Cis -restricted 3-aminopyrazole analogues of combretastatins: Synthesis from plant polyalkoxybenzenes and biological evaluation in the cytotoxicity and phenotypic sea urchin embryo assays

Article abstract of DOI:10.1021/np400310m

We have synthesized a series of novel cis-restricted 4,5-polyalkoxydiaryl- 3-aminopyrazole analogues of combretastatins via short synthetic sequences using building blocks isolated from dill and parsley seed extracts. The resulting compounds were tested in vivo in the phenotypic sea urchin embryo assay to reveal their antimitotic and antitubulin effects. The most potent aminopyrazole, 14a, altered embryonic cell division at 10 nM concentration, exhibiting microtubule-destabilizing properties. Compounds 12a and 14a displayed pronounced cytotoxicity in the NCI60 anticancer drug screen, with the ability to inhibit growth of multi-drug-resistant cancer cells.

Full text of DOI:10.1021/np400310m

Article
pubs.acs.org/jnp
cis-Restricted 3‑Aminopyrazole Analogues of Combretastatins:
Synthesis from Plant Polyalkoxybenzenes and Biological Evaluation
in the Cytotoxicity and Phenotypic Sea Urchin Embryo Assays
Dmitry V. Tsyganov,† Leonid D. Konyushkin,† Irina B. Karmanova,† Sergei I. Firgang,† Yuri A. Strelenko,†
Marina N. Semenova,‡,§ Alex S. Kiselyov,⊥ and Victor V. Semenov*,†
†N. D. Zelinsky Institute of Organic Chemistry, RAS, Leninsky Prospect, 47, 119991, Moscow, Russian Federation
‡Institute of Developmental Biology, RAS, Vavilov Street, 26, 119334, Moscow, Russian Federation
§Chemical Block Ltd., 3 Kyriacou Matsi, 3723 Limassol, Cyprus
⊥ChemDiv, Inc., 6605 Nancy Ridge Drive, San Diego, California 92121, United States
S
* Supporting Information
ABSTRACT: We have synthesized a series of novel cis-restricted 4,5-
polyalkoxydiaryl-3-aminopyrazole analogues of combretastatins via short
synthetic sequences using building blocks isolated from dill and parsley
seed extracts. The resulting compounds were tested in vivo in the phenotypic
sea urchin embryo assay to reveal their antimitotic and antitubulin effects. The
most potent aminopyrazole, 14a, altered embryonic cell division at 10 nM
concentration, exhibiting microtubule-destabilizing properties. Compounds
12a and 14a displayed pronounced cytotoxicity in the NCI60 anticancer drug screen, with the ability to inhibit growth of multi-
drug-resistant cancer cells.
ntimitotic drugs have been successfully used as chemo-
therapeutic agents for the treatment of cancer. The
microvasculature.2,8−10 Despite this promising “dual” therapeu-
tic potential, CA4P was reported to display a number of
considerable dose-limiting side effects.11,12 Several research
teams have attempted to improve the safety−efficacy profile of
this compound class.13,14
Combretastatin analogues exhibit at least three structural
commonalities. These are the trimethoxy ring A, the ring B
substituted at C3 and C4, and a cis-olefin tether of variable
length between the two rings reported to be essential for
structural rigidity and ultimately activity of these compounds
(Figure 1). A number of combretastatin analogues featuring
modified A and B rings and bridge isosteres have been
synthesized and evaluated in biological assays.13−16 Among
numerous reported cyclic analogues of combretastatins, five-
membered heterocycles have been described as active,
nonisomerizable, and metabolically stable isosteres for the cis-
olefin bridge.13,14
A
majority of known antimitotic drugs selectively target micro-
tubules, responsible for the formation of the mitotic spindle,
which is essential for proper chromosomal separation during
cell division. It is generally agreed that agents affecting tubulin
polymerization impair microtubule dynamics and consequently
arrest cells during mitosis.1−4
Natural combretastatins, namely, combretastatins A-2 and A-
4 (Figure 1; CA2, CA4), are antimitotics found in the bark of
It was reported recently that 4,5-diaryl-3-aminopyrazole
derivatives of CA4 showed strong cytotoxicity against five
human cancer cell lines, inhibited purified tubulin polymer-
ization, and caused cell cycle arrest in the G2/M phase.17
However, this study did not elaborate further on (i) the
relationship between the NH2 group position and the resulting
antimitotic activity (e.g., potency of II vs III, Figure 1) and (ii)
the optimal number and position of methoxy groups in ring A.
Moreover, tetramethoxy-, methylenedioxy-, and ethylenediox-
Figure 1. Structures of combretastatins and their 3-aminopyrazole
analogues.
Combretum caffrum Kuntze (Combretaceae).5 It is generally
recognized that these agents bind to the colchicine binding site
of tubulin and affect its assembly.6,7 Several phosphorylated
prodrugs including CA4 disodium phosphate (CA4P, Zybre-
stat) and combretastatin A-1 phosphate (Oxi4503) show
promise in the late stages of clinical trials. Notably, in addition
to affecting tubulin dynamics, they also disrupt tumor
Received: April 25, 2013
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Journal of Natural Products
Article
a
Scheme 1. Synthesis of Precursors for cis-Restricted 4,5-Polyalkoxydiaryl-3-aminopyrazoles
a
Reagents and conditions: (a) KOH, 100 °C, 40 min; ref 18; (b) O3, CHCl3−MeOH−pyridine (80:20:3 v/v), −15 °C, 1−2 h; ref 18; (c)
CO(NH2)2·H2O2, CH3OH, reflux, 1.5 h; ref 18; (d) SOCl2, MeOH, reflux 1 h; (e) NaOH, Cu, H2O, reflux, 27 h; (f) BrCH2CH2Br, K2CO3, DMF,
100−105 °C, 5 h; (g) NaBH4, MeOH, rt, 6−7 h; (h) SOCl2, C6H6, 48−50 °C, 40 min; (i) KCN, CH3CN, 50 °C, 10 h.
a
Scheme 2. Synthesis of 4,5-Polyalkoxydiaryl-3-aminopyrazoles 12 and 14
a
Reagents and conditions: (a) NaH, THF, reflux, 5−6 h; (b) N2H4·2HCl, EtOH, reflux, 12−18 h.
yaryl substituents have not been evaluated. For example, we
have shown previously that CA4, CA2, and ethylenedioxy
analogues of CA2, I (Figure 1), exhibited comparable
antimitotic microtubule-destabilizing activity in the sea urchin
embryo model.18
Considering the literature evidence, studies summarized in
this paper were aimed at (i) the development of robust
synthetic protocols for the synthesis of amino pyrazole
derivatives from natural building blocks and (ii) the assessment
of their structure−activity relationship in vivo using the
phenotypic sea urchin embryo assay. Specifically, both the
number and arrangement of OCH3 substituents on the B ring
and position of the NH2 group in the pyrazole ring were
investigated. Toward this goal, a series of conformationally
restricted 4,5-(polymethoxy)diaryl-3-aminopyrazole analogues
of CA4 and CA2 have been accessed via short synthetic
sequences from easily available dill and parsley extracts.19
RESULTS AND DISCUSSION
■
The targeted diaryl-3-aminopyrazoles I and II were conven-
iently assembled in 18−65% overall yields from the key
intermediate polyalkoxybenzoic acids 4 and respective aromatic
acetonitriles 10 (Scheme 1) following the initial base-mediated
condensation to furnish α-ketonitriles 11 and 13 (Scheme 2).
These were subsequently cyclized into pyrazoles 12 and 14
with N2H4·2HCl.17 Notably, similar reactions conducted with
N2H4·H2O resulted only in trace amounts of 12 and 14.
Hydrazides of the corresponding polymethoxybenzoic acids
B
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a
1
Table 1. H NMR Spectroscopic Data of 3-Aminopyrazoles 12 and 14
position
12a
12b
12c
12d
12e
position
14a
6.58 s
14b
14d
14e
Ring A
2″
Ring A
2′
6.71 s
6.73 d (1.5) 6.61 d (2.1) 3.39 s
3.37 s
6.49 d (1.4) 3.5 s
3.39 s
3″
4″
5″
6″
3.59 s
3.65 s
3.59 s
6.71 s
3.69 s
6.02 s
3.58 s
6.01 s
3.67 s
3.90 s
5.99 s
3′
4′
5′
6′
3.67 s
3.68 s
3.67 s
6.58 s
3.74 s
5.98 s
6.03 s
3.68 s
3.94 s
6.04 s
4.20 m
6.53 d (1.5) 6.50 d (2.1) 6.42 s
6.32 s
6.38 d (1.4) 6.32 s
6.41 s
Ring B
Ring B
2′
3′
7.30 d (8.7) 7.28 d (8.8) 7.27 d (8.7) 7.07 d (8.7) 7.07 d (8.8)
7.02 d (8.7) 7.00 d (8.8) 7.00 d (8.7) 6.86 d (8.7) 6.84 d (8.8)
2″
3″
7.35 d (8.6) 7.28 d (8.8) 7.27 d (8.7) 7.33 d (8.8)
6.98 d (8.6) 6.94 d (8.8) 6.91 d (8.7) 6.96 d (8.8)
4′
3.78 s
3.79 s
3.79 s
3.70 s
3.71 s
4″
3.77 s
3.76 s
3.74 s
3.75 s
5′
6′
NH2
7.02 d (8.7) 7.00 d (8.8) 7.00 d (8.7) 6.86 d (8.7) 6.84 d (8.8)
7.30 d (8.7) 7.28 d (8.8) 7.27 d (8.7) 7.07 d (8.7) 7.07 d (8.8)
5″
6″
NH2
6.98 d (8.6) 6.94 d (8.8) 6.91 d (8.7) 6.96 d (8.8)
7.35 d (8.6) 7.28 d (8.8) 7.27 d (8.7) 7.33 d (8.8)
b
4.33
4.37
4.30
4.34
4.45
4.42
4.40
4.38
4.42
b
NH
11.75
13.85
11.80
11.70
11.70
NH
11.82
11.91
11.87
11.90
a
b
500.13 MHz in DMSO-d6. Chemical shifts (δ) are in ppm relative to TMS. The spin coupling (J) is given in parentheses (Hz). NH2 and NH
signals were broadened due to exchange of protons in DMSO.
a
Table 2. 13C NMR Spectroscopic Data of 3-Aminopyrazoles 12 and 14
position
12a
12b
12c
12d
12e
position
14a
14b
14d
14e
Ring A
Ring A
1″
2″
124.1
105.4
121.8
108.0
119.7
104.3
113.0
135.0
59.2
118.4
144.9
60.5
1′
2′
122.5
106.5
127.2
109.1
118.3
136.1
59.3
123.8
146.0
60.6
OCH3-2″
OCH3-2′
3″
152.5
55.7
143.1
56.1
148.6
55.6
138.7
137.1
59.7
3′
152.8
55.7
143.3
56.1
138.8
137.4
59.8
OCH3-3″
OCH3-3′
4″
137.1
60.0
135.5
134.0
141.0
137.6
4′
135.5
60.0
133.3
138.8
135.4
OCH3-4″
OCH3-4′
5″
152.5
55.7
148.4
141.7
135.3
59.2
143.8
5′
152.8
55.7
148.5
135.6
56.3
143.9
OCH3-5″
OCH3-5′
6″
105.4
101.4
101.7
108.9
64.0
102.3
102.1
103.7
101.5
6′
106.5
103.4
101.1
110.4
101.5
102.8
100.9
O(CH2)nO
O(CH2)nO
Ring B
Ring B
1′
2′
3′
4′
127.6
131.5
114.1
158.6
55.2
121.5
131.4
114.3
158.8
55.1
121.8
131.2
114.4
158.9
55.2
121.4
130.5
114.1
158.7
55.0
126.0
129.1
113.7
156.9
54.8
1″
2″
3″
4″
129.3
128.7
113.8
158.8
55.2
123.2
128.7
113.9
159.0
55.2
123.2
127.8
113.8
158.9
55.1
127.8
126.4
113.4
157.1
54.9
OCH3-4′
OCH3-4″
5′
6′
3
114.1
131.5
139.5
98.7
114.3
131.4
149.0
102.9
141.4
114.4
131.2
149.4
102.6
141.7
114.1
130.5
149.2
99.3
113.7
129.1
150.0
104.5
140.0
5″
6″
3
113.8
128.7
150.5
99.9
113.9
128.7
149.6
104.2
141.5
113.8
127.8
149.8
100.5
142.1
113.4
126.4
146.0
105.8
140.1
4
4
5
149.9
142.1
5
140.0
a
1
1
125.76 MHz in DMSO-d6. Chemical shifts (δ) are in ppm relative to TMS. Assignments are based on H−1H NOESY, H−13C HMBC, and
1H−13C HSQC NMR experiments.
were the main products. The 1H NMR spectra for α-ketonitriles
11 and 13 are shown in Figures S1−S9 of the Supporting
Information. 1H and 13C NMR spectroscopic data for pyrazoles
12 and 14 are presented in Tables 1 and 2 and Figures S10−
S40 of the Supporting Information. 13C NMR signals for 12
and 14 were assigned by 1H−1H NOESY, 1H−13C HMBC, and
1H−13C HSQC techniques.
All of the resulting pyrazole derivatives were tested in the
phenotypic sea urchin embryo assay developed in our
laboratory.20 The screen has been shown to reliably and
reproducibly assess antiproliferative and microtubule-destabiliz-
ing activities of compounds. Moreover, these data were
consistent with results obtained via conventional purified
tubulin polymerization and cell-based assays.21−23 Notable
advantages of this in vivo system include the possibility to both
identify cell-permeable molecules with antimitotic or non-
specific cytotoxic activity and/or to establish their mode of
action.24 The assay allows for the simultaneous monitoring of
(i) fertilized egg test for antimitotic activity displayed by
cleavage alteration/arrest and (ii) behavioral monitoring of a
free-swimming blastula treated immediately after hatching.
During the course of the extensive validation of this assay
system at least three phenotypic parameters were observed;
namely, lack of forward movement, settlement to the bottom of
the culture vessel, and rapid spinning of an embryo around the
animal−vegetal axis correlate with a microtubule-destabilizing
activity caused by a molecule.21−23
C
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In this assay system, compounds 12a and 14a substituted
with a 3,4,5-trimethoxyaryl moiety exhibited the highest activity
(10−100 nM, Table 3). In our hands, type-III 5-amino-
and cancer cell assay systems the antiproliferative activity of
aminopyrazoles decreased in the following order: 14a > 12a >
14b > 12b ≈ 12c > 12e.
Thus, cis-restricted 4,5-polyalkoxydiaryl-3-aminopyrazole an-
alogues of natural antimitotic combretastatins were prepared
starting from the natural allylpolyalkoxybenzenes extracted
from dill and parsley seeds and from synthetic derivatives of
vanillin. Biological effects of the resulting pyrazoles were
evaluated in vivo in the phenotypic sea urchin embryo assay to
reveal their antimitotic and antitubulin activity. In these series,
pyrazoles substituted with ortho-amino and polyalkoxybenzene
moieties were consistently more potent than their symmetric
regioisomers (compare 14a and 12a; 14b and 12b), although
the exact structural rationale for this observations is not clear.
Molecules 12a and 14a, with a 3,4,5-trimethoxy ring A,
exhibited the highest antimitotic microtubule-destabilizing
effect in the sea urchin embryo assay. These compounds also
showed a considerable cytotoxicity against a panel of 60 human
cancer cell lines. In addition, both 12a and 14b inhibited
growth of multi-drug-resistant P-glycoprotein-overexpressing
ovarian cancer cells, suggestive of their potential as anticancer
agents.
Table 3. Effects of 4,5-(Polymethoxy)diaryl-3-
aminopyrazoles on Sea Urchin Embryos and Their
Cytotoxicity against Human Cancer Cells
a
EC, μM
NCI60 screen
mean GI50, mean
cleavage
alteration
cleavage
arrest
embryo
spinning
b
c
compound
μM
GI, %
d
CA4
CA2
0.002
0.002
0.002
0.1
0.01
0.01
0.01
1
0.05
0.05
0.05
>10
>5
0.0032
e
2.66
f
g
I
ND
12a
12b
12c
12d
12e
14a
14b
14d
14e
0.302
75.84
18.06
32.00
1
>4
1
>4
>5
g
4
>4
>4
ND
>4
>4
>4
3.38
89.22
45.98
0.01
0.2
0.05
>4
1
0.046
4.37
>10
>4
g
g
>4
>4
ND
ND
>4
>4
>4
a
The sea urchin embryo assay was conducted as described
EXPERIMENTAL SECTION
previously.20 Fertilized eggs and hatched blastulae were exposed to
2-fold decreasing concentrations of compounds. Duplicate measure-
ments showed no differences in effective threshold concentration (EC)
■
General Experimental Procedures. NMR spectra were collected
on a Bruker DR-500 instrument [working frequencies of 500.13 MHz
(1H) and 125.76 MHz (13C)]. Mass spectra were obtained on a
Finnigan MAT/INCOS 50 instrument (70 eV) using direct probe
injection. Elemental analysis was accomplished with the automated
Perkin-Elmer 2400 CHN microanalyzer. Compound purity was
determined by NMR, HPLC, and elemental analyses. Polyalkox-
ybenzoic styrenes 2b,d,e, aldehydes 3b,d,e, acids 4a,b,d,e, and
corresponding acid methyl esters 5b,d,e were synthesized according
to previously reported procedures.18
b
values. GI50: concentration required for 50% cell growth inhibition.
c
GI, %: inhibition of cell growth at 10 μM concentration in the single-
d
dose screen; presented for compounds with low cytotoxicity. Data
from ref 25. Mean value for seven human cancer cell lines.26 Data
e
f
g
from ref 18. ND: not determined.
pyrazoles were consistently more potent than the correspond-
ing type-II analogues (compare 14a and 12a; 14b and 12b). It
is worth noting that the majority of synthesized compounds did
not affect sea urchin embryo spinning. The only notable
exception was 14a. This molecule caused embryo spinning at 1
μM, suggesting that it is a microtubule-destabilizing agent. At
the same concentration, aminopyrazole 12a caused formation
of tuberculate arrested eggs, the effect also associated with a
microtubule disruption activity.
Substitution of trimethoxy groups in 12a and 14a with
methylenedioxy (12b and 14b) or ethylenedioxy moieties
(12c) reduced activity of the resultant compounds. Apiol and
dillapiol derivatives with tetraalkoxy ring A, 12d, 12e, 14d, and
14e, showed only modest potency in the phenotypic assay.
The outcome of the sea urchin embryo assay was supported
using a conventional cell-based screen. The cytotoxicity of
molecules 12a, 12b, 12c, 12e, 14a, and 14b was assessed in the
NCI60 anticancer drug screen (Table 3; Table S1, Supporting
Information). The most cytotoxic aminopyrazoles, 12a and
14a, exhibited mean GI50 values of 0.302 and 0.046 μM,
respectively (Table 3). The melanoma MDA-MB-435 cell line
was the most sensitive toward tested molecules. Compound
12a displayed high cytotoxicity against leukemia HL-60(TB),
K-562, and SR, colon cancer KM12 and SW-620, and ovarian
cancer OVCAR-3 cells, with GI50 values all less than 0.1 μM.
Aminopyrazole 14a significantly inhibited growth of non-small-
cell lung cancer NCI-H522 and melanoma MDA-MB-435 cells
with GI50 values less than 0.01 μM (Table S1, Supporting
Information). Notably, 12a, 14a, and 14b were more cytotoxic
against multi-drug-resistant NCI/ADR-RES ovarian cancer cells
as compared to the parent OVCAR-8 line. In both sea urchin
3,4-Dihydroxy-5-methoxybenzaldehyde (7). This was prepared as
described earlier.27 Bromovanillin 6 (105 g, 0.454 mol) was added to a
vigorously stirred suspension of NaOH (130 g, 3.25 mol) and copper
powder (550 mg, 8.65 mmol in 1.5 L of H2O). The resulting mixture
was refluxed for 27 h and cooled to room temperature; the pH was
adjusted to 7 with 1 M H2SO4 followed by the extraction with EtOAc
(7 × 100 mL). Organic extracts were combined, refluxed over
activated carbon, filtered, and dried over anhydrous Na2SO4. Solvent
was removed under reduced pressure, and the residue was crystallized
from toluene (250 mL) to yield 7 (49 g, 64% yield) as a tan powder:
mp 129−131 °C (lit.27 mp 132−133 °C); 1H NMR (DMSO-d6) δ 9.7
(1H, s, CHO), 9.50 (1H, s, ArOH), 9.44 (1H, s, ArOH), 7.04 (1H, d, J
= 1.7 Hz, ArH), 7.00 (1H, d, J = 1.7 Hz, ArH), 3.84 (3H, s, OCH3);
EIMS m/z 168 [M]+ (100), 167 (87), 125 (24), 97 (30), 53 (21), 51
(35), 39 (39); anal. C 57.25; H 4.91%, calcd for C8H8O4, C 57.14; H
4.80%.
3,4-Ethylendioxy-5-methoxybenzaldehyde (3c). Dry K2CO3 (16.8
g, 122 mmol) was added to a vigorously stirred solution of 7 (14 g, 83
mmol) and 1,2-dibromoethane (22.5 g, 10.3 mL, 120 mmol) in DMF
(110 mL). The resulting mixture was stirred for 5 h at 100−105 °C
and left overnight at room temperature. The reaction mixture was
diluted with water (600 mL), followed by extraction with EtOAc. The
EtOAc extract was washed with water, and solvent was evaporated in
vacuo. The residue (14.2 g) was crystallized from EtOH (40%) to
afford 3c (13.3 g, 68 mmol, 82% yield) as a light brown powder: mp
78−80 °C; 1H NMR (DMSO-d6) δ 9.78 (1H, s, CHO), 7.11 (1H, d, J
= 1.6 Hz, ArH), 7.10 (1H, d, J = 1.6 Hz, ArH), 4.28−4.34 (4H, octet,
OCH2CH2O), 3.85 (3H, s, OCH3); EIMS m/z 194 [M]+ (100), 193
(38), 138 (28), 123 (42), 95 (29), 66 (21), 51 (26), 39 (67), 38 (25);
anal. C 61.78; H 5.08%, calcd for C10H10O4, C 61.85; H 5.19%.
3,4-Ethylenedioxy-5-methoxybenzoic Acid (4c). Aqueous NaOH
(6 M, 18 mL) was added dropwise to a stirred solution of the urea−
hydrogen peroxide complex (1:1; 75 g) and aldehyde 3c (10 g, 5.15
D
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Article
mmol) in 200 mL of MeOH at room temperature. The resulting
mixture was stirred at 65 °C for 1 h, cooled to 40 °C, treated with 7.5 g
of fine urea−hydrogen peroxide complex (1:1), and heated at 65 °C
for an additional 1 h. The pH was adjusted to 3 with 18% HCl, and
MeOH was removed in vacuo. The residue was diluted with water
(200 mL) and extracted with CHCl3. The CHCl3 extract was washed
with water, refluxed over activated carbon, filtered, and dried over
anhydrous MgSO4. The resulting extract was concentrated in vacuo to
afford 5c (9.2 g, 4.38 mmol, 85% yield) as an off-white solid: mp 212−
213 °C; 1H NMR (DMSO-d6) δ 12.73 (1H, s, COOH), 7.11 (1H, d, J
= 1.9 Hz, ArH), 7.07 (1H, d, J = 1.9 Hz, ArH), 4.25−4.30 (4H, octet,
OCH2CH2O), 3.80 (3H, s, OCH3); EIMS m/z 210 [M]+ (100), 195
(26), 154 (31), 139 (35), 111 (35), 69 (30), 67 (23), 66 (44), 55 (29),
53 (27), 51 (28), 50 (32), 45 (26), 39 (61), 38 (50); anal. C 57.26; H
4.75%, calcd for C10H10O5, C 57.14; H 4.80%.
(43); anal. C 62.72; H 4.68; N 7.44%, calcd for C10H9NO3, C 62.82; H
4.74; N 7.33%.
2,5-Dimethoxy-3,4-methylenedioxyphenylacetonitrile (10d): 19.5
g, 88% yield; off-white solid; mp 97−98 °C (lit.18 mp 96−98 °C); 1H
NMR (CDCl3) δ 6.49 (1H, s, H-6), 5.99 (2H, s, OCH2O), 3.97 (3H,
s, OCH3), 3.87 (3H, s, OCH3-7), 3.61 (2H, s, CH2); EIMS m/z 221
[M]+ (72), 206 (80), 180 (3), 148 (10), 120 (14), 108 (11), 107 (13),
105 (12), 93 (31), 92 (20), 80 (49), 77 (79), 65 (64), 39 (100); anal.
C 59.61; H 4.95; N 6.47%, calcd for C11H11NO4, C 59.73; H 5.01; N
6.33%.
2,3-Dimethoxy-4,5-methylenedioxyphenylacetonitrile (10e): 14.4
1
g, 65% yield; beige solid; mp 60−61 °C; H NMR (CDCl3) δ 6.52
(1H, s, ArH-6), 5.94 (2H, s, OCH2O), 4.04 (3H, s, OCH3), 3.88 (3H,
s, OCH3), 3.62 (2H, s, CH2); EIMS m/z 221 [M]+ (54), 206 (100),
191 (13), 179 (12), 105 (21), 93 (16), 92 (13), 80 (27), 77 (68), 65
(30), 53 (54); anal. C 59.58; H 4.96; N 6.48%, calcd for C11H11NO4,
C 59.73; H 5.01; N 6.33%.
3,4-Ethylenedioxy-5-methoxybenzoic Acid Methyl Ester (5c). A
mixture of 4c (1g, 4.76 mmol) and SOCl2 (0.85 g, 7.12 mmol) in 15
mL of MeOH was refluxed for 1 h and concentrated in vacuo to yield
methyl ester 5c (0.96 g, 90% yield (95% purity)) as an off-white solid:
General Procedure for the Synthesis of α-Cyanoketones (11,
13). A 60% suspension of NaH (25 mmol) in mineral oil was added to
a solution of phenylacetonitrile (10.0 mmol) in anhydrous THF (20
mL) at room temperature under an argon atmosphere. The resulting
mixture was stirred for 30 min, and a solution of methyl benzoate (10
mmol) in anhydrous THF (20 mL) was added dropwise during 5 min.
The reaction mixture was refluxed for 5−6 h, cooled, quenched with
ice water (200 mL), acidified with 3 N HCl, and extracted with
CH2Cl2 (3 × 20 mL). The organic layers were combined, washed with
water, 10% NaHCO3 and water, dried over anhydrous Na2SO4, and
concentrated in vacuo to obtain the crude product. The resulting α-
cyanoketones were purified by column chromatography (SiO2,
EtOAc−hexanes).
1
mp 106−108 °C (MeOH); H NMR (DMSO-d6) δ 7.11 (1H, d, J =
1.9 Hz, ArH), 7.09 (1H, d, J = 1.9 Hz, ArH), 4.26−4.31 (4H, octet,
OCH2CH2O), 3.82 (3H, s, COOCH3), 3.81 (3H, s, OCH3); EIMS m/
z 224 [M]+ 224; anal. C 58.89; H 5.28%, calcd for C11H12O5, C 58.93;
H 5.39%.
3-Methoxy-4,5-methylenedioxybenzyl Alcohol (8b). This was
synthesized according to a literature procedure:28 mp 64−66 °C
(lit.28 mp 64−66 °C).
2,5-Dimethoxy-3,4-methylenedioxybenzyl Alcohol (8d). This was
synthesized according to a literature procedure:29 mp 88 °C (lit.29 mp
85 °C).
2,3-Dimethoxy-4,5-methylenedioxybenzyl Alcohol (8e). A sus-
pension of aldehyde 3e (10.0 g, 47.6 mmol) in MeOH (150.0 mL) was
stirred for 10 min, and NaBH4 (2.05 g, 54.7 mmol) was added in small
portions over 30 min at room temperature. After stirring for 6−7 h,
the pH was adjusted to 5 with glacial CH3COOH. Solvent was
removed in vacuo followed by the addition of water. The mixture was
filtered, washed with H2O (2 × 100 mL), and dried to afford the title
compound in 88.4% yield (8.75 g, 41.2 mmol) as colorless crystals: mp
51−53 °C; 1H NMR (DMSO-d6, 500 MHz) δ 6.60 (1H, s, H-6), 5.95
(2H, s, OCH2O), 4.98 (1H, t, J = 5.3 Hz, OH), 4.40 (2H, d, J = 5.03
Hz, CH2), 3.91 (3H, s, OCH3), 3.6 (3H, s, OCH3); EIMS m/z 212
[M]+ (87), 197 (15), 195 (21), 139 (96), 137 (50), 111 (52), 109
(33), 107 (23), 96 (23), 95 (24), 93 (34), 83 (24), 81 (29), 79 (35),
77 (29), 69 (24), 68 (50), 67 (21), 66 (28), 65 (51), 55 (51), 53 (89),
52 (21), 51 (42), 50 (28), 40 (25), 39 (100), 38 (28).; anal. C 56.71;
H 5.78%, calcd for C10H12O5, C 56.60; H 5.70%.
General Procedure for the Synthesis of Polyalkoxybenzyl
Chlorides (9b,d,e). Neat SOCl2 (8 mL, 110 mmol) was added
dropwise to a stirred solution of polyalkoxybenzyl alcohol (43.3
mmol) in dry benzene (70 mL) at room temperature. The resulting
mixture was warmed to 45−50 °C, stirred for 40 min, and
concentrated in vacuo. Traces of SO2Cl2 were removed by co-
evaporation of the residue with benzene (50 mL) in vacuo. The
resulting crude benzyl chloride (94−100% yield) was used for
synthesis of the corresponding nitriles without further purification.
General Procedure for the Synthesis of Polyalkoxyphenyla-
cetonitriles (10b,d,e). A solution of crude chloride (above, 0.1 mol)
in dry acetonitrile (120 mL) was treated with dibenzo-18-crown-6 (0.5
g, 1.4 mmol) followed by addition of dried KCN (15 g, 0.23 mol). The
reaction mixture was stirred for 10 h at 50 °C, concentrated in vacuo,
and treated with water (200 mL). The residue was filtered and
recrystallized from EtOH to yield the corresponding nitrile. 3,4,5-
Trimethoxyphenylacetonitrile (10a) was purchased from Acros.
3-Methoxy-4,5-methylenedioxyphenylacetonitrile (10b): 16.3 g,
85% yield; light brown solid; mp 88−90 °C (lit.30 mp 89−90 °C); 1H
NMR (DMSO-d6, 500 MHz) δ 6.65 (1H, d, J = 1.5 Hz, H-4), 6.60
(1H, d, J = 1.5 Hz, ArH-6), 6.00 (2H, s, OCH2O), 3.90 (2H, s, CH2),
3.83 (3H, s, OCH3); EIMS m/z 191 [M]+ (100), 190 (43), 176 (11),
160 (5), 146 (23), 133 (16), 90 (51), 78 (19), 77 (17), 64 (20), 63
2-(4-Methoxyphenyl)-3-oxo-3-(3,4,5-trimethoxyphenyl)-
propanenitrile (11a): 0.5 g, 65% yield; off-white solid; mp 94−96 °C
(lit.31 mp 90−92 °C); 1H NMR (CDCl3) δ 7.35 (2H, d, J = 8.7 Hz, H-
2′,6′), 7.18 (2H, s, H-2″,6″), 6.93 (2H, d, J = 8.7 Hz, H-3′,5′), 5.47
(1H, s, HC−CN), 3.91 (3H, s, OCH3-4′), 3.86 (6H, s, 2 × OCH3-
3″,5″), 3.80 (3H, s, OCH3-4″) (Figure S1, Supporting Information);
EIMS m/z 341 [M]+ (3), 196 (31), 195 (100), 152 (22), 147 (11),
146 (26), 137 (19), 122 (16), 109 (19), 107 (12), 92 (12), 91 (13), 81
(26), 77 (34), 76 (16), 66 (27); anal. C 66.98; H 5.67; N 3.97%, calcd
for C19H19NO5, C 66.85; H 5.61; N 4.10%.
2 - ( 4 - M e t h o x y p h e n y l ) - 3 - o x o - 3 - ( 3 - m e t h o x y - 4 , 5 -
methylenedioxyphenyl)propanenitrile (11b): 2.4 g, 59% yield; oil; 1H
NMR (CDCl3) δ 7.33 (2H, d, J = 8.7 Hz, H-2′,6′), 7.25 (1H, s, 1H, H-
4″), 7.08 (1H, s, H-6″), 6.91 (2H, d, J = 8.7 Hz, H-3′,5′), 6.06 (2H, s,
OCH2O), 5.43 (1H, s, HC−CN), 3.91 (3H, s, OCH3-4′), 3.79 (3H, s,
OCH3-3″) (Figure S2, Supporting Information); EIMS m/z 325 [M]+
(3), 180 (6), 179 (100), 151 (42), 146 (25), 103 (13), 95 (65), 93
(18), 91 (14), 78 (26), 77 (13), 65 (18), 63 (15); anal. C 66.52; H
4.69; N 4.27%, calcd for C18H15NO5, C 66.46; H 4.65; N 4.31%.
2 - ( 4 - M e t h o x y p h e n y l ) - 3 - o x o - 3 - ( 3 - m e t h o x y - 4 , 5 -
1
ethylenedioxyphenyl)propanenitrile (11c): 0.41 g, 65% yield, oil; H
NMR (CDCl3) δ 7.34 (2H, d, J = 8.7 Hz, H-2′,6′), 7.15 (1H, d, J = 2.0
Hz, H-5″), 7.13 (1H, d, J = 2.0 Hz, H-7″), 6.91 (2H, d, J = 8.7 Hz, H-
3′,5′), 5.47 (1H, s, HC−CN), 4.37 (2H, m, OCH2), 4.27 (2H, m,
OCH2), 3.89 (3H, s, OCH3-4′), 3.79 (3H, s, OCH3-3″) (Figure S3,
Supporting Information); EIMS m/z 339 [M]+ (1), 194 (20), 193
(100), 165 (18), 146 (35), 137 (12), 109 (11), 107 (19), 103 (11), 91
(11), 66 (33); anal. C 67.40; H 5.09; N 4.26%, calcd for C19H17NO5,
C 67.25; H 5.05; N 4.13%.
2-(4-Methoxyphenyl)-3-oxo-3-(2,5-dimethoxy-3,4-
methylenedioxyphenyl)propanenitrile (11d): 1.82 g, 61% yield; oil;
1H NMR (CDCl3) δ 7.27 (2H, d, J = 8.8 Hz, H-2′,6′), 6.99 (1H, s, H-
6″), 6.88 (2H, d, J = 8.8 Hz, H-3′,5′), 6.06 and 6.08 (2H, 2s, OCH2O),
5.86 (1H, s, HC−CN), 4.06 (3H, s, OCH3-4′), 3.84 (3H, s, OCH3-
2″), 3.79 (3H, s, OCH3-5″) (Figure S4, Supporting Information);
EIMS m/z 355 [M]+ (1), 210 (22), 209 (100), 194 (23), 166 (12),
146 (21), 136 (8), 121 (9), 106 (13), 103 (9), 95 (15), 93 (17), 91
(11), 78 (13), 77 (13), 65 (23); anal. C 64.33; H 4.87; N 3.83%, calcd
for C19H17NO6, C 64.22; H 4.82; N 3.94%.
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2-(4-Methoxyphenyl)-3-oxo-3-(2,3-dimethoxy-4,5-
methylenedioxyphenyl)propanenitrile (11e): 1.5 g, 65% yield; off-
white solid; mp 122−124 °C; 1H NMR (CDCl3) δ 7.27 (2H, d, J = 8.8
Hz, H-2′,6′), 6.86 (2H, d, J = 8.8 Hz, H-3′,5′), 6.75 (1H, s, H-4″), 5.98
(2H, 2s, OCH2O), 5.86 (1H, s, HC−CN), 4.03 (3H, s, OCH3-4′),
3.90 (3H, s, OCH3-2″), 3.78 (3H, s, OCH3-3″) (Figure S5, Supporting
Information); EIMS m/z 355 [M]+ (4), 209 (10), 194 (17), 166 (33),
165 (22), 146 (59), 123 (18), 121 (14), 103 (27), 95 (17), 93 (16), 91
(28), 77 (29), 76 (25), 65 (86); anal. C 64.36; H 4.88; N 3.78%, calcd
for C19H17NO6, C 64.22; H 4.82; N 3.94%.
(17), 75 (9), 63 (15), 53 (18), 51 (11), 44 (17), 43 (16), 38 (35), 36
(100); anal. C 63.86; H 5.14; N 12.44%, calcd for C18H17N3O4, C
63.71; H 5.05; N 12.38%.
4-(4-Methoxyphenyl)-5-(3-methoxy-4,5-ethylenedioxyphenyl)-
1H-pyrazol-3-amine (12c): 0.77 g, 65% yield (42% for the last two
1
steps); light brown solid; mp 216−218 °C; H and 13C NMR, see
Tables 1 and 2 and Figures S16−S20, Supporting Information; EIMS
m/z 353 [M]+ (100), 226 (17), 77 (16), 44 (30), 43 (37), 39 (16), 32
(89), 29 (57); anal. C 64.66; H 5.36; N 11.96%, calcd for C19H19N3O4,
C 64.58; H 5.42; N 11.89%.
3-(4-Methoxyphenyl)-3-oxo-2-(3,4,5-trimethoxyphenyl)-
propanenitrile (13a): 2.21 g, 65% yield; yellow oil (lit.31 mp 94−96
°C); 1H NMR (CDCl3) δ 7.95 (2H, d, J = 8.6 Hz, H-2′,6′), 6.94 (2H,
d, J = 8.6 Hz, H-3′,5′), 6.61 (2H, s, H-4″,6″), 5.46 (1H, s, HC−CN),
3.87 (3H, s, OCH3-4′), 3.85 (6H, s, 2 × OCH3-3″,5″), 3.83 (3H, s,
OCH3-4″) (Figure S6, Supporting Information); EIMS m/z 341 [M]+;
anal. C 66.92; H 5.70; N 4.01%, calcd for C19H19NO5, C 66.85; H
5.61; N 4.10%. This intermediate was used in the next steps without
further purification.
3 - ( 4 - M e t h o x y p h e n y l ) - 3 - o x o - 2 - ( 3 - m e t h o x y - 4 , 5 -
methylenedioxyphenyl)propanenitrile (13b): 1.8 g, 69% yield; off-
white solid; mp 121−123 °C; 1H NMR (CDCl3) δ 7.93 (2H, d, J = 9.0
Hz, H-2″,6″), 6.93 (2H, d, J = 9.0 Hz, H-3″,5″), 6.59 (2H, s, H-4′,6′),
5.97 (2H, s, OCH2O), 5.43 (1H, s, HC−CN), 3.89 (3H, s, OCH3-4″),
3.86 (3H, s, OCH3-3′) (Figure S7, Supporting Information); EIMS m/
z 325 [M]+ (1), 136 (13), 135 (100), 107 (17), 92 (10), 77 (16), 64
(6); anal. C 66.51; H 5.68; N 4.24%, calcd for C18H15NO5, C 66.46; H
4.65; N 4.31%.
4-(4-Methoxyphenyl)-5-(2,5-dimethoxy-3,4-methylenedioxy-
phenyl)-1H-pyrazol-3-amine (12d): 0.07 g, 22% yield (21% for the
last two steps); light brown solid; 1H and 13C NMR, see Tables 1 and
2 and Figures S21 and S22, Supporting Information; EIMS m/z 369
[M]+ (68), 238 (20), 210 (23), 198 (51), 194 (22), 184 (31), 170
(49), 169 (46), 168 (63), 156 (24), 155 (25), 154 (34), 153 (34), 152
(21), 148 (32), 147 (43), 146 (100), 142 (23), 141 (36), 140 (41),
139 (41), 135 (27), 134 (23), 128 (24), 127 (33), 126 (33), 125 (22),
116 (29), 115 (30), 83 (28), 77 (53), 76 (33), 75 (20), 69 (39), 63
(26), 45 (27), 44 (30), 43 (62), 39 (28); anal. C 61.87; H 5.24; N
11.45%, calcd for C19H19N3O5, C 61.78; H 5.18; N 11.38%.
4-(4-Methoxyphenyl)-5-(2,3-dimethoxy-4,5-methylenedioxy-
phenyl)-1H-pyrazol-3-amine (12e): 0.21 g, 24% yield (10% for the
last two steps); light brown solid; mp 182−188 °C; 1H and 13C NMR,
see Tables 1 and 2 and Figures S23− S26, Supporting Information;
EIMS m/z 369 [M]+ (100), 154 (11), 146 (29), 43 (15); anal. C
61.92; H 5.29; N 11.47%, calcd for C19H19N3O5:, C 61.78; H 5.18; N
11.38%.
3-(4-Methoxyphenyl)-3-oxo-2-(2,5-dimethoxy-3,4-
5-(4-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrazol-3-
amine (14a): 0.73 g, 68% yield (44% for the last two steps); brown
solid; mp 162−165 °C (lit.17 mp 174−176 °C); 1H and 13C NMR, see
Tables 1 and 2 and Figures S27−S30, Supporting Information; EIMS
m/z [M]+ 355; anal. C 64.15; H 5.88; N 11.90%, calcd for
C19H21N3O4, C 64.21; H 5.96; N 11.82%.
methylenedioxyphenyl)propanenitrile (13d): 1.6 g, 72% yield; off-
1
white solid; mp 97−99 °C; H NMR (CDCl3) δ 7.95 (2H, d, J = 9.0
Hz, H-2″,6″), 6.92 (2H, d, J = 9.0 Hz, H-3″,5″), 6.56 (1H, s, H-6′),
5.98 and 6.00 (2H, 2s, OCH2O), 5.94 (1H, s, HC−CN), 3.99 (3H, s,
OCH3-4″), 3.86 (3H, s, OCH3-2′), 3.83 (3H, s, OCH3-5′) (Figure S8,
Supporting Information); EIMS m/z 355 [M]+ (16), 324 (2), 220 (6),
136 (10), 135 (100), 107 (7), 92 (12), 77 (14), 43 (36); anal. C 64.29;
H 4.86; N 3.84%, calcd for C19H17NO6, C 64.22; H 4.82; N 3.94%.
3-(4-Methoxyphenyl)-3-oxo-2-(2,3-dimethoxy-4,5-
methylenedioxyphenyl)propanenitrile (13e): 1.9 g, 57% yield; off-
white solid; mp 118−120 °C; 1H NMR (CDCl3) δ 7.94 (2H, d, J = 9.0
Hz, H-2″,6″), 6.90 (2H, d, J = 9.0 Hz, H-3″,5″), 6.55 (1H, s, H-4′),
5.96 (1H, s, HC−CN), 5.89 and 5.93 (2H, 2s, OCH2O), 4.03 (3H, s,
OCH3-4″), 3.89 (3H, s, OCH3-2′), 3.85 (3H, s, OCH3-3′) (Figure S9,
Supporting Information); EIMS m/z 355 [M]+ (15), 324 (1), 220 (5),
136 (19), 135 (100), 107 (21), 104 (7), 92 (32), 77 (50), 64 (16);
anal. C 64.32; H 4.88; N 3.82%, calcd for C19H17NO6, C 64.22; H
4.82; N 3.94%.
5-(4-Methoxyphenyl)-4-(3-methoxy-4,5-methylenedioxyphenyl)-
1H-pyrazol-3-amine (14b): 0.45 g, 55% yield (38% for the last two
1
steps); light brown solid; mp 128−132 °C; H and 13C NMR, see
Tables 1 and 2 and Figures S31−S34, Supporting Information; EIMS
m/z 339 [M]+ (100), 223(12), 167 (12), 166 (11), 140 (16), 139
(15), 53 (18), 43 (16); anal. C 63.66; H 5.11; N,12.49%, calcd for
C18H17N3O4, C 63.71; H 5.05; N 12.38%.
5-(4-Methoxyphenyl)-4-(2,5-dimethoxy-3,4-methylenedioxy-
phenyl)-1H-pyrazol-3-amine (14d): 0.56 g, 35% yield (25% for the
last two steps); light brown solid; mp 132−134 °C; 1H and 13C NMR,
see Tables 1 and 2 and Figures S35−S38, Supporting Information;
EIMS m/z 369 [M]+ (35), 135 (100), 77 (19), 36 (16), 32 (36); anal.
C 61.65; H 5.07; N 11.44%, calcd for C19H19N3O5, C 61.78; H 5.18; N
11.38%.
General Procedure for the Synthesis of Diaryl-3-amino-
pyrazoles (12, 14). A suspension of α-cyanoketone (3 mmol) and
hydrazine dihydrochloride (15 mmol) in EtOH (20 mL) was refluxed
for 12−18 h with stirring until disappearance of the starting material
(TLC). After cooling, the resulting precipitate was filtered, washed
with ethanol, and dried to afford the targeted 3-aminopyrazole as a
respective hydrochloride salt. Alternatively, if precipitation was not
observed, the reaction solvent was removed in vacuo, and the residue
was treated with saturated aqueous NaHCO3 and filtered. The
resulting product was purified by SiO2 column chromatography
(EtOAc−EtOH, 2:1) to afford 12a−e and 14a,b,d,e.
5-(4-Methoxyphenyl)-4-(2,3-dimethoxy-4,5-methylenedioxy-
phenyl)-1H-pyrazol-3-amine (14e): 0.17 g, 15% yield (9% for the last
1
two steps); light brown solid; H and 13C NMR, see Tables 1 and 2
and Figures S39 and S40, Supporting Information; EIMS m/z [M]+
369; anal. C 61.85; H 5.10; N 11.31%, calcd for C19H19N3O5, C 61.78;
H 5.18; N 11.38%.
Sea Urchin Embryo Assay (ref 20). Adult sea urchins,
Paracentrotus lividus L. (Echinidae), were collected from the
Mediterranean Sea on the Cyprus coast in March−May and
October−December, 2012, and kept in an aerated seawater tank.
Gametes were obtained by intracoelomic injection of 0.5 M KCl. Eggs
were washed with filtered seawater and fertilized by adding drops of
diluted sperm. Embryos were cultured at room temperature under
gentle agitation with a motor-driven plastic paddle (60 rpm) in filtered
seawater. The embryos were observed with a Biolam light microscope
(LOMO, St. Petersburg, Russia). For treatment with the test
compounds, 5 mL aliquots of embryo suspension were transferred
to six-well plates and incubated as a monolayer at a concentration up
to 2000 embryos/mL. Stock solutions of compounds were prepared in
DMSO at 10 mM concentration followed by a 10-fold dilution with
95% EtOH. This procedure enhanced the solubility of the test
compounds in the salt-containing medium (seawater), as evidenced by
4-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-pyrazol-3-
amine (12a): 0.18 g, 18% yield (12% for the last two steps); light
yellow solid; mp 174−178 °C (lit.17 mp 169−171 °C); H and 13C
1
NMR, see Tables 1 and 2 and Figures S10 and S11, Supporting
Information; EIMS m/z 355 [M]+ (35), 340(11), 162 (13), 154 (10),
140 (10), 127 (11), 113 (12), 38 (34), 36 (100), 35 (20); anal. C
64.27; H 5.89; N 11.74%, calcd for C19H21N3O4, C 64.21; H 5.96; N
11.82%.
4-(4-Methoxyphenyl)-5-(3-methoxy-4,5-methylenedioxyphenyl)-
1H-pyrazol-3-amine (12b): 1.7 g, 40% yield (24% for the last two
steps); off-white solid; mp 191−196 °C; 1H and 13C NMR, see Tables
1 and 2 and Figures S12−S15, Supporting Information; EIMS m/z
339 [M]+ (21), 179(21), 140 (10), 139 (13), 133 (10), 77 (15), 76
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Journal of Natural Products
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microscopic examination of the samples. The maximal tolerated
concentrations of DMSO and EtOH in the in vivo assay were
determined to be 0.05% and 1%, respectively. Higher concentrations
of either DMSO (≥0.1%) or EtOH (>1%) caused nonspecific
alteration and retardation of the sea urchin embryo development
independent of the treatment stage. Combretastatins A-4 and A-2
(synthesized according to ref 18) served as reference compounds. The
antiproliferative activity was assessed by exposing fertilized eggs (8−20
min after fertilization, 45−55 min before the first mitotic cycle
completion) to 2-fold decreasing concentrations of the compound.
Cleavage alteration and arrest were clearly detected at 2.5−5.5 h after
fertilization. The effects were estimated quantitatively as an effective
threshold concentration, resulting in cleavage alteration and embryo
death before hatching or full mitotic arrest. At these concentrations all
tested microtubule destabilizers caused 100% cleavage alteration and
embryo death before hatching, whereas at 2-fold lower concentrations
the compounds failed to produce any effect. For microtubule-
destabilizing activity, the compounds were tested on free-swimming
blastulae just after hatching (8−10 h after fertilization), which
originated from the same embryo culture. Embryo spinning was
observed after 15 min to 20 h of treatment, depending on the structure
and concentration of the compound. Both spinning and lack of
forward movement were interpreted to be the result of the
microtubule-destabilizing activity of a molecule. Video illustrations
are available at http://www.chemblock.com. Both sea urchin embryo
assay and DTP NCI60 cell line activity data are available free of charge
via the Internet at http://www.zelinsky.ru.
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ASSOCIATED CONTENT
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S
* Supporting Information
1H NMR spectra of α-cyanoketones 11 and 13 (Figures S1−
1
1
1
1
S9). H, 13C, H−1H NOESY, H−13C HMBC, and H−13C
HSQC NMR spectra of 3-aminopyrazoles 12 and 14 (Figures
S10−S40). Cell growth inhibition of compounds 12a and 14b
in 60 human cancer cell lines (Table S1). This material is
available free of charge via the Internet at http://pubs.acs.org.
(22) Kiselyov, A. S.; Semenova, M. N.; Chernyshova, N. B.; Leitao,
A.; Samet, A. V.; Kislyi, K. A.; Raihstat, M. M.; Oprea, T.; Lemcke, H.;
Lantow, M.; Weiss, D. G.; Ikizalp, N. N.; Kuznetsov, S. A.; Semenov,
V. V. Eur. J. Med. Chem. 2010, 45, 1683−1697.
(23) Semenova, M. N.; Kiselyov, A. S.; Tsyganov, D. V.; Konyushkin,
L. D.; Firgang, S. I.; Semenov, R. V.; Malyshev, O. R.; Raihstat, M. M.;
Fuchs, F.; Stielow, A.; Lantow, M.; Philchenkov, A. A.; Zavelevich, M.
P.; Zefirov, N. S.; Kuznetsov, S. A.; Semenov, V. V. J. Med. Chem.
2011, 54, 7138−7149.
AUTHOR INFORMATION
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Corresponding Author
*Tel: +7 916 620 9584. Fax: +7 499 137 2966. E-mail: vs@
zelinsky.ru.
Notes
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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This work was supported by a grant from Chemical Block Ltd.
We thank the National Cancer Institute (NCI) (Bethesda, MD,
USA) for screening compounds 12a, 12b, 12c, 12e, 14a, and
14b by the Developmental Therapeutics Program at NCI
(Anticancer Screening Program; http://dtp.cancer.gov).
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