Synthesis and cytotoxic activity of derivatives of the tert-butyl ester of 7Z-acetylmethylene-3-methyl-3-cephem-4-carboxylic acid

Article abstract of DOI:10.1007/s10593-009-0254-2

The condensation of the acetylmethylene group in the tert-butyl esters of 7Z-acetylmethylene-3-methyl-3-cephem-4-carboxylic acid and 7Z-acetylmethylene-3- methyl-1,1-dioxo-3-cephem-4-carboxylic acid and in 7Z-acetylmethylene-3- methylene-1,1-dioxo-3-cephe

Full text of DOI:10.1007/s10593-009-0254-2

Chemistry of Heterocyclic Compounds, Vol. 45, No. 2, 2009
SYNTHESIS AND CYTOTOXIC ACTIVITY
OF DERIVATIVES OF THE tert-BUTYL ESTER
OF 7Z-ACETYLMETHYLENE-3-METHYL-
3-CEPHEM-4-CARBOXYLIC ACID
I. Potorocina1, M. Vorona1, G. Veinberg1*, I. Shestakova1, I. Kanepe1,
M. Petrova1, E. Liepinsh1, and E. Lukevics1
The condensation of the acetylmethylene group in the tert-butyl esters of 7Z-acetylmethylene-3-methyl-
3-cephem-4-carboxylic acid and 7Z-acetylmethylene-3-methyl-1,1-dioxo-3-cephem-4-carboxylic acid
and in 7Z-acetylmethylene-3-methylene-1,1-dioxo-3-cephem with arylmethoxyamines and O-alkylation
of the tert-butyl ester of 7Z-(2-hydroxyimino)propylidene-3-methyl-1,1-dioxo-3-cephem-4-carboxylic
acid using substituted benzyl bromides as well as pyridylmethyl chlorides gave arylmethoxyimino and
pyridylmethoxyimino derivatives of these compounds in the syn and anti isomeric forms. The Vilsmaier
reagent was used to introduce the N,N-dimethylaminomethylene group at C-2 of the cephem system in the
tert-butyl esters of 7Z-[2-(arylmethoxyimino)propylidene]-3-methyl-1,1-dioxo-3-cephem-4-carboxylic acid.
Subsequent transformation of the N,N-dimethylaminomethylene cephems using hydroxylamine led to
3Z-[2-(anti-arylmethoxyimino)propylidene]-tert-butoxycarbonylmethyl-4-(5-methyl-4-isoxazolylsulfonyl)-
azetidin-2-ones. Condensation of the acetyl group in the tert-butyl ester of 7Z-acetylmethylene-
3-methyl-1,1-dioxo-3-cephem-4-carboxylic acid with 4-bromophenylhydrazine gave a cephem with a
2-(4-bromophenylhydrazono)propylidene group at C-7. Acylation of the tert-butyl ester of 7Z-(2-
hydroxyimino)propylidene-3-methyl-1,1-dioxo-3-cephem-4-carboxylic acid by 2-bromobenzoyl chloride
gave a cephem with a 2-(2-bromo-benzoyloxyimino)propylidene group at C-7. Biological screening of
these products towards to malignant and normal cells in vitro showed that their antitumor activity and
cytotoxic selectivity towards to malignant and normal cells depend on the structure and configuration of
the arylmethoxyimino and pyridylmethoxyimino groups in the 7-alkylidene substituent as well as on the
presence or absence of N,N-dimethylaminomethylene and carboxyl groups, respectively, at C-2 and C-4
of the cephem system.
Keywords: 3Z-[2-(arylmethoxyimino)propylidene]-1-tert-butoxycarbonylmethyl-4-(5-methyl-4-isoxazolyl-
sulfonyl)azetidin-2-ones, tert-butyl ester of 7Z-[2-(arylmethoxyimino)propylidene]-3-methyl-3-cephem-
4-carboxylic acid, tert-butyl ester of 7Z-[2-(2-bromobenzoyloxyimino)propylidene]-3-methyl-1,1-dioxo-
_______
* To whom correspondence should be addressed, e-mail: veinberg@osi.lv
1Latvian Institute of Organic Synthesis, Riga LV1006, Latvia; e-mail: max@osi.lv
__________________________________________________________________________________________
Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 2, pp. 284-303, February, 2009. Original
article submitted November 12, 2008.
228
0009-3122/09/4502-0228©2009 Springer Science+Business Media, Inc.

3-cephem-4-carboxylic acid, tert-butyl ester of 7Z-[2-(4-bromophenylhydrazono)propylidene]-
3-methyl-1,1-dioxo-3-cephem-4-carboxylic acid, tert-butyl ester of 7Z-[2-(hydroxyimino)propylidene]-
3-methyl-1,1-dioxo-3-cephem-4-carboxylic acid, tert-butyl esters of 7Z-[2-(arylmethoxyimino)-
propylidene]-2-(dimethylaminomethylene)-3-methyl-1,1-dioxo-3-cephem-4-carboxylic acid, tert-butyl
esters of 7Z-(arylmethoxyimino)propylidene]-3-methyl-1,1-dioxo-3-cephem-4-carboxylic acid, tert-butyl
esters of 3-methyl-1,1-dioxo-7Z-[2-(pyridylmethoxyimino)propylidene]-3-cephem-4-carboxylic acid.
In a continuation of a study of the relation between the structure and antitumor properties of
cephalosporin derivatives, we have modified the cephem system and substituents in the previously synthesized
tert-butyl ester of 7Z-acetylmethylene-3-methyl-1,1-dioxo-3-cephem-4-carboxylic acid (1) [1].
The transformation of compound 1 and its arylmethoxyimino and pyridylmethoxyimino derivatives 6a-k
was carried out by two methods:
A. Condensation of the arylmethylene group with the hydrochloride salts of arylmethoxyamines 2a-g in
methanol in the presence of sodium acetate.
B. Oximination of the acetyl group in ester 1 with subsequent replacement of the hydrogen atom in the
hydroxyimino group of the tert-butyl ester of 7Z-(2-(hydroxyimino)propylidene]-3-methyl-1,1-dioxo-3-cephem-
4-carboxylic acid (3) by sodium and O-aralkylation of the intermediate 3-cephem 4 by substituted benzyl
bromides 5h,i as well as 3- and 4-pyridylmethyl chlorides 5j,k.
N
O
O
Me
Me
NH2
O
O
Ar
O
Ar
S2
S2
H
H
7
6
2a–g
2
3
1
N
N
Me
Me
8
5
O
O
4
COOBu-t
t
COOBu-
6a–k
1
Ar
X
HONH2
Me
5h–k
HON
H
NaON
H
Me
O
O
S2
S2
NaH
N
N
Me
COOBu-
Me
O
O
t
t
COOBu-
3
4
2, 6 a Ar = Ph, b Ar = 2-BrC6H4, c Ar = 3-BrC6H4, d Ar = 4-BrC6H4, e Ar = 2-ClC6H4,
f Ar = 2-FC6H4, g Ar = 2-CF3C6H4; 5, 6 h Ar = 2,6-Cl2C6H3, i Ar = 3,4-Cl2C6H3,
j Ar = 3-Py, k Ar = 4-Py; 5 h,i X = Br, j,k X = Cl
The cephalosporin derivatives 6a-k synthesized by the two methods are formed as mixtures of syn and anti
isomers. Esters 6a,b,e,f,g were separated by column chromatography into pure isomers. The structural assignment of
the syn and anti isomers was carried out using 1H NMR 2D-NOESY spectroscopy for cephems 6b and 3.
The spectra of the syn isomers of both these compounds show cross peaks between the protons of the
9-CH and CH2OH groups in cephem 6b and, correspondingly, between the protons of the 9-CH and HON
groups in cephem 3. This effect is accompanied by a downfield shift of the signal for 9-CH in comparison with
the analogous signal in anti-6b and anti-3, for which an Overhauser effect is not observed.
229

Br
H
O
O
Br
N
N
N
N
O
O
Me
Me
Me
Me
H
9
H
H
H
H
7
O
O
O
O
syn-3
anti-3
anti-6b
syn-6b
To establish the biological activity on the oxidation degree of heterocyclic sulfur atom from tert-butyl
ester of 7Z-acetylmethylene-3-methyl-3-cephem-4-carboxylic acid (7) [1], according to method A, its individual
2-brombenzyloxyimino derivatives of syn-8b and anti-8b were obtained.
Br
O
O
Br
N
Me
N
O
Me
Me
2b
S
H
S
H
S
H
+
N
N
N
Me
O
Me
Me
O
O
COOBu-t
COOBu-t
COOBu-
t
7
anti-8b
syn-8b
The condensation of previously synthesized 7Z-acetylmethylene-3-methyl-1,1-dioxo-3-cephem (9) [2]
with arylmethoxyamines 2a-f was carried out according to method A to evaluate the effect of esterification of
the carboxyl group on the antitumor properties of cephems. The resultant mixtures of the anti and syn isomers of
decarboxylated cephems 10a-f were separated by column chromatography. The 1H NMR spectra of
cephems 10a-f show a signal for H-4 at 6.56 ppm in accord with their structure.
Ar
O
N
N
O
O
Me
Me
Me
O
O
O
S 2
S2
Ar
S 2
H
H
H
2a–f
+
N
N
N
Me
Me
Me
O
O
O
H
H
H
9
syn-10a–f
anti-10a–f
2, 10 a Ar = Ph, b Ar = 2-BrC6H4, c Ar = 3-BrC6H4, d Ar = 4-BrC6H4,
e Ar = 2-ClC6H4, f Ar = 2-FC6H4
230

The introduction of an N,N-dimethylaminomethylene group at C-2 in the cephem system of the mixtures of
the syn and anti isomers of 6a-f using the Vilsmaier reagent was carried out according to our previous procedure [3].
Fractionation of the reaction products by column chromatography gave the syn-11a-f and anti-11a-f
isomers as pure compounds. The presence of a double bond in this substituent provides the preconditions for
finding the N,N-dimethylaminomethylene group in the Z- and E-isomeric forms.
Ar
O
N
N
O
Me
Me
H
H
O2
S
O2
S
Ar
+
H
H
–
NMe2
NMe2
Me2N=CHCl Cl
+
6a–f
N
N
Me
Me
O
O
t
COOBu-
COOBu-
t
syn-11a–f
anti-11a–f
6, 11 a Ar = Ph, b Ar = 2-BrC6H4, c Ar = 3-BrC6H4, d Ar = 4-BrC6H4,
e Ar = 2-ClC6H4, f Ar = 2-FC6H4
1
In accord with the spectral data for this type of compounds by H NMR 2D-NOESY spectroscopy [3],
we found the close proximity of the sulfone and dimethylamino groups in isomer 2Z-11a-f leads to a
characteristic downfield shift of the signals of the NMe2 protons in comparison with the analogous signals for
the 2E-11a-f isomers.
H
NMe2
O
O
S2
S2
NMe2
H
N
N
Me
Me
COOBu-
t
COOBu-
t
2E-11a–f
2Z-11a–f
The action of hydroxylamine hydrochloride on a mixture of the syn and anti isomers of
N,N-dimethylaminomethylenecephems 11a,b-d in acetonitrile at 40-50°C for 2 h led to opening of the double
bond in the 3-cephem system and formation of 2-azetidinones 14a,b,d substituted at C-4
N
O
Me
H
H
O
O
S2
Ar
S 2
H
HONH2
NMe2
NHOH
N
N
∆
Me
Me
O
t
COOBu-
COOBu-
t
12
11a,b,d
N
O
Me
O2
S
O
S2
Ar
H
N
N
O
N
O
N
Me
COOBu-t
H
O
Me
t
- BuOOC
14a,b,d
13
11, 14 a Ar = Ph, b Ar = 2-BrC6H4, d Ar = 4-BrC6H4,
231

of the 5-methylisoxazolyl-4-sulfonyl system. According to our previous investigation [3], the mechanism of this
reaction involves formation of two intermediates, namely, the product of replacement of the N,N-dimethylamino
group by a hydroxyamino group 12 and the unstable tricyclic system 13 formed as a result of the addition of a
hydroxyamino group to the C(3)=C(4) bond in 3-cephem 12.
In order to establish the role of the –CH2O- fragment in the 7-alkylidene sidechain of these cephems on
their biological activity, we replaced this fragment by imino and carbonyl groups.
In the former case, we condensed the acetyl group in starting cephem 1 with 4-bromophenylhydrazine,
leading to cephem 15 with a 2-anti-(4-bromophenylhydrazono)propylidene group at C-7, as indicated by the
signal for the =CHC(Me) group at 6.97 ppm.
3
1
4-BrC6H4NHNH2
2-BrC6H4COCl
Br
Br
NH
O
O
N
Me
N
O2
S
Me
H
O2
S
H
N
Me
O
N
Me
COOBu-
COOBu-t
O
t
anti-16
anti-15
In the latter case, we achieved our goal by acylation of the hydroxyimino group in cephem 3 using
2-bromobenzoyl chloride. In this case, only cephem 16 with anti configuration of the acyloxyimino group was
isolated.
Biological screening of these products in vitro included determination of their cytotoxic properties
towards to monolayer lines of HT-1080 (human fibrosarcoma) and MG-22A (murine hepatoma) malignant cells
in comparison to normal 3T3 cells (murine embryonic fibroblasts). Coloration of the 3T3 fibroblasts by a neutral
red dye permitted us to calculate the expected LD50 toxicity for the compounds tested using a special equation
without recourse to experiments in vivo [4].
The screening data for esters 6a-k given in Table 1 indicate that the biological properties of these
compounds depend both on the structure of the aromatic fragment and configuration of the arylmethoxyimino
and pyridylmethoxyimino groups in the 7-alkylidene substituent.
Thus, the presence of one or two halogen atoms in the benzene ring in esters 6b-i, as a rule, enhances the
cytotoxic effect of these compounds not only relative to malignant but also normal cells in comparison with the
analogous effect shown by cephems 6a,j,k, which have a phenyl or pyridyl group as the aromatic fragment. The
anti or syn configuration of the arylmethoxyimino group also is a significant factor determining the selectivity of
the cytotoxic action of the compounds tested, primarily in regard to only the malignant cells. The syn isomers of
esters 6a,b,e,f (with the exception of syn-6g) isolated as pure compounds proved less toxic in comparison with
their anti isomers toward normal 3T3 cells than toward malignant HT-1080 and MG-22A cells. In the case of
ester 6f, the LD50 data show that this difference is two-fold, while in the case of ester 6a, this difference is
three-fold.
232

TABLE 1. Biological Properties of tert-Butyl Esters of 7Z-[2-(Aryl-
and Pyridylmethoxyimino)propylidene]-3-methyl-1,1-dioxo-3-cephem-
4-carboxylic Acid 6*
LC50, µg/ml
Compound
LD50, mg/kg
HT-1080
MTT
MG-22A
MTT
3T3
NR
CV
TG100
CV
TG100
anti-6a
3
3
3
2
250
350
54
3
2
3
300
233
23
7
100
2
313
982
194
289
294
315
240
289
279
557
309
309
309
309
671
716
syn-6a
3
45
3
anti-6b
24
3
18
3
23
3
syn-6b
200
250
800
50
300
250
700
16
5
anti/syn-6c
anti/syn-6d
anti-6e
3
3
2
2
5
3
3
1
1
6
33
3
28
2
100
2
89
2
4
syn-6e
250
200
200
250
250
200
133
350
550
300
200
250
150
133
350
82
5
anti-6f
3
2
3
3
6
syn-6f
14
1.5
2
14
1.5
2
3
3
28
6
anti-6g
3
2
syn-6g
3
3
6
anti/syn-6h
anti/syn-6i
anti/syn-6j
anti/syn-6k
0.8
1.5
2.1
2.6
1.3
2.6
0.8
1.5
1.8
0.04
0.8
1.5
3.2
0.03
0.5
2.0
5.8
4.8
6
400
267
54
_______
* LC50 is the concentration providing death of 50% of cells, CV indicates
coloration by crystal violet, MTT indicates coloration by
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, TG100
is the specific NO generating activity of the compound, NR indicates
coloration by Neutral Red, and LD50 is the calculated expected toxicity.
The data in Table 2 reflect the diminution of the biological effect following modification of the tert-butyl
esters of 7Z-[2(arylmethoxyimino)propylidene]-3-methyl-1,1-dioxo-3-cephem-4-carboxylic acid through
replacement of the sulfone moiety by a sulfide moiety and removal of the carboxyl group. In the former case,
isomeric anti-8b and syn-8b show significantly weaker cytotoxic activity. However, in the latter case,
independently of the configuration of the substituent at C-7, enhancement of the cytotoxic activity of
cephems 10a-f was observed, mainly, relative to normal cells reflected in a marked drop in the LD50 values.
According to the data in Table 3, the introduction of an N,N-dimethylaminomethylene group at C-2 of
the cephem system leads to a significant increase in the cytotoxic selectivity of the corresponding derivatives of
the tert-butyl ester of 7Z-[2-(arylmethoxyimino)propylidene]-3-methyl-1,1-dioxo-3-cephem-4-carboxylic
acid 11a-f. Comparison of the biological screening data of the anti and syn isomers indicates that better
selectivity of ester 11a is found for the anti isomer, while better selectivity of esters 11c,d,f is found for the syn
isomers. Opening of the cephem system at the C(3)=C(4) bond to give azetidinones 14a-c is accompanied by
weakening of the anticancer activity in vitro. Analogous properties were seen for cephem 15 with a
2-(4-bromophenylhydrazono)propylidene group at C-7. On the other hand, replacement of the methylene group
by a carbonyl group in the 2-bromobenzyl moiety of cephem anti-16 may hold promise for modification of
biological activity.
The cytotoxic effect of most of the compounds tested is related to their capacity to generate nitric oxide
in cellular media. As a rule, high toxicity accompanies a high level of NO generation and vice versa.
233

TABLE 2. Biological Properties of tert-Butyl Esters of 7Z-[2-(2-Bromo-
benzyloxyimino)propylidene]-3-methyl-3-cephem-4-carboxylic Acid 8 and
7Z-[2-(2-Arylmethoxyimino)propylidene]-3-methyl-1,1-dioxo-3-cephems 10
LC50, µg/ml
LD50,
mg/kg
Compound
HT-1080
MTT
MG-22A
MTT
3T3
NR
CV
TG100
CV
TG100
anti-8b
syn-8b
60
100
2
56
>100
1
9
5
70
81
1
63
>100
2
11
7
>1000
2812
2714
233
234
210
170
272
183
235
235
160
160
146
219
922
4
anti-10a
syn-10a
anti-10b
syn-10b
anti-10c
syn-10c
anti-10d
syn-10d
anti-10e
syn-10e
anti-10f
syn-10f
400
250
350
250
300
300
300
500
200
200
200
100
300
250
450
100
300
300
300
350
150
60
2
2
2
1
5
2
0.2
2
2
0.4
2
2
0.2
1
2
0.3
1
3
2
5
0.6
1
0.5
1
0.3
0.3
2
0.6
0.3
1
2
3
1
2
3
1
1
2
2
2
2
0.2
0.2
0.2
0.7
0.2
0.3
0.2
0.3
0.2
0.3
0.3
0.3
120
100
2
5
TABLE 3. Biological Properties of Cephems 11a-f, 15, 16, and
Azetidinones 14a-c
LC50, µg/ml
LD50,
Compound
HT-1080
MTT
MG-22A
MTT
3T3
NR
mg/kg
CV
TG100
CV
TG100
anti-11a
syn-11a
anti-11b
anti-11c
syn-11c
anti-11d
syn-11d
anti-11е
syn-11е
anti-11f
syn-11f
anti-14а
anti-14b
anti-14с
anti-15
3
3
3
3
1000
500
200
67
2
3
2
2
1000
150
37
87
1003
417
12
534
100
920
30
90
10
10
3
37
10
11
3
3
3
2380
1161
2961
639
3
3
200
300
300
850
300
533
64
175
500
800
100
325
300
250
200
450
150
250
150
2
2
1
2
3
3
2
2
151
100
72
1335
1072
965
4
4
2
2
1
3
1
1
3
3
4
3
23
572
10
32
7
22
30
10
20
26
1
4
6
250
350
267
300
19
100
167
16
1091
1273
512
23
3
29
6
21
21
1
19
49
1
17
49
<1
100
100
13
1080
1072
432
anti-16
200
EXPERIMENTAL
1
The H NMR spectra were taken on a Varian Mercury-200 spectrometer at 200 MHz in CDCl3 for 6, 8,
11, 14, and 16, in DMSO-d6 for 15, and on a Varian Mercury-400 spectrometer at 400 MHz in DMSO-d6 for 3
with HMDS as the internal standard (δ 0.05 ppm). The elemental analysis was carried out on a Carlo Erba 1108
analyzer. The ESI-MS mass spectra were taken on a Micromass Quatro MicroTM API inductively coupled
234

plasma mass spectrometer in MeCN for 6, 10, anti-11a, anti-11b, anti-11f, 15, and 16, MeOH + HCO2H for
syn-11a, and MeOH for 11c-11e and 14-16. The reaction course was monitored by thin-layer chromatography
on Merck Kieselgel plates with development in UV light. Preparative column chromatography was carried out
using Merck Kieselgel (0.060-0.200 mm). The reagents and materials used in these experiments were obtained
from Acros and Aldrich.
1
The 2D spectra were taken with a 4096 × 1024 database, which provided τ2max = 250 msec for H for
recording along the F2 axis and τ1max = 100 msec along the F1 axis. In order to improve the signal-to-noise ratio,
the data matrix prior to the Fourier transformation was twice supplemented with zeros and multiplied by the
cosine function. The mixing time in the 2D-NOESY was 1 sec.
The optical density in the biological tests carried out in a 96-well panel was determined on a
Tetretek Multiscan MCC/340 horizontal spectrophotometer.
3:1 Mixture of tert-Butyl Ester of 7Z-[2-(anti-Hydroxyimino)propylidene]-3-methyl-1,1-dioxo-
3-cephem-4-carboxylic Acid (anti-3) and tert-Butyl Ester of 7Z-[2-syn-Hydroxyimino)propylidene-
3-methyl-1,1-dioxo-3-cephem-4-carboxylic Acid (syn-3). Hydroxylamine hydrochloride (188 mg, 2.7 mmol)
and sodium acetate (224 mg, 2.7 mmol) were added to a solution of tert-butyl ester 7Z-acetyl-
methylene-3-methyl-1,1-dioxo-3-cephem-4-carboxylic acid (715 mg, 2.1 mmol) in methanol (30 ml). The
reaction mixture was stirred for 24 h at room temperature and evaporated at reduced pressure. The residue was
fractionated on a silica gel column. The fractions with Rf 0.34 (1:4 ethyl acetate–hexane) gave 495 mg (66%)
1
mixture of anti-3 and syn-3. H NMR spectrum, δ, ppm (J, Hz): anti-3) 1.46 (9H, s, C4H9); 1.92 (3H, s, CH3);
2.02 (3H, s, CH3C=N); 4.18-4.40 (2H, m, SO2CH2); 6.20 (1H, br. s, H-6); 6.88 (1H, d, 4J = 1.2, =CHC(Me)=N);
12.32 (1H, s, OH); syn-3) 1.46 (9H, s, C4H9); 1.92 (3H, s, CH3); 2.10 (3H, s, CH3C=N); 4.18-4.40 (2H, m,
4
SO2CH2); 6.19 (1H, s, H-6); 7.41 (1H, d, J = 1.2, =CHC(Me)=N); 11.32 (1H, s, OH). Found, %: C 50.65;
H 5.78; N 7.91. C15H20N2O6S. Calculated, %: C 50.55; H 5.66; N 7.87.
Preparation of tert-Butyl Esters of 7Z-[2-(Arylmethoxyimino)propylidene]-3-methyl-1,1-dioxo-
3-cephem-4-carboxylic Acids 6a-g. Method A. Arylmethoxyamine hydrochloride 2a-g (3.26 mmol) and
sodium acetate (267 mg, 3.26 mmol) were added to tert-butyl ester of 7Z-acetylmethylene-
3-methyl-1,1-dioxo-3-cephem-4-carboxylic acid (857 mg, 2.51 mmol) in methanol (30 ml). The reaction
mixture was stirred for 24 h at room temperature and evaporated at reduced pressure. The residue was
fractionated on a silica gel column to give cephems 6a-g.
2:1 Mixture of tert-Butyl Ester of 7Z-[2-anti-(Benzyloxyimino)propylidene]-3-methyl-1,1-dioxo-
3-cephem-4-carboxylic Acid (anti-6a) and tert-Butyl Ester of 7Z-[2-syn-(Benzyloxyimino)propylidene]-
3-methyl-1,1-dioxo-3-cephem-4-carboxylic Acid (syn-6a) was obtained in 89% yield using benzyloxyamine
hydrochloride. Mass spectrum, m/z: 469 [M+Na+]. Found, %: C 58.90; H 6.13; N 6.57. C22H26N2O6S.
Calculated, %: C 59.18; H 5.87; N 6.27.
The fractions with Rf 0.71 (1:1 ethyl acetate–hexane) gave anti-6a. 1H NMR spectrum, δ, ppm (J, Hz): 1.53
(9H, s, C4H9); 2.07 (3H, s, CH3); 2.10 (3H, s, CH3C=N); 3.64, 3.87 (2H, two d, AB system, 2J = 18, SO2CH2); 5.25
(2H, s, CH2Ph); 5.37 (1H, br. s, H-6); 6.90 (1H, d, 4J = 1.2, =CHC(Me)=N); 7.31-7.40 (5H, m, C6H5).
1
The fractions with Rf 0.63 (1:1 ethyl acetate–hexane) gave syn-6a. H NMR spectrum, δ, ppm (J, Hz):
4
1.53 (9H, s, C4H); 2.07 (3H, s, CH3); 2.19 (3H, s, CH3C=N); 3.64, 3.87 (2H, two d, AB system, J = 18,
4
SO2CH2); 5.18 (2H, s, CH2Ph); 5.28 (1H, br. s, H-6); 7.56 (1H, d, J = 1.2, =CHC(Me)=N); 7.31-7.40 (5H, m,
C6H5).
4:1 Mixture of tert-Butyl Ester of 7Z-[2-anti-(2-Bromobenzyloxyimino)propylidene]-3-methyl-
1,1-dioxo-3-cephem-4-carboxylic Acid (anti-6b) and tert-Butyl Ester of 7Z-[2-syn-(2-Bromobenzyl-
oxyimino)propylidene]-3-methyl-1,1-dioxo-3-cephem-4-carboxylic Acid (syn-6b) was obtained in 75% yield
using 2-bromobenzyloxyamine hydrochloride.
1
The fractions with Rf 0.61 (1:1 ethyl acetate–hexane) gave anti-6b in 60% yield. H NMR spectrum, δ,
ppm (J, Hz): 1.54 (9H, s, C4H9); 2.08 (3H, s, CH3); 2.14 (3H, s, CH3C=N); 3.65, 3.89 (2H, two d, AB system,
235

4
2J = 18, SO2CH2); 5.35 (2H, s, CH2Ph); 5.39 (1H, br. s, H-6); 6.90 (1H, d, J = 1.2, =CHC(Me)=N); 7.10-7.44
(3H, m, H-4, H-5, H-6 C6H4); 7.57 (1H, d, 3J = 7.3, H-3 C6H4). Mass spectrum, m/z: 547/549 [M+Na+].
1
The fractions with Rf 0.53 (1:1 ethyl acetate–hexane) gave syn-6b in 15% yield. H NMR spectrum, δ,
ppm (J, Hz): 1.54 (9H, s, C4H9); 2.08 (3H, s, CH3); 2.20 (3H, s, CH3C=N); 3.65, 3.89 (2H, two d, AB system,
2J = 18, SO2CH2); 5.27 (2H, s, CH2Ph); 5.35 (1H, br. s, H-6); 7.11-7.44 (3H, m, H-4, H-5, H-6 C6H4); 7.56 (1H,
d, 3J = 7.8, H-3 C6H4); 7.62 (1H, d, 4J = 1.2, =CHC(Me)=N). Mass spectrum: 547/549 [M+Na+].
2:3 Mixture of tert-Butyl Ester of 7Z-[2-anti-(3-Bromobenzyloxyimino)propylidene]-3-methyl-
1,1-dioxo-3-cephem-4-carboxylic Acid (anti-6c) and tert-Butyl Ester of 7Z-[2-syn-(3-bromobenzyl-
oxyimino)propylidene]-3-methyl-1,1-dioxo-3-cephem-4-carboxylic acid (syn-6c) was obtained in 64% yield
using 3-bromobenzyloxyamine hydrochloride. The fractions with Rf 0.70 (1:1 ethyl acetate–hexane) gave an
isomer mixture of anti-6c and syn-6c. 1H NMR spectrum, δ, ppm (J, Hz): anti-6c) 1.53 (9H, s, C4H9); 2.06 (3H,
s, CH3); 2.10 (3H, s, CH3C=N); 3.64, 3.89 (2H, two d, AB system, 2J = 18, SO2CH2); 5.20 (2H, s, CH2Ph); 5.37
(1H, br. s, H-6); 6.87 (1H, d, 4J = 1.2, =CHC(Me)=N); 7.16-7.53 (4H, m, C6H4); syn-6c) 1.53 (9H, s, C4H9); 2.06
(3H, s, CH3); 2.18 (3H, s, CH3C=N); 3.64, 3.87 (2H, two d, AB system, 2J = 18, SO2CH2); 5.12 (2H, s, CH2Ph);
4
5.31 (1H, br. s, H-6); 7.16-7.53 (4H, m, C6H4); 7.54 (1H, d, J = 1.2, =CHC(Me)=N). Found, %: C 49.99;
H 4.55; N 5.16. C22H25BrN2O6S. Calculated, %: C 50.29; H 4.80; N 5.33.
Mixture of tert-Butyl Ester of 7Z-[2-anti-(4-Bromobenzyloxyimino)propylidene]-3-methyl-
1,1-dioxo-3-cephem-4-carboxylic Acid (anti-6d) and tert-Butyl Ester of 7Z-[2-syn-(4-bromobenzyl-
oxyimino)propylidene]-3-methyl-1,1-dioxo-3-cephem-4-carboxylic acid (syn-6d) was obtained in 62% yield
using 4-bromobenzyloxyamine. The fractions with Rf 0.70 (1:1 ethyl acetate–hexane) gave an isomer mixture of
anti-6d and syn-6d. 1H NMR spectrum, δ, ppm (J, Hz): anti-6d) 1.53 (9H, s, C4H9); 2.07 (3H, s, CH3); 2.09 (3H,
2
s, CH3C=N); 3.65, 3.90 (2H, two d, AB system, J = 18, SO2CH2); 5.19 (2H, s, CH2Ph); 5.39 (1H, br. s, H-6);
4
3
3
6.87 (1H, d, J = 1.5, =CHC(Me)=N); 7.23 (2H, d, J = 8.8, H-2, H-6 C6H4); 7.48 (2H, d, J = 8.8, H-3,
H-5 C6H4); syn-6d) 1.53 (9H, s, C4H9); 2.07 (3H, s, CH3); 2.17 (3H, s, CH3C=N); 3.65, 3.90 (2H, two d,
AB system, 2J = 18, SO2CH2); 5.11 (2H, s, CH2Ph); 5.34 (1H, br. s, H-6); 7.23 (2H, d, 3J = 8.8, H-2, H-6 C6H4);
7.48 (2H, d, 3J = 8.8, H-3, H-5 C6H4); 7.54 (1H, d, 4J = 1.4, =CHC(Me)=N). Mass spectrum, m/z: 548 [M+Na+].
Found, %: C 50.31; H 4.70; N 5.33. C22H25BrN2O6S. Calculated, %: C 50.29; H 4.80; N 5.33.
Mixture of tert-Butyl Ester of 3-Methyl-1,1-dioxo-7Z-[2-anti-(2-chlorobenzyloxyimino)propyli-
dene]-3-cephem-4-carboxylic Acid (anti-6e) and tert-Butyl Ester of 3-Methyl-1,1-dioxo-7Z-[2-syn-
(2-chlorobenzyloxyimino)propylidene]-3-cephem-4-carboxylic Acid (syn-6e) was obtained using
2-chlorobenzyloxyamine hydrochloride.
The fractions with Rf 0.63 (1:1 ethyl acetate–hexane) gave anti-6e in 43% yield; mp 190-192°C.
1H NMR spectrum, δ, ppm (J, Hz): 1.53 (9H, s, C4H9); 2.07 (3H, s, CH3); 2.13 (3H, s, CH3C=N); 3.64, 3.88 (2H,
2
4
two d, AB system, J = 18, SO2CH2); 5.37 (2H, s, CH2Ph); 5.40 (1H, br. s, H-6); 6.89 (1H, d, J = 1.3,
=CHC(Me)=N); 7.22-7.43 (4H, m, C6H4). Mass spectrum, m/z: 503 [M+Na+].
The fractions with Rf 0.57 (1:1 ethyl acetate–hexane) gave syn-6e in 30% yield; mp 168-170°C.
1H NMR spectrum, δ, ppm (J, Hz): 1.53 (9H, s, C4H9); 2.07 (3H, s, CH3); 2.19 (3H, s, CH3C=N); 3.66, 3.90 (2H,
two d, AB system, 2J = 18, SO2CH2); 5.30 (2H, s, CH2Ph); 5.33 (1H, br. s, H-6); 7.22-7.44 (4H, m, C6H4); 7.61
(1H, d, 4J = 1.4, =CHC(Me)=N). Mass spectrum, m/z: 503 [M+Na+].
Mixture of tert-Butyl Ester of 3-Methyl-1,1-dioxo-7Z-[2-anti-(2-fluorobenzyloxyimino)propyli-
dene]-3-cephem-4-carboxylic Acid (anti-6f) and tert-Butyl Ester of 3-Methyl-1,1-dioxo-7Z-[2-syn-
(2-fluorobenzyloxyimino)propylidene]-3-cephem-4-carboxylic Acid (syn-6f) was obtained using
2-fluorobenzyloxyamine.
The fractions with Rf 0.63 (1:1 ethyl acetate–hexane) gave anti-6f in 60% yield; mp 162-166°C.
1H NMR spectrum, δ, ppm (J, Hz): 1.53 (9H, s, C4H9); 2.06 (3H, s, CH3); 2.08 (3H, s, CH3C=N); 3.64, 3.88 (2H,
2
4
two d, AB system, J = 18, SO2CH2); 5.32 (2H, s, CH2Ph); 5.41 (1H, br. s, H-6); 6.89 (1H, d, J = 1.3,
=CHC(Me)=N); 6.99-7.44 (4H, m, C6H4). Mass spectrum: 487 [M+Na+].
236

1
The fractions with Rf 0.54 (1:1 ethyl acetate–hexane) give syn-6f in 24% yield; mp 167-172°C. H NMR
spectrum, δ, ppm (J, Hz): 1.54 (9H, s, C4H9); 2.07 (3H, s, CH3); 2.18 (3H, s, CH3C=N); 3.65, 3.89 (2H, two d,
AB system, 2J = 18, SO2CH2); 5.24 (2H, s, CH2Ph); 5.31 (1H, br. s, H-6); 6.99-7.45 (4H, m, C6H4); 7.56 (1H, d,
4J = 1.3, =CHC(Me)=N). Mass spectrum, m/z: 487 [M+Na+].
Mixture of tert-Butyl Ester of 3-Methyl-1,1-dioxo-7Z-[2-anti-(2-trifluoromethylbenzyloxyimino)-
propylidene]-3-cephem-4-carboxylic Acid (anti-6g) and tert-Butyl Ester of 3-Methyl-1,1-dioxo-
7Z-[2-syn-(2-trifluoromethylbenzyloxyimino)propylidene]-3-cephem-4-carboxylic Acid (syn-6g) was
obtained using 2-trifluoromethylbenzyloxyamine.
The fractions with Rf 0.66 (1:1 ethyl acetate–hexane) gave anti-6f in 48% yield; mp 152-156°C.
1H NMR spectrum, δ, ppm (J, Hz): 1.53 (9H, s, C4H9); 2.06 (3H, s, CH3); 2.12 (3H, s, CH3C=N); 3.64, 3.88 (2H,
2
4
two d, AB system, J = 18, SO2CH2); 5.40 (1H, br. s, H-6); 5.46 (2H, s, CH2Ph); 6.89 (1H, d, J = 1.4,
3
=CHC(Me)=N); 7.35-7.48 (2H, m, H-5, H-6 C6H4); 7.54 (1H, m, H-4 C6H4); 7.66 (1H, d, J = 7.3, H-3 C6H4).
Mass spectrum, m/z: 537 [M+Na+].
The fractions with Rf 0.54 (1:1 ethyl acetate–hexane) gave syn-6g in 31% yield; mp 155-158°C.
1H NMR spectrum, δ, ppm (J, Hz): 1.54 (9H, s, C4H9); 2.08 (3H, s, CH3); 2.20 (3H, s, CH3C=N); 3.66, 3.90 (2H,
2
two d, AB system, J = 18, SO2CH2); 5.33 (1H, br. s, H-6); 5.39 (2H, s, CH2Ph); 7.35-7.49 (2H, m, H-5,
4
3
H-6 C6H4); 7.51-7.59 (1H, m, H-4 C6H4); 7.62 (1H, d, J = 1.5, =CHC(Me)=N); 7.66 (1H, d, J = 7.8,
H-3 C6H4). Mass spectrum, m/z: 537 [M+Na+].
Preparation of tert-Butyl Esters of 7Z-[2-(Aryl- and 7Z-(2-Pyrimidylmethoxyimino)propylidene]-
3-methyl-1,1-dioxo-3-cephem-4-carboxylic Acids 6h-k. Method B. 60% NaH (34 mg, 0.85 mmol) and
arylmethyl bromide 5h,i or pyridylmethyl bromide 5j,k (0.20 mmol) were added to a solution of mixture of
anti and syn isomers of the tert-butyl ester of 7Z-[2-(hydroxyimino)propylidene]-3-methyl-1,1-dioxo-3-cephem-
4-carboxylic acid (60 mg, 0.17 mmol) in DMF (5 ml) at 0°C. The mixture was warmed to room temperature,
stirred for 3 h, then diluted with 50 ml ethyl acetate, and washed with two portions of 5% aqueous NaCl
(50 ml). The organic layer was dried over anhydrous sodium sulfate, and evaporated. The residue was
fractionated on a silica gel column to give cephems 6h-k.
1:6 Mixture of tert-Butyl Ester of 3-Methyl-1,1-dioxo-7Z-[2-anti-(2,6-Dichlorobenzyloxyimino)-
propylidene]-3-cephem-4-carboxylic Acid (anti-6h) and tert-Butyl Ester of 3-Methyl-1,1-dioxo-
7Z-[2-syn-(2,6-dichlorobenzyloxyimino)propylidene]-3-cephem-4-carboxylic Acid (syn-6h) was obtained
using 2,6-dichlorobenzyl bromide. The fractions with Rf 0.22 (1:4 ethyl acetate–hexane) gave the ester mixture
1
in 55% yield. H NMR spectrum, δ, ppm (J, Hz): anti-6h) 1.53 (9H, s, C4H9); 2.05 (3H, s, CH3); 2.10 (3H, s,
CH3C=N); 3.63, 3.88 (2H, two d, AB system, 2J = 18, SO2CH2); 5.30 (2H, s, CH2Ph); 5.53 (1H, br. s, H-6); 6.90
4
(1H, d, J = 1.4, =CHC(Me)=N); 7.16-7.36 (3H, m, C6H3); syn-6h) 1.53 (9H, s, C4H9); 2.07 (3H, s, CH3); 2.19
2
(3H, s, CH3C=N); 3.64, 3.88 (2H, two d, AB system, J = 18, SO2CH2); 5.30 (2H, s, CH2Ph); 5.53 (1H, br. s,
4
H-6); 7.16-7.36 (3H, m, C6H3); 7.54 (1H, d, J = 1.4, =CHC(Me)=N). Found, %: C 51.35; H 4.72; N 5.36.
C22H24Cl2N2O6S. Calculated, %: C 51.27; H 4.69; N 5.44.
4:1 Mixture of tert-Butyl Ester of 3-Methyl-1,1-dioxo-7Z-[2-anti-(3,4-dichlorobenzyloxyimino)-
propylidene]-3-cephem-4-carboxylic Acid (anti-6i) and tert-Butyl Ester of 3-Methyl-1,1-dioxo-
7Z-[2-syn-(3,4-dichlorobenzyloxyimino)propylidene]-3-cephem-4-carboxylic Acid (syn-6i) was obtained
using 3,4-dichlorobenzyl bromide. The fractions with Rf 0.20 (4:1 ethyl acetate–hexane) gave the ester mixture
1
in 48% yield. H NMR spectrum, δ, ppm (J, Hz): anti-6i) 1.53 (9H, s, C4H9); 2.07 (3H, s, CH3); 2.09 (3H, s,
CH3C=N); 3.64, 3.87 (2H, two d, AB system, 2J = 18, SO2CH2); 5.09 (2H, s, CH2Ph); 5.18 (1H, br. s, H-6); 6.85
4
(1H, d, J = 1.4, =CHC(Me)=N); 7.14-7.25 (1H, m, H-2 C6H3); 7.37-7.54 (2H, m, H-5, H-6 C6H3); syn-6i) 1.53
(9H, s, C4H9); 2.07 (3H, s, CH3); 2.17 (3H, s, CH3C=N); 3.64, 3.87 (2H, two d, 2J = 18, SO2CH2); 5.31 (1H, br.
s, H-6); 5.35 (2H, s, CH2Ph); 7.14-7.25 (1H, m, H-2 C6H3); 7.37-7.54 (2H, m, H-5, H-6 C6H3); 7.54 (1H, d,
4J = 1.4, =CHC(Me)=N). Found, %: C 51.42; H 4.75; N 5.50. C22H24Cl2N2O6S. Calculated, %: C 51.27; H 4.69;
N 5.44.
237

2:3 Mixture of tert-Butyl Ester of 3-Methyl-1,1-dioxo-7Z-[2-anti-(3-pyridylmethoxyimino)-propylidene]-
3-cephem-4-carboxylic Acid (anti-6j) and tert-Butyl Ester of 3-Methyl-1,1-dioxo-7Z-[2-syn-(3-pyridyl-
methoxyimino)propylidene]-3-cephem-4-carboxylic acid (syn-6j) was obtained using 3-pyridylmethyl
1
chloride. The fractions with Rf 0.08 (3:1 ethyl acetate–hexane) gave the ester mixture in 15% yield. H NMR
spectrum, δ, ppm (J, Hz): anti-6j) 1.53 (9H, s, C4H9); 2.08 (3H, s, CH3); 2.08 (3H, s, CH3C=N); 3.64, 3.96 (2H,
2
4
two d, AB system, J = 18, SO2CH2); 5.19 (2H, s, CH2Py); 5.28 (1H, br. s, H-6); 6.82 (1H, d, J = 1.0,
=CHC(Me)=N); 7.30-7.45 (1H, m, H-5 Py); 7.70-7.85 (1H, m, H-4 Py); 8.50-8.80 (2H, br. s, H-2, H-6 Py);
syn-6j) 1.53 (9H, s, C4H9); 2.08 (3H, s, CH3); 2.17 (3H, s, CH3C=N); 3.64, 3.90 (2H, two d, AB system, 2J = 18,
4
SO2CH2); 5.30 (1H, br. s, H-6); 5.37 (2H, s, CH2Py); 7.30-7.45 (1H, m, H-5 Py); 7.52 (1H, d, J = 1.0,
=CHC(Me)=N); 7.70-7.85 (1H, m, H-4 Py); 8.50-8.80 (2H, br. s, H-2, H-6 Py). Found, %: C 56.51; H 5.73; N
9.42. C21H25N3O6S. Calculated, %: C 56.36; H 5.63; N 9.39.
1:2 Mixture of tert-Butyl Ester of 3-methyl-1,1-dioxo-7Z-[2-anti-(4-pyridylmethoxyimino)propyli-
dene]-3-cephem-4-carboxylic Acid (anti-6k) and tert-Butyl Ester of 3-Methyl-1,1-dioxo-7Z-[2-syn-
(4-pyridylmethoxyimino)propylidene]-3-cephem-4-carboxylic Acid (syn-6k) was obtained using 4-pyridyl-
methyl chloride.
1
The fractions with Rf 0.08 (3:1 ethyl acetate–hexane) gave the ester mixture in 15% yield. H NMR
spectrum, δ, ppm (J, Hz): anti-6k) 1.53 (9H, s, C4H9); 2.08 (3H, s, CH3); 2.15 (3H, s, CH3C=N); 3.65, 3.85 (2H,
3
4
two d, AB system, J = 18, SO2CH2); 5.21 (1H, br. s, H-6); 5.36 (2H, s, CH2Py); 6.84 (1H, d, J = 1.0,
=CHC(Me)=N); 7.33 (2H, br. s, H-3, H-5 Py); 8.61 (2H, br. s, H-2, H-6 Py); syn-6k) 1.53 (9H, s, C4H9); 2.08
(3H, s, CH3); 2.20 (3H, s, CH3C=N); 3.66, 3.91 (2H, two d, AB system, 2J = 18, SO2CH2); 5.21 (1H, br. s, H-6);
4
5.36 (2H, s, CH2Py); 7.33 (2H, br. s, H-3, H-5 Py); 7.60 (1H, d, J = 1.0, =CHC(Me)=N); 8.61 (2H, br. s, H-2,
H-6 Py). Found, %: C 56.47; H 5.70; N 9.45. C21H25N3O6S. Calculated, %: C 56.36; H 5.63; N 9.39.
2:1 Mixture of tert-Butyl Ester of 7Z-[2-anti-(2-Bromobenzyloxyimino)propylidene]-3-methyl-
3-cephem-4-carboxylic Acid (anti-8b) and tert-Butyl Ester of 7Z-[2-syn-(2-Bromobenzyloxyimino)-
propylidene]-3-methyl-3-cephem-4-carboxylic Acid (syn-8b) was obtained by Method A from the tert- butyl
ester of 7Z-acetylmethylene-3-methyl-3-cephem-4-carboxylic acid 7 and 2-bromobenzyloxyamine
hydrochloride. Found, %: C 53.67; H 5.20; N 5.75. C22H25BrN2O4S. Calculated, %: C 53.55; H 5.11; N 5.68.
1
The fractions with Rf 0.57 (1:3 ethyl acetate–hexane) gave anti-8b in 41% yield. H NMR spectrum, δ,
ppm (J, Hz): 1.53 (9H, s, C4H9); 2.03 (3H, s, CH3); 2.07 (3H, s, CH3C=N); 3.14, 3.50 (2H, two d, AB system,
2J = 18, SCH2); 5.26 (1H, br. s, H-6); 5.31 (2H, s, CH2Ph); 6.68 (1H, d, 4J = 1.4, =CHC(Me)=N); 7.12-7.43 (3H,
m, H-4, H-5, H-6 C6H4); 7.56 (1H, d, 3J = 7.8, H-3 C6H4).
1
The fractions with Rf 0.48 (1:3 ethyl acetate–hexane) gave syn-8b in 21% yield. H NMR spectrum, δ,
ppm (J, Hz): 1.53 (9H, s, C4H9); 2.03 (3H, s, CH3); 2.13 (3H, s, CH3C=N); 3.18, 3.53 (2H, two d, AB system,
2J = 18, SCH2); 5.24 (2H, s, CH2Ph); 5.27 (1H, br. s, H-6); 7.10-7.41 (3H, m, H-4, H-5, H-6 C6H4); 7.45 (1H, d,
4J = 1.4, =CHC(Me)=N); 7.55 (1H, d, 3J = 8.8, H-3 C6H4).
7Z-[2-anti-(Benzyloxyimino)propylidene]-3-methyl-1,1-dioxo-3-cephem (anti-10a) and 7Z-[2-syn-
(Benzyloxyimino)propylidene]-3-methyl-1,1-dioxo-3-cephem (syn-10a) were obtained by Method A from
7Z-acetylmethylene-3-methyl-1,1-dioxo-3-cephem and benzyloxyamine hydrochloride in 47% yield. Found, %:
C 59.03; H 5.32; N 8.15. C17H18N2O4S. Calculated, %: C 58.94; H 5.24; N 8.09.
1
The fractions with Rf 0.68 (1:1 ethyl acetate–hexane) gave anti-10a in 28% yield. H NMR spectrum, δ,
2
ppm (J, Hz): 1.80 (3H, s, CH3); 2.11 (3H, s, CH3C=N); 3.46, 3.96 (2H, two d, AB system, J = 17.6, SO2CH2);
4
5.25 (2H, s, CH2Ph); 5.38 (1H, br. s, H-6); 6.55 (1H, br. s, H-4); 6.92 (1H, d, J = 1.5, =CHC(Me)=N);
7.31-7.41 (5H, m, C6H5).
1
The fractions with Rf 0.60 (1:1 ethyl acetate–hexane) gave syn-10a in 19% yield. H NMR spectrum, δ,
2
ppm (J, Hz): 1.80 (3H, s, CH3); 2.19 (3H, s, CH3C=N); 3.46, 3.93 (2H, two d, AB system, J = 18.1, SO2CH2);
5.17 (2H, s, CH2Ph); 5.26 (1H, br. s, H-6); 6.55 (1H, br. s, H-4); 7.31-7.41 (5H, m, C6H5); 7.54 (1H, d, 4J = 1.4,
=CHC(Me)=N).
238

Mixture of 7Z-[2-anti-(2-Bromobenzyloxyimino)propylidene]-3-methyl-1,1-dioxo-3-cephem (anti-10b)
and 7Z-[2-syn-(2-bromobenzyloxyimino)propylidene]-3-methyl-1,1-dioxo-3-cephem (syn-10b) was obtained
from 7Z-acetylmethylene-3-methyl-1,1-dioxo-3-cephem and 2-bromobenzyloxyamine hydrochloride.
1
The fractions with Rf 0.48 (1:1 ethyl acetate–hexane) gave anti-10b in 34% yield. H NMR spectrum, δ,
2
ppm (J, Hz): 1.80 (3H, s, CH3); 2.15 (3H, s, CH3C=N); 3.45, 3.95 (2H, two d, AB system, J = 18.1, SO2CH2);
4
5.35 (2H, s, CH2Ph); 5.39 (1H, br. s, H-6); 6.54 (1H, br. s, H-4); 6.91 (1H, d, J = 1.4, =CHC(Me)=N);
7.12-7.41 (3H, m, H-4, H-5, H-6 C6H4); 7.56 (1H, d, 3J = 7.9, H-3 C6H4). Mass spectrum: 447/449 [M+Na+].
1
The fractions with Rf 0.37 (1:1 ethyl acetate–hexane) gave syn-10b in 24% yield. H NMR spectrum, δ,
2
ppm (J, Hz): 1.81 (3H, s, CH3); 2.20 (3H, s, CH3C=N); 3.46, 3.96 (2H, two d, AB system, J = 17.6, SO2CH2);
5.27 (2H, s, CH2Ph); 5.32 (1H, br. s, H-6); 6.56 (1H, br. s, H-4); 7.12-7.41 (3H, m, H-4, H-5, H-6 C6H4); 7.56
(1H, d, 3J = 8.3, H-3 C6H4); 7.60 (1H, d, 4J = 1.0, =CHC(Me)=N). Mass spectrum, m/z: 447/449 [M+Na+].
Mixture of 7Z-[2-anti-(3-Bromobenzyloxyimino)propylidene]-3-methyl-1,1-dioxo-3-cephem (anti-10c)
and 7Z-[2-syn-(3-Bromobenzyloxyimino)propylidene]-3-methyl-1,1-dioxo-3-cephem (syn-10c) was obtained
in 60% yield by Method A from 7Z-acetylmethylene-3-methyl-1,1-dioxo-3-cephem and 3-bromo-
benzyloxyamine hydrochloride. Found, %: C 48.21; H 4.11; N 6.64. C17H17BrN2O4S. Calculated, %: C 48.01;
H 4.03; N 6.59.
1
The fractions with Rf 0.48 (1:1 ethyl acetate–hexane) gave anti-10c in 37% yield. H NMR spectrum, δ,
2
ppm (J, Hz): 1.80 (3H, s, CH3); 2.11 (3H, s, CH3C=N); 3.46, 3.96 (2H, two d, AB system, J = 17.6, SO2CH2);
4
5.21 (2H, s, CH2Ph); 5.36 (1H, br. s, H-6); 6.55 (1H, br. s, H-4); 6.88 (1H, d, J = 1.4, =CHC(Me)=N);
7.17-7.32 (2H, m, H-5, H-6 C6H4); 7.44 (1H, d, 3J = 7.4, H-4 C6H4); 7.51 (1H, s, H-2 C6H4).
1
The fractions with Rf 0.36 (1:1 ethyl acetate–hexane) gave syn-10c in 23% yield. H NMR spectrum, δ,
2
ppm (J, Hz): 1.80 (3H, s, CH3); 2.18 (3H, s, CH3C=N); 3.46, 3.95 (2H, two d, AB system, J = 18.1, SO2CH2);
5.11 (2H, s, CH2Ph); 5.27 (1H, s, H-6); 6.55 (1H, br. s, H-4); 7.16-7.32 (2H, m, H-5, H-6 C6H4); 7.44 (1H, d,
3J = 7.3, H-4 C6H4); 7.49 (1H, br. s, =CHC(Me)=N); 7.51 (1H, s, H-2 C6H4).
Mixture of 7Z-(2-anti-(4-Bromobenzyloxyimino)propylidene]-3-methyl-1,1-dioxo-3-cephem (anti-10d)
and 7Z-(2-syn-(4-Bromobenzyloxyimino)propylidene]-3-methyl-1,1-dioxo-3-cephem (syn-10d) was
obtained in 61% yield from 7Z-acetylmethylene-3-methyl-1,1-dioxo-3-cephem and 4-bromobenzyloxyamine
hydrochloride. Found, %: C 48.15; H 4.17; N 6.63. C17H17BrN2O4S. Calculated %: C 48.01; H 4.03; N 6.59.
1
The fractions with Rf 0.48 (1:1 ethyl acetate–hexane) gave anti-10d in 36% yield. H NMR spectrum, δ,
2
ppm (J, Hz): 1.80 (3H, s, CH3); 2.10 (3H, s, CH3C=N); 3.45, 3.94 (2H, two d, AB system, J = 17.6, SO2CH2);
5.19 (2H, s, CH2Ph); 5.36 (1H, br. s, H-6); 6.55 (1H, br. s, H-4); 6.88 (1H, d, 4J = 1.5, =CHC(Me)=N); 7.23 (2H,
d, 3J = 8.3, H-2, H-6 C6H4); 7.48 (2H, d, 3J = 8.3, H-3, H-5 C6H4).
1
The fractions with Rf 0.36 (1:1 ethyl acetate–hexane) gave syn-10d in 25% yield. H NMR spectrum, δ,
2
ppm (J, Hz): 1.81 (3H, s, CH3); 2.18 (3H, s, CH3C=N); 3.46, 3.94 (2H, two d, AB system, J = 17.6, SO2CH2);
5.11 (2H, s, CH2Ph); 5.27 (1H, br. s, H-6); 6.55 (1H, br. s, H-4); 7.22 (2H, d, 3J = 8.3, H-2, H-6 C6H4); 7.48 (2H,
d, 3J = 8.3, H-3, H-5 C6H4); 7.53 (1H, d, 4J = 1.4, =CHC(Me)=N).
Mixture of 3-Methyl-1,1-dioxo-7Z-[2-anti-(2-chlorobenzyloxyimino)propylidene]-3-cephem (anti-10e)
and 3-Methyl-1,1-dioxo-7Z-[2-syn-(2-chlorobenzyloxyimino)propylidene-3-cephem (syn-10e) was obtained
by Method A from 7Z-acetylmethylene-3-methyl-1,1-dioxo-3-cephem and 2-chlorobenzyloxyamine hydro-
chloride.
1
The fractions with Rf 0.43 (1:1 ethyl acetate–hexane) gave anti-10e in 64% yield. H NMR spectrum, δ,
2
ppm (J, Hz): 1.79 (1H, s, CH3); 2.14 (3H, s, CH3C=N); 3.45, 3.95 (2H, two d, AB system, J = 18, SO2CH2);
4
5.36 (2H, s, CH2Ph); 5.40 (1H, br. s, H-6); 6.54 (1H, br. s, H-4); 6.90 (1H, d, J = 1.5, =CHC(Me)=N);
7.22-7.42 (4H, m, C6H4). Mass spectrum, m/z: 381/383 [M+].
1
The fractions with Rf 0.31 (1:1 ethyl acetate–hexane) gave syn-10e in 24% yield. H NMR spectrum, δ,
ppm (J, Hz): 1.81 (3H, s, CH3); 2.20 (3H, s, CH3C=N); 3.46, 3.95 (2H, two d, AB system, 2J = 18, SO2CH2);
239

5.29 (2H, s, CH2Ph); 5.37 (1H, br. s, H-6); 6.56 (1H, br. s, H-4); 7.22-7.42 (4H, m, C6H4); 7.58 (1H, d, 4J = 1.4,
=CHC(Me)=N). Mass spectrum, m/z: 381/383 [M+].
Mixture of 3-Methyl-1,1-dioxo-7Z-[2-anti-(2-fluorobenzyloxyimino)propylidene]-3-cephem (anti-10f)
and 3-methyl-1,1-dioxo-7Z-[2-syn-(2-fluorobenzyloxyimino)propylidene]-3-cephem (syn-10f) was obtained
by Method A from 7Z-acetylmethylene-3-methyl-1,1-dioxo-3-cephem and 2-fluorobenzyloxyamine hydro-
chloride.
The fractions with Rf 0.40 (1:1 ethyl acetate–hexane) gave anti-10f in 73% yield; mp 120-122°C.
1H NMR spectrum, δ, ppm (J, Hz): 1.79 (3H, s, CH3); 2.09 (3H, s, CH3C=N); 3.44, 3.95 (2H, two d, AB system,
4
2J = 18, SO2CH2); 5.32 (2H, s, CH2Ph); 5.40 (1H, br. s, H-6); 6.54 (1H, br. s, H-4); 6.90 (1H, d, J = 1.5,
=CHC(Me)=N); 6.99-7.44 (4H, m, C6H4). Mass spectrum, m/z: 387/388 [M+Na+].
The fractions with Rf 0.33 (1:1 ethyl acetate–hexane) gave syn-10f in 24% yield; mp 122-124°C.
1H NMR spectrum, δ, ppm (J, Hz): 1.80 (3H, s, CH3); 2.19 (3H, s, CH3C=N); 3.46, 3.95 (2H, two d, AB system,
2J = 18, SO2CH2); 5.24 (1H, s, CH2Ph); 5.29 (1H, br. s, H-6); 6.55 (1H, br. s, H-4); 6.99-7.44 (4H, m, C6H4);
7.53 (1H, d, 4J = 1.3, =CHC(Me)=N). Mass spectrum, m/z: 387/388 [M+Na+].
Preparation of tert-Butyl Esters of 7Z-[2-(Arylmethoxyimino)propylidene]-2-(N,N-dimethyl-
aminomethylene)-3-methyl-1,1-dioxo-3-cephem-4-carboxylic Acids 11a-f. Typical procedure. A mixture of
oxalyl chloride (234 µl, 2.687 mmol) and DMF (209 µl, 2.687 mmol) in acetonitrile (15 ml) was added to a
stirred suspension of
tert-butyl ester
of 7Z-[2-(arylmethoxyimino)-propylidene]-3-methyl-1,1-dioxo-
3-cephem-4-carboxylic acid (0.672 mmol) in acetonitrile (15 ml) at -5°C in an argon atmosphere. The reaction
mixture was stirred for 1.5 h at 0°C, neutralized by adding dry pyridine (217 µl, 2.687 mmol), diluted by adding
100 ml 5% aqueous NaCl, and extracted with two 30-ml portions of ethyl acetate. The organic phase was dried
over anhydrous Na2SO4. The solvent was evaporated at reduced pressure. The residue was fractionated on a
silica gel column with ethyl acetate–hexane as the eluent (the solvent ratio was varied from 1:10 to 1:2) to give
cephems 11a-f.
tert-Butyl Ester of 7Z-[2-anti-(Benzyloxyimino)propylidene]-2(E/Z)-(N,N-dimethylaminomethylene)-
3-methyl-1,1-dioxo-3-cephem-4-carboxylic Acid (anti-11a) and tert-Butyl Ester of 7Z-[2-syn-(Benzyl-
oxyimino)propylidene]-2(E/Z)-(N,N-dimethylaminomethylene)-3-methyl-1,1-dioxo-3-cephem-4-carboxylic
acid (syn-11a) were obtained from a mixture of the anti and syn isomers of the tert-butyl ester of
7Z-[2-(benzyloxyimino)propylidene]-3-methyl-1,1-dioxo-3-cephem-4-carboxylic acid.
The fractions with Rf 0.60 (1:1 ethyl acetate–hexane) gave 195 mg (58%) of a 4:1 mixture of 2E- and 2Z-
isomers of anti-11a; mp 96-98°C. 1H NMR spectrum, δ, ppm (J, Hz): 1.53 (9H, s, C4H9); 2.14 (3H, s, CH3); 2.23
(3H, s, CH3C=N); 3.04 (4.8H, s, N(CH3)2 2E-isomer); 3.32 (1.2H, s, N(CH3)2 2Z-isomer); 5.23 (2H, s, CH2Ph);
4
5.42 (1H, J = 1.0, H-6); 6.85 (1H, br. s, =CHC(Me)=N); 6.93 (0.2H, s, =CHNMe2 2Z-isomer); 7.23 (0.8H, s,
=CHNMe2 2E-isomer); 7.31-7.40 (5H, m, C6H5). Mass spectrum, m/z: 399 [M+-CO2C(CH3)3].
The fractions with Rf 0.48 (1:1 ethyl acetate–hexane) gave 88 mg (26%) of a 4:1 mixture of a
1
2E/2Z-isomers of syn-11a; mp 90-92°C. H NMR spectrum, δ, ppm (J, Hz): 1.54 (9H, s, C4H9); 2.21 (3H, s,
CH3); 2.22 (3H, s, CH3C=N); 3.05 (4.8H, s, N(CH3)2 2E-isomer); 3.33 (1.2H, s, N(CH3)2 2Z-isomer); 5.16 (2H,
s, CH2Ph); 5.34 (1H, s, H-6); 6.95 (0.2H, s, =CHNMe2 2Z-isomer); 7.25 (0.8H, s, =CHNMe2 2E-isomer);
7.31-7.40 (5H, m, C6H5); 7.50 (1H, s, =CHC(Me)=N). Mass spectrum, m/z: 399 [M+-CO2C(CH3)3].
tert-Butyl Ester of 7Z-[2-anti-(2-Bromobenzyloxyimino)propylidene]-2(E/Z)-(N,N-dimethylamino-
methylene)-3-methyl-1,1-dioxo-3-cephem-4-carboxylic Acid (anti-11b) and tert-Butyl Ester of 7Z-[2-syn-
(2-bromobenzyloxyimino)propylidene]-2(E/Z)-(N,N-dimethylaminomethylene)-3-methyl-1,1-dioxo-3-cephem-
4-carboxylic acid (syn-11b) were obtained using a mixture of the anti and syn isomers of the tert-butyl ester of
7Z-[2-(2-bromobenzyloxyimino]propylidene-2(E/Z)-3-methyl-1,1-dioxo-3-cephem-4-carboxylic acid.
The fractions with Rf 0.78 (2:1 ethyl acetate–hexane) gave 33 mg (30%) of a 4:1 mixture of 2E- and
1
2Z-isomers of anti-11b; mp 134-136°C. H NMR spectrum, δ, ppm (J, Hz): 1.53 (9H, s, C4H9); 2.18 (3H, s,
CH3); 2.23 (3H, s, CH3C=N); 3.05 (4.8H, s, N(CH3)2) 2E-isomer); 3.32 (1.2H, s, N(CH3)2 2Z-isomer); 5.32 (2H,
240

s, CH2Ph); 5.42 (1H, d, 4J = 1.0, H-6); 6.84 (1H, d, 4J = 1.0, =CHC(Me)=N); 6.93 (0.2H, s,
3
=CHNMe2 2Z- isomer); 7.09-7.44 (3.8H, m, =CHMe2 2E-isomer, H-4, H-5, H-6 C6H4); 7.56 (1H, d, J = 7.8,
H-3 C6H4). Mass spectrum, m/z: 602/604 [M+-CO2C(CH3)3].
The fractions with Rf 0.62 (2:1 ethyl acetate–hexane) gave 21 mg (19%) of a 4:1 mixture of the 2E- and
1
2Z-isomers of syn-11b; mp 130-132°C. H NMR spectrum, δ, ppm (J, Hz): 1.53 (9H, s, C4H9); 2.22 (3H, s,
CH3); 2.22 (3H, s, CH3C=N); 3.05 (4.8H, s, N(CH3)2 2E-isomer); 3.33 (1.2H, s, N(CH3)2 2Z-isomer); 5.25 (2H,
s, CH2Ph); 5.36 (1H, s, H-6); 6.96 (0.2H, s, =CHNMe2 2Z-isomer); 7.10-7.44 (3.8H, m, =CHMe2 2E-isomer,
3
H-4, H-5, H-6 C6H4); 7.54 (1H, d, J = 7.4, H-3 C6H4); 7.55 (1H, s, =CHC(Me)=N). Mass spectrum, m/z:
602/604 [M+-CO2C(CH3)3].
tert-Butyl Ester of 7Z-[2-anti-(3-Bromobenzyloxyimino)propylidene]-2(E/Z)-(N,N-dimethylamino-
methylene)-3-methyl-1,1-dioxo-3-cephem-4-carboxylic Acid (anti-11c) and tert-Butyl Ester of
7Z-[2-syn-(3-Bromobenzyloxyimino)propylidene]-2(E/Z)-(N,N-dimethylaminomethylene)-3-methyl-1,1-dioxo-
3-cephem-4-carboxylic Acid (syn-11c) were obtained using a mixture of anti and syn isomers of the tert-butyl
ester of 7Z-[2-(3-bromobenzyloxyimino)propylidene]-3-methyl-1,1-dioxo-3-cephem-4-carboxylic acid.
The fractions with Rf 0.28 (1:2 ethyl acetate–hexane) gave 40 mg (36%) of a 4:1 mixture of the 2E- and
1
2Z-isomers of anti-11c; mp 82-84°C. H NMR spectrum, δ, ppm (J, Hz): 1.53 (9H, s, C4H9); 2.14 (3H, s, CH3);
2.22 (3H, s, CH3C=N); 3.04 (4.8H, s, N(CH3)2 2E-isomer); 3.32 (1.2H, s, N(CH3)2 2Z-isomer); 5.17 (2H, s,
CH2Ph); 5.41 (1H, s, H-6); 6.82 (1H, s, =CHC(Me)=N); 6.94 (0.2H, s, =CHNMe2 2Z-isomer); 7.17-7.33 (2.8H,
3
m, =CHMe2 2E-isomer); H-5, H-6 C6H4); 7.44 (1H, d, J = 7.4, H-4 C6H4); 7.51 (1H, br. s, H-2 C6H4). Mass
spectrum: 603 [M+Na+].
The fractions with Rf 0.48 (1:2 ethyl acetate–hexane) gave 21 mg (19%) of a 4:1 mixture of the 2E- and
1
2Z-isomers of syn-11c; mp 73-75°C. H NMR spectrum, δ, ppm (J, Hz): 1.53 (9H, s, C4H9); 2.20 (3H, s, CH3);
2.22 (3H, s, CH3C=N); 3.05 (4.8H, s, N(CH3)2 2E-isomer); 3.32 (1.2H, s, N(CH3)2 2Z-isomer); 5.10 (2H, s,
CH2Ph); 5.35 (1H, s, H-6); 6.96 (0.2H, s, =CHNMe2 2Z-isomer); 7.15-7.34 (2.8H, m, =CHNMe2 2E-isomer,
3
H-5, H-6 C6H4); 7.44 (1H, d, J = 7.3, H-4 C6H4); 7.48 (2H, br. s, =CHC(Me)=N, H-2 C6H4). Mass spectrum,
m/z: 603 [M+Na+].
tert-Butyl Ester of 7Z-[2-anti-(4-Bromobenzyloxyimino)propylidene]-2(E/Z)-(N,N-dimethylamino-
methylene)-3-methyl-1,1-dioxo-3-cephem-4-carboxylic Acid (anti-11d) and tert-Butyl Ester of 7Z-[2-syn-
(4-Bromobenzyloxyimino)propylidene]-2(E/Z)-(N,N-dimethylaminomethylene)-3-methyl-1,1-dioxo-3-cephem-
4-carboxylic Acid (syn-11d) was obtained from a mixture of the anti and syn isomers of the tert-butyl ester of
7Z-[2-(4-bromobenzyloxyimino)propylidene]-3-methyl-1,1-dioxo-3-cephem-4-carboxylic acid.
The fractions with Rf 0.43 (2:1 ethyl acetate–hexane) gave 47 mg (43%) of a 4:1 mixture of the 2E- and
1
2Z-isomers of anti-11d. H NMR spectrum, δ, ppm (J, Hz): 1.53 (9H, s, C4H9); 2.13 (3H, s, CH3); 2.23 (3H, s,
CH3C=N); 3.04 (4.8H, s, N(CH3)2 2E-isomer); 3.32 (1.2H, s, N(CH3)2 2Z-isomer); 5.16 (2H, s, CH2Ph); 5.41
(1H, s, H-6); 6.83 (1H, s, =CHC(Me)=N); 6.93 (0.2H, s, =CHNMe2 2Z-isomer); 7.23 (0.8H, s,
3
3
=CHNMe2 2E-isomer); 7.23 (2H, d, J = 8.3, H-2, H-6 C6H4); 7.48 (2H, d, J = 8.3, H-3, H-5 C6H4). Mass
spectrum: 602/604 [M+Na+].
The fractions with Rf 0.37 (2:1 ethyl acetate–hexane) gave 32 mg (29%) of a 4:1 mixture of the 2E- and
1
2Z-isomers of syn-11d. H NMR spectrum, δ, ppm (J, Hz): 1.53 (9H, s, C4H9); 2.20 (3H, s, CH3); 2.22 (3H, s,
CH3C=N); 3.05 (4.8H, s, N(CH3)2 2E-isomer); 3.33 (1.2H, s, N(CH3)2 2Z-isomer); 5.09 (2H, s, CH2Ph); 5.33
(1H, s, H-6); 6.95 (0.2H, s, =CHNMe2 2Z-isomer); 7.17-7.25 (2.8H, m, =CHNMe2 2E-isomer); H-2, H-6 C6H4);
7.46 (1H, br. s, =CHC(Me)=N); 7.48 (2H, d, 3J = 8.3, H-3, H-5 C6H4). Mass spectrum, m/z: 602/604 [M+Na+].
tert-Butyl Ester of 2(E/Z)-(N,N-Dimethylaminomethylene)-3-methyl-1,1-dioxo-7Z-[2-anti-(2-chloro-
benzyloxyimino)propylidene]-3-cephem-4-carboxylic Acid (anti-11e) and tert-Butyl Ester of 2(E/Z)-(N,N-
Dimethylaminomethylen-7Z-[2-syn-(2-chlorobenzyloxyimino)propylidene]-3-cephem-4-carboxylic
Acid
(syn-11e) were obtained from a mixture of the anti and syn isomers of the tert-butyl ester of 3-methyl-1,1-dioxo-
7Z-(2-(2-chlorobenzyloxyimino)propylidene]-3-cephem-4-carboxylic acid.
241

The fractions with Rf 0.30 (1:1 ethyl acetate–hexane) gave 22 mg (36%) of a 4:1 mixture of the 2E- and
1
2Z-isomers of anti-11e; mp 134-136°C. H NMR spectrum, δ, ppm (J, Hz): 1.53 (9H, s, C4H9); 2.17 (3H, s,
CH3); 2.23 (3H, s, CH3C=N); 3.04 (4.8H, s, N(CH3)2 2E-isomer); 3.32 (1.2H, s, N(CH3)2 2Z-isomer); 5.34 (2H,
s, CH2Ph); 5.42 (1H, br. s, H-6); 6.84 (1H, br. s, =CHC(Me)=N); 6.93 (0.2H, s, =CHNMe2 2Z-isomer);
7.20-7.43 (4.8H, m, =CHNMe2 2E-isomer, C6H4). Mass spectrum, m/z: 558/560 [M+Na+].
The fractions with Rf 0.21 (1:1 ethyl acetate–hexane) gave 31 mg (51%) of a 4:1 mixture of the 2E- and
1
2Z-isomers of syn-11e; mp 130-132°C. H NMR spectrum, δ, ppm (J, Hz): 1.53 (9H, s, C4H9); 2.23 (6H, br. s,
CH3, CH3C=N); 3.05 (4.8H, s, N(CH3)2 2E-isomer); 3.33 (1.2H, s, N(CH3)2 2Z-isomer); 5.28 (2H, s, CH2Ph);
5.36 (1H, s, H-6); 6.96 (0.2H, s, =CHNMe2 2Z-isomer); 7.20-7.44 (4.8H, m, =CHNMe2 2E-isomer, C6H4); 7.54
(1H, br. s, =CHC(Me)=N). Mass spectrum, m/z: 558/560 [M+Na+].
tert-Butyl Ester of 2(E/Z)-(N,N-Dimethylaminomethylene)-3-methyl-1,1-dioxo-7Z-[2-anti-(2-fluoro-
benzyloxyimino)propylidene]-3-cephem-4-carboxylic Acid (anti-11f) and tert-Butyl Ester of 2(E/Z)-(N,N-Di-
methylaminomethylene)-3-methyl-1,1-dioxo-7Z-[2-syn-(2-fluorobenzyloxyimino)propylidene]-3-cephem-
4-carboxylic Acid (syn-11f) were obtained from a mixture of the anti and syn isomers of the tert-butyl ester of
3-methyl-1,1-dioxo-7Z-[2-(2-fluorobenzyloxyimino)propylidene]-3-cephem-4-carboxylic acid.
The fractions with Rf 0.29 (1:1 ethyl acetate–hexane) gave 8 mg (35.5%) of a 4:1 mixture of the 2E- and
1
2Z-isomers of anti-11f; mp 85-87°C. H NMR spectrum, δ, ppm (J, Hz): 1.53 (9H, s, C4H9); 2.13 (3H, s, CH3);
2.23 (3H, s, CH3C=N); 3.04 (4.8H, s, N(CH3)2 2E-isomer); 3.32 (1.2H, s, N(CH3)2 2Z-isomer); 5.29 (2H, s,
CH2Ph); 5.42 (1H, br. s, H-6); 6.84 (1H, br. s, =CHC(Me)=N); 6.93 (0.2H, s, =CHNMe2 2Z isomer); 6.97-7.19
(2H, m, H-4, H-5 C6H4); 7.23 (0.8H, s, =CHNMe2 2E-isomer); 7.27-7.45 (2H, m, H-3, H-6 C6H4). Mass spectra,
m/z: 520 [M]+.
The fractions with Rf 0.20 (1:1 ethyl acetate–hexane) gave 10 mg (45.6%) of a 4:1 mixture of the 2E-and
1
2Z-isomers of syn-11f; mp 81-83°C. H NMR spectrum, δ, ppm (J, Hz): 1.53 (9H, s, C4H9); 2.21 (3H, s, CH3);
2.22 (3H, s, CH3C=N); 3.04 (4.8H, s, N(CH3)2 2E-isomer); 3.32 (1.2H, s, N(CH3)2 2Z-isomer); 5.22 (2H, s,
CH2Ph); 5.34 (1H, br. s, H-6); 6.95 (0.2H, s, =CHNMe2 2Z-isomer); 6.98-7.19 (2H, m, H-4, H-5 C6H4); 7.24
(0.8H, s, =CHNMe2 2E-isomer); 7.27-7.44 (2H, m, H-3, H-6 C6H4); 7.49 (1H, br. s, =CHC(Me)=N). Mass
spectrum, m/z: 520 [M]+.
3Z-[2-anti-(Arylmethoxyimino)propylidene]-1-tert-butoxycarbonylmethyl-4-(5-methyl-4-isoxazolyl-
sulfonyl)azetidin-2-ones (anti-14a,b,d). Typical procedure. Hydroxylamine hydrochloride (22 mg,
0.319 mmol) was added to a solution of a mixture of the syn and anti isomers of tert-butyl ester of
7Z-[2-(arylmethoxyimino)propylidene]-2(E/Z)-(N,N-dimethylaminomethylene)-3-methyl-1,1-dioxo-3-cephem-
4-carboxylic acid (80 mg, 0.159 mmol) in acetonitrile (25 ml). The reaction mixture was stirred for 2 h at
40-50°C and evaporated at reduced pressure. The residue was fractionated on a silica gel with ethyl acetate–
hexane as the eluent (the solvent ratio was varied from 1:5 to 1:2) to give azetidinones 14a, 14b, and 14d.
3Z-[2-anti-(2-Benzyloxyimino)propylidene]-1-tert-butoxycarbonylmethyl-4-(5-methyl-4-isoxazolyl-
sulfonyl)azetidine-2-one (anti-14a) was obtained from a mixture of the syn and anti isomers of the tert-butyl ester
of 7Z-[2-(benzyloxyimino)propylidene]-2(E/Z)-(N,N-dimethylaminomethylene)-3-methyl-1,1-dioxo-3-cephem-
4-carboxylic acid.
The fractions with Rf 0.33 (1:1 ethyl acetate–hexane) gave anti-14a in 64% yield; mp 76-78°C. 1H NMR
spectrum, δ, ppm (J, Hz): 1.47 (9H, s, C4H9); 2.15 (3H, s, CH3C=N); 2.50 (3H, s, CH3 isoxazole); 4.05, 4.40
(2H, two d, AB system, 2J = 18, NCH2CO2); 5.25 (2H, s, CH2Ph); 5.75 (1H, s, H-4); 6.65 (1H, s, =CH(Me)=N);
7.30-7.44 (5H, m, C6H5); 8.17 (1H, s, H-3 isoxazole). Mass spectrum, m/z: 512 [M+Na+].
3Z-[2-anti-(2-Bromobenzyloxyimino)propylidene]-1-tert-butoxycarbonylmethyl-4-(5-methyl-4-isoxazole-
sulfonyl)azetidin-2-one (anti-14b) was obtained from a mixture of the syn and anti isomers of the tert-butyl
ester of 7Z-[2-(2-bromobenzyloxyimino)propylidene]-(2E/Z)-(N,N-dimethylaminomethylene)-3-methyl-1,1-dioxo-
3-cephem-4-carboxylic acid.
242

1
The fractions with Rf 0.43 gave an oily product in 15% yield. H NMR spectrum, δ, ppm (J, Hz): 1.47
2
(9H, s, C4H9); 2.19 (3H, s, CH3C=N); 2.54 (3H, s, CH3 isoxazole); 4.05, 4.41 (2H, two d, AB system, J = 18,
NCH2CO2); 5.34 (2H, s, CH2Ph); 5.77 (1H, s, H-4); 6.65 (1H, s, =CHC(Me)=N); 7.12-7.38 (3H, m, H-4, H-5,
H-6 C6H4); 7.58 (1H, d, 3J = 7.4, H-3 C6H4); 8.21 (1H, s, H-3 isoxazole). Mass spectrum, m/z: 590 [M+Na+].
3Z-[2-anti-(4-Bromobenzyloxyimino)propylidene]-1-tert-butoxycarbonylmethyl-4-(5-methyl-4-isoxazole-
sulfonyl)azetidin-2-one (anti-14d) was obtained from a mixture of the syn and anti isomers of the tert-butyl
ester of 7Z-[2-(4-bromobenzyloxyimino)propylidene]-(2E/Z)-(N,N-dimethylaminomethylene)-3-methyl-1,1-dioxo-
3-cephem-4-carboxylic acid.
1
The fractions with Rf 0.39 gave an oily product in 8% yield. H NMR spectrum, δ, ppm (J, Hz): 1.47
2
(9H, s, C4H9); 2.14 (3H, s, CH3C=N); 2.52 (3H, s, CH3 isoxazole); 4.05, 4.40 (2H, two d, AB system, J = 18,
3
NCH2CO2); 5.19 (2H, s, CH2Ph); 5.75 (1H, s, H-4); 6.62 (1H, s, =CHC(Me)=N); 7.24 (2H, d, J = 8.4, H-2,
3
H-6 C6H4); 7.50 (2H, d, J = 8.4, H-3, H-5 C6H4); 8.18 (1H, s, H-3 isoxazole). Mass spectrum, m/z: 590
[M+Na+].
tert-Butyl Ester of 7Z-[2-anti-(4-Bromophenylhydrazono)propylidene]-3-methyl-1,1-dioxo-3-cephem-
4-carboxylic Acid (anti-15). 4-Bromophenylhydrazine hydrochloride (85 mg, 0.381 mmol) and sodium acetate
(32 mg, 0.381 mmol) were added to a solution of tert-butyl ester 7Z-acetylmethylene-3-methyl-1,1-dioxo-
cephem-4-carboxylic acid (100 mg, 0.293 mmol) in methanol (10 ml). The reaction mixture was stirred for 24 h at
room temperature and then evaporated at reduced pressure. The residue was fractionated on a silica gel column. The
1
fractions with Rf 0.23 (1:1 ethyl acetate–hexane) gave cephem anti-15 in 68% yield; mp 210-213°C. H NMR
spectrum, δ, ppm (J, Hz): 1.48 (9H, s, C4H9); 1.91 (3H, s, CH3); 2.12 (3H, s, CH3C=N); 4.16, 4.34 (2H, two d,
2
3
AB system, J = 17.6, SO2CH2); 6.25 (1H, s, H-6); 6.97 (1H, s, =CHC(Me)=N); 7.24 (2H, d, J = 8.8, H-2, H-6
C6H4); 7.43 (2H, d, 3J = 8.8, H-3, H-5 C6H4); 10.15 (1H, s, NH). Mass spectrum, m/z: 534 [M+Na+].
tert-Butyl Ester of 7Z-[2-anti-(2-Bromobenzoyloxyimino)propylidene]-3-methyl-1,1-dioxo-3-cephem-
4-carboxylic Acid (anti-16). 2-Bromobenzoic acid chloride (36 mg, 0.168 mmol) and pyridine (4 mg,
0.056 mmol) were added to a solution of tert-butyl ester of a mixture of syn and anti isomers of
Z-[2-(hydroxyimino)propylidene]-3-methyl-1,1-dioxo-3-cephem-4-carboxylic acid (20 mg, 0.056 mmol) in
10 ml acetonitrile. The reaction mixture was stirred for 72 h at room temperature, diluted with 100 ml
5% aqueous NaCl, and extracted with two 30-ml portions of ethyl acetate. The organic layer was dried over
anhydrous sodium sulfate. The solvent was evaporated at reduced pressure. The residue was fractionated on a
silica gel column. The fractions with Rf 0.57 (1:1 ethyl acetate–hexane) gave cephem anti-16 in 54% yield;
1
mp 128-132°C. H NMR spectrum, δ, ppm (J, Hz): 1.54 (9H, s, C4H9); 2.10 (3H, s, CH3); 2.35 (3H, s,
CH3C=N); 3.65, 3.93 (2H, two d, AB system, 2J = 17.6, SO2CH2); 5.58 (1H, s, H-6); 7.09 (1H, s,
=CHC(Me)=N); 7.34-7.48 (2H, m, H-4, H-5 C6H4); 7.66-7.75 (1H, m, H-3 C6H4); 7.77-7.86 (1H, m, H-6 C6H4).
Mass spectrum, m/z: 561/563 [M+Na+].
Determination of Cytotoxic Activity in vitro. The cytotoxic properties of the products relative to
monolayer malignant and normal cells at c = (2-5)·104 cells/ml: HT-1080 (human fibrosarcoma); MG-22A
(murine hepatoma); 3T3 (embryonal murine fibroblasts) were determined in 96-well panels using CV, MTT, and
NR dyes according to a standard procedure [5].
Cellular Generation of NO Radicals. The determination of the nitric oxide radicals in acid media
according to Gracey [6] was carried out in plastic 96-well panels. The NO radical concentration (in nmoles) in
the culture medium with the surviving cells after incubation for 72 h in the presence of the test compound at
c = 50 µg/ml in the 200-µl well was 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, C is the percentage of surviving cells
determined by their coloration with CV.
243

This work has been partly supported by the European Social Fund within the National Programme
"Support for the carrying out doctoral study programm's and post-poctoral researches" project "Support for the
development of doctoral Studies at Riga Technical University".
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