Synthesis and antimitotic properties of ortho-substituted polymethoxydiarylazolopyrimidines

Article abstract of DOI:10.24820/ark.5550190.p010.031

Ortho-substituted polymethoxydiarylazolopyrimidines were synthesized using polymethoxysubstituted benzaldehydes and acetophenones as starting material. X-ray crystallography data clearly confirmed that the subsequent cyclization of 3-amino-1,2,4-triazole

Full text of DOI:10.24820/ark.5550190.p010.031

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Paper
Archive for
Organic Chemistry
Arkivoc 2017, part iii, 151-165
Synthesis and antimitotic properties of ortho-substituted
polymethoxydiarylazolopyrimidines
a
a
b,c
a
Natalia B. Chernyshova, Dmitry V. Tsyganov, Victor N. Khrustalev, Mikhail M. Raihstat, Leonid D.
a
a
d
a
Konyushkin, Roman V. Semenov, Marina N. Semenova, and Victor V. Semenov*
a
d
N. D. Zelinsky Institute of Organic Chemistry RAS, 47 Leninsky Prospect, 119991 Moscow, Russian Federation
b
A. N. Nesmeyanov Institute of Organoelement Compounds, 28 Vavilov Street, 119991 Moscow, Russian
Federation
c
Peoples’ Friendship University of Russia (RUDN University), Moscow, Russian Federation
N. K. Koltzov Institute of Developmental Biology RAS, 26 Vavilov Street, 119334 Moscow, Russian Federation
E-mail: vs@zelinsky.ru
Dedicated to Professor Oleg A. Rakitin on the occasion of his 65th birthday
Received 01-25-2017
Accepted 05-03-2017
Published on line 06-20-2017
Abstract
Ortho-substituted polymethoxydiarylazolopyrimidines were synthesized using polymethoxysubstituted
benzaldehydes and acetophenones as starting material. X-ray crystallography data clearly confirmed that the
subsequent cyclization of 3-amino-1,2,4-triazole with ketoaldehydes yielded polymethoxyphenylsubstituted
6,7-diaryl-[1,2,4]triazolo[1,5-a]pyrimidines as single isomers. All compounds were evaluated in vivo using
phenotypic sea urchin embryo assay. 6-(4-Methoxyphenyl)-7-(3,4,5-trimethoxyphenyl)pyrazolo[1,5-
a]pyrimidine showed antimitotic microtubule destabilizing activity. The importance of aryl rings substituents
in diaryltriazolopyrimidines for their antiproliferative antitubulin effect has been suggested.
O
X
O
N
CH3
B
R
R
A
N
H
NH2
R = OH, (OCH3)n
X = H, N
X
N
N
N
Sea urchin embryo:
antimitotic microtubule
destabilizing activity
R
A
B
R
Keywords: Diarylazolopyrimidines, chalcone, sea urchin embryo, antimitotic, tubulin
DOI: https://doi.org/10.24820/ark.5550190.p010.031
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Introduction
The pyrimidine core, a building unit of DNA, RNA and nucleosides, can be found in a number of natural
products representing pharmacologically attractive chemical scaffold. Various pyrimidine derivatives were
1
,2
3
4
reported to exhibit diverse biological activities including antimicrobial,
antiviral, and antitumor effects.
antitubercular, antifungal,
5
6,7
Notably, polymethoxysubstituted diarylpyrimidines displayed dual
antimicrobial-anticancer properties. As an example, a pyrimidin-2-one (Figure 1, I) demonstrated antifungal
activity against Candida albicans comparable with that of clotrimazole as well as inhibited growth of a panel of
8
60 human cancer cell lines with mean GI50, TGI and LC50 values of 3.39, 17.4, and 61.7 microMol, respectively.
O
H CO
3
HN
NH
H CO
OCH3
OCH3
3
H CO
3
H CO
3
H CO
OH
OCH3
3
OCH3
I
II
N
Het
N
H CO
3
N
N
1
H CO
3
R
H CO
3
2
OH
R
OCH3
III
IV
R , R = Alk, OMe, Hal, CF , etc.
1
2
3
Figure 1. Structures of polymethoxysubstituted diarylpyrimidines, combretastatin A-4 (II) and its heterocyclic
derivatives.
Diarylpyrimidines can be considered as structural analogues of a potent natural antimitotic combretastatin
9
A-4 (CA4, Figure 1, II) isolated from the bark of African willow tree Combretum caffrum. The cis-configuration
of CA4 is essential for its antiproliferative antitubulin effect. Spontaneous isomerization to the trans-double
10
bond observed both in vitro and in vivo causes a dramatic decrease of biological activity. To stabilize the
1
1
11,12
11
active cis-conformation, several heteroaromatic rings, such as pyrazole, imidazole,
thiazole,
13,14
15
11,16,17
11,18–20
isoxazole,
1,2,3-thiadiazole, isomeric triazoles,
and tetrazole
have been introduced as a non-
isomerizable and metabolically stable isosteric equivalent of cis-double bond (Figure 1, III), resulting in highly
active microtubule destabilizing antimitotic agents. Replacement of the double bond by triazolopyrimidine
yielded diaryl-o-substituted-triazolopyrimidines (Figure 1, IV) with pronounced cytotoxicity against human
21
cancer cells both in vitro and in mouse xenograft model. However, in contrast to CA4 derivatives III, these
compounds promoted purified tubulin polymerization and stimulated aggregation of mitotic spindle
21
microtubules in cells.
For the design of potential anticancer molecules we synthesized a series of ortho-substituted
polymethoxydiarylpyrimidines (Scheme 1, 7,8,9aa). 4-Methoxy, 3,4,5-trimethoxy, and 3-hydroxy-4-methoxy
substituted aromatic aldehydes and ketones were used as starting material to prepare azolopyrimidines
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structurally similar to CA4 and its potent heterocyclic derivatives. The resulting compounds were evaluated for
antimitotic activity in a sea urchin embryo model. Selected molecules were also screened against a panel of
human cancer cell lines (NCI60 anticancer drug screen, http://dtp.cancer.gov).
Results and Discussion
Chemistry. Simple condensation of 3,4,5-trimethoxyacetophenone (1a) with 4-methoxybenzaldehyde (2a)
resulted in chalcone 3aa (Scheme 1). Rearrangement of chalcone 3aa mediated by phenyliodine diacetate in
22
the presence of H
2
SO afforded ketoaldehydediacetal 4aa in high yield, according to published procedure.
4
Targeted diarylpyrimidines 7–9 were synthesized by condensation in acidic media of diacetal 4aa with the
respective amines: urea, 3-aminopyrazole, or 3-amino-1,2,4-triazole. However, the yield of the desired
products 7–9 was reduced by formation of byproducts 5 and 6. Small quantity of 5 and 6 were detected during
the reaction by NMR-analysis and TLC. According to literature data, dimethylacetals (ex., 4aa) are easily
22
23
deformylated to benzyl ketones in acidic and alkaline media. Pure benzyl ketone 6 was specially
synthesized from dimethylacetal 4aa in a quantitative yield.
O
O
H CO
H CO
3
3
(i)
CH3
O
+
H CO
OCH3
H CO
3
OCH3
3
OCH3
OCH3
1a
2a
3aa 78%
(ii)
O
HN
N
OCH3
OCH3
O
O
O
H CO
3
H CO
H3CO
H3CO
H N
NH2
3
2
H CO
3
(iii)
H CO
O
3
H CO
OCH3
H CO
3
3
H CO
H3CO
3
4
aa 81%
5
7
34% OCH3
N
(iii)
N
(iii)
N
(iv)
N
H
NH2
N
H
NH2
N
OCH3
O
N
N
N
N
N
N
H CO
3
H CO
H CO
3
3
H CO
3
H CO
H CO
3
3
H CO
3
6
100%
H CO
H CO
3
3
8
45%
OCH3
9aa 61%
OCH3
Scheme 1. Synthesis of diarylpyrimidines 7–9. Reagents and conditions: (i) NaOH–EtOH, rt, 24 h; (ii) PhI(OAc)2 ,
+
+
MeOH–H , rt, 8 h; (iii) EtOH–H , refux, 2–6.5 h; (iv) KOH–EtOH, reflux, 4 h.
Triazolopyrimidines 9ab and 9bb with 3-hydroxy-4-methoxybenzene ring were prepared from
acetophenones 1a,b and isovanillin (2b) (Scheme 2). Intermediate chalcones 3ab and 3bb were acylated and
rearranged to ketoacetals 4ab and 4bb accompanied by simultaneous removal of acetyl group. Further
condensation of 4ab and 4bb with 3-amino-1,2,4-triazole afforded targeted diarylazolopyrimidines 9ab and
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9
bb. Diarylazolopyrimidine 9ca derived from 3-acetoxy-4-methoxyacetophenone (1с) and anisaldehyde (2a)
24
was synthesized from guaiacol (Scheme 3).
O
O
R
H CO
OCH3
OH
R
OH
(i)
+
H CO
OCH3
3
3
R
O
R
1
a: R = OCH3
b: R = H
3ab: R = OCH3
3bb: R = H
56-61%
2b
Isovanillin
1
(ii)
N
N
OCH3
OH
O
N
N
O
O
R
R
R
H CO
O
(iv)
(iii)
H CO
H CO
OCH3
3
3
3
H CO
OCH3
R
3
R
R
OH
OCH3
3-65%
9
9
ab
bb
3ab-Ac
3bb-Ac
72-81%
4ab
bb
in situ 70-80%
4
6
Scheme 2. Synthetic route for preparation of compounds 9ab and 9bb. Reagents and conditions: (i) NaOH–
+
+
EtOH, rt, 24 h; (ii) AcCl–Pyridine, CH
2
Cl
2
, rt, 3 h; (iii) PhI(OAc)
2
, MeOH–H , rt, 8 h; (iv) EtOH–H , reflux, 5 h.
O
O
O
O
HO
HO
O
OCH3
2a
AcOH-POCl3
Ref 21
(i)
H CO
H CO
3
OCH3
3
H CO
3
Guaiacol
1c
3ca
57%
(ii)
N
N
OCH3
O
N
O
O
N
N
N
N
H
NH2
HO
O
HO
(iii)
(iv)
H CO
H CO
OCH3
3
3
H CO
3
H CO
OCH3
3
9ca 24%
4ca in situ 84%
3ca-Ac 88%
OCH3
Scheme 3. Preparation of compound 9ca from guaiacol. Reagents and conditions: (i) NaOH–EtOH, rt, 24 h; (ii)
+
+
AcCl–Pyridine, CH
2
Cl
2
, rt, 3 h; (iii) PhI(OAc) , MeOH–H , rt, 8 h; (iv) EtOH–H , reflux, 5 h.
2
Generally, during the reaction between 3-amino-1,2,4-triazoles and aliphatic ketoaldehydes (or their
derivatives) cyclization can proceed in four different ways (Scheme 4).
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N
N
N
N
N
N
N
N
O
1
R
O
1
1
R
R
2
2
2
R
R
R
A
B
+
N N
N N
N
H
1
N
NH2
N
N
N
N
N
4
1
1
R
R
2
2
R
R
C
D
Scheme 4. Isomeric triazolopyrimidines which could be formed during condensation of ketoaldehydes with 3-
amino-1,2,4-triazole.
2
5
Type A: Connection of the aldehyde group with the amino group and of the ketone moiety with N1 atom.
Type B: Coupling of the aldehyde group with N1 atom and of the ketone moiety with the amino group.
2
6–28
Type C: Binding of the aldehyde group with the amino group and of the ketone moiety with N4 atom. Up to
date, there are no literature data on the formation of a pure type C isomer.
2
8
Type D: Connection of the aldehyde group with N4 atom and of the ketone moiety with the amino group.
26,27
29
The formation of mixtures of two isomers A+B
or A+C was published as well. Only two examples of
21,27
or D30
diarylketoaldehydes cyclization with 3-amino-1,2,4-triazoles that proceeded according to type A
have been reported.
X-ray structure determination. Literature data25–30 together with our results suggest that the structure of key
pyrimidine products 9 could not be unambiguously confirmed by NMR analysis. In the present study X-ray
crystallography was carried out in order to determine the precise structure of 6-(4-methoxyphenyl)-7-(3,4,5-
trimethoxyphenyl)[1,2,4]triazolo[1,5-a]pyrimidine (9aa). The analysis confirmed type A cyclization with the
formation of compound 9aa as isomer A (Figure 2).
The yellow prismatic crystal of 9aa (C21
20
H N
4
O , M = 392.41) was triclinic, space group, P-1, at T = 120 K: a
4
=
1
11.0658(7) Å, b = 12.6659(8) Å, c = 13.8403(9) Å, α = 94.490(1), β = 101.040(1),  = 90.963(1), V =
3
3
–1
897.1(2) Å , Z = 4, dcalc = 1.374 g/cm , F(000) = 824, μ = 0.097 mm . 25375 total reflections (11062 unique
)-
reflections, Rint = 0.040) were measured on a three-circle Bruker APEX II CCD diffractometer ((MoK
radiation, graphite monochromator,  and  scan mode, 2θmax = 60.0°) and corrected for absorption. The
structure was solved by direct methods and refined by a full-matrix least squares technique on F with

31
2
anisotropic displacement parameters for non-hydrogen atoms. One of the eight methoxy groups was
disordered over two sites with the occupancies of 0.8:0.2. The hydrogen atoms were placed in calculated
positions and refined within the riding model with fixed isotropic displacement parameters (Uiso(H) = 1.5Ueq(C)
for the CH
3
-group and Uiso(H) = 1.2Ueq(C) for the other groups). The final divergence factors were R
= 0.132 for all independent reflections, S = 1.010. All
1
= 0.049 for
7812 independent reflections with I > 2(I) and wR
2
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32
calculations were carried out using the SHELXTL program. Full crystallographic data for compound 9aa
Tables S1–S6, Supplementary Material) have been deposited with the Cambridge Crystallographic Data
Center, CCDC 1525158. Copies of this information may be obtained free of charge from the Director, CCDC, 12
(
33
Union Road, Cambridge CB2 1EZ, UK.
Figure 2. Molecular structure of 6-(4-methoxyphenyl)-7-(3,4,5-trimethoxyphenyl)[1,2,4]triazolo[1,5-
a]pyrimidine (9aa) (isomer A). Two crystallographically independent molecules distinguished by the
conformations of methoxy groups are shown. The alternative position of the disordered methoxy group is
depicted by dashed lines. Selected geometrical parameters (Å and deg.): N1–C2 1.3286(17), N1–N8
1.3694(15), C2–N3 1.3562(18), N3–C3A 1.3363(18), C3A–N4 1.3452(17), C3A–N8 1.3859(16), N4–C5
1.3165(18), C5–C6 1.4305(17), C6–C7 1.3824(18), C7–N8 1.3668(16), C2–N1–N8 101.29(11), N1–C2–N3
117.29(13), C3A–N3–C2 102.27(11), N3–C3A–N4 128.76(12), N3–C3A–N8 109.47(11), N4–C3A–N8 121.77(12),
C5–N4–C3A 115.67(11), N4–C5–C6 125.67(12), C5–C6–C7 117.65(12), N8–C7–C6 115.97(11) (Supplementary
Material).
Biological evaluation. The targeted diarylazolopyrimidines 7–9 and by-product 6 were evaluated for
34
antimitotic activity in a phenotypic sea urchin embryo assay. The assay provides rapid information on
antiproliferative, embryotoxic, and microtubule destabilizing activities of tested molecules. The results are
presented in Table 1 with CA4 as a positive control. For comparison, EC and GI50 values for intermediate
chalcones 3aa, 3ab, 3d, and 3e were included. Compounds 3d and 3e were synthesized as described in the
Experimental Section.
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Table 1. Effects of diarylazolopyrimidines on sea urchin embryos and human cancer cells
O
O
OCH3
OCH3
HO
OCH3
OCH3
H CO
H CO
3
3
3d
OCH3
3e
OCH3
Compound
Sea urchin embryo effects, EC (microMol)a
Cell growth inhibition
b
Cleavage
alteration
Cleavage
arrest
Embryo
spinning
>5
GI50 (microMol)
aac
0.2
0.02
2
4
4
1 (TE)
0.2 (TE)
d
0.3 (K562)e
0.0043 (K562)
3
3
3
c
d
e
ab
0.5
>4
>4
>4
>4
>5
c
e
d
>4
>4
>4
>4
4.5 (K562)
f
3e
ND
ND
ND
f
6
7
8
f
4
d
g
0.5
>4
>4
>4
>4
0.002
4 (TE)
4.07
9aa
>4
>4
>4
>4
>10
f
9ab
ND
ND
ND
f
9
bb
ca
CA4
>4
>4
f
9
>4
>4
h
0.01
0.05
0.00768
a
34
The sea urchin embryo assay was conducted as described previously. Fertilized eggs and hatched blastulae
were exposed to 2-fold decreasing concentrations of compounds. Duplicate measurements showed no
differences in effective threshold concentration (EC) values.
b
GI50: concentration required for 50% cell growth inhibition.
EC values from Ref. 35.
TE: tuberculate eggs typical of microtubule destabilizing agents.
Data from Ref. 36 for K562 human leukemia cell line.
ND: not determined.
Mean value for 60 human cancer cell lines.
Mean value for 60 human cancer cell lines, NSC 613729.
c
d
e
f
g
h
As shown from Table 1, intermediate chalcones 3aa and 3ab exhibited antimitotic activity in the sea urchin
embryo assay. Embryo spinning after the exposure to 3ab, a full analog of CA4, and tuberculate eggs
characteristic for 3aa suggested their microtubule destabilizing mode of action. Notably, the interchange of
substituted aryl rings in chalcones 3aa and 3ab yielded significantly less active compounds 3d and 3e,
respectively. Therefore we paid attention to the synthesis of azolopyrimidine isomers 8, 9aa, and 9ab from
intermediate chalcones 3aa and 3ab. Cyclization of chalcones 3 into dyarylazolopyrimidines 8 and 9 resulted in
decrease of antimitotic effect. Only pyrazolopyrimidine 8 exhibited antiproliferative and cytotoxic effects both
in sea urchin embryos and human cancer cells. Compound 8 could be considered as microtubule targeting
agent since it caused formation of tuberculate arrested eggs typical for microtubule destabilizers. In contrast
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21
to microtubule stabilizing diaryl-o-substituted-triazolopyrimidines (Figure 1, IV), the antimitotic effect of
pyrazolopyrimidine 8 was likely associated with microtubule disassembly. Pyrimidine derivative 7 as well as
by-product 6 displayed weak antiproliferative activity. All four triazolopyrimidines 9 failed to affect cell division
(cleavage) up to 4 microMol concentration. Generally, introduction of N-containing 5-membered heterocycles
as linkers between two aryl rings in CA4 analogues (Figure 1, III) resulted in potent cytotoxic compounds with
11–20
antimitotic microtubule destabilizing mode of action.
Similarly, CA4 analogues with bicyclic
triazolopyrimidine linker, halogen substituted triazolopyrimidines (Figure 1, IV), displayed cytotoxicity against
21
human cancer cell lines with IC50 in nanomolar concentration range. In the present study, polymethoxy-
hydroxy-substituted diaryltriazolopyrimidines 9 were found to be inactive up to 4 microMol concentration.
The results indicated the importance of aryl rings substituents in diaryltriazolopyrimidine derivatives of CA4
for their antimitotic antitubulin effects.
Conclusions
Ortho-substituted polymethoxydiarylazolopyrimidines were synthesized using condensation of
polymethoxysubstituted benzaldehydes and acetophenones. Further cyclization of 3-amino-1,2,4-triazoles
with aliphatic ketoaldehydes yielded triazolopyrimidines 9 as a single set of isomers. X-ray crystallography
analysis exemplified by 6-(4-methoxyphenyl)-7-(3,4,5-trimethoxyphenyl)[1,2,4]triazolo[1,5-a]pyrimidine (9aa)
confirmed that the cyclization of 3-amino-1,2,4-triazole with ketoaldehyde followed the reaction route to
isomer A. Sea urchin embryo screen identified pyrazolopyrimidine 8 as antimitotic microtubule destabilizing
agent, whereas diaryltriazolopyrimidines 9 were inactive. Aryl rings substituents in triazolopyrimidine
analogues of CA4 are likely crucial for their antiproliferative antitubulin efficiency.
Experimental Section
General. Melting points were measured on a Boetius melting point apparatus and were uncorrected. Reaction
1
mixtures were stirred magnetically. H NMR spectra were recorded on a Bruker DRX-500 (500.13 MHz)
instrument. Chemical shifts were stated in parts per million (ppm) and referenced to the appropriate NMR
1
solvent peak(s). Spin–spin coupling constants (J) were reported in hertz (Hz). Original H NMR spectra for
azolopyrimidines 7, 8, and 9 are presented in Supplementary Material. Low resolution mass spectra (m/z)
were recorded on a Finnigan MAT/INCOS 50 mass spectrometer at 70 eV using direct probe injection.
Elemental analysis was performed on the automated PerkinElmer 2400 CHN microanalyzer. Flash
chromatography was carried out on silica gel (Acros 0.035–0.070 mm, 60 Å). TLC was performed on Merck 60
F
254 plates.
37
General procedure for the synthesis of chalcones 3. NaOH (60 mmol) was added to a vigorously stirred
solution containing arylacetophenones 1 (20 mmol) and benzaldehydes 2 (20 mmol) in EtOH (150 mL) at 5 C.
The reaction mixture was stirred at room temperature for 24 h and the solvent was evaporated. The residue
was diluted with water (130 mL), neutralized with 15% HCl, and extracted with CH
2
Cl (3  80 mL). Organic
2
extracts were washed with brine (2  80 mL), dried with Na
2
SO
4
, and evaporated. The residue was crystallized
from EtOAc-petroleum (1:1) and dried to afford chalcones 3.
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(
2E)-3-(4-Methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-2-propen-1-one (3aa). Bright yellow crystals; Yield 5.12
38
1
g, 78%; mp 99–101 C (Lit. 100–101 C); H NMR (DMSO-d
6
): δ 7.88 (2H, d, J 8.8 Hz, H-2'',6''), 7.81 (1H, d, J
1
5.5 Hz, H-2), 7.72 (1H, d, J 15.5 Hz, H-3), 7.42 (2H, s, H-2',6'), 7.03 (2H, d, J 8.8 Hz, H-3'',5''), 3.90 (6H, s, OCH -
3
+
+
3',5'), 3.83 (3H, s, OCH
3
), 3.77 (3H, s, OCH ); EIMS m/z 329 [M+1] (19), 328 [M] (100), 327 (13), 314 (12), 313
3
(
59), 297 (29), 285 (20), 195 (31), 171 (12), 161 (65), 133 (44), 118 (22), 109 (14), 89 (27), 77 (35); Anal. Calcd.
: C, 69.50; H, 6.14. Found: C, 69.62; H, 6.18.
2E)-3-(3-Hydroxy-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-2-propen-1-one (3ab). Yellow crystals; Yield
for C19
(
4
2
6
H O
20 5
3
9
1
.2 g, 61%; mp 126–128 C (Lit. 127–129 C); H NMR (DMSO-d
), 7.62 (1H, d, J 15.4 Hz, H-3), 7.40 (2H, s, H-2',6'), 7.39 (1H, d, J 2 Hz, H-2''), 7.30 (1H, dd, J 8.3 Hz, J 2 Hz, H-
''), 7.00 (1H, d, J 8.3 Hz, 5''), 3.90 (6H, s, OCH -3',5'), 3.84 (3H, s, OCH ), 3.76 (3H, s, OCH ); EIMS m/z 345
6
): δ 9.19 (1H, s, OH), 7.71 (1H, d, J 15.4 Hz, H-
3
3
3
+
+
[M+1] (21), 344 [M] (100), 330 (12), 329 (58), 313 (25), 301 (17), 195 (37), 177 (34), 156 (24), 134 (18), 117
(17), 106 (10), 105 (13), 89 (28), 77 (26); Anal. Calcd. for C19 : C, 66.27; H 5.85. Found: C, 66.40; H, 5.88.
(2E)-3-(3-Hydroxy-4-methoxyphenyl)-1-(4-methoxyphenyl)-2-propen-1-one (3bb). The reaction mixture was
H O
20 6
40
1
stirred at room temperature for 72 h. Yellow crystals; Yield 3.18 g, 56%; mp 149–151 C (Lit. 150–152 C); H
NMR (DMSO-d ): δ 9.10 (1H, s, OH), 8.13 (2H, d, J 8.7 Hz, H-2',6'), 7.69 (1H, d, J 15.5 Hz, H-2), 7.59 (1H, d, J 15.5
Hz, H-3), 7.31 (1H, s, H-2''), 7.28 (1H, d, J 8.3 Hz, H-6''), 7.07 (2H, d, J 8.7 Hz, H-3',5'), 7.0 (1H, d, J 8.3 Hz, 5''),
3
1
5
6
+
+
.87 (3H, s, OCH
3
), 3.84 (3H, s, OCH ); EIMS m/z 285 [M+1] (10), 284 [M] (51), 283 (16), 269 (32), 241 (10),
3
77 (11), 135 (83), 117 (14), 107 (27), 105 (20), 92 (71), 89 (41), 77 (100); Anal. Calcd. for C17
H
16
O : C, 71.82; H,
4
.67. Found: C, 71.88; H, 5.70.
(
2E)-1-(3-Hydroxy-4-methoxyphenyl)-3-(4-methoxyphenyl)-2-propen-1-one (3ca). Acetyl group was
hydrolyzed during the condensation of 5-acetyl-2-methoxyphenyl acetate (1c) and anisaldehyde (2a) (Scheme
1
3
). Yellow crystals; Yield 3.24 g, 57%; mp 106–108 C; H NMR (DMSO-d
6
): δ 9.35 (1H, s, OH), 7.82 (2H, d, J 8.7
Hz, H-2'',6''), 7.74 (1H, d, J 15.5 Hz, H-2), 7.73 (1H, dd, J 8.5 Hz, J 2.0 Hz, H-6'), 7.65 (1H, d, J 15.5 Hz, H-3), 7.52
(
1H, d, J 2.0 Hz, H-2'), 7.05 (1H, d, J 8.5 Hz, H-5'), 7.01 (2H, d, J 8.5 Hz, H-3'',5''), 3.88 (3H, s, OCH
3
), 3.82 (3H, s,
); EIMS m/z 285 [M+1] (19), 284 [M] (100), 283 (23), 269 (53), 253 (20), 161 (46), 151 (37), 133 (25), 108
: C, 71.82; H, 5.67. Found: C, 71.96; H, 5.72%.
2E)-1-(4-Methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-2-propen-1-one (3d). Yellow crystals; Yield 4.86 g,
+
+
OCH
3
(19), 89 (24), 77 (25); Anal. Calcd. for C17
H O
16 4
(
36
1
7
4%; mp 132–134 C (Lit. 131–133 C); H NMR (DMSO-d
6
): δ 8.18 (2H, d, J 8.9 Hz, H-2',6'), 7.89 (1H, d, J 15.5
),
); EIMS m/z 329 [M+1] (19), 328 [M] (84), 327 (13), 314 (6), 313
30), 298 (8), 297 (40), 285 (18), 225 (10), 171 (15), 135 (86), 133 (10), 128 (14), 120 (11), 119 (10), 107 (30), 77
100); Anal. Calcd. for C, 69.50; H, 6.14. Found: C19 , C, 69.59; H, 6.17.
2E)-1-(3-Hydroxy-4-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-2-propen-1-one (3e). Acetyl group was
Hz, H-2), 7.66 (1H, d, J 15.5 Hz, H-3), 7.22 (2H, s, H-2'',6''), 7.10 (2H, d, J 8.9 Hz, H-3',5'), 3.88 (3H, s, OCH
3
(
(
(
3
+
+
.87 (6H, s, OCH
3
-3'',5''), 3.72 (3H, s, OCH
3
H O
20 5
hydrolyzed during the condensation of 5-acetyl-2-methoxyphenyl acetate (1c) and 3,4,5-
39
1
trimethoxybenzaldehyde. Yellow crystals;Yield 4.34 g, 63%; mp 129–131 C (Lit. 130–132 C); H NMR
DMSO-d ): δ 9.39 (1H, s, OH), 7.83 (1H, d, J 15.5 Hz, H-2), 7.76 (1H, dd, J 8.5 Hz, J 1.8 Hz, H-6'), 7.62 (1H, d, J
5.4 Hz, H-3), 7.54 (1H, d, J 1.8 Hz, H-2'), 7.20 (2H, s, H-2'',6''), 7.07 (1H, d, J 8.5 Hz, 5'), 3.88 (3H, s, OCH ), 3.87
); EIMS m/z 345 [M+1] (19), 344 [M] (100), 330 (8), 329 (40), 314 (9),
13 (42), 301 (13), 157 (5), 151 (13), 127 (6); Anal. Calcd. for C19 : C, 66.27; H, 5.85. Found: C, 66.37; H,
.89.
(
6
1
3
+
+
(6H, s, OCH
3
-3'',5''), 3.71 (3H, s, OCH
3
3
5
H O
20 6
General procedure for the synthesis of chalcone acetates 3ab-Ac, 3bb-Ac, and 3ca-Ac. The mixture of AcCl
(1.18 g, 15 mmol) and pyridine (1.19 g, 15 mmol) in abs. CH
2
Cl
2
(15 mL) was added by small portions to a
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Chernyshova, N. B. et al.
stirred solution of a correcponding chalcone 3 (10 mmol) and pyridine (0.79 g, 10 mmol) in abs. CH
at 20 C. The reaction mixture was stirred at room temperature for 3 h, diluted with water (50 mL), and the
organic layer was separated. Water layer was extracted by CH Cl (30 mL). Combined organic extracts were
washed with water (3  50 mL), dried with Na SO and evaporated. The residue was crystallized from EtOAc-
petroleum ether (1:1) and dried to afford chalcone acetates 3a-Ac, 3b-Ac, and 3c-Ac.
2
Cl
2
(50 mL)
2
2
2
4
2
-Methoxy-5-[(1E)-3-oxo-3-(3,4,5-trimethoxyphenyl)-1-propenyl]phenyl acetate (3ab-Ac). Yellow crystals;
1
Yield 2.1 g, 72%; mp 114–116 C; H NMR (DMSO-d
6
): δ 7.84 (1H, d, J 15.5 Hz, H-2), 7.82 (1H, d, J 2.2 Hz, H-6'),
7
.74 (1H, dd, J 8.6 Hz, J 2.2 Hz, H-4'), 7.71 (1H, d, J 15.5 Hz, H-1), 7.43 (2H, s, H-2'',6''), 7.21 (1H, d, J 8.6 Hz, H-
+
3'), 3.91 (6H, s, OCH
3
-3'',5''), 3.85 (3H, s, OCH
3
), 3.77 (3H, s, OCH
3
), 2.30 (3H, s, CH C(O)); EIMS m/z 386 [M]
3
(15), 344 (28), 329 (21), 313 (9), 301 (7), 195 (18), 177 (15), 156 (13), 89 (12), 77 (16), 43 (100); Anal. Calcd. for
C
21 22 7
H O
: C, 65.28; H, 5.74. Found: C, 65.37; H, 5.78.
2
-Methoxy-5-[(1E)-3-(4-methoxyphenyl)-3-oxo-1-propenyl]phenyl acetate (3bb-Ac). Yellow crystals; Yield
1
0.46 g, 81%; mp 136–138 C (EtOAc/petr=1/1); H NMR (DMSO-d
6
): δ 8.17 (2H, d, J 8.9 Hz, H-2'',6''), 7.85 (1H,
d, J 15.5 Hz, H-2), 7.79 (1H, d, J 2.1 Hz, H-6'), 7.72 (1H, dd, J 8.6 Hz, J 2.1 Hz, H-4'), 7.66 (1H, d, J 15.5 Hz, H-1),
7
CH
.20 (1H, d, J 8.6 Hz, H-3'), 7.08 (2H, d, J 8.9 Hz, H-3'',5''), 3.87 (3H, s, OCH
3
), 3.84 (3H, s, OCH ), 2.30 (3H, s,
3
+
3
C(O)); EIMS m/z 326 [M] (6), 284 (14), 269 (9), 135 (22), 92 (13), 77 (25), 43 (100); Anal. Calcd. for
C
19
H
18
O : C, 69.93; H, 5.56. Found: C, 69.99; H, 5.59.
5
2
8
7
3
-Methoxy-5-[(2E)-3-(4-methoxyphenyl)-2-propenoyl]phenyl acetate (3ca-Ac). Yellow crystals; Yield 2.9 g,
1
9%; mp 126–128 C; H NMR (DMSO-d
6
): δ 8.13 (1H, dd, J 8.7 Hz, J 2.0 Hz, H-4'), 7.93 (1H, d, J 2.0 Hz, H-6'),
.86 (2H, d, J 8.7 Hz, H-2'',6''), 7.81 (1H, d, J 15.5 Hz, H-2), 7.71 (1H, d, J 15.5 Hz, H-3), 7.30 (1H, d, J 8.7 Hz, H-
'), 7.02 (2H, d, J 8.7 Hz, H-3'',5''), 3.89 (3H, s, OCH ), 3.83 (3H, s, OCH ), 2.31 (3H, s, CH C(O); EIMS m/z 327
3
3
3
+
+
[M+1] (16), 326 [M] (78), 285 (20), 284 (100), 283 (30), 269 (43), 253 (16), 161 (41), 151 (21), 133 (14), 89 (7),
7
3
5
7 (8); Anal. Calcd. for C19
,3-Dimethoxy-2-(4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1-propanone (4aa). Solution of H
.5 mL) in MeOH was added dropwise to the vigorously stirred suspension of chalcone 3aa (6.57g, 20 mmol)
(6.86 g, 21.3 mmol) in MeOH (93 mL) and stirred for 4 h at room temperature. The reaction
solution was diluted with water (150 mL) and extracted twice by CH Cl (150 mL and 50 mL). The organic layer
was washed with brine, water (2  50 mL), and dried over anhydrous Na SO . The solvent was evaporated; the
H
18
O
5
: C, 69.93; H, 5.56. Found: C, 69.99; H, 5.59.
2
SO (50%,
4
and PhI(OAc)
2
2
2
2
4
remainder oil was diluted with MeOH (20 mL), boiled till formation of transparent solution, and kept overnight
at 2–6 C. Crystals were filtered, washed with cold EtOH (2  3 mL), petroleum ether (3 mL), and dried to
1
afford dimethylacetal 4aa. Colorless crystals; Yield 6.32 g, 81%; mp 104–106 C; H NMR (CDCl
3
) δ 7.32 (2H, d,
J 8.3 Hz, H-2'',6''), 7.26 (6H, s, H-2',6'), 6.85 (2H, d, J 8.3 Hz, H-3'',5''), 5.05 (1H, d, J 8.2 Hz, H-2), 4.73 (1H, d, J
8
.2 Hz, H-3), 3.87 (9H, s, OCH
3
-3',4',5'), 3.76 (3H, s, OCH
3
-4''), 3.46 (3H, s, OCH
3
), 3.23 (3H, s, OCH ); EIMS m/z
3
+
+
391 [M+1] , 390 [M] ; Anal. Calcd. for C21 : C, 64.60; H, 6.71. Found: C, 64.57; H, 6.66.
H O
26 7
General rearrangement procedure for synthesis of dimethylketoacetals 4ab, 4bb, and 4ca with simultaneous
removal of acetyl group. Solution of H SO (50%, 5 mL) in MeOH was added dropwise to the vigorously stirred
suspension of chalcone acetate 3ab-Ac, 3bb-Ac, or 3ca-Ac (20 mmol) and PhI(OAc) (30 mmol) in MeOH (100
mL), stirred for 8 h at room temperature and kept overnight at at 2–6 C. The reaction mixture was diluted
with water (300 mL) and extracted by CH Cl (3  100 mL). The organic layer was washed with brine (2 ×80
mL), water (50 mL), and dried over anhydrous Na SO . The solvent was evaporated; the remainder oil was
roughly separated by column chromatography (EtOAc-hexane = 1:4) to afford 4ab, 4bb, and 4ca. 1-(3,4,5-
2
4
2
2
2
2
4
Trimethoxyphenyl)-3,3-dimethoxy-2-(3-hydroxy-4-methoxyphenyl)-1-propanone
(4ab)
and
1-(4-
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Chernyshova, N. B. et al.
methoxyphenyl)-3,3-dimethoxy-2-(3-hydroxy-4-methoxyphenyl)-1-propanone (4bb) were further used
without additional purification.
1
-(3-Hydroxy-4-methoxyphenyl)-3,3-dimethoxy-2-(4-methoxyphenyl)-1-propanone (4ca). White solid; Yield
1
5.82 g, 84%; mp 104–107 C ( EtOAc/petr); H NMR (CDCl
3
): δ 7.56 (1H, dd, J 8.2 Hz, J 2.1 Hz, H-6'), 7.55 (1H, d,
J 2.1 Hz, H-2'), 7.32 (2H, d, J 8.7 Hz, H-2'',6''), 6.84 (1H, d, J 8.2 Hz, H-5'), 6.83 (2H, d, J 8.7 Hz, H-3'',5''), 5.61
(
1H, s, OH), 5.07 (1H, d, J 8.5 Hz, H-2), 4.77 (1H, d, J 8.5 Hz, H-3), 3.91 (3H, s, OCH
3
), 3.75 (3H, s, OCH
3
), 3.43
+
+
(3H, s, OCH
3
), 3.21 (3H, s, OCH
3
); EIMS m/z 347 [M+1] , 346 [M] ; Anal. Calcd. for C19 : C, 65.88; H, 6.40.
H O
22 6
Found: C, 66.02; H, 6.46.
-(4-Methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)ethanone (6). KOH (0.9 g, 16 mmol) was added to the
23
2
stirred suspension of dimethylacetal 4aa (3.12 g, 8 mmol) in EtOH (72 mL) and water (8 mL), and refluxed
under argon with stirring for 4 h (TLC control). The reaction solution was acidified with HCl (1N), evaporated,
and diluted with CH
Na SO . The solvent was evaporated; the residue was dried in vacuo under P
solid; Yield 2.53 g, 100%; mp 85–87 C (Lit. 86–88 C); H NMR (CDCl
2
Cl
2
(150 mL). Organic layer was washed by water (3  30 mL) and dried over anhydrous
at 60–90 C to afford 6. White
): δ 7.29 (2H, s, H-2',6'), 7.18 (2H, d, J
), 3.90 (3H, s, OCH ), 3.89 (6H, s, OCH -3',5'),
2
4
O
2 5
41
1
3
8
3
1
.1 Hz, H-2'',6''), 6.87 (2H, d, J 8.1 Hz, H-3'',5''), 4,19 (2H, s, CH
2
3
3
+
3
.79 (3H, s, OCH ); EIMS m/z 316 [M] (8), 196 (16), 195 (100), 167 (5), 152 (11), 137 (12), 135 (8), 122 (11),
21 (45), 109 (10), 98 (8), 81 (15), 78 (22), 77 (26); Anal. Calcd. for C18
H
20
O : C, 68.34; H, 6.37. Found: C, 68.40;
5
H, 6.41.
-(4-Methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-2(3H)-pyrimidinone (7). The mixture of dimethylacetal 4aa
0.39 g, 1 mmol), urea (0.09 g, 1.5 mmol), and HCl (36%, 10 drops) in EtOH (2 mL) was refluxed for 6.5 h, and
5
(
then evaporated to dryness. The deposit was triturated with EtOAc (1 mL), filtered and dried in vacuo at 60 C
to afford hydrochloride of 7 as bright yellow crystals (0.27 g, 66%; mp 150–160 C). The crystals were
triturated with NH
4
OH (12%), washed with water and dried in vacuo under P
2
O
5
to afford targeted
): δ 13.0 (1H, br,
1
pyrimidinone 7. Yellowish crystals; Yield 0.125 g, 34%; mp 131–133dec.C; H NMR (DMSO-d
6
NH), 8.16 (1H, s, H-6), 7.06 (2H, d, J 8.4 Hz, H-2'',6''), 6.88 (2H, d, J 8.4 Hz, H-3'',5''), 6.71 (2H, s, H-2',6'), 3. 87
+
+
(
3H, s, OCH
3
), 3.81 (3H, s, OCH
53 (10), 337 (20), 326 (11), 135 (11), 77 (7); Anal. Calcd. for C20
5.33; H, 5.51; N, 7.50.
-(4-Methoxyphenyl)-7-(3,4,5-trimethoxyphenyl)pyrazolo[1,5-a]pyrimidine
3
), 3.65 (6H, s, OCH
3
-3', 5'); EIMS m/z 369 [M+1] (21), 368 [M] (100), 367 (53),
3
6
6
20
H N
O
2 5
: C, 65.21; H, 5.47; N, 7.60. Found: C,
(8).
The
mixture
of
dimethylacetal 4aa (0.39 g, 1 mmol), 3-aminopyrazole (0.125 g, 1.5 mmol) and HCl (36%, 4 drops) in EtOH (1.6
mL) was refluxed for 2 h (TLC control), then evaporated to dryness. The deposit was triturated with NH
12%), filtered, washed with water, EtOH (3  0.4 mL), and dried in vacuo at 60 C to afford pyrimidinone 8.
4
OH
(
1
Yellowish crystalls; Yield 0.18 g, 45%; mp 165–167 C (EtOH); H NMR (DMSO-d
6
): δ 8.61 (1H, s, H-5), 8.20 (1H,
d, J 2.2 Hz, H-2), 7.21 (2H, d, J 8.6 Hz, H-2',6'), 6.91 (2H, d, J 8.6 Hz, H-3',5'), 6.84 (1H, d, J 2.2 Hz, H-3), 6.80 (2H,
+
s, H-2'',6''), 3.74 (3H, s, OCH
[
3
), 3.70 (3H, s, OCH
3
), 3.59 (6H, s, OCH
3
-3'', 5''); EIMS m/z 392 [M+1] (24), 391
+
M] (100), 390 (53), 344 (11), 290 (11), 262 (11), 219 (19), 218 (15), 192 (10), 180 (11), 165 (11), 164 (16), 152
(
10), 151 (11), 139 (10), 126 (10), 89 (14), 77 (11); Anal. Calcd. for C22
21
H N
3
O : C, 67.51; H, 5.41; N, 10.74.
4
Found: C, 67.60; H, 5.45; N, 10.67%.
-(4-Methoxyphenyl)-7-(3,4,5-trimethoxyphenyl)[1,2,4]triazolo[1,5-a]pyrimidine (9aa). The mixture of
6
dimethylacetal 4aa (0.39 g, 1 mmol), 3-amino-1,2,4-triazole (0.125 g, 1.5 mmol), and HCl (36%, 4 drops) in
EtOH (1.6 mL) was refluxed for 3 h (TLC control), then evaporated to dryness. The deposit was triturated with
abs. ether (1 mL), filtered and dried in vacuo to afford hydrochloride of 9aa as yellow crystals (0.28 g, 66%).
Hydrochloride of 9aa was triturated with NH OH (12%), filtered, washed with water and dried in vacuo at 60
4
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Chernyshova, N. B. et al.
1

C to afford pyrimidinone 9aa. White powder; Yield 0.24 g, 61%; mp 190–192 C; H NMR (DMSO-d
6
): δ 8.94
(
3
1H, s, H-2, 8.66 (1H, s, H-5), 7.24 (2H, d, J 8.7 Hz, H-2',6'), 6.95 (2H, d, J 8.7 Hz, H-3',5'), 6.85 (2H, s, H-2'',6''),
+
+
3
.76 (3H, s, OCH ), 3.71 (3H, s, OCH
3
), 3.60 (6H, s, OCH -3'', 5''); EIMS m/z 393 [M+1] (20), 392 [M] (100), 391
3
(22), 377 (18), 361 (10), 346 (10), 317 (16), 275 (16), 274 (12), 221 (10), 220 (12), 195 (16), 193 (13), 181 (9),
1
80 (13), 166 (10), 165 (13), 164 (11), 151 (11), 139 (10), 89 (7), 77 (7); Anal. Calcd. for C21
20
H N
4
O : C, 64.28; H,
4
5.14; N, 14.28. Found: C, 64.33; H, 5.17; N, 14.21.
General procedure for synthesis of triazolopyrimidines 9ab, 9bb, and 9ca from crude ketoacetals 4ab, 4bb,
and 4ca. The mixture of dimethylacetal 4ab, 4bb, or 4 ca (3.65 mmol), 3-amino-1,2,4-triazole (0.31 g, 3.65
mmol) and HCl (36%, 10 drops) in EtOH (10 mL) was refluxed for 5 h and kept overnight at 2–6 C. The deposit
was filtered and washed with hot EtOAc (3  10 mL) to afford hydrochlorides of triazolopyrimidines 9ab, 9bb,
4
and 9ca. Then they were stirred with NH OH (20%, 10 mL), filtered, reprecipitated from DMF by EtOH, and
dried to afford targeted pyrimidinones 9ab, 9bb, and 9ca.
6
-(3-Hydroxy-4-methoxyphenyl)-7-(3,4,5-trimethoxyphenyl)[1,2,4]triazolo[1,5-a]pyrimidine (9ab). White
1
crystals; Yield 0.94 g, 63%; mp 228–232 C; (9ab-hydrochloride: mp 161–164dec.C); H NMR (DMSO-d
6
): δ 9.05
(
8
1H, br, OH), 8.90 (1H, s, H-2), 8.64 (1H, s, H-5), 6.93 (1H, d, J 8.2 Hz, H-5'), 6.87 (2H, s, H-2'',6''), 6.73 (1H, dd, J
.2 Hz, J 2.0 Hz, H-6'), 6.71 (1H, d, J 2.0 Hz, H-2'), 3.76 (3H, s, OCH ), 3.72 (3H, s, OCH ), 3.61 (2H, s, H-3'',5'');
EIMS m/z 409 [M+1] (22), 408 [M] (100), 407 (6), 378 (6), 329 (9), 188 (10), 173 (8), 165 (9), 151 (8), 127 (9),
3
3
+
+
8
4 (49); Anal. Calcd. for C21
20
H N
4
O : C, 61.76; H, 4.94; N, 13.72. Found: C, 61.78; H, 4.98; N, 13.62%.
5
6
-(3-Hydroxy-4-methoxyphenyl)-7-(4-methoxyphenyl)[1,2,4]triazolo[1,5-a]pyrimidine (9bb). White crystals;
1
Yield 0.83 g, 65%; mp 258–260 C; H NMR (DMSO-d
6
): δ 9.07 (1H, s, OH), 8.86 (1H, s, H-2), 8.61 (1H, s, H-5),
7
.47 (2H, d, J 8.7 Hz, H-2'',6''), 6.99 (2H, d, J 8.7 Hz, H-3'',5''), 6.90 (1H, d, J 7.9 Hz, H-5'), 6.69 (1H, s, H-2'), 6.68
+
+
(1H, d, J 7.9 Hz, H-6'), 3.80 (3H, s, OCH
3
), 3.76 (3H, s, OCH
3
); EIMS m/z 349 [M+1] (21), 348 [M] (100), 347
: C, 65.51; H, 4.63;
(77), 333 (6), 332 (9), 305 (8), 152 (11), 135 (17), 92 (13), 77 (20); Anal. Calcd. for C19
16
H N
O
4 3
N, 16.08. Found: C, 65.58; H, 4.66; N, 16.02.
7
-(3-Hydroxy-4-methoxyphenyl)-6-(4-methoxyphenyl)[1,2,4]triazolo[1,5-a]pyrimidine (9ca). White crystals;
1
Yield 0.3 g, 24%; mp 278–282 C; H NMR (DMSO-d
6
): δ 9.22 (1H, s, OH), 8.89 (1H, s, H-2), 8.61 (1H, s, H-5),
7
3
3
4
.22 (2H, d, J 8.7 Hz, H-2',6'), 6.99 (1H, d, J 2.1 Hz, H-2'), 6.95 (1H, d, J 8.4 Hz, H-5'), 6.92 (2H, d, J 8.7 Hz, H-
+
',5'), 6.85 (1H, dd, J 8.4 Hz, J 2.1 Hz, H-6'), 3.80 (3H, s, OCH
48 [M] (100), 347 (71), 315 (10), 304 (5), 164 (10), 152 (12), 139 (.9). Anal. Calcd. for C19
3
), 3.76 (3H, s, OCH
3
); EIMS m/z 349 [M+1] (17),
: C, 65.51; H,
+
H
16
N
4 3
O
.63; N, 16.08. Found: C, 65.55; H, 4.68; N, 16.03.
34
Phenotypic sea urchin embryo assay. Adult sea urchins, Paracentrotus lividus L. (Echinidae), were collected
from the Mediterranean Sea on the Cyprus coast 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 96% EtOH. This procedure enhanced the solubility of the test compounds in the salt-
containing medium (seawater), as evidenced by 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%,
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Chernyshova, N. B. et al.
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. Combretastatin A-4
42
(
synthesized as reported previously ) served as a positive control. The antiproliferative activity was assessed
by exposing fertilized eggs (8–15 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, when control embryos reached 8-cell and early blastula stages, respectively. 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. Experiments with the sea urchin
embryos fulfill the requirements of biological ethics. The artificial spawning does not cause animal death,
embryos develop outside the female organism, and both post spawned adult sea urchins and the excess of
intact embryos are returned to the sea, their natural habitat.
Acknowledgements
The authors thank Dr. Ju. A. Strelenko from N. D. Zelinsky Institute of Organic Chemistry RAS for the
development a software for NMR spectra presentation.
References
1
. Habib, N. S.; Soliman, R.; El-Tombary, A. A.; El-Hawash, S. A.; Shaaban, O. G. Arch. Pharm. Res. 2007, 30,
511.
1
https://doi.org/10.1007/BF02977319
2
3
4
5
. Bazgira, A.; Khanaposhtani, M. M.; Soorki, A. A. Bioorg. Med. Chem. Lett. 2008, 18, 5800.
https://doi.org/10.1016/j.bmcl.2008.09.057
. Johar, M.; Manning, T.; Kunimoto, D. Y.; Kumara, R. Bioorg. Med. Chem. 2005, 13, 6663.
https://doi.org/10.1016/j.bmc.2005.07.046
. Fathalla, O. A.; Awad, S. M.; Mohamed, M. S. Arch. Pharm. Res. 2005, 28, 1205.
https://doi.org/10.1007/BF02978199
. Cosimelli, B.; Greco, G.; Ehlardo, M.; Novellino, E.; Da Settimo, F.; Taliani, S.; La Motta, C.; Bellandi, M.;
Tuccinardi, T.; Martinelli, A.; Ciampi, O.; Trincavelli, M. L.; Martini, C. J. Med. Chem. 2008, 51, 1764.
https://doi.org/10.1021/jm701159t
6
. Li, J.; Zhao, Y. F.; Zhao, X. L.; Yuan, X. Y.; Gong, P. Arch. Pharm. Chem. Life Sci. 2006, 339, 593.
https://doi.org/10.1002/ardp.200600098
Page 163
©ARKAT USA, Inc

Arkivoc 2017, iii, 151-165
Chernyshova, N. B. et al.
7
8
. Shallal, H. M.; Russu, W. A. Eur. J. Med. Chem. 2011, 46, 2043.
https://doi.org/10.1016/j.ejmech.2011.02.057
. Rostom, S. A. F.; Badr, M. H.; Abd El Razik, H. A.; Ashour, H. M. A.; Abd El Wahab, A. E. Arch. Pharm. Chem.
Life Sci. 2011, 344, 572.
https://doi.org/10.1002/ardp.201100077
9
1
1
. Pettit, G. R.; Singh, S. B.; Hamel, E.; Lin, C. M.; Alberts, D. S.; Garcia-Kendall, D. Experientia 1989, 45, 209.
https://doi.org/10.1007/BF01954881
0. Tron, G. C.; Pyrali, T.; Sorba, G.; Pagliai, F.; Busacca, S.; Genazzani, A. A. J. Med. Chem. 2006, 49, 3033.
https://doi.org/10.1021/jm0512903
1. Ohsumi, K.; Hatanaka, T.; Fujita, K.; Nakagawa, R.; Fukuda, Y.; Nihei, Y.; Suga, Y.; Morinaga, Y.; Akiyama, Y.;
Tsuji, T. Bioorg. Med. Chem. Lett. 1998, 8, 3153.
https://doi.org/10.1016/S0960-894X(98)00579-4
1
1
1
2. Bonezzi, K.; Taraboletti, G.; Borsotti, P.; Bellina, F.; Rossi, R.; Giavazzi, R. J. Med. Chem. 2009, 52, 7906.
https://doi.org/10.1021/jm900968s
3. Kaffy, J.; Pontikis, R.; Carrez, D.; Croisy, A.; Monneret, C.; Florent, J. C. Bioorg. Med. Chem. 2006, 14, 4067.
https://doi.org/10.1016/j.bmc.2006.02.001
4. Lee, S.; Kim, J. N.; Lee, H. K.; Yoon, K. S.; Shin, K. D.; Kwon, B. M.; Han, D. C. Bioorg. Med. Chem. Lett. 2011,
21, 977.
https://doi.org/10.1016/j.bmcl.2010.12.044
1
1
5. Wu, M.; Sun, Q.; Yang, C.; Chen, D.; Ding, J.; Chen, Y.; Lin, L.; Xie, Y. Bioorg. Med. Chem. Lett. 2007, 17, 869.
https://doi.org/10.1016/j.bmcl.2006.11.060
6. Romagnoli, R.; Baraldi, P. G.; Cruz-Lopez, O.; Lopez Cara, C.; Carrion, M. D.; Brancale, A.; Hamel, E.; Chen,
L.; Bortolozzi, R.; Basso, G.; Viola, G. J. Med. Chem. 2010, 53, 4248.
https://doi.org/10.1021/jm100245q
1
7. Demchuk, D. V.; Samet, A. V.; Chernysheva, N. B.; Ushkarov, V. I.; Stashina, G. A.; Konyushkin, L. D.;
Raihstat, M. M.; Firgang, S. I.; Philchenkov, A. A.; Zavelevich, M. P.; Kuiava, L. M.; Chekhun, V. F.; Blokhin,
D. Yu.; Kiselyov, A. S.; Semenova, M. N.; Semenov V. V. Bioorg. Med. Chem. 2014, 22, 738.
https://doi.org/10.1016/j.bmc.2013.12.015
1
1
2
8. Beale, T. M.; Allwood, D. M.; Bender, A.; Bond, P. J.; Brenton, J. D.; Charnock-Jones, D. S.; Ley, D. V.; Myers,
R. M.; Shearman, J. W.; Temple, J.; Unger, J.; Watts, C. A.; Xian, J. ACS Med. Chem. Lett. 2012, 3, 177.
https://doi.org/10.1021/ml200149g
9. Romagnoli, R.; Baraldi, P. G.; Salvador, M. K.; Preti, D.; Tabrizi, M. A.; Brancale, A.; Fu, X. H.; Li, J.; Zhang, S.
Z.; Hamel, E.; Bortolozzi, R.; Basso, G.; Viola, G. J. Med. Chem. 2012, 55, 475.
https://doi.org/10.1021/jm2013979
0. Jedhe, G. S.; Paul, D.; Gonnade, R. G.; Santra, M. K.; Hamel, E.; Nguyen, T. L.; Sanjayan, G. J. Bioorg. Med.
Chem. Lett. 2013, 23, 4680.
https://doi.org/10.1016/j.bmcl.2013.06.004
2
2
2
1. Schmitt, M. R.; Kirsch, D. R.; Harris, J. E.; Beyer, C. F.; Pees, K.-J.; Carter, P. A.; Pfrengle, W.; Albert, G. U.S.
Patent 20020068744, 2002.
2. Yusubov, M. S.; Zholobova, G. A.; Filimonova, I. L.; Chi, K. W. Russ. Chem. Bull. 2004, 53, 1735; Izvestiya
Akademii Nauk, Seriya Khimicheskaya 2004, 53, 1669.
3. Vu, B.; Mezey-Vandor, G.; Nogradi, M. Liebigs Ann. Chem. 1984, 4, 734.
https://doi.org/10.1002/jlac.198419840410
Page 164
©ARKAT USA, Inc

Arkivoc 2017, iii, 151-165
Chernyshova, N. B. et al.
2
4. Coulthard, E. C.; Marshall, J.; Pyman, F. L. J. Chem. Soc. 1930, 280.
https://doi.org/10.1039/JR9300000280
2
2
5. Petrov; A. A.; Kasatochkin, A. N. Russ. J. Org. Chem. 2013, 49, 502.
6. Allen, C. F. H.; Beilfuss, H. R.; Burness, D. M.; Reynolds, G. A.; Tinker, J. F.; VanAllan, J. A. J. Org. Chem.
959, 24, 787.
1
https://doi.org/10.1021/jo01088a014
2
2
2
7. Sirakawa, K. Yakugaku Zasshi 1959, 79, 1487.
https://doi.org/10.1248/yakushi1947.79.12_1487
8. Cook, J. W.; Gentles, R. P.; Tucker, S. H. Recl. Trav. Chim. Pay-B. 1950, 69, 343.
https://doi.org/10.1002/recl.19500690314
9. Cox, J. M.; Chu, H. D.; Kuethe, J. T.; Gao, Y.-D.; Scapin, G.; Eiermann, G.; He, H.; Li, X.; Lyons, K. A.; Metzger,
J.; Petrov, A.; Wu, J. K.; Xu, S.; Sinha-Roy, R.; Weber, A. E.; Biftu, T. Bioorg. Med. Chem. Lett. 2016, 26, 2622.
https://doi.org/10.1016/j.bmcl.2016.04.020
3
3
0. Zhang, Z.-T.; Xie, J.; Zhu, M.-L.; Xue, D. Synlett 2010, 12, 1825.
https://doi.org/10.1055/s-0030-1258105
1. Sheldrick, G. M. SADABS, v. 2.03, Bruker/Siemens Area Detector Absorption Correction Program, Bruker
AXS, Madison, Wisconsin, 2003.
3
3
3
2. Sheldrick, G. M. Acta Cryst. 2015, C71, 3.
3. Fax: +44 1223 336033; e-mail: deposit@ccdc.cam.ac.uk or www.ccdc.cam.ac.uk.
4. Semenova, M. N.; Kiselyov, A. S.; Semenov, V. V. BioTechniques 2006, 40, 765.
https://doi.org/10.2144/000112193
3
3
5. Semenov, V. V.; Semenova, M. N. Russ. Chem. Rev. 2015, 84, 134.
https://doi.org/10.1070/RCR4449
6. Ducki, S.; Rennison, D.; Woo, M.; Kendall, A.; Chabert, J. F. D.; McGown, A. T.; Lawrence, N. J. Bioorg. Med.
Chem. 2009, 17, 7698.
https://doi.org/10.1016/j.bmc.2009.09.039
3
7. Saxena, H. O.; Faridi, U.; Srivastava, S.; Kumar, J. K.; Darokar, M. P.; Luqman, S.; Chanotiya, C. S.; Krishna,
V.; Negi, A. S.; Khanuja, S. P. S. Bioorg. Med. Chem. Lett. 2008, 18, 3914.
https://doi.org/10.1016/j.bmcl.2008.06.039
3
3
8. Gutsche, C. D.; Jason, E. F.; Coffey, R. S.; Johnson, H. E. J. Am. Chem. Soc. 1958, 80, 5756.
https://doi.org/10.1021/ja01554a043
9. Kong, Y.; Wang, K.; Edler, M. C.; Hamel, E.; Mooberry, S. L.; Paige, M. A.; Brown, M. L. Bioorg. Med. Chem.
2010, 18, 971.
https://doi.org/10.1016/j.bmc.2009.11.003
4
0. Raghavan, S.; Manogaran, P.; Kuppuswami, B. K.; Venkatraman, G.; Narasimha, K. K. G. Med. Chem. Res.
2015, 24, 4157.
https://doi.org/10.1007/s00044-015-1453-2
4
4
1. Acharya, A.; Kumar, S. V.; Ila, H. Chem.Eur. J. 2015, 21, 17116.
https://doi.org/10.1002/chem.201501828
2. Semenov, V. V.; Kiselyov, A. S.; Titov, I. Y.; Sagamanova, I. K.; Ikizalp, N.; Chernysheva, N. B.; Tsyganov, D.
V.; Konyushkin, L. D.; Firgang, S. I.; Semenov, R. V.; Karmanova, I. B.; Semenova, M. N. J. Nat. Prod. 2010,
73, 1796.
https://doi.org/10.1021/np100427
Page 165
©ARKAT USA, Inc