Synthesis, cytotoxic activity, and thermal studies of novel N-[(1,3-diphenylpyrazol-4-yl)methyl] α-amino acids

Article abstract of DOI:10.1002/jhet.400

(Chemical Equation Presented) New N-[(1,3-diphenylpyrazol-4-yl)methyl]a- amino acids (1a-i) have been synthesized and tested in vitro for their antiproliferative activity against human myelogenous leukemia K562, colon adenocarcinoma HT-29, cervix carcinoma HeLa, and normal fetal lung fibroblasts, MRC-5. Compounds derived from both phenylalanine enantiomer precursors appeared to be the most active against myelogenous leukemia K562 cell lines with a high cytotoxic potential.

Full text of DOI:10.1002/jhet.400

850
Synthesis, Cytotoxic Activity, and Thermal Studies of Novel
N-[(1,3-Diphenylpyrazol-4-yl)methyl] a-Amino Acids
Vol 47
a
Milan D. Joksovic, * Gordana Bogdanovic, Vesna Kojic,
b
b
´
´ ´
´
´
c
c
b
´
´
Katalin Meszaros Szecsenyi, Vukadin M. Leovac, Dimitar Jakimov,
d
a
a
ˇ
´ ´ ´
Snezana Trifunovic, Violeta Markovic, and Ljubinka Joksovic
aDepartment of Chemistry, Faculty of Sciences, University of Kragujevac, 34000 Kragujevac, Serbia
bInstitute of Oncology Sremska Kamenica, Institutski put 4, 21204 Sremska Kamenica, Serbia
cDepartment of Chemistry, Faculty of Sciences, University of Novi Sad, 21000 Novi Sad, Serbia
dFaculty of Chemistry, University of Belgrade, 11000 Belgrade, Serbia
*E-mail: mjoksovic@kg.ac.rs
Received October 19, 2009
DOI 10.1002/jhet.400
Published online 8 June 2010 in Wiley InterScience (www.interscience.wiley.com).
New N-[(1,3-diphenylpyrazol-4-yl)methyl]a-amino acids (1a–i) have been synthesized and tested
in vitro for their antiproliferative activity against human myelogenous leukemia K562, colon adenocar-
cinoma HT-29, cervix carcinoma HeLa, and normal fetal lung fibroblasts, MRC-5. Compounds derived
from both phenylalanine enantiomer precursors appeared to be the most active against myelogenous leu-
kemia K562 cell lines with a high cytotoxic potential.
J. Heterocyclic Chem., 47, 850 (2010).
INTRODUCTION
view of these observations and as a continuation of our
interest toward synthesis and biological activity of aryl-
pyrazole derivatives [14,15], some new N-[(1,3-diphe-
nylpyrazol-4-yl)methyl] a-amino acids were prepared
with the aim to have promising antiproliferative proper-
ties against several kinds of human tumor cell lines.
Arylpyrazole derivatives play an important role in
biologically active compounds and therefore represent
an interesting template for medicinal chemistry. These
compounds displayed diverse biological properties such
as antiparasitic [1], antifungal [2], antibacterial [3], and
antidiabetic [4]. Moreover, arylpyrazole derivatives have
shown several biological activities as seen in cyclooxy-
genase-2 [5], p38 MAP kinase [6], and nonnucleoside
HIV-1 reverse transcriptase inhibitors [7]. In the
research for antitumor agents, arylpyrazole derivatives
exhibited promising antiproliferative properties against
several kinds of human tumor cell lines [8–10].
RESULTS AND DISCUSSION
In this work, condensation of the optically active L-
and D-amino acids (except 1a) with 1,3-diphenylpyra-
zole-4-carboxaldehyde in the presence of NaOH led to
the formation of Schiff base intermediates. As a-amino
acids are sparingly soluble in alcoholic solvents, the
Schiff base formation takes long time, and the reaction
gives lower yields even under reflux conditions. Thus, the
condensation reaction was accelerated using solventless
method [16] combining aldehyde, a-amino acids, and
NaOH in a porcelain mortar with pestle and aggregating
the dry solid mixture until a white powder was formed.
The condensation was completed by an additional heating
at reflux for 2 h in dry methanol (Scheme 1).
The cytotoxic drugs, such as doxorubicin, 5-fluoroura-
cil, and camptothecins, can damage DNA or affect cel-
lular metabolic pathways causing indirect block of the
cell cycle. Unfortunately, these agents produce an irre-
versible damage to both normal and tumor cells, result-
ing in a significant toxicity and side effects [11–13]. In
this respect, it is desirable to synthesize new highly spe-
cific antitumor agents with comparable efficacy and
reduced toxicity than the currently available drugs. In
C
V
2010 HeteroCorporation

July 2010
Synthesis, Cytotoxic Activity, and Thermal Studies of
Novel N-[(1,3-Diphenylpyrazol-4-yl)methyl] a-Amino Acids
851
Scheme 1. Reagents and conditions: (a) NaOH, continuous aggregating at rt, then MeOH, reflux, 2 h; (b) NaBH4, 0–5ꢁC, then rt, 12 h, AcOH.
The stability of the formed Schiff base products
depends on several factors such as amino acid side chain
polarity [17], the metal, pH, solvent, and temperature
[18]. The problem with Schiff base instability can be
overcome by their reduction to give a more flexible
amine and not constrained compounds. In the light of
these facts, we have performed out the reduction with
an excess of NaBH4 without isolation of Schiff base to
afford the novel N-[(1,3-diphenylpyrazol-4-yl)methyl]
a-amino acids.
the presence of characteristic singlet peak attributed to
pyrazole ring proton appeared between 7.97 and 8.67
depending on the applied solvent system. The complete
assignment of all reported signals (1H and 13C) in the
experimental part was carried out by means of 1D and
2D homo- and heteronuclear correlated NMR
spectroscopy.
The newly synthesized compounds 1a–i were eval-
uated in vitro for their antiproliferative activity against
human myelogenous leukemia K562, colon adenocarci-
noma HT-29, cervix carcinoma HeLa, and normal fetal
lung fibroblasts, MRC-5 using sulforhodamine B (SRB)
assay [21] and doxorubicin (Dox) as reference drug. The
results are presented in Table 1.
The structures of the new compounds were confirmed
1
using different spectral data (IR, H, and 13C NMR) and
elemental analysis. The IR spectra revealed the presence
of NHþ2 stretching frequencies between 2650 and 2300
cmꢀ1 in the form of broad band with multiple peaks on
the low frequency wing, which continue until about
2200 cmꢀ1, confirming their zwitterionic nature. The mas
(COO) was related to the strong absorption band appear-
The malignant cell line K562 was the most sensitive,
where the pronounced cytotoxicity was achieved by
seven of nine tested samples. The compounds with ben-
zyl group 1d and 1e showed the most potent cytotoxic
activity against human myelogenous leukemia K562 cell
lines. The replacement of the benzyl group in 1d by a
hydrophobic isobutyl group leading to 1c would retain
potential hydrophobic bonding while creating spatial
property differences between the benzyl and isobutyl
group. The comparison of the cytotoxicity of 1d or 1e
with 1c indicates that 1d and 1e are more active in inhi-
bition of these cells. When the side chain was replaced
with an isopropyl group 1b, a complete inactivity
against K562 was observed. The moderate activity was
achieved when an additional heteroatom was
ing in the spectra between 1619 and 1601 cmꢀ1
,
whereas the symmetric carboxylate stretches ms (COO)
correspond to the medium–strong peaks about 1410
cmꢀ1 [19]. The other characteristic very strong absorp-
tion bands in the IR spectra were those attributed to the
pyrazole ring: m (C¼¼C) and m (C¼¼N) between 1601
and 1547 cmꢀ1 as well as d (C¼¼C) about 1505 cmꢀ1
[20].
1
The H NMR spectra of all compounds exhibited a
characteristic AB system except 1a and ABX system for
1
1d, 1e, 1g, 1h, and 1i. The H NMR spectra revealed
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

´ ´ ´
M. D. Joksovic, G. Bogdanovic, V. Kojic, K. Meszaros Szecsenyi, V. M. Leovac, D. Jakimov,
´
´ ´ ´
S. Trifunovic, V. Markovic, and L. Joksovic
´
´ ´
852
Vol 47
Table 1
important in biological systems [24]; the thermal proper-
ties of these new amino acid derivatives may be of spe-
cial interest.
In vitro cytotoxicity of compounds 1a–i.
IC50 (lM)a
In the series of nine new compounds, two of them are
pure N-substituted amino acids, whereas the others crys-
tallize with different number of water molecules.
Thermal measurements reveal that the stability of
N-substituted amino acids 1a and 1b is rather high in both
atmospheres (in N2 200 and 205ꢁC, respectively). Most
probably as a consequence of some sensitivity toward oxi-
dation, in air the thermal stability of the compounds is
somewhat less. The decomposition mechanism in inert
and oxidative atmosphere also differs (see Fig. 1).
The dehydration pattern of the hydrates does not
depend on the atmosphere. The dehydrated compounds
1c, 1g, 1h, and 1i are stable to above 200ꢁC, with the
previous remark: their stability in air is less with about
5ꢁC. Compounds 1d, 1e, and 1f decompose further
after dehydration, without giving a stable anhydrous
intermediate.
Compounds
K562
HeLa
HT29
MRC-5
1a
1b
1c
1d
1e
8.89
>100
4.17
1.02
0.97
6.37
>100
4.69
3.25
0.36
>100
9.21
>100
>100
72.31
8.31
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.32
>100
14.43
11.97
>100
7.64
6.45
1f
>100
>100
>100
67.32
0.51
1g
1h
1i
96.34
>100
1.17
Dox
a IC50 is the concentration of compound required to inhibit the cell
growth by 50% compared to an untreated control.
incorporated into the side chain 1f. These differences in
cytotoxicity could be related to their conformation and
size of the substitutes. The bulky isopropyl group 1b
and voluminous sulfur atom 1g closely to aminocarbox-
ylate skeleton induce the decrease of cytotoxic activity,
probably preventing the formation of intermolecular
interaction with cell receptors. Comparison of the cyto-
toxicity of compounds derived from different enantiom-
ers of phenylalanine indicates that D-phenylalanine
demonstrated similar activity with respect to L-phenylal-
anine analog. We could also suggest that 1d and 1e are
the most cytotoxic compounds because of their planarity
and hydrophobic properties. These results indicate that
spatial effects and side chain length should be an impor-
tant factor in the design of future molecules. It is inter-
esting to note that the compounds 1h and 1i having po-
lar hydroxyl groups proved to be less active than 1d and
1e, suggesting that electronic character could be dimin-
ished in comparison with hydrophobic requirements of
amino acid part of molecule. Compounds 1g and 1e
were also active against HeLa and HT29 cell lines,
respectively, but their activity was significantly lower
compared with doxorubicin. On the other hand, all com-
pounds were devoid of any cytotoxicity against the nor-
mal fetal lung fibroblasts MRC-5. Generally, the hydro-
phobic character and steric effects in amino acid moiety
appear to be the main factors for the growth suppressing
potential against K562 cell lines. Further modification of
compounds 1d and 1e, especially the transformation of
carboxylate group and identification of its molecular tar-
get, is under investigation.
The decomposition of all samples was accompanied
by melting, observed visually. The decomposition mech-
anism of the first decomposition step of dry compounds
is proposed only on the basis of the mass loss. The frag-
mentation begins with that of the side chain, e.g., the
mass loss to the minimum in DTG curve of 1a amounts
14.5% in N2 that agrees well with the loss of CO2 mole-
cule (calcd. 14.32%). The valine 1b derivative most
probably decomposes by the loss of the methyl groups.
However, it is pointless to give a decomposition mecha-
nism in lack of coupled measurements. It is more impor-
tant to have a closer look to the dehydration process of
the hydrates from both the theoretical and practical point
of view. The mass loss with the corresponding tempera-
ture of the dehydration is given in Table 2 together with
the decomposition temperature of the anhydrous
compound.
Thermal analysis. Thermal analysis of pharmaceuti-
cal compounds is a very reliable method for purity con-
trol, and it is a necessary part of the characterization of
new compounds with potential bioactivity [22,23]. As
amino acid–water–protein interface interactions are very
Figure 1. Thermal decomposition curves of 1b in air and nitrogen.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

July 2010
Synthesis, Cytotoxic Activity, and Thermal Studies of
Novel N-[(1,3-Diphenylpyrazol-4-yl)methyl] a-Amino Acids
853
Table 2
Mass loss of the dehydration, the dehydration temperature, and the onset temperature of the decomposition of the anhydrous compound in N2.
Dehydration, Dm (%)
Compound
Exp.
Calcd.
tDEHYD. (ꢁC)
tDECOMP. (ꢁC)
Comments
1c
1d, 1e
1f
1g
1h
4.2
7.3
4.72
8.31
10.17
4.67
5.34
4.36
<105
<130
<135
<50
<150
<30
215
–
Stable anhydrous compound
Stepwise dehydration, continuous decomposition
Stepwise dehydration, continuous decomposition
Crystal water
10.4
–
1.3
2.2
0.5
203
225
235
Structural water
Loosely bond crystal water
1i
As the decomposition of the compounds 1d, 1e, and
1f is continuous to obtain better separated decomposi-
tion steps, quasi-isothermal [25] (SWI) measurement
was carried out. Figure 2 illustrates SWI curve of the
compound 1f. The five water molecules evaporate to
150ꢁC (exp. 15.8%, calcd. 16.95%) in five clearly distin-
guished steps, not one by one, but through a complex
process governed by macroscopic properties of the sam-
ple (e.g., the rate of the diffusion) as well as by their
different bonding energy. The dehydration of this com-
pound is most probable followed by the evaporation of
CH3COOH molecule. The difference in experimentally
determined mass loss and the theoretical one is most
probable because of the evaporation of the crystal water
at room temperature. The dehydration of 1d and 1e
derivatives shows similar complexity of the dehydration.
Derivative 1h, according to elemental analysis data, con-
tains both crystal and structural water, belonging to
more than one molecule. By TA only the structural
water is detected, because in air the compound lost its
crystal water completely. Compound 1i has some crystal
water left, while most of it has evaporated during the
storage time.
the distance of bulky substitutes from aminocarboxylate
part of molecule appeared to play an important role in
antiproliferative activity against different malignant cell
lines. The dehydration temperature is related to the
water bond energy and might be connected with the
active sites of some bioactive molecules.
EXPERIMENTAL
All chemicals were obtained from commercial sources
(Aldrich) and used as supplied. 1,3-Diphenylpyrazole-4-car-
boxaldehyde was synthesized by Vilsmeier-Haack reaction
[27].
Melting points were determined on a Mel-Temp capillary
melting points apparatus, model 1001, and are uncorrected.
Optical rotations were measured on a Rudolph Research Ana-
lytical automatic polarimeter Autopol IV. Elemental (C, H, N,
and S) analysis of the samples was carried out by standard
micromethods in the Center for Instrumental Analysis, Faculty
of Chemistry, Belgrade. IR spectra were recorded on a Perkin
Elmer Spectrum One FTIR spectrometer with a KBr disc. 1H
and 13C NMR spectra were recorded on a Varian Gemini 200
(200 MHz) and a Bruker Avance III spectrometer operating at
500 MHz. As a consequence of the poor solubility in DMSO-
d6, the NMR spectra were recorded in pyridine-d5/D2O and
NaOD/D2O solvent system for 1h and 1i. Thermal measure-
ments were conducted using SDT Q600 TA Instruments’
Taking into account the importance of the interactions
in the relation of amino acid–water–protein, the cytotox-
icity of the compounds might be related to H-bond-
forming capability in the molecules. This, indirectly,
may be investigated using TA data of hydrates. How-
ever, the correlation is not straightforward. TA data may
refer only to the preferences of an amino acid to bind
water molecules and the strength of the bond between
them [26].
CONCLUSIONS
In summary, a new class of N-[(1,3-diphenylpyrazol-
4-yl)methyl] a-amino acids were synthesized, and their
antiproliferative activity against human myelogenous
leukemia K562, colon adenocarcinoma HT-29, cervix
carcinoma HeLa, and normal fetal lung fibroblasts,
MRC-5 was evaluated. The nature, spatial effects and
Figure 2. SWI decomposition curve of 1g, illustrating the complexness
of its dehydration.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

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M. D. Joksovic, G. Bogdanovic, V. Kojic, K. Meszaros Szecsenyi, V. M. Leovac, D. Jakimov,
´
´ ´ ´
S. Trifunovic, V. Markovic, and L. Joksovic
´
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854
Vol 47
thermal analyzer with about 2 mg sample masses and a heating
rate of 20ꢁC/min in air and nitrogen atmospheres. The sample
holder and the reference cells were made of alumina.
m
(C¼¼C)Ar
,
1560
m
(C¼¼N)Ar
,
1503
d
(C¼¼C)Ar
, 1454
d (C¼¼N)Ar, 1412 ms (COOꢀ), 1076 d (CAH)ip, 755 d
(CAH)oop;
1H NMR (200 MHz, pyridine-d5/D2O, 9/1 v/v):
General procedure for the preparation of 1a–i. 1,3-
Diphenylpyrazole-4-carboxaldehyde (0.62 g, 2.5 mmol),
a-amino acid (2.75 mmol), and NaOH (0.11 g, 2.75 mmol, for
1i 0.22g, 5.50 mmol) were milled using a porcelain mortar and
pestle to obtain a homogenous white powder until the release
of water was observed. This mixture was transferred into
50 cm3 of dry methanol and heated to reflux for 2 h. After
cooling in an ice bath, sodium borohydride (0.11 g, 3 mmol)
was added in several portions with stirring. The solution was
stirred for additional 2 h at room temperature, then diluted
with 50 cm3 of deionized water, and left for 12 h, after which
time the white precipitate had formed by addition of glacial
acetic acid. The crude product was purified by dissolving in
1M NaOH and precipitation with glacial acetic acid. Recrystal-
lized compound was collected by filtration, washed with plenty
of water, and dried over anhydrous CaCl2.
0.92 (d, 3H, J ¼ 6.72 Hz, CH3), 0.96 (d, 3H, J ¼ 6.74 Hz,
CH3), 1.89 (m, 2H, CHACH2ACH), 2.16 (m, 1H, (CH3)2CH),
3.87 and 3.90 (2d, 1H, J ¼ 6.36 Hz and J ¼ 6.60 Hz,
NHACHACOO), dA ¼ 4.42 and dB ¼ 4.15 (AB system, 2H,
JAB ¼ 13.50 Hz, PzACH2), 7.32–7.61 (m, 6H, 1H at C-4, 1H
at C-40, 2H at C-3/5, and 2H at C-30/50), 8.03 (d, 2H at C-2/6,
J ¼ 8.00 Hz), 8.36 (d, 2H at C-20/60, J ¼ 8.00 Hz), 8.67 (s,
1H, Pz); 13C NMR (pyridine-d5/D2O, 9/1 v/v): 21.17 (CH3),
21.94 (CH3), 24.14 ((CH3)2CH), 41.38 (CHACH2ACH), 41.62
(PzACH2), 59.50 (CHACOO), 117.67 (C-2/6), 118.20 (C-4,
Pz), 125.36 (C-4), 127.23 (C-40), 127.43 (C-20/60), 127.67 (C-
30/50), 128.22 (C-5, Pz), 128.71 (C-3/5), 132.66 (C-10), 139.28
(C-3, Pz), 150.78 (C-1), 176.47 (COO); Anal. Calcd. for
C22H27N3O3 (381.48 g/mol): C, 69.27; H, 7.13; N, 11.02;
Found: C, 68.99; H, 7.21; N, 10.89.
N-[(1,3-Diphenylpyrazol-4-yl)methyl]-L-phenylalanine
dihydrate (1d). White powder; yield: 0.95 g (88%); mp 185–
186ꢁC (Dec.); [a]D20 ¼ ꢀ1.82 (c ¼ 1.100ꢃꢂ 10ꢀ3 g/cm3, pyri-
dine/H2O, 9/1 v/v); IR (KBr, cmꢀ1): 3062 m (CAH)Ar, 2972
and 2853 m (CAH)Al, 2620–2380 m (NHþ2 ), 1619 mas (COOꢀ),
1599 m (C¼¼C)Ar, 1553 m (C¼¼N)Ar, 1503 d (C¼¼C)Ar, 1453 d
N-[(1,3-Diphenylpyrazol-4-yl)methyl]glycine
(1a). White
powder; yield: 0.56 g (73%); mp 190–191ꢁC (Dec.); IR (KBr,
cmꢀ1): 3063 m (CAH)Ar, 2960 and 2820 m (CAH)Al, 2650–
2400 m (NHþ2 ), 1626 mas (COOꢀ), 1601 m (C¼¼C)Ar, 1547 m
(C¼¼N)Ar
,
1502
d
(C¼¼C)Ar
,
1453
d
(C¼¼N)Ar
, 1412 ms
(COOꢀ), 1066 d (CAH)ip, 758 d (CAH)oop
;
1H NMR (200
(C¼¼N)Ar, 1412 ms (COOꢀ), 1072 d (CAH)ip, 754 d (CAH)oop
;
1H NMR (200 MHz, pyridine-d5/D2O, 9/1 v/v): dA ¼ 3.42, dB
¼ 3.21, and dX ¼ 4.04 (ABX system, 3H, JAB ¼ 13.59 Hz,
JAX ¼ 5.15 Hz, JBX ¼ 8.26 Hz, CHACH2), dA ¼ 4.29 and
dB ¼ 3.99 (AB system, 2H, JAB ¼ 13.50 Hz, PzACH2),
7.28–7.62 (m, 11H, ArAH), 7.93 (d, 2H at C-2/6, J ¼
8.00 Hz), 8.20 (d, 2H at C-20/60, J ¼ 8.00 Hz), 8.29 (s, 1H,
Pz); 13C NMR (pyridine-d5/D2O, 9/1 v/v): 40.10 (PhACH2),
42.88 (PzACH2), 63.30 (CHACOO), 118.77 (C-2/6), 120.66
(C-4, Pz), 126.36 (C-400), 126.78 (C-4), 128.19 (C-40), 128.50
(C-20/60), 128.69 (C-300/500), 128.75 (C-5, Pz), 128.96 (C-30/50),
129.78 (C-3/5), 130.05 (C-200/600), 134.18 (C-10), 139.35
(C-100), 140.48 (C-3, Pz), 151.76 (C-1), 176.98 (COO); Anal.
Calcd. for C25H27N3O4 (433.51 g/mol): C, 69.27; H, 6.28; N,
9.69; Found: C, 69.51; H, 6.31; N, 9.74.
MHz, pyridine-d5/D2O, 9/1 v/v): 4.19 (s, 2H, CH2ACOO),
4.74 (s, 2H, PzACH2), 7.40–7.71 (m, 6H, 1H at C-4, 1H at C-
40, 2H at C-3/5, and 2H at C-30/50), 7.97 (d, 2H at C-2/6, J ¼
8.00 Hz), 8.00 (d, 2H at C-20/60, J ¼ 8.00 Hz), 8.94 (s, 1H,
Pz); 13C NMR (pyridine-d5/D2O, 9/1 v/v): 41.87 (PzACH2),
50.13 (CH2ACOO), 113.09 (C-4, Pz), 119.15 (C-2/6), 127.31
(C-4), 128.66 (C-20/60), 129.08 (C-40), 129.46 (C-30/50), 130.02
(C-3/5), 130.40 (C-5, Pz), 132.42 (C-10), 139.72 (C-3, Pz),
152.50 (C-1), 170.83 (COO); Anal. Calcd. for C18H17N3O2
(307.35 g/mol): C, 70.34; H, 5.58; N, 13.67; Found: C, 70.12;
H, 5.62; N, 13.55.
N-[(1,3-Diphenylpyrazol-4-yl)methyl]-L-valine (1b). White
powder; yield: 0.72 g (82%); mp 198ꢁC (Dec.); [a]D20
¼
þ12.77 (c ¼ 1.096 ꢂ 10ꢀ3 g/cm3, pyridine/H2O, 9/1 v/v); IR
(KBr, cmꢀ1): 3059 m (CAH)Ar, 2965 and 2876 m (CAH)Al,
N-[(1,3-Diphenylpyrazol-4-yl)methyl]-D-phenylalanine
dihydrate (1e). Yield: 0.93 g (86%); [a]2D0 ¼ þ1.82 (c ¼
1.100ꢃꢂ 10ꢀ3 g/cm3, pyridine/H2O, 9/1 v/v).
2600–2400 m (NHþ2 ), 1614 mas (COOꢀ), 1600 m (C¼¼C)Ar
;
(COOꢀ), 1069 d (CAH)ip, 756 d (CAH)oop 1H NMR (200
,
1550 m (C¼¼N)Ar, 1504 d (C¼¼C)Ar, 1451 d (C¼¼N)Ar, 1411 ms
N-[(1,3-Diphenylpyrazol-4-yl)methyl]-L-methionine acetate
pentahydrate (1f). White powder; yield: 1.18 g (89%); mp
185–186ꢁC (Dec.); [a]D20 ¼ ꢀ6.94 (c ¼ 1.296ꢃꢂ 10ꢀ3 g/cm3,
pyridine/H2O, 9/1 v/v); IR (KBr, cmꢀ1): 3291 m (OAH,
broad), 3060 m (CAH)Ar, 2916 and 2853 m (CAH)Al, 2600–
2440 m (NHþ2 ), 1681 m (C¼¼O), 1607 mas (COOꢀ), 1598 m
MHz, pyridine-d5/D2O, 9/1 v/v): 1.16 (d, 3H, J ¼ 6.62 Hz,
CH3), 1.19 (d, 3H, J ¼ 6.56 Hz, CH3), 2.31 (m, 1H,
(CH3)2CH), 3.55 (d, 1H, J ¼ 5.46 Hz, CHACOO), dA ¼ 4.31
and dB ¼ 4.01 (AB system, 2H, JAB ¼ 13.50 Hz, PzACH2),
7.33–7.62 (m, 6H, 1H at C-4, 1H at C-40, 2H at C-3/5, and 2H
at C-30/50), 8.02 (d, 2H at C-2/6, J ¼ 8.00 Hz,), 8.36 (d, 2H at
C-20/60, J ¼ 8.00 Hz), 8.55 (s, 1H, Pz); 13C NMR (pyridine-
d5/D2O, 9/1 v/v): 18.81 (CH3), 19.95 (CH3), 31.78
((CH3)2CH), 43.25 (PzACH2), 67.49 (CHACOO), 118.80
(C-2/6), 120.14 (C-4, Pz), 126.46 (C-4), 128.33 (C-40), 128.56
(C-20/60), 128.98 (C-30/50), 129.14 (C-5, Pz), 129.85 (C-3/5),
134.13 (C-10), 140.46 (C-3, Pz), 151.93 (C-1), 177.01 (COO);
Anal. Calcd. for C21H23N3O2 (349.43 g/mol): C, 72.18; H,
6.63; N, 12.03; Found: C, 72.02; H, 6.68; N, 11.86.
(C¼¼C)Ar
,
1558
m
(C¼¼N)Ar
,
1504
d
(C¼¼C)Ar
, 1452 d
(C¼¼N)Ar, 1412 ms (COOꢀ), 1067 d (CAH)ip, 756 d (CAH)oop
;
1H NMR (200 MHz, pyridine-d5/D2O, 9/1 v/v): 2.04 (s, 3H,
CH3AS), 2.43 (s, 3H, CH3ACOO), 2.53 (m, 2H, CHACH2),
3.00 (t, 2H, J ¼ 6.09 Hz, CH2AS); 4.13 (t, 1H, J ¼ 5.60 Hz,
CHACH2), dA ¼ 4.58 and dB ¼ 4.35 (AB system, 2H, JAB
¼
13.50 Hz, PzACH2), 7.36–7.70 (m, 6H, 1H at C-4, 1H at C-40,
2H at C-3/5, and 2H at C-30/50), 8.03 (d, 2H at C-2/6, J ¼
8.00 Hz), 8.21 (d, 2H at C-20/60, J ¼ 7.52 Hz); 8.66 (s, 1H,
Pz); 13C NMR (pyridine-d5/D2O, 9/1 v/v): 14.92 (CH3AS),
23.38 (CH3ACOO), 30.91 (SACH2), 32.70 (SACH2ACH2),
42.28 (PzACH2), 62.92 (CHACOO), 117.35 (C-4, Pz), 118.95
(C-2/6), 126.74 (C-4), 128.59 (C-20/60), 128.64 (C-40), 129.18
N-[(1,3-Diphenylpyrazol-4-yl)methyl]-L-leucine
monohy-
drate (1c). White powder; yield: 0.82 g (86%); mp 184ꢁC
(Dec.); [a]2D0 ¼ ꢀ6.09 (c ¼ 1.313ꢃꢂ 10ꢀ3 g/cm3, pyridine/
H2O, 9/1 v/v); IR (KBr, cmꢀ1): 3065 m (CAH)Ar, 2956 and
2869 m (CAH)Al, 2550–2300 m (NHþ2 ), 1612 mas (COOꢀ), 1600
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

July 2010
Synthesis, Cytotoxic Activity, and Thermal Studies of
Novel N-[(1,3-Diphenylpyrazol-4-yl)methyl] a-Amino Acids
855
(C-30/50), 129.90 (C-3/5), 130.05 (C-5. Pz), 133.35 (C-10),
140.11 (C-3, Pz), 152.10 (C-1), 177.02 (COO), 177.39
(CH3ACOO); Anal. Calcd. for C23H37N3O9S (531.63 g/mol):
C, 51.96; H, 7.02; N, 7.90; S, 6.03; Found: C, 51.86; H, 6.94;
N, 7.82; S, 5.93.
(d, 2H at C-20/60, J ¼ 8.00 Hz), 7.97 (s, 1H, Pz); 13C NMR
(500 MHz, NaOD/D2O): 40.84 (PhACH2), 43.62 (PzACH2),
67.60 (CH), 121.38 (300), 121.64 (C-4, Pz), 122.24 (C-2/6),
126.28 (100), 129.81 (C-4), 130.72 (C-20/60), 131.35 (C-40),
131.63 (C-30/50), 132.15 (C-5, Pz), 132.39 (C-3/5), 133.03
(200), 134.60 (C-10), 141.83 (C-3, Pz), 154.77 (C-1), 167.28
(400), 184.13 (COO); Anal. Calcd. for C25H25N3O4 (431.48 g/
mol): C, 69.59; H, 5.84; N, 9.74; Found: C, 69.69; H, 5.82; N,
9.74.
Biological evaluation. Three human tumor cell lines and one
human nontumor cell line were used in this study: K562 (chronic
myelogenous leukemia), HeLa (epitheloid carcinoma of cervix),
HT-29 (colon adenocarcinoma), and MRC5 (lung fetal fibro-
blasts). The cells were grown in RPMI 1640 (K562 cells) or
Dulbecco’s modified Eagle’s medium (DMEM) with 4.5% of
glucose (HeLa, HT-29, and MRC5 cells). Media were supple-
mented with 10% of fetal calf serum (FCS, NIVNS) and antibi-
otics: 100 IU/mL of penicillin and 100 lg/mL of streptomycin
(ICN Galenika). All cell lines were cultured in flasks (Costar,
25 cm2) at 37ꢁC in the 100% humidity atmosphere and 5% of
CO2. Only viable cells were used in the assay. Viability was
determined by dye exclusion assay with Trypan blue.
Cytotoxicity was evaluated by colorimetric SRB assay as
described by Skehan et al. [21]. Briefly, single cell suspension
was plated into 96-well microtiter plates (Costar, flat bottom):
1 ꢂ 104 of K562 and 5 ꢂ 103 of HeLa, HT29, and MRC5
cells, per 180 mL of medium. Plates were preincubated for
24 h at 37ꢁC, 5% CO2. Tested substances at concentration
ranging from 10ꢀ8 to 10ꢀ4M were added to all wells except to
the control ones. After incubation period (48 h/37ꢁC/5% CO2),
SRB assay was carried out as follows: 50 lL of 80% trichloro-
acetic acide (TCA) was added to all wells; an hour later, plates
were washed with distillate water, and 75 lL of 0.4% SRB
was added to all wells; half an hour later, plates were washed
with citric acid (1%) and dried at room temperature. Finally,
200 lL of 10 mmol TRIS (pH ¼ 10.5) basis was added to all
wells. Absorbance was measured on the microplate reader
(Multiscan MCC340, Labsystems) at 540/690 nm. The wells
without cells, containing compete medium only, acted as
blank.
N-[(1,3-Diphenylpyrazol-4-yl)methyl]-S-methyl-L-cysteine
monohydrate (1g). White powder; yield: 0.88 g (92%); mp
187ꢁC (Dec.); [a]D20 ¼ ꢀ11.04 (c ¼ 1.177ꢃꢂ 10ꢀ3 g/cm3, pyri-
dine/H2O, 9/1 v/v); IR (KBr, cmꢀ1): 3051 m (CAH)Ar, 2923
and 2853 m (CAH)Al, 2601–2388 m (NHþ2 ), 1628 mas (COOꢀ),
1599 m (C¼¼C)Ar, 1549 m (C¼¼N)Ar, 1504 d (C¼¼C)Ar, 1451 d
(C¼¼N)Ar, 1413 ms (COOꢀ), 1067 d (CAH)ip, 757 d (CAH)oop
;
1H NMR (500 MHz, pyridine-d5/D2O, 9/1 v/v): 2.20 (s, 3H,
CH3AS), dA ¼ 3.25, dB ¼ 3.16, and dX ¼ 4.01 (ABX system,
3H, JAB ¼ 13.50 Hz, JAX ¼ 5.73 Hz, JBX ¼ 7.27 Hz,
CHACH2), dA ¼ 4.34 and dB ¼ 4.14 (AB system, 2H, JAB
¼
13.50 Hz, PzACH2), 7.29 (t, 1H, J ¼ 7.50 Hz, C-4), 7.44 (t,
1H, J ¼ 7.50 Hz, C-40), 7.50 (t, 2H, J ¼ 7.50 Hz, C-3/5), 7.57
(t, 2H, J ¼ 7.50 Hz, C-30/50), 8.01 (d, 2H, J ¼ 8.00 Hz, C-2/
6), 8.35 (d, 2H, J ¼ 8.00 Hz, C-20/60), 8.53 (s, 1H, Pz); 13C
NMR (pyridine-d5/D2O, 9/1 v/v): 16.35 (CH3AS), 38.08
(SACH2), 43.02 (PzACH2), 61.60 (CH), 118.93 (C-2/6),
120.69 (C-4, Pz), 126.52 (C-4), 128.41 (C-40), 128.74 (C-20/
60), 128.98 (C-5, Pz), 129.12 (C-30/50), 129.95 (C-3/5), 134.43
(C-10), 140.75 (C-3, Pz), 151.96 (C-1), 175.98 (COO); Anal.
Calcd. for C20H23N3O3S (385.48 g/mol): C, 62.32; H, 6.01; N,
10.90; S, 8.32; Found: C, 62.49; H, 5.99; N, 10.92; S, 8.35.
N-[(1,3-Diphenylpyrazol-4-yl)methyl]-L-serine monohydrate
(1h). White powder; yield: 0.71 g (80%); mp 190–191ꢁC
(Dec.); [a]2D0 ¼ þ3.96 (c ¼ 1.263ꢃꢂ 10ꢀ3 g/cm3, pyridine/
H2O, 9/1 v/v); IR (KBr, cmꢀ1): 3445 m (OAH, broad), 3055 m
(CAH)Ar, 2961 and 2853 m (CAH)Al, 2600–2390 m (NHþ2 ),
1628 mas (COOꢀ), 1600 m (C¼¼C)Ar, 1547 m (C¼¼N)Ar, 1504 d
(C¼¼C)Ar, 1452 d (C¼¼N)Ar, 1417 ms (COOꢀ), 1074 d (CAH)ip,
752 d (CAH)oop
;
1H NMR (500 MHz, NaOD/D2O): dA
¼
¼
¼
3.71, dB ¼ 3.65, and dX ¼ 3.17 (ABX system, 3H, JAB
11.50 Hz, JAX ¼ 4.66 Hz, JBX ¼ 6.09 Hz, CHACH2), dA
3.37 and dB ¼ 3.62 (AB system, 2H, JAB ¼ 13.50 Hz,
PzACH2), 7.35 (t, 1H, J ¼ 7.50 Hz, C-4), 7.44–7.52 (m, 5H,
2H at C-3/5, 1H at C-40, and 2H at C-30/50), 7.58 (d, 2H at C-
2/6, J ¼ 8.00 Hz), 7.60 (d, 2H at C-20/60, J ¼ 8.00 Hz), 8.05
(s, 1H, Pz); 13C NMR (NaOD/D2O): 43.88 (PzACH2); 65.67
(CH2AOH), 67.22 (CH), 121.65 (C-4, Pz), 122.09 (C-2/6),
129.75 (C-4), 130.69 (C-20/60), 131.32 (C-40), 131.58 (C-30/50),
132.13 (C-5, Pz), 132.33 (C-3/5), 134.68 (C-10), 141.78 (C-3,
Pz), 154.57 (C-1), 181.84 (COO); Anal. Calcd. for
C19H21N3O4 (355.39 g/mol): C, 64.21; H, 5.96; N, 11.82; S,
8.32; Found: C, 64.40; H, 5.94; N, 11.81.
Cytotoxicity was calculated according to the formula:
ð1 ꢀ ATEST=ACONTROLÞ ꢂ 100
and expressed as a percent of cytotoxicity (CI %).
Acknowledgment. The work was financed by the Ministry for
Science of the Republic of Serbia (Grant Nꢁ 142028) and the Pro-
vincial Secretariat for Science and Technological Development
of Vojvodina.
N-[(1,3-Diphenylpyrazol-4-yl)methyl]-L-tyrosine monohy-
drate (1i). White powder; yield: 0.85 g (79%); mp 190–191ꢁC
(Dec.); [a]2D0 ¼ ꢀ6.00 (c ¼ 1.166ꢃꢂ 10ꢀ3 g/cm3, pyridine/
H2O, 9/1 v/v); IR (KBr, cmꢀ1): 3418 m (OAH, broad), 3061 m
(CAH)Ar, 2957 and 2812 m (CAH)Al, 2622–2400 m (NHþ2 ),
1611 mas (COOꢀ), 1597 m (C¼¼C)Ar, 1559 m (C¼¼N)Ar, 1504 d
(C¼¼C)Ar, 1451 d (C¼¼N)Ar, 1410 ms (COOꢀ), 1072 d (CAH)ip,
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