Synthesis and cytotoxicity of epoxide and pyrazole analogs of the combretastatins

Article abstract of DOI:10.1016/j.bmc.2005.06.028

Twenty-six epoxide and corresponding pyrazole derivatives, of the structurally related chalcones and combretastatin A-4 (CA-4), were synthesized and tested for in vitro cytotoxicity. These molecules were synthesized by epoxidation of the relevant chalcone

Full text of DOI:10.1016/j.bmc.2005.06.028

Bioorganic & Medicinal Chemistry 13 (2005) 6025–6034
Synthesis and cytotoxicity of epoxide and pyrazole analogs
of the combretastatins
Regan LeBlanc,a John Dickson,a Toni Brown,a Michelle Stewart,a Hari N. Pati,a
Don VanDerveer,c Hadi Arman,c Jeff Harris,c William Pennington,c
Herman L. Holt, Jr.b and Moses Leea,*
aDepartment of Chemistry, Furman University, Greenville, SC 29613, USA
bDepartment of Chemistry, University of North Carolina, Asheville, NC 28804, USA
cDepartment of Chemistry, Clemson University, Clemson, SC 29634-0973, USA
Received 26 April 2005; revised 2 June 2005; accepted 9 June 2005
Available online 1 August 2005
Abstract—Twenty-six epoxide and corresponding pyrazole derivatives, of the structurally related chalcones and combretastatin A-4
(CA-4), were synthesized and tested for in vitro cytotoxicity. These molecules were synthesized by epoxidation of the relevant chal-
cones, followed by reaction with hydrazine. The structures of epoxides 3 and 7, and pyrazole 17, were confirmed by X-ray diffraction
studies. The relatively coplanar conformation of a 30,300,40,400,50,500-hexamethoxypyrazole 17 was in good agreement with the shape
for 30,300,40,400,50-pentamethoxypyrazole 16, which was determined from molecular mechanics optimization. In vitro cytotoxicity of
each class of compounds was obtained using a 72 h continuous exposure MTT assay against two murine cancer cell lines; B16 mel-
anoma and L1210 leukemia. The effect of substitution in the A-ring is addressed: three methoxy groups versus two, generally
increased cytotoxicity across both cell lines. In the majority of cases, the pyrazoles are generally more active than the epoxides, with
the most active, 5-(300-amino-400-methoxyphenyl)-3-(30,40,50-trimethoxyphenyl)pyrazole 21, possessing an IC50 value of 5 and 2.4 lM
(B16 and L1210, respectively). Due to their planar conformations, the pyrazoles are typically less active than the corresponding chal-
cones, which adopt angular conformations similar to CA-4. B-ring modifications confirmed that in general the amino compounds
are more active than the corresponding nitro compounds. Varying the number and orientation of methoxy groups on the A-ring did
not produce any significant differences in toxicity in the cell lines studied.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
tumor growth is also delayed and inhibited by the
obstruction of blood flow in tumor microvasculature.7
Research into the antitumor properties of chalcones has
received significant attention over the last few years,
particularly with the discovery that these compounds
possess a similar mode of action to the structurally relat-
ed combretastatins.1 Combretastatin A-4 (CA-4, Fig. 1)
is isolated from the African willow tree (Combretum
caffrum)1b and binds to the colchicine site of tubulin,
thus preventing tubulin polymerization.2–5 Further-
more, CA-4 interrupts metaphase of the cell cycle: the
lack of microtubules (primarily composed of tubulin)
prevent the formation of mitotic spindles.6 Moreover,
The development of CA-4 as a potential antitumor
drug has been compromised by its poor solubility in
biological media and low bioavailability. This physio-
chemical limitation led to the development of a water
soluble phosphate derivative (CA-4P, Fig. 1), designed
to improve in vivo efficacy. This prodrug is currently
undergoing clinical evaluation for the treatment of
cancer.8 Another derivative of CA-4 that shows excel-
lent potency and improved solubility in biological
media is AC-7739 (Fig. 1), which has an amino group
in place of the hydroxyl moiety in CA-4.3 The afore-
mentioned limitations of the combretastatins have
fuelled further research into structural analogs of these
compounds with the goal of enhancing bioavailability
and antitumor activity. A wide range of structural
analogs have been reported, which include substitution
of the A- and/or B-ring in the combretastatin frame-
Keywords: Combretastatins; Chalcones; Epoxides; Pyrazoles; Antican-
cer; Cytotoxicity.
*
Corresponding author. Tel.: +1 864 294 3368; fax: +1 864 294
3559; e-mail: moses.lee@furman.edu
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.06.028

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R. LeBlanc et al. / Bioorg. Med. Chem. 13 (2005) 6025–6034
Figure 1. Structure of combretastatin A-4 (CA-4), the water soluble prodrug (CA-4P), and an amino derivative (AC-7739).
work with different heterocycles.9 B-ring modifications
have also included aroylindoles,10 quinoline, and qui-
noxaline moieties.11 Replacing the stilbene core with
different functionality has also been attempted by
several groups, including our own laboratory, for
example, enones (the chalcones).12 We have recently
described the synthesis and in vitro cytotoxicity of
methoxy-substituted chalcones, comparing the effect
of nitro and amino groups on both the A- and B-
ring.12 Other functional groups that bridge the two
aryl moieties have also been investigated, and they in-
clude carbonyl10 and different heterocycles, including,
for example, furanones,13 isoxazoles,14 imidazoles,15
triazoles16, and azetidinone17 compounds; all of which
possess varying levels of cytotoxicity. The rationale
behind the central heterocyclic ring is to ꢀlockꢁ the
molecule into the cis-orientation; identified previously
as crucial for activity.
2. Results and discussion
2.1. Synthesis
Synthesis of the chalcones was achieved in high chemical
yields using the Claisen–Schmidt condensation of func-
tionalized acetophenones and benzaldehydes using the
published method.12 The general method used in the
synthesis of the epoxides and the subsequent pyrazoles
is depicted in Scheme 1. Chalcones were treated with
powdered potassium carbonate in methanol followed
by excess hydrogen peroxide. The epoxides (1–11,
Table 1) were isolated as pure solids (in most cases) in
good yields (67–89%). The functionalized pyrazoles were
synthesized by dissolving the epoxide in xylenes and
treating with p-toluenesulfonic acid and hydrazine hy-
drate and the solution stirred at reflux until the product
precipitated (12–18, 20, 22, 23, 25, Table 1). Removal of
the xylenes and washing with hexane produced the pure
products in good yields (43–95%). The pyrazole synthe-
sis, via the epoxide intermediate, allows relatively large
numbers of compounds to be produced readily without
the need for tedious purification, as the crude product in
most cases, is sufficiently pure and can be used directly.
The synthetic strategy reported in this paper improves
on yield, reaction time, and purification compared to
reported approaches.13,19
During our design of novel derivatives of chalcones, as
analogs of the combretastatins, with tubulin binding
and antitumor properties, it was apparent that hetero-
cyclic derivatives of chalcones had not been systemati-
cally investigated for this purpose. Eleven chalcone
epoxide precursors (1–11), and the fifteen 3,5-diarylpy-
razoles (12–26), were synthesized and studied to estab-
lish the cytotoxicity. These compounds were designed
to test any structure–activity relationships arising from
the number and position of methoxy groups in the A-
and B-rings, and the effect of a nitro versus an amino
group on cytotoxicity. Both classes of compounds were
assessed for biological activity against the growth of
two murine cell lines (B16 and L1210) grown in cul-
ture. Several 3,5-diarylpyrazoles have been prepared
and studied as ligands in transition metal chemistry18
and some have been proposed to have herbicidal prop-
erties.19 Substituted 3,5-diarylpyrazole compounds are
particularly intersting as they can be readily prepared
from their corresponding chalcones, via the corre-
sponding chalcone epoxide, di- or a-bromochal-
cones.19,20 They can also be synthesized by 1,3-
dipolar cycloadditions of diazo compounds generated
in situ.21
Several attempts were made to prepare the amino epox-
ide compounds by reduction of the nitro precursors (10,
11, 23, 25) with stannous chloride dihydrate (Scheme 2).
In all cases, the amino products were unstable and
decomposed during the work-up procedures. The amin-
opyrazoles (19, 21, 24, 26) were prepared in 78%–95%
yields by atmospheric pressure catalytic hydrogenation
over 5% palladium-on-carbon (Scheme 2). The struc-
tures of all compounds described in this study were
1
characterized using FT-IR, 500 MHz H NMR, mass
spectrometry, and accurate mass measurements.
Crystals of epoxides 3 and 7, and pyrazole 17 were ob-
tained following recrystallization from hexane/ethyl ace-
tate and their structures were unequivocally confirmed
Scheme 1. Synthesis of epoxides and pyrazoles from the starting chalcones. Reagents and conditions: (i) K2CO3, MeOH, room temperature, H2O2
(ii) Hydrazine hydrate, p-toluenesulfonic acid, xylenes, D.

R. LeBlanc et al. / Bioorg. Med. Chem. 13 (2005) 6025–6034
Table 1. Cytotoxicity data for compounds 1–26
6027
IC50 (lM)
B16
L1210
B =
>100 (1)
55 (12)
40 (1)
15 (12)
25 (2)
38 (13)
46 (14)
5 (2)
24 (13)
30 (14)
>100 (3)
>100 (3)
>100 (4)
>100 (5)
40 (15)
35 (16)
45 (4)
81 (5)
40 (15)
24 (16)
38 (6)
34 (17)
32 (6)
24 (17)
29 (7)
>100 (18)
>100 (19)
3.9 (7)
>100 (18)
43 (19)
N/Aa
N/Aa
31 (8)
32 (20)
5 (21)
5.3 (8)
37 (20)
2.4 (21)
N/Aa
N/Aa
59 (9)
50 (22)
>100 (9)
22 (22)
>100 (10)
>100 (23)
40 (24)
>100 (10)
>100 (23)
35 (24)
N/Aa
N/Aa
>100 (11)
>100 (25)
>100 (26)
>100 (14)
>100 (25)
>100 (26)
N/Aa
N/Aa
a Compounds not tested as the epoxide–amine could not be synthesized due to decomposition of the amine before isolation.

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R. LeBlanc et al. / Bioorg. Med. Chem. 13 (2005) 6025–6034
Scheme 2. Synthesis of amino compounds. Reagents: (i) SnCl2Æ2H2O, EtOAc, D. (ii) H2, 5% Pd/C, THF.
Figure 2. X-ray structures of epoxides, 7 (A) and 3 (B), and pyrazole 17, aerial view (C) and side view (D).
by single crystal X-ray analysis (Fig. 2). The X-ray struc-
ture of compound 17 affirmed the high degree of coplan-
arity between the aromatic rings. This conformation of
pyrazole 17 is in agreement with an MM2 (Cache 3.9)
optimized structure of pyrazole 16 (Fig. 3), which differ
by one methoxy group (five cf. six). The coplanar con-
Figure 3. MM2 structures of chalcone 27 and pyrazole 16. The calculations were performed using CAChe 3.9 software and augmented MM2
parameters. Optimization of each structure was accomplished using a conjugate gradient procedure and a convergence to 0.001 kcal molꢀ1. The
cytotoxicity IC50 values for both compounds with B16 and L1210 cells are given in the figure.

R. LeBlanc et al. / Bioorg. Med. Chem. 13 (2005) 6025–6034
6029
formation of compounds 16 and 17 is dramatically dif-
ferent from the angular shape of CA-4.22 Interestingly,
the MM2 optimized structure of the pentamethoxychal-
cone 27, which exhibits appreciable cytotoxicity, also
adopts an angular conformation (Fig. 3), similar to
CA-4. The difference in conformation between the pla-
nar pyrazole compounds 16 and 17 from angular CA-4
and chalcone 27 could influence their ability to bind to
tubulin and inhibit cell growth, assuming the former
compounds to have a similar mechanism of action as
the chalcones.23
nitro-pyrazole 20 (cf. > 10012–32 lM, respectively).
However, the amino-chalcone 29 (Fig. 4) is highly cyto-
toxic and it has an IC50 of 0.24 lM12 in B16 cells, com-
pared to lower activity for amino pyrazole 21 (IC50 of
5 lM). A similar relationship can be observed if chal-
cones 30 and 31 are considered (Fig. 4), when compared
with their respective pyrazoles 18 and 19 (Table 1). This
difference in cytotoxicity could be related to their con-
formation; the pyrazole being coplanar and the chal-
cones twisted. Guided by the shape of CA-4 and its
conformational relationship to colchicine,27 it is reason-
able to suggest that the chalcones are more cytotoxic
than the pyrazoles because they prefer a twisted shape,
which allows them to fit more snugly into the binding
site of tubulin.23 These results indicate that shape should
be an important factor in the design of future molecules.
Despite their angular conformation, and in contrast to
the relevant chalcones, the epoxides were less cytotoxic
than their corresponding pyrazoles. For example, epox-
ide 5 was 3–4 times less active than pyrazole 16, which in
turn was 5–15 times less active than the relevant
chalcone.
2.2. Cytotoxicity
The epoxides and pyrazoles were subjected to cytotoxic-
ity testing to determine if a structure–activity relation-
ship could be identified. In vitro cytotoxicity
experiments were performed on all synthesized com-
pounds using a 72 h continuous exposure MTT assay24
with murine melanoma and leukemia cells (B16 and
L1210, respectively). Ninety-six-well plates were treated
with the compound of interest dissolved firstly in
DMSO, at concentrations of 1.75 · 10ꢀ2 M, then diluted
1 in 10 with media to final concentrations of 1.0 · 10ꢀ4
–
When an amino group is present in the meta position of
the B-ring, increased activity is seen, in both cell lines,
compared with an amine in the para position for the tri-
methoxy compounds [e.g., IC50 = >100 and 43 vs 5 and
2.4 lM for pyrazoles 19 and 21 in B16 and L1210,
respectively (Table 1)]. The same trend can be observed
for the dimethoxy compounds [e.g., IC50 = >100 vs 40
and 35 lM for pyrazoles 26 and 24 in B16 and L1210,
respectively (Table 1)]. This finding correlates with the
potency of CA-4 which has a hydroxy group in the meta
position25 and AC-7739 that also has an amino group in
the meta position.3,28
1.0 · 10ꢀ12 M. Concentrations of compounds that
inhibited the growth of tumor cells by 50% relative to
an untreated control, or IC50 (lM) values, for both cell
lines are shown in Table 1 (compounds 1–26). The re-
sults show that for both the pyrazoles and the epoxides
(in both cell lines) the compounds containing three
methoxy groups in the A-ring are generally more active
than those containing two methoxy groups [e.g., epoxide
6 and pyrazole 16 vs epoxide 9 and pyrazole 21
(Table 1)]. This discovery is consistent with the activity
of the combretastatins, such as CA-4 (IC50 = 7 lM in
L1210 cells),25 which also has three similarly positioned
methoxy substituents in the A-ring.26 Comparison of the
cytotoxicity of CA-4 with the L1210 data shown in Ta-
ble 1 indicates that several of the epoxides are more ac-
tive in these cells; namely compounds 2, 7, and 8. From
the pyrazole series compound 21 is the most active with
an IC50 of 2.4 lM. The data highlights that both series
of compounds are worthy of further investigation.
Consistent with the chalcone series,12 increased cytotox-
icity is observed when the nitro groups of the epoxides
and pyrazoles are reduced to the amines. However,
when the amine is present in the para position no change
in activity is observed for the 20,50-dimethoxy com-
pounds (IC50 = >100 lM for both 25 and 26 in both cell
lines, Table 1). When the 30,40,50-trimethoxy compounds
are considered, no change in activity is seen in the B16
cell line (IC50 = >100 lM for 18 and 19, Table 1), how-
ever, in the L1210 cell line the activity of the amine com-
pound 19 is increased compared to the nitro, 18
(IC50 = 43 and 100 lM, respectively). In general the
L1210 cell line appears more sensitive to these two clas-
ses of compounds when compared with the B16 results.
This was particularly evident within the epoxides, for
example, an epoxide 2 with a 300-methoxy group
Amino chalcones are in general more active than the
corresponding pyrazoles.12 For example, the IC50 values
of pyrazole 16 for B16 and L1210 cancer cells were 35
and 24 lM, respectively. In contrast, the cytotoxicity
of corresponding chalone 27 (Fig. 3) against the same
cancer cells was 4.6 and 2.6 lM, respectively.12 More-
over, if B16 cells were considered, nitro-chalcone 28
(Fig. 4) shows less activity than the corresponding
Figure 4. Structures and cytotoxicity of representative chalcones, 28–31. The respective IC50 values for B16 cells are given inside the parentheses.

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R. LeBlanc et al. / Bioorg. Med. Chem. 13 (2005) 6025–6034
produced IC50 values of 25 and 5 lM for B16 and
L1210. This difference in activity between cell lines could
be due to the nature of the cells. In general leukemia
cells (e.g., L1210) are more sensitive to antiproliferative
agents than solid tumor cells (B16) which are known to
be chemoresistant, shown in the literature with other
anticancer agents, particularly the duocarmycins.29
pounds were purified by silica gel column chromatogra-
phy using a 10%–30% gradient ethyl acetate/petroleum
ether solvent.
3.2.1.
2,3-Epoxy-1-(30,40,50-trimethoxyphenyl)-3-(200-
methoxyphenyl)propanone (1). White solid (210 mg,
67%), mp 124–126 ꢁC: 1H NMR (CDCl3) d 3.84 (s,
3H), 3.90 (s, 6H), 3.93 (s, 3H), 4.11 (d, 1H,
J = 1.95 Hz), 4.40 (d, 1H, J = 1.95 Hz), 6.90–6.93 (m,
1H), 6.98–7.04 (m, 1H), 7.29–7.32 (m, 2H), 7.34 (s,
2H); IR (KBr) m 2936, 2833, 1679, 1581, 1498, 1456,
The significant enhancement in the activity of amino
compounds over their respective nitro counterparts
could be useful in the quest for bioreductively active
prodrugs. The nitro group could be reduced in vivo to
the amino compound, reducing problems with stability
and potential nonselective toxicity. For example,
CB1954, a nitro-compound substrate for DT-diapho-
rase is reduced in vivo and is active against the growth
of cancer cells.30 Similarly, the nitro moiety of a 4-
nitrobenzyloxycarbonyl prodrug of seco-cyclopropy-
lindoline (CI) of CC-1065 was effectively reduced by
nitroreductase, thereby releasing the active drug.31
1415, 1342, 1249, 1160, 1124, 1020, 1000, 756 cmꢀ1
;
MS (EI) m/z (rel intensity) 344 (M+, 36). 195 (100).
3.2.2.
2,3-Epoxy-1-(30,40,50-trimethoxyphenyl)-3-(300-
methoxyphenyl)propanone (2). White solid (280 mg,
1
89%), mp 79–81 ꢁC: H NMR (CDCl3) d 3.83 (s, 3H),
3.89 (s, 3H), 3.93 (s, 3H), 4.08 (d, 1H, J = 1.8 Hz),
4.18 (d, 1H, J = 1.8 Hz), 6.86–6.95 (m, 3H), 7.25 (s,
2H), 7.26–7.29 (m, 1H); IR (KBr) m 3620, 3536, 2939,
2834, 1678, 1583, 1494, 1458, 1416, 1337, 1227, 1164,
1128, 1038, 1002, 885 cmꢀ1; MS (EI) m/z (rel intensity)
344 (M+, 50), 195 (100).
3. Experimental
3.1. Synthesis
3.2.3.
2,3-Epoxy-1-(30,40,50-trimethoxyphenyl)-3-(400-
methoxyphenyl)propanone (3). Clear oil (170 mg, 54%):
1H NMR (CDCl3) d 3.82 (s, 3H), 3.88 (s, 6H), 3.92 (s,
3H), 4.04 (d, 1H, J = 1.5 Hz), 4.20 (d, 1H, J = 1.5 Hz),
7.00 (d, 2H, J = 9.0 Hz), 7.30 (s, 2H), 7.83 (d, 2H,
J = 9.0 Hz); IR (KBr) m 3007, 2941, 2838, 1714, 1678,
1585, 1510, 1461, 1415, 1333, 1252, 1168, 1126, 1029,
1001, 832, 760 cmꢀ1; MS (EI) m/z (rel intensity) 344
(M+, 55), 195 (100).
Solvents and organic reagents were purchased from
Aldrich or Fisher, and were used without further purifi-
cation. Melting points (mp) were performed using a
Mel-temp instrument and are uncorrected. Infrared
(IR) spectra were recorded using a Perkin-Elmer Para-
gon 500 FT-IR instrument as films on KBr discs, unless
1
otherwise stated. H NMR spectra were obtained using
a Varian Unity Inova 500 instrument unless otherwise
stated. Chemical shifts (d) are reported at 20 ꢁC in parts
per million (ppm) downfield from internal tetramethylsi-
lane (Me4Si). High-resolution mass spectra (HRMS)
and low-resolution mass spectra (LRMS) were provided
by the Mass Spectrometry Laboratory, University of
South Carolina, Columbia. Reaction progress was as-
sessed by thin-layer chromatography (TLC) using
Merck silica gel (60 F254) on aluminium plates unless
otherwise stated. Visualisation was achieved with UV
light at 254 nm and/or 366 nm, I2 vapor staining, and
ninhydrin spray.
3.2.4. 2,3-Epoxy-1-(30,40,50-trimethoxyphenyl)-3-(200,500-
dimethoxyphenyl)propanone (4). White crystalline solid
(516 mg, 82%), mp 81 ꢁC: H NMR (CDCl3) d 3.79 (s,
3H), 3.80 (s, 3H), 3.89 (s, 3H), 3.93 (s, 3H), 4.08 (d,
1H, J = 2.0 Hz), 4.38 (d, 1H, J = 2.0 Hz), 6.85 (s, 1H),
6.86 (s, 1H), 6.88 (s, 1H), 7.26 (s, 1H), 7.33 (s, 1H); IR
(KBr) m 2999, 2936, 2833, 1679, 1586, 1503, 1461,
1415, 1337, 1269, 1217, 1160, 1124, 1041, 1020, 880,
808, 735 cmꢀ1; MS (EI) m/z (rel intensity) 374 (M+,
46), 195 (100).
1
3.2.5. 2,3-Epoxy-1-(30,40,50-trimethoxyphenyl)-3-(300,400-
dimethoxyphenyl)propanone (5). Yellow oil (461 mg,
3.2. General preparation of substituted chalcone epoxides
(1–11)
1
74%): H NMR (CDCl3) d 3.88 (s, 3H), 3.89 (s, 3H),
3.92 (s, 3H), 3.94 (s, 3H), 3.96 (s, 3H), 4.04 (d, 1H,
J = 1.95 Hz), 4.17 (d, 1H, J = 1.95 Hz), 6.83–6.99 (m,
2H), 7.25 (s, 2H), 7.40–7.47 (m, 1H); IR (KBr) m 2999,
2936, 2833, 1679, 1586, 1513, 1461, 1415, 1332, 1269,
1238, 1124, 1020, 859, 808, 756 cmꢀ1; MS (EI) m/z (rel
intensity) 374 (M+, 42), 195 (100); HRMS [M+] for
C20H27O7: obsd 374.1536, calcd 374.1566.
Powdered K2CO3 (332 mg, 3 mol equiv) was added to a
suspension of the required chalcone (300 mg, 1 mol
equiv) in MeOH (30 cm3), followed by excess aqueous
hydrogen peroxide (35%, 1 cm3, 10 mol equiv); added
over 10 min. The mixture was stirred at room tempera-
ture for 3 h and reaction progress was monitored by
TLC (30:70 v/v EtOAc/hexanes). Upon completion,
the MeOH was removed under reduced pressure and
the resulting residue dissolved in CH2Cl2 (50 cm3), and
washed with H2O (20 cm3). The organic phase was sep-
arated, dried (Na2SO4) and the solvent removed under
reduced pressure to yield the corresponding epoxide.
The compounds were generally pure by TLC and
500 MHz 1H NMR analyses. If required, the com-
3.2.6.
2,3-Epoxy-1-(30,40,50-trimethoxyphenyl)-3-
(300,400,500-trimethoxyphenyl)propanone (6). Purified by
flash column chromatography using silica (30:70 v/v
EtOAc–petroleum ether). Yellow solid (416 mg, 80%),
mp 83 ꢁC: 1H NMR (CDCl3) d 3.82 (s, 3H), 3.84
(s, 6H), 3.86 (s, 3H), 3.90 (s, 3H), 4.02 (d, 1H,
J = 1.5 Hz), 4.12 (d, 1H, J = 1.5 Hz), 6.57 (s, 4H); IR

R. LeBlanc et al. / Bioorg. Med. Chem. 13 (2005) 6025–6034
6031
(KBr) m 3005, 2941, 2837, 1681, 1588, 1504, 1462, 1416,
3.3. General preparation of 3,5-diarylpyrazoles (12–18,
20, 22, 23, 25)
1327, 1236, 1165, 1127, 1003, 884, 855, 755 cmꢀ1; MS
(EI) m/z (rel intensity) 404 (M+, 63), 195 (100); HRMS
[M+] for C21H24O8: obsd 404.1469, calcd 404.1471.
The required epoxide (170 mg, 1 mol equiv) was dis-
solved in xylenes (4 cm3). CH2Cl2 (2 cm3) was added,
if required, to achieve dissolution. p-Toluenesulfonic
acid monohydrate (22 mg) and hydrazine hydrate
(0.23 cm3, 3 mol equiv) were then added to the epoxide
solution. The reaction mixture was stirred under reflux-
ing conditions for 3 h until a yellow precipitate formed.
The xylenes were removed under reduced pressure and
the obtained solid washed with hexanes and air dried
for 12–14 h to yield the corresponding pyrazole com-
3.2.7.
2,3-Epoxy-1-(30,40,50-trimethoxyphenyl)-3-(400-
nitrophenyl)propanone (7). Yellow solid (462 mg,
78%), mp 140 ꢁC: 1H NMR (CDCl3) d 3.91 (s, 6H),
3.95 (s, 3H), 4.18 (d, 1H, J = 1.75 Hz), 4.23 (d, 1H,
J = 1.75 Hz), 7.30 (s, 2H), 7.56 (d, 2H, J = 8.25 Hz),
8.28 (d, 2H, J = 8.25 Hz); IR (KBr) m 3397, 2936,
2843, 1679, 1583, 1520, 1456, 1415, 1342, 1162,
1126, 994, 849 cmꢀ1; MS (EI) m/z (rel intensity) 359
(M+, 55), 195 (100); HRMS [M+] for C18H17NO7:
obsd 359.1010, calcd 359.1005.
1
pound. Analyses by TLC and 500 MHz H NMR con-
firmed their purity. However, in a few cases the
products were purified by flash column chromatography
using silica gel (2:98 v/v MeOH–CHCl3).
3.2.8.
2,3-Epoxy-1-(30,40,50-trimethoxyphenyl)-3-(400-
methoxy-300-nitrophenyl)propanone (8). Yellow solid
1
3.3.1. 3-(30,40,50-Trimethoxyphenyl)-5-(200-methoxyphe-
nyl)pyrazole (12). Yellow solid (131 mg, 63%), mp 78–
(0.23 g, 74%), mp 148 ꢁC: H NMR (CDCl3) d 3.90 (s,
6H), 3.94 (s, 3H), 3.99 (s, 3H), 4.10 (d,
1H,J = 1.80 Hz), 4.17 (d, 1H, J = 1.80 Hz), 7.13 (d,
1H, J = 8.7 Hz), 7.26 (s, 2H), 7.56 (dd, 1H, J = 8.7 Hz,
2.4 Hz), 7.85 (d, 1H, J = 2.4 Hz); IR (KBr) m 2999,
2943, 2840, 1679, 1622, 1582, 1534, 1503, 1459, 1416,
1346, 1278, 1163, 1126, 1005, 882, 818, 756 cmꢀ1; MS
(EI) m/z (rel intensity) 389 (M+, 35), 195 (100); HRMS
[M+] for C19H19NO8: obsd 389.1106, calcd 389.1111.
1
81 ꢁC: H NMR (CDCl3) d 3.89 (s, 3H), 3.95 (s, 6H),
4.01 (s, 3H), 6.90 (s, 1H), 7.03–7.10 (m, 2H), 7.11 (s,
2H), 7.31–7.37 (m, 1H), 7.74–7.77 (m, 1H); IR (KBr) m
3195, 2934, 2836, 1899, 1584, 1488, 1466, 1438, 1427,
1381, 1299, 1237, 1176, 1121, 1020, 1000, 852,
751 cmꢀ1; MS (EI) m/z (rel intensity) 340 (M+, 100).
3.3.2. 3-(30,40,50-Trimethoxyphenyl)-5-(300-methoxyphe-
nyl)pyrazole (13). Yellow semisolid (148 mg, 83%): H
3.2.9. 2,3-Epoxy-1-(20,50-dimethoxyphenyl)-3-(300,400,500-
trimethoxyphenyl)propanone (9). White solid (250 mg,
78%), mp 130–132 ꢁC: 1H NMR (CDCl3) d 3.61 (s, 3H),
3.81 (s, 3H), 3.86 (s, 3H), 3.88 (s, 6H), 3.96 (d, 1H,
J = 1.8 Hz), 4.28 (d, 1H, J = 1.8 Hz), 6.61 (s, 2H), 6.88
(d, 1H, J = 9.0 Hz), 7.09 (dd, 1H, J = 9.0, 3.3 Hz), 7.35
(d, 1H, J = 3.3 Hz); IR (KBr) m 2995, 2941, 2835, 1663,
1589, 1491, 1454, 1418, 1276, 1265, 1230, 1221, 1189,
1
NMR (CDCl3) d 3.62 (s, 3H), 3.66 (s, 3H), 3.82 (s,
3H), 6.63 (s, 1H), 6.74–6.78 (m, 1H), 6.85 (s, 2H),
7.12–7.26 (m, 3H); IR (KBr) m 3107, 3000, 2938, 2835,
1590, 1494, 1466, 1430, 1391, 1314, 1233, 1170, 1123,
1039, 1001, 858, 729 cmꢀ1; MS (ES+) m/z (rel intensity)
340 (M+, 100); HRMS [M+] for C19H20N2O4: obsd
340.1425, calcd 340.1423.
1168, 1121, 1022, 1005, 978, 912, 882, 819, 725 cmꢀ1
;
MS (EI) m/z (rel intensity) 374 (M+, 45), 165 (100).
3.3.3. 3-(30,40,50-Trimethoxyphenyl)-5-(400-methoxyphe-
nyl)pyrazole (14). Light yellow solid (80 mg, 48%), mp
3.2.10. 2,3-Epoxy-1-(20,50-dimethoxyphenyl)-3-(400-meth-
oxy-300-nitrophenyl)propanone (10). White-yellow pow-
der (177 mg, 67%), mp 179 ꢁC: 1H NMR (CDCl3) d
3.65 (s, 3H), 3.82 (s, 3H), 3.99 (s, 3H), 4.01 (d, 1H,
J = 1.8 Hz), 4.32 (d, 1H,J = 1.8 Hz), 6.09 (d, 1H,
J = 9.0 Hz), 7.11 (dd, 1H, J = 9.0 Hz, 3.3 Hz), 7.13 (d,
1H, J = 8.8 Hz), 7.35 (d, 1H, J = 3.3 Hz), 7.57 (dd, 1H,
J = 8.8 Hz, 2.3 Hz), 7.88 (d, 1H, J = 2.3 Hz); IR (KBr)
m 3061, 2939, 2833, 1672, 1609, 1577, 1541, 1498, 1452,
1418, 1353, 1268, 1226, 1168, 1041, 1018, 888, 836,
730 cmꢀ1; MS (EI) m/z (rel intensity) 359 (M+, 27).
165 (100); HRMS [M+] for C18H17NO7: obsd
359.1008, calcd 359.1005.
1
230 ꢁC: H NMR (CDCl3) d 3.84 (s, 3H), 3.85 (s, 3H),
3.86 (s, 6H), 6.84 (s, 1H), 6.98 (d, 1H, J = 8.5 Hz),
7.02 (s, 2H), 7.14 (d, 1H, J = 7.5 Hz), 7.69 (d, 1H,
J = 8.5 Hz), 7.72 (d, 1H, J = 8.5 Hz); IR (KBr) m 3392,
2948, 1171, 1459, 1376, 1215, 1167, 761 cmꢀ1; MS (EI)
m/z (rel intensity) 340 (M+, 100).
3.3.4. 3-(30,40,50-Trimethoxyphenyl)-5-(200,500-dimethoxy-
phenyl)pyrazole (15). Yellow solid (81 mg, 53%), mp
1
99 ꢁC: H NMR (CDCl3) d 3.84 (s, 3H), 3.88 (s, 3H),
3.89 (s, 3H), 3.95 (s, 6H), 6.82 (d, 1H, J = 8.5 Hz),
6.87 (s, 1H), 6.96 (d, 1H, J = 8.5 Hz), 7.11 (s, 2H),
7.69 (s, 1H); IR (KBr) m 3341, 2999, 2936, 2833, 1622,
1591, 1508, 1461, 1264, 1233, 1124, 1020, 808,
756 cmꢀ1; MS (EI) m/z (rel intensity) 370 (M+, 100).
3.2.11. 2,3-Epoxy-1-(20,50-dimethoxyphenyl)-3-(400-nitro-
phenyl)propanone (11). White-yellow powder (735 mg,
87%), mp 152 ꢁC: 1H NMR (CDCl3) d 3.60 (s, 3H),
3.82 (s, 3H), 4.11 (d, 1H, J = 1.8 Hz), 4.31 (d, 1H,
J = 1.8 Hz), 6.89 (d, 1H, J = 9.0 Hz), 7.13 (dd, 1H,
J = 9.0 Hz, 3.1 Hz), 7.36 (d, 1H, J = 3.1 Hz), 7.56 (d,
2H, J = 8.7 Hz), 8.27 (d, 2H, J = 8.7 Hz); IR (KBr) m
3082, 2936, 2833, 1677, 1604, 1517, 1496, 1463, 1416,
3.3.5. 3-(30,40,50-Trimethoxyphenyl)-5-(300,400-dimethoxy-
phenyl)pyrazole (16). Yellow solid (96 mg, 78%), mp
1
68 ꢁC: H NMR (CDCl3) d 3.65 (s, 6H), 3.66 (s, 3H),
3.82 (s, 3H), 3.84 (s, 3H), 6.56 (s, 1H), 6.68–6.71 (m,
1H), 6.87 (s, 2H), 7.14–7.18 (m, 2H); IR (KBr) m 3331,
3123, 3009, 2936, 2833, 1586, 1508, 1467, 1430, 1249,
1129, 1025, 865, 756 cmꢀ1; MS (EI) m/z (rel intensity)
1346, 1278, 1224, 1169, 1044, 893, 840, 804, 729 cmꢀ1
;
MS (EI) m/z (rel intensity) 329 (M+, 43), 165 (100).

6032
R. LeBlanc et al. / Bioorg. Med. Chem. 13 (2005) 6025–6034
370 (M+, 100); HRMS [M+] for C20H22N2O5: obsd
370.1523, calcd 370.1529.
(M+, 100); HRMS [M+] for C18H17N3O5: obsd
355.1170, calcd 355.1168.
3.3.6. 3-(30,40,50-Trimethoxyphenyl)-5-(300,400,500-trimeth-
oxyphenyl)pyrazole (17). Brown-yellow solid (82 mg,
43%), mp 200 ꢁC: H NMR (CDCl3) d 3.73 (s, 12H),
3.4. General preparation of aminopyrazoles (19, 21, 24,
26)
1
3.85 (s, 6H), 6.59 (s,1H), 6.90 (s, 4H); IR (KBr) m
3130, 3005, 2938, 2834, 1591, 1503, 1472, 1429, 1389,
1328, 1241, 1187, 1166, 1128, 1005, 864, 838,
754 cmꢀ1; MS (EI) m/z (rel intensity) 400 (M+, 100);
HRMS [M+] for C21H24N2O6: obsd 400.1636, calcd
400.1634.
A suspension of a nitro-3,5-diarylpyrazole (20 mg) and
5% Pd/C (10 mg) in THF was hydrogenated at atmo-
spheric pressure and room temperature for 4 h. The cat-
alyst was removed by filtration over Celite and the
desired products were obtained by concentration of
1
the filtrate. TLC and 500 MHz H NMR analyses indi-
cated that the compounds were homogeneous.
3.3.7.
3-(30,40,50-Trimethoxyphenyl)-5-(400-nitrophen-
yl)pyrazole (18). Yellow solid (174 mg, 64%), mp
3.4.1.
5-(400-Aminophenyl)-3-(30,40,50-trimethoxyphe-
1
192 ꢁC: H NMR (CDCl3) d 3.86 (s, 6H), 3.88 (s, 3H),
nyl)pyrazole (19). Tan solid (20 mg, 95%), mp 90 ꢁC:
1H NMR (CDCl3) d 3.89 (s, 3H), 3.95 (s, 6H), 6.68 (s,
1H), 6.76 (d, 2H, J = 8.5 Hz), 7.01 (s, 2H), 7.46 (d,
2H, J = 8.5 Hz); IR (KBr) m 2999, 2935, 1619, 1589,
1506, 1465, 1368, 1238, 1179, 1126, 1001, 833,
756 cmꢀ1; MS (EI) m/z (rel intensity) 325 (M+, 100);
HRMS [M+] for C18H19N3O3: obsd 325.1430, calcd
325.1426.
6.85 (s, 2H), 6.87 (s, 1H), 7.91 (d, 2H, J = 8.7 Hz),
8.23 (d, 2H, J = 8.7 Hz); IR (KBr) m 3310, 2925, 2843,
1592, 1517, 1464, 1415, 1341, 1240, 1126, 1000, 855,
754 cmꢀ1; MS (EI) m/z (rel intensity) 355 (M+, 100);
HRMS [M+] for C18H17N3O5: obsd 355.1170, calcd
355.1168.
3.3.8. 3-(30,40,50-Trimethoxyphenyl)-5-(400-methoxy-300-
nitrophenyl)pyrazole (20). Yellow solid (0.10 g, 60%),
3.4.2.
methoxyphenyl)pyrazole
5-(300-Amino-400-methoxyphenyl)-3-(30,40,50-tri-
(21). Yellow semisolid
1
mp 82 ꢁC: H NMR (DMSO-d6) d 3.72 (s, 3H), 3.84
1
(s, 6H), 3.92 (s, 3H), 6.85 (s, 1H), 7.00 (s, 2H), 7.16 (d,
1H, J = 9 Hz), 7.98 (dd, 1H, J = 9 Hz, 2.1 Hz), 8.22 (d,
1H, J = 2.1 Hz); IR (KBr) m 3009, 2936, 2833, 1621,
(22.6 mg, 78%): H NMR (CDCl3) d 3.90 (s, 3H), 3.91
(s, 3H), 3.95 (s, 6H), 6.69 (s, 1H), 6.73–6.75 (m, 1H),
6.84–6.86 (m, 1H), 7.01 (s, 2H), 7.03–7.07 (m, 1H); IR
(KBr) m 3368, 2949, 2834, 1620, 1589, 1510, 1458,
1227, 1122, 1028, 907 cmꢀ1; MS (EI) m/z (rel intensity)
355 (M+, 100); HRMS [M+] for C19H21N3O4: obsd
355.1538, calcd 355.1532.
1581, 1531, 1462, 1353, 1276, 1126, 1009, 755 cmꢀ1
;
MS (EI) m/z (rel intensity) 385 (M+, 100); HRMS
[M+] for C19H19N3O6: obsd 385.1273, calcd 385.1274.
3.3.9. 3-(20,50-Dimethoxyphenyl)-5-(300,400,500-trimethoxy-
phenyl)pyrazole (22). Yellow semisolid (225 mg, 94%):
1H NMR (CDCl3) d 3.85 (s, 3H), 3.89 (s, 3H), 3.96 (s,
6H), 3.97 (s, 3H), 6.86 (s, 1H), 6.87 (dd, 1H, J = 9.0,
3.0 Hz), 6.94 (d, 1H, J = 9.0 Hz), 7.11 (s, 2H), 7.27 (d,
1H, J = 3.0 Hz); IR (KBr) m 3714, 2247, 3002, 2939,
2834, 2352, 1589, 1500, 1463, 1227, 1122, 1038,
1002 cmꢀ1; MS (EI) m/z (rel intensity) 370 (M+, 100);
HRMS [M+] for C20H22N2O5: obsd 370.1529, calcd
370.1529.
3.4.3. 5-(300-Amino-400-methoxyphenyl)-3-(20,50-dimeth-
oxyphenyl)pyrazole (24). Tan solid (12 mg, 94%), mp
73 ꢁC: H NMR (CDCl3) d 3.85 (s, 3H), 3.90 (s, 3H),
3.96 (s, 3H), 6.83–6.89 (m, 4H), 6.96–6.98 (m, 2H),
7.22–7.24 (m, 1H); IR (KBr) m 2931, 2843, 1617, 1498,
1461, 1254, 1227, 1171, 1025, 909, 802, 735 cmꢀ1; MS
(EI) m/z (rel intensity) 325 (M+, 100); HRMS [M+] for
C18H19N3O3: obsd 325.1430, calcd 325.1426.
1
3.4.4. 5-(400-Aminophenyl)-3-(20,50-dimethoxyphenyl)pyr-
azole (26). Brown-yellow solid (24 mg, 88%), mp 115 ꢁC:
1H NMR (CDCl3) d 3.85 (s, 3H), 3.98 (s, 3H), 6.75 (d,
2H, J = 8.5 Hz), 6.84 (s, 1H), 6.90 (dd, 1H, J = 8.5,
3.0 Hz), 6.97 (d, 1H, J = 8.5 Hz), 7.27 (d, 1H,
J = 3.0 Hz), 7.66 (d, 2H, J = 8.5 Hz); IR (KBr) m 2962,
2843, 1619, 1497, 1453, 1262, 1227, 1179, 1088, 1043,
910, 803, 734 cmꢀ1; MS (EI) m/z (rel intensity) 295
(M+, 100).
3.3.10.
3-(20,50-Dimethoxyphenyl)-5-(400-methoxy-300-
nitrophenyl)pyrazole (23). Brown solid (183 mg, 83%),
1
mp 57 ꢁC: H NMR (CDCl3) d 3.86 (s, 3H), 3.98 (s,
3H), 4.01 (s, 3H), 6.91 (s, 1H), 7.15 (d, 1H,
J = 8.7 Hz), 8.08 (dd, 1H, J = 8.7 Hz, 2.3 Hz), 8.33 (d,
1H, J = 2.3 Hz); IR (KBr) m 2923, 2843, 1622, 1537,
1493, 1456, 1351, 1279, 1226, 1176, 1041, 1019, 805,
745 cmꢀ1; MS (EI) m/z (rel intensity) 355 (M+, 100);
HRMS [M+] for C18H17N3O5: obsd 355.1170, calcd
355.1168.
4. X-ray crystallography
3.3.11. 3-(20,50-Dimethoxyphenyl)-5-(400-nitrophenyl)pyr-
azole (25). Brown solid (161 mg, 83%), mp 184 ꢁC: H
NMR (CDCl3) d 3.86 (s, 3H), 4.00 (s, 3H), 6.91 (dd,
1H, J = 8.8, 3.0 Hz), 7.00 (d, 1H, J = 8.8 Hz), 7.01 (s,
1H), 7.26 (d, 1H, J = 3.0 Hz), 8.04 (d, 2H, J = 8.5 Hz),
8.30 (d, 2H, J = 8.5 Hz); IR (KBr) m 2999, 2940, 2833,
1601, 1515, 1338, 1226, 1176, 1109, 1041, 984, 953,
854, 797, 750 cmꢀ1; MS (EI) m/z (rel intensity) 355
1
The molecular structure of derivative 17, C21H24N2O6,
was determined by X-ray diffraction methods. The sub-
stance crystallizes in the monoclinic space group P21
˚
˚
˚
with a = 14.950(3) A, b = 43.390(9) A, c = 14.980(3) A,
b = 116.76(3)ꢁ, and Z = 16 refined to an agreement R1
factor of 0.0862. The hydrogen atoms were positioned
in ideal positions and refined with the riding model.

R. LeBlanc et al. / Bioorg. Med. Chem. 13 (2005) 6025–6034
6033
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Intramolecular bond distances and angles, along with
other crystallographic data, are given as supporting
information. The structure of compound 17 has been
deposited in the Cambridge Crystallographic Data Cen-
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compounds 3 and 7 were determined using a similar
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