Synthesis and antitumour effect of the melanogenesis-based antimelanoma agent N-Propionyl-4-S-cysteaminylphenol

Article abstract of DOI:10.1016/S0006-2952(98)00090-2

Chemotherapy of malignant melanoma is still a great challenge, as no effective drugs are available. The development of melanogenesis-based drugs is a promising area of research because melanogenesis is a unique biochemical pathway operating only in melano

Full text of DOI:10.1016/S0006-2952(98)00090-2

Biochemical Pharmacology, Vol. 55, pp. 2023–2029, 1998.
© 1998 Elsevier Science Inc. All rights reserved.
ISSN 0006-2952/98/$19.00 ϩ 0.00
PII S0006-2952(98)00090-2
Synthesis and Antitumour Effect of the
Melanogenesis-based Antimelanoma Agent
N-Propionyl-4-S-cysteaminylphenol
Manju Tandon,* Panakkezhum D. Thomas, Mohammed Shokravi, Shradha Singh,
Satinder Samra, Daniel Chang and Kowichi Jimbow*†‡
*DIVISION OF DERMATOLOGY & CUTANEOUS SCIENCES, FACULTY OF MEDICINE, UNIVERSITY OF ALBERTA,
EDMONTON, ALBERTA, CANADA; AND †DEPARTMENT OF DERMATOLOGY, SAPPORO MEDICAL UNIVERSITY,
SAPPORO, JAPAN
ABSTRACT. Chemotherapy of malignant melanoma is still a great challenge, as no effective drugs are
available. The development of melanogenesis-based drugs is a promising area of research because melanogenesis
is a unique biochemical pathway operating only in melanoma cells (and their normal counterparts) so that the
tumour can be targeted. We have been using cysteinylphenol, a sulphur-containing analogue of tyrosine, and
derivatives for that purpose. N-Acetyl-4-S-cysteaminylphenol was found to have the best antimelanoma effect
in cell culture systems and in mice bearing B16 melanoma tumours. It also caused depigmentation of the skin,
suggesting the possibility of use as a hypopigmenting agent. To improve the efficiency of the drug, we thought
of replacing the acetyl group in N-acetyl-4-S-cysteaminylphenol with a propionyl group in the hope that
increased hydrophobicity would increase the cellular uptake of the drug. N-Propionyl-4-S-cysteaminylphenol
was synthesized by condensing 4-hydroxythiophenol with 2-ethyl-2-oxazoline. The drug showed both cytostatic
and cytocidal effects in a human melanotic melanoma cell line. The drug was found to be a good depigmenting
agent for the black hair follicles of C57 black mice when given s.c. for 14 days. A 10-day treatment with
N-propionyl-4-S-cysteaminylphenol at 300 mg/kg body weight reduced the growth rate of B16 melanoma s.c.
tumours in mice by 36%. The propionyl derivative was found to increase the life span of mice bearing melanoma
more effectively than did the acetyl derivative. BIOCHEM PHARMACOL 55;12:2023–2029, 1998. © 1998 Elsevier
Science Inc.
KEY WORDS. tyrosinase; cysteaminylphenol; depigmenting potency; melanocytotoxicity; melanoma chemo-
therapy
Melanocytes and their neoplastic counterpart, malignant
melanoma cells, possess a unique metabolic pathway, mel-
anin synthesis, that is highly accelerated in the latter [1].
Currently, our research group has been involved in the
development of depigmenting and antimelanoma agents by
exploiting this unique metabolic pathway of melanocytes
and melanoma cells. The key enzyme of this pathway is
tyrosinase, which is always retained in vivo in melanoma
cells, and, hence, melanin metabolites are always being
synthesized, even though the whole tumour becomes non-
pigmented and amelanotic. In our earlier studies, we dem-
onstrated that melanin/melanosomes, per se, if overpro-
duced, is/are toxic to melanocytes and may kill them [2].
This cytotoxicity due to melanin synthesis may be under-
stood if one looks at 1) the role of tyrosinase in the melanin
biosynthesis pathway, in which initial oxidation involves
hydroxylation of tyrosine to form DOPA§ and 2) the
oxidation of DOPA to form o-quinone (DOPA quinone),
which is converted subsequently into dihydroxyindole de-
rivatives through auto-oxidation/hydroxylation. The major
melanin polymers and metabolites, therefore, can promote
the oxidation of some compounds and the reduction of
others. Products of tyrosinase action (melanin metabolites)
can thus exhibit useful chemical cytotoxicity for melanoma
cells, providing a rational basis for the development of
targeted chemotherapy using tyrosine analogues [1, 3].
We recently synthesized the sulphur homologue of ty-
rosine, cysteinylphenol, and its amine derivative, cysteami-
nylphenol (4-S-CAP), for the purpose of melanoma che-
motherapy. 4-S-CAP showed a potent depigmenting and
antimelanoma effect, but it was not stable and caused
non-specific cytotoxicity affecting even non-melanocytic
‡ Corresponding author: Kowichi Jimbow, M.D., Ph.D., FRCPC, Division
of Dermatology & Cutaneous Sciences, 260G Heritage Medical Research
Centre, University of Alberta, Edmonton, Alberta T6G 2S2, Canada. Tel.
(403) 492-2425; FAX (403) 492-7715.
§ Abbreviations: DOPA, 3,4-dihydroxyphenyl alanine; ILS, increased life
span; 4-S-CAP, 4-S-cysteaminylphenol; N-acetyl-4-S-CAP, N-acetyl-4-S-
cysteaminylphenol; and N-propionyl-4-S-CAP, N-propionyl-4-S-cys-
teaminylphenol.
Received 5 September 1997; accepted 4 February 1998.

2024
M. Tandon et al.
cells rich in monoamine oxidase [4]. To overcome this
difficulty, the N-acetyl derivative of 4-S-CAP, N-acetyl-4-
S-CAP, was synthesized. Our in vivo melanocytotoxicity
studies [5, 6] indicated that N-acetyl-4-S-CAP would be a
potent melanocytotoxic agent in vivo. Furthermore, the i.p.
injection of N-acetyl-4-S-CAP inhibited the lung colony
formation by B16F10 melanoma cells in C57 black mice. It
reduced the lung colony formation by 25% of the mean
control [7]. This in vivo antimelanoma effect of N-acetyl-
4-S-CAP was confirmed with human tumour xenograft in
nude mice. We found that 400 mg/kg of N-acetyl-4-S-CAP
induced significant inhibition of melanoma growth by 17
days when injected i.p. at 3-day intervals, but no effect on
the ovarian tumour, JAM, was observed [8]. There was no
in vivo cytotoxicity to albino melanocytes in which there
was no production of active tyrosinase, indicating again the
importance of the melanin biosynthesis pathway for selec-
tive melanocyte toxicity [6]. Importantly, our biodistribu-
tion study of N-acetyl-4-S-CAP, using the 14C-labelled
form, indicated that the label is selectively incorporated
into and accumulated in melanoma tissues at 48 hr post-
injection [9].
Although these studies have shown clearly that
N-acetyl-4-S-CAP is a promising antimelanoma agent, our
recent study indicates that it would require additional
treatment (e.g. depleting of glutathione in tissue) to
achieve a highly efficient melanoma therapy [9]. Therefore,
we have synthesized a propionyl derivative of 4-S-CAP,
N-propionyl-4-S-CAP, with the expectation that melano-
cytotoxicity and the antimelanoma effects of N-acetyl-4-S-
CAP would be enhanced because of the more hydrophobic
structure of the drug, leading to increased penetration into
melanoma cells. Here, we present the chemical synthesis,
chemical characterization, and the preliminary screening of
the in vitro and in vivo melanocytotoxicity and antimela-
noma effects of N-propionyl-4-S-CAP.
Chemical Co. N-Acetyl-4-S-CAP was synthesized accord-
ing to our previous report [9].
Synthesis of N-propionyl-4-S-CAP
A mixture of 4-hydroxythiophenol (25 g; 0.198 mol) and
2-ethyl-2-oxazoline (19.6 g; 0.198 mol) was heated at 160°
under an inert atmosphere for 4 hr. A chromatographic
examination of the reaction mixture showed no evidence of
the starting reagents. Overnight cooling of the reaction
mixture resulted in solidification of the crude product,
which was purified on a silica gel column using CH2Cl2:
MeOH (9.8:0.2, v/v) as eluent. This purified product, after
recrystallization from dichloromethane, yielded 43.5 g
(97%) of pure N-propionyl-4-S-CAP. Rf 0.34; m.p., 83°; 1H
NMR (CD3COCD3 ϩ D2O) Ϫ 1.04 (t, JCH -CH ϭ 7.7 Hz,
3
2
3H, CH3), 2.14 (q, J
ϭ 7.7 Hz, 2H, propionyl CH2),
CH3-CH2
2.88 (t, J
ϭ 6.3 Hz, 2H, S-CH2), 3.3 (t, J
ϭ
CH2-CH2
CH2-CH2
6.3 Hz, 2H, CH2 Ϫ NH), 6.79 (d, J2,3 ϭ J6,5 ϭ 8.6 Hz, 2H,
aromatic H-3 and H-5) and 7.28 (d, J3,2 ϭ J5,6 ϭ 8.6 Hz,
2H, aromatic H-2 and H-6); 13C NMR (CD3COCD3) Ϫ
10.14 (CH3), 35.70 (S-CH2), 39.68 (CH2-NH), 116.94
(aromatic C-3 and C-5), 124.87 (C-4), 134.48 (C-2 and
C-6 aromatic), 158.04 (C-1), and 174.45 (CAO); anal. for
C11H15NO2S; calcd., C, 58.63; H, 6.71; N, 6.21%; found,
C, 58.88; H, 6.76; and N, 6.13%.
Activity as Tyrosinase Substrate
Murine B16F10 tumour cells were grown in Dulbecco’s
modified Eagle’s medium (Life Technologies) supple-
mented with 10% fetal bovine serum, penicillin, and
streptomycin (Life Technologies). The cells were harvested
with trypsin, washed with PBS, and homogenized by brief
sonication in PBS. Equal volumes of the homogenate and 1
mM of N-propionyl-4-S-CAP in 50 mM of potassium
phosphate buffer, pH 6.8, were mixed and incubated at 37°,
and the absorbance at 360 nm was measured at different
time intervals, as indicated.
MATERIALS AND METHODS
Kinetic parameters for tyrosinase action on test com-
pounds were determined following our previous reports [5,
10]. Mushroom tyrosinase (3670 units/mg; Sigma Chemical
Co.) was used for enzyme tyrosinase kinetics with some
modification. Briefly, the reaction mixture consisted of the
substrate at different concentrations (10 ␮M to 1 mM), and
mushroom tyrosinase (20 units) in 1.0 mL of 50 mM of
sodium phosphate buffer, pH 6.8, at 37°. The enzyme
reactions were stopped by cooling in ice. Product formation
was measured spectrophotometrically at 360 nm.
Analytical Methods and Chemicals
Melting points were determined on a Buchi capillary
apparatus and are uncorrected. Nuclear magnetic resonance
spectra (1H NMR and 13C NMR) were recorded on a
Brucker AM-300 spectrometer. 1H NMR assignments were
confirmed by double irradiation experiments and the chem-
ical shifts are provided in ␦ ppm downfield with respect to
tetramethyl silane (TMS) as internal standard. 13C NMR
resonances were assigned by using the J-spin echo modula-
tion (Jmod) technique to determine the number of attached
hydrogen atoms. The purity of the compounds was checked
on Whatman MK6F (250-␮m thickness) silica gel micro-
slides using dichloromethane:methanol (9:1, v/v; system A)
as developing solvent. Silica gel column chromatography
was carried out using Merck silica gel (100–200 mesh
particle size). 4-Hydroxythiophenol, propionic anhydride,
and 2-ethyl-2-oxazoline were purchased from the Aldrich
In Vitro Melanocytotoxicity Assays
The cytotoxicity of N-propionyl-4-S-CAP was assessed in
vitro in a melanoma cell line using two different methods:
hexosaminidase assay and ATP assay. SK-MEL-23, a hu-
man melanotic melanoma cell line (gift from Dr. A.
Houghton, Sloan–Kettering Cancer Center) was grown in

Melanogenesis-based Antimelanoma Agents
2025
FIG. 1. Action of B16F10 homogenate on
N-propionyl-4-S-CAP. The reaction con-
ditions are given in Materials and Meth-
ods. The reaction mixtures consisted of
0.684 mg/mL of cellular protein, 0.5 mM
of N-propionyl-4-S-CAP and 10 ␮M of
DOPA without (}) and with (■) 0.2 mM
of phenyl thiourea. Values (means ؎ SD)
from triplicate measurements are given.
RPMI 1640 medium (BioWhittaker) supplemented with
10% fetal bovine serum, penicillin (100 units/mL), and
streptomycin (100 ␮g/mL) and passaged with trypsin. For
the hexosaminidase assay, 2000 cells/well were seeded into
96-well plates. After 24 hr, the medium was replaced by
medium containing the predissolved drugs at the indicated
concentrations and incubated for 96 hr at 37° in a humid-
ified atmosphere of 5% CO2 in air. Then the plates were
processed for hexosaminidase measurement [11]. Absor-
bance at 405 nm was measured using an ELISA plate reader
(Easy Reader EAR 400AT, SLT-LAB Instruments).
For the ATP assay, 3–5 ϫ 105 cells/well were seeded into
24-well plates. After 24 hr, the medium was replaced by
medium containing the predissolved drugs at the indicated
concentrations and incubated for 96 hr as above. After
incubation, the medium was removed, the cells were
washed twice with ice-cold PBS, and ATP was measured
using the ATP Bioluminescence Assay Kit (Sigma Chem-
ical Co.) according to the instructions given. Briefly, cells,
after washing and being kept on ice, were suspended in 0.4
mL (per well) of ice-cold ATP Releasing Solution; aliquots
(10–20 ␮L) were mixed with the diluted assay reagent, and
light emission was measured immediately using a Lumat
9501 luminometer. Aliquots of 100–200 ␮L from each well
were measured for protein [12].
In Vivo Melanocytotoxicity in Black Hair Follicles
Black hair follicles of C57BL/6J black mice were plucked
manually from the dorsal skin in order to initiate new
anagen hair growth and induce tyrosinase activity as well as
melanin synthesis in follicular melanocytes. N-Propionyl-
4-S-CAP or normal saline (for control) was injected i.p.
daily for 14 days, starting on day 2 after plucking the hair
follicles on the dorsal side. The dose of injection (i.p.)
followed that of our previous study with N-acetyl-4-S-CAP,
300 mg/kg of body weight, and the volume of injection was
1 mL.
In Vivo Growth Inhibition of Melanoma Tumour and
Increased Life Span of Melanoma Bearing Mice
The murine B16F10 melanoma cell line was used for this
study. B16F10 melanoma cells were grown in Dulbecco’s
modified Eagle’s medium (Life Technologies) supple-
mented with 10% fetal bovine serum (Life Technologies),
penicillin (100 units/mL), and streptomycin (100 ␮g/mL).
Cells were incubated at 37° in a humidified atmosphere of
5% CO2 in air. When the cells reached the exponential
growth phase, they were scraped and inoculated s.c. into
C57BL/6J black mice (8-week-old females, 17.0 g). Seven
mice were used in each experimental group. They were
weighed and randomized at day 1 after s.c. inoculation of
B16 melanoma cells (1 ϫ 105 cells in 0.1 mL of normal
saline). From day 5, N-propionyl-4-S-CAP (300 mg/kg of
body weight), N-acetyl-4-S-CAP, and normal saline were
administered i.p. in a volume of 1 mL daily for 10 days. The
test compounds were dissolved in normal saline and steril-
ized by heating to 100° for 2 min. They were stable under
this treatment, as judged by HPLC study [10]. The in vivo
antimelanoma effects of the synthetic compounds, N-pro-
pionyl-4-S-CAP and N-acetyl-4-S-CAP, were evaluated by
percent growth inhibition of the s.c. inoculated tumor (%
Measurement of LD
50
Breeding pairs of C57BL/6J black mice were used in the
study when they were 8 weeks old (weighing approximately
17.0 g). The LD50 for N-propionyl-4-S-CAP was established
by a single i.p. injection of this drug dissolved in 1 mL of
normal saline solution, in the dose range of 100–1400
mg/kg of body weight. The LD50 was found to be 1000 mg/kg
of body weight for 4-propionyl-4-S-CAP. The LD of
50
N-acetyl-4-S-CAP was reported to be 1300 mg/kg [10].

2026
M. Tandon et al.
FIG. 2. Hexosaminidase assay for the melanocytotoxic action of cysteaminylphenol derivatives. The assay was carried out with
SK-MEL-23 cells as described in Materials and Methods. Absorbance at 405 nm (mean ؎ SD from 7 readings) is plotted as a function
of concentration of the drug for N-acetyl-4-S-CAP (}) and N-propionyl-4-S-CAP (■).
GI) and percent increase of life span (% ILS). The percent
GI was computed from the mean tumor volume at day 12 or
13 of the experiment, i.e. 100 ϫ (1 Ϫ treated group/control
group). Tumor volume was calculated using the formula:
natural substrate (data not given). A Lineweaver–Burk plot
gave Km and Vmax values of 340 ␮M and 5.4 ␮mol/min/mg
of protein, respectively, for N-propionyl-4-S-CAP.
2
1
long axis ϫ (short axis) ϫ ⁄ . The size of the tumor, i.e.
2
long and short axes (mm) of tumor, was measured every
other day through the skin externally.
In Vitro Melanocytotoxicity of N-Propionyl-4-S-CAP
The hexosaminidase assay results given in Fig. 2 indicate
that N-propionyl-4-S-CAP was cytotoxic to melanotic
melanoma cells, as expected. The effectiveness of the acetyl
and propionyl derivatives was comparable in this assay. The
results of ATP measurements in SK-MEL-23 cells treated
with the drugs (Fig. 3) showed ATP depletion at high
concentration of the drugs. The propionyl derivative
seemed to be slightly more effective than the acetyl
derivative in this assay.
RESULTS
N-Propionyl-4-S-CAP as Tyrosinase Substrate
Figure 1 gives the time course for the action of B16F10
homogenate, a source of mammalian tyrosinase, on N-pro-
pionyl-4-S-CAP in the presence of 10 ␮M of DOPA as a
cofactor. There was a time-dependent increase in the
absorbance, suggesting that N-propionyl-4-S-CAP is uti-
lized as a substrate by murine tyrosinase. That this reaction
was due to tyrosinase was confirmed by the almost 100%
inhibition of the reaction by 0.2 mM of phenyl thiourea, an
inhibitor of tyrosinase.
In Vivo Depigmenting Potency of N-Propionyl-4-S-CAP
for Black Hair Follicles
After the plucking of hair follicles, N-propionyl-4-S-CAP
was injected s.c. for 10 consecutive days into the dorsal
surface of C57 black mice where hair follicles were plucked
manually. The regrowing new hair follicles at the site of
hair plucking showed a complete depigmentation of hair
follicles, starting (visible) at day 20 after initiation of the
injection. The depigmenting effect is clearly seen from the
pictures of control and treated mice given in Fig. 4. The
extent of depigmentation was almost identical to that of
N-acetyl-4-S-CAP, as reported previously [7].
Enzyme kinetics with mushroom and melanoma tyrosi-
nase for 4-S-CAP and N-acetyl-4-S-CAP were reported
previously [10, 13]. The Km values for 4-S-CAP and
N-acetyl-4-S-CAP were found to be 117 and 375 ␮M,
respectively, whereas Vmax values were 39.0 and 8.3 ␮mol/
min/mg protein, respectively. Similarly, the kinetic param-
eters for our new synthetic compound, N-propionyl-4-S-
CAP, were measured along with those for N-acetyl-4-S-
CAP using mushroom tyrosinase. Both the compounds are
oxidized by mushroom tyrosinase faster than is tyrosine, the

Melanogenesis-based Antimelanoma Agents
2027
FIG. 3. ATP assay for the melanocytotoxic
action of cysteaminylphenol derivatives. The
assay was carried out with SK-MEL-23 cells
as described in Materials and Methods. The
ATP levels (means ؎ SD from triplicates)
are plotted as a function of concentration of
the drug for N-acetyl-4-S-CAP (}) and
N-propionyl-4-S-CAP (■).
In Vivo Growth Inhibition of Melanoma Tumour and
Increased Life Span of Melanoma Bearing Mice by
N-Propionyl-4-S-CAP
DISCUSSION
As a part of our efforts to develop potent, yet safe antimela-
noma agents, we have been focusing on various derivatives
of cysteaminylphenol based on the hypothesis that these
compounds will be taken up by the cell and will be
converted into cytotoxic species by the action of the
enzyme tyrosinase present only in melanoma cells (and
normal melanocytes). Earlier studies have given promising
results with N-acetyl-4-S-CAP, as this compound seemed
to be better than 5-(3,3-dimethyl-1-triazeno) imidazole-4-
carboxamide (DTIC), the drug used presently for mela-
noma chemotherapy, in animal studies [1]. However, the
potency was not spectacular. Thus, we are seeking more
potent derivatives of cysteaminylphenol to develop an
effective targeted chemotherapy of melanoma. One of our
ideas was to improve upon N-acetyl-4-S-CAP by effecting
structural changes that would enhance the cellular uptake
of the drug. We thought that replacing the acetyl group by
a propionyl group would increase the hydrophobicity and,
hence, the lipophilicity of the drug molecule, which in turn
was expected to increase the penetration of the drug into
the cell through the cell membrane.
Both N-acetyl-4-S-CAP and N-propionyl-4-S-CAP caused
a significant reduction in the tumour volume of melanoma
tissues inoculated s.c. into C57 black mice, their percent GI
being 42 and 36%, respectively (Table 1). The difference in
tumour size between control and treatment groups was
found to be statistically significant by t-test. There was also
an increase in the life span of melanoma-bearing animals,
their percent ILS being 27 and 39% in N-acetyl- and
N-propionyl-4-S-CAP treated animals, respectively.
N-Propionyl-4-S-CAP was synthesized by condensing
4-hydroxythiophenol with 2-ethyl-2-oxazoline and was
1
characterized by H NMR, 13C NMR, elemental analysis,
and melting point. The compound was found to be very
stable as a solid for several months at room temperature and
as a solution in cell culture medium for several days.
Targeted chemotherapy exploiting the melanin pathway
requires that the compound be a tyrosinase substrate. This
was tested using murine tyrosinase (as B16F10 cell homog-
enate) and mushroom tyrosinase (as a model), and the
results confirmed that N-propionyl-4-S-CAP is a very good
FIG. 4. Depigmenting effect of N-propionyl-4-S-CAP on
C57BL/6J black mice hair follicles. The drug administration
protocol is given in Materials and Methods. (A) a control mouse
and (B) an N-propionyl-4-S-CAP-treated mouse.

2028
M. Tandon et al.
TABLE 1. Antimelanoma effect of cysteaminylphenol derivatives in mice-bearing B16 melanoma
Tumour volume
(mm3)
Median survival
time (days)
Treatment
% GI
% ILS
Saline
N-Acetyl-4-S-CAP
N-Propionyl-4-S-CAP
8977 Ϯ 3778
5221 Ϯ 2336
5744 Ϯ 2155
0
42
36
16.5
21.0
23.0
0
27
39
% GI (growth inhibition of tumour) and % ILS (increased life span) of mice bearing B16 melanoma tumour were assessed as given in Materials and Methods. The differences
in tumour volumes (given as means Ϯ SD, N ϭ 7) between saline and drug-treated groups were found to be significant at the level of P Ͻ 0.05 for both N-acetyl-4-S-CAP and
N-propionyl-4-S-CAP.
substrate for tyrosinase. The enzyme kinetic parameters
were found to be similar to those of the homologue,
N-acetyl-4-S-CAP.
The in vitro cytotoxicity of substituted phenols has been
shown to increase with the length of the side chain [14], in
agreement with the present results. Thus, it is clear from
these studies that N-propionyl-4-S-CAP will be a better
choice than 4-acetyl-4-S-CAP for melanoma chemother-
apy. Further studies to characterize the action in cell culture
systems and the in vivo antimelanoma effect as well as
biodistribution of the compound are in progress.
The melanocytotoxicity and antimelanoma effects were
assessed in vitro and in vivo by different methods. The in vitro
studies on the melanotic melanoma cell line SK-MEL-23
(Figs. 2 and 3) gave evidence for the melanocytotoxicity of
N-propionyl-4-S-CAP. But the hexosaminidase and ATP
assays gave widely different ranges for the toxic dose. This
may be explained based on the fact that ATP depletion
occurs only when there is cell killing (leaking), whereas
hexosaminidase activity is determined by both cell killing
and inhibition of cell proliferation. Thus, these results
suggest that N-propionyl-4-S-CAP has both cytostatic and
cytocidal activities, the latter requiring higher concentra-
tions. The trend seen in Fig. 3 suggests that N-propionyl-
4-S-CAP may have a better cytocidal effect than N-acetyl-
4-S-CAP.
This study was supported by research grants from the National Cancer
Institute of Canada (NCI 4145), the Alberta Cancer Board (R1-91),
the Ministry of Education, Japan (08407022), and Stiefel Laboratories
International Division. K. J. is an Alberta Heritage Foundation for
Medical Research (AHFMR) Scientist.
References
The in vivo effects of N-propionyl-4-S-CAP were assessed
by two different tests: depigmenting effect on black hair
follicles and the inhibition of melanoma tumour growth in
mice. The depigmentation of hair follicles in black mice
will be a direct assay for melanocytotoxicity and an indirect
assay for antimelanoma effect, if depigmentation occurs as
result of cytostatic or cytocidal effect. Our earlier studies
with the parent compounds and N-acetyl-4-S-CAP showed
that depigmentation results from a specific degeneration of
follicular melanocytes [6, 7]. Hence, we used this simpler
test as the first in vivo test for antimelanoma action.
N-Propionyl-4-S-CAP was found to be a potent depigment-
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and N-acetyl-4-S-CAP-induced depigmentation occurs as a
result of melanocyte degeneration, it is quite likely that
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was supported by the ATP-depleting effect of the drug. In
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of tumour and life span of mice bearing B16 melanoma
tumour. The results (Table 1) showed an antimelanoma
effect of the drug, both in reducing the size of the tumour
and in increasing the life span of the treatment group. The
acetyl and propionyl derivatives were comparable in growth
inhibitory effect, but the propionyl derivative was consid-
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life span of mice bearing melanoma.
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