Synthesis of novel pyrazole derivatives using organophosphorus, stibine, and arsine reagents and their antitumor activities

Article abstract of DOI:10.1515/znb-2015-0187

The reactions of 5-azido-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde (azidopyrazole) with several classes of organophosphorus reagents: phosphonium ylides, Wittig-Horner reagents, dialkylphosphonates, trialkylphosphites, tris(dialkylamino)phosphanes, triphenylstibane, triphenylarsane, and Lawesson's reagent are reported. Structural reasoning for the new products was based on compatible analytical and spectral data. The cytotoxic activity of most of the new products was evaluated against human breast carcinoma cell line (MCF7) and human hepatocellular carcinoma cell line(HepG2). Certain tested compounds showed promising results.

Full text of DOI:10.1515/znb-2015-0187

Z. Naturforsch. 2016; aop
Naglaa F. El-Sayed*, Ewies F. Ewies, Marwa El-Hussieny, Leila S. Boulos and
ElSayed M. Shalaby
Synthesis of novel pyrazole derivatives using
organophosphorus, stibine, and arsine reagents
and their antitumor activities
DOI 10.1515/znb-2015-0187
toward phosphonium ylides 2a–c, Wittig-Horner reagents
Received October 12, 2015; accepted December 19, 2015
3a,b, diethylphosphonate 4, trialkylphosphites 5a,b,
Abstract: The reactions of 5-azido-3-methyl-1-phenyl- tris(dialkylamino)phosphanes 5c,d, triphenylstibane 6a,
1H-pyrazole-4-carbaldehyde (azidopyrazole) with several triphenylarsane 6b, and Lawesson’s reagent (LR) 7 (Fig. 1).
classes of organophosphorus reagents: phosphonium
ylides, Wittig-Horner reagents, dialkylphosphonates, tri-
alkylphosphites, tris(dialkylamino)phosphanes, triph-
2 Results and discussion
enylstibane, triphenylarsane, and Lawesson’s reagent are
reported. Structural reasoning for the new products was
based on compatible analytical and spectral data. The 2.1 Chemistry
cytotoxic activity of most of the new products was evalu-
ated against human breast carcinoma cell line (MCF7) and When azidopyrazole 1 was treated with two molar equiva-
human hepatocellular carcinoma cell line(HepG2). Cer- lents of (cyanomethylene)triphenylphosphorane (2a) in
tain tested compounds showed promising results.
dry THF at ambient temperature for 1 h, the triphenyl phos-
phoranylidenesuccinonitrile 8a was obtained (85% yield).
Triphenylphosphane oxide (TPPO) was also isolated and
identified (Scheme 1). Structure 8a is confirmed by a
correct elemental analysis, IR, H, C, P NMR, and mass
spectra. Its IR spectrum revealed the presence of a strong
band at 2182 cm−1, which is ascribed to the N3 absorption
[13]. A possible explanation for the course of the forma-
Keywords: antitumor; azidopyrazole; crystal structure;
organophosphorus; stibine.
1
13
31
1 Introduction
The easily prepared pyrazole ring is an interesting tem- tion of compound 8a is shown in Scheme 1. Azidopyrazole
plate for combinatorial [1, 2] and medicinal chemistry [3, 1 reacted with P-ylide 2a to give the intermediate A with
4] as well as a prominent structural motif found in numer- expulsion of TPPO. Moreover, intermediate A reacted with
ous pharmaceutically active compounds [5]. Moreover, another molecule of the ylide 2a yielding intermediate B,
the pyrazole derivatives are known to have pharmaco- which cyclized to intermediate C. Rearrangement of the
logical, antimicrobial, antifungal [6], and antitumor [7] latter gave the new ylide 8a (Scheme 1). Furthermore, when
activities. In view of this and in continuation of our work azidopyrazole 1 was allowed to react with (acetylmethyl-
in organophosphorus chemistry [8–12], it was of consider- ene)triphenylphosphorane (2b) in dry THF at room tem-
able interest to investigate the behavior of azidopyrazole 1 perature, product 8b was separated as colorless crystals
with 75% yield (Scheme 1). Elemental and mass spectral
analysis of 8b led to an empirical formula C14H13N5O. The IR
*Corresponding author: Naglaa F. El-Sayed, Department of
spectrum displays absorption bands at ν ꢀ=ꢀ 1682 (Cꢀ=ꢀO) and
1593 (cyclic Cꢀ=ꢀN) cm−1 but lacks the azido function group
around at 2180 cm−1 [13]. The 1H NMR spectrum of 8b gave
signals at δ ꢀ=ꢀ 1.96 (s, 3H, CH3), at 2.64 ppm (s) for a methyl
group attached to the triazole ring, at 7.34–7.54 ppm (m,
6H, Harom, ꢀ=ꢀCH), and a singlet at 9.66 ppm for the aldehydic
proton. The 13C NMR spectrum of 8b added a good support
for the proposed structure and revealed signals at 8.3 and
Organometallic and Organometalloid Chemistry, National Research
Centre, 31-El-Bohouth Street, 12622 Dokki, Giza, A. R. Egypt,
e-mail: naglaa2778@yahoo.com
Ewies F. Ewies, Marwa El-Hussieny and Leila S. Boulos: Department
of Organometallic and Organometalloid Chemistry, National
Research Centre, 31-El-Bohouth Street, 12622 Dokki, Giza, A. R. Egypt
ElSayed M. Shalaby: X-Ray Crystallography Laboratory, Physics
Division, National Research Centre, 31-El-Bohouth Street, 12622
Dokki, Giza, A. R. Egypt
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ꢀꢀꢀꢁ
2ꢀ
ꢀN.F. El-Sayed et al.: Novel pyrazole derivatives and their antitumor activities
Ph
R
O
P
: B
Me
CHO
N3
O
P
EtO
EtO
Ph
P
EtO
EtO
R2
R2
R1
− H
Ph
N
N
3a, b
2a−c
Ph
a, R = H, R1 = CN
b, R = H, R1 = COMe
c, R = R1 = Br
a, R2 = CN
b, R2 = C6H4-OMe-4
1
O
P
EtO
EtO
EtO
EtO
R
R
S
S
S
H
P
OH
Ph
Ph
P
R
Ar
P
2
Ar
P
X
P
Ar
S
S
Ph
4
S
7
5a−d
6a−c
Ar = C6H4-OMe-4 (LR)
a, X = Sb
b, X = As
c, X = P
a, R = OMe
b, R = O-i Pr
c, R = N(Me)2
d, R = N(Et)2
Fig. 1:ꢀAzidopyrazole 1, organophosphorus reagents 2–6 and LR 7.
The purpose of this study was to determine the preferential site of attack by these reagents and synthesize new P-containing pyrazole
derivatives with anticipated biological activities.
H
Ph
CN
H
Ph3P
CN
H
CN
Ph3P
CN
H
Ph
P
Me
Me
Me
Ph
H
+2a
CN
N3
CN
N3
2a
N
N3
N
N
N
N
N
− TPPO
Ph
A
Ph
Ph
C
B
Ph
Ph3P
COMe
Me
CHO
Me
CHO
Me
CHO
N3
Ph
P
CN
Me
Ph
2b
N
N
N
N
N
O
N
N
CN
N
N
N
N
N
N
N3
Ph
− TPPO
N
Ph
Ph
1
PPh3
Me
Me
Ph
8a
D
8b
Ph
Br
Br
Br
Br
Ph
P
Me
Ph
2c
N
N3
N
− TPPO
Ph
8c
Scheme 1:ꢀThe behavior of azidopyrazole 1 toward stabilized phosphonium ylides 2a–c.
13.7 ppm for the two methyl groups, 136.3 and 136.6 ppm results of the structure refinement are presented in Table 2
(2C, triazole), 123.2–133.2 ppm (aromatic CH), 117.4, 136.7, (cf. Experimental Section).
and 151.6 ppm (quaternary carbon atoms C-4, C-3, C-5 of
Similarly, the Wittig reaction of (dibromomethylene)
pyrazole), and 183.4 ppm for a carbonyl group. The reac- triphenylphosphorane (2c) with azidopyrazole 1 afforded
tion presumably proceeds via 1,3-dipolar cycloaddition of 4-(2,2-dibromovinyl)pyrazole 8c (Scheme 1). The IR spec-
the azide group of azidopyrazole 1 to the Cꢀ=ꢀC bond of the trum of 8c revealed the presence of absorption bands at
ylide 2b followed by loss of TPPO from the cyclic intermedi- ν ꢀ=ꢀ 2125 (N3) [13] and 626 (C–Br) [17] cm−1. The structure
13
ate D to give 1,2,3-triazole derivative 8b [14–16] (Scheme 1). of 8c was also deduced from its NMR (1H, C) and mass
The molecular structure of 8b was also confirmed by X-ray spectra together with correct microanalyses (cf. Experi-
crystallographic analysis. An Ortep diagram of compound mental Section).
8b is shown in Fig. 2. The crystal data and experimental
Next, when azidopyrazole 1 was allowed to react
parameters used for intensity data collection and the final with two molar equivalents of diethyl(cyanomethyl)
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ꢁꢀꢀꢀ
ꢂ3
N.F. El-Sayed et al.: Novel pyrazole derivatives and their antitumor activitiesꢂ
via intermediate E. The assigned product 9b presumably
was formed via intermediate F. Under the influence of the
base present in the reaction medium, elimination of N2
was followed by hydrolysis F to give 9b and dialkyl phos-
phite (Scheme 2). The dialkyl phosphite was detected in
the water layer by the development of a violet color on
addition of 3,5-dinitrobenzoic acid [18]. The assigned cis
configuration for 9b is supported by the chemical shift
and coupling constant of the two protons CHꢀ=ꢀCHCN
(JHH ꢀ=ꢀ 7.6 Hz) (Scheme 2).
The reaction of azidopyrazole 1 with 4-methoxyben-
zylphosphonate 3b was also investigated. We have found
that the reaction of 1 with a molar equivalent of Wittig-
Horner reagent 3b in the presence of sodium ethoxide in
ethanol afforded the pure 9c (Scheme 2). The structural
assignment for compound 9c was based upon elemen-
tal analysis and spectroscopic data (cf. Experimental
Section). Product 9c can be obtained via intracyclization
of azide 1. It has been reported that o-azido-carbaldehyde
compounds can be intracyclized with good yield under
Fig. 2:ꢀOrtep view of 8b with crystallographic atom numbering. Dis-
placement parameters are drawn at the 30% probability level, H as
spheres with arbitrary radii. Selected bond lengths (Å) and angles
(deg): N1–N2 1.372(3), N3–N4 1.374(4), N4–N5 1.294(4); N1–N2–C6
120.2(2), N1–N2–C11 111.1(2), N3–N4–N5 106.8(3).
phosphonate (3a) in an ethanolic sodium ethoxide solu- the influence of a basic medium [19, 20].
tion at room temperature, products 9a and 9b were
The reaction of azidopyrazole 1 with diethyl phospho-
obtained (Scheme 2). The most important feature of struc- nate (diethyl hydrogen phosphite) (4) in dry toluene at
ture 9a is the presence of a signal at δ ꢀ=ꢀ 23.03 (s) ppm room temperature gave 5-amino-pyrazole-4-carbaldehyde
31
in its P NMR spectrum and the presence of an IR band 10 with 75% yield (Scheme 4). The assignment of structure
at 2114 cm−1, which is ascribed to the N3 absorption [13]. 10 was based on correct elemental analysis and molecular
Moreover, the 1H NMR spectrum of 9a revealed a triplet at weight determination (MS), H NMR, and IR comparison
1
δ ꢀ=ꢀ 1.32 ppm, which indicates CH3 protons of ethyl group with an authentic specimen [21].
with JHH ꢀ=ꢀ 13.4 Hz, singlet at 2.63 ppm (3H, CH3), doublet
A mechanism that accounts for the reaction of azi-
at 2.72 ppm with 2JHP ꢀ=ꢀ 22.89 Hz, doublet at 3.47 ppm with dopyrazole 1 with diethyl phosphonate (4) is depicted in
3JHP ꢀ=ꢀ 12.5 Hz (CHOH), and two quartet at 4.22 ppm (4H, Scheme 4. The initial attack of the phosphorus lone pair
3JHP ꢀ=ꢀ 12.5 Hz) for CH2 protons of ethyl group.
at the azide group with expulsion of a nitrogen molecule
A possible explanation for the course of the reaction gives intermediate G, which decomposes due to the una-
of 1 with Wittig-Horner reagent 3a is shown in Scheme 3. voidable moisture to give the aminopyrazole 10 and dieth-
The initial attack of the anion derived from phosphonic ylphosphite (Scheme 4) [21].
ester 3a on the most reactive center of 1 gave product 9a
We have found that the reaction of trimethyl phosphite
(5a) with azidopyrazole 1 in dry toluene proceeded at room
temperature to give chromatographically pure adduct 11a
(Scheme 5), which was separated as brown crystals (75%
yield). The results of elemental analysis and molecular
weight determination (MS) of this product correspond
to C14H18N3O4P. The 31P NMR spectrum has one signal at
δ ꢀ=ꢀ 23.2 ppm. The IR spectrum reveals the absence of an
N3 band [13] and the presence of both the (Cꢀ=ꢀN) band at
1558 and Cꢀ=ꢀO band at 1658 cm−1. The structure of 11a has
also been assigned on the basis of 1H and 13C NMR spectra
(cf. Experimental Section). Similarly, when 1 was reacted
with 5b in dry toluene at room temperature, the phos-
phorane imine product 11b was obtained with 85% yield
(Scheme 5). Structure elucidation of 11b was derived from
O
P
O
P
OEt
OEt
EtO
EtO
HO
NC
Me
CN
Me
O
P
3a
CN
OEt
OEt
+
+
HO
N
EtONa, EtOH
N
N3
NH2
N
N
Ph
9a
Ph
Me
CHO
9b
N
N3
N
Ph
1
OMe
O
P
O
EtO
EtO
Me
NH
N
3b
N
N
N
EtONa, EtOH
Ph
9c
Scheme 2:ꢀThe behavior of azidopyrazole 1 toward W-H reagents 3a,b. its spectral data (cf. Experimental Section).
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ꢀꢀꢀꢁ
4ꢀ
ꢀN.F. El-Sayed et al.: Novel pyrazole derivatives and their antitumor activities
O
O
P
OEt
OEt
O
P
EtO
EtO
OEt
OEt
O
CN
P
HO
Me
N
CHO
N3
Me
Me
3a
CN
CN
N
EtONa, EtOH
N
N3
O
P
N
N
N
N
Ph
1
N
N
Ph
− HO
OEt
OEt
Ph
9a
E
CN
CN
O
P
Me
Me
unavoidable moisture
hydrolysis, − N2
OEt
+
HO
OEt
N
N
NH2
N
N
N
O
N
Ph
Ph
N
P
OEt
OEt
9b
F
Scheme 3:ꢀFormation mechanism of 9a,b.
O
P
EtO
Me
CHO
N
EtO
EtO
Me
CHO
N3
Me
N
CHO
N
P
OH
H
EtO
OH
P
4
N
OEt
OEt
N
OH
OEt
N
− N2
N
Staudinger-like reaction
N
Ph
N
P
Ph
1
Ph
OEt
G
N
Me
CHO
H2O
O
N
N
HO
+
P
OEt
OEt
NH2
Ph
10
Scheme 4:ꢀThe behavior of azidopyrazole 1 toward diethylphosphonate 4.
PR3
5a, b
also been assigned on the basis of elemental analysis,
Me
CHO
− N2
1
13
31
MS, IR, H, C, P NMR spectral data (cf. Experimental
Section). The structure of 11c was also confirmed by
X-ray crystallographic analysis. An Ortep diagram of
the molecular structure of compound 11c in the crystal
is shown in Fig. 3. Furthermore, this study has been
extended to include the reaction of azidopyrazole 1 with
triphenylstibane (6a) and triphenylarsane (6b) to estab-
lish whether they would behave in a similar manner as
triphenylphosphane (6c) [22–25]. A different behavior is
observed in the reaction of 1 with 6a and 6b, yielding
5-azido-4-(triphenylstiboranylidene)methyl-1H-pyrazole
12a in 75% yield and 4-(triphenylarsoranylidene)methyl-
1H-pyrazole 12b in 45% yield with extrusion triphenyl-
stibane oxide and triphenylarsane oxide, respectively
(Scheme 6).
R
N
N P
R
N
a, R= OMe
b, R= O-i Pr
R
Ph
11a, b
Me
CHO
N3
N
N
Ph
1
Me
CHO
N N
PR3
R
5c, d
N
N
P
R
N
R
c, R = N(Me)2
d, R= N(Et)2
Ph
11c, d
Scheme 5:ꢀThe behavior of azidopyrazole 1 toward trialkylphosphites
5a,b and trisdialkylaminophosphines 5c,d.
When the pyrazole-4-carbaldehyde 1 was stirred at
ambient temperature with LR, the 5-sulfido-1,2,3,4,5-
thiatriazaphosphole 13 was obtained in good yield
Meanwhile, performing the reaction of 1 with through cycloaddition reaction between the monomeric
tris(dialkylamino)phosphanes 5c,d led to the forma- form of LR 7 and the azido group of 1 [26, 27] (Scheme 7).
tion of the [tris(dialkylamino)phosphoranylidene]tria- The elemental microanalysis, H, 13C, 31P NMR, and MS
1
zenes 11c,d (Scheme 5). The structures of 11c,d have data agreed with structure 13.
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ꢁꢀꢀꢀ
N.F. El-Sayed et al.: Novel pyrazole derivatives and their antitumor activitiesꢂ ꢂ5
The structure assigned to 13 was also based on a 13C NMR
spectrum that shows signals of 55.7 (OCH3), 105.0 (quater-
nary C-4, pyrazole), 113.9–145.5 (aromatic C-H, C-3, and C-5
of pyrazole), and 161.6, 161.7 (Cꢀ=ꢀO) ppm.
2.2 Description of the crystal and molecular
structures of 8b and 11c
Ortep plots of the molecular structures of compounds 8b
and 11c are shown in Figs. 2 and 3, respectively. Both com-
pounds crystallize in the monoclinic system possessing
four molecules in the unit cell with space groups Cc for 8b
and P2/n for 11c. The asymmetric unit in the two structures
contains only one molecule. In 8b, there is a significant
twist between the pyrazole ring and both attached triazole
and benzene rings with the dihedral angles being 49.9(2)°
and 62.3(2)°, respectively. The same is observed in com-
pound 11c, with a significant twist between the pyrazole
ring and the attached benzene ring with the dihedral angle
being 38.4(2)°. An important feature to note in 8b is that
the molecules are linked into chains by one weak hydro-
gen bond of the C–H···O type and a set of H···π and O···π
interactions as listed in Table 1 and illustrated in Fig. 4.
Molecules 11c are linked into chains by one hydrogen bond
of the C–H···O type listed in Table 2 and illustrated in Fig. 5.
Fig. 3:ꢀOrtep view of 11c with crystallographic atom numbering.
Displacement parameters are drawn at the 30% probability level,
H atoms as spheres with arbitrary radii. Selected bond lengths (Å) and
angles (deg): P1–N5 1.579(4), P1–N6 1.709(4), P1–N7 1.714(4), P1–N8
1.704(4); N5–P1–N6 113.2(4), N5–P1–N7 110.8(4), N6–P1–N7 110.3(4),
N5–P1–N8 106.1(4), N6–P1–N8 109.9(4), N7–P1–N8 106.3(4).
The IR spectrum of structure compound 13 revealed
the absence of an N3 absorption band [13] and showed 2.3 Anticancer screening
bands at 625 cm−1 (Pꢀ=ꢀS), 1585 cm−1 (Cꢀ=ꢀN), 1696 cm−1 (Cꢀ=ꢀO).
The 1H NMR shifts of compound 13 were at δ ꢀ=ꢀ 2.46 (s, 3H, Cancer is one of the diseases with a high mortality rate [28].
CH3), 3.76 (s, 3H, OCH3), and 6.95–7.65 (m, 9H aromatic). The current trend of research focuses on investigating the
Ph
Ph
Ph
Ph
P
X
Me
CHO
N
Ph
Ph
6c
Ph
6a,b
Me
CHO
N3
Me
X
Ph
Ph
Ph
Ph
N3
N
P
Ph
N
N
a, X = Sb
b, X= As
N
N
N
Ph
Ph
Ph
12c
1
12a, b
Scheme 6:ꢀThe behavior of azidopyrazole 1 toward TPP, TPSb, TPAs 7a–c.
S
S
S
S
Me
CHO
N3
Ar
P
S
Me
CHO
2
Ar
P
P
Ar
S
7
N
N
N
N
S
N
N
N
Ar = C6H4-OMe-4
P
S
Ph
1
Ph
Ar
13, Ar = C6H4-OMe-4
Scheme 7:ꢀThe behavior of azidopyrazole 1 toward LR 8.
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ꢀꢀꢀꢁ
6ꢀ ꢀN.F. El-Sayed et al.: Novel pyrazole derivatives and their antitumor activities
Table 1:ꢀHydrogen bond geometry and non-bonding contacts (Å,
deg) of compound 8b.a
D–H···A
ꢁ
D–H
ꢁ
H···A
ꢁ
D···A
ꢁ
D–H···A
C14–H142···O1iꢃ
0.95
0.95
Y–X
ꢃ
ꢃ
ꢃ
2.55
2.99
X···Cg
ꢃ
ꢃ
ꢃ
3.499(5)ꢃ
3.469(4)ꢃ
175
C1–H11···Cg2
Y–X···Cg
ꢃ
ꢃ
ꢃ
113
Y···Cg
ꢃ
Y–X···Cg
90.7(2)
C10–O1··· Cg1i
1.207(4)ꢃ
3.195(3)ꢃ
3.430(4)ꢃ
aSymmetry codes: (i) x, 1 – y, –1/2 + z; Cg1 ꢃ=ꢃ N1–N2–C11–C9–C7;
Cg2 ꢃ=ꢃ N3–N4–N5–C12–C13.
Fig. 5:ꢀPacking of the molecules in the unit cell of compound 11c
showing weak hydrogen bonds as dashed lines.
Fig. 4:ꢀPacking of the molecules in the unit cell of compound 8b
showing various types of intermolecular interactions as dashed
lines.
Table 3:ꢀCytotoxic activity of azidopyrazole derivatives against
MCF7 and HepG2.
Compounds ꢁ
Doxorubicinb ꢃ
MCF7 IC50 (μm)ꢁ
HepG2 IC50 (μm)a
Table 2:ꢀHydrogen bond geometry and non-bonding contacts (Å,
deg) of compound 11c.a
10.2 0.13ꢃ
21.27 0.85ꢃ
10.12 0.65ꢃ
1.012 0.13ꢃ
8.56 0.23ꢃ
8.36 0.11ꢃ
19.31 1.65ꢃ
13.33 0.28ꢃ
24.33 0.17ꢃ
24.78 1.75ꢃ
15.22 0.92ꢃ
26.23 0.88ꢃ
24.09 0.60ꢃ
32.23 0.38ꢃ
5.61 0.037
18.94 2.14
12.52 0.220
24.1 1.78
1
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
8a
D–H···A
ꢁ
D–H ꢁ
H···Aꢁ
D···A
ꢁ
D–H···A
8b
C17–H173···O1iꢁ
0.95ꢁ
2.34ꢁ
3.273(14)ꢁ
165
9a
20.1 0.42
9b
16.88 0.29
14.85 0.39
25.96 1.39
12.99 4.81
19.42 1.29
16.31 0.23
36.46 0.86
12.58 5.34
37.6 0.13
aSymmetry code: (i) 3/2 – x, 1/2 + y, 3/2 – z.
11a
11b
11c
11d
12a
12b
12c
13
biological activity of newly synthesized compounds, aiming
to develop new anticancer drugs. Derivatives of pyrazole
have received considerable attention owing to their diverse
chemotherapeutic potentials including their versatile anti-
cancer activities [7]. Thus, the current study was tailored
to screen in vitro cytotoxic and growth inhibitory activi-
ties of most of the newly synthesized azidopyrazole com-
pounds on human breast carcinoma cell line (MCF7) and
human hepatocellular carcinoma cell line (HepG2). MCF7
aIC50 value was defined as the concentration at which 50% survival
of cells was observed and it is expressed as mean SE. DMSO was
used as negative control. bDoxorubicin was used a reference drug.
and HepG2 cells were cultured as monolayers and treated showed less antitumor activity against HepG2 cells with
with our compounds for 48 h. The rate of proliferation was higher IC50 than doxorubicin (IC50 ꢀ=ꢀ 5.61 μm). The study
assessed by the Alamar blue reduction assay. According to revealed that azidopyrazole derivatives have a potential
current data, compounds 8a, 8b, 9a, and 9b demonstrate a antitumor activity against MCF7 cells; however, those
higher antitumor activity against MCF7 cells with low cyto- derivatives are less potent against HepG2 cells. In addition,
toxic activity (IC50 ꢀ=ꢀ 10.12, 1.012, 8.56, and 8.36 μm, respec- the current data stressed that compounds 9a, 9b, and 8b
tively) than that of the reference drug (doxorubicin, IC50 ꢀ=ꢀ have the most effective antitumor activity. Compound 8b
10.2 μm) (Table 3). Meanwhile, azidopyrazole derivatives has a triazole ring that was previously reported to have a
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N.F. El-Sayed et al.: Novel pyrazole derivatives and their antitumor activitiesꢂ
potentially broad range of biological and pharmacological Varian MAT 311A spectrometer. Elemental analyses were
activities [29]. Additionally, compounds 9a and 9b have a performed using an Elementar Vario E1 instrument. The
significant antitumor activity that may be associated with reported yields are of pure isolated materials obtained by
presence of the nitrile (CN) group, which has previously column chromatography on silica gel 60 (Merk). 5-Azido-
been reported to have a strong biological activity due to 3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde (1) was
their strong hydrogen bond acceptor leading to strong ver- prepared according to a reported method [31, 32].
satile interactions with different proteins [30]; therefore,
compounds 9a, 9b, and 8b have promising therapeutic
effects that can be further studied in vivo and in vitro.
4.2 Reaction of (cyanomethylene)triph-
enylphosphorane (2a) with 1
3 Conclusion
A mixture of 2a (0.60 g, 2 mmol) and 0.27 g (1 mmol) of 1
in dry THF (30 mL) was stirred for 1h. The volatile mate-
The reaction of 5-azido-3-methyl-1-phenyl-1H-pyrazole- rial was evaporated under reduced pressure. The residue
4-carbaldehyde (1) with phosphonium ylides 2a–c led to was subjected to silica gel column chromatography to give
different products 8a–c depending on the nature of the product 8a. TPPO was also isolated and identified.
phosphonium ylides as well as the stability of the addition
products. The reaction of 1 with Wittig-Horner reagents 3a
and 3b gave different product 9a,b and 9c depending on 4.2.1 2-((5-Azido-3-methyl-1-phenyl-1H-pyrazol-
the nature of the methane phosphonate anion as well as
the stability of the addition products. In the reaction of 1
with diethyl phosphonate, 4,5-amino-3-methyl-1-phenyl-
4-yl)methyl)-3-(triphenylphosphoranylidene)
succinonitrile (8a)
1H-pyrazole-4-carbaldehyde (10) and dialkyl phosphite Eluent: petroleum ether-ethyl acetate (70:30, v/v). Product
were the sole reaction products. The reaction of azidopyra- 8a was separated as deep red crystals, yield 85%; m.p.
zole 1 with trialkyl phosphites 5a,b and tris(dialkylamino) 262°C–263°C. – IR (KBr): ν ꢀ=ꢀ 1587 (cyclic Cꢀ=ꢀN), 2182 (N3),
1
phosphines 5c,d yielded different products. These pro- 2235, 2364 (2 CN) cm−1. – H NMR (300 MHz, DMSO):
cesses can be considered as new and simple routes for the δ ꢀ=ꢀ 1.76 (s, 3 H, CH3), 1.99 (t, 1H, CH), 3.60 (d, 2 H, CH2),
preparation of various organophosphorus and heterocy- 7.65–7.84 (m, 20H, Harom) ppm. – 13C NMR (75 MHz, DMSO):
clic compounds that cannot be obtained by other conven- δ ꢀ=ꢀ 14.6 (CH3), 25.6 (CH2), 67.5 (CH), 99.9 (C-4, pyrazole),
tional methods. The compounds have shown promising 115.8, 118.21 (2 CN), 121.5, 121.6 (Cꢀ=ꢀP, Jc,p ꢀ=ꢀ 98.5 Hz),118.4–
anticancer activities against the human breast cancer cell 144.2 (aromatic C–H, C-3, C-5 of pyrazole) ppm. – 31P NMR:
+
lines (MCF7) at very low concentrations.
δ ꢀ=ꢀ 21.88 ppm. – MS (EI, 70 eV): m/z (%) ꢀ=ꢀ 551(5) [M] , 384
+
(100) [M–167] . – C33H26N7P (551.58): calcd. C 71.86, H 4.75,
N 17.78, P 5.62; found C 71.89, H 4.77, N 17.98, P 5.72.
4 Experimental section
4.3 Reaction of (acetylmethylene)triph-
enylphosphorane (2b) with 1
4.1 Chemistry
Melting points were determined in open glass capillar- A mixture of 1 mmol of 2b (0.31 g) and 1 mmol of 1 (0.27 g)
ies using an Electrothermal IA 9100 series digital melting in dry THF (30 mL) was stirred for 2 h. The volatile mate-
point apparatus (Electorthermal, Essex, UK), IR spectra rial was evaporated under reduced pressure. The residue
were measured as KBr pellets with a Perkin-Elmer infra- was subjected to silica gel column chromatography to give
1
13
cord spectrophotometer model 157. The H and C NMR product 8b. TPPO was also isolated and identified.
spectra were recorded in CDCl3 or [D6]dimethyl sulfoxide
(DMSO) as solvents on a Jeol-500 spectrometer (1H: 500
MHz; 13C: 125 MHz), and the chemical shifts are recorded 4.3.1 3-Methyl-5-(5-methyl-1H-1,2,3-triazol-1-yl)-1-
31
in δ values relative to TMS as internal reference. The P
phenyl-1H-pyrazole-4-carbaldehyde (8b)
NMR spectra were taken with a Varian CFT-20 spectrome-
ter (vs. external 85% H3PO4 as standard). The mass spectra Eluent: petroleum ether-ethyl acetate (80:20, v/v). Product
were recorded at 70 eV with a Kratos MS equipment or 8b was separated as colorless crystals, yield 75%; m.p.
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8ꢀ ꢀN.F. El-Sayed et al.: Novel pyrazole derivatives and their antitumor activities
99°C–101°C. – IR (KBr): ν ꢀ=ꢀ 1593 (Cꢀ=ꢀN), 1682 (Cꢀ=ꢀO) cm−1. ethyl acetate, dried over anhydrous sodium sulfate, and
– 1H NMR (500 MHz, CDCl3): δ ꢀ=ꢀ 1.96 (s, 3H, CH3), 2.64 (s, the extracts were evaporated under reduced pressure. The
3 H, CH3), 7.34–7.54 (m, 6H, Harom, ꢀ=ꢀCH), 9.66 (s, 1H, CHO) residue was applied to silica gel column chromatography
ppm. – 13C NMR (125 MHz, CDCl3): δ ꢀ=ꢀ 8.3 (CH3), 13.7 (CH3), to give compounds 9a,b and/or 9c.
136.3, 136.6 (2C, triazole), 123.2–133.2 (aromatic CH), 117.4,
136.7, 151.6 (C-4, C-3, C-5of pyrazole), 183.4 (CHO) ppm. –
+
+
MS (EI, 70 eV): m/z (%) ꢀ=ꢀ 268 (10) [M+1] , 267(8) [M] . – 4.5.1 Diethyl 2-(5-azido-3-methyl-1-phenyl-1H-pyrazol-4-
C14H13N5O (267.29): calcd. C 62.91, H 4.90, N 26.20; found C
62.95, H 4.87, N 26.18.
yl)-1-cyano-2-hydroxyethylphosphonate (9a)
Eluent: petroleum ether-ethyl acetate (80:20, v/v). Product
9a was separated as pale yellow crystals, yield 20%; m.p.
86°C–87°C. – IR (KBr): ν ꢀ=ꢀ 1024 (Pꢀ=ꢀO), 1592 (Cꢀ=ꢀN), 2114
(N3), 2359 (CN), 3441 (OH) cm−1. – 1H NMR (500 MHz, CDCl3):
δ ꢀ=ꢀ 1.32 (t, 6H, JHH ꢀ=ꢀ 13.4 Hz, P–OCH2CH3), 2.63 (s, 3H, CH3),
4.4 Reaction of (dibromomethylene)triph-
enylphosphorane (2c) with 1
Triphenylphosphane (0.5 g, 0.1 mmol) was added to a well- 2.72 (d, 1H, CH–P, 2JHP ꢀ=ꢀ 22.9 Hz), 3.47 (d, 1H, 3JHP ꢀ=ꢀ 12.5 Hz,
3
stirred solution of carbon tetrabromide (0.3 g, 0.05 mL) in CHOH), 4.22 (2q, 4H, JHP ꢀ=ꢀ 12.5 Hz, P–O–CH2),7.92–8.72
DCM (3 mL). When the solution became orange (i.e. 2c was (m, 5H, Harom), 8.96 (s, 1H, OH) ppm. – 13C NMR (125 MHz,
formed) [33], pyrazole 1 (0.27 g, 0.1 mmol) was added, and CDCl3): δ ꢀ=ꢀ 13.6 (CH3), 16.4 (O–CH2–CH3), 40.5 (C–P, JC P ꢀ=ꢀ
the mixture was stirred at room temperature for 8 h. After 125 Hz), 63.58, 64.49 (CH2), 69.73 (CH–OH), 101.9 (CN),
evaporation of the volatile material, the buff residual sub- 104.5 (C-4, pyrazole), 124.2–144.6 (aromatic C–H, C-3, C-5
stance was chromatographed on a silica gel column. TPPO of pyrazole) ppm. – 31P NMR: δ ꢀ=ꢀ 23.03 ppm. – MS (EI, 70
+
was also isolated and identified.
eV): m/z (%) ꢀ=ꢀ 386 (100) [M–OH] . – C17H21N6O4P (404.36):
calcd. C 50.50, H 5.23, N 20.78, P 7.66; found C 50.48, H 5.11,
N 20.66, P 7.93.
4.4.1 5-Azido-4-(2,2-dibromovinyl)-3-methyl-1-phenyl-
1H-pyrazole (8c)
4.5.2 (Z)-3-(5-Amino-3-methyl-1-phenyl-1H-pyrazol-4-yl)
Eluent: petroleum ether-ethyl acetate (80:20, v/v). Product
8c was separated as buff powder, yield 35%. – m.p.
acrylonitrile (9b)
158°C–161°C. – IR (KBr): ν ꢀ=ꢀ 626 (C–Br), 1593 (Cꢀ=ꢀN), 2125 Eluent: petroleum ether-ethyl acetate (70:30, v/v). Product
(N3) cm−1. – 1H NMR (500 MHz, [D6]DMSO): δ ꢀ=ꢀ 2.04 (s, 3H, 9b was separated as buff crystals, yield 40%; m.p. 165°C–
13
CH3), 7.58–7.99 (m, 6H, Harom), 8.59 (s, 1H, CH) ppm. – C 166°C. – IR (KBr): ν ꢀ=ꢀ 1599 (Cꢀ=ꢀN), 2362 (CN), 3362, 3302,
NMR (125 MHz, [D6]DMSO): δ ꢀ=ꢀ 13.8 (CH3), 99.8 (ꢀ=ꢀC(Br)2), 3205 (NH2) cm−1. – 1H NMR (500 MHz, CDCl3): δ ꢀ=ꢀ 2.55 (s, 3H,
100.2 (C-4, pyrazole), 124.8–140.3 (aromatic C–H), 135.2, CH3), 6.47 (d, 1H, J ꢀ=ꢀ 7.6 Hz, CHCN), 7.76 (1H, CHꢀ=ꢀCH, JHH ꢀ=ꢀ 7.6
150.3 (C-3, C-5 of pyrazole) ppm. – MS (EI, 70 eV): m/z Hz), 7.92–8.72 (m, 7H, Harom, NH2) ppm. – 13C NMR (125 MHz,
(%) ꢀ=ꢀ 380, 382, 384 [1:2:1] (15). – C12H9Br2N5 (383.04): calcd. CDCl3): δ ꢀ=ꢀ 13.8 (CH3), 98.5 (C–CN), 112.5 (C-4, pyrazole),
C 37.63, H 2.37, Br 41.72, N 18.28; found C 37.71, H 2.23, Br 123.2–149.6 (aromatic C–H, C-3, C-5 of pyrazole) ppm. – MS
+
41.64, N 18.19.
(EI, 70 eV): m/z (%) ꢀ=ꢀ 224(5) [M] . – C13H12N4 (224.26): calcd.
C 69.62, H 5.39, N 24.98; found C 69.73, H 5.42, N 25.01.
4.5 Reaction of diethyl cyanomethylphos-
phonate (3a) or diethyl 4-methoxyben-
zylphosphonate (3b) with 1
4.5.3 5-Methyl-7-phenyl-3H-pyrazolo[3,4-d][1,2,3]
triazin-4(7H)-one (9c)
A solution of sodium ethoxide (0.14 g, 2 mmol) in absolute Eluent: petroleum ether-ethyl acetate (95:5, v/v). Product
ethanol (30 mL) was treated with an equimolar amount 9c was separated as orange crystals, yield 65%; m.p.
of diethyl (cyanomethyl)phosphonate (3a) or diethyl 160°C–162°C. – IR (KBr): ν ꢀ=ꢀ 3334 (NH), 1672 (Cꢀ=ꢀO), 1665
4-(methoxybenzyl)phosphonate (3b) (2 mmol). Azidopyra- (Nꢀ=ꢀN), 1619 (Cꢀ=ꢀC), 1596 (Cꢀ=ꢀN) cm−1. – 1H NMR (500 MHz,
zole 1 (0.27 g, 1 mmol) was added, and the reaction mixture CDCl3): δ ꢀ=ꢀ 2.36 (s, 3 H, CH3), 4.51 (s, 1 H, NH, exchange-
13
was stirred at room temperature for 4 h (TLC). The mixture able with D2O), 7.45–7.57 (m, 5 H, CHarom) ppm. – C NMR
was poured on a small amount of water, extracted with (CDCl3): δ ꢀ=ꢀ 14.3 (CH3), 96.3, 152.5 (cyclic Cꢀ=ꢀC), 124.9–138.7
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N.F. El-Sayed et al.: Novel pyrazole derivatives and their antitumor activitiesꢂ
(aromatic, C–H), 149.2 (Cꢀ=ꢀN), 162.4 (Cꢀ=ꢀO) ppm. – MS (EI, 4.7.2 Triisopropyl (4-formyl-3-methyl-1-phenyl-1H-
+
70 eV): m/z (%) ꢀ=ꢀ 227 (50) [M] . – C11H9N5O (227.22): calcd.
pyrazol-5-yl)phosphorimidate (11b)
C 58.14, H 3.99, N 30.82; found: C 58.12, H 4.01, N 30.74.
Product 11b was separated as brown crystals (benzene),
yield 85%; m.p. 257°C–259°C. – IR (KBr): ν ꢀ=ꢀ 1082 (P–O–
4.6 Reaction of diethyl hydrogenphosphite
(4) with 1
1
alkyl), 1592 (Cꢀ=ꢀN), 1655 (Cꢀ=ꢀO) cm−1. – H NMR (500 MHz,
CDCl3): δ ꢀ=ꢀ1.26 (m, 18H, 3 CH(CH3)2), 2.46 (s, 3H, CH3), 3.96
(m, 3H, CH, 3 CH(CH3)2), 7.22–7.83 (m, 5H, Harom), 10.11 (s, H,
CHO) ppm. – 13C NMR (125 MHz, CDCl3): δ ꢀ=ꢀ 13.9 (CH3), 22.8
(CH3, 3 CH(CH3)2), 74.2 (d, 2JCP ꢀ=ꢀ 11 Hz, P–O–CH), 104.5 (C-4,
pyrazole), 123.3–152.5 (aromatic C–H, C-3, C-5 of pyrazole),
179.4 (Cꢀ=ꢀꢀ=ꢀO) ppm. – 31P NMR: δ ꢀ=ꢀ 23.7 ppm. – MS (EI,
A mixture of 1 mmol of 4 (0.17g) and 1 mmol of 1 (0.27 g) in
dry toluene (30 mL) was stirred for 30 min. After evapora-
tion of the volatiles under reduced pressure, the residue
was subjected to silica gel column chromatography to
yield structure 10.
+
70 eV): m/z (%) ꢀ=ꢀ 407 (100) [M] . – C20H30N3O4P (407.44):
calcd. C 58.96, H 7.42, N 10.31, P 7.60; found C 59.00, H, 7.22,
N, 10.52, P7.61.
4.6.1 5-Amino-3-methyl-1-phenyl-1H-pyrazole-4-
carbaldehyde (10)
4.8 Reaction of tris(dialkylamino)phos-
phanes 5c,d with 1
Eluent: petroleum ether-ethyl acetate (80:20, v/v). Product
10 was separated as colorless crystals, yield 75%; m.p.
96°C–98°C. – IR (KBr): ν ꢀ=ꢀ 1543 (Cꢀ=ꢀN), 1606 (Cꢀ=ꢀC), 1644
A mixture of 5c or 5d (1 mmol) and 0.27 g (1 mmol) of 1
in dry benzene (30 mL) was stirred for 3 h. After evapora-
tion of the volatile material under reduced pressure, the
residue was subjected to silica gel column chromatogra-
phy to yield 11c,d.
1
(Cꢀ=ꢀO), 3398, 3288, 3187 (NH2) cm−1. – H NMR (500 MHz,
CDCl3): δ ꢀ=ꢀ 2.64 (s, 3 H, CH3), 5.32 (s, 2H, NH2 exchangeable
with D2O), 7.34–7.54 (m, 5H, Harom), 9.66 (s, 1H, CHO) ppm.
+
– MS (EI, 70 eV): m/z (%) ꢀ=ꢀ 201.22 (100) [M] . Compound
10 gave a correct elemental analysis and it was character-
ized by TLC analysis (one spot) and IR comparison with an
authentic specimen [21].
4.8.1 3-Methyl-1-phenyl-5-[(tris(dimethylamino)
phosphoranylidene]triaz-1-en-1-yl)-1H-pyrazole-4-
carbaldehyde (11c)
4.7 Reaction of trialkylphosphite (5a,b) with 1
A mixture of 1 mmol of 5a or 5b and 1 mmol of 1 in dry Eluent: petroleum ether-ethyl acetate (80:20, v/v). Product
toluene (30 mL) was stirred for 1–2 h (TLC). After evapora- 11c was separated as yellow crystals, yield 75%; m.p.
tion of the volatile material, the residue was recrystallized 140°C–142°C. – IR (KBr): ν ꢀ=ꢀ1599 (Cꢀ=ꢀN), 1651 (Cꢀ=ꢀO), 1665
1
from a proper solvent to yield compound 11a,b.
(Nꢀ=ꢀN) cm−1. – H NMR (300 MHz, CDCl3): δ ꢀ=ꢀ 2.36 (s, 3 H,
3
CH3), 2.50, 2.64, 3.36 (d, JHP ꢀ=ꢀ 9.3 Hz, 18H, P[N(CH3)2]3),
13
7.33–7.69 (m, 5H, Harom), 9.71 (s, H, CHO) ppm. – C NMR
4.7.1 Trimethyl (4-formyl-3-methyl-1-phenyl-1H-pyrazol-
(75 MHz, CDCl3): δ ꢀ=ꢀ 14.8 (CH3), 37.0 (N(CH3)2), 110.8 (C-4,
5-yl)phosphoramidate (11a)
pyrazole), 124.7–149.6 (aromatic C–H, C-3, C-5 of pyrazole),
31
186.9 (Cꢀ=ꢀO) ppm. – P NMR: δ ꢀ=ꢀ 29.8 ppm. – MS (EI, 70
+
Product 11a was separated as brown crystals (benzene), eV): m/z (%) ꢀ=ꢀ 390 (10) [M] . – C17H27N8OP (390.42): calcd.
yield 75%; m.p. 238°C–239°C. – IR (KBr): ν ꢀ=ꢀ 1063 (P–O– C 52.30, H 6.97, N 28.70, P 7.93; found C 52.23, H 6.82, N
1
alkyl), 1558 (Cꢀ=ꢀN), 1658 (Cꢀ=ꢀO) cm−1. – H NMR (500 MHz, 28.73, P 7.87.
CDCl3): δ ꢀ=ꢀ 2.66 (s, 3H, CH3), 3.36–3.52 (d, 3JHP ꢀ=ꢀ 11.0 Hz, 9H,
P(OCH3)3), 7.25–7.97 (m, 5H, Harom), 10.24 (s, H, CHO) ppm.
13
– C NMR (125 MHz, CDCl3): δ ꢀ=ꢀ 13.9 (CH3), 54.9, 55.1 (d, 4.8.2 3-Methyl-1-phenyl-5-[(tris(diethylamino)
2JCP ꢀ=ꢀ 11 Hz, P–(OCH3)3), 104.5 (C-4, pyrazole), 123.2–152.6
(aromatic C–H, C-3, C-5 of pyrazole), 179.4 (Cꢀ=ꢀO) ppm. –
phosphoranylidene]triaz-1-en-1-yl)-1H-pyrazole-4-
carbaldehyde (11d)
+
MS (EI, 70 eV): m/z (%) ꢀ=ꢀ 323 (35) [M] . – 31P NMR δ ꢀ=ꢀ 23.2
ppm. – C14H18N3O4P (323.28): calcd. C 52.01, H 5.61, N 13.00, Eluent: petroleum ether-ethyl acetate (80:20, v/v). Product
P 9.58; found C 52.21, H 5.66, N 13.11, P 9.32.
11d was separated as colorless crystals, yield 60%; m.p.
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ꢀꢀꢀꢁ
10ꢀ ꢀN.F. El-Sayed et al.: Novel pyrazole derivatives and their antitumor activities
153°C–155°C. – IR (KBr): ν ꢀ=ꢀ 1599 (Cꢀ=ꢀN), 1664 (Nꢀ=ꢀN), 1728 was evaporated under reduced pressure. The precipi-
1
(Cꢀ=ꢀO) cm−1. – H NMR (500 MHz, CDCl3): δ ꢀ=ꢀ 1.06–1.19 tate was filtered off and washed with diethyl ether, then
(m, 18H, CH3), 2.48 (s, 3 H, CH3), 2.99–3.03 (q, 12H, CH2), recrystallized from benzene to afford product 13.
13
7.47–7.96 (m, 5H, Harom), 9.93 (s, H, CHꢀ=ꢀO) ppm. – C NMR
(125 MHz, CDCl3): δ ꢀ=ꢀ 12.21 (CH3), 19.34 (N(CH3)2), 46.73
4.10.1 5-[5-(4-Methoxyphenyl)-5-sulfido-1,2,3,4,5-
(N(CH2)2), 118.9 (C-4, pyrazole), 126.0–152.6 (aromatic
thiatriazaphosphol-2(5H)-yl]-3-methyl-1-phenyl-
1H-pyrazole-4-carbaldehyde (13)
31
C–H, C-3, C-5 of pyrazole), 167.7 (Cꢀ=ꢀO) ppm. – P NMR:
+
δ ꢀ=ꢀ 25.8 ppm. – MS (EI, 70 eV): m/z (%) ꢀ=ꢀ 474 (15) [M] . –
C23H39N8OP (474.58): calcd. C 58.21, H 8.28, N 23.61, P 6.53;
Compound 13 was isolated as buff powder, yield 65%;
found C 58.31, H 8.31, N 23.54, P 6.23.
m.p. 144°C–146°C. – IR (KBr): ν ꢀ=ꢀ 625 (Pꢀ=ꢀS), 1585 (Cꢀ=ꢀN),
1696 (Cꢀ=ꢀO) cm−1. – 1H NMR (500 MHz, [D6]DMSO): δ ꢀ=ꢀ 2.46
4.9 Reaction of triphenylstibane/tripheny-
larsane 6a,b with 1
(s, 3H, CH3), 3.76 (s, 3H, OCH3), 6.95–7.65 (m, 9H, Harom
)
13
ppm. – C NMR (125 MHz, [D6]DMSO): δ ꢀ=ꢀ 12.1 (CH3), 55.7
(OCH3), 105.0 (C-4, pyrazole), 113.9–145.5 (aromatic C–H,
31
A mixture of 6a or 6b (1 mmol) and 0.27 g (1 mmol) of 1 C-3, C-5 of pyrazole), 161.6, 161.7 (Cꢀ=ꢀO) ppm. – P NMR:
+
in dry toluene (30 mL) was warmed for 1–4 h (TLC). After δ ꢀ=ꢀ 15.0 ppm. – MS (EI, 70 eV): m/z (%) ꢀ=ꢀ 429(5) [M] .
evaporation of the volatile material, the buff residual sub- – C18H16N5O2PS2 (429.46): calcd C 50.34, H 3.76, N, 16.31, P
stance was recrystallized from benzene to yield 12a,b and 7.21, S 14.93; found: C 55.30, H 3.78, N 16.32, P 7.23, S 14.91.
triphenylstibane oxide, triphenylarsane oxide were iso-
lated and identified (m.p. and mixed m.p.).
4.11 Crystal structure determinations of 8a
and 11c
4.9.1 5-Azido-3-methyl-1-phenyl-4-
[(triphenylstiboranylidene)methyl]-1H-pyrazole (12a) Single crystals of 8a and 11c were grown by crystallization
from benzene. The diffraction data were collected on an
Product 12a was separated as buff crystals (benzene), yield Enraf-Nonius 590 diffractometer with a Kappa CCD detector
75%; m.p. 236°C–238°C. – IR (KBr): ν ꢀ=ꢀ1593 (Cꢀ=ꢀN), 2195 (N3) using graphite-monochromatized MoKα (λ ꢀ=ꢀ 0.71073 Å) radi-
cm−1. – 1H NMR (500 MHz, [D6]DMSO):δ ꢀ=ꢀ 1.99 (s, 3 H, CH3), ation at National Research Center of Egypt [34, 35]. Reflec-
13
6.90 (s, 1H, CH), 7.33–7.68 (m, 20H, Harom) ppm. – C NMR tion data have been recorded in the rotation mode using the
(75 MHz, [D6]DMSO): δ ꢀ=ꢀ 14.5 (CH3), 70.0 (CH), 117.8 (C-4, φ and ω scan technique with θmax ꢀ=ꢀ 34.98° for 8a and 30.04°
pyrazole), 120.5–148.6 (aromatic C–H, C-3, C-5 of pyrazole), for 11c. Unit cell parameters were determined from least-
+
166.7 (Cꢀ=ꢀO) ppm. – MS (EI, 70 eV): m/z (%) ꢀ=ꢀ 564(5) [M] , squares refinement with θin the range 4 ꢀꢀ≤ꢀꢀ θꢀꢀ≤ꢀꢀ 34°for 8a and
+
429 (15) [M–135] . – C29H24N5Sb (564.29): Sb 21.58, Sb 21.60.
3 ꢀꢀ≤ꢀꢀ θ ꢀꢀ≤ꢀꢀ 30° for 11c. The structures of 8a and 11c were solved
by Direct Methods using Superflip [36] implemented in the
program suit Crystals [37]. The refinement was carried out
by full-matrix least-squares method on the positional and
anisotropic temperature parameters of all non-hydrogen
atoms based on F2 using Crystals. All hydrogen atoms were
positioned geometrically and were initially refined with
soft restraints on the bond lengths and angles to regular-
ize their geometry (C–H in the range 0.93–0.98 Å and N–H
in the range 0.86–0.89 Å) and Uiso(H) ꢀ=ꢀ 1.2–1.5 ꢀ×ꢀ Ueq of the
parent atom. Then, the positions were refined with riding
constraints [38]. The general-purpose crystallographic tool
Platon [39] was used for the structure analysis and pres-
entation of the results. The molecular graphics were done
using Ortep-iii for Windows [40] and Diamond [41]. Details
of the data collection conditions and the parameters of the
refinement process for 8a and 11c are given in Table 4.
CCDC 1415329 (8a) and 1415326 (11c) contain the sup-
4.9.2 5-Azido-3-methyl-1-phenyl-4-
((triphenylarsoranylidene)methyl)-1H-pyrazole (12b)
Product 12b was separated as buff crystals (benzene),
yield 45%; m.p. 211°C–213°C. – IR (KBr): ν ꢀ=ꢀ1592 (Cꢀ=ꢀN),
2196 (N3) cm−1. – H NMR (500 MHz, CDCl3): δ ꢀ=ꢀ 2.36(s, 3
1
13
H, CH3), 7.25–7.57 (m, 21H, Harom, CH) ppm. – C NMR (125
MHz, CDCl3): δ ꢀ=ꢀ 14.2 (CH3), 70.07 (CH), 117.9 (C-4, pyra-
zole), 122.5–149.6 (aromatic C–H, C-3, C-5 of pyrazole),
+
169.7 (Cꢀ=ꢀO) ppm. – MS (EI, 70 eV): m/z (%) ꢀ=ꢀ 517(5) [M] .
– C29H24AsN5 (517.46): As 14.48, As 14.50.
4.10 Reaction of LR 7 with 1
A mixture of 0.20 g (0.5 mmol) of 7 and 0.27 g (1 mmol) of 1
was stirred for 15 min in dry benzene. The volatile material plementary crystallographic data for this paper. These
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ꢁꢀꢀꢀ
N.F. El-Sayed et al.: Novel pyrazole derivatives and their antitumor activitiesꢂ
ꢂ11
data can be obtained free of charge from The Cambridge ml), and streptomycin (100 mg/ml). The culture was main-
Crystallographic Data Centre via www.ccdc.cam.ac.uk/ tained at 37°C in an atmosphere of 5% CO2 and 95% relative
data_request/cif.
humidity. The cells were transferred to a new flask every
2 days and treated with trypsin–EDTA to detach them from
the flask. Cells were counted under a microscope using a
hemacytometer (Hausser Scientific). Cell solutions were
diluted with growth medium to a concentration of 1 ꢀ×ꢀ 105
cells/mL, transferred to a 96-well plate, and treated with
gradient concentrations of test compounds.
4.12 Anticancer evaluation
4.12.1 Cell culture and treatment
All reagents were handled as previously reported [42–44] in
a sterile fume hood. DMEM medium and fetal bovine serum
(FBS) were purchased from Gibco; phosphate-buffered 4.12.2 Resazurin cell growth inhibition assay
saline (PBS), pH 7.4, and trypsin-EDTA were obtained from
Sigma-Aldrich. Alamar blue or Resazurin (Promega, Man- Alamar blue or Resazurin (Promega) reduction assay was
nheim, Germany) reduction assay was used to assess the used to assess the cytotoxicity of the studied samples. The
cytotoxicity of the studied samples. The growth medium assay tests the cellular viability and mitochondrial func-
(DMEM medium with 10% FBS, 100 U/mL penicillin, and tion. Briefly, adherent cells were grown in tissue culture
100 mg/L streptomycin) and Alamar blue were stored at flasks, and then harvested by treating the flasks with
48°C, whereas trypsin-EDTA and FBS were stored frozen 0.025%trypsinand0.25mmEDTAfor5min. Oncedetached,
at –80°C and thawed before use; PBS was stored at room cells were washed and counted, and an aliquot (5 ꢀ×ꢀ 103
temperature. The MCF7 and HepG2 were obtained from cells) was placed in each well of a 96-well cell culture plate
the German Cancer Research Center (DKFZ).Cells were with a total volume of 100 μL. Cells were allowed to attach
cultured in 50-cm2 culture flasks (Corning) using DMEM overnight and then treated with samples. The final con-
medium supplemented with 10% FBS, penicillin (100 IU/ centration of samples ranged from 0 to 100 μm. After 48 h,
Table 4:ꢀCrystal data and experimental parameters of data collection and structure refinement of compounds 8b and 11c.
ꢁ
8b
ꢁ
11c
Crystal data
ꢂChemical formula
ꢂMr
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
C14H13N5O
267.292
298
ꢃ
ꢃ
ꢃ
C17H27N8OP
390.43
ꢂTemperature, K
ꢂCrystal size, mm3
ꢂCrystal system, space group
ꢂa, Å
298
0.12 ꢃ×ꢃ 0.13 ꢃ×ꢃ 0.15ꢃ
0.14 ꢃ×ꢃ 0.15 ꢃ×ꢃ 0.18
Monoclinic, P21/n
10.8101(4)
13.3926(5)
15.6394(8)
107.0286(12)
2164.93(16)
4
Monoclinic, Cc
11.9497(10)
14.8959(13)
7.7132(8)
96.245(5)
1364.8(2)
4
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢂb, Å
ꢂc, Å
ꢂβ, deg
ꢂV, Å3
ꢂZ
ꢂRadiation type
MoK
ꢂμ, mm−1
0.09α
ꢃ
0.15
Data collection
ꢂDiffractometer
ꢂAbsorption correction
ꢂTmin/Tmax
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
Nonius Kappa CCD diffractometer
Multi-scan
0.85/1.00
2843/2621
1097
ꢃ
ꢃ
ꢃ
ꢃ
0.83/1.00
6100/3100
1425
ꢂRefl. measured/unique
ꢂRefl. obeserved [I ꢀ>ꢀ 2.0 σ(I)]
ꢂ(sinθ/λ)max, Å−1
Refinement
0.807
0.704
ꢂNo. of reflections/parametersꢃ
1097/153
0.043/0.090
–3(6)
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
1425/142
0.056/0.131
–
0.90
0.25/–0.29
ꢂR[F2 ꢃ>ꢃ 2 σ(F2)]/wR (F2)
ꢂx (Flack)
ꢃ
ꢃ
ꢃ
ꢃ
ꢂS
0.95
ꢂΔρmax/min, e Å−3
0.16/–0.15
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ꢀꢀꢀꢁ
12ꢀ ꢀN.F. El-Sayed et al.: Novel pyrazole derivatives and their antitumor activities
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20 μL of Resazurin 0.01% w/v solution was added to each
well, and the plates were incubated at 37°C for 1–2 h. Fluo-
rescence was measured on an automated 96-well Infinite
M2000 Pro™ plate reader (Tecan, Crailsheim, Germany)
using an excitation wavelength of 544 nm and an emission
wavelength of 590 nm. After 48 h incubation, plates were
treated with Resazurin solution as mentioned above. Doxo-
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0.13 μm against MCF7 cells and 5.61 0.037 μm against
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Acknowledgments: The authors thank the National
Research Centre (NRC), for the financial support through
scientific project no. 10050002, the Cell Culture Unit at
Ain-Shams University, and Associate Professor M. Youns,
Biochemistry Department, Faculty of Pharmacy, Helwan
University, for antitumor screening.
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Z. Naturforsch.ꢂ
ꢂ2016 | Volume x | Issue x(b)
Graphical synopsis
Naglaa F. El-Sayed, Ewies F. Ewies,
Marwa El-Hussieny, Leila S. Boulos
and ElSayed M. Shalaby
Synthesis of novel pyrazole derivatives
using organophosphorus, stibine, and
arsine reagents and their antitumor
activities
DOI 10.1515/znb-2015-0187
Z. Naturforsch. 2016; x(x)b: xxx–xxx
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