Synthesis and anticancer properties of 7α-chloro-3-methyl-1,1- dioxoceph-3-em-4-carboxylic acid esters

Article abstract of DOI:10.1007/s10593-007-0033-x

The tert-butyl esters of 3-azidomethyl-, 3-isocyanatomethyl-, 3-chloromethyl-, and 3-p-nitrophenylvinyl-7α-chloro-1,1-dioxoceph-3-em-4- carboxylic acid, and also esters of 7α-chloro-3-methyl-1,1-dioxoceph-3-em- 4-carboxylic acid, and of 7α-chloro-3-methyl

Full text of DOI:10.1007/s10593-007-0033-x

Chemistry of Heterocyclic Compounds, Vol. 43, No. 2, 2007
SYNTHESIS AND ANTICANCER PROPERTIES OF 7α-CHLORO-
3-METHYL-1,1-DIOXOCEPH-3-EM-4-CARBOXYLIC ACID ESTERS
M. Vorona, G. Veinberg, I. Shestakova, I. Kanepe, I. Potorochina,
K. Dikovskaya, R. Bokaldere, M. Petrova, E. Liepinsh, and E. Lukevics
The tert-butyl esters of 3-azidomethyl-, 3-isocyanatomethyl-, 3-chloromethyl-, and 3-p-nitrophenylvinyl-
7α-chloro-1,1-dioxoceph-3-em-4-carboxylic acid, and also esters of 7α-chloro-3-methyl-1,1-dioxoceph-
3-em-4-carboxylic acid, and of 7α-chloro-3-methyl-2-dimethylaminomethylene-1,1-dioxoceph-3-em-
4-carboxylic acid have been synthesized. Results of cytotoxic screening of these compounds in relation
to cancer and normal cells in vitro are correlated and analyzed.
Keywords: esters of 7α-chloro-3-methyl-2-dimethylaminomethylene-1,1-dioxoceph-3-em-4-carboxylic
acid, esters of 7α-chloro-3-methyl-1,1-dioxoceph-3-em-4-carboxylic acid, cytotoxic activity.
The directed structural modification of substituents in penicillin, cephalosporin, and 2-azetidinone
carried out in the past 20 years has led to the discovery of compounds with anti-inflammatory, antiviral,
anticancer, anticoagulant, and other activities "unplanned by nature". Their mechanism of action at the molecular
level consists of the inhibition of specific serine- and cysteine-containing proteases as a result of acylation of
hydroxyl or mercapto groups found in their active centers by the β-lactam ring [1].
The literature data referring to this investigation indicate that the structural variations of substituents in
the β-lactam pharmacophore directed towards achieving effective inhibition of the target protease are
accompanied by analogous, although less marked, effects on one or several related enzymes [2-5]. The negative
side of this phenomenon consists of the probability of displaying undesirable secondary activity, but the positive
is the possibility of using it for the targeted development of substances with new biological properties. Such an
interpretation of the secondary activity of clavulanic acid ester, which is a specific inhibitor of the bacterial
enzyme β-lactamase, in relation to Human Leucocyte Elastase (HLE) enabled the design of anti-inflammatory
analogs of cephalosporin [6].
We encountered an analogous secondary effect on studying the biological properties of structural
analogs of the tert-butyl ester of 7α-chloro-1,1-dioxoceph-3-em-4-carboxylic acid 1 and 2 [7].
O
O
O
O
O
O
Cl
Cl
O
Cl
S
S
S
1
2
3
7
8
6
N 5
O
R1
4
N
O
N
O
Me
Me
O
O
O
R 2
t
OBu-
t
OBu-
O
3
2
1
__________________________________________________________________________________________
Latvian Institute of Organic Synthesis, Riga LV-1006; e-mail: veinberg@osi.lv Translated from
Khimiya Geterotsiklicheskikh Soedinenii, No. 2, pp. 259-273, February, 2007. Original article
submitted December 4, 2006.
0009-3122/07/4302-0207©2007 Springer Science+Business Media, Inc.
207

According to the data of Table 1 the presence of an acetoxymethyl group in position 3 of the cephem
nucleus (in agreement with the data of [6]) provided the high inhibitory effect of compound 1 in relation to
Porcine Pancreas Elastase (PPE) and a weak cytotoxic activity in vitro in relation to monolayer tumor cell lines
HT-1080 (human fibrosarcoma) and MG-22a (mouse hepatoma). The deacetoxycephalosporin 2, differing from
cephalosporin 1 in the absence of the acetoxy group at position 3, was characterized by the reverse intensity of
inhibitory and cytotoxic effects.
The substancial similarity of both compounds enabled a similar mechanism of action to be proposed for
them at the molecular level, consisting of inhibition of the serine protease belonging to elastase family. In this
way HLE is the main target for compound 1 [6]. The secondary targets are the specific elastases promoting
growth and proliferation of cancer cells [8-10], and they are main for compound 2. Consequently the bicyclic
condensed system oxidized at position 1, and also substituted at positions 3, 4, and 7α by methyl, a carbonyl
group, and halogen respectively, and represented by structure 3, is a potential pharmacophore for anticancer
activity.
In favor of this hypothesis evidence the negative results on testing the cytotoxic properties in vitro of
resynthesized compounds 4a,b, and also of the previously obtained structural analogs of cephalosporin 5-7
[7, 11, 12] with significant differences in the pharmacophore fragment of the molecule.
O
O
O
O
O
O
O
S
n
R3M
O
R
Cl
O
Cl
S
S
S
Cl
N
O
N
O
N
O
N
O
Me
Me
Me
O
O
Me
t
OBu-
OCHPh2
OBu-t
t
OBu-
5 a,b
6 a–c
7
4 a,b
4 a n = 0, b n = 1; 5 a R = H, b R = I; 6 a R = SiEt3, b R = SiMe2Bu-t, c R = SiMe2Ph
Thus a reduction in the degree of oxidation of the heterocyclic sulfur atom in 4a,b, substitution of
chlorine by hydrogen, iodine, or a trisubstituted silyl group in 5a,b, 6a-c, reduction of the double bond in the
cephem nucleus and introduction of a chlorine atom at position 3 in compound 7 is accompanied by a significant
decline or complete disappearance of the cytotoxic properties of these compounds in relation to cancer cells HT-
1080 and MG-22A in comparison with compound 2.
TABLE 1. Biological Properties of tert-Butyl Esters
of 7α-Chlorocephalosporanic Acid Sulfone
Cytotoxic activity in vitro, LC50, µg/ml *
Compound
IC50, µmol
HT-1080
MTT
MG-22A
CV
CV
MTT
33
6
40
6
32
6
25
2
1
2
0.16±0.02
11±0,9
_______
* LC50 is the concentration causing 50% cell death; CV staining with
Crystal Violet; MTT staining with 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl-2H-tetrazolium bromide; IC50 is the concentration causing
50% inhibition of the amidolytic activity of Porcine Pancreas Elastase in
relation to the p-nitroanilide of N-methoxysuccinyl-Ala-Ala-Pro-Val as
substrate.
208

It proved to be more promising in this respect to substitute the methyl group in position 3 of the tert-
butyl ester of 7α-chloro-1,1-dioxoceph-3-em-4-carboxylic acid (2). Synthesis of compounds of this type was
effected from the tert-butyl ester of 3-bromomethyl-7α-chloro-1,1-dioxoceph-3-em-4-carboxylic acid (8),
obtained by the allylic bromination of the methyl group in compound 2 [7]. Nucleophilic substitution of bromine
in 8 by the triethylammonium salts of hydroxy- and acetoxy-substituted benzoic acids 9a-d [13], by sodium
azide or ammonium isothiocyanate, led to the preparation of the cephalosporin analogs 10a-d, 11, and 12. The
use of the Wittig reaction enabled introduction of a p-nitrobenzylidene substituent into the methyl group of
compound 13.
O
N
O
R1
Cl
S
R
O
O
O
OBu-t
O
O
O
Cl
10a–d
S
–
+
N
O
N3
O
OEt3NH
O
OBu -t
R
11
NaN3
R 1
9a–d
O
O
O
O
Cl
Cl
O
S
S
NBS, hν
NH4NCS
2
N
O
NCS
N
Br
O
OBu-t
OBu-t
O
8
12
O
2.
1. PPh3
O
N
O
Na2CO3
O
O
Cl
O
S
N
O
N
O
OBu-t
O
13
9,10 а R = 2-OH, R1 = H; b R = 2-OH, R1 = 4-OH; c R= 2-OAc, R1 = H;
d R = 2-OAc, R1 = 4-OAc
209

Exchange of the bromine in compound 8 by hydroxyl with ammonium trifluoroacetate with subsequent
hydrolysis of the intermediate trifluoroacetoxy group led to the preparation of the 3-hydroxymethyl analog of
cephalosporin 14. With the aid of phosphorus pentachloride or chloroformates 14 was transformed into
compounds 15 and 17 containing in position 3 of the cephem nucleus a methyl group substituted by chlorine or
by a carbonate group respectively [14].
Data on the biological screening of compounds 8, 10-15, and 17, given in Table 2, indicate that with the
exception of compounds 8 and 10a, they are characterized by moderate cytotoxicity in relation to cancer test
cultures.
O
O
O
O
CF3COONH4
H2O
Cl
Cl
S
S
N
OH
N
O
Br
O
O
O
OBu-t
OBu-t
Cl
8
O
O
14
PCl5
S
ROCOCl
16a–c
N
Cl
O
O
O
Cl
S
O
OBu-t
N
O
O
O
15
R
O
O
OBu-t
17a–c
16,17 a R = CCl3CH2, b R = BrCH2CH2, c R = 4-O2NC6H4
TABLE 2. Biological Activity of the tert-Butyl Ester of 7α-Chloro-
1,1-dioxo-ceph-3-em-4-carboxylic Acid Modified in Position 3
Cytotoxic activity in vitro, LC50, µg/ml
Com-
pound
IC50, µmol
References
HT-1080
MG-22A
CV
MTT
CV
MTT
8
12
2
5
2
6
2
1
6
—
4.1 (41*)
5.0 (68*)
0.35
[7]
[13]
[13]
[13]
[13]
—
10a
10b
10c
10d
11
50
46
52
18
33
59
18
10
39
37
53
50
42
53
3
37
>50
31
9
37
>50
40
11
7
15 (6.3*)
—
12
10
61
4
18
52
56
72
12
48
11
11
46
45
45
—
—
13
53
10
10
58
42
62
—
—
14
15
24
—
—
—
17a
17b
17c
13
[14]
[14]
[14]
0.040
0.047
_______
* Inhibition (%) of the amidolytic activity of Porcine Pancreas Elastase in
relation to the p-nitroanilide of N-methoxysuccinyl-Ala-Ala-Pro-Val as
substrate.
210

However the high inhibitory effect in relation to PPE displayed by individual representatives of this type of
compounds (10a, 17b, and 17c) indicates that modification of the methyl group represents little promise since in
principle it contributes to a reduction of the selectivity of anticancer action of this type of cephalosporin.
Modification of the ester group was therefore selected as the next subject of investigation of the
dependence of structure and anticancer properties of of 7α-chloro-3-methyl-1,1-dioxoceph-3-em-4-carboxylic
acid analogs.
Me
Me
N
O
O
O
O
Me
Cl
Cl
S
S
N
+
N
O
N
O
Me
Me
O
N
O
Me
O
O
Cl
S
OBu-t
OBu-t
Me
Z-25
E-25
O
19
+
–
[Me2N=CHCl]Cl
20
Et3N
O
S
O
O
O
Cl
Cl
S
CF3COOH
N
O
N
O
Me
Me
OH
O
O
OBu-t
18
2
20
O
O
N
O
Cl
S
O
O
Me
Cl
O
S
Me
N
Cl
21
N
O
Me
Me
O
ROH
22 a–m
ROH
O
O
O
22 n
Cl
S
N
Me
O
O
O
O
OR
Et
23 a–m
24
22, 23 a R = Me, b R = Et, c R = i-Pr, d R = n-Bu, e R = CH2CH=CH2, f R = CH2CH2Cl,
g R = CH2CCl3, h R = n-C5H11, i R = n-C7H15, j R = Ph, k R = Bn,
O
,
n R =
m R =
l R =
,
O
O
Et
211

Esters of 7α-chloro-3-methyl-1,1-dioxoceph-3-em-4-carboxylic acid 23a-m and also their analogs
24 and 25, containing a dimethylaminomethylene group at position 2, were synthesized starting from the tert-
butyl ester 2. Its treatment with trifluoroacetic acid, as given in [15], led to the preparation of 7α-chloro-
3-methyl-1,1-dioxoceph-3-em-4-carboxylic acid (18). Compound 18 is stable in acidic medium, but in neutral
and in basic media it is subject to rapid decarboxylation with the formation of 7α-chloro-3-methyl-1,1-
dioxoceph-3-em (19). The action of the Vilsmeier reagent 20 on the carboxyl group of cephem 18, in a medium
of methylene chloride according to [15], led to conversion into the acid chloride 21, which without isolation was
subjected to treatment with the appropriate alcohol. The object esters 23a-m were isolated from the reaction
mixture by column chromatography on silica gel. The use of the ethyl ester of p-hydroxybenzoic acid for
esterification of 21 led to the preparation of ester 24, having a dimethylaminomethylene group at position 2. The
analogous reaction, based on the alkylating properties of the Vilsmeier reagent in relation to the cephem nucleus
was noted in [16]. Treatment of tert-butyl ester 2 with this reagent led to the preparation of a product, substituted
with dimethylaminomethylene in position 2, as a mixture of isomers E-25 and Z-25 in a ratio of 3 : 1.
The structures of isomers E-25 and Z-25 were determined by 2D-NOESY two-dimensional
spectroscopy. The presence of a NOE between the dimethylamino and 3-methyl groups (indicating their
closeness in space) and the absence of one between the 3-methyl group and the =CH proton in the predominant
isomer confirms the E-orientation of the latter. In its turn, a NOE was observed in the minor isomer between the
3-methyl group and the =CH proton but was absent for the dimethylamino and 3-methyl groups thereby
demonstrating the Z-orientation of the minor isomer.
The close disposition of the sulfone group leads to a displacement towards low field of the resonance
signals of the neighboring dimethylamino group in the Z-25 isomer and of the =CH proton in the E-25 isomer.
Me
O
Me
3.35 ppm.
7.00 ppm
7.29 ppm
N
H
O
O
O
Cl
Cl
S
S
Me
N
H
3.01 ppm
N
O
N
O
Me
Me
Me
2.22 ppm
O
O
2.21 ppm
OBu-t
OBu-t
E-25
Z-25
Biological screening of the synthesized esters of 7α-chloro-3-methyl-1,1-dioxoceph-3-em-4-carboxylic
acid 23a-m, 24, and 25, and the decarboxylated product 19 in vitro included determination of their cytotoxic
properties in relation to monolayer cancer cell lines HT-1080 and MG-22A. Also assessed was their ability to
initiate biosynthesis of nitric oxide radicals, the high reactivity of which is one of the components of the
cytotoxic effect. Calculation of the specific NO-generating activity of compounds, consisted of determination of
the NO ion concentration (nmol) in cellular medium in panel wells of volume 200 µliter after incubation for
72 h, in the presence of the substance being tested at a concentration of 50 µg/ml by the method of [18], then
converting the value obtained to 100% of the amount of cells contained in the medium at the beginning of the
experiment:
TG100 = G ·100/C (nmol/µliter)
where TG100 is the specific NO-generating activity of a compound; G the NO concentration (nmol) generated in
the culture medium of volume 200 µliter by the surviving cells; C the percentage of surviving cells determined
by CV-staining.
212

TABLE 3. Biological Activity of Esters of 7α-Chloro-3-methyl-1,1-dioxo-
ceph-3-em-4-carboxylic Acid
Cytotoxic activity in vitro, LC50, µg/ml
Com-
pound
HT-1080
MTT
MG-22A
MTT
CV
TG100
CV
TG100
23d
23g
28
9
16
14
23
15
21
20
12
32
135
800
1050
450
850
340
400
450
18
18
13
28
13
12
25
18
22
20
14
24
8
350
400
200
250
200
350
200
250
23h
23i
17
32
14
25
16
18
23j
23k
23l
16
26
21
25a/25b
The tested esters may be divided into two groups according to the displayed anticancer effect. The n-
butyl (23d), n-pentyl (23h), n-heptyl (23i), 2,2,2-trichloroethyl (23g), phenyl (23j), benzyl (23k), and cinnamyl
(23l) esters, and also the isomeric mixture of tert-butyl esters E-25/Z-25 substituted in position 2, which are
characterized by LC50 values in the range 10-30 µg/ml and by active generation of nitric oxide radicals in the cell
medium, fall into the first group (Table 3).
The second group of compounds, consisting of tert-butyl ester 2, methyl, ethyl, isopropyl, allyl,
chloroethyl, and furfuryl esters 23a-c, 23e, 23f, 23m, and also the p-ethoxycarbonylphenyl ester 24 substituted
in position 2, which displayed a higher cytotoxic activity in relation to both or one of the cell cultures HT-1080
and MG-22A, were tested additionally in relation to B16 (mouse melanoma) and Neuro 2A (mouse
neuroblastoma) cancer cells, and also normal cells 3T3 (mouse embryo fibroblasts) and BHK cells (golden
hamster kidney fibroblasts) (Table 4).
Staining of 3T3 fibroblasts with Neutral Red (NR) enabled calculation of the value of the expected
toxicity LD50 for the tested compounds, without resorting to experiments in vivo, with the aid of the
equation [17]
log LD50 (mg/kg) = 0.435 x log LC50 (mM) + 0.625
According to the data of Table 4 the methyl ester 23a and the decarboxylated cephem 19 are
characterized by a high cytotoxic effect in relation to both cancer cells and to normal cells. Such an absence of
selectivity is reflected in the low LD50 values of 236 and 252 mg/kg for these compounds. An increase in the
carbon chain in the ester group of compounds 23b, 23c, and 23e-g to 2 or 3 carbon atoms enabled a
strengthening of the toxic effect in relation to cancer and a weakening of it in relation to normal cells. The
selectivity developed led to a more than twofold reduction in the toxicity index (LD50 490 to 626 mg/kg). The
leading position in effectiveness and selectivity of anticancer effect in this group belongs to tert-butyl ester 2
with LD50 1162 mg/kg. The cytotoxic effect of compounds is connected with the intense generation of nitric
oxide radicals by the cancer cells (see Table 4).
The results of the investigation carried out indicate that modification of the ester group in 7α-chloro-3-
methyl-1,1-dioxoceph-3-em-4-carboxylic acid is promising in the search for new anticancer substances
permitting action both on the cytotoxic effectiveness of compounds and on their selectivity in relation to cancer
and normal cells.
213

214

EXPERIMENTAL
1
The H NMR spectra were recorded on Bruker WH90/DS (90 MHz) and Varian Mercury 400 (400
MHz) spectrometers in CDCl3, internal standard was TMS. Elemental analysis was carried out on a Carlo Erba
1108 analyzer and the difference of experimental and calculated values was ±0.4%. The IR spectra were taken
on a Perkin-Elmer 580B spectrometer in nujol. A check on the progress of reactions was effected by TLC on
Merck Kieselgel plates with visualization in UV light. The HPLC data were obtained on a Du-Pont Model 8800
instrument fitted with a UV detector (λ = 254 nm) and column (4.6×250 mm) packed with Symmetry C18 phase
or Ultrasphere octyl in the system acetonitrile–water or acetonitrile–0.1 N phosphate buffer, pH 2.5 (60 : 40),
rate 0.8-1.5 ml/min. Silica gel type Merck Kieselgel (0.063-0.230 mm) was used for preparative column
chromatography. Reagents and materials from Acros, Aldrich, and Sigma were used in experiments. The two-
dimensional 2D-NOESY spectra for compounds E-25 and Z-25 were recorded on a Varian Mercury 400 (400
MHz) spectrometer in CDCl3 at 24°C using impulse gradient technology. When recording 2D-spectra a data
1
matrix of size 4096×1024 was used, which provided τ2max = 250 msec for H when recording along the F2 axis
1
and τ1max = 100 msec for H along the F1 axis. To optimize the signal-to-noise ratio the data matrix before
Fourier transformation was supplemented with zeros twice and multiplied by the cosine function. The duration
of the mixing time in 2D-NOESY was 1 sec. Optical density in the biological tests carried out in 96-well panels
was determined with a Tetretek Multiscan MCC/340 horizontal spectrophotometer.
tert-Butyl Ester of 3-Azidomethyl-7α-chloroceph-3-em-4-carboxylic Acid Sulfone (11). Sodium
azide (18 mg, 0.25 mmol) was added to a solution of the tert-butyl ester of 3-bromomethyl-7α-chloroceph-3-em-
4-carboxylic acid sulfone [6] (100 mg, 0.25 mmol) in DMF (10 ml). The mixture was stirred for 24 h at room
temperature, then diluted with water (50 ml) and diethyl ether (50 ml). The organic layer was separated and dried
over anhydrous Na2SO4. The solvent was evaporated at reduced pressure. The residue was fractionated on a
chromatographic column of silica gel (eluent ethyl acetate–hexane, 1 : 3). Fractions with Rf 0.33 were combined
and evaporated. Mp 124-126°C, yield 7 mg (8%). IR spectrum: 2100 (N3), 1810 (β-lactam), 1720 cm-1 (C=O).
1H NMR spectrum, δ, ppm (J, Hz): 1.55 (9H, s, C4H9); 3.77 and 4.06 (2H, two d, AB system, 2J = 19, SO2CH2);
4.08 and 4.35 (2H, two d, AB system, 2J = 15, CH2N3); 4.83 (1H, br. s, H-6); 5.33 (1H, d, 3J = 1.5, H-7). Found,
%: C 40.05; H 4.26; N 15.39. C12H15ClN4O5S. Calculated, %: C 39.73; H 4.17; N 15.44.
tert-Butyl Ester of 7α-Chloro-3-isothiocyanatomethylceph-3-em-4-carboxylic acid Sulfone (12).
Ammonium thiocyanate (28 mg, 0.30 mmol) was added to a solution of the tert-butyl ester of 3-bromomethyl-
7α-chloroceph-3-em-4-carboxylic acid sulfone (100 mg, 0.25 mmol) in DMF (10 ml). The mixture was stirred
for 24 h at room temperature, diluted with water (50 ml) and diethyl ether (50 ml). The organic phase was
separated, and dried over anhydrous Na2SO4. The solvent was evaporated at reduced pressure. The residue was
fractionated on a chromatographic column of silica gel (eluent was ethyl acetate–hexane, 1 : 3). Fractions with Rf
0.22 were combined and evaporated. The oily substance had >95% content of the main substance, according to
1
HPLC analysis, yield 30 mg (32%). H NMR spectrum, δ, ppm (J, Hz): 1.55 (9H, s, C4H9); 3.62 and 4.44 (2H,
2
2
two d, AB-system, J = 14, CH2NCS); 3.77 and 4.22 (2H, two d, AB system, J = 19, SO2CH2); 4.84 (1H, m,
3
H-6); 5.31 (1H, d, J = 1.5, H-7). Found, %: C 41.48; H 4.09; N 7.28. C13H15ClN2O5S2. Calculated, %: C 41.21;
H 3.99; N 7.39.
Mixture of tert-Butyl Esters of cis-7α-Chloro-3-(4-nitrophenylvinyl)ceph-3-em-4-carboxylic Acid
Sulfone and trans-7α-Chloro-3-(4-nitrophenylvinyl)ceph-3-em-4-carboxylic Acid Sulfone (13). p-Nitrobenz-
aldehyde (17 mg, 0.11 mmol) and 5% Na2CO3 solution (5 ml) were added to a solution of the tert-butyl ester of
7α-chloro-3-(triphenylphosphonium)methylceph-3-em-4-carboxylic acid bromide [6] (30 mg, 0.045 mmol) in
dichloromethane (10 ml). The mixture was stirred for 2 h at room temperature, washed with 5% NaHSO3
solution (50 ml), and dried over Na2SO4. The solvent was evaporated at reduced pressure. The residue was
fractionated on a chromatographic column of silica gel (eluent ethyl acetate–hexane, 1 : 1). Fractions with Rf
0.51 were combined and evaporated. Yield 10 mg (49%). 1H NMR spectrum, δ, ppm (J, Hz): cis-isomer – 1.55
215

(9H, s, C4H9); 3.51 and 3.77 (2H, two d, AB system, 2J = 19, SO2CH2); 4.80 (1H, br. s, H-6); 5.31 (1H, d, 3J = 2,
3
3
3
H-7); 6.62 and 6.82 (2H, two d, J = 10, CH=CH); 7.48 (2H, d, J = 9, C6H4); 8.24 (2H, two d, J = 9, C6H4);
trans-isomer – 1.64 (9H, s, C4H9); 4.06-4.26 and 3.77 (2H, m, AB system, 2J = 17, SO2CH2); 4.86 (1H, m, H-6);
5.37 (1H, d, 3J = 2, H-7); 6.53 and 7.86 (2H, two d, 3J = 14, CH=CH); 7.60 and 8.26 (4H, two d, 3J = 9, C6H4).
Found, %: C 49.83; H 4.34; N 5.77. C19H19ClN2O7S·0.25 H2O. Calculated, %: C 49.67; H 4.27; N 6.09.
tert-Butyl Ester of 7α-Chloro-3-chloromethylceph-3-em-4-carboxylic Acid Sulfone (15). Phosphorus
pentachloride (50 mg, 0.19 mmol) was added to a solution of the tert-butyl ester of 7α-chloro-3-hydroxymethylceph-
3-em-4-carboxylic acid sulfone [14] (50 mg, 0.15 mmol) in dichloromethane (20 ml) at 0°C. The mixture was stirred
for 30 min at room temperature, diluted with dichloromethane (20 ml), the solution was washed with 5% Na2CO3
solution (40 ml), and dried over anhydrous Na2SO4. The solvent was evaporated at reduced pressure. The residue was
fractionated on a chromatographic column of silica gel (eluent ethyl acetate–hexane, 1 : 4). Fractions with Rf 0.31
were combined and evaporated. Mp 142-144°C, yield 33 mg (62%). 1H NMR spectrum, δ, ppm (J, Hz): 1.66 (9H, s,
3
2
C4H9); 3.75 and 4.17 (2H, two d, AB system, J = 18, SO2CH2); 4.26 and 4.62 (2H, two d, AB system, J = 12,
3
CH2Cl); 4.80 (1H, br. s, H-6); 5.28 (1H, d, J = 1.5, H-7). Found, %: C 40.52; H 4.30; N 3.81. C12H15Cl2NO5S.
Calculated, %: C 40.46; H 4.24; N 3.93.
7α-Chloro-3-methyl-1,1-dioxoceph-3-em (19). Trifluoroacetic acid (2 ml, 38 mmol) was added to a
solution of the tert-butyl ester of 7α-chloro-3-methylceph-3-em-4-carboxylic acid (342 mg, 1.11 mmol) in
dichloromethane (30 ml) at 0°C. The mixture was stirred for 1 h at room temperature, diluted with dichloromethane
(100 ml), and with water (100 ml). The organic phase was separated, and extracted with 5% Na2CO3 solution (40 ml).
The aqueous extract was acidified with conc. HCl to pH 2, and extracted with ethyl acetate. The extract was
evaporated under reduced pressure. The residue was crystallized from diethyl ether. 7α-Chloro-3-methyl-
1,1-dioxoceph-3-em-4-carboxylic acid (18) (254 mg, 90%) was obtained and was dissolved in a mixture of acetone
(20 ml) and triethylamine (2 ml). The solution was stirred at room temperature for 1 h, and the solvent was
evaporated under reduced pressure. The residue was fractionated on a chromatographic column of silica gel (eluent
ethyl acetate–hexane, 1 : 1). Fractions with Rf 0.48 were combined and evaporated. Mp 122-124°C, yield 195 mg
1
2
(90%). H NMR spectrum, δ, ppm (J, Hz): 1.80 (3H, s, 3-CH3); 3.48 and 3.96 (2H, two d, AB system, J = 18,
3
SO2CH2); 4.73 (1H, m, H-6); 5.31 (1H, d, J = 1.8, H-7); 6.45 (1H, m, H-4). Found, %: C 38.03; H 3.77; N 6.29.
C7H8ClNO3S. Calculated, %: C 37.93; H 3.64; N 6.32.
Preparation of Esters of 7α-Chloro-3-methylceph-3-em-4-carboxylic Acid Sulfone and 7α-Chloro-
2-(dimethylaminomethylene)-3-methylceph-3-em-4-carboxylic Acid Sulfone (23, 24) (Typical Procedure). A
mixture of oxalyl chloride (98 µliter) and DMF (10µliter) was added with stirring at room temperature to a
suspension of 7α-chloro-3-methylceph-3-em-4-carboxylic acid sulfone (100 mg, 0.375 mmol) in dichloromethane
(20 ml). The reaction mixture was heated at 40°C for 20 min, cooled to room temperature, and concentrated at
reduced pressure. The residue, containing the acid chloride (21) of 7α-chloro-3-methyl-1,1-dioxoceph-3-em-
4-carboxylic acid, was dissolved in dichloromethane (15 ml). A mixture consisting of alcohol 22 (0.75 mmol) and
triethylamine (130 µliter, 0.93 mmol) was added to the obtained solution cooled to -5°C. The reaction mixture was
stirred for 20 min, diluted with dichloromethane (20 ml), washed with saturated NH4Cl solution (40 ml), and dried
over anhydrous Na2SO4. The solvent was evaporated under reduced pressure. The residue was fractionated on a
chromatographic column of silica gel, and esters 23, 24 were obtained.
Methyl Ester of 7α-Chloro-3-methylceph-3-em-4-carboxylic Acid Sulfone (23a) was obtained by the
typical procedure using methanol and was isolated from fractions (eluent ethyl acetate–hexane, 1 : 1) with Rf 0.42.
Mp 159-161°C, yield 24 mg (23%). 1H NMR spectrum, δ, ppm (J, Hz): 2.11 (3H, s, 3-CH3); 3.54 and 3.97 (2H, two
d, AB system, 2J = 18, SO2CH2); 3.88 (3H, s, OCH3); 4.73 (1H, m, H-6); 5.28 (1H, d, 3J = 1.5, H-7). Found, %: C
38.81; H 3.69; N 4.91. C9H10ClNO5S. Calculated, %: C 38.65; H 3.60; N 5.01.
Ethyl Ester of 7α-Chloro-3-methylceph-3-em-4-carboxylic Ester Sulfone (23b) was obtained by the
typical procedure using ethanol and was isolated from fractions (eluent ethyl acetate–hexane, 1 : 1) with Rf 0.52. Mp
1
3
106-107°C, yield 64 mg (58%). H NMR spectrum, δ, ppm (J, Hz): 1.33 (3H, t, J = 7, CH3); 2.09 (3H, s,
216

3-CH3); 3.68 and 3.97 (2H, two d, AB system, 2J = 18, SO2CH2); 4.28 and 4.44 (2H, two q, 3J = 7, CH2); 4.77 (1H,
m, H-6); 5.29 (1H, d, 3J = 1.5, H-7). Found, %: C 41.01; H 4.21; N 4.71. C10H12ClNO5S. Calculated, %: C 40.89; H
4.12; N 4.77.
Isopropyl Ester of 7α-Chloro-3-methylceph-3-em-4-carboxylic Acid Sulfone (23c) was obtained by the
typical procedure using 2-propanol and was isolated from fractions (eluent ethyl acetate–hexane, 1 : 1) with Rf 0,60.
Mp 161-163°C, yield 57 mg (50%). 1H NMR spectrum, δ, ppm (J, Hz): 1.44 (6H, d, 3J = 6, (CH3)2CH); 2.08 (3H, s,
3-CH3); 3.66 and 4.00 (2H, two d, AB system, 2J = 18, SO2CH2); 4.80 (1H, m, H-6); 5.17 (1H, m, 3J = 6, Me2CH);
5.26 (1H, d, 3J = 1.5, H-7). Found, %: C 43.04; H 4.67; N 4.41. C11H14ClNO5S. Calculated, %: C 42.93; H 4.59; N
4.55.
n-Butyl ester of 7α-Chloro-3-methylceph-3-em-4-carboxylic Acid Sulfone (23d) was obtained by the
typical procedure using n-butanol and was isolated from fractions (eluent ethyl acetate–hexane, 1 : 1) with Rf 0.70.
Mp 81-83°C, yield 46 mg (38%). 1H NMR spectrum, δ, ppm (J, Hz): 0.93 (3H, t, 3J = 6, (CH2)3CH3) 1.55-1.88 (4H,
m, 2CH2); 2.09 (3H, s, 3-CH3); 3.69 and 4.00 (2H, two d, AB system, 2J = 18, SO2CH2); 4.31 (2H, t, 3J = 6, CH2);
4.77 (1H, m, H-6); 5.27 (1H, d, 3J = 1.5, H-7). Found, %: C 44.90; H 5.11; N 4.24. C12H16ClNO5S. Calculated, %:
C 44.79; H 5.01; N 4.35.
Allyl Ester of 7α-Chloro-3-methylceph-3-em-4-carboxylic Acid Sulfone (23e) was obtained by the
typical procedure using allyl alcohol and was isolated from fractions (eluent ethyl acetate–hexane, 1 : 1) with Rf 0.70.
Mp 128-130°C, yield 55 mg (48%). 1H NMR spectrum, δ, ppm (J, Hz): 2.11 (3H, s, 3-CH3); 3.66 and 4.00 (2H, two
d, AB system, 2J = 18, SO2CH2); 4.80-4.83 (2H, m, CH2CH=CH2); 4.88 (1H, m, H-6); 5.27 (1H, d, 3J = 2, H-7); 5.27
(1H, d, J = 11, cis-CH=CH2); 5.44 (1H, d, J = 18, trans-CH=CH2); 5.89 (1H, ddt, J = 18, J = 11, J = 6, CH=CH2).
Found, %: C 43.38; H 4.11; N 4.42. C11H12ClNO5S. Calculated, %: C 43.21; H 3.96; N 4.58.
2-Chloroethyl Ester of 7α-Chloro-3-methylceph-3-em-4-carboxylic Acid Sulfone (23f) was obtained by
the typical procedure using 2-chloroethanol and was isolated from fractions (eluent ethyl acetate–hexane 1 : 1) with
Rf 0.75. Mp 136-138°C, yield 65 mg (53%). 1H NMR spectrum, δ, ppm (J, Hz): 2.11 (3H, s, 3-CH3); 3.71 and 3.95
2
3
3
(2H, two d, AB system, J = 18, SO2CH2); 3.81 (2H, t, J = 6, CH2CH2Cl); 4.45 and 4.60 (2H, two t, J = 6,
3
CH2CH2Cl); 4.80 (1H, m, H-6); 5,31 (1H, d, J = 2, H-7). Found, %: C 36.74; H 3.45; N 4.21. C10H11Cl2NO5S.
Calculated, %: C 36.60; H 3.38; N 4.27.
2,2,2-Trichloroethyl Ester of 7α-Chloro-3-methylceph-3-em-4-carboxylic Acid Sulfone (23g) was
obtained by the typical procedure using 2,2,2-trichloroethanol and was isolated from fractions (eluent ethyl acetate–
hexane, 1 : 3) with Rf 0.48. Mp 168-170°C, yield 74 mg (50%). 1H NMR spectrum, δ, ppm (J, Hz): 2.20 (3H, s, 3-
2
CH3); 3.73 and 4.00 (2H, two d, AB system, J = 18, SO2CH2); 4.80 (1H, m, H-6); 4.82 and 5.29 (2H, two d, AB
system, 2J = 12, CH2CCl3); 5.20 (1H, d, 3J = 2, H-7). Found, %: C 30.39; H 2.35; N 3.43. C10H9Cl4NO5S. Calculated,
%: C 30.25; H 2.28; N 3.53.
n-Pentyl Ester of 7α-Chloro-3-methylceph-3-em-4-carboxylic Acid Sulfone (23h) was obtained by the
typical procedure using n-pentanol and was isolated from fractions (eluent ethyl acetate–hexane, 1 : 1) with Rf 0.58.
Mp 62-63°C, yield 65 mg (52%). 1H NMR spectrum, δ, ppm (J, Hz): 0.91 (3H, t, 3J = 6, (CH2)4CH3); 1.17-1.91 (6H,
m, CH2(CH2)3CH3); 2.13 (3H, s, 3-CH3); 3.68 and 3.97 (2H, two d, AB system, 2J = 18, SO2CH2); 4.28 (2H, t, 3J = 6,
3
CH2C4H9); 4.75 (1H, m, H-6); 5.29 (1H, d, J = 2, H-7). Found, %: C 46.72; H 5.56; N 4.11. C13H18ClNO5S.
Calculated, %: C 46.50; 5.40; N 4.17.
n-Heptyl Ester of 7α-Chloro-3-methylceph-3-em-4-carboxylic Acid Sulfone (23i) was obtained by the
typical procedure using n-heptanol and was isolated from fractions (eluent ethyl acetate–hexane, 1 : 1) with Rf 0.58.
1
3
Mp 92°C, yield 61 mg (45%). H NMR spectrum, δ, ppm (J, Hz): 0.84 (3H, t, J = 6, CH2(CH2)5CH3); 1.00-1.75
(10H, m, CH2(CH2)5CH3); 2.11 (3H, s, 3-CH3); 3.69 and 3.98 (2H, two d, AB system, 2J = 18, SO2CH2); 4.26 (2H, t,
3
3J = 6, CH2(CH2)5CH3); 4.77 (1H, m, H-6); 5.11 (1H, d, J = 2, H-7). Found, %: C 49.82; H 6.29; N 3.71.
C15H22ClNO5S. Calculated, %: C 49.51; H 6.09; N 3.85.
Phenyl Ester of 7α-Chloro-3-methylceph-3-em-4-carboxylic Acid Sulfone (23j) was obtained by the
typical procedure using phenol and was isolated from fractions (eluent ethyl acetate–hexane, 1 : 1) with Rf 0.33.
217

Mp 141-143°C, yield 20 mg (15%). 1H NMR spectrum, δ, ppm (J, Hz): 2.17 (3H, s, 3-CH3); 3.73 and 4.04 (2H, two
d, AB system, 2J = 18, SO2CH2); 4.84 (1H, m, H-6); 5.37 (1H, d, 3J = 2, H-7); 7.15-7.57 (5H, m, C6H5). Found, %: C
49.31; H 3.60; N 4.07. C14H12ClNO5S. Calculated, %: C 49.20; H 3.54; N 4.10.
Benzyl Ester of 7α-Chloro-3-methylceph-3-em-4-carboxylic Acid Sulfone (23k) was obtained by the
typical procedure using benzyl alcohol and was isolated from fractions (eluent ethyl acetate–hexane, 1 : 1) with
Rf 0.80. Mp 143-145°C, yield 60 mg (42%). 1H NMR spectrum, δ, ppm (J, Hz): 2.06 (3H, s, 3-CH3); 3.66 and 3.93
(2H, two d, AB system, 2J = 18, SO2CH2); 4.73 (1H, m, H-6); 5.01 and 5.24 (2H, two d, AB system, 2J = 12, CH2Ph);
5.31 (1H, d, 3J = 2, H-7); 7.40 (5H, m, C6H5). Found, %: C 50.69; H 4.02; N 3.8. C15H14ClNO5S. Calculated, %: C
50.64; H 3.97; N 3.94.
Cinnamyl Ester of 7α-Chloro-3-methylceph-3-em-4-carboxylic Acid Sulfone (23l) was obtained by the
typical procedure using cinnamyl alcohol and was isolated from fractions (eluent ethyl acetate–hexane, 1 : 1) with Rf
0.66. Mp 130-132°C, yield 69 mg (48%). 1H NMR spectrum, δ, ppm (J, Hz): 2.08 (3H, s, 3-CH3); 3.62 and 3.95 (2H,
two d, AB system, 2J = 18, SO2CH2); 4.75 (1H, m, H-6); 4.93 (2H, d, 3J = 5, CH2CH=CH); 5.23 (1H, d, 3J = 2, H-7);
6.31 (1H, dt, 3J = 16, 3J = 5, CH2CH=CH); 6.75 (1H, d, 3J = 16, CH2CH=CH); 7.20-7.58 (5H, m, C6H5). Found, %: C
53.31; H 4.82; N 3.60. C17H18ClNO5S. Calculated, %: C 53.19; H 4.73; N 3.65.
Tetrahydrofurfuryl Ester of 7α-Chloro-3-methylceph-3-em-4-carboxylic Acid Sulfone (23m) was
obtained by the typical procedure using tetrahydrofurfuryl alcohol and was isolated from fractions (eluent ethyl
acetate–hexane, 1 : 1) with Rf 0.37. Mp 140-142°C, yield 11 mg (3%). 1H NMR spectrum, δ, ppm (J, Hz): 1.71-2.04
(6H, m, 3CH2, C4H7O); 2.15 (3H, s, 3-CH3); 3.55-3.73 (1H, m, 2-CH, C4H7O); 3.71 and 4.01 (2H, two d, AB system,
2J = 19, SO2CH2); 4.15-4.31 (2H, m, COOCH2); 4.84 (1H, m, H-6); 5.26 (1H, d, 3J = 2, H-7). Found, %: C 43.07; H
4.32; N 4.07. C12H14ClNO6S. Calculated, %: C 42.93; H 4.20; N 4.17.
4-Ethoxycarbonylphenyl Ester of 2E-7α-Chloro-2-(dimethylamino-methylene)-3-methylceph-3-em-4-
carboxylic Acid Sulfone (24) was obtained by the typical procedure using 4-ethoxycarbonylphenol and was isolated
1
from fractions (eluent ethyl acetate–hexane, 1 : 1) with Rf 0.08. Mp 173-175°C, yield 30 mg (17%). H NMR
spectrum, δ, ppm (J, Hz): 1.33 (3H, t, 3J = 7, CH3), 2.51 (3H, s, 3-CH3); 3.21 (6H, s, N(CH3)2); 4.37 (2H, q, 3J = 7,
3
3
3
CH2); 4.77 (1H, d, J = 2, H-6); 5.24 (1H, d, J = 2, H-7); 7.28 and 8.06 (4H, two d, J = 9, C6H4); 7.31 (1H, s,
=CHNMe2). Found, %: C 50.82; H 4.53; N 5.65. C20H21ClN2O7S. Calculated, %: C 51.23; H 4.51; N 5.97.
Mixture of tert-Butyl Ester of 2E-7α-Chloro-2-(dimethylaminomethylene)-3-methylceph-3-em-
4-carboxylic Acid Sulfone and tert-Butyl Ester of 2Z-7α-Chloro-2-(dimethylaminomethylene)-3-methylceph-3-
em-4-carboxylic Acid Sulfone 25a and 25b. A mixture consisting of oxalyl chloride (162 µliter, 1.86 mmol) and
DMF (48 µliter, 0.62 mmol) was added at room temperature in an atmosphere of argon to a stirred suspension of the
tert-butyl ester of 7α-chloro-3-methylceph-3-em-4-carboxylic acid sulphone (200 mg, 0.65 mmol) in
dichloromethane (20 ml). The reaction mixture was heated at 40°C for 20 min, cooled to room temperature, and
concentrated under reduced pressure. The residue was dissolved in dichloromethane (15 ml). Triethylamine
(130 µliter, 0.93 mmol) was added to the obtained solution, the mixture was stirred for 20 min, diluted with
dichloromethane (20 ml), washed with saturated NH4Cl solution (40 ml), and dried over anhydrous Na2SO4. The
solvent was evaporated under reduced pressure. The residue was fractionated on a chromatographic column of silica
gel (eluent ethyl acetate–hexane, 2 : 1). Fractions with Rf 0.44 were combined and evaporated. Yield 134 mg (58%).
1
According to the NMR spectrum (400 MHz) the substance is a mixture of E- and Z-isomers in a ratio of 3 : 1. H
NMR spectrum, δ, ppm (J, Hz): Z-25 – 1.53 (9H, s, C4H9); 2.22 (3H, s, 3-CH3); 3.35 (6H, s, N(CH3)2); 4.55 (1H, d, 3J
= 1.5, H-6); 5.24 (1H, d, 3J = 1.5, H-7); 7.02 (1H, s, =CHNMe2); E-25 – 1.53 (9H, s, C4H9); 2.21 (3H, s, 3-CH3); 3.01
3
3
(6H, s, N(CH3)2); 4.47 (1H, d, J = 1.5, H-6); 5.06 (1H, d, J = 1.5, H-7); 7.29 (1H, s, =CHNMe2). Found, %: C
47.99; H 5.75; N 7.40. C15H21ClN2O5S. Calculated, %: C 47.81; H 5.62; N 7.43.
Biological Screening. The cytotoxic properties of the synthesized substances were measured in vitro in
relation to monolayers of cancer cells HT-1080 (human fibrosarcoma), MG-22A (mouse hepatoma), B16 (mouse
melanoma), Neuro 2A mouse neuroblastoma) and of normal cells 3T3 (Swiss Albino mouse fibroblasts) and BHK
218

cells (golden hamster baby kidney fibroblasts), and also the concentration of nitric oxide radicals in Griess method
was determined in 96-well plastic panels using CV, MTT, and NR dyestuffs in accordance with the procedures [18,
19] tested by us previously [7].
REFERENCES
1.
2.
G. Veinberg, M. Vorona, I. Shestakova, I. Kanepe, and E. Lukevics, Curr. Med. Chem., 10, 1741 (2003).
J. B. Doherty, B. M. Ashe, L. W. Argenbright, P. L. Barker, R. J. Bonney, G. O. Chandler, M. E. Dahlgren,
C. P. Dorn, Jr., P. E. Finke, R. A. Firestone, D. Fletcher, W. K. Hagmann, R. Mumford, L. O’Grady,
A. L. Maycock, J. M. Pisano, S. K. Shah, K. R. Thompson, and M. Zimmerman, Nature, 322, 192 (1986).
W. T. Han, A. K. Trehan, J. J. K. Wright, M. E. Federici, S. M. Seller, and N. A. Meanwell, Bioorg, Med.
Chem., 3, 1123 (1995).
A. D. Borthwick, G. Weingarten, T. M. Haley, M. Tomaszewski, W. Wang, Z. Hu, J. Bedard, H. Jin,
L. Yuen, and T. S. Mansour, Bioorg. Med. Chem. Lett., 8, 365 (1998).
N. E. Zhou, D. Guo, G. Thomas, A. V. N. Reddy, J. Kaleta, E. Prisima, R. Menard, R. G. Micetich, and
R. Singh, Bioorg. Med. Chem. Lett., 13, 139 (2003).
3.
4.
5.
6.
J. B. Doherty, B. M. Ashe, P. L. Barker, T. J. Blacklock, J. W. Butcher, G. O. Chandler, M. E. Dahlgren,
P. Davies, C. P. Dorn, Jr., P. E. Finke, R. A. Firestone, W. K. Hagmann, T. Halgren, W. B. Knight,
A. L. Maycock, M. A. Navia, L. O’Grady, J. M. Pisano, S. K. Shah, K. R. Thompson, H. Weston, and
M. Zimmerman, J. Med. Chem., 33, 2513 (1990).
7.
8.
G. A. Veinberg, I. Shestakova, N. Grigan, D. Musel, I. Kanepe, I. Domrachova, V. Griroryeva, O. Zharkova,
I. Turovskis, I. Kalvinsh, A. Strakovs, and E. Lukevics, Eur. J. Med. Chem., 33, 755 (1998).
K. Taniguchi, P. Yang, J. Jett, E. Bass, R. Meyer, Y. Wang, C. Deschamps, and W. Liu, Clin. Cancer Res., 8,
1115 (2002).
9.
F. Nozawa, M. Hirota, A. Okabe, M. Shibata, T. Iwamura, Y. Haga, and M. Ogawa, J. Surg. Res., 94, 153
(2000).
10.
11.
12.
K. Iwatsuki, E. Kumara, T. Yoshimine, H. Nakagawa, M. Sato, and T. Hayakawa, Neurol. Res., 22, 465
(2000).
G. Veinberg, M. Vorona, D. Musel, H. Kazhoka, I. Shestakova, I. Kanepe, I. Domracheva, A. Strakovs, and
E. Lukevits, Khim. Geterotsikl. Soedin., 1504, (1998). [Chem. Heterocycl. Comp., 34, 1276 (1998)].
G. Veinberg, R. Bokaldere, K. Dikovskaya, M. Vorona, D. Musel, H. Kazhoka, I. Turovskis, I. Shestakova,
I. Kanepe, I. Domracheva, and E. Lukevics, Khim. Geterotsikl. Soedin., 1494 (1998). [Chem. Heterocycl.
Comp., 34, 1266 (1998)].
13.
14.
15.
N. Grigan, G. Veinberg, I. Shestakova, I. Kanepe, and E. Lukevics, Khim. Geterotsikl. Soedin., 1424 (2000).
[Chem. Heterocycl. Comp., 36, 1232 (2000)].
G. Veinberg, M. Vorona, N. Grigan, I. Kanepe, I. Shestakova, A. Strakovs, and E. Lukevics, Khim.
Geterotsikl. Soedin., 847 (2000). [Chem. Heterocycl. Comp., 36, 744 (2000)].
M. Alpegiani, A. Baici, P. Bissolino, P. Carminati, G. Cassinelli, S. Del Nero, G. Franceschi, P. Orezzi, E.
Perrone, V. Rizzo, and N. Sacchi, Eur. J. Med. Chem., 27, 875 (1992).
16.
17.
W. H. W. Lunn and P. A. Hipskind, Tetrahedron Lett., 33, 6291 (1992).
US Department of Health and Human Services, Guidance Document on Using in vitro Data to Estimate in
vivo Starting Doses for Acute Toxicity, National Institute of Health (2001).
18.
19.
D. J. Fast, R. C. Lynch, and R. W. Leu, J. Leukocyt. Biol., 52, 255 (1992).
P. J. Freshney, in Culture of Animal Cells (A Manual of Basic Technique), Wiley-Liss, New York (1994).
p. 296.
219