Structure-activity relationships of quaternary protoberberine alkaloids having an antimalarial activity

Article abstract of DOI:10.1016/S0223-5234(99)00127-0

Seventeen quaternary protoberberine alkaloids related to berberine 1 were tested for antimalarial activity in vitro against Plasmodium falciparum and structure-activity relationships are proposed. The activity of the protoberberine alkaloids was influenced by the type of the oxygen substituents on rings A, C and D and the position of the oxygen functions on ring D. The position of the oxygen functions on ring D and the type of the oxygen substituents at the C-13 position (ring C) strongly influenced the activity. Shifting the oxygen functions at C-9 and C-10 to C-10 and C-11 on ring D resulted in a significant increase in the activity. Compounds bearing a methylenedioxy function at C-2 and C-3 (ring A) or C-9 and C-10 (ring D) showed higher activity than those which have methoxy groups at the same positions. Introduction of a methoxy group into the C-1 position (ring A) decreased the activity. Replacement of a hydroxy group at C-2 or C-3 (ring A) by a methoxy group led to a reduction in the activity. Displacement of a hydroxy function at C-13 (ring C) by the oxygen substituents such as OMe, OEt, OCOOEt, and OCON(Me)₂ reduced the activity. In the same replacement at C-9 (ring D), the activity depended upon the type of the oxygen function. Six protoberberines displayed more potent activity than berberine 1. The activity decreased in the order: 10, 11, 17 and 18 > 7 and 8 > 1.

Full text of DOI:10.1016/S0223-5234(99)00127-0

Eur. J. Med. Chem. 34 (1999) 1077−1083
© 1999 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
1077
Short communication
Structure-activity relationships of quaternary protoberberine alkaloids having an
antimalarial activity
Kinuko Iwasaa*, Yumi Nishiyamaa, Momoyo Ichimarua, Masataka Moriyasua, Hye-Sook Kimb,
Yusuke Watayab, Takao Yamoric, Turuo Takashid, Dong-Ung Leee
aKobe Pharmaceutical University, 4-19-1 Motoyamakita, Higashinada-ku, Kobe 658-8558, Japan
bFaculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, Japan
cDivision of Experimental Chemotherapy, Cancer Chemotherapy Center, 1-37-1 kamiikebukuro, Toyoshima-ku, Tokyo 170-8455, Japan
dInstitute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 yayoi, bunkyo-ku, Tokyo 113-0000, Japan
eDepartment of Biochemistry, College of Natural Science, Dongguk University, Kyongju Kyongbuk 780-714, Korea
(Received 20 April 1999; revised 28 June 1999; accepted 30 June 1999)
Abstract – Seventeen quaternary protoberberine alkaloids related to berberine 1 were tested for antimalarial activity in vitro against
Plasmodium falciparum and structure-activity relationships are proposed. The activity of the protoberberine alkaloids was influenced by the
type of the oxygen substituents on rings A, C and D and the position of the oxygen functions on ring D. The position of the oxygen functions
on ring D and the type of the oxygen substituents at the C-13 position (ring C) strongly influenced the activity. Shifting the oxygen functions
at C-9 and C-10 to C-10 and C-11 on ring D resulted in a significant increase in the activity. Compounds bearing a methylenedioxy function
at C-2 and C-3 (ring A) or C-9 and C-10 (ring D) showed higher activity than those which have methoxy groups at the same positions.
Introduction of a methoxy group into the C-1 position (ring A) decreased the activity. Replacement of a hydroxy group at C-2 or C-3 (ring
A) by a methoxy group led to a reduction in the activity. Displacement of a hydroxy function at C-13 (ring C) by the oxygen substituents such
as OMe, OEt, OCOOEt, and OCON(Me)2 reduced the activity. In the same replacement at C-9 (ring D), the activity depended upon the type
of the oxygen function. Six protoberberines displayed more potent activity than berberine 1. The activity decreased in the order: 10, 11, 17
and 18 > 7 and 8 > 1. © 1999 Éditions scientifiques et médicales Elsevier SAS
in vitro antimalarial activity / structure-activity relationships / protoberberinium salts
1. Introduction
In the present study, the antimalarial activity of 17
protoberberinium salts, in which the type and/or position
of the oxygen substituents such as hydroxy, methylene-
dioxy, methoxy, ethoxycarbonyloxy, and N, N-dimethyl-
carbamoyloxy functions on the aromatic rings A, C and D
are different from 1 or 2 (table I), were examined using
the selectivity indices as an indication of the activity. The
results on the study of the structure-antimalarial activity
relationships of the tested alkaloids are discussed.
In Japan, and other Asian countries, berberine 1 and the
extracts of Phellodendri cortex (Obaku in Japanese) and
Coptidis rhizoma (Oren in Japanese) are used in the
treatment of diarrhoea and other gastrointestinal diseases.
Berberine and its relatives exhibit several types of bio-
logical activity [1]. We found that some of the 8- and
13-alkylderivatives of berberine 1 and palmatine 2 pos-
sessed antimicrobial and antimalarial activities [2–6]. We
have recently shown that a 13-hydroxy derivative of 1
had similar antimalarial activity to that of 1 [6].
2. Chemistry
The protoberberinium salts 3–13 and 17–20 have been
prepared or isolated from natural sources previously [2,
5, 7, 8]. Palmatrubine 14 was prepared according to the
method applied to the synthesis of 5 [2]. The carboethoxy
*Correspondence and reprints

1078
Table I. Protoberberinium salts.
Table III. Mass spectral data and melting points of the protober-
berinium salts (15, 16, 21 and 22).
Com- M.p. °C
Formula
LSIMS
HR-LSIMS
found
m/z [M – Cl]+ calcd.
pound
(dec)
15
160–166
C
22H20NO6
394
393
424
423
394.1289 394.1310
393.1449 393.1458
424.1395 424.1404
423.1556 423.1565
16
21
22
210–214 C22H21N2O5
155–158 C23H22NO7
194–200 C23H23N2O6
and N,N-dimethylcarbamoyl derivatives of berberrubine
5 and 13-hydroxyberberine 17 (15 and 21, and 16 and 22,
respectively) were prepared by the reaction with ethyl
chloroformate and N,N-dimethylcarbamoyl chloride, re-
spectively, in the presence of triethylamine or pyridine.
1H-NMR (table II), LSIMS (table III), and HR-LSIMS
(table III) data were consistent with the assigned struc-
tures.
R1
R2
R3
R4
R5
R6
R7
1
2
3
4
5
6
7
8
H
H
H
H
H
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH2O
OMe
OMe H
OMe H
OMe H
OMe H
OMe H
OMe H
H
H
H
Me
Me
H
H
H
Me
Me
OMe OMe OMe
OCH2O OMe
OMe OMe OMe
OCH2O
OCH2O
OCH2O
OH
OMe
OCH2O
OCH2O
OCH2O
OCH2O
H
H
9
OMe OMe
OCH2O
3. Results and discussion
10
11
12
13
14
15
16
17
18
19
20
21
22
H
H
OMe
OMe OMe H
OMe OMe Me
OMe H
OMe H
OMe H
OMe H
OCH2O
Protoberberinium salts 6–22 were tested in vitro
against human malaria Plasmodium falciparum FCR-3.
The antimalarial activity of each compound was deter-
mined as a percentage reduction. The compound concen-
tration required to inhibit cell growth by 50% was
expressed as IC50 (table IV). From the evaluation of the
toxicity of the compounds for mammalian cells, the
concentration causing a 50% reduction of cell growth
(GI50) of mouse mammary FM3A cells, a model of the
host, was determined (table IV). The GI50/IC50 ratios for
the compounds were calculated as selectivity indices
(table IV). These ratios were used as an evaluation of
antimalarial activity. The results are presented in table IV.
OMe OH
OH
OMe OMe OH
H
Me
H
H
OMe OMe
OCH2O
OCH2O
OCH2O
OCH2O
OCH2O
OCH2O
OCH2O
OCH2O
OCOOEt
OCON(Me)2 OMe H
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe H
OMe H
OMe H
OMe H
OMe H
OMe H
OH
O–
OMe
OEt
OCOOEt
OCON(Me)2
Table II. 1H-NMRa data of the protoberberinium salts 14–16, 21 and 22.
C-9 or C-13
C-9 or C-13 H-5
OCON(CH3)2
H-6
2-OMe 3-OMe 10-OMe 9-OMe OCH2O H-4
H-1
H-11
7.96
H-12
7.71
H-13
8.65
H-8
OCOOCH2CH3
14
3.25
4.87
4.00
3.92
4.05
7.00
7.58
9.73
2H, t (6.0)
2H, t (6.0)
3H, s 3H, s 3H, s
1H, s 1H, s 1H, d (9.0) 1H, d (9.0)
1H, s 1H, s
15 4.40
2H, q (7.0)
1.43
3.26
4.94
4.11
6.11
6.97
7.69
8.24*
8.21*
8.82
9.79
3H, t (7.0)
2H, t (6.5)
2H, t (6.5)
3H, s
2H, s
1H, s 1H, s 1H, d (9.0) 1H, d (9.0)
1H, s 1H, s
16
3.09 3.31 3.26
4.95
4.09
6.11
6.97
7.68
8.19*
8.18*
8.80
9.73
3H, s 3H, s 2H, t (6.5)
2H, t (6.5)
3H, s
2H, s
1H, s 1H, s 1H, d (9.0) 1H, d (9.0)
1H, s 1H, s
21 4.31
1.31
3.21
4.95
4.13*
3H, s
4.24* 6.12
3H, s 2H, s
7.03
7.67
8.19*
8.01*
9.88
2H, q (7.0)
3H, t (7.0)
2H, t (6.0)
overlap
1H, s 1H, s 1H, d (9.0) 1H, d (9.0)
1H, s
22
2.98 3.42 3.20
4.92
4.12*
3H, s
4.22* 6.11
3H, s 2H, s
7.00
7.63
8.17
7.96
9.84
3H, s 3H, s 2H, t (6.0)
overlap
1H, s 1H, s 1H, d (9.0) 1H, dd (9.0, 0.5)
1H, d (0.5)
*
aCoupling constants (Hz in parentheses). Assignments may be interchanged.

1079
Table IV. In vitro antimalarial activity of protoberberinium salts 1–22.
50% growth inhibition (µM)
Selectivity indexes
Distribution of selectivity indexes
Plasmodium falciparum
mouse mammary mouse mammary cells/
human tumour cells/
Plasmodium falciparum
the number (selectivity indexes: GI50a/IC50)
cells
Plasmodium falciparum
FCR-3
IC50
FM3A
GI50
GI50/IC50
< 150
150–300 > 300
1
2
3
4
5
6
7
8
9
0.27b
6.4b
2.1b
7.0b
2.2b
0.60
0.25
0.26
0.50
0.18
> 12b
> 27b
> 11b
> 25b
5.0b
12
14
> 14
> 14
> 19
> 44b
> 4.0b
> 5.2b
> 3.5b
2.3b
20
56
> 54
> 28
>106
27
38
38
38
38
7
4 (> 370)
23
6
7
7
2 (300–400) 6 (> 400)
10
1 (300–400) 5 (400–550) 19
(> 634)
11
12
13
14
15
16
17
18
19
20
21
22
0.29
0.67
1.0
0.90
2.2
> 15
> 17
> 16
5.0
> 52
> 25
> 16
5.6
4.0
2.0
1.9
> 41
> 38
> 41
20
0.16
17
> 22
> 68
> 53
12
2
25
0.56
0.78
2.4
0.084
0.68
5.3
38 (> 454)
37
7
Quinine 0.11
100
910
20 (< 38)
17 (> 1100)
b
aGI50 values presented in table V. These data have been previously reported [6].
Introduction of a methoxy group at the C-1 position
(ring A) of 1 caused a decrease in antimalarial activity
(compare 6 with 1) (table IV). Replacement of methoxy
groups at the C-9 and C-10 positions (ring D) of 1 or 3 by
a methylenedioxy group increased the activity (compare
7 with 1 or 8 with 3) (table IV) and the same replacement
at the C-2 and C-3 positions (ring A) of 9 resulted in an
increase in the activity (compare 9 with 8) (table IV).
Shifting methoxy groups at C-9 and C-10 of 1 or 3 to
C-10 and C-11 caused a significant increase in the activity
(compare 10 with 1 or 11 with 3) (table IV). Displace-
ment of a methoxy group at C-3 (ring A) of 2 by a
hydroxy function increased the activity and the same
replacement at the C-2 position (ring A) of 4 also
increased the activity (compare 12 with 2 and 13 with 4)
(table IV). Substitution of a methoxy group at C-9 (ring
D) of 2 by a hydroxy group increased the activity
(compare 14 with 2) (table IV), though the same displace-
ment at C-9 of 1 resulted in a decrease in the activity
(compare 5 with 1) as shown in the previous data [6].
Replacement of a hydroxy group at C-9 (ring D) of 5 by
OCOOEt did not notably change its activity (compare 15
with 5) (table IV), whereas replacing a hydroxy function
at C-9 of 5 by OCON(Me)2 caused an increase in the
activity (compare 16 with 5) (table IV).
We have found that the inhibitory effect of 13-
hydroxyberberine 17 was comparable to that of berberine
1 [6]. However, the inhibitory effects of phenolbetaine
form 18 have been reported to be much less than that of
1 [9]. Thus, a comparison of the inhibitory effect between
17 and 18 was carried out. As a result, both 17 and 18
displayed a similar inhibitory effect in vitro. Substitution
of a hydroxy group at the C-13 position of 17 by OMe or
OEt or OCOOEt or OCON(Me)2 showed a reduction in
the activity (compare 19–22 with 17) (table IV).
Among the tested salts, 7, 8, 10, 11, 17 and 18
exhibited much more potent activity (selectivity index
over 50) than berberine 1.
The selectivity indexes (table IV) of 1, 7, 10 and 17
were also determined using the 50% growth-inhibitory
concentration (GI50, in table V) derived from the re-
sults [10] of in vitro cytotoxity tests on 38 human tumour

1080
Table V. Cytotoxicities of protoberberinium salts 1–5, 7, 10 and 17 against various human cell lines.
Human tumour
cell line
Compounds / Cytotoxicity (GI50 in µM)a
1
2
3
4
5
7
10
17
Quinine
Breast
HBC-4
BSY-1
HBC-5
MCF-7
MDA-MB-231
CNS
4.4
5.2
30
24
50
76
27
b
b
b
5.1
2.8
29
10
47
33
12
99
50
b
35
23
39
20
56
19
10
11
12
39
17
7.1
79
27
b
b
b
b
b
b
b
35
b
55
12
U251
SF-268
SF-295
SF-539
SNB-75
SNB-78
Colon
6.9
7.7
4.8
23
30
35
55
48
b
b
b
5.7
14
1.8
17
30
37
76
91
b
81
b
28
30
27
25
22
26
39
27
b
37
5.9
52
35
27
b
b
42
b
b
b
b
b
b
b
b
b
b
37
b
b
55
b
HCC2998
KM-12
HT-29
20
4.3
41
42
b
b
71
b
b
b
16
4.6
31
34
b
96
35
b
b
b
51
27
43
35
50
32
38
7.6
79
53
b
44
33
b
b
b
b
b
b
b
b
b
76
b
41
WiDr
HCT-15
HCT-116
Lung
b
43
30
b
27
b
37
b
NCI-H23
NCI-H226
NCI-H522
NCI-H460
A549
DMS273
DMS114
Melanoma
LOX-IMVI
Ovarian
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
Renal
8.2
8.1
5.8
12
41
5.9
3.9
b
7.1
14
4.3
16
77
4.2
2.1
62
40
28
b
b
40
25
48
19
37
11
22
25
42
18
36
12
19
48
20
6.0
81
b
16
b
b
56
20
b
b
b
b
b
b
b
87
29
100
86
b
58
28
b
b
b
52
85
31
33
b
25
b
32
40
b
b
31
6.2
31
39
17
b
46
b
b
b
b
5.2
22
29
14
b
34
b
b
b
b
44
47
31
40
49
21
36
31
32
b
29
77
b
91
b
b
b
b
b
b
b
b
72
b
b
RXF-631L
ACHN
59
b
b
b
64
b
b
b
26
17
b
b
b
b
b
b
b
79
Stomach
St-4
MKN1
b
b
b
b
95
b
83
42
32
13
28
16
b
b
84
76
b
36
38
31
27
35
31
b
b
b
39
b
34
96
b
b
b
b
b
b
30
74
38
79
b
76
26
14
28
16
93
4.2
11
24
9.8
MKN7
MKN28
MKN45
MKN74
53
b
71
aThe cytotoxicity GI50 values are the concentrations corresponding to 50% growth inhibition. GI50 values were not presented previously [10].
bGI50 value is > 100 µM.
cell lines (seven lung, six colon, six CNS, six stomach,
five ovarian, five breast, two renal, and one melanoma),
the hosts for FM3A cells. The selectivity indexes of
inactive 2–5 and the antimalarial drug quinine were also
determined as the control experiments. 13-
Hydroxyberberine 17 and compound 10 displayed a

1081
significant activity, coptisine 7 less potent activity, ber-
berine 1 much less activity, and compounds 2–5 inactiv-
ity.
Analyses were made on a Cosmosil 5C18-AR reversed-
phase column (20 i.d. × 250 mm or 4.6 i.d. × 150 mm)
eluting with H2O (0.05% TFA)/MeOH (0.05% TFA),
A/B, initial (30% of B), 20 min (100% of B).
Berberine 1 and palmatine 2 were purchased. Proto-
berberinium salts 3 [7], 4 [7], 5 [2], 6 [5], 7 [5], 9–11 [5],
and 17–20 [5] had previously been prepared. Corysamine
8 was obtained by oxidation of tetrahydrocorysamine [11]
isolated from Corydalis incisa Pers.
Columbamine 12 [8] and dehydrocorybulbine 13 [8]
were natural products isolated from C. turtschaninovii
Besser forma yanhusuo Y.H. Chou and C.C. Hsu.
4. Conclusion
From the structure-activity point of view, some fea-
tures can be pointed out. On the basis of the results
derived from examinations with protoberberinium salts, it
appears that the position and the type of the oxygen
substituents on rings A, C and D had an influence on the
antimalarial activity. The activity was strongly influenced
by the position of the oxygen functions on ring D and the
type of the oxygen substituents at the C-13 position (ring
C). Compounds with the oxygen functions at C-10 and
C-11 on ring D displayed more activity than those which
bear the same substituents at C-9 and C-10. Compounds
bearing a methylenedioxy function at C-2 and C-3 or C-9
and C-10 on ring A or D showed higher activity than
those which have methoxy groups at the same position.
Introduction of a methoxy group into the C-1 position on
ring A caused a decrease in the activity. Replacement of
a hydroxy group at C-2 or C-3 on ring A by a methoxy
group led to a reduction in the activity. Displacement of
a hydroxy function at C-13 on ring C by an OMe, OEt,
OCOOEt or OCON(Me)2 function decreased the activity.
In the same replacement at C-9 on ring D, the activity
depended on the type of the oxygen substituents. Among
the potent protoberberinium salts under investigation, the
activity decreased in the order: 10, 11, 17 and 18 > 7 and
8 > 1. The most active compound may be comparable
with the antimalarial drug quinine in activity. Studies on
in vivo antimalarial activity of the most potent alkaloids
are in progress.
5.1.1. Preparation of palmatrubine 14
Palmatine (1 g) was heated at 240–250 °C in a dry
oven under vacuum (20–30 mm Hg) for 10 min. The
crude product was recrystallized from EtOH to give 14
(870 mg, 90%), m.p. 288–295 °C (dec.); LSIMS m/z 338.
1
For H-NMR data see table II.
5.1.2. Preparation of the ethoxycarbonyl derivative of
berberrubine 15
A solution of ClCOOEt (1.2 g) in CHCl3 (3 mL) was
added dropwise to a mixture of berberrubine 5
(300 mg) [2] and (Et)3N (900 mg) in CHCl3 (50 mL).
The mixture was stirred at room temperature for 30 min
and evaporated in vacuo. (Me)2CO was added to the
residue and the resulting crystals were collected, which
recrystallized from MeOH-(Me)2CO to give 15 (229 mg,
1
64%). For H-NMR, LSIMS, and HR-LSIMS data and
melting point see tables II and III.
5.1.3. Preparation of N,N-dimethylcarbamoyl derivative
of berberrubine 16
A solution of (Me)2NCOCl (0.7 mL) in CHCl3 (2 mL)
was added dropwise to a mixture of berberrubine 5
(200 mg) and (Et)3N (0.5 mL) in CHCl3 (50 mL). The
mixture was stirred at room temperature for 5 h. To the
reaction mixture (Me)2NCOCl (0.5 mL) was further
added and the mixture was allowed to stir at room
temperature overnight. (Me)2NCOCl (0.5 mL) was added
to the solution which was further stirred overnight.
Solvent was removed under reduced pressure and
(Me)2CO was added to the residue. The resulting crystals
were recrystallized from MeOH-(Me)2CO to give 16
5. Experimental protocols
5.1. Chemistry
Melting points were determined on a Yanako Mi-
cromelting Point apparatus and are uncorrected. 1H-NMR
spectra were recorded on a Varian VXR-500 (500 MHz)
spectrometer using tetramethylsilane as an internal stan-
dard and CD3OD as solvent. Mass spectra were deter-
mined on a Hitachi M-4100 instrument. The secondary
ion mass spectra (LSIMS) were measured using glycerol
as matrix. Preparative HPLC and HPLC analyses were
performed on a Hitachi M-6250 Intelligent Pump (6 mL/
min) and a Hitachi M-6200 Intelligent Pump (1 mL/min),
respectively, and a Hitachi L-4000 UV detector (280 nm).
1
(196 mg, 82%). For H-NMR, LSIMS, and HR-LSIMS
data and melting point see tables II and III.
5.1.4. Preparation of ethoxycarbonyl derivative of 13-
hydroxyberberine 21
A solution of ClCOOEt (0.7 mL) in CHCl3 (2 mL) was
added dropwise to a mixture of 13-hydroxyberberine 17
(200 mg) [5] and (Et)3N (0.5 mL) in CHCl3 (20 mL). The

1082
mixture was stirred at room temperature for 1 h and
evaporated in vacuo. (Me)2CO was added to the residue
and the resulting crystals were recrystallized from H2O-
MeOH to give 21 (100 mg, 46%). For 1H-NMR, LSIMS,
and HR-LSIMS data and melting point see tables II and
III.
numbers were measured using a microcell counter CC-
130 (Toa Medical Electric Co., Japan). The GI50 value
refers to the concentration of the compound necessary to
inhibit the increase in cell density at 48 h by 50% of
control. Selectivity refers to the mean of the GI50 value
for FM3A cells per the mean of the IC50 value for P.
falciparum.
5.1.5. Preparation of N,N-dimethylcarbamoyl derivative
of 13-hydroxyberberine 22
5.2.4. Selective toxicity using human tumour cells
The in vitro cytotoxicity assay was carried out accord-
ing to the method [10, 16] of a modified National Cancer
Institute protocol [17]. We screened eight compounds,
1–5, 7, 10, and 17 against a panel of 38 human cancer cell
lines (seven lung, six CNS, six colon, six stomach, five
breast, five ovarian, two renal, and one melanoma). The
50% growth-inhibitory concentration (GI50) for any
single cell line is an index of cytotoxicity or cytostasis.
The selective toxicity was estimated from the GI50/IC50
ratio between the malaria parasites and human tumour
cells which served as a model host.
A solution of (Me)2NCOCl (0.5 mL) in CHCl3 (2 mL)
was added dropwise to a mixture of 13-hydroxyberberine
17 (50 mg) and (Et)3N (0.5 mL) in CHCl3 (20 mL). The
mixture was stirred at room temperature overnight. The
reaction was followed by HPLC. To the solution 3 drops
of pyridine was added and the mixture was allowed to stir
at room temperature for 3 days. The solvent was evapo-
rated under reduced pressure. The residue was purified by
1
preparative HPLC to give 22 (20 mg, 36%). For H-
NMR, LSIMS, and HR-LSIMS data and melting point
see tables II and III.
5.2. In vitro antimalaria screening
Acknowledgements
5.2.1. Parasites and mammalian cells
This work was supported in part by a Grants-in-Aid for
Scientific Research on Priority Areas (08281105) from
the Ministry of Education, Science, Culture and Sports,
Japan.
Plasmodium falciparum (ATCC 30932, FCR-3 strain)
was used in our study. P. falciparum was cultivated by a
modification of the method of Trager and Jensen [12].
Mouse mammary tumour FM3A cells (wild-type, sub-
clone F28-7) in culture were used as a control for
mammalian cell cytotoxicity [13].
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