Synthesis, antitumor evaluation and crystal structure of hydroxyurea derivatives

Article abstract of DOI:10.1248/cpb.58.94

A series of hydroxyurea derivatives have been synthesized and elucidated by means of FT-IR, ¹H-, ¹³C-NMR and MS. The exact stereostructures of representative compounds have been determined by X-ray crystal structure analysis. In the crystals, inversion dimers linked by pairs of N-H...O hydrogen bonds occurred, and further N-H...O links led to chains of molecules. In vitro antitumor activities against Tca8113 human tongue cancer cells and L1210 murine leukemia cells were evaluated. A total of 8 of the 12 compounds had higher inhibitory activities than hydroxyurea against L1210 cells. Among them, the most promising compounds were 3e, 3d, 3a and 2d.

Full text of DOI:10.1248/cpb.58.94

94
Notes
Chem. Pharm. Bull. 58(1) 94—97 (2010)
Vol. 58, No. 1
Synthesis, Antitumor Evaluation and Crystal Structure of Hydroxyurea
Derivatives
c
c
c
Xi MAI,a,b,c Xiaosan LU, Hongying XIA, Yusheng CAO, ,a,b Yijing LIAO,c and Xiaolan LV
*
a State Key Laboratory of Food Science and Technology, Nanchang University; b Sino-Germany Joint Research Institute,
Nanchang University; and c Department of Pharmacy, Medical College, Nanchang University; Nanchang 330006, China.
Received February 25, 2009; accepted September 30, 2009; published online October 27, 2009
A series of hydroxyurea derivatives have been synthesized and elucidated by means of FT-IR, 1H-, 13C-NMR
and MS. The exact stereostructures of representative compounds have been determined by X-ray crystal struc-
…
ture analysis. In the crystals, inversion dimers linked by pairs of N–H O hydrogen bonds occurred, and further
…
N–H O links led to chains of molecules. In vitro antitumor activities against Tca8113 human tongue cancer
cells and L1210 murine leukemia cells were evaluated. A total of 8 of the 12 compounds had higher inhibitory ac-
tivities than hydroxyurea against L1210 cells. Among them, the most promising compounds were 3e, 3d, 3a and
2d.
Key words synthesis; antitumor evaluation; crystal structure; hydroxyurea
Hydroxyurea (HU) has been used in cancer chemotherapy Results and Discussion
for many years. It has been of manifold pharmacological in-
Synthesis The target compounds were prepared using
terest, so it has been used for the treatment of melanoma, the reaction sequence in Chart 1. The compounds 2a—f were
chronic myelocytic leukemia, and recurrent, metastatic, or synthesized in good yield by condensation of HU with vari-
inoperable ovarian cancer. HU is also used in therapy of ous benzyls (1a—f) in the presence of potassium hydroxide
squamous cell carcinomas in the head and neck and relapsed under reflux. The condensation of 2a—f with HU afforded
metastasis ovarian cancer,1) and people have found that it has compounds 3a—f. The chemical structures of the synthe-
certain effect on sickle cell anemia,2) beta-thalassemia,3) and sized compounds (2a—f and 3a—f) were confirmed by
psoriasis.4) It is also reported that HU has been used for the spectroscopic methods, and exact stereostructures of com-
treatment of AIDS in combination with didanosine, showing pounds 2a and 3f have been determined by X-ray crystal
no viral rebound after one year treatment.5) HU causes an im- structure analysis.
mediate inhibition of DNA synthesis by acting on the R2 sub-
X-Ray Crystal Structure Analysis The crystallographic
unit of the ribonucleotide reductase.6—8) Therapeutic applica- data of 2a are summarized in Table 1. The selected bond
tion of HU has several disadvantages such as short half-life lengths, angles and torsion angles are given in Table 2.
(1.9—3.9 h) in patients due to its small molecular size ORTEP drawings of the compounds 2a and 3f12) are illus-
(MWꢀ76.06) and extremely polar nature (Clog Po/wꢀ trated in Fig. 1. Crystallographic data and the structure
ꢁ1.80), the necessity of using a high dosage (80 mg/kg every analysis of compound 3f have been outlined in the previous
third day or 20—30 mg/kg daily), and the rapid development paper.12) Conformations of the C8ꢀO2 double bond and
of resistance.1,9—11)
N1–O1 bond in 2a are the opposite of each other, similar to
In this study, structure modification of HU based on in- that observed in 3f (C1ꢀO2 double bond and N1–O1 bond),
creasing its hydrophobic nature and molecular size has been N-hydroxyurea13—17) and other hydroxyurea derivates.18) The
adopted to obtain a more potent compound. A series of length of the carbonyl bond (C8ꢀO2) in 2a is in the normal
monosubstituents and disubstituents of HU with different range of 1.19—1.23 Å, similar to that observed in 3f,12) but
benzyls were synthesized and their structures were elucidated obviously shorter than N-hydroxyurea, 1-hydroxy-1-methyl-
using spectrometry along with X-ray crystal structures analy- urea and 1-hydroxy-3-methylurea (ꢂ1.25 Å). This may be
sis for representative compounds. The antitumor activity tests related to the hydroxyl etherification. The average distance
in vitro for human tongue cancer cell line and murine between the carbon atom and the coordination nitrogen atom
leukemia cell line were evaluated.
is 1.300 (3) Å. The group N–(CꢀO)–N urea planar forms a
dihedral angle of 71.48 (15)° with the benzyl group, and the
N–O bonds are twisted by about 20° out of the N–(CꢀO)–N
Chart 1. Synthesis and Structures of Compounds 2a—f and 3a—f
© 2010 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: weireizzz123@yahoo.com.cn

January 2010
95
Table 1 Crystal and Experimental Data
Empirical formula: C8H10N2O2
Formula weightꢀ166.18
Wavelengthꢀ0.71073 Å
Crystal system: monoclinic
Space group: P21/c
aꢀ12.456 (3) Å
bꢀ5.0081 (13) Å
cꢀ13.681 (4) Å
bꢀ96.017 (4)°
Volumeꢀ848.7 (4) Å3
Zꢀ4
Dxꢀ1.301 g/cm3
Absorption coefficient: 0.10 mmꢁ1
F(000)ꢀ352
Crystal size: 0.80ꢃ0.41ꢃ0.16 mm
q range for data collection: 2.3 to 26.8°
Limiting indices: ꢁ14ꢄhꢄ14, ꢁ5ꢄkꢄ5, ꢁ16ꢄlꢄ16
Reflections collected/unique: 4629/1453 [Rintꢀ0.020]
2qmaxꢀ50.0° with MoKa
Absorption correction: multi-scan
Max. and min. transmission: 0.99 and 0.94
Goodness-of-fit on F2: 1.04
Final R indices [Iꢂ2s(I)]: R1ꢀ0.057, wR2ꢀ0.166
(Dr)minꢀꢁ0.42 e Åꢁ3
(Dr)maxꢀ0.46 e Åꢁ3
(D/s)maxꢅ0.001
No. of reflections usedꢀ1173
Measurement: Bruker APEX-II area-detector diffractometer
Program system: SHELXL 97
Structure determination: direct method
Refinement: full matrix least-squares on F2
Fig. 1. ORTEP Structures of 2a and 3f, Showing 30% Probability Ellip-
soids
Table 2. Selected Bond Distances (Å), Angles (°), and Torsion Angles (°)
for 2a
C1–O1
C8–N2
C8–O2
C2–C1–O1
C8–N1–O1
N1–C8–N2
C1–C2–C3–C4
C1–C2–C7–C6
C2–C1–O1–N1
C1–O1–N1–C8
1.441 (3)
1.326 (3)
1.220 (3)
105.3 (2)
112.71 (17)
116.4 (2)
179.5 (3)
ꢁ179.5 (3)
71.5 (2)
N1–C8
O1–N1
1.274 (3)
1.406 (2)
N1–O1–C1
O2–C8–N1
O2–C8–N2
N2–C8–N1–O1
O1–C1–C2–C3
O1–C1–C2–C7
O2–C8–N1–O1
107.48 (18)
115.16 (19)
128.36 (19)
ꢁ20.0 (3)
55.5 (3)
ꢁ125.1 (3)
163.84 (19)
105.1 (2)
urea planes. In the crystal structure, molecules are linked to
antiparallel chains running along the b axis by intermolecular
N–H O hydrogen bonding (Fig. 2). The hydrogen bonding
Fig. 2. Perspective View of the Three-Dimensional Structure of 2a
Table 3. Hydrogen Bonding Geometry (Å, °) of 2a
…
parameters are summarized in Table 3.
Antitumor Evaluation The prepared compounds were
evaluated for their cytotoxicity against cancer in vitro using
HU as positive control. Firstly, compounds 2a—d and 3a—d
were evaluated for their cytotoxic activity in vitro against
human tongue cancer cell line (Tca8113). The results are
summarized in Table 4. As shown in Table 4, compounds 3a,
3b and 3d are the most active compounds to Tca8113 cells
…
…
…
…
D–H A
D–H
A
D–H
H
A
D
A
i
…
N2–H2B O2
0.86
0.86
2.29
1.90
2.950 (3)
2.733 (2)
134
163
ii
…
N2–H2A O2
Symmetry codes: (i) x, yꢆ1, z; (ii) ꢁxꢆ1, yꢆ1/2, ꢁzꢆ5/2.
with IC50 values ranging from 5.29ꢃ10ꢁ4ꢃ2.87 mM, with 3d ꢂ3aꢂ3dꢂ2dꢂ3dꢂ3cꢂ3dꢂ3bꢂ3dꢂ3fꢂ3dꢂ2eꢂ3dꢂHU
exhibiting the most potent activity (IC50 is 5.29ꢃ10ꢁ4 mM). ꢂ3dꢂ2aꢂ3dꢂ2cꢂ3dꢂ2bꢂ3dꢂ2f. Among them, the most
Because of the good results, we continued to synthesize promising compounds were 3e, 3d, 3a and 2d. The IC50 ra-
the hydroxyurea derivatives 2e—f and 3e—f, and evaluated tios of HU over compounds 3e, 3d, 3a and 2d range from 10
the cytotoxicity by the 3-(4,5-dimethylthiazol-2-yl)-2,5- to 40, indicating that the four compounds are 10 to 40 fold
diphenyltetrazolium bromide (MTT) assay for all the target more potent than HU against L1210 cells. 3c, 3b and 3f also
products using murine leukemia cell line L1210. The ranking had remarkable activity.
order of the cytotoxicity of all the compounds is 3eꢂ3d
The approximate partition coefficient Clog P of each de-

96
Vol. 58, No. 1
Table 4. Cytotoxicity Results of Compounds
d6) d: 4.75 (2H, s, –CH2–), 6.38 (2H, s, –NH2), 7.40 (5H, m, Ar-H), 9.06
(1H, s, –NH–). 13C-NMR (DMSO-d6) d: 77.73, 128.43, 128.66, 129.16,
137.06, 161.27. IR (KBr) cmꢁ1: 3394.5 (NH), 3222.8 (NH), 1631.7 (CꢀO),
1107.1 (C–O). MS m/z: 167.1 (MꢆH)ꢆ (Calcd for C8H10N2O2: 166.18).
4-Methylbenzyloxyurea (2b): Yield 82%. mp 124—126 °C. 1H-NMR
(DMSO-d6) d: 2.31 (3H, s, –CH3), 4.67 (2H, s, –CH2–), 6.31(2H, s, –NH2–),
7.17 (2H, d, Jꢀ8.0 Hz, Ar-H ), 7.30 (2H, d, Jꢀ7.6 Hz, Ar-H), 8.98(1H, s,
–NH–). 13C-NMR (DMSO-d6) d: 21.26, 77.57, 129.19, 129.31, 133.96,
137.66, 161.22. IR (KBr) cmꢁ1: 3384.8 (NH), 3197.8 (NH), 2916.2 (–CH3),
1662.5 (CꢀO), 1118.6 (C–O). MS m/z: 181.1 (MꢆH)ꢆ (Calcd for
C9H12N2O2: 180.20).
Tca8113
(IC50 mM)
L1210
(IC50 mM)
Compounds
Clog P
ꢁ1.8
HU
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
3e
3f
31.03
3.51ꢃ104
15.64
20.10
31.36
nda)
71.70
161.10
481.09
195.73
6.90
66.59
959.73
6.24
1.704
2.203
1.623
2.417
2.567
1.847
2.994
3.992
2.832
4.42
4-Methoxylbenzyloxyurea (2c): Yield 84%. mp 121—123 °C. 1H-NMR
(DMSO-d6) d: 3.76 (3H, s, –OCH3), 4.63 (2H, s, –CH2–), 6.27 (2H, s,
–NH2–), 6.92 (2H, d, Jꢀ8.4 Hz, Ar-H), 7.34 (2H, d, Jꢀ8.4 Hz, Ar-H), 8.94
(1H, s, –NH–). 13C-NMR (DMSO-d6) d: 55.54, 77.38, 114.02, 128.90,
130.93, 130.98, 161.18. IR (KBr) cmꢁ1: 3404.1 (NH), 3201.6 (NH), 2837.1
(–OCH3), 1664.5 (CꢀO), 1172.6 (C–O). MS m/z: 197.2 (MꢆH)ꢆ (Calcd for
C9H12N2O3: 196.20).
nd
2.87
1.85
21.68
15.17
1.94
8.48ꢃ102
5.29ꢃ10ꢁ4
nd
1.81
4.72
3.28
nd
28.84
2-Chlorobenzyloxyurea (2d): Yield 72%. mp 130—132 °C. 1H-NMR
(DMSO-d6) d: 4.72 (2H, s, –CH2–), 6.42 (2H, s, –NH2–), 7.38 (3H, t,
Jꢀ4.8 Hz, Ar-H), 7.52 (1H, s, Ar-H), 9.05 (1H, s, –NH–). 13C-NMR
(DMSO-d6) d: 76.67, 127.60, 128.25, 128.78, 130.48, 133.34,
139.71,161.22. IR (KBr) cmꢁ1: 3406.1 (NH), 3197.8 (NH), 1662.5 (CꢀO),
1112.9 (C–O). MS m/z: 201.1 (MꢆH)ꢆ (Calcd for C8H9ClN2O2: 200.62).
4-Bromobenzyloxyurea (2e): Yield 76%. mp 163—164 °C. 1H-NMR
(DMSO-d6) d: 4.68 (2H, s, –CH2–), 6.37 (2H, s, –NH2–), 7.38 (2H, d,
Jꢀ8.4 Hz, Ar-H), 7.56 (2H, d, Jꢀ8.0 Hz, Ar-H), 9.01 (1H, s, –NH–). 13C-
NMR (DMSO-d6) d: 76.75, 121.59, 131.34, 131.52, 136.55, 161.18. IR
(KBr) cmꢁ1: 3394.5 (NH), 3197.8 (NH),1666.4 (CꢀO), 1114.8 (C–O). MS
m/z: 245.2 (Mꢆ) (Calcd for C8H9BrN2O2: 245.07).
a) ndꢀnot determined.
rivative compound was calculated, and the values are in-
cluded in Table 4. The higher Clog P value corresponds to
the stronger hydrophobic or weaker hydrophilic nature of the
compound. After the chemical modification, all of the HU
derivatives possessed higher Clog P values than HU. Notably,
disubstituents 3 with higher Clog P showed higher cytotoxity
than the corresponding monosubstituents 2 with lower
Clog P, suggesting that the stronger hydrophobic nature of
the HU derivatives might favor the cytotoxic activity.
In conclusion, the desired HU derivatives were prepared.
From the data of antitumor activity tests in vitro, some of
them showed high or medium cytotoxicity against the cancer
cell lines Tca8113 and L1210. Among them, the most prom-
ising compounds were 3e, 3d, 3a and 2d. To assess the po-
tentials of these new compounds as cancer chemotherapeutic
1-(2-Fluorobenzyloxy)urea (2f): Yield 73%. mp 150—152 °C. 1H-NMR
(DMSO-d6) d: 4.78 (2H, s, –CH2–), 6.36 (2H, s, –NH2–), 7.22 (2H, d,
Jꢀ7.2 Hz, Ar-H), 7.41 (1H, d, Jꢀ6.0 Hz, Ar-H), 7.54 (1H, t, Jꢀ9.4 Hz, Ar-
H), 9.10 (1H, s, –NH–). 13C-NMR (DMSO-d6) d: 71.24, 124.74, 124.77,
130.88, 130.97, 132.14, 132.18, 161.20. IR (KBr) cmꢁ1: 3398.3 (NH),
3226.7 (NH), 1631.7 (CꢀO), 1230.5 (C–O). MS m/z: 206.8 (MꢆNa)ꢆ
(Calcd for C8H9FN2O2: 184.17).
Production of 3a—f. General Procedure Potassium hydroxide
(17 mmol) and benzyl chloride or benzyl bromide with different substituents
on phenyl (1a—f) (13 mmol) were added to a solution of compounds 2a—f
agents, further in vivo activity and toxicity studies are (13 mmol) in methanol (80 ml). After refluxing for 14—18 h (as evidenced
by TLC), solvent was removed under reduced pressure at 35 °C. The residue
needed. The results obtained from this study can be used as
was extracted with ether and the extraction solution was concentrated under
guidelines for further development.
reduced pressure. The crude product was column chromatographed on silica
using acetone/chloroform (1 : 4) as eluent, solvent was eliminated from the
elution under reduced pressure and then recrystallized in an acetone and
chloroform mixture (5 : 1.5) to get 3a—f as colorless crystals.
Experimental
Materials The starting compounds (1a—f) were purchased from
Shanghai Darei Finechemical Co., Ltd., China. Hydroxyurea was purchased
from Lingyi Furei Finechemical Co., Ltd., China. All reagents were obtained
from commercial sources and used without further purification unless stated.
Methanol was dried over calcium chloride and distilled. Acetone was dried
over magnesium sulphate and distilled.
Apparatus Melting points (mp) were determined using a capillary
method and were uncorrected. IR spectra were recorded on a Shimadzu FT-
IR 8400 spectrometer (KBr pellets). 1H- and 13C-NMR spectra were
recorded on a Bruker AV 400 MHz spectrometer. Mass spectra were
recorded on a Waters 2695 LC- ZQ4000 system. Crystal data were collected
by a Bruker APEX-II area-detector diffractometer.
Cell Lines The human tongue cancer cell line Tca8113 was provided by
the Institute of Medical Sciences in Jiangxi province, China and the lympho-
cytic murine leukemia cell line L1210 was purchased from Nanjing Keygen
Biotech. Co., Ltd., China. The two cell lines were cultured in RPMI-1640
medium supplemented with 10% heat-inactivated fetal bovine serum (FBS),
100 IU/ml penicillin G and 100 IU/ml streptomycin sulfate at 37 °C in a hu-
midified atmosphere containing 5% CO2 atmosphere.
1-Benzyl-1-benzyloxyurea (3a): Yield 32%. mp 97—98 °C. 1H-NMR
(DMSO-d6) d: 4.65 (2H, s, –CH2–), 4.66 (2H, s, –CH2–), 5.35 (2H, s,
–NH2), 7.35 (10H, m, Ar-H). 13C-NMR (DMSO-d6) d: 52.76, 77.04, 127.68,
128.45, 128.72, 128.92, 129.05, 129.31, 134.97, 136.76, 160.94. IR (KBr)
cmꢁ1: 3402.2 (NH), 3209.3 (NH), 1654.8 (CꢀO), 1209.3 (C–O). MS m/z:
257.2 (MꢆH)ꢆ (Calcd for C15H16N2O2: 256.30).
1-(4-Methylbenzyl)-1-(4-methylbenzyloxy)urea (3b): Yield 34%. mp
128—130 °C. 1H-NMR (DMSO-d6) d: 2.27 (3H, s, –CH3), 2.30 (3H, s,
–CH3), 4.46 (2H, s, –CH2–), 4.68 (2H, s, –CH2–), 6.47 (2H, s, –NH2), 7.10
(2H, d, Jꢀ8.0 Hz, Ar-H), 7.16 (4H, d, Jꢀ10.4 Hz, Ar-H), 7.27 (2H, d,
Jꢀ8.0 Hz, Ar-H); 13C-NMR (DMSO-d6) d: 21.15, 21.27, 51.29, 75.99,
129.01, 129.10, 129.20, 129.97, 133.18, 134.95, 136.62, 138.04, 160.75. IR
(KBr) cmꢁ1: 3377.1 (NH), 3199.7 (NH), 2947.0 (–CH3), 1666.4 (CꢀO),
1118.6 (C–O). MS m/z: 286.1 (MꢆH)ꢆ (Calcd for C17H20N2O2: 284.35).
1-(4-Methoxylbenzyl)-1-(4-methoxylbenzyloxy)urea (3c): Yield 36%. mp
99—101 °C. 1H-NMR (DMSO-d6) d: 3.33 (6H, s, –OCH3, –OCH3), 4.53
(2H, s, –CH2–), 4.74 (2H, s, –CH2–), 6.55 (2H, s, –NH2), 7.31 (8H, m, Ar-
H). 13C-NMR (DMSO-d6) d: 20.64, 20.73, 51.80, 76.56, 128.63, 128.78,
Production of 2a—f. General Procedure Potassium hydroxide
(34 mmol) and benzyl chloride or benzyl bromide with different substituents
on phenyl (1a—f) (26 mmol) were added to a solution of hydroxyurea
(26 mmol) in methanol (80 ml). The reaction mixture was refluxed and
checked by TLC until HU was consumed; solvent was removed under re-
duced pressure at 35 °C. The resulting crude solid was filtered and washed in
chloroform, then recrystallized in acetone and chloroform (5 : 2) to get 2a—
f as colorless crystals.
128.91, 129.15, 129.70, 133.03, 134.86, 136.81, 160.86. IR (KBr) cmꢁ1
:
3404.1 (NH), 3209.3 (NH), 2945.1(–OCH3), 1654.8 (CꢀO), 1209.3 (C–O).
MS m/z: 317.3 (MꢆH)ꢆ (Calcd for C17H20N2O4: 316.35).
1-(2-Chlorobenzyl)-1-(2-chlorobenzyloxy)urea (3d): Yield 24%. mp 81—
1
83 °C. H-NMR (DMSO-d6) d: 4.56 (2H, s, –CH2–), 4.78 (2H, s, –CH2–),
6.70 (2H, s, –NH2), 7.35 (8H, m, Ar-H). 13C-NMR (DMSO-d6) d: 50.97,
75.13, 127.56, 127.60, 128.49, 128.67, 128.73, 128.80, 129.71, 130.50,
133.22, 133.28, 138.54, 140.49, 160.66. IR (KBr) cmꢁ1: 3467.8 (NH),
1-(Benzyloxy)urea (2a): Yield 80%. mp 140—142 °C. 1H-NMR (DMSO-

January 2010
97
3195.8 (NH), 1689.5 (CꢀO), 1010.6 (C–O). MS m/z: 325.0 (Mꢆ) (Calcd for
C15H14Cl2N2O2: 325.19).
Acknowledgements This work was financially supported by the Na-
tional Key S&T Special Project of China: Grand New Drug R&D (NO.
2009ZX09103-087) and the Grand Science and Technology Special Project
of Jiangxi Province (20041A0300201). The authors also thank Jing Gang-
Shan College and Professor Xiao-Niu Fang for assistance with the crystallo-
graphic data collections and refinements of compounds 2a and 3f.
1-(4-Bromobenzyl)-1-(4-bromobenzyloxy)urea (3e): Yield 28%. mp
141—142 °C. 1H-NMR (DMSO-d6) d: 4.50 (2H, s, –CH2–), 4.70 (2H, s,
–CH2–), 6.60 (2H, s, –NH2), 7.21 (2H, d, Jꢀ8.4 Hz, Ar-H), 7.36 (2H, d,
Jꢀ8.4 Hz, Ar-H), 7.50 (2H, d, Jꢀ8.4, Ar-H), 7.54 (2H, d, Jꢀ8 Hz, Ar-H).
13C-NMR (DMSO-d6) d: 31.48, 51.06, 75.24, 120.76, 122.08, 131.18,
131.56, 132.08, 135.54, 137.36, 160.64. IR (KBr) cmꢁ1: 3404.1 (NH),
3186.2 (NH), 1670.2 (CꢀO), 1070.4 (C–O). MS m/z: 414.9 (Mꢆ) (Calcd for
C15H14Br2N2O2: 414.09).
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1
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9) Gwilt P. R., Tracewell W. G., Clin. Pharmacokinet., 34, 347—358
(1998).
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McKenna C. E., Csipke C., Tokes Z. A., Lien E. J., J. Med. Chem., 45,
410—419 (2002).
12) Mai X., Xia H. Y., Cao Y. S., Lu X. S., Fang X. N., Acta Cryst., E65,
o442 (2009).
X-Ray Crystallography Colorless needle-shaped single crystals of the
compound 2a suitable for X-ray structure analysis were obtained by slow
evaporation from the mixed solvent acetone and N-hexane (9 : 11) at room
temperature for one week. The X-ray data were collected on a diffractometer
equipped with graphite-monochromated MoKa radiation (lꢀ0.71073 Å) at
296(2) K. The structure was determined by direct methods and refined on F2
by full-matrix least-squares using the program SHELXTL-97.19) An X-ray
diffraction study of the compound showed that it was crystallized in a mono-
clinic system with the P21/c space group. Non-hydrogen atoms were refined
with anisotropic displacement parameters. H atoms were calculated and al-
lowed to ride. Computer programs: structure solution, SHELXS-97,19) re-
finement, SHELXS-97,20) molecular diagrams, ORTEP.21)
Pharmacology The newly synthesized compounds were evaluated for
their in vitro cytotoxicity by growth-inhibition studies using two cancer-cell
lines: human tongue cancer cell line (Tca8113) and murine leukemia cell
line (L1210). Sulforhodamine B (SRB) assay22) was used for study of
Tca8113 cells, while MTT assay23) was used of L1210 cells.
13) Howard W., Shields P. J., Hamrick J., Welby R., Chem. Phys., 46,
(1967).
SRB Method Tumor cells (1ꢃ105 cells mlꢁ1) were inoculated in 96-
well culture plates (100 ml/well). After culture for 24 h at 37 °C in a 5% CO2
atmosphere, 50 ml of culture medium containing synthetic compound of var-
ious concentrations was added to the wells, the cells were then incubated for
65 h, and the medium was removed by suction. The cells were fixed with
10% cold trichloroacetic acid (TCA) and the plates were kept at 4 °C for 1 h.
The TCA was removed by suction, and the plates were rinsed with water re-
peatedly 5 times and stained with SRB 0.4% (w/v) in 1% acetic acid for
15 min. Excess dye was washed out by 1% acetic acid repeatedly 5 times, air
dried and the bound stain was subsequently solubilized with 150 ml Tris base
(tris(hydroxy-methyl)aminomethane). The absorbance of SRB solution was
measured at 490 nm with a microplate reader.
14) Thiessen W. E., Levy H. A., Flaig B. D., Acta Cryst., B34, 2495
(1978).
15) Armagan N., Richards J. P. G., Uraz A. A., Acta Cryst., B32, 1042
(1976).
16) Helen B., Kim S. H., Acta Cryst., 23, 180 (1967).
17) Ingrid K. L., Bodil J., Acta Chem. Scand., 20, 983—991 (1966).
18) Bettina B. N., Karla, F., Ingrid K. L., Acta Cryst., C49, 1018—1022
(1993).
19) Sheldrick G. W., SHELXS-97, “Program for Crystal Structure Solu-
tion,” University of Göttingen, Germany, 1997.
20) Sheldrick G. W., SHELXS-97, “Program for Crystal Structure Refine-
ment,” University of Göttingen, Germany, 1997.
21) Johnson C. K., “Report ORNL-5138,” OAK Ridge National Labora-
tory,OAK Ridge, TN, 1976.
22) Monks A., Scudiero D., Skehan P., Shoemaker R., Paull K., Vistica D.,
Hose C., Langley J., Cronise P., Vaigro-Wolff A., Gray-Goodrich M.,
Campbell H., Mayo J., Boyd M., J. Natl. Cancer Inst., 83, 757—766
(1991).
23) Chen X. Z., Xie M. J., Liu W. P., Ye Q. S., Yao Y., Inorg. Chim. Acta,
360, 285—298 (2007).
MTT Method Tumor cells (1ꢃ106 cells mlꢁ1) were inoculated in 96-
well culture plates (100 ml/well). After cultured for 24 h, 50 ml of culture
medium containing synthetic compound of various concentrations was
added to the wells, then the cells were incubated for 65 h. Twenty microliters
of MTT was added at a final concentration of 5 mg/ml and after 4 h incuba-
tion, 150 ml of DMSO was added and the optical density was measured at
490 nm.