Synthesis and cytotoxic properties of derivatives of the tert-butyl ester of 7-alkylidene-3-methyl-3-cepheme-4-carboxylic acid

Article abstract of DOI:10.1007/s10593-008-0099-0

Sulfones of the tert-butyl esters of 7-arylmethylene-and 7-(2-furyl)methylene-3-methyl-3-cepheme-4-carboxylic acid were obtained by the condensation of the tert-butyl ester of 3-methyl-7-oxo-3-cepheme-4-carboxylic acid with arylmethylene-and 2-furylidenetriphenylphosphoranes and subsequent oxidation of the intermediate products by meta-chloroperbenzoic acid. The combination of the tert-butyl esters of 7E-bromomethylene-and 7,7-dibromomethylene-3-methyl-1,1-dioxo-3-cepheme-4-carboxylic acids with trimethylsilylacetylene under conditions of the Sonogashira reaction gave the tert-butyl esters of 3-methyl-1,1-dioxo-7E-(3-trimethylsilyl-2-propynylidene)-3- cepheme-4-carboxylic acid and 3-methyl-1,1-dioxo-7-[1,5-bis(trimethylsilyl)-1,4- pentadiyn-3-ylidene]-3-cepheme-4-carboxylic acid. The Vilsmeier reagent was used to incorporate the dimethylaminomethylene group at C-2 of the 7Z-and 7E-isomers of the tert-butyl ester of 7-(4-chlorophenyl)methylene-3-methyl-1,1-dioxo-3- cepheme-4-carboxylic acid. The cytotoxic properties of the derivatives of the tert-butyl ester of 7-alkylidene-3-methyl-3-cepheme-4-carboxylic acid in regard to cancer and normal cells in vitro depends on the structure and 7Z-or 7E-isomerism of the substituent in the 7-alkylidene group as well as the presence of a dimethylaminomethylene group at C-2 of the 3-cepheme system.

Full text of DOI:10.1007/s10593-008-0099-0

Chemistry of Heterocyclic Compounds, Vol. 44, No. 6, 2008
SYNTHESIS AND CYTOTOXIC PROPERTIES
OF DERIVATIVES OF THE tert-BUTYL ESTER
OF 7-ALKYLIDENE-3-METHYL-3-CEPHEME-
4-CARBOXYLIC ACID
M. Vorona, I. Potorocina, G. Veinberg, I. Shestakova,
I. Kanepe, and E. Lukevics
Sulfones of the tert-butyl esters of 7-arylmethylene- and 7-(2-furyl)methylene-3-methyl-3-cepheme-
4-carboxylic acid were obtained by the condensation of the tert-butyl ester of 3-methyl-7-oxo-
3-cepheme-4-carboxylic acid with arylmethylene- and 2-furylidenetriphenylphosphoranes and
subsequent oxidation of the intermediate products by meta-chloroperbenzoic acid. The combination of
the tert-butyl esters of 7E-bromomethylene- and 7,7-dibromomethylene-3-methyl-1,1-dioxo-3-cepheme-
4-carboxylic acids with trimethylsilylacetylene under conditions of the Sonogashira reaction gave the
tert-butyl esters of 3-methyl-1,1-dioxo-7E-(3-trimethylsilyl-2-propynylidene)-3-cepheme-4-carboxylic acid
and 3-methyl-1,1-dioxo-7-[1,5-bis(trimethylsilyl)-1,4-pentadiyn-3-ylidene]-3-cepheme-4-carboxylic acid.
The Vilsmeier reagent was used to incorporate the dimethylaminomethylene group at C-2 of the 7Z- and
7E-isomers of the tert-butyl ester of 7-(4-chlorophenyl)methylene-3-methyl-1,1-dioxo-3-cepheme-
4-carboxylic acid. The cytotoxic properties of the derivatives of the tert-butyl ester of 7-alkylidene-
3-methyl-3-cepheme-4-carboxylic acid in regard to cancer and normal cells in vitro depends on the
structure and 7Z- or 7E-isomerism of the substituent in the 7-alkylidene group as well as the presence of
a dimethylaminomethylene group at C-2 of the 3-cepheme system.
Keywords: 7Z-acetylmethylene-3-methyl-1,1-dioxo-3-cepheme, tert-butyl esters of 7-arylmethylene-
3-methyl-1,1-dioxo-3-cepheme-4-carboxylic acids, tert-butyl ester of 7-(2-furyl)-methylene-3-methyl-
1,1-dioxo-3-cepheme-4-carboxylic acid, tert-butyl ester of 3-methyl-1,1-dioxo-7E-(3-trimethylsilyl-
2-propynylidene)-3-cepheme-4-carboxylic acid, tert-butyl ester of 3-methyl-1,1-dioxo-7-[1,5-bis-
(trimethylsilyl)-1,4-pentadiyn-3-ylidene]-3-cepheme-4-carboxylic acid, tert-butyl ester of 7Z-(4-chloro-
phenyl)methylene-2-(dimethylaminomethylene)-3-methyl-1,1-dioxo-3-cepheme-4-carboxylic acid, tert-
butyl ester of 7E-(4-chlorophenyl)methylene-2-dimethylaminomethylene-3-methyl-1,1-dioxo-3-cepheme-
4-carboxylic acid, cytotoxic activity.
We have already shown that the introduction of alkylidene group at C-7 in the tert-butyl ester of
3-acetoxy-1,1-dioxo-3-cepheme-4-carboxylic acid 1a-g is conducive to high cytotoxic activity relative to cancer
cells in vitro [1]. In subsequent work [2], we synthesized and studied the physicochemical properties of
__________________________________________________________________________________________
Latvian Organic Synthesis Institute, LV-1006 Riga, Latvia; e-mail: amx@oxi.lv, e-mail:
veinberg@osi.lv. Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 6, pp. 918-931, June, 2008.
Original article submitted April 10, 2008.
0009-3122/08/4406-0739©2008 Springer Science+Business Media, Inc.
739

1R- and 1S-sulfoxides as well as the sulfones of the tert-butyl esters of 7Z-acetylmethylene-3-methyl-
3-cepheme-4-carboxylic acid 2a,b and 3-acetoxymethyl-7Z-(3-trimethylsilyl-2-propynylidene)-3-cepheme-
4-carboxylic acid 3a,b.
O
R
O2
S
H
H
RHC
S
H
H
7
N
N
CH2X
CH2OAc
O
O
COOBu-
t
COOBu- t
1a–g
1S, 7Z - 2a,b
R
O
R
O2
S
H
N
H
S
H
H
H
N
CH2X
CH2X
COOBu-
O
O
COOBu- t
t
7Z-3a,b
1R,7Z-2a,b
1 a R = t-BuOCO, b R = MeOCO, c R = MeCO, d R = Ph, e R = 4-O2NC6H4,
f R = 4-pyridyl, g R = 2-furyl; 2, 3 a R = Ac, X = H; b R = Me3SiC≡C, X = OAc
In a continuation of this investigation, we have synthesized new analogs of the tert-butyl ester of
7-alkylidene-1,1-dioxo-3-methyl-3-cepheme-4-carboxylic acid and subjected these products to cytotoxic
screening, including 2,3a,b relative to cancer and normal cells in vitro in order to establish a link between their
structure and anti-carcinogenic properties.
7-Arylmethylene and 7-(2-furyl)methyl derivatives of tert-butyl esters of 3-cepheme-4-carboxylic acids
6a-d as chromatographically inseparable mixtures of the 7E- and 7Z-isomers were synthesized according to
methods described in previous work [1-3] by the condensation of the tert-butyl ester of 7-oxo-3-methyl-
3-cepheme-4-carboxylic acid (4) with phosphoranes 5a-d. The oxidation of the resultant cephemes 6a-d to the
corresponding sulfones 7a-d was carried out using meta-chloroperbenzoic acid (MCPBA). The presence of an
oxidized sulfur atom in the mixtures of sulfones 7a-d facilitated their separation into pure 7E- and 7Z-isomers
by column chromatography. Structural identification of these products by 1H NMR spectroscopy was carried out
in accord with the literature data [2, 3]. Thus, 3-cephemes 7Z-7a-d are characterized by a downfield shift of the
signal for H-9 in comparison to the signal for 7E-7a-d due to deshielding by the β-lactam carbonyl.
In our preceding work [2], we found that the sulfoxides and sulfone of the tert-butyl ester of
3-acetoxymethyl-7Z-(3-trimethylsilyl-2-propynylidene)-3-cepheme-4-carboxylic acid (2b,3b) are obtained
predominantly as the 7Z-isomer due to stereospecific features of the Wittig reaction. Thus, in order to obtain the
other isomer, we adapted a procedure for the synthesis of 7E-alkylidene derivatives of cephalosporin [3]. The
tert-butyl ester of 3-methyl-7-oxo-3-cepheme-4-carboxylic acid (4) was converted to the tert-butyl ester of
7-dibromomethyl-3-methyl-3-cepheme-4-carboxylic acid (8) by the action of dibromomethylenephosphorane.
Ester 8 was stereoselectively reduced by Zn/NH4Cl to 7E-monobromide 9. The conversion of bromide 9 to the
tert-butyl ester of 3-methyl-1,1-dioxo-7E-(3-trimethylsilyl-2-propynylidene)-3-cepheme-4-carboxylic acid
(7E-12) involved oxidation of the sulfur heteroatom and coupling of the 7E-bromomethylene group of
740

R
H
9
O
O
RCH=PPh3
S
S
S
H
R
2
3
1
7
8
5a–d
6
N5
2MCPBA
4
N
N
+
Me
Me
Me
COOBu-
O
O
COOBu-
t
t
COOBu-
t
4
7E-6a–d
7Z- 6a–d
R
H
O2
S
O
H
S2
R
+
N
N
Me
COOBu- t
7Z-7a–d
Me
O
O
COOBu-
t
7E-7a–c
5–7 a R = 4-ClC6H4, b R = 4-NCC6H4, c R = 2-BrC6H4, d R =
O
intermediate sulfone 10 with trimethylsilylacetylene (11) under conditions of the catalytic Sonogashira reaction.
The use of the sulfone of the 7-dibromo derivative of 3-cepheme 13 as the starting compound in this reaction
with two equivalents of trimethylsilylacetylene (11) led to the tert-butyl ester of 3-methyl-1,1-dioxo-7-[1,5-bis-
(trimethylsilyl)-1,4-pentadiyn-3-ylidene]-3-cepheme-4-carboxylic acid (14).
The introduction of a dimethylaminomethylene group at C-2 of the 7Z- and 7E-isomers of the tert-butyl
ester of 7-(4-chlorophenyl)methylene-3-methyl-1,1-dioxo-3-cepheme-4-carboxylic acid (7Z-7a, 7E-7a) was
achieved using the Vilsmeier reagent according to the method given in our previous work [4].
H
H
O2
S
O2
S
Me3SiC CH
Br
11
Me3Si
N
N
CuI, Pd(dba)2, i-Pr2NH
Me
COOBu-
Me
O
O
t
COOBu-
t
12
10
MCPBA
S
CBr4 + 2PPh3
Br
H
S
Br
Br
Zn/NH4Cl
Me
Br2C=PPh3 + Br2PPh3
4
+
N
N
Me
COOBu-
O
O
t
COOBu-
t
9
8
SiMe3
MCPBA
Br
O2
S
O2
S
11
Br
Me3Si
N
N
Me
COOBu-
O
Me
COOBu-
O
t
t
14
13
741

1
Analysis of the H NMR spectra of 16 showed that the dimethylaminomethylene group in these
compounds exists as 2E- and 2Z-isomers. The deshielding effect of the sulfone oxygen atoms in 3-cephemes
2E,7Z-16 and 2E,7E-16 leads to a downfield shift of the signal for the =HCNMe2 proton relative to the
analogous signal for isomers 2Z,7Z-16 and 2Z,7E-16. An analogous deshielding effect is observed for the signal
of the six N(CH3)2 protons in the 2Z,7Z-16 and 2Z,7E-16 isomers.
Cl
Cl
Me2NCHO + (COCl)2
NMe2
H
H
O2
S
O2
S
+
–
H
H
Me2N=CHCl
Cl
NMe2
2
2
+
15
N
N
7Z-7a
Me
Me
O
O
COOBu-
COOBu-
t
t
2Z,7Z-16
2E,7Z-16
H
H
NMe2
H
O
O2
S
S2
15
2
H
2
NMe2
7E-7a
Cl
+
Cl
N
N
Me
COOBu-
Me
COOBu-
O
O
t
t
2Z,7E-16
2E,7E-16
The conversion of the tert-butyl ester of the sulfone of 7Z-acetylmethylene-3-methyl-1,1-dioxo-
3-cepheme-4-carboxylic acid (7Z-3a) to 7Z-acetylmethylene-3-methyl-1,1-dioxo-3-cepheme (7Z-18) involved
cleavage of the tert-butyl group using trifluoroacetic acid and decarboxylation of the intermediate carboxylic
acid 17 in the presence of N-methylmorpholine.
O
O
Me
Me
O
O
S2
S2
MeN
O
H
H
CF3COOH
7Z-3a
N
N
–CO2
Me
Me
COOH
O
O
H
18
17
Biological screening of these products in vitro included determination of their cytotoxic properties
relative to monolayer lines of HT-1080 cancer cells (human fibrosarcoma) and MG-22A cancer cells (murine
hepatoma) in comparison with those of normal 3T3 cells (embryonic muscle fibroblasts). Coloring of the 3T3
fibroblasts by Neutral Red permitted us to calculate the expected toxicity LD50 for the compounds tested using a
special formula without in vivo experiments [5].
The screening data shown in Table 1 indicate that the structural variation including Z- and E-isomerism
of the 7-alkylidene group, 1S- and 1R-isomerism of the sulfoxide oxygen, and inclusion of an N,N-dimethyl-
aminomethylene group at C-2 in the 3-cepheme system has a significant effect on these parameters. Thus, the
comparative analysis of the biological properties of the stereoisomeric sulfoxides of the tert-butyl ester of
7Z-acetylmethylene-3-methyl-3-cepheme-4-carboxylic acids 1S,7Z-2a and 1R,7Z-2a showed that the two
742

TABLE 1. Biological Properties of Structural Analogs of the tert-Butyl
Ester of 7-Alkylidene-3-methyl-3-cepheme-4-carboxylic Acid*
Cytotoxic activity, LC50, µkg/ml
Com-
pound
LD50,
HT-1080
MTT
MG-22A
MTT
3T3
NR
mg/kg
CV
TG100
CV
TG100
1S,7Z-2а
1R,7Z-2а
7Z-3a
7Z-3b
7Z-18
7E-7a
7Z-7a
7E-7b
7Z-7b
7E-7c
7Z-7c
7Z-7d
7E-12
14
2.3
2.3
2.5
0.05
2.2
6
4.8
3.3
5.3
0.07
2.6
5
350
450
500
1050
400
100
150
450
175
233
200
250
550
350
400
150
1.5
2.8
2.4
0.45
1.2
1.7
9
1.6
2.8
2.7
0.50
1
450
450
400
700
700
56
11
33
14
316
521
358
−
200
1476
738
42
10*2
6
2
294
57
16
2
12
2
9
150
425
433
400
200
250
300
550
200
150
2
3
0.08
14
2
1
2
3
392
318
363
1023
206
256
1488
930
3
2.5
1.6
19
9
2.5
2.3
19
23
4
3.2
2.7
16
26
0.3
2
7.1
9.3
142
3.2
4
1.1
21
19
2
2
7E-16
7Z-16
9
8
3
250
84
10
17
8.0
10
_______
* LC50 is the concentration providing for annihilation of 50% of cells, CV
is coloration by Crystal Violet, MTT is coloration by 3-(4,5-dimethyl-
2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, TG50 is the specific
NO generating activity of a compound, NR is coloration by Neutral Red,
and LD50 is the dose of a substance providing for annihilation of 50% of
cells.
*2 Coloration by CV.
compounds have virtually identical cytotoxic activity in vitro relative to cancer cells but differ significantly in
their cytotoxic activity relative to normal cells, reflected in a 1.5-fold greater LD50 value for the R-isomer. The
oxidation of the sulfur atom in 1S,7Z-2a and 1R,7Z-2a to the sulfone did not alter the cytotoxic effect of
3-cepheme 7Z-3a but reduced its selectivity relative to cancer and normal cells. An analogous tendency was
found for a structural analog of this compound, 7Z-18, with elimination of the tert-butoxycarbonyl fragment
from C-4.
3-Cephemes 7Z-3b, 7E-12, and 14, which differ in the structure of the substituent at C-3, 7Z- and
7E-isomerism of the trimethylsilyl fragments and their number in the 7-alkylidene group, have high toxicity
relative to normal cells. Ester 7Z-3b suppressed the growth of cancer cells at much lower concentrations than
7E-12 and 14.
The biological properties of 7a-d containing a monosubstituted aromatic or furan system at C-7, depend
both on the isomerism of the alkylidene group and the structure of the aromatic system. Although the 7Z- and
7E-isomers of 3-cepheme 7a with a para-chlorophenyl substituent in the alkylidene group have similar
cytotoxic activity relative to cancer cells, the 7E-7a isomer is only half as toxic as the 7Z-7a isomer. In the case
of a para-cyanophenyl substituent, the toxicity of the 7E-7b isomer proved nine times greater than the toxicity
of the 7Z-7b isomer. The cytotoxic properties of the 7Z-7c and 7E-7c isomers with an ortho-bromophenyl group
relative to cancer and normal cells were found to be virtually identical. The introduction of a furan heterocycle
into the alkylidene group of 7Z-7d led to a substantial decrease in its toxicity relative to cancer and normal cells.
743

In comparison with starting 3-cephemes 7E-7a and 7Z-7a, esters 7E-16 and 7Z-16, containing an
N,N-dimethylaminomethylene group at C-2, displayed similar cytotoxic activity relative to cancer cells and
reduced cytotoxic activity relative to normal cells.
The cytotoxic effect of the compounds tested, similar to previously studied tert-butyl esters of
7-alkylidene derivatives of cephalosporins [1], is interrelated to their capacity to their capacity to generate NO
radicals in the cell medium. High cytotoxicity of these compounds is accompanied, as a rule, by enhanced NO
radical generation and vice versa.
EXPERIMENTAL
1
The H NMR spectra were taken on a Bruker WH90/DS spectrometer at 90 MHz and Varian
Mercury-200 spectrometer at 200 MHz in CDCl3 as the internal standard. The elemental analysis was carried out
on a Carlo Erba 1108 analyzer. The ESI (electrospray ionization) mass spectrometry was carried out on a
Micromass Quatro MicroTM API. The high-performance liquid chromatography was carried out on a
Dupont Model 8800 chromatograph equipped with a UV detector (λ = 254. nm) and 4.6×250-mm column
packed with µPorasil using 20:80 ethyl acetate–hexane as the eluent, Altima C18 using 50:50 acetonitrile–
0.1 M phosphate buffer (pH 2.5) as the eluent, and Zorbax R×C18 with 60:40 acetonitrile-water as the eluent.
The reaction course was monitored by thin-layer chromatography on Merck Kieselgel plates with UV detection.
Merck Kieselgel (0.063-0.230 mm) was used for the preparative column chromatography. Reagents and
materials supplied by Acros, Aldrich, and Sigma were used in the experiments. The optical density in the
biological tests carried out on 96-well panels was determined using a horizontal Tetretek Multiscan MCC/340
spectrophotometer.
tert-Butyl Ester of 7Z-(4-Chlorophenyl)methylene-3-methyl-1,1-dioxo-3-cepheme-4-carboxylic
Acid (7Z-7a) and tert-Butyl Ester of 7E-(4-Chlorophenyl)methylene-3-methyl-1,1-dioxo-3-cepheme-
4-carboxylic Acid (7E-7a). A sample of butyllithium in hexane (2.2 ml, 1.6 M) was added with stirring to a
solution of para-chlorobenzyltriphenylphosphonium chloride (694 mg, 1.64 mmol) in absolute THF (15 ml) at
0°C. The solution was stirred for 30 min at 10°C, cooled to -78°C, and then, 479 mg (1.64 mmole) tert-butyl
ester of 3-methyl-7-oxo-3-cepheme-4-carboxylic acid [5] in dichloromethane (10 ml) was added. The mixture
obtained was stirred for 30 min at -78°C and poured into saturated aqueous NH4Cl (100 ml) with ice. The
mixture was stirred until the ice melted and extracted with two 50 ml portions of dichloromethane. The organic
phase was washed with cold aq. NH4Cl and dried over anhydrous sodium sulfate. the solvent was evaporated off
at reduced pressure and the residue was fractionated on a silica gel column using 1:2.5 ethyl acetate–
petroleum ether as the eluent. The fractions with Rf 0.68 were combined and evaporated off to give 500 mg
(80%) of an oily product. HPLC analysis indicate that this product consisted of a 1:1 mixture of the tert-butyl
ester of 7Z-(4-chlorophenyl)methylene-3-methyl-3-cepheme-4-carboxylic acid (7Z-6a) and the tert-butyl ester
of 7E-(4-chlorophenyl)methylene-3-methyl-3-cepheme-4-carboxylic acid (7E-6a).
A sample of 70% 3-chloroperbenzoic acid (815 mg, 3.31 mmol) was added with stirring to a solution
mixture of isomers 7Z-6a and 7E-6a (500 mg, 1.32 mmol) in dichloromethane (20 ml) at 0°C. The mixture was
stirred at room temperature for 4 h, diluted by adding 20 ml dichloromethane, washed with 5% aq. Na2SO3
(50 ml) followed by two 50 ml portions of 5% aq. Na2CO3, and dried over anhydrous sodium sulfate. The
solvent was evaporated off at reduced pressure and the residue was fractionated on a silica gel column using
1:2 ethyl acetate–petroleum ether as the eluent.
1
The fractions with Rf 0.28 gave 230 mg (42%) 7Z-7a, mp 40-41°C. H NMR spectrum (90 MHz), δ,
2
ppm (J, Hz): 1.51 (9H, s, C4H9); 2.06 (3H, s, CH3); 3.63 and 3.98 (2H, two d, AB system, J = 18, SO2CH2);
5.51 (1H, br. s, H-6); 7.34 (1H, br. s, H-9); 7.38 and 7.76 (4H, two d, 3J = 7, C6H4); Found, %: C 55.90; H, 5.08;
N 3.53. C19H20ClNO5S. Calculated, %: C 55.68; H 4.92; N 3.42.
744

1
The fractions with Rf 0.11 gave 187 mg (34%) 7E-7a, mp 185-186°C. H NMR spectrum (90 MHz), δ,
2
ppm (J, Hz): 1.53 (9H, s, C4H9); 2.04 (3H, s, CH3); 3.58 and 3.95 (2H, two d, AB system, J = 18, SO2CH2);
5.17 (1H, br. s, H-6); 6.82 (1H, br. s, H-9); 7.95 and 8.08 (4H, two d, 3J = 7, C6H4). Found, %: C 55.87; H 5.00;
N 3.58. C19H20ClNO5S. Calculated, %: C 55.68; H 4.92; N 3.42.
tert-Butyl Ester of 7Z-(4-Cyanophenyl)methylene-3-methyl-1,1-dioxo-3-cepheme-4-carboxylic
Acid (7Z-7b) and tert-Butyl Ester of 7E-(4-Cyanophenyl)methylene-3-Methyl-1,1-dioxo-3-cepheme-
4-carboxylic acid (7E-7b). A sample of butyllithium (2.2 ml, 1.6 M) in hexane was added with stirring to a
solution of para-cyanobenzyltriphenylphosphonium chloride (559 mg, 1.35 mmol) in absolute THF (15 ml) at
0°C. The solution was stirred for 30 min at 10°C, cooled to -78°C, and then tert-butyl (479 mg, 1.64 mmol) ester
of 3-methyl-7-oxo-3-cepheme-4-carboxylic acid in dichloromethane (10 ml) was added. The mixture obtained
was stirred for 30 min at -78°C and poured into saturated aqueous NH4Cl (100 ml) with ice. The mixture was
stirred until the ice melted and extracted with two 50 ml portions of dichloromethane. The organic phase was
washed with cold aq. NH4Cl and dried over anhydrous sodium sulfate. The solvent was evaporated off at
reduced pressure and the residue was fractionated on a silica gel column with 1:2.5 ethyl acetate-petroleum ether
as the eluent. The fractions with Rf 0.68 were combined and evaporated to give 380 mg (76%) of an oily
product. HPLC analysis indicated that this product consisted of a mixture of the tert-butyl ester of
7Z-(4-cyanophenyl)methylene-3-methyl-3-cepheme-4-carboxylic acid (7Z-6b) and the tert-butyl ester of
7E-(4-cyanophenyl)methylene-3-methyl-3-cepheme-4-carboxylic acid (7E-6b).
A sample of 70% 3-chloroperbenzoic acid (665 mg, 2.70 mmol) was added to a solution of the mixture
of isomers 7Z-6b and 7E-6b (380 mg, 1.03 mmol) in dichloromethane (20 ml) at 0°C. The mixture was stirred at
room temperature for 4 h, diluted by adding 20 ml dichloromethane, washed with 50 ml 5% aq. Na2SO3
followed by two 50 ml portions of 5% aq. Na2CO3,and dried over anhydrous sodium sulfate. The solvent was
evaporated at reduced pressure and the residue was fractionated on a silica gel column with 1:4 ethyl acetate–petro-
leum ether as the eluent.
1
The fractions with Rf 0.42 gave 91 mg (22%) 7Z-7b, mp 153-154°C. H NMR spectrum (90 MHz), δ,
2
ppm (J, Hz): 1.55 (9H, s, C4H9); 2.16 (3H, s, CH3); 3.68 and 3.95 (2H, two d, AB system, J = 18, SO2CH2);
5.53 (1H, br. s, H-6), 7.39 (1H, br. s, H-9); 7.74 and 8.00 (4H, two d, 3J = 8, C6H4). Found, %: C 59.99; H 5.03;
N 7.00. C20H20N2O5S. Calculated, %: C 60.09; H 5.08; N 7.11.
The fractions with Rf 0.24 gave 57 mg (14%) 7E-7b, mp 118-120°C. H NMR spectrum (200 MHz), δ,
ppm (J, Hz): 1.57 (9H, s, C4H9); 2.17 (3H, s, CH3); 3.62 and 3.90 (2H, two d, AB system, J = 18, SO2CH2);
1
2
3
5.17 (1H, br. s, H-6); 6.88 (1H, br. s, H-9); 7.98 and 8.07 (4H, two d, AB system, J = 7, C6H4). Found, %:
C 60.13; H 5.23; N 7.12. C20H20N2O5S. Calculated, %: C 60.09; H 5.08; N 7.11.
tert-Butyl Ester of 7Z-(2-Bromophenyl)methylene-3-methyl-1,1-dioxo-3-cepheme-4-carboxylic
Acid (7Z-7c) and tert-Butyl Ester of 7E-(2-Bromophenyl)methylene-1,1-dioxo-3-cepheme-4-carboxylic
Acid (7E-7c). A sample of sodium tert-butylate (234 mg, 2.44 mmol) was added with stirring to a solution of
2-bromobenzyltriphenylphosphonium chloride (1140 mg, (2.44 mmol) in absolute THF (15 ml) at 0°C. The
solution was stirred for 30 min at 10°C, cooled to -78°C, and, then, tert-butyl ester (479 mg, 1.64 mmol) of
3-methyl-7-oxo-3-cepheme-4-carboxylic acid in dichloromethane (10 ml) was added. The mixture obtained was
stirred for 30 min at -78°C and poured into saturated aqueous NH4Cl (100 ml) with ice. The mixture was stirred
until the ice melted and then extracted with two 50 ml portions of dichloromethane. The organic phase was
washed with cold aq. NH4Cl and dried over anhydrous sodium sulfate. The solvent was evaporated off at
reduced pressure and the residue was fractionated on a silica gel column with 1:4 ethyl acetate–petroleum ether
as the eluent. The fractions with Rf 0.68 were combined and evaporated to give 400 mg (39%) of an oily
product. HPLC indicated that this product was a 1:1 mixture of the tert-butyl ester of 3-methyl-7Z-(2-bromo-
phenyl)methylene-3-cepheme-4-carboxylic acid (7Z-6c) and the tert-butyl ester of 3-methyl-7E-(2-bromo-
phenyl)-3-cepheme-4-carboxylic acid (7E-6c).
745

A sample of 70% 3-chloroperbenzoic acid (566 mg, 2.30 mmol) was added with stirring to a solution of
the mixture of isomers 7Z-6c and 7E-6c (400 mg) in dichloromethane (20 ml) at 0°C. The mixture was stirred at
room temperature for 4 h, diluted by adding dichloromethane (20 ml), washed with 5% aq. Na2SO3 (50 ml)
followed by two 50 ml portions of 5% aq. Na2CO3, and dried over anhydrous sodium sulfate. The solvent was
evaporated at reduced pressure and the residue was fractionated on a silica gel column with 1:3 ethyl acetate–petro-
leum ether as the eluent.
1
The fractions with Rf 0.24 gave 84 mg (19%) 7Z-7c, mp 118-119°C. H NMR spectrum (200 MHz), δ,
2
ppm (J, Hz): 1.56 (9H, s, C4H9); 2.06 (3H, s, CH3); 3.67 and 3.93 (2H, two d, AB system, J = 18, SO2CH2);
5.44 (1H, s, H-6), 7.21-7.44 (2H, m, C6H4), 7.61-7.68 (1H, m, C6H4), 7.81 (1H, s, H-9), 7.85-7.91 (1H, m,
C6H4). Found, %: C 50.31; H 4.53; N 3.14. C19H20BrNO5S. Calculated, %: C 50.23; H 4.44; N 3.08.
The fractions with Rf 0.14 gave 121 mg (28%) 7E-7c, mp 140-141°C. 1H NMR spectrum (200 MHz), δ,
2
ppm (J, Hz): 1.55 (9H, s, C4H9); 2.07 (3H, s, CH3); 3.62 and 3.89 (2H, two d, AB system, J = 18, SO2CH2);
5.19 (1H, s, H-6); 7.20-7.46 (2H, m, C6H4); 7.32 (1H, s, H-9); 7.58-7.66 (1H, m, C6H4); 8.49-8.55 (1H, m,
C6H4). Found, %: C 50.43; H 4.49; N 3.28. C19H20BrNO5S. Calculated, %: C 50.23; H 4.44; N 3.08.
tert-Butyl Ester of 7Z-(2-Furyl)methylene-3-methyl-1,1-dioxo-3-cepheme-4-carboxylic Acid
(7Z-7d). A sample of butyllithium in hexane (1 ml, 1.6 M) was added with stirring to a solution of
2-furyltriphenylphosphonium chloride (604 mg, 1.60 mmol) in THF (10 ml) at 10°C. The mixture was stirred
for 30 min at 10°C, cooled to -78°C and then, tert-butyl ester (523 mg, 1.60 mmol) of 3-methyl-7-oxo-
3-cepheme-4-carboxylic acid in absolute THF (5 ml) was added. The mixture was stirred for 30 min at -78°C
and poured into saturated aqueous NH4Cl (100 ml) with ice. The mixture was stirred until the ice melted and
extracted with two 20 ml portions of dichloromethane. The organic phase was washed with cold aqueous
NH4Cland dried over anhydrous sodium sulfate. The solvent was evaporated at reduced pressure and the residue
was fractionated on a silica gel column using 1:3 ethyl acetate–petroleum ether as the eluent. The fractions with
Rf 0.50 were combined and evaporated to give 350 mg (39%) of an oily product showed by HPLC analysis to
consist of a 10:1 mixture of the tert-butyl ester of 7Z-(2-furyl)methylene-3-methyl-3-cepheme-4-carboxylic acid
(7Z-6d) and the tert-butyl ester of 7Z-(2-furyl)methylene-3-methyl-3-cepheme-4-carboxylic acid (7E-6d).
A sample of 70% 3-chloroperbenzoic acid (815 mg, 3.31 mmol) was added with stirring to a solution of
the mixture of isomers of 7Z-6d and 7E-6d (350 mg, 1.04 mmol) in dichloromethane (20 ml) at 0°C. The
mixture was stirred at room temperature for 4 h, diluted by adding 20 ml dichloromethane, washed with
50 ml 5% aq. Na2SO3 followed by two 50 ml portions of 5% aq. Na2CO3, and dried over anhydrous sodium
sulfate. The solvent was evaporated at reduced pressure and the residue was fractionated on a silica gel column
using 1:2 ethyl acetate–petroleum ether as the eluent. The fractions with Rf 0.14 gave 87 mg (23%) 7Z-7d,
mp 103-105°C.
1H NMR spectrum (200 MHz); δ, ppm (J, Hz): 1.54 (9H, s, C4H9); 2.13 (3H, s, CH3); 3.70 and 3.95 (2H,
2
4
3
3
two d, AB system, J = 18, SO2CH2); 5.69 (1H, d, J = 0.25, H-6); 6.64 (1H, two d, J = 2, J = 4, C-4 furan);
7.41 (1H, d, 3J = 4, C-3 furan); 7.56 (1H, d, 3J = 2, C-5 furan); 7.70 (1H, d, 4J = 0.25, H-9). Found, %: C 56.01;
H 5.43; N 3.94. C17H19NO6S. Calculated, %: C 55.88; H 5.24; N 3.83.
tert-Butyl Ester of 7,7-Dibromomethylene-3-methyl-3-cepheme-4-carboxylic Acid (8). A sample of
tetrabromomethane (3.78 g, 11.4 mmol) was added in one portion to a solution of triphenylphosphine (6.00 g,
22.8 mmol) in dichloromethane (40 ml) cooled to -10°C in an argon atmosphere. The solution was stirred for
10 min at room temperature, cooled to -70°C and then, a solution of tert-butyl ester (3.00 g, 11.4 mmol) of
3-methyl-7-oxo-3-cepheme-4-carboxylic acid in dichloromethane previously (40 ml) cooled to -40°C was
added. The solution was warmed to -40°C and stirred for 15 min. The solution was then warmed to -10°C and
poured into saturated aqueous NH4Cl (100 ml) with ice. The mixture was stirred until the ice melted and
extracted with two 50 ml portions of dichloromethane. The organic phase was washed with cold aqueous NH4Cl
and dried over anhydrous sodium sulfate. The solvent was evaporated at reduced pressure and the residue was
fractionated on a silica gel column using 1:4 ethyl acetate–petroleum ether as the eluent. The fractions with
746

Rf 0.61 were combined and evaporated to give 1.2 g (24%) 8, mp 133-135°C. The product contained 90% major
1
compound as indicated by HPLC analysis on a Zorbax R×C18 column. H NMR spectrum (90 MHz), δ, ppm
(J, Hz): 1.57 (9H, s, C4H9), 2.09 (3H, s, 3-CH3), 3.13 and 3.49 (2H, two d, AB system, 2J = 18, SCH2), 5.17 (1H,
s, H-6).
tert-Butyl Ester of 7E-Bromomethylene-3-methyl-3-cepheme-4-carboxylic Acid (9). Ammonium
chloride (50 mg, 0.94 mmol) and zinc powder (31 mg, 0.48 mmol) were added to a solution of tert-butyl ester
(100 mg, 23.5 mmol) 7-dibromomethylene-3-cepheme-4-carboxylic acid (8) in 2:1 methanol-tetrahydrofuran
cooled (6 ml) to 0°C. The solution was stirred for 30 min at 0°C for an additional 30 min at room temperature,
filtered, and evaporated at reduced pressure. The residue was treated with diethyl ether (60 ml), washed
with aqueous NaCl (30 ml), dried over anhydrous sodium sulfate, and evaporated at reduced pressure. The
residue was fractionated on a silica gel column using 1:3 ethyl acetate–petroleum ether as the eluent. The
fractions with Rf 0.17 were combined and evaporated to give 20 mg (24%) 9, mp 106-108°C. The product
1
contains 97% of the desired compound as indicated by HPLC analysis on a Zorbax×C18 column. H NMR
spectrum (90 MHz), δ, ppm (J, Hz): 1.53 (9H, s, C4H9);2.04 (3H, s, 3-CH3); 3.11 and 3.49 (2H, two d,
AB system, 2J = 18, SCH2); 5.13 (1H, br. s, H-6); 6.66 (1H, br.s, H-9).
tert-Butyl Ester of 7E-Bromomethylene-3-methyl-1,1-dioxo-3-cepheme-4-carboxylic Acid (10). A
sample of 70% 3-chloroperbenzoic acid (4.84 g, 19.65 mmol) was added with stirring to a solution of tert-butyl
ester (1.7 g, 4.91 mmol) of 7E-bromomethylene-3-methyl-3-cepheme-4-carboxylic acid in dichloromethane
(25 ml) at 0°C. The mixture was stirred at room temperature for 4 h, diluted by adding dichloromethane
(25 ml), washed with 50 ml 5% aqueous Na2SO3 followed by two 50 ml portions of 5% aqueous Na2CO3, and
dried over anhydrous sodium sulfate. The solvent was evaporated on a silica gel column using 1:1 ethyl acetate–
petroleum ether as the eluent. The fractions with Rf 0.52 were combined and evaporated to give 1.30 g (70%) 10,
1
mp 88-90°C. H NMR spectrum (90 MHz), δ, ppm (J, Hz): 1.51 (9H, s, C4H9); 2.04 (3H, s, 3-CH3); 3.55 and
2
4
4
3.77 (2H, two d, AB system, J = 18, SO2CH2);5.09 (1H, d, J = 0.5, H-6); 6.93 (1H, d, J = 0.5, H-9). Found,
%: C 41.37; H 4.31, N 3.87. C13H16BrNO3S. Calculated, %: C 41.28; H 4.26; N 3.70.
tert-Butyl Ester of 3-Methyl-1,1-dioxo-7-(3-trimethylsilylpropyn-2-ylidene)-3-cepheme-4-carboxylic
Acid (7E-12). A sample of trimethylsilylacetylene (31 µl, 0.22 mmol) and i-Pr2NH (27 µl, 0.37 mmol) were
added to a suspension of tert-butyl ester (70 mg, 18.5 mmol) of 7E-bromomethylene-1,1-dioxo-3-methyl-
3-cepheme-4-carboxylic acid, CuI (70 mg, 0.37 mmol), and adduct of tris(dibenzylidenacetone)dipalladium(0)
with chloroform (17 mg) in N-methylpyrrolidone (10 ml) in an argon atmosphere. The solution was stirred for
24 h at room temperature, diluted by adding diethyl ether (80 ml), and washed with 30 ml 5% aqueous K2CO3
and then 30 ml 5% NaCl, dried over anhydrous sodium sulfate, and evaporated at reduced pressure. The residue
was fractionated on a silica gel column using 1:10 ethyl acetate–petroleum ether. The fractions with Rf 0.11 were
combined and evaporated to give 20 mg (40%) 7E-12. The product contains 97% desired compound as indicated
by HPLC analysis. 1H NMR spectrum(90 MHz), δ, ppm (J, Hz): 0.20 (9H, s, (CH3)3Si); 1.55 (9H, s, C4H9); 2.04
(3H, s, 3-CH3); 3.55 and 3.91 (2H, two d, AB system, 2J = 18, SO2CH2); 5.11 (1H, s, H-6); 6.09 (1H, s, H-9).
tert-Butyl Ester of 7,7-Dibromomethylene-3-methyl-1,1-dioxo-3-cepheme-4-carboxylic Acid (13). A
sample of 70% 3-chloroperbenzoic acid (1.62 g, 6.60 mmol) was added with stirring to a solution of tert-butyl ester
(1.12 g, 2.64 mmol) of 7,7-dibromomethylene-3-methyl-3-cepheme-4-carboxylic acid in dichloromethane
(25 ml) at 0°C. The mixture was stirred at room temperature for 4 h, diluted by adding dichloromethane (25 ml),
washed with 50 ml 5% aqueous Na2SO3 followed by two 50-ml portions of 5% aqueous Na2CO3, and dried over
anhydrous sodium sulfate. The solvent was evaporated at reduced pressure and the residue was fractionated on a
silica gel column using 1:2 ethyl acetate–petroleum ether as the eluent. The fractions with Rf 0.27 were
1
combined and evaporated to give 0.70 g (58%) 13, mp 108-110°C. H NMR spectrum (90 MHz), δ, ppm
747

2
(J, Hz): 1.53 (9H, s, C4H9); 2.06 (3H, s, 3-CH3); 3.11 and 3.49 (2H, two d, AB system, J = 18, SO2CH2); 5.17
(1H, s, H-6). Found, %: C 34.25; H 3.46; N 3.15. C13H15Br2NO5S Calculated, %: C 34.16; H 3.31; N 3.06.
tert-Butyl
Ester
of
3-Methyl-1,1-dioxo-7-[1,5-bis(trimethylsilyl)-1,4-pentadiyn-3-ylidene]-
3-cepheme-4-carboxylic Acid (14). A sample of trimethylsilylacetylene (62 µl, 0.44 mmol) and i-Pr2NH
(27 µl, 0.37 mmol) were added to a suspension of tert-butyl ester (100 mg, 22 mmol) of 7-dibromomethylene-
3-methyl-1,1-dioxo-3-cepheme-4-carboxylic acid, CuI (130 mg, 0.68 mmol), and adduct of tris(dibenzyliden-
acetone)dipalladium(0) with chloroform (20 mg) in N-methylpyrrolidone (10 ml) in an argon atmosphere. The
solution was stirred for 24 h at room temperature, diluted by adding diethyl ether (80 ml), and washed with
30 ml 5% aq. K2CO3, then 30 ml 5% aq. NaCl, dried over anhydrous sodium sulfate, and evaporated at reduced
pressure. The residue was fractionated on a silica gel column using 1:10 ethyl acetate–petroleum ether as the
eluent. The fractions with Rf 0.36 were combined and evaporated to give 31 mg (28%) 14, mp 48-50°C. This
1
product contained 97.5% desired compound as indicated by HPLC analysis. H NMR spectrum (90 MHz), δ,
ppm (J, Hz): 0.22 (18H, s, 2(CH3)3Si); 1.53 (9H, s, C4H9); 2.00 (3H, s, 3-CH3); 3.55 and 3.88 (2H, two d,
AB system, 2J = 18, SO2CH2); 5.17 (1H, s, H-6).
Mixture of tert-Butyl Ester of 7Z-(4-Chlorophenyl)methylene-2E-dimethylaminomethylene-
3-methyl-1,1-dioxo-3-cepheme-4-carboxylic Acid (2E,7Z-16) and tert-Butyl Ester of 7Z-(4-Chloro-
phenyl)methylene-2Z-dimethylaminomethylene-3-methyl-1,1-dioxo-3-cepheme-4-carboxylic acid (2Z,7Z-16).
A sample of tert-butyl ester (100 mg, 0.24 mmol) of 7Z-(4-chlorophenyl)methylene-3-methyl-1,1-dioxo-
3-cepheme-4-carboxylic acid was added to a stirred solution of DMF (76 µl, 0.97 mmol) and oxalyl chloride
(85 µl, 0.97 mmol) in dichloromethane (10 ml) at 0°C in an argon atmosphere. After 30 min stirring at 0°C,
pyridine (79 µl, 0.79 mmol) was added to the reaction mixture, which was then stirred at room temperature for
1 h. The reaction mixture was evaporated at reduced pressure. The residue was dissolved in 20 ml
dichloromethane. The solution obtained was washed with 40 ml saturated aqueous NH4Cl and dried over
anhydrous sodium sulfate. The solvent was evaporated at reduced pressure. The residue was fractionated on a
silica gel column using 2:1 ethyl acetate–hexane as the eluent. The fractions with Rf 0.61 were combined and
1
evaporated to give 76 mg (68%) of a 3:1 mixture of the 2E- and 2Z-somers as indicated by H NMR
1
spectroscopy at 200 MHz. H NMR spectrum (200 MHz), δ, ppm (J, Hz): 2Z,7Z-16) 1.58 (9H, s, C4H9); 2.22
(3H, s, 3-CH3); 3.33 (6H, s, N(CH3)2); 5.17 (1H, d, 4J = 0.2, H-6); 7.18 (1H,s, =CHNMe2); 7.26 (1H, d, 4J = 0.2,
3
H-9); 7.38 and 7.60 (4H, two d, J = 8, C6H4); 2E,7Z-16) 1.54 (9H, s, C4H9); 2.22 (3H, s, CH3); 3.06 (6H, s,
4
4
N(CH3)2); 5.17 (1H, d, J = 0.2, H-6); 7.26 (1H, d, J = 0.2, H-9); 7.28 (1H, s, =CHNMe2); 7.38 and 7.60 (4H,
two d, 3J = 8, C6H4). ESI-MS (MeCN): 487 [MNa+].
Mixture of tert-Butyl Ester of E-(4-Chlorophenyl)methylene-2E-dimethylaminomethylene-
3-methyl-1,1-dioxo-73-cepheme-4-carboxylic Acid (2E,7E-16) and tert-Butyl Ester of 7E-(4-Chloro-
phenyl)methylene-2Z-dimethylaminomethylene-3-methyl-1,1-dioxo-3-cepheme-4-carboxylic
Acid
(2Z,7E-16) was obtained according to the above procedure using the tert-butyl ester of 7Z-(4-chloro-
phenyl)methylene-3-methyl-1,1-dioxo-3-cepheme-4-carboxylic acid and separated from the fractions with
Rf 0.48 upon chromatography using 2:1 ethyl acetate–hexane as the eluent. The yield of this mixture was 57 mg
(51%). The 1H NMR spectrum at 200 MHz indicated that the product was a 4:1 mixture of the indicated 2E- and
2Z-isomers. 1H NMR spectrum (200 MHz), δ, ppm (J, Hz): 2Z,7Z-16) 1.55 (9H, s, C4H9); 2.26 (3H, s, 3-CH3); 3.32
(6H, s, N(CH3)2); 5.12 (1H, s, H-6); 6.73 (1H, s, H-9); 7.17 (1H, s, =CHNMe2); 7.40 and 7.94 (4H, two d, 3J = 8,
C6H4); 2E,7Z-16) 1.55 (9H, s, C4H9); 2.26 (3H, s, 3-CH3); 3.02 (6H, s, N(CH3)2); 5.12 (1H, s, H-6); 6.73 (1H, s,
H-9); 7.24 (1H, s, =CHNMe2); 7.40 and 7.94 (4H, two d, 3J = 8, C6H4). ESI-MS (MeCN): 487 [MNa+].
7Z-Acetylmethylene-3-methyl-1,1-dioxo-3-cepheme (7Z-18). A sample of trifluoroacetic acid (2 ml,
38 mmol) was added to a solution of tert-butyl ester (341 mg, 1.00 mmol) of 7Z-acetylmethylene-3-methyl-
1,1-dioxo-3-cepheme-4-carboxylic acid in dichloromethane (30 ml)at 0°C. The mixture was stirred for 1 h at
room temperature, diluted by adding dichloromethane (100 ml) and water (100 ml). The organic phase was
748

separated and extracted with 40 ml 5% aqueous Na2CO3. The aqueous extract was brought to pH 2 by adding
concentrated hydrochloric acid and extracted with 100 ml ethyl acetate. The extract was evaporated at reduced
pressure. The residue was dissolved in a mixture of 20 ml acetone and 2 ml N-methylmorpholine. The solution
was stirred at room temperature for 1 h and the solvent was evaporated at reduced pressure. The residue was
fractionated on a silica gel column using 1:2 ethyl acetate–hexane as the eluent. The fractions with Rf 0.12 were
1
combined and evaporated to give 181 mg (75%) 7Z-18, mp 135-137°C. H NMR spectrum (200 MHz), δ, ppm
2
(J, Hz): 1.82 (3H, s, 3-CH3); 2.42 (2H, s, CH3CO); 3.48 and 4.03 (2H, two d, AB system, J = 18, SO2CH2);
5.58 (1H, s, H-6), 6.57 (1H, s, H-9); 6.85 (1H, s, 4-H). Found, %: C 49.91; H 4.77; N 5.89. C10H11NO4S.
Calculated, %: C 49.78; H 4.60; N 5.81.
Determination of the in vitro Cytotoxic Activity. The cytotoxic activity of the products relative to
cultures of monolayer cancer and normal cells at c = (2-5)·104 cells/ml [HT-1080 (human fibrosarcoma),
MG-22A (murine hepatoma), 3T3 (embryonic muscle fibroblasts)] was determined on 96-well plastic panels.
The NO radical concentrations (nmoles) in the culture medium with surviving cells after incubation for 72 h in
the presence of the compound tested at c = 50 µg/ml in a 200 µl well were used to calculate the specific
NO generating activity of the compounds (TG100):
TG100 = G 100/C (nmol/µl),
where G is the NO concentration (nmoles) in 200 µl culture medium with surviving cells, while C is the
percentage of surviving cells determined by their CV coloration.
This work was carried out with the cooperation of the European Social Fund in the framework of the
Doctoral Student Support Project in the National Program for Cooperation for Doctoral and Post-Doctoral
Programs.
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749