Synthesis and biological activity of alkylidene-substituted cephems and penams

Article abstract of DOI:10.1007/s10593-011-0832-y

The condensation of tert-butyl esters of 3-methyl-7-oxoceph-3-em-4- carboxylic and 6-oxopenicillanic acids with a series of 2- oxoalkylidene(triphenyl)phosphoranes gave tert-butyl esters of new cephalosporin and penicillin analogs with an alkylidene substituent in the β-lactam ring. Most of these products were oxidized by meta-chloroperbenzoic acid to the corresponding sulfones. The cephemes and penams synthesized including the oxidized products displayed high cytotoxicity relative to cancer cells in vitro. Some of the alkylidene-substituted cephems as the free acids, similar to Tazobactam, inhibit the catalytic activity of Enterobacter cloacae penicillinase.

Full text of DOI:10.1007/s10593-011-0832-y

Chemistry of Heterocyclic Compounds, Vol. 47, No. 6, September 2011 (Russian original Vol. 47, No. 6, June, 2011)
SYNTHESIS AND BIOLOGICAL ACTIVITY
OF ALKYLIDENE-SUBSTITUTED
CEPHEMS AND PENAMS
I. Potorocina1, M. Vorona1, I. Shestakova1,
I. Domracheva1, E. Liepinsh1, and G. Veinberg1*
The condensation of tert-butyl esters of 3-methyl-7-oxoceph-3-em-4-carboxylic and 6-oxopenicillanic
acids with a series of 2-oxoalkylidene(triphenyl)phosphoranes gave tert-butyl esters of new cephalospo-
rin and penicillin analogs with an alkylidene substituent in the ꢀ-lactam ring. Most of these products
were oxidized by meta-chloroperbenzoic acid to the corresponding sulfones. The cephemes and penams
synthesized including the oxidized products displayed high cytotoxicity relative to cancer cells in vitro.
Some of the alkylidene-substituted cephems as the free acids, similar to Tazobactam, inhibit the catalytic
activity of Enterobacter cloacae penicillinase.
Keywords: tert-butyl esters of (6Z)-alkylidene-1,1-dioxopenicillanic acids, tert-butyl esters of (7Z)-alky-
lidene-3-methyl-1,1-dioxoceph-3-em-4-carboxylic acids, (6Z)-(N-methyl-N-methoxycarbamoylmethy-
lidene)-1,1-dioxopenicillanic acid, 3-methyl-(7Z)-(N-methyl-N-methoxycarbamoylmethylidene)-1,1-di-
oxoceph-3-em-4-carboxylic acid, cyclic sulfones, condensation, oxidation by m-chloroperbenzoic acid.
Continuing a study of the relations between structure and biological activity of cephalosporin and peni-
cillin derivatives [1], we report here the synthesis of new analogs of these antibiotics with an alkylidene
substituent in the ꢀ-lactam ring as well as their 1,1-dioxo derivatives and the anticancer activity of these
compounds in vitro. The effect of the tert-butyl carboxyl protective group on the capacity of some of these
cephalosporin and penicillin analogs to inhibit ꢀ-lactamase was clarified.
The desired tert-butyl esters of 7-alkylidene-3-methyl-1,1-dioxoceph-3-em-4-carboxylic acids 3a–c
were synthesized by condensation of the tert-butyl ester of 7-oxo-3-methylceph-3-em-4-carboxylic acid (1) with
N-methoxycarbamoyl-N-methylmethylidene- (2a), 3-ethoxycarbonyl-2-oxopropylidene- (2b), and 2-(2-furyl)-
2-oxoethylidene(triphenyl)phosphorane (2c), respectively. Products 3a–c were oxidized using meta-chloro-
perbenzoic acid (MCPBA) to give the corresponding sulfones 4a–c.
_______________
*To whom correspondence should be addressed, e-mail: veinberg@osi.lv.
Latvian Institute of Organic Synthesis, 21 Aizkraukles St., Riga LV-1006 , Latvia.
__________________________________________________________________________________________
Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 6, pp. 928–938, June, 2011. Original
article submitted February 3, 2011. Submitted after revision April 11, 2011.
0009-3122/11/4706-0767©2011 Springer Science+Business Media, Inc.
767
ꢀ

R
O
R
O
O
O
H
H
N
H
S
O
O
9
H
S
R
S
MCPBA
H
1
O
6
N
+
8
O
Me
CO2Bu-t
4
N
Me
-t
Me
-t
O
PPh3
2a–c
CO2Bu
CO2Bu
1
3a–c
4a–c
2ꢁ4 a R = N(OMe)Me, b R = CH2COOEt, c R = 2-furyl
The 1H NMR spectral data for 3a,c and 4 a,c indicate that their alkylidene substituent has (7Z)-configu-
ration: the singlet for H-9 proton in these compounds is found at 6.6–7.2 ppm and is characteristic for
(7Z)-methylidenecephalosporins [2, 3]. The signals of the alkylidene substituent protons in 3b and 4b, which
can undergo keto-enol tautomerization, are seen as three one-proton singlets at 5.4–5.5, 6.4–6.6, and 11.7–
11.8 ppm. The signal for the 11-CH2 group protons is lacking, while the signal for H-9 proton is shifted upfield
relative to the analogous signal in the spectra of 3a,c and 4a,c. These features suggest a predominantly enol form
for this substituent in CDCl3 solution.
CO2Et
CO2Et
OH
O
11
10
H
H
N
9
7
8
H
O
H
N
O
3b,4b
The condensation of ester 1 with 1-formylmethylidene- (5a) and 1-formylethylidene(triphenyl)phos-
phorane (5b) did not proceed with such high stereospecificity. The reaction products, namely, the tert-butyl
esters of 3-methyl-7-(1-formylmethylidene)- (6a) and 3-methyl-7-(1-formyl-1-ethylidene)ceph-3-em-4-carboxy-
lic acids (6b), were obtained as 3:2 mixtures of the (7Z) and (7E) isomers as indicated by 1H NMR spectroscopy.
The mixtures were separated by preparative column chromatography.
H
O
10
R
H
H
N
O
H
R
O
S
R
S
10
+
1
+
H
N
PPh3
Me
CO2Bu-t
(7Z)-6a,b
Me
CO2Bu-t
O
O
5a,b
(7E)-6a,b
5, 6 a R = H, b R = Me
1
The position of aldehydic proton H-10 is a distinguishing feature in the H NMR spectra of (7E)-iso-
mers 6a and 6b. This signal is shifted downfield relative to the analogous signal of the (Z)-isomers (by 0.51 ppm
for ester 6a and 0.63 ppm for ester 6b) due to hydrogen bonding between H-10 proton and the lactam ring C=O
group.
768
ꢀ

Condensation of the tert-butyl ester of 6-oxopenicillanic acid (7) with phosphoranes 2a and 2b led to the
formation of tert-butyl esters of 6-alkylidenepenicillanates 8a and 8b with predominantly (Z)-configuration of
the alkylidene fragment and its enolic form in product 8b. Oxidation of these products using MCPBA gave the
corresponding sulfones 9a and 9b.
Me
Me
N
O
N
O
MeO
MeO
O
H
N
8
H
O
Me
Me
CO2Bu-t
S
Me
H
MCPBA
S
H
2a
Me
N
O
H
N
O
-t
CO2Bu
Me
O
O
S
8a
9a
Me
CO2Bu
-t
CO2Et
2b
CO2Et
7
OH
H
OH
O
O
H
8
Me
Me
CO2Bu
MCPBA
S
Me
S
H
H
O
Me
CO2Bu -t
N
N
-t
O
8b
9b
The steric specificity discovered for the condensation of cephem 1 and penam 7 with phosphoranes 2a–
2c and the lack of such specificity in the analogous reactions of cephem 1 with phosphoranes 5a and 5b
probably should be attributed to differences in the structures responsible for formation of intermediate [2+2]
cycloaddition products according to the mechanism of the Wittig reaction [4]. In the case of phosphoranes 2a–c
there is possible formation of forms A and B or in the case of phosphoranes 5a and 5b – forms C and D. These
intermediate forms determine the configuration of the final products.
R
H
H
Ph3P
O
H
H
Ph3P
O
Ph3P
O
O
H
Ph3P
O
R
O
H
O
O
6
6
6
R
6
N
H
H
O
N
R
N
N
O
O
O
A
B
C
D
Thus, the virtually quantitative formation of cephems 3a–c and penams 8a and 8b in the (7Z)-isomeric
form may be attributed to the predominant formation of form A due to mutual repulsion of the C=O substituent
in the oxaphosphoethane ring and C=O group of the lactam ring in form B.
The formation of 7-(1-formylmethylidene)cephems 6a and 6b as a 3:2 mixture of the (7Z)- and (7E)-
1
isomers as indicated by H NMR spectroscopy suggests a competing effect of hydrogen bonding between the
formyl group carbonyl and H-6 proton of the azetidine ring in form C and also between the formyl group proton
and ꢀ-lactam carbonyl in form D.
Cleavage of the ester bond using trifluoroacetic acid in cephem 4a and penam 9a gave acids 10 and 11,
respectively.
769
ꢀ

Me
N
Me
N
O
MeO
O
O
O
H
N
MeO
CF3CO2H
O
O
H
S
H
Me
Me
CO2H
CF3CO2H
S
H
4a
9a
Me
N
O
O
CO2H
10
11
Attempts to use a solution of ZnBr2 in dichloromethane [5] for the analogous cleavage of esters 4a and
9a led to the formation of unidentified products mixture.
Evaluation of the cytotoxic activity of the synthesized cephems in vitro entailed its determination rela-
tive to monolayer human fibrosarcoma cell lines HT-1080 and murine hepatoma cell lines MG22A in
comparison with the analogous activity relative to normal embryonic murine fibroblast cells 3T3. Staining of the
3T3 fibroblasts with neutral red permitted calculation of the expected toxicity LD50 for the compounds tested
using a correlation equation developed at the US National Institutes of Health without recourse to in vitro
experiments in the initial state of biological testing [6].
The screening data for cephems 3a,b, 4a–4c, (7Z)-6b, and (7E)-6b (Table 1) showed that these com-
pounds have high and virtually identical cytotoxicity relative to cancer cells (independently of the structure of
the alkylidene fragment, its configuration, and extent of oxidation of the sulfur atom) and the capacity to
generate NO radicals in the cell medium, which inhibit division of the cancer cells (as the result of a chemical
reaction with their proteins and nucleic acids [7]). Moderate toxicity was found for cephem 3a, its sulfone 4a,
and sulfone 4c, while identically high toxicity was found for the (7Z)- and (7E)-isomers of cephem 6a.
Bacterial ꢀ-lactamases from Bacillus cereus and Enterobacter cloacae were used to evaluate the inhibit-
ing properties of the synthesized alkylidenecephems and alkylidenepenams with esterified and free carboxylic
acid groups as well as of 2-oxopropylidenepenams 12a,2b and 2-oxopropylidenecephems 13a,b [1, 8, 9].
TABLE 1. Cytotoxic Properties of tert-Butyl Esters of Alkylidene-
cephems
IC50, μg/ml*
Compound
LD50, mg/kg
HT-1080
MTT
MG-22A
MTT
3T3
NR
CV
TG
CV
TG
3a
3b
3
2
2
4
2
1
2
3
3
3
3
2
4
4
9
2
3
3
100
67
3
3
2
3
3
3
1
3
3
3
3
3
3
3
3
1
3
3
100
50
19
15
14
10
18
4
425
343
383
331
395
177
177
278
371
4a
100
250
46
100
250
41
4b
4c
(7Z)-6a
(7E)-6a
(7Z)-6b
83
43
100
40
60
4
100
100
10
17
(7E)-6b
67
_________
*IC50 is the concentration providing the annihilation of 50% of cells, CV
is the staining with crystal violet, MTT is staining with 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide, TG is the specific NO
generating activity of the compound, NR is staining with neutral red, and
LD50 is the calculated expected toxicity.
770
ꢀ

Me
H
O
Me
H
O
O
O
O
O
O
O
H
H
H
S
Me
Me
S
N
Me N
CO2H
S
N
N
N
N
O
Me
O
O
CO2R
CO2R
13a,b
12a,b
Tazobactam
a R = H, b R = t-Bu
H
H
N
N
S
NO2
O
S
O
CO2H
Nitrocephin
NO2
Prior to the addition of the standard Nitrocefin substrate, penicillinase was incubated with the tested
compound for 10 min. The inhibiting action of the compounds and of the standard Tazobactam inhibitor was
determined spectrophotometrically relative to the change in intensity of absorption at 486 nm over 5 min in
comparison with the control consisting of nitrocefin and ꢀ-lactamase solution.
The testing of the synthesized compounds showed that they all lack inhibiting properties relative to Ba-
cillus cereus penicillinase in contrast to tazobactam, which lowered the catalytic activity of this enzyme by 50%
at a concentration of 0.36 ꢂM.
The screening data relative to Enterobacter cloacae penicillinase given in Table 2 show that acids 10
and 13a, in contrast to their tert-butyl esters 4a and 13b, reduce the catalytic activity of this enzyme by 50% in
concentrations of 14.4 and 8.2 ꢂg/ml comparable with Tazobactam at a concentration of 3.3 ꢂg/ml.
Thus, (6Z)-( N-methoxycarbamoyl-N-methyl methylidene)-1,1-dioxopenicillanic acid 11 and its tert-bu-
tyl ester 9a display a weak inhibiting effect only in concentrations of 200 ꢂg/ml. The tert-butyl esters of alky-
lidenecephalosporanates 4b, (7Z)-6b, (7E)-6b, and penicillanates 9a and 12b as well as penicillanic acid 12a
lack an inhibiting effect.
These results show that the tert-butyl esters of 7-alkylidene-3-methyl-1,1-dioxoceph-3-em-4-carboxy-
lic acids, which possess anticancer activity, may be transformed into ꢀ-lactamase inhibitors upon their deeste-
rification.
TABLE 2. Inhibiting Activity of Alkylidene-Substituted Cephems and
Penams Relative to Enterobacter cloacae ꢀ-Lactamase
Enzyme catalytic activity inhibition, %
Conc.,
μg/ml*
Tazo-
bactam
4a
10
13b
13a
9a
11
12b
12a
200
50
ꢁ15 0
ꢁ20 9
12 1
0 10
4 3
82 4
84 7
51 3
36 5
23 3
29 11
14.4
ꢁ1 1
2 1
10 4
9 7
4 7
9 1
ꢁ
87 2
74 3
56 2
30 6
18 3
6 2
48 3
28 3
8 1
31 7
13 4
1 2
ꢁ9 5
3 4
2 4
1 3
2 3
2 1
ꢁ
13 3
0 5
3 0
3 3
7 9
3 0
ꢁ
100 3
96 6
89 1
44 3
12 1
4 1
12.5
3.13
0.78
0.2
0 10
ꢁ10 4
ꢁ17 2
200
ꢁ9 10
ꢁ8 5
4 1
7 1
IC50*
ꢁ
8.2
>200
3.3
_________
*IC50 is the concentration providing for 50% inhibition of the catalytic
activity of the enzyme.
771
ꢀ

EXPERIMENTAL
1
The H NMR spectra were taken on a Varian Mercury-400 spectrometer at 400 MHz in CDCl3 (for
compounds 4a–c, 6a,b, 9a,b) and DMSO-d6 (for compounds 10 and 11) with HMDS as internal standard. The
ESI mass spectra were taken on a Micromass Quatro MicroTM API instrument in acetonitrile solution. The
reaction course was monitored by thin-layer chromatography on Merck Kieselgel plates with development in
UV light. Preparative column chromatography (CC) was carried out on Merck Kieselgel silica gel (0.060–
0.200 mm) using a mixture of ethyl acetate (EA) and petroleum ether (PE) as the eluent. The reagents and
materials were obtained from Acros and Aldrich.
The optical density in the biological tests in 96-well microtiter plates was determined using a Tetretek
Multiscan MC C/340 horizontal spectrophotometer.
tert-Butyl Ester of (7Z)-(N-Methoxycarbamoyl-N-methylmethylidene)-3-methyl-1,1-dioxoceph-3-em-
4-carboxylic Acid ((7Z)-4a). Triphenylphosphorane 2a (445 mg, 1.22 mmol) was added to a solution of ester 1
(330 mg, 1.22 mmol) in dichloromethane (20 ml) at 0°C. The mixture was stirred for 2 h at 0°C and then
evaporated at reduced pressure. Column chromatography of the residue using 1:5 EA–PE as the eluent gave
198 mg (46%) of tert-butyl ester of (7Z)-(N-methoxycarbamoyl-N-methylmethylidene)-3-methyl-ceph-
3-em-4-carboxylic acid ((7Z)-3a) as an oil; Rf 0.40 (1:1 EA–PE). 1H NMR spectrum, ꢃ, ppm (J, Hz): 1.55 (9H,
2
s, C(CH3)3); 2.09 (3H, s, CH3); 3.17 and 3.56 (2H, two d, AB system, J = 18.6, SCH2); 3.28 (3H, s, NCH3);
3.75 (3H, s, OCH3); 5.51 (1H, s, H-6); 6.97 (1H, s, H-9). ESI-MS mass spectrum: Found: m/z 377. C16H22N2O5S.
Calculated: M = 354. [M+Na]+ = 377.
70% MCPBA (238 mg, 1.38 mmol) was added to a solution of ester (7Z)-3a (195 mg, 0.55 mmol) in
dichloromethane (20 ml) at 0°C under stirring. The mixture was stirred at room temperature for 16 h, diluted by
20 ml dichloromethane adding, washed with 50 ml 5% aqueous sodium sulfite, two 50 ml portions of
5% aqueous sodium carbonate, and dried over anhydrous sodium sulfate. The solvent was evaporated at reduced
pressure. Column chromatography of the residue using 1:5 EA–PE as the eluent gave 165 mg (78%) of desired
1
ester (7Z)-4a; Rf 0.15 (1:1 EA–PE). H NMR spectrum, ꢃ, ppm (J, Hz): 1.53 (9H, s, C(CH3)3); 2.11 (3H, s,
2
CH3); 3.28 (3H, s, NCH3); 3.76 (3H, s, OCH3); 3.69 and 3.99 (2H, two d, AB system, J = 17.6, SCH2); 5.70
(1H, s, H-6); 7.19 (1H, s, H-9). ESI-MS mass spectrum: Found: m/z 386. C16H22N2O7S. Calculated: M = 386.
Found, %: C 49.92; H 5.49; N 6.87. C16H22N2O7S. Calculated, % : C 49.73; H 5.74; N 7.25.
tert-Butyl Ester of (7Z)-(3-Ethoxycarbonyl-2-hydroxyprop-2-enylidene)-3-methyl-1,1-dioxo-
ceph-3-em-4-carboxylic acid ((7Z)-4b). A solution of sodium hydroxide (30 mg, 0.75 mmol) in water (5 ml)
was added with stirring to a solution of 3-ethoxycarbonyl-2-oxopropylidene(triphenyl)phosphonium chloride
(317 mg, 0.743 mmol) in dichloromethane (10 ml) at 0°C. The solution was stirred for 30 min at 10°C. The
organic phase containing phosphorane 2b was separated, dried over sodium sulfate, and then added to a solution
of 200 mg (0.743 mmol) ester 1 in 20 ml dichloromethane. The reaction mixture was stirred for 2 h at 0°C, then
solvent was evaporated at reduced pressure. Column chromatography of the residue with 1:5 EA–PE as the
eluent gave 225 mg (80%) tert-butyl ester of (7Z)-(3-ethoxycarbonyl-2-hydroxyprop-2-enylidene)-3-methyl-
ceph-3-em-4-carboxylic acid ((Z)-3b) as an oil; Rf 0.65 (1:3 EA–PE). 1H NMR spectrum, ꢃ, ppm (J, Hz): 1.30
(3H, t, J = 7.1, CH2CH3); 1.55 (9H, s, C(CH3)3); 2.07 (3H, s, CH3); 3.16 and 3.55 (2H, two d, AB system,
2J = 19.0, SCH2); 42.4 (2H, q, J = 7.1, CH2CH3); 5.32 (1H, s, H-6); 5.44 (1H, s, H-9); 6.39 (1H, s, H-11); 11.76
(1H, s, OH). ESI-MS mass spectrum: Found: m/z 404. C18H23NO6S. Calculated: M = 381. [M+Na]+ = 404.
According to the procedure for the synthesis of ester (7Z)-4a, a solution of ester (7Z)-3b (225 mg,
0.59 mmol) in dichloromethane (20 ml) was oxidized using 70% MCPBA (255 mg, 1.48 mmol) for 4 h. After
work-up of the reaction mixture, column chromatography of the residue with 1:4 EA–PE as the eluent gave
1
112 mg (46%) ester (7Z)-4b; Rf 0.36 (1:2 EA–PE); mp 136–140°C. H NMR spectrum, ꢃ, ppm (J, Hz): 1.30
(3H, t, J = 7.1, CH3CH2); 1.53 (9H, s, C(CH3)3); 2.10 (3H, s, CH3); 3.68 and 3.90 (2H, two d, AB system,
2J = 17.4, SCH2); 4.24 (2H, q, J = 7.1, CH2CH3); 5.37 (1H, s, H-6); 5.51 (1H, s, H-9); 6.62 (1H, s, H-11); 11.67
(1H, s, OH). Found, %: C 49.94; H 5.66; N 3.52. C18H23NO8S·H2O. Calculated, %: C 50.10; H 5.84; N 3.25.
772
ꢀ

tert-Butyl Ester of (7Z)-[2-(2-Furyl)-2-oxoethylidene]-3-methyl-1,1-dioxoceph-3-em-4-carboxylic
Acid ((7Z)-4c). A solution of NaOH (0.3 g, 7.5 mmol) in water (20 ml) was added with stirring to a solution of
2-(2-furyl)-2-oxoethylidene(triphenyl)phosphonium bromide (3.25 g, 7.2 mmol) in dichloromethane (20 ml) at
0°C. The solution was stirred for 30 min at 10°C, then extracted with ethyl acetate, and dried over sodium
sulfate. The extract containing phosphorane 2c was evaporated and the residue was dissolved in dichloro-
methane (10 ml) and added to the solution of ester 1 (1.94 g, 7.2 mmol) in dichloromethane (20 ml) at 0°C. The
reaction mixture was stirred for 2 h at 0°C and then evaporated. Column chromatography of the residue using
1:5 EA–PE as the eluent gave an oily product with Rf 0.31 (1:3 EA–PE), which was dissolved in dichloro-
methane (20 ml). Then, 70% MCPBA (4.4 g, 18 mmol) was added to the obtained solution under stirring. The
reaction and work-up of the reaction mixture were carried out as described above for ester 4b. After evaporation,
column chromatography of the residue using 1:3 EA–PE as the eluent gave 1.24 g (44%) amorphous ester
(7Z)-4c, Rf 0.30 (1:1 EA–PE). 1H NMR spectrum, ꢃ, ppm (J, Hz): 1.54 (9H, s, C(CH3)3); 2.13 (3H, s, CH3); 3.70
2
and 3.95 (2H, dd, AB system, J = 17.6, SCH2); 5.69 (1H, br. s, H-6); 6.61–6.67 (1H, m, H-9); 7.42 (1H, d,
3
3J = 4.4, H furyl); 7.56 (1H, d, J = 1.5, H furyl); 7.70 (1H, m, H furyl). ESI-MS mass spectrum: Found:
m/z 394. Calculated: M = 393. [M+H]+ = 394. Found, %: C 54.77; H 4.88; N 3.71. C18H19NO7S. Calculated, %:
C 54.95; H 4.87; N 3.56.
tert-Butyl Ester of (7E)-(1-Formylmethylidene)-3-methylceph-3-em-4-carboxylic acid ((7E)-6a) and
tert-Butyl Ester of (7Z)-(1-Formylmethylidene)-3-methylceph-3-em-4-carboxylic acid ((7Z)-6a). Employing
the procedure for the preparation of ester (7Z)-4b, a solution of 1-formylmethylidene(triphenyl)phosphonium
chloride (222 mg, 0.65 mmol) in dichloromethane (10 ml) was treated with a solution of sodium hydroxide
(26 mg, 0.65 mmol) in water (5 ml). The resulting solution of phosphorane 5a in dichloromethane (10 ml) was
added to ester 1 (175 mg, 0.65 mmol) in dichloromethane (20 ml). After evaporation, column chromatography
of the residue using 1:5 EA–PE as the eluent gave 142 mg (74%) of the (7E- and (7Z)-isomers of ester 6a
mixture as an amorphous substance; Rf 0.57 (1:2 EA–PE). Found, %: C 56.73; H 5.83; N 4.71. C14H17NO4S.
Calculated, %: C 56.93; H 5.80; N 4.74.
This mixture of isomers was separated by column chromatography using 1:20 EA–PE as the eluent to
give 49 mg (25%) (E)-isomer (7E)-6a and 71 mg (37%) (Z)-isomer (7Z)-6a.
1
Ester (7E)-6a was obtained as a crystalline compound; Rf 0.54 (1:2 EA–PE); mp 102–122°C. H NMR
spectrum, ꢃ, ppm (J, Hz): 1.56 (9H, s, C(CH3)3); 2.11 (3H, s, CH3); 3.22 and 3.55 (2H, two d, AB system,
2J = 18.2, SCH2); 5.27 (1H, s, H-6); 6.22 (1H, d, 3J = 7.9, H-9); 10.31 (1H, d, 3J = 7.9, CHO).
1
Ester (7Z)-6a was obtained as a crystalline compound; Rf 0.57 (1:2 EA–PE); mp 82–99°C. H NMR
spectrum, ꢃ, ppm (J, Hz): 1.55 (9H, s, C(CH3)3); 2.12 (3H, s, CH3); 3.23 and 3.60 (2H, two d, AB system,
2J = 28.2, SCH2); 5.46 (1H, s, H-6); 6.55 (1H, d, 3J = 6.4, H-9); 9.80 (1H, d, 3J = 6.4, CHO).
tert-Butyl Ester of (7E)-(1-Formylethylidene)-3-methylceph-3-em-4-carboxylic acid ((7E)-6b) and
tert-Butyl Ester of (7Z)-(1-Formylethylidene)-3-methylceph-3-em-4-carboxylic acid ((7Z)-6b). Employing the
procedure for the preparation of ester (7Z)-4a, ester 1 (174 mg, 0.647 mmol) in dichloromethane (25 ml) and 1-for-
mylethylidene(triphenyl)phosphorane (5b) (206 mg, 0.647 mmol) gave a residue after evaporation of the reaction
mixture. Column chromatography using 1:6 EA–PE as the eluent gave 195 mg (98%) of the (7E)- and (7Z)-isomers
of ester 6b mixture. Found, %: C 57.85; H 5.98; N 4.44. C15H19NO4S. Calculated, %: C 58.23; H 6.19; N 4.53.
The isomers mixture obtained was separated by column chromatography using 1:20 EA–PE as the elu-
1
ent. The fraction with Rf 0.49 (1:3 EA–PE) gave 70 mg (35%) (E)-isomer (7E)-6b; mp 146–150°C. H NMR
spectrum, ꢃ, ppm (J, Hz): 1.56 (9H, s, C(CH3)3); 1.92 (3H, s, 3-CH3); 2.09 (3H, s, 9-CH3); 3.21 and 3.56 (2H,
2
two d, AB system, J = 18.3, SCH2); 5.29 (1H, s, H-6); 10.36 (1H, s, CHO). ESI-MS mass spectrum: Found:
m/z 332. C15H19NO4S. Calculated : M = 309. [M+Na]+ = 332.
The fraction with Rf 0.51 (1:3 EA–PE) gave 102 mg (51%) (Z)-isomer (7Z)-6b; mp 132–152°C.
1H NMR spectrum, ꢃ, ppm (J, Hz): 1.57 (9H, s, C(CH3)3); 2.10 (3H, s, 3-CH3); 2.13 (3H, s, 9-CH3); 3.21 and
2
3.59 (2H, two d, AB system, J = 18.5, SCH2); 5.41 (1H, s, H-6); 9.73 (1H, s, CHO). ESI-MS mass spectrum:
Found: m/z 310. C15H19NO4S. Calculated: M = 309, [M+H]+ = 310.
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tert-Butyl Ester of (6Z)-(N-Methoxycarbamoyl-N-methylmethylidene)-1,1-dioxopenicillanic Acid
(9a). Triphenylphosphorane 2a (363 mg, 1.0 mmol) was added to a solution of tert-butyl ester of 6-oxopenicil-
lanic acid (7) (271 mg, 1.0 mmol) in dichloromethane (20 ml) at 0°C. The reaction mixture was stirred for 2 h at
0°C and maintained for 16 h at –20°C. Then, the mixture was evaporated at reduced pressure. Column
chromatography of the residue with 1:8 EA–PE as the eluent gave 350 mg (98%) tert-butyl ester of
1
(6Z)-(N-methoxycarbamoyl-N-methylmethylidene)penicillanic acid (8a); Rf 0.29 (1:2 EA–PE). H NMR
spectrum, ꢃ, ppm (J, Hz): 1.50 (9H, s, C(CH3)3); 1.52 (3H, s, 2-CH3); 1.57 (3H, s, 2-CH3); 3.26 (3H, s, NCH3);
3.73 (3H, s, OCH3); 4.42 (1H, s, H-3); 6.04 (1H, s, H-5); 6.85 (1H, s, H-8).
According to the procedure for compound (7Z)-4a, a solution of the ester obtained 8a (350 mg,
0.98 mmol) in dichloromethane (20 ml) was oxidized using 70% MCPBA (530 mg, 2.45 mmol) for 16 h. After
work-up of the reaction mixture, column chromatography using 1:5 EA–PE as the eluent gave 201 mg (53%)
1
penicillanic acid 9a; Rf 0.29 (1:1 EA–PE); mp 133–135°C. H NMR spectrum, ꢃ, ppm (J, Hz): 1.47 (3H, s,
2-CH3); 1.52 (9H, s, C(CH3)3); 1.59 (3H, s, 2-CH3); 3.30 (3H, s, NCH3); 3.76 (3H, s, OCH3); 4.36 (1H, s, H-3);
5.56 (1H, s, H-5); 7.18 (1H, s, H-8). Found, %: C 49.63; H 6.16; N 7.05. C16H24N2O7S. Calculated, %: C 49.47;
H 6.23; N 7.21.
tert-Butyl Ester of (6Z)-(3-Ethoxycarbonyl-2-hydroxyprop-2-enylidene)-1,1-dioxopenicillanic
Acid (9b). According to the procedure described for compound (7Z)-4b, the action of sodium hydroxide
(40 mg) in water (5 ml) on 3-ethoxycarbonyl-2-oxopropylidene(triphenyl)phosphonium chloride (426 mg,
1.0 mmol) gave phosphorane 2b, which, without purification, was dissolved in dichloromethane (10 ml). The
resultant solution was added to ester 7 (271 mg, 1.0 mmol) in dichloromethane (20 ml). The reaction mixture
was stirred for 2 h at 0°C, maintained for 16 h at –20°C, and evaporated at reduced pressure. Column
chromatography using 1:10 EA–PE as the eluent gave 92 mg (24%) tert-butyl ester of (6Z)-(3-ethoxycarbo-
nyl-2-hydroxyprop-2-enylidene)penicillanic acid (8b) as an amorphous substance with Rf 0.71 (1:2 EA–PE).
1H NMR spectrum, ꢃ, ppm (J, Hz): 1.29 (3H, t, J = 7.4, CH2CH3); 1.49 (3H, s, 2-CH3); 1.50 (9H, s, C(CH3)3);
1.53 (3H, s, 2-CH3); 4.23 (2H, q, J = 7.4, CH2CH3); 4.42 (1H, s, H-3); 5.29 (1H, s, H-5); 6.03 (1H, s, H-8); 6.27
(1H, s, H-10); 11.66 (1H, s, OH).
Employing the procedure for compound (7Z)-4a, resultant ester 8b (91 mg, 0.237 mmol) was oxidized
by MCPBA (102.4 mg, 0.593 mmol). After work-up of the reaction mixture, column chromatography using
1
1:5 EA–PE as the eluent gave 36.8 mg (37%) 9b; Rf 0.50 (1:2 EA–PE); mp 136–140°C. H NMR spectrum, ꢃ,
ppm (J, Hz): 1.30 (3H, t, J = 7.4, CH2CH3); 1.46 (3H, s, 2-CH3); 1.52 (9H, s, C(CH3)3); 1.58 (3H, s, 2-CH3);
4.24 (2H, q, J = 7.4, CH2CH3); 4.34 (1H, s, H-3); 5.38 (1H, s, H-5); 5.46 (1H, s, H-8); 6.62 (1H, s, H-10); 11.69
(1H, s, OH). Found, %: C 52.18; H 6.25; N 3.37. C18H25NO8S. Calculated, %: C 52.04; H 6.07; N 3.37.
(7Z)-(N-Methoxycarbamoyl-N-methylmethylidene)-3-methyl-1,1-dioxoceph-3-em-4-carboxylic
Acid (10). Trifluoroacetic acid (1.5 ml) was added to a solution of ester 4a (113.5 mg, 0.294 mmol) in
dichloromethane (15 ml). The reaction mixture was stirred at room temperature for 24 h and evaporated under
reduced pressure. Column chromatography of the residue using 3:1 EA–PE as the eluent gave 54.5 mg (56%)
1
acid 10, Rf 0.22 (3:1 EA–PE). H NMR spectrum, ꢃ, ppm (J, Hz): 1.96 (3H, s, CH3); 3.19 (3H, s, NCH3); 3.74
(3H, s, OCH3); 4.16 and 4.31 (2H, two d, AB system, 2J = 19.2, SO2CH2); 6.07 (1H, s, H-6); 7.17 (1H, s, H-9);
13.67 (1H, br. s, CO2H). Found, %: C 43.38; H 4.25; N 8.39. C12H14N2O7S. Calculated, %: C 43.63; H 4.27;
N 8.48.
(6Z)-(N-methoxycarbamoyl-N-methylmethylidene)-1,1-dioxopenicillanic Acid (11). Trifluoroacetic
acid (1.5 ml) was added to a solution of ester 9a (70 mg, 0.18 mmol) in dichloromethane (15 ml). The reaction
mixture was stirred at room temperature for 120 h and evaporated at reduced pressure to give 53 mg (82%) of
acid 11; Rf 0.20 (3:1 EA–PE). 1H NMR spectrum, ꢃ, ppm (J, Hz): 1.45 (3H, s, 2-CH3); 1.70 (3H, s, 2-CH3); 3.21
(3H, s, NCH3); 3.75 (3H, s, OCH3); 5.08 (1H, s, H-3); 6.87 (1H, s, H-5); 7.31 (1H, s, H-8); 13.53 (1H, br. s,
CO2H). Found, %: C, 43.18; H, 4.65; N, 8.19. C12H16N2O7S. Calculated, %: C, 43.37; H, 4.85; N, 8.43.
Determination of Cytotoxic Activity of Alkylidene-substituted Cephems in vitro. The cytotoxic
properties of the compounds synthesized relative to cultures of monolayer cancer and normal cells at c from
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2·104 to 5·104 cells/ml (human fibrosarcoma HT-1080, murine hepatoma MG-22A, murine embryonal
fibroblasts 3T3) were determined in 96-well microtiter plates using CV, MTT, and NR staining according to
standard procedures [10].
Generation of NO Radicals by Cells. The determination of the nitric oxide radical concentration in the
Gries cell medium according to [11] for the alkylidene-substituted cephems was carried out in 96-well plastic
panels (well volume 200 ꢂl, concentration of the compound tested 50 ꢂg/ml). The concentrations of the NO
radicals (in nmol) in the culture medium with cells surviving after incubation in the presence of the tested
compound over 72 h using the following formula for calculating the specific NO generating activity for the
compounds (TG):
TG = G 100/C (nmol/ꢂl),
where G is the NO concentration (nmol) in the culture medium (200 ꢂl) with surviving cells and C is the
percentage of surviving cells determined upon their CV staining.
Spectrophotometric Determination of the Inhibiting Properties of Alkylidene-substituted Ce-
phems and Penams Relative to ꢀ-Lactamase. The total volume of the reaction mixture in each well of the
96-well plate was 200 ꢂl and was obtained by adding 20 ꢂl solution of Bacillus cereus ꢀ-lactamase (Sigma,
P0389) (the final concentration in the reaction mixture was 0.8 E) or Enterobacter cloacae ꢀ-lactamase (Sigma,
P4524) in 0.1 M phosphate buffer (pH 7.0) (the final concentration in the reaction mixture was 0.3 U). Then,
160 ꢂl phosphate buffer (pH 7.0) containing the compound tested or Tazobactam in amounts sufficient to
achieve concentrations of 0.2, 0.78, 3.13, 12.5, 50, and 200 ꢂM was added. The mixture was incubated for
10 min and then 20 ꢂl 0.97 ꢂM (final concentration in the reaction mixture was 0.097 ꢂM) solution of
Nitrocefin in a 1:19 mixture of DMSO and 0.1 M phosphate buffer (pH 7.0) was added. The measurement of the
optical density at 486 nm was carried out over 5 min and the result obtained was compared with the result of a
control experiment, which was carried out without addition of the compound tested and Tazobactam to the
solution of penicillinase and Nitrocefin. The IC50 values characterizing the concentration of the inhibitor
reducing the activity of the enzyme by 50% was calculated using the Graph Pad Prism®.
REFERENCES
1.
2.
3.
I. Potorocina, M. Vorona, G. Veinberg, I. Shestakova, I. Kanepe, M. Petrova, E. Liepinsh, and
E. Lukevics, Khim. Geterotsikl. Soedin., 284 (2009). [Chem. Heterocycl. Comp., 44, 228 (2009)].
J. D. Buynak, A. S. Rao, G. P. Ford, C. Carver, G. Adam, B. Geng, B. Bachmann, S. Shobassy, and
S. Lackey, J. Med. Chem., 40, 3423 (1997).
G. Veinberg, M. Vorona, I. Shestakova, I. Kanepe, O. Zharkova, R. Mezapuke, I. Turovskis,
I. Kalvinsh, and E. Lukevics, Bioorg. Med. Chem., 8, 1033 (2000).
4.
5.
E. Vedejs, C. F. Marth, and R. Ruggeri, J. Am. Chem. Soc., 110, 3940 (1988).
Y.-G. Wu, D. C. Limburg, D. E. Wilkinson, M. J. Vaal, and G. S. Hamilton, Tetrahedron Lett., 41, 2847
(2000).
Guidance Document on Using In Vitro Data to Estimate In Vivo Starting Doses for Acute Toxicity by
Interagency Coordinating Committee on the Validation of Alternative Methods, Research Triangle Park,
NC: National Toxicology Program, US Dept. of Health and Human Services, Public Health Service,
National Institutes of Health, 2001, http://www.epa.gov/hpv/pubs/general/nih2001b.
J. F. Kerwin, Jr., J. R. Lancaster, and P. L. Feldman, J. Med. Chem., 38, 4343 (1995).
G. Veinberg, I. Shestakova, M. Vorona, I. Kanepe, and E. Lukevics, Bioorg. Med. Chem. Lett., 14, 1007
(2004).
6.
7.
8.
9.
10.
11.
M. Vorona, G. Veinberg, S. Belyakov, M. Petrova, E. Liepinsh, and E. Lukevics, Khim. Geterotsikl.
Soedin., 618 (2008). [Chem. Heterocycl. Comp., 44, 486 (2008)].
P. J. Freshney, in: Culture of Animal Cells (A Manual of Basic Techniques), Wiley-Liss, New York
(1994), p. 296.
D. J. Fast, R. C. Lynch, and R. W. Leu, J. Leukocyte Biol., 52, 255 (1992).
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