johnson, E. F.; Stoll, V. S.; Mamo, M.; Li, Q.; Rosenberg, S. H.;
Giranda, V. L. Bioorg. Med. Chem. 2007, 15, 2441.
For Pim-1 and Pim-3 models, the position of hydrogen atoms
and amino-acid residue side chains were then optimized by per-
forming an energy minimization calculation (Tripos force field,
MMF94 charges, dielectric constant set to 4.0, conjugate gradient
method), before minimization without any constraint.
Docking experiments were performed using Sybylx2.0. After
superimposition of Pim-1 or Pim-3 models with 3JPV structure, a
protomol was created around 1DR ligand (threshold and bloat val-
ues set to 0.05 and 10, respectively). The Surflex-Dock Geom X
method was used, with the option allowing H and heavy atom
movements. The best docking solutions were selected from the
analysis of the molecular database generated by Sybylx. In partic-
ular, Polar versus Total_Score, Crash versus Total_Score and Strain
versus Total_Score 2D-graphs were produced and analyzed. The
best docking solution for each ligand was then minimized (Tripos
force field, MMF94 charges, dielectric constant set to 78, conjugate
gradient method).
Acknowledgments
23. Slade, D. J.; Pelz, N. F.; Bodnar, W.; Lampe, J. W.; Watson, P. S. J. Org. Chem.
2009, 74, 6331.
The authors are grateful for the financial support of ANR (ANR-
08-JCJC-0131-CSD 3) and the French Ministère de l’Enseignement
Supérieur et de la Recherche (ES).
24. Rotzler, J.; Vonlanthen, D.; Barsella, A.; Boeglin, A.; Fort, A.; Mayor, M. Eur. J.
Org. Chem. 2010, 1096.
25. Al-Zoubi, R. M.; Hall, D. G. Org. Lett. 2010, 12, 2480.
26. Bain, J.; Plater, L.; Elliott, M.; Shpiro, N.; Hastie, J.; McLauchlan, H.; Klervernic,
I.; Arthur, S. C.; Alessi, D. R.; Cohen, P. Biochem. J. 2007, 408, 297.
27. http://www.icsn.cnrs-gif.fr.
References and notes
28. Sali, A.; Blundell, T. L. J. Mol. Biol. 1993, 234, 779.
29. Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.;
Meng, E. C.; Ferrin, T. E. J. Comput. Chem. 2004, 25, 1605.
30. Meng, E. C.; Pettersen, E. F.; Couch, G. S.; Huang, C. C.; Ferrin, T. E. BMC
Bioinformatics 2006, 7, 339.
31. Yang, Z.; Lasker, K.; Schneidman-Duhovny, D.; Webb, B.; Huang, C. C.;
Pettersen, E. F.; Goddard, T. D.; Meng, E. C.; Sali, A.; Ferrin, T. E. J. Struct. Biol.
2012, 179, 269.
1. Anizon, F.; Shtil, A. A.; Danilenko, V. N.; Moreau, P. Curr. Med. Chem. 2010, 17,
4114.
2. Morwick, T. Exp. Opin. Ther. Patents 2010, 20, 193.
3. Schenone, S.; Tintori, C.; Botta, M. Curr. Pharm. Des. 2010, 16, 3964.
4. Brault, L.; Gasser, C.; Bracher, F.; Huber, K.; Knapp, S.; Schwaller, J.
Haematologica 2010, 95, 1004.
5. Isaac, M.; Siu, A.; Jongstra, J. Drug Resist. Update 2011, 14, 203.
6. Tsuhako, A. L.; Brown, D. S.; Koltun, E. S.; Aay, N.; Arcalas, A.; Chan, V.; Du, H.;
Engst, S.; Franzini, M.; Galan, A.; Huang, P.; Johnston, S.; Kane, B.; Kim, M. H.;
32. Sybylx2.0, Tripos International, 1699 South Hanley Rd., St. Louis, Missouri,
63144, USA.