Synthesis and cytotoxic properties of 7α-chloro-3-methyl-1,1- dioxoceph-3-ems, substituted with amide or keto group at position 4

Article abstract of DOI:10.1007/s10593-007-0242-3

7α-Chloro-3-methyl-1,1-dioxoceph-3-ems with amide or keto group at position 4 have been synthesized by structural modification of 7α-chloro-3-methyl-1,1-dioxoceph-3-em-4-carboxylic acid. Screening of these compounds for cytotoxic activity revealed compoun

Full text of DOI:10.1007/s10593-007-0242-3

Chemistry of Heterocyclic Compounds, Vol. 43, No. 12, 2007
SYNTHESIS AND CYTOTOXIC
PROPERTIES OF 7α-CHLORO-
3-METHYL-1,1-DIOXOCEPH-3-EMS,
SUBSTITUTED WITH AMIDE
OR KETO GROUP AT POSITION 4
M. Vorona, G. Veinberg, I. Shestakova, I. Kanepe, M. Petrova,
E. Liepinsh, and E. Lukevics
7α-Chloro-3-methyl-1,1-dioxoceph-3-ems with amide or keto group at position 4 have been synthesized
by structural modification of 7α-chloro-3-methyl-1,1-dioxoceph-3-em-4-carboxylic acid. Screening of
these compounds for cytotoxic activity revealed compounds with specific activity against cancer cells in
vitro, capable of effective inhibition of the growth of sarcoma S-180 in vivo.
Keywords: amides of 7α-chloro-3-methyl-1,1-dioxoceph-3-em-4-carboxylic acid, amides of 2-(N,N-
dimethylaminomethylene)-7α-chloro-3-methyl-1,1-dioxoceph-3-em-4-carboxylic acids, 4-aryl-7α-chloro-
3-methyl-1,1-dioxoceph-3-ems, cytotoxic activity.
We have shown previously that the intensity and selectivity of cytotoxic properties in vitro of esters of
7α-chloro-3-methyl-1,1-dioxoceph-3-em-4-carboxylic acid relative to cancer and normal cells depend on the
structure of the substituent at position 4 [1]. In a continuation of this work we have synthesized and studied the
cytotoxic activity of 7α-chloro-3-methyl-1,1-dioxoceph-3-ems with amide and keto groups in position 4 some
representatives of, which according to literature data, are effective inhibitors of human leucocyte elastases [2, 3].
Amides of 7α-chloro-3-methyl-1,1-dioxoceph-3-em-4-carboxylic acid 5a-o were obtained, starting from
7α-chloro-3-methyl-1,1-dioxoceph-3-em-4-carboxylic acid (1) by conversion into the acid chloride using the
Vilsmeier reagent. The latter was treated with the corresponding mono- or disubstituted amines 4a-o without
isolation (Scheme). Along with the amides of 7α-chloro-3-methyl-1,1-dioxoceph-3-em-4-carboxylic acid 5a-o,
the cephems 6a,d,f,g,l, containing N,N-dimethylaminomethyl group at position 2, were obtained from the
reaction mixture when tert-butylamine 4a, benzhydrylamine 4d, 3-pyridylmethylamine 4f, para-
methoxycarbonylaniline 4g, and piperidine 4l were used.
1
By analogy with the H NMR spectra of esters of 2,N,N-dimethylaminomethylene-7α-chloro-3-methyl-
1,1-dioxoceph-3-em-4-carboxylic acid [1], the characteristic shift of the resonance signals of the protons of the
=CN(CH3)2 group in the E-isomers to the 3.00-3.10 ppm region and of the Z-isomers to the 3.30-3.35 ppm
region, indicates that in the cephems 6a,d,l the group =CN(CH3)2 is a mixture of the E- and Z-isomers, whereas
in cephems 6f,g it is predominantly in the form of the E-isomer.
__________________________________________________________________________________________
Latvian Institute of Organic Synthesis, Riga, LV-1006; e-mail: veinberg@osi.lv, e-mail: max@osi.lv.
Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 12, 1849-1859, December 2007. Original article
submitted October 18, 2007.
0009-3122/07/4312-1567©2007 Springer Science+Business Media, Inc.
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Conversion of the 7α-chloro-3-methyl-1,1-dioxoceph-3-em-4-carbonyl chloride (3) into the 4-oxo-
substituted cephems 8a-d using the Grignard reagents 7a-d was carried out by a published method [3]. In
addition to the previously synthesized compounds 8b and 8c, the new cephems 8a and 8d with
isopropylcarbonyl and 2-thienylcarbonyl substituents at position 4 were obtained (Scheme).
The biological studies of the synthesized amides and ketones 5, 6, and 8 included:
a) screening for cytotoxic activity in vitro relative to monolayer lines of cancer cells HT-1080 (human
fibrosarcoma) and MG-22A (mouse hepatoma), B16 (mouse melanoma), and Neuro 2A (mouse neuroblastoma);
b) screening for cytotoxic activity in vitro relative to monolayer lines of normal 3T3 cells (mouse
embryonic fibroplasts) and BHK (Baby Hamster Kidney – fibroplasts of golden hamster kidneys);
c) determination of the specific ability of compounds to initiate the biosynthesis of nitrogen oxide
radicals in a cellular medium;
(d) calculation of the expected toxicity of the compounds tested, LD50 (µg/kg) [4];
(e) investigation of the anticancer activity in experiments in vivo in mice with grafted fast growing B16
tumor (melanoma) and slow growing S-180 tumor (sarcoma).
On the basis of the data on cytotoxic screening in vitro relative to cancer and normal cells, and also the
calculated values of the expected toxicity, the compounds tested were separated into three groups.
Group I – compounds of low activity, the cytotoxic activity of which with respect to the tested cancer
cells (LC50) fell within 15 to 100 µg/kg range. The 7α-chloro-3-methyl-1,1-dioxoceph-3-ems 5b and 5i, the
amide group of which contains tert-octyl- and 6-methylpyridyl groups, and also 2-N,N-dimethylamino-
methylene-7α-chloro-3-methyl-1,1-dioxoceph-3-ems 6g and 6i with amide groups synthesized with the help of
4-methoxycarbonylaniline and piperidine, belong to this group.
Table 1. Cytotoxic Activity in vitro of Amides of 7α-Chloro-3-methyl-
1,1-dioxoceph-3-em-4-carboxylic acid*
Cytotoxic activity, LC50, µg/ml
Com-
pound
LD50,
mg/kg
HT-1080
MTT
MG-22A
MTT
3T3
NR
CV
TG100
CV
TG100
5a
5c
5d
6d
5e
5k
5l
1
3
0.4
3
2
5
0.6
2
200
700
500
400
50
2
2
2
3
3
2
250
350
650
300
100
250
100
150
500
49
12
21
14
29
30
40
27
15
597
344
487
438
537
481
559
486
373
3
2
2
2
2
7
6
12
3
500
15
2
2
3
0.4
9
2
0.2
7
1
5m
5n
15
6
12
7
100
650
_______
* Here and later: LC50 is the concentration causing destruction of 50% of the
cells; CV – coloring by crystal violet; MTT – coloring by 3-(4,5-dimethyl-
thiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide; TG100 – specific NO
generating activity of the compound; NR – coloring by neutral red.
Group II (Table 1) – cephems 5a,c-e,k-n, 6d – synthesized using tert-butylamine, benzylamine,
benzhydrylamine, methyl R-phenylglycine, diethylamine, piperidine, 2-methylpiperidine, and hexamethylene-
imine, show high cytotoxicity both with respect to HT-1080 and MG-22A cancer cells, and normal 3T3 cells
and, consequently show characteristically low values of LD50 (344-597 µg/kg).
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Table 3. Therapeutic Activity in vivo of 7α-Chloro-3-methyl-1,1-dioxo-
4-(thienylcarbonyl)ceph-3-em (8d)
Slowing of growth of the tumor, GI%
S-180 B-16
Schedule
of injection
on days
Daily dose, Total dose,
mg/kg
mg
9 days
11 days
9 days
11 days
1, 2, 3, 4, 7, 8
1, 2, 3, 4, 7, 8
10
5
60
30
83
78
76
75
51
25
32
17
Group III (Table 2) – 2-substituted and unsubstituted 7α-chloro-3-methyl-1.1-dioxoceph-3-ems 6a,f,
5g,h,j,o, the amide groups of which were obtained by using tert-butylamine, 3-pyridylmethylamine, para-
methoxycarbonylaniline, para-cyanoaniline, dimethylamine, and morpholine. Their cytotoxicity with respect to
all or some of the test cultures of cancer cells considerably exceeded those for normal cells BNK-21 and 3T3.
The appearance of selectivity lead to a decreased toxicity of these compounds to values of LD50 equal to
718-1376 mg/kg.
The cytotoxic activity of the tested amides 5 and 6 is similar to that of the previously studied esters of
7α-chloro-3-methyl-1,1-dioxoceph-3-em-4-carboxylic acid [1], coupled with their ability to generate NO free
radicals in the cell medium. High cytotoxicity of compounds is accompanied, as a rule, by increased generation
of NO radicals and the opposite.
All 4-oxo-substituted 7α-chloro-4-isopropylcarbonyl-3-methyl-1,1-dioxoceph-3-ems 8a-d, are
characterized by a highly selective cytotoxic effect in relation to cancer and normal cells, with a consequent low
toxicity, LD50 617-1313 mg/kg (Table 2). This is also indicated by data on the anticancer activity of 7α-chloro-
3-methyl-1,1-dioxo4-(2-thienylcarbonyl)ceph-3-em (8d) in vivo in experiments on mice inoculated with tumors.
It is seen from the data of Table 3 that in the case of slowly growing sarcoma S-180 a daily injection into the
mouse of the test substance at doses of 5 and 10 mg/kg slowed the growth of the tumor by 75-76%. However the
therapeutic effect of compound 8d on the fast growing melanoma B16 was 2-4 times less at these doses.
The results of these studies indicate that conversion of carboxyl groups at position 4 of 7α-chloro-
3-methyl-1,1-dioxoceph-3-ems into amide or keto group is a potential direction for the creation of anticancer
substances, the mechanism of which, as a result of their structural similarity to inhibitors of leucocytic elastases
of man, may be ascribed to the inhibition of specific elastases which facilitate the growth and proliferation of
cancer cells [5-7].
EXPERIMENTAL
13
1H and C NMR spectra of CDCl3 solutions with TMS as internal standard were recorded on Bruker
1
1
WH90/DS (90 MHz for H), Varian-Mercury BB (200 MHz for H), and Varian-Mercury Plus (400 and 100
MHz for H and 13C respectively). Elemental analyses were carried out with a Carlo Erba analyzer with
1
differences between experimental and calculated values of ±0.4%. Experiments were monitored by TLC on
Merck Kieselgel plates with development with UV light. HPLC was carried out with a Du Pont Model 8800
equipped with a UV detector (λ = 254 nm) and a column (4.6×250 nm) filled with Symetry C18 or Ultrasphere
octyl phases in acetonitrile–water or acetonitrile–0.1 N phosphate buffer (pH 2.5) (60:40) systems, rate
0.8-1.5 ml/min. Silica gel (Merck Kieselgel 0.063-0.230 mm) was used for preparative column chromatography.
Material from Acros, Aldrich, and Sigma were used as experimental reagents. Optical density in the biological
tests were carried out in 96 hole panels and determined with a Tetretek Multiscan MCC/340 horizontal
spectrophotometer.
1571

Preparation of Amides of 7α-Chloro-3-methyl-1,1-dioxoceph-3-em-4-carboxylic Acid (5) and
2E-(N,N-Dimethylaminomethylene)-7a-chloro-3-methyl-1,1-dioxoceph-3-em-4-carboxylic acid (6) (General
Method). To a stirred suspension of the sulfone of 7α-chloro-3-methylceph-3-em-4-carboxylic acid (300 mg,
1.13 mmol) in dichloromethane (20 ml) at room temperature a mixture of oxalyl chloride (434 µl, 3.39 mmol) in
DMF (4 µl) was added. The mixture was warmed at 40°C over 20 min, cooled to room temperature and
concentrated under reduced pressure. The residue, containing 7α-chloro-3-methylceph-3-em-4-carbonyl chloride,
was dissolved in dichloromethane (15 ml). An amine 4 (2.8 mmol) was added to the solution cooled to -5°C. The
reaction mixture was stirred for 30 min at room temperature and for 30 min at 40°, diluted with dichloromethane
(40 ml), washed with 5% HCl (2×20 ml), and dried over anhydrous Na2SO4. The solvent was evaporated under
reduced pressure. The residue was fractionated on a silica gel chromatographic column to give amides 5 and also
their derivatives 6, containing an N,N-dimethylaminomethylene group in position 2.
4-N-tert-Butylaminocarbonyl-7α-chloro-3-methyl-1,1-dioxoceph-3-em (5a) was obtained using
tert-butylamine 4a in the general method and isolated from fraction (eluent 1:1 ethyl acetate–hexane) with Rf 0.33.
Yield 47 mg (13%); mp 100-102°C. 1H NMR spectrum (90 MHz), δ, ppm (J, Hz): 1.40 (9H, s, C4H9); 1.95 (3H, s,
3-CH3); 3.62 and 4.00 (2H, two d, AB system, 2J = 18, SO2CH2); 4.88 (1H, br. s, H-6); 5.31 (1H, d, 3J =0.5, H-7);
6.44 (1H, br. s, NH). Found, %: C 44.98; H 5.44; N 8.80. C12H17ClN2O4S. Calculated, %: C 44.93; H 5.34; N 8.73.
Mixture of 2(E)-4-N-tert-butylaminocarbonyl-2-(N,N-dimethylaminomethylene)-7a-chloro-3-methyl-
1,1-dioxoceph-3-em (E-6a) and 2(Z)-4-N-tert-butylaminocarbonyl-2-(N,N-dimethylaminomethylene)-
3-methyl-1,1-dioxo-7a-chloroceph-3-em (Z-6a) (4:1) was obtained by using tert-butylamine in the general
method and isolated from fraction (eluent 1:1 ethyl acetate–hexane) with Rf 0.18. Yield 64 mg (15%). 1H NMR
spectrum (400 MHz), δ, ppm (J, Hz): E-6a – 1.41 (9H, s, C4H9); 2.08 (3H, s, 3-CH3); 3.05 (6H, s, N(CH3)2);
3
3
13
4.77 (1H, d, J = 2, H-6); 5.08 (1H, d, J = 2, H-7); 6.15 (1H, br. s, NH); 7.21 (1H, s, =CHNMe2). C NMR
spectrum, δ, ppm: 21.40 (CH3); 28.46 (C(CH3)2); 43.32 ((N(CH3)2); 52.10 (CMe3); 55.63 (C-7); 74.39 (C-6);
98.51 (C-2); 126.42 (C-4); 131.11 (C-3); 148.46 (CHNMe2); 160.91 (CONH); 162.75 (C-8). 1H NMR spectrum,
δ, ppm (J, Hz): Z-6a – 1.41 (9H, s, C4H9); 2.22 (3H, s, 3-CH3); 3.29 (6H, s, N(CH3)2); 4.55 (1H, br. s, H-6); 5.25
(1H, br. s, H-7); 6.88 (1H, br. s, NH); 6.89 (1H, s, =CHNMe2). 13C NMR spectrum, δ, ppm: 17.86 (CH3); 28.46
(C(CH3)3); 46.67 (N(CH3)2); 52.09 (CMe3); 55.60 (C-7); 71.09 (C-6); 99.74 (C-2); 117.82 (C-4); 124.74 (C-3);
149.34 (CHNMe2); 159.48 (C-8); 161.49 (CONH). Found, %: C 48.05; H 6.11; N 11.22. C15H22ClN3O4S.
Calculated, %: C 47.93; H 5.90; N 11.18.
4-N-(1,1,3,3-Tetramethylbutyl)aminocarbonyl-7α-chloro-3-methyl-1,1-dioxoceph-3-em (5b) was
obtained by use of tert-octylamine in the general method and isolated from fraction (eluent 1:1 ethyl acetate–
1
hexane) with Rf 0.47. Yield 255 mg (60%); mp 102-104°C. H NMR spectrum (90 MHz); δ, ppm (J, Hz): 1.00
2
(9H, s, 3-CH3 octyl); 1.46 (6H, s, 2-CH3 octyl); 1.77 (2H, d, J = 6, CH2 octyl); 2.02 (3H, s, 3-CH3); 3.57 and
3.86 (2H, two d, AB system, 2J = 18, SO2CH2); 4.71 (1H, br. s, H-6); 5.26 (1H, d, 3J = 2, H-7); 6.42 (1H, br. s,
NH). Found, %: C 51.22; H 6.80; N 7.40. C16H25ClN2O4S. Calculated, %: C 50.99; H 6.69; N 7.43.
4-N-Benzylaminocarbonyl-7α-chloro-3-methyl-1,1-dioxoceph-3-em (5c) was obtained from
benzylamine by the general method and isolated from ractions (eluent 1:1 ethyl acetate–hexane) with Rf = 0.08.
Yield 100 mg (25%); mp 71-73°C. 1H NMR spectrum (90 MHz); δ, ppm (J, Hz): 2.00 (3H, s, 3-CH3); 3.63 and
3.89 (2H, two d, AB system, 2J = 18, SO2CH2); 4.51 (2H, d, 3J = 6, CH2 benzyl); 4.73 (1H, br. s, H-6); 5.22 (1H,
3
d, J = 2, H-7); 7.06 (1H, br. s, NH); 7.33 (5H, s, C6H5). Found, %: C 50.89; H 4.31; N 7.95. C15H15ClN2O4S.
Calculated, %: C 50.78; H 4.26; N 7.90.
4-N-Benzhydrylaminocarbonyl-7α-chloro-3-methyl-1,1-dioxoceph-3-em (5d) was obtained by using
benzhydrylamine 4d in the general method and was isolated from fractions (eluent 1:3 ethyl acetate–hexane)
1
with Rf 0.43. Yield 195 mg (40%); mp 152-154°C. H NMR spectrum (90 MHz), δ, ppm (J, Hz): 1.93 (3H, s,
3-CH3); 3.33 and 3.88 (2H, two d, AB system, 2J =18, SO2CH2); 4.73 (1H, br. s, H-6); 5.28 (1H, d, 3J = 2, H-7);
3
6.28 (1H, d, J =8, CHPh2); 7.08-7.60 (11H, m, 2C6H5, NH). Found, %: C 58.72; H 4.63; N 6.57.
C21H19ClN2O4S. Calculated, %: C 58.53; H 4.44; N 6.50.
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A mixture of 2(E)-4-N-benzhydrylaminocarbonyl-2-(N,N-dimethylaminomethylene)-7α-chloro-
3-methyl-1,1-dioxoceph-3-em (E-6d) and 2(Z)-4-N-benzhydrylaminocarbonyl-2-(N,N-dimethylamino-
methylene)-7α-chloro-3-methyl-1,1-dioxoceph-3-em (Z-6d) (2:1) was obtained using benzhydrylamine 4d in
the general method and isolated from fractions (eluent1:1 ethyl acetate–hexane) with Rf 0.18. Yield 71 mg
1
(13%). H NMR spectrum (90 MHz), δ, ppm (J, Hz): E-6d – 2.26 (3H, s, 3-CH3); 3.04 (6H, s, N(CH3)2); 4.77
3
3
3
(1H, d, J =1, H-6); 5.12 (1H, d, J = 8, H-7); 6.26 (1H, d, J = 8, CHPh2); 6.80-7.66 (12H, m, 2C6H5, NH,
=CHNMe2). Z-6d – 2.31 (3H, s, 3-CH3); 3.33 (6H, s, N(CH3)2); 4.55 (1H, br. s, H-6); 5.26 (1H, br. s, H-7); 6.26
3
(1H, d, J =8, CHPh2); 6.80-7.66 (12H, m, 2C6H5, NH, =CHNMe2). Found, %: C 59.55; H 5.09; N 8.71.
C24H24ClN3O4S. Calculated, %: 59.31; H 4.98; N 8.65.
4-N-(1(R-Methoxycarbonyl)benzylaminocarbonyl-7α-chloro-3-methyl-1,1-dioxoceph-3-em
(5e)
was obtained from methyl R-phenylglycine in the general method and isolated from a fraction (eluent 1:1 ethyl
acetate–hexane) with Rf 0.34. An amorphous substance, containing 98% of the basic material according to
HPLC, yield 28 mg (6%). 1NMR spectrum (200 MHz); δ, ppm (J, Hz): 2.04 (3H, s, 3-CH3); 3.63 and 3.90 (2H,
3
3
two d, AB system, J = 18, SO2CH2); 3.75 (3H, s, OCH3); 4.77 (1H, br. s, H-6); 5.29 (1H, d, J = 2, H-7); 5.63
(1H, d, 3J = 7, CHPh); 7.38 (5H, s, C6H5); 7.60 (1H, d, 3J = 7, NH).
4-N-(3-Pyridylmethyl)aminocarbonyl-7α-chloro-3-methyl-1,1-dioxoceph-3-em (5f) was obtained
using 3-pyridylmethylamine in the general method and isolated from fractions (eluent 3:1 ethyl acetate–hexane)
with Rf 0.32. An amorphous substance, containing 98% of the basic material according to HPLC, yield 36 mg
(9%). 1H NMR spectrum (90 MHz), δ, ppm (J, Hz): 2.02 (3H, s, 3-CH3); 3.60 and 3.95 (2H, two d, AB system,
3
2J = 19, SO2CH2); 4.55 (2H, d, J = 6, CH2Py); 4.77 (1H, br. s, H-6); 5.29 (1H, br. s, H-7); 7.26-7.42 (1H, m,
H-5 Py); 7.51-7.80 (2H, m, NH, H-4 Py); 8.37-8.66 (2H, H-2,6 Py). Found, %: C 47.39; H 4.06; N 11.95.
C14H14ClN3O4S. Calculated, %: C 47.26; H 3.97; N 11.81.
2(E)-(N,N-Dimethylaminomethylene)-7α-chloro-3-methyl-1,1-dioxoceph-3-em (E-6f) was obtained
by using 3-pyridylmethylamine in the general method and isolated from fractions (eluent (1:1 CH2Cl2–EtOH)
with Rf 0.55. An oily substance, containing 98% of the basic material according to HPLC, yield 32 mg (7%).
3
1H NMR spectrum (90 MHz), δ ppm (J, Hz): 2.24 (3H, s, 3-CH3); 3.08 (6H, s, N(CH3)2); 4.48 (2H, d, J = 8,
CH2Py); 4.77 (1H, br. s, H-6); 5.06 (1H, br. s, H-7); 7.11-7.33 (2H, m, =CHNMe and H-5 Py); 7.51-7.80 (2H, m,
NH, H-4 Py); 8.35-8.64 (2H, m, H-2,6 Py). Found, %: C 49.78; H 4.51; N 13.70. C17H19ClN4O4S. Calculated, %:
C 49.69; H 4.66; N 13.64.
4-N-(4-Methoxycarbonylphenyl)aminocarbonyl-7α-chloro-3-methyl-1,1-dioxoceph-3-em (5g) was
obtained by using 4-methoxycarbonylaniline 4g in the general method and isolated from fractions (eluent 1:1
ethyl acetate–hexane) with Rf 0.16. Yield 63 mg (14%); mp 226-228°C. 1H NMR spectrum (90 MHz), δ, ppm (J,
2
Hz): 2.11 (3H, s, 3-CH3); 3.68 and 4.00 (2H, two d, AB system, J = 18, SO2CH2); 3.88 (3H, s, OCH3); 4.80
3
3
(1H, br. s, H-6); 5.37 (1H, d, J = 0.5, H-7); 7.64 and 8.04 (4H, two d, J = 8, C6H4); 9.13 (1H, br. s, NH).
Found, %: C 48.23; H 3.84; N 7.11. C16H15ClN2O6S. Calculated, %: C 48.19; H 3.79; N 7.02.
2(E)-(N,N-dimethylaminomethylene)-4-N-(4-methoxycarbonylphenyl)aminocarbonyl-7a-chloro-
3-methyl-1,1-dioxoceph-3-em (E-6g) was obtained by using 4-methoxycarbonylaniline 4g in the general
method and isolated from fractions (eluent 1:1 ethyl acetate–hexane) with Rf = 0.05. Yield 31 mg (6%);
1
mp 207-210°C. H NMR spectrum (90 MHz), δ, ppm (J, Hz): 2.60 (3H, s, 3-CH3); 3.08 (6H, s, N(CH3)2); 3.38
(3H, s, OCH3); 4.84 (1H, br. s, H-6); 5.18 (1H, br. s, H-7); 7.24 (1H, s, CHNMe2); 7.64 and 7.97 (4H, two d,
3J = 8, C6H4); 8,84 (1H, br. s, NH). Found, %: C 50.36, H 4.52, N 9.11. C19H20ClN3O6S. Calculated, %: C 50.28;
H 4.44; N 9.26.
4-N-(4-Cyanophenyl)aminocarbonyl-7α-chloro-3-methyl-1,1-dioxoceph-3-em (5h) was obtained
using 4-cyanoaniline in the general method and isolated from fraction (eluent 1:1 ethyl acetate–hexane) with
Rf = 0.18. Yield 45 mg (11%); mp 220°C. 1H NMR spectrum (90 MHz, DMSO-d6); δ, ppm (J, Hz): 1.86 (3H, s,
3
3-CH3); 4.26 (2H, s, SO2CH2); 5.53 (1H, br. s, H-6); 5.88 (1H, d, J = 0.5, H-7); 7.82 (4H, br. s, C6H4); 11.02
(1H, br. s, NH). Found, %: C 49.25; H 3.31; N 11.55. C15H12ClN3O4S. Calculated, %: C 50.28; H 4.44; N 11.49.
1573

4-N-(6-Methyl-2-pyridyl)aminocarbonyl-7α-chloro-3-methyl-1,1-dioxo-ceph-3-em (5i) was obtained
by using 2-amino-6-methylpyridine in the general method and isolated from fractions (eluent 1:1 ethyl acetate–
hexane) with Rf = 0.24. Oily substance, containing >97% of the basic material according to HPLC, yield 28 mg
(7%). 1H NMR spectrum (90 MHz), δ, ppm (J, Hz): 1.98 (3H, s, 3-CH3); 2.37 (3H, s, CH3 at Py); 3.62 and 4.02
(2H, two d, AB system, 2J = 18, SO2CH2); 4.84 (1H, br. s, H-6); 5.37 (1H, d, 3J =0.5, H-7); 6.95 (1H, d, 3J = 8,
H-5 Py); 7.62 (1H, t, 3J = 8, H-4 Py); 8.04 (1H, d, 3J = 8, H-3 Py); 9.70 (1H, br. s, NH).
4-N,N-Dimethylaminocarbonyl-7α-chloro-3-methyl-1,1-dioxoceph-3-em (5j) was obtained by using
dimethylamine in the general method and isolated from fraction (eluent 3:1 ethyl acetate–hexane) with Rf = 0.42.
Yield 46 mg (14%); mp 133°C (dec.). 1H NMR spectrum (200 MHz), δ, ppm (J, Hz): 2.16 (3H, s, 3-CH3); 3.02
and 3.07 (6H, two s, N(CH3)2); 3.51 and 3.97 (2H, two d, AB system, 2J = 18, SO2CH2); 4.87 (1H, d, 3J = 1.6, H-
3
6); 5.31 (1H, d, J = 1.6, H-7). Found, %: C 41.28, H 4.61, N 9.65. C10H13ClN2O4S. Calculated, %: C 41.03; H
4.48; N 9.57.
4-N,N-Diethylaminocarbonyl-7α-chloro-3-methyl-1,1-dioxoceph-3-em (5k) was obtained by using
diethylamine in the general method and isolated from a fraction (eluent 1:1 ethyl acetate–hexane) with Rf = 0.25.
Yield 94 mg (26%); mp 198-201°C (dec.). 1H NMR spectrum (90 MHz), δ, ppm (J, Hz): 1.08 and 1.20 (6H, two
3
t, J = 7, N(CH2CH3)2); 1.77 (3H, s, 3-CH3); 3.17-3.55 (4H, m, N(CH2CH3)2); 3.48 and 4.02 (2H, two d, AB
system, 2J = 18, SO2CH2); 4.86 (1H, br. s, H-6); 5.28 (1H, d, 3J = 1.5, H-7). Found, %: C 45.16; H 5.48; N 8.80.
C12H17ClN2O4S. Calculated, %: C 44.93; H 5.34; N 8.73.
7α-Chloro-3-methyl-1,1-dioxo-4-piperidinocarbonylceph-3-em (5l) was obtained by using piperidine
4l in the general method and isolated from fractions (eluent ethyl acetate) with Rf = 0.42. Yield 45 mg (12%); mp
1
187-189°C. H NMR spectrum (90MHz); δ, ppm (J, Hz): 1.45-1.70 (6H, m, 3-CH2, 4-CH2, and 5-CH2
piperidine); 1.75 (3H, s, 3-CH3); 3.26-3.77 (4H, m, 2-CH2 and 6-CH2 piperidine); 3.57 and 4.05 (2H, two d, AB
system, 2J = 18, SO2CH2); 4.94 (1H, br. s, H-6); 5,33 (1H, d, 3J = 1.5, H-7). Found, %: C 47.17; H 5.25; N 8.49.
C13H17ClN2O4S. Calculated, %: C 46.92; H 5.15; N 8.42.
Mixture of 2-(E)-(N,N-dimethylaminomethylene)-7α-chloro-3-methyl-1,1-dioxo-4-piperidino-
carbonylceph-3-em (E-6l) and 2-(Z)-(N,N-dimethylaminomethylene)-7α-chloro-3-methyl-1,1-dioxo-
4-piperidinocarbonylceph-3-em (Z-6l) (3:1) was obtained by using piperidine 4l in the general method and
1
isolated as fractions (eluent ethyl acetate) with Rf = 0.14. Yield 22 mg (5%). H NMR spectrum (90 MHz), δ,
ppm (J, Hz): E-6l – 1.34-1.70 (6H, m, 3-CH2, 4-CH2, and 5-CH2 piperidine); 1.73 (3H, s, 3-CH3); 3.04 (6H, s,
3
3
N(CH3)2); 3.20-3.71 (4H, m, 2-CH2 and 6-CH2 piperidine); 4.73 (1H, d, J = 1.5, H-6); 5.06 (1H, d, J = 1.5,
H-7); 7.24 (1H, s, =CHNMe2). Z-6l – 1.34-1.70 (6H, m, 3-CH2, 4-CH2, and 5-CH2 piperidine); 1.90 (3H, s,
3-CH3); 3.25 (6H, s, N(CH3)2); 3.20-3.71 (4H, m, 2-CH2 and 6-CH2 piperidine); 4.73 (1H, br. s, H-6); 5.23 (1H,
d, 3J = 1.5, H-7); 6.78 (1H, s, =CHNMe2 ). Found, %: C 49.66; H 5.81; N 10.78. C16H22ClN3O4S. Calculated, %:
C 49.54; H 5.72; N 10.83.
7α-Chloro-3-methyl-1,1-dioxo-4-(2-methylpiperidino)carbonylceph-3-em (5m) was obtained by
using 2-methylpiperidine in the general method and isolation from fractions (eluent 2:1 ethyl acetate–hexane)
1
with Rf = 0.22. Yield 47 mg (12%); mp 128-130°C. H NMR spectrum (90 MHz), δ, ppm (J, Hz): 1.04-1.33
(4H, m, 3-CH2 and 4-CH2 piperidine); 1.33-1.68 (5H, m, 5-CH2, 2-CH3 piperidine); 1.71 (3H, s, 3-CH3);
2.62-3.26 (2H, m, 6-CH2 piperidine); 3.44 and 4.00 (2H, two d, AB system, 2J = 18, SO2CH2); 4.20-4.58 (1H, m,
2-CH piperidine); 4.88 (1H, br. s, H-6); 5.24 (1H, br. s, H-7). Found, %: C 48.60; H 5.71; N 8.15.
C14H19ClN2O4S. Calculated, %: C 48.48; H 5.52; N 8.08.
4-N-Hexamethyleneiminocarbonyl-7α-chloro-3-methyl-1,1-dioxoceph-3-em (5n) was obtained by
using hexamethyleneimine in the general method and isolation from fractions (eluent 1:1 ethyl acetate–hexane)
1
with Rf = 0.08. Yield 153 mg, 39%; mp 179-181°C. H NMR spectrum (90 MHz), δ, ppm (J, Hz): 1.46-2.00
(8H, m, –(CH2)4– hexamethyleneimine); 1.75 (3H, s, CH3); 3.33-3.73 (4H, m, CH2NCH2, hexamethylene-imine);
3.53 and 4.02 (2H, two d, AB system, 2J = 18, SO2CH2); 4.91 (1H, br. s, H-6); 5.35 (1H, 3J = 1.0, H-7). Found,
%: C 48.63; H 5.59; N 8.21. C14H19ClN2O4S. Calculated, %: C 48.48; H 5.52; N 8.08.
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7α-Chloro-3-methyl-4-morpholinocarbonyl-1,1-dioxoceph-3-em (5o) was obtained by using
morpholine in the general method and isolation from fractions (eluent ethyl–acetate) with Rf = 0.08. Yield 23 mg
(6%); mp 153-155°C. 1H NMR spectrum, (90 MHz, CDCl3), δ, ppm (J, Hz): 1.75 (3H, s, 3-CH3); 3.31-4.00 (8H,
m, 4CH2, morpholine); 3.80 and 4.00 (2H, two d, AB system, 2J = 18, SO2CH2); 4.88 (1H, br. s, H-6); 5.31 (1H,
d, 3J = 1.0, H-7). Found, %: C 43.28; H 4.70; N 8.45. C12H15ClN2O5S. Calculated, %: C 43.05; H 4.52; N 8.37.
Preparation of 4-Acyl-7α-chloro-3-methyl-1,1-dioxoceph-3-ems (8) (General Method). A mixture of
oxalyl chloride (98 µl) and DMF (10µl) was added to a stirred suspension of the sulfone of a 7α-chloro-
3-methylceph-3-em-4-carboxylic acid (300 mg, 1.13 mmol) in dichloromethane (20 ml) at room temperature.
The reaction mixture was stirred at 40°C for 20 min, cooled to room temperature and concentrated under reduced
pressure. The residue, containing 7α-chloro-3-methyl-1,1-dioxoceph-3-em-4-carbonyl chloride, was dissolved in
THF (15 ml). CuI (215 mg, 1.13 mmol) was added to the solution. The suspension was cooled to -100°C and an
ethereal solution of a Grignard reagent (10 ml) (prepared by addition of the corresponding alkyl, phenyl, or
thienyl bromide (0.376 mmol) to magnesium) was added under argon over 30 min. The temperature of the
reaction mixture was raised to -40°C and poured into a mixture of ice, ether, and 30% aqueous NH4Cl solution
(200 ml). The organic layer was separated, washed with 5% NaHCO3 (2×20 ml) and NaCl and dried over
anhydrous Na2SO4. The solvent was evaporate under reduced pressure. The residue was fractionated on a
chromatographic column containing silica gel to obtain 4-acyl-substituted cephems 8.
7α-Chloro-4-isopropylcarbonyl-3-methyl-1,1-dioxoceph-3-em (8a) was prepared by the use of
isopropyl bromide in the general method and isolated from fractions (eluent 1:1 ethyl acetate–hexane) with
1
Rf = 0.75. Yield 20 mg (6%); mp 160-162°C. H NMR spectrum (90 MHz), δ, ppm (J, Hz): 1.40 and 1.48 (6H,
two d, 3J = 6, 2CH3); 2.11 (3H, s, CH3); 3.64 and 3.95 (2H, two d, AB system, 2J = 18, SO2CH2); 4.73 (1H, br. s,
3
H-6); 5.01-5.32 (1H, m, CHMe2); 5.26 (1H, d, J = 1.5, H-7). Found, %: C 45.36; H 4.91; N 4.86.
C11H14ClNO4S. Calculated, %: C 45.28; H 4.84; N 4.80.
4-tert-Butylcarbonyl-7α-chloro-3-methyl-1,1-dioxoceph-3-em (8b) was prepared by using tert-butyl
1
bromide by a published method [3]. Yield 45 mg (13%). H NMR spectrum (200 MHz), δ ppm (J, Hz): 1.53
2
(9H, s, C4H9); 2.07 (3H, s, 3-CH3); 3.65 and 3.88 (2H, two d, AB system, J = 18, SO2CH2); 4.71 (1H, br. s,
H-6); 5.26 (1H, br. s, H-7).
4-Benzoyl-7α-chloro-3-methyl-1,1-dioxoceph-3-em (8c) was prepared by using bromobenzene by a
1
published method [3]. Yield 26 mg (7%). H NMR spectrum (200 MHz), δ, ppm (J, Hz): 1.69 (3H, s, 3-CH3);
3.65 and 4.04 (2H, two d, AB system, 2J = 18, SO2CH2); 4.93 (1H, d, 3J = 1.5, H-6); 5.34 (1H, d, 3J = 1.5, H-7);
7.46-7.77 (3H, m, H-3, H-5, Ph); 7.87-8.05 (2H, m H-2, H-6, Ph).
7α-Chloro-3-methyl-1,1-dioxo-4-(2-thienylcarbonyl)ceph-3-em (8d) was prepared by using 2-bromo-
thiophene in the general method and isolation from fractions (eluent 1:3 ethyl acetate–hexane) with Rf 0.08.
Yield 64 mg (17%); mp 183-188°C. 1H NMR spectrum (200 MHz); δ ppm (J, Hz): 1.80 (3H, s, 3-CH3); 3.66 and
4.00 (2H, two d, AB system, 2J = 18, SO2CH2); 4.90 (1H, d, 3J = 1.5, H-6); 5.35 (1H, d, 3J = 1.5, H-7); 7.15-7.22
(1H, m, H-4, thienyl); 7.71-7.84 (2H, m H-3, H-5, thienyl). Found, %: C 43.56; H 3.21; N 4.18. C12H10ClNO4S2.
Calculated, %: C 43.44; H 3.04; N 4.22.
Determination of Cytotoxic Activity in vitro. The cytotoxic properties in vitro of the substances
synthesized against the monolayer cancer cells HT-1080 (human fibrosarcoma), MG-22A (mouse hepatoma),
B16 (mouse melanoma), Neuro2A (mouse neuroblastoma), and normal cells 3T3 (mouse embryonic fibroblasts)
and BNK (kidney fibroblasts of the golden hamster) were determined on 96 holes plastic panels using the
dyestuffs CV, MTT and NR by the corresponding methodology [8, 9].
Calculation of the Expected Toxicity [4]. The values of the expected toxicity LD50 (mg/kg) for the
compounds tested were calculated from the following equation:
lg LD50 (mg/kg) = 0.435 × lg LC50 (mmol/l) + 0.625,
where LC50 is the concentration of the tested substance (mmol/l) causing death of 50% of the fibroblasts 3T3
with cells dyed with NR.
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Generation of NO Radicals by Cells. Determination of the concentration of nitric oxide radicals in cell
media by Greis’ method [8] was carried out on 96 holes in plastic panels. The concentrations of NO radicals
obtained in this way in culture media with surviving cells after incubation for 72 h in the presence of the test
substances with a concentration 50 µg/ml in the holes with a volume of 200 µl were used to calculate the values
of the specific NO generating activities of the compounds (TG100):
TG100 = G 100/C (nmol/µl)
where G is the concentration of NO (nmol) in the cultural medium with a volume of 200 µl with surviving cells;
C is the percent of living cells, determined by the coloring by CV.
Determination of the Anticancer Activity in vivo. The therapeutic activity of a compound was
determined with tumors of the sarcoma S-180 and melanoma B16. Cancer cells in a quantity of 106 were
introduced subcutaneously into male mice (CBA/DBH 2F line). The number of mice in the experimental groups
was from 3 to 6. The test substances for the group of experimental mice were introduced intraperitonally as a 2%
solution in DMSO with the addition of 0.3% of agarose. The control group analogously were injected with 0.2
ml DMSO with the addition of 0.3% agarose without the test substance. The effective retardation (GI) was
determined as the difference in the volume of the tumors in the control and experimental groups at 9 and 11 days
after implantation of the cancer cells according to the formula:
GI, % = 100(1.00 – E/V);
where E is the volume of the tumor in the experimental group of animals, and V is the volume of the tumor in the
control group of animals.
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