Synthesis and anticancer activity comparison of phenylalkyl isoselenocyanates with corresponding naturally occurring and synthetic isothiocyanates

Article abstract of DOI:10.1021/jm800993r

Synthesis and identification of novel phenylalkyl isoselenocyanates (ISCs), isosteric selenium analogues of naturally occurring phenylalkyl isothiocyanates (ITCs), as effective cytotoxic and antitumor agents are described. The structure - activity relationship comparison of ISCs with ITCs and effect of the increasing alkyl chain length in inhibiting cancer cell growth were evaluated on melanoma, prostate, breast, glioblastoma, sarcoma, and colon cancer cell lines. IC₅₀ values for ISC compounds were generally lower than their corresponding ITC analogues. Similarly, in UACC 903 human melanoma cells, the inhibition of cell proliferation and induction of apoptosis were more pronounced with ISCs compared to ITCs. Further, ISCs and ITCs effectively inhibited melanoma tumor growth in mice following intraperitoneal xenograft. A similar reduction in tumor size was observed at 3 times lower doses of ISCs compared to corresponding ITCs.

Full text of DOI:10.1021/jm800993r

7820
J. Med. Chem. 2008, 51, 7820–7826
Synthesis and Anticancer Activity Comparison of Phenylalkyl Isoselenocyanates with
Corresponding Naturally Occurring and Synthetic Isothiocyanates
Arun K. Sharma,*,†,‡,§ Arati Sharma,†,§ Dhimant Desai,†,‡,§ SubbaRao V. Madhunapantula,†,§ Sung Jin Huh,†
Gavin P. Robertson,†,§ and Shantu Amin†,‡,§
Department of Pharmacology, H072, Chemical Carcinogenesis and ChemopreVention Program of Penn State Hershey Cancer Institute, and
Penn State Melanoma Therapeutics Program, Penn State Hershey College of Medicine, Hershey, PennsylVania 17033
ReceiVed August 6, 2008
Synthesis and identification of novel phenylalkyl isoselenocyanates (ISCs), isosteric selenium analogues of
naturally occurring phenylalkyl isothiocyanates (ITCs), as effective cytotoxic and antitumor agents are
described. The structure-activity relationship comparison of ISCs with ITCs and effect of the increasing
alkyl chain length in inhibiting cancer cell growth were evaluated on melanoma, prostate, breast, glioblastoma,
sarcoma, and colon cancer cell lines. IC50 values for ISC compounds were generally lower than their
corresponding ITC analogues. Similarly, in UACC 903 human melanoma cells, the inhibition of cell
proliferation and induction of apoptosis were more pronounced with ISCs compared to ITCs. Further, ISCs
and ITCs effectively inhibited melanoma tumor growth in mice following intraperitoneal xenograft. A similar
reduction in tumor size was observed at 3 times lower doses of ISCs compared to corresponding ITCs.
Introduction
shorter alkyl chain length.17 Collectively, these data indicate
that ITCs with a longer alkyl chain persist longer at the target
organ site.
Isothiocyanates (ITCs) are naturally occurring compounds that
are stored as thioglucoside conjugates, termed glucosinolates,
in plants and cruciferous vegetables such as watercress, brussels
sprouts, broccoli, cabbage, cauliflower, radish, turnip, etc.1-3
They are among the most effective naturally occurring cancer
chemopreventive agents4 that inhibit carcinogenicity in animal
models.5 In addition, epidemiological studies have demonstrated
that the human consumption of isothiocyanates in vegetables
decreases cancer risk.6,7 This fact is supported by strong
literature data that suggest that ITCs are effective chemopre-
ventive agents for specific human cancers. ITCs have been
shown to exhibit the anticarcinogenic effects through dual
mechanisms occurring at the level of initiation of carcinogenesis
by blocking phase I enzymes (cytochrome P-450) that activate
procarcinogens and by inducing phase II enzymes that detoxify
electrophilic metabolites generated by phase I enzymes.8-13
ITCs are also known to block cell-cycle progression and induce
apoptosis in human cancer cells, suggesting that these agents
act also at the postinitiation and progression stages.12-16
The activity of ITCs has been shown to vary with varying
alkyl chain length. While some reports show the increase in
potency with increasing alkyl chain length,17-19 others have
even reported a reverse phenomenon.20 Structure-activity
studies have also demonstrated that increased lipophilicity or
chain length of ITCs increases inhibitory potency against NNK-
induced lung tumorigenesis in A/J mice.21,22 Conaway et al.
have shown that synthetic ITCs can be generated with enhanced
lipophilicity by increasing alkyl chain length up to six carbons.17
In addition, the elimination half-life (T1/2e) of ITCs with longer
alkyl chain length has been shown to be higher than those with
The well established chemopreventive properties of ITCs
warranted a structure-activity study of synthetically modified
ITC analogues to enhance their chemopreventive and chemo-
therapeutic efficacy. We hypothesized that isosteric replacement
of sulfur in ITCs by selenium would result in more effective
anticancer agents. The hypothesis was based on the observation
that in comparison to the sulfur structural analogues, selenium
compounds are much more active in cancer prevention.23
Furthermore, selenium supplementation is recognized for phar-
macological intervention, especially in the clinical domain of
cancer chemoprevention, and its use is not limited only to correct
nutritional deficiencies. This is evident from the observation
that epidemiological studies carried out for past 3 decades
provide evidence of an inverse relationship between selenium
intake and cancer mortality, and selenium supplementation in
the diet or drinking water has been shown to inhibit neoplasms
of the liver, skin, pancreas, colon, and mammary glands.24,25
Furthermore, two-thirds of animal studies showed reduced
incidence of tumors triggered by chemical carcinogens or viruses
following selenium supplementation.26 The organoselenium
compounds have also been reported to inhibit initiation and
postinitiation stages of chemical carcinogenesis.27 Mechanisti-
cally, organoselenium compounds have been suggested to
activate certain proapoptotic genes linked to p53, NFκB, and
stress signal pathways, thereby preventing tumorigenesis.28 In
addition, selenium supplementation could provide significant
therapeutic potential, since patients with melanoma, colon,
breast, ovary, pancreas, as well as head and neck cancers show
decreased levels of selenium in whole blood or serum than do
healthy controls. Thus, in view of the known anticancer
properties of ITCs and oraganoselenium coumpounds, we have
developed isosteric selenium analogues [the phenylalkyl isos-
elenocyanates (ISCs), 2] of ITCs (1) (Figure 1). The objectives
were to (a) determine whether replacing sulfur in ITCs with
selenium increases the cancer inhibitory potency and (b)
determine the optimal chain length for maximal anticancer
* To whom correspondence should be addressed. Address: Department
of Pharmacology, H072, Penn State Milton S. Hershey Medical Center,
Penn State College of Medicine, 500 University Drive, Hershey, PA 17033.
Phone: 717-531-0003, extension 285016. Fax: 717-531-0244. E-mail:
aks14@psu.edu.
†
Department of Pharmacology.
Chemical Carcinogenesis and Chemoprevention Program of Penn State
‡
Hershey Cancer Institute.
§
Penn State Melanoma Therapeutics Program.
10.1021/jm800993r CCC: $40.75
2008 American Chemical Society
Published on Web 11/18/2008

Isothiocyanates and Isoselenocyanates
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7821
In Vitro Studies on Melanoma Cells. For melanoma cells
(UACC 903), an increase in chain length or a change from ITCs
to ISCs by replacement of sulfur with selenium led to an
insignificant change in IC50 values (Table 1), indicating that
both ISCs and ITCs are capable of inhibiting melanoma cell
growth equally. Cell viability was measured using the MTS
assay (Figure 2), which determines the capacity of mitochondrial
dehydrogenase in viable cells to transform tetrazolium salt into
colorimetric formazan.34 UACC 903 human melanoma cells
(5 × 103) were plated in a 96-well plate and 24 h later exposed
to DMSO or increasing concentrations (5, 10, and 15 µM) of
ITCs (1) or ISCs (2) for 24 h. MTS assay showed a dose
dependent decrease in cell viability for both ISC and ITC series
of compounds. There was no significant difference in activities
between the two series; longer alkyl chain analogues ISC-4 (at
10 and 15 µM) and ISC-6 (at 10 µM) were found to be a little
more effective than the corresponding ITC derivatives. To
establish the potency of ISCs in comparison to ITCs and to
quantify the effect of increasing chain length of these agents,
cell proliferation and apoptosis were carried out in UACC 903
human melanoma cells. Cellular proliferation following ISC and
ITC derivatives treatment was measured using bromodeoxyuri-
dine (BrdU) incorporation, which is a convenient tool to monitor
DNA synthesis within the cells. A dose dependent decrease in
number of proliferating cells was observed upon treatment with
ITC or ISC derivatives (Figure 3). In general, ISC derivatives
were more effective compared to corresponding ITCs with
similar alkyl chain length, especially at higher concentrations.
The caspase 3/7 activity, which is reflective of apoptosis, was
determined using Apo-ONE homogeneous caspase-3/7 assay kit
(Promega Corporation, Madison, WI). A dose dependent
increase in caspase-3/7 activity was observed for both ITCs and
ISCs. In comparison to ITCs, ISC derivatives exhibited much
higher caspase activity. While the difference was not significant
between BITC and ISC-1, there was a striking difference
between longer alkyl chain derivatives of ITCs and ISCs,
especially at higher concentrations of 10 and 15 µM (Figure
4). ISC-4 and ISC-6 were found to be the most effective inducer
of apoptosis among all other compounds tested.
Figure 1. General structures of phenylalkyl isothiocyanates (1) and
phenylalkyl isoselenocyanates (2).
activity. The overall goal was to establish whether altered
chemical reactivity and lipophilicity caused by the outlined
structural modifications will affect potency of compounds and
hence to identify structural requirements necessary for optimal
activity against multiple cancers. Thus, we synthesized a variety
of selenium isostere of naturally occurring and synthetic ITCs
and evaluated their effect on in vitro cell viability of melanoma,
prostate, colon, glioblastoma, sarcoma, and breast cancers cell
lines. The two classes of compounds were also evaluated for
their ability to inhibit melanoma cell proliferation and induction
of apoptosis in vitro and to inhibit tumor development in
preclinical melanoma xenograft.
Results
Synthesis of Isoselenocyanates. The first synthesis of isos-
elenocyanates was reported by Barton et al.29 starting from the
corresponding formamides. Several modifications30 in the
procedure have been made since then to improve yields and
avoid polymerization31 of intermediate compounds.29,32 We have
used the method of Fernandez-Bolanos et al.,33 which conve-
niently uses solid triphosgene, instead of phosgene,29 in a one-
pot dehydration of the formamides in refluxing dichloromethane
(Scheme 1). The synthetic strategy involved the formylation of
phenylalkylamines, followed by treatment with triphosgene and
selenium powder in the presence of triethylamine to furnish the
desired phenylalkyl isoselenocyanates (2) in good yields.
Compounds were purified by silica gel column chromatography
1
and characterized by H NMR and high-resolution MS.
SAR Study on Cancer Cell Lines. The ITCs (1) and the
corresponding ISCs (2) were tested for their ability to inhibit
cell growth in six cancer cell lines, e.g., melanoma (UACC 903),
breast (MDA-MB-231), glioblastoma (T98G), fibrosarcoma
(HT-1080), colon (Caco-2), and prostate (PC-3) cancer cell lines.
The IC50 values for compounds 1 and 2 are depicted in Table
1. The IC50 values consistently decreased with increasing alkyl
chain length of ITCs in the cases of glioblastoma, breast, and
prostate cancer cell lines but showed no particular trend in
fibrosarcoma, colon, and melanoma cells. In the case of the
melanoma cell line, the IC50 values remained essentially the
same for all ITCs while in the case of the colon cancer cell line
the IC50 values actually increased with increasing carbon chain
length of ITCs. Among ISC derivatives, ISC-1 was least
effective at killing cancer cells compared to higher alkyl chain
analogues ISC-2 to ISC-6. The difference was more striking in
glioblastoma, prostate, and breast cancer cell lines, while in other
cancer cell lines, the difference between ISC-1 and higher alkyl
chain ISCs was not significant. Among ISC-2, ISC-4, and ISC-6
there was no particular trend. In almost all the cases (except
for comparable values of ISC-1 and ISC-2 with BITC and
PEITC in UACC 903 cells) the ISC derivatives had lower IC50
values than corresponding ITCs. This suggested superiority of
ISC compounds over ITCs and that lower concentrations of
selenium analogues would be required for similar therapeutic
efficacy.
ISCs Reduce Melanoma Tumor Development More Ef-
fectively Than ITCs. The in vitro studies in general suggested
the superiority of ISC compounds over ITC derivatives. To
further establish the effect in vivo, the efficacy of ISC and ITC
compounds for inhibiting tumor development was evaluated in
preclinical mouse model of melanoma. Nude mice were subcutane-
ously injected with UACC 903 cells, and tumor development was
allowed to occur for 6 days, by which time tumors have undergone
vascularization (angiogenesis). Mice were then treated ip with ISC
and ITC derivatives three times a week on Mondays, Wednesdays,
and Fridays. At a dose of 0.76 µM (3 ppm of selenium), ISC-2,
ISC-4, and ISC-6 showed a 30-45% reduction in tumor size
(Figure 5A). ISC-1 had no effect at this concentration. Notably,
none of the ITC derivatives were effective in reducing tumor
size at 0.76 µM (data not shown). However, at a dose of 2.5
µM (∼3 times that used for ISC compounds) a 40-60%
reduction in tumor size was observed (Figure 5C). No evidence
of systemic toxicity was observed at the doses used for any of
the ISC or ITC derivatives (parts B and D of Figure 5).
Interestingly, a reverse trend of chain length effect was observed;
i.e., the potency actually decreased with increasing alkyl chain
length in ITCs, with BITC being the most effective. A similar
trend in behavior was observed by Xiao et al.
20 in a study where
they observed BITC to be more effective compared to PEITC
in breast cancer cell lines.

7822 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Scheme 1. Synthesis of ITCs 1 and ISCs 2a
Sharma et al.
a Reagents and conditions: (a) C2H5OCHO, -20 °C to reflux; (b) Et3N, triphosgene, Se powder, CH2Cl2, reflux; (c) CSCl2, NaOH.
Table 1. IC50 (µM) of ITC and ISC Derivatives on Different Cancer Cells
cancer cell line, IC50 (µM)a
compd
breast MDA-MB-231
glioblastoma T98G
prostate PC-3
fibrosarcoma HT-1080
colonb Caco-2
melanoma UACC 903
1a (BITC)
1b (PEITC)
1c (PBITC)
1d (PHITC)
2a (ISC-1)
2b (ISC-2)
2c (ISC-4)
2d (ISC-6)
42 ( 3
38 ( 6
27 ( 2
24 ( 2
29 ( 2
20 ( 3
21 ( 6
22 ( 2
>100
>50
>50
15 ( 2
14 ( 2
27 ( 2
49 ( 9
13 ( 3
11 ( 1
12 ( 3
10 ( 1
15 ( 3
12 ( 1
16 ( 1
15 ( 2
16 ( 3
12 ( 4
10 ( 3
10 ( 1
>100
24 ( 2
24 ( 2
17 ( 1
24 ( 1
16 ( 1
19 ( 1
14 ( 1
15 ( 1
15 ( 1
29 ( 3
13 ( 3
12 ( 3
11 ( 1
12 ( 1
35 ( 1
26 ( 2
43 ( 4
24 ( 1
27 ( 1
23 ( 2
a Values are mean values ( SE. b Drug treatment for 72 h.
Figure 2. MTS assay showing the viability of UACC 903 cells after treatment with ISC and ITC derivatives. Both ITCs (BITC, PEITC, PBITC,
and PHITC) and ISCs (ISC-1, ISC-2, ISC-4, and ISC-6) effectively reduced cell viability compared to DMSO control. The average value is represented
as the percentage of control DMSO treated cells. The values for all experiments represent mean values, with bars indicating SEM from three
independent experiments.
Synthesis and Cell Viability of N-Acetylcysteine (NAC)
Conjugates. The activity of ITCs is reported to be through the
formation of NAC conjugates, and presumably the ISCs may
follow a similar mechanism of action. To determine that, we
synthesized NAC conjugates of representative ISC-4 and the
corresponding PBITC by treating them with NAC in THF in
the presence of NaOH (Scheme 2). The conjugates were
characterized on the basis of 1H NMR and high resolution MS
spectra. The cell viability measurements on UACC 903 cells
using MTS assay revealed an IC50 value of 17 ( 0.5 µM for
the ISC-4-NAC conjugate (6) compared to 10 ( 3 µM for
ISC-4 and a value of 24 ( 1 µM for the PBITC-NAC conjugate
(5) compared to 16 ( 1 µM for PBITC.
Figure 3. Proliferation profile of UACC 903 cells after treatment with
ISC and ITC derivatives. The 5 × 103 cells were treated with DMSO
or increasing concentrations of ITCs or ISCs for 24 h and proliferating
cells measured using BrdU labeling. The average value is represented
as the percentage of control DMSO treated cells. The values for all
experiments represent mean values, with bars indicating SEM from
three independent experiments.
Discussion
ITCs (R-NdCdS) are electrophilic compounds and are
known to react predominantly with thiols and to a much lesser
extent with NH2 and OH groups.35 Therefore, the major route

Isothiocyanates and Isoselenocyanates
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7823
Figure 4. Levels of caspase-3/7 activity (an indicator of apoptosis) in cells exposed to ITCs or ISCs were measured using the Apo-ONE homogeneous
caspase-3/7 assay kit. Results show fold increase in caspase-3/7 activity relative to DMSO vehicle treated cells. Results represent the average of
three independent experiments; bars represent SEM. Compared to DMSO control, increasing concentrations of ISCs or ITCs elevated caspase-3/7
activity in a dose dependent manner. ISC-2, ISC-4, and ISC-6 showed significantly higher caspase-3/7 activity compared to their corresponding
ITCs.
Figure 5. In vivo melanoma tumor inhibition using ISC and ITC derivatives. Six days after subcutaneous injection of UACC 903 cells, mice were
treated ip with ISC or ITC derivatives thrice per week. Both ISC and ITC derivatives significantly reduced tumor development; however, the
concentration of ISC (0.76 µmol) was 3-fold less than ITCs (2.5 µmol) (A, C). At a dose of 0.76 µmol, ISC-2, ISC-4, and ISC-6 showed about
30-45% reduction in tumor size (A). ISC-1 failed to show any effect at this concentration. ITC derivatives were also effective in reducing the
tumor size at 2.5 µmol (∼3 times higher than ISC compound) (C). Parts B and D show the body weights of mice treated with ISC and ITC
derivatives, respectively, compared to the control DMSO vehicle treated mice. No significant difference in weights was detected between groups,
demonstrating negligible systemic toxicity.
of metabolism and elimination of ITCs36 from the body is the
mercapturic acid pathway, i.e., by formation of nonenzymatic
and enzymatic conjugation with glutathione (GSH) to give thiol
conjugates. Stepwise enzymatic hydrolysis of GSH conjugates
of ITCs yields L-cysteine (Cys) conjugate (ITC-Cys), which
on subsequent acetylation gives N-acetyl-L-cysteine (NAC)
conjugates of ITCs (NAC-ITCs).37,38 NAC conjugates of ITCs
have been detected as the main metabolite in the urine of rodents
and humans.38 NAC and thiol conjugates of ITCs have shown
a chemopreventive efficacy against TSNA and PAH-induced
lung tumorigenesis in rodents.39-41 Conaway et al. have also
shown that ITC conjugates have longer half-lives than the parent
isothiocyanates.39 ISCs, being structurally similar to ITCs, are
expected to follow a similar metabolic and elimination pathway.
Thus, like ITCs, the activity of ISCs may also be due to the
formation of NAC-ISC conjugate metabolites. Our results have
indicated that the IC50 value of the ISC-4-NAC conjugate (6)
(17 ( 0.5 µM) compared to that of the ISC-4 (10 ( 3 µM) and

7824 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Scheme 2. Synthesis of NAC Conjugates of PBITC and ISC-4
Sharma et al.
have been developed. We have demonstrated that by isosterically
replacing sulfur with selenium, we were able to significantly
improve antitumor activity of ITCs. The ISC compounds were
more efficient in inhibiting cell growth in melanoma, glioblas-
toma, fibrosarcoma, colon, breast, and prostate cancers cells
compared to corresponding ITC analogues. Furthermore, in
general, the efficacy increased with increasing alkyl chain length
in the cases of ITC and ISC compounds. In the preclinical
melanoma xenograft model, the ISC compounds showed similar
tumor inhibition at 3 times lower doses compared to ITC
derivatives without any measurable systemic toxicity. Interest-
ingly, tumor inhibitory effect decreased with increasing chain
length in ITCs while it increased with increasing chain length
in case of ISCs. Collectively, on the basis of both in vitro and
in vivo experiments, ISC-4 proved to be the most effective agent
among the series of ISC and ITC analogues tested. ISC-4 thus
holds promise of being an effective chemopreventive/chemo-
therapeutic agent.
Table 2. Calculated log P a (CLogP) Values of ITC and ISC
Compounds
ITCs
CLogP
ISCs
CLogP
Experimental Section
1a (BITC)
3.204
3.263
4.171
5.229
2a (ISC-1)
2b (ISC-2)
2c (ISC-4)
2d (ISC-6)
3.177
3.506
4.414
5.472
General. Melting points were recorded on a Fischer-Johns
melting point apparatus and are uncorrected. NMR spectra were
recorded using a Varian 300 MHz NMR spectrometer or a Bruker
Avance 500 MHz spectrometer. Chemical shifts (δ) were reported
in parts per million downfield from the internal standard. The signals
are quoted as s (singlet), d (doublet), t (triplet), m (multiplet), and
dt (doublet of triplet). HRMS results were determined at the
Chemistry Instrumentation Center, State University of New York
at Buffalo, NY. Thin-layer chromatography (TLC) was developed
on aluminum-supported precoated silica gel plates (EM industries,
Gibbstown, NJ). Column chromatography was conducted on silica
gel (60-200 mesh). Benzyl isothiocyanate (BITC, 1a) and phen-
ethyl isothiocyanate (PEITC, 1b) were obtained from commercial
sources. Phenylbutyl isothiocyanate (PBITC, 1c) and phenylhexyl
isothiocyanate (PHITC, 1d) were synthesized according to a
literature method.21
1b (PEITC)
1c (PBITC)
1d (PHITC)
a Calculated log P values were estimated using ChemDraw Ultra 9.0.
the IC50 value of the PBITC-NAC conjugate (5) (24 ( 1 µM)
compared to that of the PBITC (16 ( 1 µM) were consistent
with the trend reported in the literature for ITC and their
corresponding conjugates,42 where NAC conjugates have been
shown to be slightly less effective than the parent ITC. However,
the comparable activity of ISC-4 and the corresponding NAC
conjugate indicates that, similar to ITCs, the main route of
metabolism and hence the in vivo activity of ISCs may be
mediated by metabolic transformation to GSH conjugates.
Literature reports have attributed the increased potency with
the increasing chain length of ITCs to the increase in lipophi-
licity.17 It has been suggested that increasing lipophilicity
decreases the reactivity of ITCs with glutathione (GSH),
resulting in a slowing of excretion and thus enhancing the
potency of the compound.43 In an attempt to correlate ITC and
ISC data to their lipophilicity, we calculated the log P value of
both ITC and ISC series of compounds using ChemDraw 9.0
Ultra. The log P values increased with increasing chain length
in both series, with ISC compounds in general having a slightly
higher value than the corresponding ITC analogues (Table 2).
The observed reverse trend of tumor inhibition decreasing with
increasing alkyl chain length in melanoma model (Figure 5C)
suggests that lipophilicity is not the only determining factor in
the case of ITCs. This reverse trend is in agreement with the
Xiao et al. results in breast cancer cells.20 ISC compounds,
however, showed increased efficacy both in vitro and in vivo
with increasing lipophilicity. Overall, the results suggest the
participation of other pharmacokinetic and pharmacodynamic
factors that influence the efficacy of these compounds. Both
ISC-4 and ISC-6 were equally effective in vitro and in inhibiting
tumor growth. However, the higher lipophilicity of ISC-6 (log P
) 5.472), just like PHITC, makes it less suitable as a probable
chemopreventive/therapeutic agent. Higher lipophilicity of
PHITC has been linked to the observed induction of esophageal
carcinogenesis in Fisher 344 rats.44 In view of this, ISC-4 is
the most suitable agent for evaluation as a drug.
General Method for Synthesis of Phenylalkylformamides.
Formamides (4b-d) were synthesized following a literature
method.45 Briefly, ethyl formate (120 mmol) was added dropwise
to phenylalkylamine (40 mmol) at room temperature, and the result-
ing mixture was refluxed for 4-6 h. The excess ethyl formate was
removed under reduced pressure to yield the corresponding phe-
nylakylformamide as colorless viscous oils.
Phenylethylformamide (4b). Yield, 96%; viscous oil; 1H NMR
(CDCl3, 300 MHz) δ 2.84 (t, 2H, J ) 6.9 Hz), 3.57 (dt, 2H, J )
6.9 and 6.6 Hz), 5.68 (br d, 1H, NH), 7.15-7.35 (m, 5H), 8.12 (s,
1H, CHO). HRMS (EI) calcd for C9H11NO, 149.0835; found,
149.0839.
Phenylbutylformamide (4c). Yield, 98%; viscous oil; 1H NMR
(CDCl3, 500 MHz) δ 1.56-1.62 (m, 2H), 1.66-1.72 (m, 2H), 2.66
(t, 2H, J ) 6.5 Hz), 3.35 (dt, 2H, J ) 7.0 and 6.5 Hz), 5.92 (br s,
1H), 7.18-7.24 (m, 3H), 7.29-7.33 (m, 2H), 8.19 (s, 1H). HRMS
(EI) calcd for C11H15NO, 177.1148; found, 177.1149.
Phenylhexylformamide (4d). Yield, 91%; viscous oil; 1H NMR
(CDCl3, 500 MHz) δ 1.36-1.41 (m, 4H), 1.52-1.58 (m, 2H),
1.61-1.67 (m, 2H), 2.63 (t, 2H, J ) 7.5 Hz), 3.30 (dt, 2H, J ) 7.0
and 6.5 Hz), 5.58 (br s, 1H), 7.18-7.21 (m, 3H), 7.28-7.31 (m,
2H), 8.19 (s, 1H). HRMS (EI) calcd for C13H19NO, 205.1461; found,
205.1462.
Benzyl Isoselenocyanate (ISC-1, 2a). To a refluxing mixture
of the benzyl formamide (1.35 g, 10.0 mmol), triethylamine (4.35
g, 6.0 mL, 43.0 mmol) in CH2Cl2 (35 mL), and 4 Å molecular
sieves was added dropwise a solution of triphosgene (1.48 g, 5.0
mmol) in CH2Cl2 (15 mL) for a period of 1 h. After the addition
was complete, the mixture was refluxed for an additional 2.5 h.
Selenium powder (1.58 g, 20 mmol) was then added, and the
resulting mixture was refluxed for an additional 7 h. The mixture
was cooled and filtered, and the solvent was evaporated to yield
the crude mixture, which was purified by silica gel column
Conclusion
In summary, ISCs, the isosteric selenium analogues of well-
known naturally occurring and synthetic anticancer agents ITCs,

Isothiocyanates and Isoselenocyanates
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7825
chromatography (EtOAc/hexanes 3:97) to afford 1.21 g (62%) of
using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-
2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (Promega, Madison,
WI). In brief, (2.5-5) × 103 cells per well in 100 µL of DMEM
containing 10% FBS were grown in a 96-well plate for 24 h and
treated with either control DMSO vehicle or increasing concentra-
tions (2.5-100 µM) of ITC and ISC for 24 h. The percentages of
viable cells compared to control DMSO treated cells were
determined using MTS assay and IC50 values calculated using
GraphPad Prism, version 4.01 (GraphPad software, San Diego, CA).
The IC50 value for each compound was determined by at least three
independent experiments and represented with a standard error
(Table 1).
Cellular proliferation and apoptosis rates were measured by
seeding 5 × 103 human melanoma cell line UACC 903 in a 96-
well plate, followed by treatment for 24 h with ITCs or ISCs.
Proliferation and apoptosis rates were measured using a BrdU
ELISA kit (Roche Applied Sciences, Indianapolis, IN) or Apo-
ONE homogeneous caspase-3/7 assay kit (Promega Corporation,
Madison, WI), respectively.
Tumorigenicity Assessment. Animal experimentation was
performed according to protocols approved by the Institutional
Animal Care and Use Committee at The Pennsylvania State
University College of Medicine. Tumor kinetics were measured
by subcutaneous injection of 5 × 106 UACC 903 melanoma cells
in 0.2 mL of DMEM supplemented with 10% FBS above both left
and right rib cages of 4-6 week old female athymic nude mice
(Harlan Sprague-Dawley, Indianapolis, IN). Six days later mice
were randomly divided into control (DMSO) and experimental
(ISC-1, ISC-2, ISC-4, ISC-6, BITC, PEITC, PBITC, PHITC) groups
(5 mice/group, 2 tumors/mouse). Six days after subcutaneous
injection of UACC 903 melanoma cells, mice were treated ip with
ITC (2.5 µmol) or ISC (0.76 µmol, equivalent to 3 ppm selenium)
three times per week (Mondays, Wednesdays, and Fridays). Control
mice received an equal volume of the vehicle. The dimensions of
the developing tumors (using calipers) and body weight were
measured three times a week (Mondays, Wednesdays, and Fridays),
and the size was estimated in cubic millimeters.
1
2a as a viscous oil. H NMR (CDCl3, 300 MHz) δ 4.81 (s, 2H,
CH2), 7.30-7.44 (m, 5H). HRMS (EI) calcd for C8H7NSe,
196.9738; found, 196.9741.
Phenylethyl Isoselenocyanate (ISC-2, 2b). A mixture of phen-
ethylformamide (0.67 g, 4.5 mmol), triethylamine (1.94 g, 2.67 mL,
19.2 mmol), 4 Å molecular sieves, triphosgene (0.65 g, 2.2 mmol),
and selenium powder (0.71 g, 9.0 mmol) in CH2Cl2 (35 mL) was
refluxed and worked up as mentioned above for 2a. The crude
residue thus obtained was purified by silica gel column chroma-
tography (EtOAc/hexanes 5:95) to give 0.62 g (65%) of 2b as an
1
oil. H NMR (CDCl3, 300 MHz) δ 3.03 (t, 2H, J ) 6.9 Hz), 3.81
(t, 2H, J ) 6.9 Hz), 7.20-7.38 (m, 5H). HRMS (EI) calcd for
C9H9NSe, 210.9895; found, 210.9892.
Phenylbutyl Isoselenocyanate (ISC-4, 2c). A mixture of phe-
nylbutylformamide (1.77 g, 10.0 mmol), triethylamine (4.35 g, 6.0
mL, 43 mmol), 4 Å molecular sieves, triphosgene (1.48 g, 5.0
mmol), and selenium powder (1.58 g, 20.0 mmol) in CH2Cl2 (50
mL) was refluxed and worked up as mentioned above for 2a. The
crude residue thus obtained was purified by silica gel column
chromatography (EtOAc/hexanes 5:95) to give 1.7 g (71%) of 2c
as an oil. 1H NMR (CDCl3, 300 MHz) δ 1.74-1.76 (m, 4H), 2.66
(t, 2H, J ) 6.6 Hz), 3.60 (t, 2H, J ) 5.6 Hz), 7.15-7.32 (m, 5H).
HRMS (EI) calcd for C11H13NSe, 239.0208; found, 239.0211.
Phenylhexyl Isoselenocyanate (ISC-6, 2d). A mixture of
phenylbutylformamide (1.64 g, 8.0 mmol), triethylamine (3.49 g,
4.8 mL, 34.5 mmol), 4 Å molecular sieves, triphosgene (1.19 g,
4.0 mmol), and selenium powder (1.26 g, 16.0 mmol) in CH2Cl2
(40 mL) was refluxed and worked up as mentioned for 2a. The
crude residue thus obtained was purified by silica gel column
chromatography (EtOAc/hexanes 3:97) to give 1.35 g (63%) of 2d
as an oil. 1H NMR (CDCl3, 500 MHz) δ 1.37-1.43 (m, 2H),
1.45-1.51 (m, 2H), 1.64-170 (m, 2H), 1.72-1.78 (m, 2H), 2.64
(t, 2H, J ) 7.6 Hz), 3.60 (t, 2H, J ) 6.7 Hz), 7.19-7.25 (m, 3H),
7.32-7.40 (m, 2H). HRMS (EI) calcd for C13H17NSe, 267.0521;
found, 267.0529.
PBITC-NAC Conjugate (5). To a solution of 1c (382 mg, 2.0
mmol) in THF (25 mL) at 0 °C was added a solution of
N-acetylcysteine (326 mg, 2.0 mmol) in water (15 mL), and to this
mixture was added 2% NaOH (0.1 mL). The reaction mixture was
allowed to warm to room temperature and stirred for 12 h. The
mixture was washed with hexane (2 × 5 mL), and the aqueous
layer was acidified with 2 N HCl and extracted with ethyl acetate.
The organic layer was separated, dried (MgSO4), and concentrated
in vaccuo to give 5 as a viscous oil in quantitative yield. 1H NMR
(CD3OD, 500 MHz) δ 1.67-1.70 (m, 4H), 1.96 (s, 3H), 2.01-2.03
(m, 2H), 2.64-2.67 (m, 2H), 3.69-3.74 (m, 2H), 4.67 (dd, 1H, J
) 8.5 and 4.5 Hz), 7.14-7.22 (m, 3H), 7.25-7.28 (m, 2H). HRMS
(ESI) calcd for C16H22N2O3S2 ·H+, 355.1145; found, 354.1144.
ISC-4-NAC Conjugate (6). To a solution of 2c (238 mg, 1.0
mmol) in THF (15 mL) at 0 °C was added a solution of
N-acetylcysteine (163 mg, 1.0 mmol) in water (7.0 mL), and to
this mixture was added 2% NaOH (50 µL). Following similar
reaction conditions and workup as mentioned for 5 gave 6 as a
Statistical Analysis. Statistical analysis was undertaken using
the one-way ANOVA followed by an appropriate post hoc test.
Results were considered significant at a P value of <0.05.
Acknowledgment. This study was supported by grants from
the the Elsa U. Pardee Foundation, Melanoma Research
Foundation, and NIH (Grant CA-127892-01A). The project was
also funded, in part, under a grant from the Pennsylvania
Department of Health using Tobacco Settlement Funds. The
authors thank Dr. Jyh-Ming Lin, Solution Phase NMR Facility
at Core Research Facilities of the Penn State Hershey College
of Medicine, for recording of NMR spectra.
Supporting Information Available: 1H NMR spectra for
1
compounds 1c, 1d, 2a-d, 4b-d, 5, and 6 and H NMR data for
compounds 1c and 1d. This material is available free of charge via
the Internet at http://pubs.acs.org.
1
viscous oil in quantitative yield. H NMR (CD3OD, 500 MHz) δ
1.73-1.78 (m, 2H), 1.80-1.84 (m, 4H), 1.98 (s, 3H), 2.69 (t, 2H,
J ) 8.0 Hz), 3.63-3.67 (m, 2H), 4.69-4.73 (m, 1H), 7.17-7.30
(m, 5H), 7.67 (d, 1H, J ) 7.0 Hz), 9.92 (br s, 1H). HRMS (ESI)
calcd for C16H22N2O3SSe·H, 403.0589; found, 403.0576.
Cell Lines and Culture Conditions. Colon adenocarcinoma cell
line (Caco-2, ATCC no. HTB-37) was grown in advanced DMEM
supplemented with 10% heat treated (56 °C for 30 min) FBS and
L-glutamine. Fibrosarcoma (HT-1080, ATCC no. CCL-121), pros-
tate adenocarcinoma (PC-3, ATCC no. CRL-1435), breast adeno-
carcinoma cell line (MDA-MB-231, ATCC no. HTB-26), glioblas-
toma cell line (T98G, ATCC no. CRL-1690), and human melanoma
cell line UACC 903 were grown in DMEM supplemented with
10% FBS.
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