Substituted 6-amino-4H-[1,2]dithiolo[4,3-b]pyrrol-5-ones: Synthesis, structure-activity relationships, and cytotoxic activity on selected human cancer cell lines

Article abstract of DOI:10.1016/j.bmc.2007.04.017

An efficient synthesis and the cytotoxic activity of a series of substituted 6-amino-4H-[1,2]dithiolo[4,3-b]pyrrol-5-ones 1a-q is described. The synthesis was accomplished in an expedient manner (seven-steps) from commercially available starting materials

Full text of DOI:10.1016/j.bmc.2007.04.017

Bioorganic & Medicinal Chemistry 15 (2007) 4601–4608
Substituted 6-amino-4H-[1,2]dithiolo[4,3-b]pyrrol-5-ones:
Synthesis, structure–activity relationships, and cytotoxic activity
on selected human cancer cell lines
Bin Li,* Michael P. A. Lyle,* Genhui Chen, Jason Li, Kaiji Hu,
Liren Tang, Moulay A. Alaoui-Jamalià and John Webster
Welichem Biotech Incorporated, Technology Place, 316-4475 Wayburne Drive, Burnaby, BC, Canada V5G 3L1
Received 3 March 2007; revised 29 March 2007; accepted 4 April 2007
Available online 19 April 2007
Abstract—An efficient synthesis and the cytotoxic activity of a series of substituted 6-amino-4H-[1,2]dithiolo[4,3-b]pyrrol-5-ones 1a–
q is described. The synthesis was accomplished in an expedient manner (seven-steps) from commercially available starting materials.
Several of the derivatives tested demonstrated significant in vitro cytotoxic activity against the human cancer cell lines H460
(P7 nM) and LCC6 (P28 nM). Following SAR and pharmacokinetic studies a derivative was further evaluated for its in vivo
anti-tumor activity against a highly angiogenic human melanoma xenograft where it demonstrated significant efficacy as a
mono-therapy and in combination with Taxol and Cisplatin.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
displaying antimycobacterial activity, thiolutin 3 (R1
and R2 = Me) has been shown to suppress tumor-in-
duced angiogenesis via the inhibition of focal adhesion
kinase (FAK).5 Thiomarinol A 4 is another structurally
related and potent antibiotic which was isolated from
marine bacteria Alteromonas rava.6 Hall and co-workers
have recently reported the asymmetric total synthesis of
a thiomarinol antibiotic.7 The total syntheses of holo-
mycin 2 (R1 = H and R2 = Me) and thiolutin 3 (R1
and R2 = Me) have been reported in the literature.8
However, the reported synthetic routes were not amend-
able to the preparation of derivatives with aromatic sub-
stituents on the pyrrolone nitrogen atom (R1 = Ar),
which were required for the present study. In this paper,
we report the synthesis, structure–activity relationships,
and cytotoxic activity on selected human cancer cell
lines of a series of substituted 6-amino-4H-[1,2]dithiolo-
[4,3-b]pyrrol-5-ones 1a–q.
Xenorhabdus sp. are bacterial symbionts of soil-living,
entomopathogenic nematodes and have been a rich
source of biologically active compounds, most notably
antibiotics.1 It has been theorized that the antibiotics
produced by the bacteria act to minimize competition
from secondary microbial contaminants within their
host and thus provide an optimal environment for
growth.2 Recently, while evaluating the bioactivity of
small molecule metabolites isolated from Xenorhabdus
sp. we demonstrated that the substituted 6-amino-4H-
[1,2]dithiolo[4,3-b]pyrrol-5-ones 1a–q (Fig. 1) have sig-
nificant anticancer activity.3 There are several known
natural products with related molecular structures such
as the antibiotics holomycin 2 (R1 = H and R2 = Me)
and thiolutin 3 (R1 and R2 = Me), both of which were
isolated from the bacteria Streptomyces sp.4 As well as
Keywords: Synthesis; 6-Amino-4H-[1,2]dithiolo[4,3-b]pyrrol-5-ones;
Cytotoxicity; Anti-tumor.
2. Results and discussion
2.1. Chemistry
*
Corresponding authors. Tel.: +1 604 432 1703; fax: +1 604 432
1704; e-mail addresses: bli@welichem.com; mlyle@welichem.com
Present address: Celestial Pharmaceuticals (Shenzhen) Ltd, Guang-
dong 518057, China.
The synthesis of the substituted 6-amino-4H-[1,2]dithi-
olo[4,3-b]pyrrol-5-ones 1a–q was accomplished in six
steps from 1,3-bis(tert-butylsulfanyl)propan-2-one 6
(Scheme 1). The 1,3-bis(tert-butylsulfanyl)propan-2-
à
Address: Lady Davis Institute for Medical Research, Departments
of Medicine, Oncology, Pharmacology and Therapeutics, McGill
University Faculty of Medicine, Montreal, Que., Canada.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.04.017

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B. Li et al. / Bioorg. Med. Chem. 15 (2007) 4601–4608
O
F C
3
S
2
S
R
CF
H
3
N
O
H
N
O
H
S
N
H
O
O
N
S
O
O
HO
N
S
Me
1
S
O
R
HO
HO
N
O
OMe
1
2
1a-q (R and R = various)
Holomycin 2 (R = H, R = Me)
1
2
OH
Me
1j
1
2
Thiolutin 3 (R and R = Me)
OMe
Me
Thiomarinol A 4
Figure 1. Substituted 6-amino-4H-[1,2]dithiolo[4,3-b]pyrrol-5-ones 1a–q, compound 1j, and the structurally related natural products holomycin 2
(R1 = H, R2 = Me), thiolutin 3 (R1 and R2 = Me), and thiomarinol A 4.
OH
ButS
O
O
a
b
c
ButS
StBu
O
Cl
Cl
N
R
1
ButS
5
6
1
7a (R = 4-methylphenyl)
1
7b (R = 4-methoxyphenyl)
1
7c (R = 2,4-dimethoxyphenyl)
1
7d (R = 4-iso-propylphenyl)
1
7e (R = benzyl)
CF
3
H
X
NH
2
ButS
ButS
N
ButS
O
O
e
d
O
O
+
N
H
N
R
N
1
1
ButS
ButS
R
ButS
9 (X = NH )
10 (X = OH)
2
1
1
8a (R = 4-methylphenyl)
8b (R = 4-methoxyphenyl)
8c (R = 2,4-dimethoxyphenyl)
8d (R = 4-iso-propylphenyl)
11a (R = 4-methylphenyl)
11b (R = 4-methoxyphenyl)
11c (R = 2,4-dimethoxyphenyl)
11d (R = 4-iso-propylphenyl)
1
1
1
1
1
1
1
1
8e (R = benzyl)
11e (R = benzyl)
2
CF
3
R
H
H
NH
HCl
N
2
N
O
O
O
S
S
f
g
S
S
O
S
S
O
N
N
N
1
1
R
R
1
R
1
1
13a (R = 4-methylphenyl)
1a-q (See Table 1)
12a (R = 4-methylphenyl)
1
1
13b (R = 4-methoxyphenyl)
12b (R = 4-methoxyphenyl)
1
1
13c (R = 2,4-dimethoxyphenyl)
12c (R = 2,4-dimethoxyphenyl)
1
1
13d (R = 4-iso-propylphenyl)
12d (R = 4-iso-propylphenyl)
1
1
13e (R = benzyl)
12e (R = benzyl)
Scheme 1. Synthesis of the substituted 6-amino-4H-[1,2]dithiolo[4,3-b]pyrrol-5-ones 1a–q. Reagents and conditions: (a) tert-butylmercaptan, NaOH
(aq), DMF, rt; (b) R1NH2, TiCl4, THF, 0 ꢁC then oxalyl chloride, NEt3, 0 ꢁC–rt (60–70% yield, over two steps); (c) ammonium butanoate, 100 ꢁC
(80–96% yield); (d) trifluoroacetic anhydride, THF, rt (90–95% yield); (e) Hg(OAc)2, TFA, rt then I2, CH2Cl2, rt (50–75% yield); (f) HCl (concd),
MeOH, reflux (65–80% yield); (g) R2COCl, NEt3, THF, rt (90–95% yield).
one 6 was prepared on a kilogram scale from commer-
cially available 1,3-dichloroacetone 5 upon reaction with
two equivalents of tert-butylmercaptan in the presence
of sodium hydroxide.9 The ketone 6 was reacted with
the appropriate primary amine in tetrahydrofuran in
the presence of titanium tetrachloride and triethylamine,

B. Li et al. / Bioorg. Med. Chem. 15 (2007) 4601–4608
4603
and the corresponding imines were subsequently reacted
(without isolation) with oxalyl chloride to afford the cyc-
lic enols 7a–e in good yield (60–70% yield, over two
steps). In all instances the cyclic enols 7a–e were ob-
tained as a single geometrical isomer. The cyclic enols
7a–e were then converted to the cyclic enamines 8a–e
upon heating in molten ammonium acetate at 150 ꢁC.
However, under these reaction conditions significant
amounts of the reaction byproducts 9 and 10 (up to
90% combined yield) were isolated along with the de-
sired reaction products 8a–e. The quantity of the
byproducts isolated was substrate dependent and purifi-
cation of the reaction mixtures required tedious chroma-
tography. It was subsequently determined that using
ammonium butanoate at 100 ꢁC led to significantly im-
proved yields of the desired reaction products 8a–e (up
to 95% yield).10 Reaction of the enamines 8a–e with tri-
fluoroacetic anhydride in tetrahydrofuran afforded the
corresponding trifluoroacetates 11a–e in high yields.
The tert-butyl protecting groups were then removed
upon treatment with mercuric acetate in trifluoroacetic
acid and the resultant dithiols were directly cyclized to
the dithiopyrrolones 12a–e under oxidative conditions
upon treatment with iodine. Hydrolysis of the trifluoro-
acetate moieties with concentrated hydrochloric acid
and methanol afforded the bicyclic enamines 13a–e in
good yield. It was essential to isolate the bicyclic enam-
ines 13a–e as their hydrochloride salts as they were
unstable in their neutral form. In the final step of the
synthesis, the bicyclic enamines 13a–e were elaborated
to the desired substituted 6-amino-4H-[1,2]dithiolo[4,3-
b]pyrrol-5-ones 1a–q upon treatment with the appropriate
acyl chloride in the presence of triethylamine.
Compounds 1a–q were isolated as highly stable and
brightly colored crystalline solids.
2.2. Biological results I (in vitro cytotoxicity)
For evaluation of their antiproliferative activity,
the substituted 6-amino-4H-[1,2]dithiolo[4,3-b]pyrrol-5-
ones 1a–q were tested in vitro against the actively grow-
ing human cancer cell lines H460 (lung) and LCC6
(breast). The effects are expressed as IC50 values below
(Table 1). A number of the derivatives tested displayed
notable cytotoxic activity against H460 (P7 nM) and
LCC6 (P28 nM). The natural product 1a (R1 = H,
R2 = 1-methylpentyl, entry 1), which was isolated from
Xenorhabdus sp., exhibited IC50 values of 200 nM for
both H460 and LCC6. Significant enhancements of
cytotoxic activity, particularly against H460, were ob-
served with aromatic substituents on the pyrrolone
nitrogen atom. Compound 1f (R1 = 2,4-dimethoxyphe-
nyl, R2 = methyl, entry 6) exhibited IC50 values of 30
and 190 nM for H460 and LCC6, respectively. Incorpo-
ration of a second aromatic substituent on the exocyclic
amide moiety led to a further increase in potency. Com-
pound 1h (R1 = 2,4-dimethoxyphenyl, R2 = 4-trifluo-
romethylphenyl, entry 8) exhibited IC50 values of 7
and 85 nM for H460 and LCC6, respectively. Decreases
in cytotoxicity were observed when benzyl substituents
were incorporated on the pyrrolone nitrogen atom.
Compound 1q (R1 = benzyl, R2 = 3,5-bistrifluorometh-
ylphenyl, entry 17) exhibited IC50 values of 706 and
1180 nM for H460 and LCC6, respectively. In general
it was observed that aromatic subsituents on the pyrro-
lone nitrogen atom as well as the exocyclic amide moiety
Table 1. IC50 values of the substituted 6-amino-4H-[1,2]dithiolo[4,3-b]pyrrol-5-ones 1a–q
R2
O
H
N
S
1a-q
S
O
N
R1
R1
R2
a
Cytotoxicity IC50 (nM)
Entry
Compound
H460
200 29
53
190 24
44
170 29
LCC6
1
2
1a
1b
1c
1d
1e
1f
H
1-Methylpentyl
Methyl
200 25
220 31
220 35
220 19
4-Methoxyphenyl
4-Methoxyphenyl
4-Methylphenyl
2,4-Dimethoxyphenyl
2,4-Dimethoxyphenyl
2,4-Dimethoxyphenyl
2,4-Dimethoxyphenyl
2,4-Dimethoxyphenyl
2,4-Dimethoxyphenyl
2,4-Dimethoxyphenyl
2,4-Dimethoxyphenyl
4-iso-Propylphenyl
2,4-Dimethoxyphenyl
Benzyl
6
3
Trifluoromethyl
Methyl
4
7
5
Trifluoromethyl
Methyl
60
9
6
30
11
7
6
1
1
1
190 43
251 31
85 11
7
1g
1h
1i
2,4-Dimethoxyphenyl
4-Trifluoromethylphenyl
3,5-Difluorophenyl
3,5-Bis-trifluoromethylphenyl
2-Furan
8
9
11
349 36
160 22
10
11
12
13
14
15
16
17
1j
130 19
1k
1l
50
8
5
1
28
210 34
40
70 13
2
2-Thiophene
1m
1n
1o
1p
1q
3,5-Dihydroxy-4-iso-propylphenyl
3,5-Dihydroxy-4-iso-propylphenyl
Methyl
150 27
72 11
7
900 137
170 32
706 143
—
250 48
1180 168
Benzyl
Benzyl
3,5-Dihydroxy-4-iso-propylphenyl
3,5-Bis-trifluoromethylphenyl
a IC50 values are reported as means SE of three independent determinations.

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B. Li et al. / Bioorg. Med. Chem. 15 (2007) 4601–4608
led to compounds with significantly increased potency
with respect to the natural product 1a (R1 = H,
R2 = 1-methylpentyl, entry 1).
2.3. Biological results (pharmacokinetics study)
To assess the pharmacokinetic (PK) properties of this
class of substituted 6-amino-4H-[1,2]dithiolo[4,3-b]pyr-
rol-5-ones 1a–q, compounds 1j and 1n were adminis-
tered by intraperitoneal injection (IP) to female mice.
These two compounds were chosen due to their superior
in vitro cytotoxicity profiles that were observed in the
NCI’s extensive cell line screening. The mean plasma
concentrations were plotted versus time following an
IP dose of 20 mg/kg (Fig. 2).
Following the preliminary investigations in our
laboratory against the H460 and LCC6 cancer cell lines,
the substituted 6-amino-4H-[1,2]dithiolo[4,3-b]pyrrol-5-
ones 1a–q were tested on the National Cancer Institute’s
(NCI) panel of 60 cancer cell lines. It was demonstrated
that the cytotoxicity of the substituted 6-amino-4H-
[1,2]dithiolo[4,3-b]pyrrol-5-ones 1a–q was selective for
solid cancer cells over hematological cancer cells since
the LC50 values were significantly lower (ꢀ100-fold) in
all of the solid cancer cell lines with respect to leukemia
and lymphoma cell lines. Similar selectivity was not
observed in the IC50 values which measure growth
inhibition and thus, as anticancer agents, the substituted
6-amino-4H-[1,2]dithiolo[4,3-b]pyrrol-5-ones 1a–q may
be most effective against solid tumors. During the NCI
testing, compounds 1j and 1n emerged as the derivatives
which demonstrated the most promising overall in vitro
cytotoxicity (NCI data not shown).
Each of the compounds 1j and 1n was absorbed rapidly
into the blood stream and their Cmax values (57 and
106 lM, respectively) were reached within 30 min of
administration. Over the next 8 h, the plasma concentra-
tions of 1j and 1n decreased significantly. The plasma
concentrations of compounds 1j and 1n exceeded their
corresponding IC50 values for both the H460 and
LCC6 cell lines for the entire duration of the PK exper-
iments (8 h). The overall IP pharmacokinetic profiles of
compounds 1j and 1n are summarized below (Table 2).
The systemic plasma clearances (CL) for compounds
The NCI’s COMPARE algorithm did not yield any sig-
nificant activity correlations with any other compounds
in the database which indicates that the substituted
6-amino-4H-[1,2]dithiolo[4,3-b]pyrrol-5-ones 1a–q may
exert their cytotoxic effect via a unique mode of action.
Of note, we have demonstrated that the substituted
6-amino-4H-[1,2]dithiolo[4,3-b]pyrrol-5-ones 1a–q in-
duce apoptosis in both human endothelial cells and hu-
man caner cells using the Annexin V apoptosis assay.
Moreover, the substituted 6-amino-4H-[1,2]dithiol-
o[4,3-b]pyrrol-5-ones 1a–q do not affect cell-cycle pro-
gression and thus the demonstrated cytotoxic effect is
not simply due to the inhibition of cell proliferation.
Table 2. The IP pharmacokinetic profile of compounds 1j and 1n in
female micea
Parameter
Unit
Compound 1j
Compound 1n
Dose
Cmax
lmol/kg
lM
min
36.8
57
38.2
106
3
Tmax
30
160
T1/2
min
lM min
mL/min/kg
94
6097
6.3
AUC(0–480)
CL
12,629
2.9
a Reported values are means of two mice/time/concentration data
points.
110
100
Compound 1n
Compound 1j
90
80
70
60
50
40
30
20
10
0
0
50
100
150
200
250
300
350
400
450
500
Time (min)
Figure 2. A plot of the mean plasma concentration versus time for compounds 1j and 1n following intraperitoneal injection (20 mg/kg body weight).
The error bars represent the overall data distribution ( SD/n1/2).

B. Li et al. / Bioorg. Med. Chem. 15 (2007) 4601–4608
4605
1j and 1n were 2.9 mL/min/kg and 6.3 mL/min/kg,
respectively. The calculated half-life (T1/2) for com-
pounds 1j and 1n was 160 min and 94 min, respectively.
In the combination-therapy experiments, compound 1j
was administered by intraperitoneal injection daily for
24 days (5 and 10 mg/kg body weight) and in addition
either Taxol (10 mg/kg body weight) or Cisplatin
(3 mg/kg body weight) was administered by intraperito-
neal injection every second day for 24 days (total of 12
doses). In combination with Taxol, compound 1j at
10 mg/kg every day inhibited SKMEL-28 V+ tumor
growth by approximately 87% compared to vehicle mice
(Taxol alone induced approximately 37% inhibition,
Fig. 3). In combination with Cisplatin, compound 1j
at 10 mg/kg every day inhibited SKMEL-28 V+ growth
by approximately 76% (Cisplatin alone induced approx-
imately 26% inhibition, Fig. 3).
Similar pharmacokinetic profiles were observed when
compounds 1j and 1n were orally administered. High
calculated oral bioavailabilities (Fpo) of greater than
70% were noted. Following an analysis of the pharma-
cokinetic and in vitro cytotoxicity data, compound 1j
was selected for advancement into in vivo anti-tumor
efficacy studies.
2.4. Biological results (in vivo anti-tumor activity)
The in vivo anti-tumor activity of compound 1j was
evaluated as a mono-therapy and as a combination-ther-
apy with the commercial chemotherapy drugs Taxol or
Cisplatin in male mice (Mus musculus) implanted subcu-
taneously with a human SKMEL-28 V+ tumor xeno-
graft. SKMEL-28 V+ tumor cells are a variant of
SKMEL-28 cells that have been engineered to overex-
press vascular endothelial growth factor (VEGF) and
thus display high angiogenic activity. This particular tu-
mor was chosen due to the hypothesis that compound 1j
may inhibit tumor-induced angiogenesis.4 In the mono-
therapy experiments, compound 1j was administered by
intraperitoneal injection daily for 24 days (5 and 10
mg/kg body weight) or every second day (20 mg/kg body
weight). Upon completion of the experiment, compound
1j had demonstrated significant in vivo anti-tumor activ-
ity in the absence of gross (e.g., loss of body weight) or
histological indications of toxicity. Dose–response
efficacy was observed and, at the highest dose of
20 mg/kg given every second day, tumor growth was
inhibited by approximately 58% as compared to vehicle
mice (Fig. 3).
2.5. Conclusion
We have described the efficient synthesis and prelimin-
ary biological evaluation of a novel class of substi-
tuted 6-amino-4H-[1,2]dithiolo[4,3-b]pyrrol-5-ones 1a–q
as potential anticancer agents. SAR studies demon-
strated that aromatic substituents on the pyrrolone
nitrogen atom as well as the exocyclic amide moiety
led to compounds with significant in vitro antiprolifer-
ative activity against H460 (P7 nM) and LCC6
(P28 nM) cancer cell lines while displaying favorable
pharmacokinetic profiles. Ultimately, compound 1j
was selected for in vivo anti-tumor activity studies
upon human SKMEL-28 V+ xenografts as both a
mono-therapy and in combination with commercial
Cisplatin or Taxol where strong efficacy was
demonstrated. Work to determine the molecular tar-
get(s) responsible for the anti-cancer activity of the
substituted 6-amino-4H-[1,2]dithiolo[4,3-b]pyrrol-5-
ones 1a–q is currently underway and will be reported
in due course.
7
Vehicle
1j (5 mg/kg)
6
1j (10 mg/kg)
1j (20 mg/kg)
5
taxol (10 mg/kg)
1j (5 mg/kg) + taxol (10 mg/kg)
4
1j (10 mg/kg) + taxol (10 mg/kg)
3
2
1
0
0
5
10
15
20
25
Time Post Intial Treatment (days)
Figure 3. The in vivo efficacy of compound 1j (5, 10, and 20 mg/kg) as a mono-therapy and in combination with Taxol (10 mg/kg) or Cisplatin (3 mg/
kg) on SKMEL-28 V+ human tumor xenograft growth compared to vehicle. The error bars represent the overall data distribution ( SD/n1/2).

4606
B. Li et al. / Bioorg. Med. Chem. 15 (2007) 4601–4608
3. Experimental
compounds 1n and 1j were 8.13 min and 5.51 min,
respectively.
3.1. General experimental
3.1.4. Tumor animal models and treatment. The animal
experiments were approved by McGill University Ethics
Committee and were conducted at the Lady Davis Insti-
tute Animal Facility in accordance with the Canadian
Guide for the Care and Use of Laboratory Animals,
and the Animal Welfare Act. Clinical grade Cisplatin
and Taxol were purchased from the Oncology Pharmacy
at the Jewish General Hospital (McGill University) and
stored at room temperature or at 4 ꢁC, respectively. To
induce tumor formation in mice, SKMEL-28 cells (orig-
inally from the ATCC) were engineered to overexpress
mouse VEGF and were grown to 60% confluence in
DMEM that was purchased from Invitrogen and sup-
plemented with 10% FBS and then harvested using tryp-
sin–EDTA solution. The cells were centrifuged and
washed twice with phosphate-buffered saline solution
and then re-suspended at a dilution of 1–2 · 106 cells/
0.1 mL. The cell viability was confirmed by Trypan blue
staining. Only those cells with >95% viability and ‘nor-
mal’ morphology were used for the in vivo experiments.
Approximately 2 million cells were used to induce pri-
mary tumors in five mice. Once the tumor size reached
approximately 1 cm3, the tumor was isolated, dissected
to remove non-tumor tissue, washed three times with
phosphate-buffered saline (PBS), and then sliced into
small pieces of approximately 1 mm3. Tissue explants
were then inoculated subcutaneously into individual
mice. Once the tumors became palpable, the mice were
randomized into experimental groups and the treatment
was initiated. All drugs were administered by intraperi-
toneal injection daily or every second day. Primary tu-
mor growth was monitored every second to fifth day
using a caliper. Relative tumor volume (cm3) was esti-
mated using the formula [(length(cm) · width(cm)2)/2].
At the end of the experiment, the mice were sacrificed
3.1.1. Chemistry. All non-aqueous reactions were per-
formed under an atmosphere of dry nitrogen and in
flame- or oven-dried glassware unless stated otherwise.
The reaction temperatures stated were those of the
external bath. All solvents and reagents were purified
according to standard procedures or used as sup-
plied.11 Silica gel chromatography (‘flash chromatogra-
phy’) was carried out using Merck silica gel 60 (230–
400 mesh).12 All proton and carbon nuclear magnetic
resonance spectra (1H and 13C NMR, respectively)
were recorded on a Varian 400 FT spectrometer at
ambient temperature. The chemical shifts for all com-
pounds are listed in parts per million downfield from
tetramethylsilane using the NMR solvent as an inter-
nal reference. The reference values used for deuterated
1
chloroform (CDCl3) were 7.26 and 77.16 ppm for H
and 13C NMR spectra, respectively. Low resolution
mass spectra (MS) were recorded on a Hewlett–Pack-
ard 5985 GC-mass spectrometer. The mode of ioniza-
tion used was chemical ionization (CI) with
isobutane. Microanalyses were performed on a Carlo
Erba Model 1106 CHN analyzer.
3.1.2. Determination of IC50 values. The human cancer
cell lines, H460 and LCC6, were purchased from the
American Type Culture Collection (ATCC) and were
cultured in RPMI 1640 containing 10% fetal bovine ser-
um (FBS) which was purchased from Invitrogen. The
drug solutions were prepared in DMSO and the cells
were treated with the appropriate concentrations
for 48 h. The cells were then incubated with MTT
(0.5 mg/mL) for 4 h, the resultant MTT color precipi-
tates were dissolved in DMSO, and the absorbance
was determined at 470 nm. The percentage of cell
growth versus log concentration was plotted to generate
a linear equation. The IC50 value of the compound is the
concentration that gives a 50% growth.13
by cervical dislocation and
conducted.
a full autopsy was
3.2. Chemical experimental
3.1.3. Pharmacokinetic studies. The pharmacokinetic
studies with the substituted 6-amino-4H-[1,2]dithiolo-
[4,3-b]pyrrol-5-ones 1n and 1j were conducted in con-
scious male mice (n = 2 per study). The compounds 1n
and 1j were administered by either intraperitoneal injec-
tion in a vehicle of 5% chremophore in saline at a dose
of 20 mg/kg or orally via gavage in a vehicle of soybean
oil at a dose of 20 mg/kg. Blood samples of 300 lL were
drawn from the tail vein for determination of the plasma
concentrations of the substituted 6-amino-4H-[1,2]dithi-
olo[4,3-b]pyrrol-5-ones 1n and 1j at multiple time points
up to 8 h after administration. Calibration standards
were prepared via serial dilution from a 5 lg/mL aceto-
nitrile standard. Blood samples of 100 lL were centri-
fuged and the resultant plasma was extracted with
300 lL of cold acetonitrile. HPLC analysis for quantifi-
cation was performed on a Waters 2695 instrument with
a Waters l Boundpack C18 column. The mobile phase
was comprised of 30% water (containing 0.2% triethyl-
amine and 0.5% phosphoric acid) and 70% acetonitrile
at a flow rate of 1.0 mL/min. The retention times for
3.2.1. 4-tert-Butylsulfanyl-5-tert-butylsulfanylmethylene-
1-(2,4-dimethoxyphenyl)-3-hydroxy-1,5-dihydro-pyrrol-2-
one (7c). To a stirred solution of 1,3-bis(tert-butylsulfa-
nyl)propan-2-one 6 (2.34 g, 10.0 mmol) in tetrahydrofu-
ran (100 mL) were added 2,4-dimethoxyaniline (1.53 g,
10.0 mmol) and triethylamine (2.8 mL, 20 mmol) and
the resultant solution was cooled to 0 ꢁC. A solution
of titanium tetrachloride (1.04 g, 5.50 mmol) in hexanes
(15 mL) was added dropwise over the course of 30 min.
After the addition was complete, the reaction mixture
was allowed to warm to room temperature and was then
heated at reflux temperature for 2 h. The reaction mix-
ture was cooled to À10 ꢁC and oxalyl chloride
(0.84 mL, 10 mmol) was added followed by the dropwise
addition of
a solution of triethylamine (2.8 mL,
20 mmol) in tetrahydrofuran (100 mL) over the course
of 30 min. After the addition was complete, the reaction
mixture was allowed to warm to room temperature and
was stirred for 15 h. The solution was then filtered and
the precipitate was washed with ether (250 mL). The

B. Li et al. / Bioorg. Med. Chem. 15 (2007) 4601–4608
4607
combined filtrate was washed with water (3· 50 mL),
dried over anhydrous sodium sulfate, and concentrated
in vacuo to afford the crude product. The crude product
was recrystallized from ethyl acetate/hexanes to afford
the title compound 7c (2.97 g, 70%) as a light yellow
crystalline solid. Mp >190 ꢁC, ethyl acetate/hexanes;
1H NMR (CDCl3) d 1.30 (9H, s, C(CH3)3), 1.41 (9H,
s, C(CH3)3), 3.76 (3H, s, OCH3), 3.88 (3H, s, OCH3),
6.45 (1H, s, vinyl CH), 6.53–6.60 (2H, m, ArH),
7.09–7.17 (1H, m, ArH); MS (CI) m/z (rel. intensity)
424 (M+H, 100); Anal. Calcd for C21H29NO4S2:
C, 59.54; H, 6.90; N, 3.31. Found: C, 59.34; H, 6.97;
N, 3.19.
anes/dichloromethane (1:1:1) as the eluant afforded
the title compound 12c (1.36 g, 67%) as a yellow crys-
talline solid. Mp 151–152 ꢁC, ethyl acetate/hexanes/
dichloromethane; 1H NMR (CDCl3) d 3.73 (3H, s,
OCH3), 3.81 (3H, s, OCH3), 6.52 (1H, s, vinyl CH),
6.53–6.56 (2H, m, ArH), 7.14–7.16 (1H, m, ArH),
8.43 (1H, s, NHCOCF3); MS (CI) m/z (rel. intensity)
405 (M+H, 100); Anal. Calcd for C15H11F3N2O4S2:
C, 44.55; H, 2.74; N, 6.93. Found (V2O5 added): C,
44.35; H, 2.96; N, 6.69.
3.2.4.
N-[4-(2,4-Dimethoxyphenyl)-5-oxo-4,5-dihydro-
[1,2]-dithiolo[4,3-b]pyrrol-6-yl]-3,5-bis-trifluoromethyl benz-
amide (1j). To a stirred solution of the trifluoroaceta-
mide 12c (1.00 g, 2.47 mmol) in methanol (150 mL)
was added concentrated hydrochloric acid (5 mL) and
the resultant solution was heated at reflux temperature
for 2 h. The reaction mixture was allowed to cool to
room temperature and was then concentrated in vacuo
to afford the hydrochloride salt 13c (760 mg, 89%) as a
green crystalline solid. This material was taken up in tet-
rahydrofuran (30 mL) and was treated with 3,5-bis-trif-
luoromethylbenzoyl chloride (622 mg, 2.25 mmol).
Triethylamine (0.55 mL, 4.2 mmol) was then added
dropwise over the course of 2 min and the resultant solu-
tion was stirred at room temperature for 30 min. The
reaction mixture was concentrated in vacuo to afford
the crude product. Flash chromatography using ethyl
acetate/hexanes/dichloromethane (1:1:1) afforded the
title compound 1j (977 mg, 81%) as a bright orange crys-
talline solid. Mp 135–138 ꢁC, ethyl acetate/hexanes/
dichloromethane; 1H NMR (CDCl3) d 3.78 (3H, s,
OCH3), 3.82 (3H, s, OCH3), 6.53–6.57 (3H, m, vinyl
CH and ArH), 7.21 (1H, d, J = 7 Hz, ArH), 8.06 (1H,
s, ArH), 8.48 (2H, s, ArH), 8.89 (1H, s, NHCO); MS
(CI) m/z (rel. intensity) 549 (M+H, 100); Anal. Calcd
for C22H14F6N2O4S2: C, 48.18; H, 2.57; N, 5.11. Found:
C, 47.90; H, 2.90; N, 4.73.
3.2.2. 4-tert-Butylsulfanyl-5-tert-butylsulfanylmethylene-
1-(2,4-dimethoxyphenyl)-3-amino-1,5-dihydro-pyrrol-2-
one (8c). A mixture of the enol 7c (2.12 g, 5.00 mmol)
and ammonium butanoate (50 g, 478 mmol) was heated
to 100 ꢁC and held at that temperature for 15 min. The
reaction mixture was allowed to cool to approximately
50 ꢁC and was poured into water (100 mL) and the
resultant mixture was extracted with ether (3·
100 mL). The combined organic extracts were washed
with an aqueous solution of sodium hydroxide (10%
w/w, 25 mL) and then with water (2· 25 mL). The or-
ganic solution was then dried over anhydrous sodium
sulfate and concentrated in vacuo to afford the crude
product. Flash chromatography using hexanes/ethyl
acetate (1:1) as the eluant afforded the title compound
8c (2.01 g, 95%) as a yellow crystalline solid. Mp 123–
124 ꢁC, ethyl acetate/hexanes; 1H NMR (CDCl3) d
1.28 (9H, s, C(CH3)3), 1.40 (9H, s, C(CH3)3), 3.77
(3H, s, OCH3), 3.88 (3H, s, OCH3), 4.43 (2H, broad s,
NH2), 6.14 (1H, s, vinyl CH), 6.52–6.58 (2H, m, ArH),
7.13–7.16 (1H, m, ArH); MS (CI) m/z (rel. intensity)
423 (M+H, 100); Anal. Calcd for C21H30N2O3S2: C,
59.68; H, 7.16; N, 6.63. Found: C, 59.30; H, 7.27; N,
6.52.
3.2.3. N-[4-(2,4-Dimethoxyphenyl)-5-oxo-4,5-dihydro-[1,
2]dithiolo[4,3-b]pyrrol-6-yl]-2,2,2-trifluoroacetamide
(12c). To a stirred solution of the enamine 8c (2.11 g,
5.00 mmol) in tetrahydrofuran (20 mL) was added tri-
fluoroacetic anhydride (0.76 mL, 5.5 mmol) and the
resultant solution was stirred for 30 min. The reaction
mixture was concentrated in vacuo to afford the triflu-
oroacetamide 11c (2.60 g, 100%) as a yellow crystal-
line solid. This material was taken up in
trifluoroacetic acid (50 mL) and was treated with mer-
curic acetate (1.59 g, 5.00 mmol) and the resultant
solution was stirred at room temperature for 1 h.
The reaction mixture was concentrated in vacuo and
the residue was taken up in acetonitrile (100 mL).
Hydrogen sulfide was then bubbled through the reac-
tion mixture for 1 h. The reaction mixture was then
degassed of excess hydrogen sulfide via bubbling nitro-
gen through the reaction mixture for 30 min. A solu-
tion of iodine (1.27 g, 5.00 mmol) in dichloromethane
(50 mL) was then added dropwise over the course of
10 min and the reaction mixture was then stirred at
room temperature for 30 min. The reaction mixture
was concentrated in vacuo to afford the crude prod-
uct. Flash chromatography using ethyl acetate/hex-
Acknowledgments
We are grateful to the National Research Council of
Canada (NRC) for financial support (NRC-IRAP
#611050). MPAL thanks the National Sciences and
Engineering Research Council of Canada (NSERC)
for an industrial postdoctoral research fellowship. We
also thank the National Cancer Institute (NCI) for
in vitro screening and Simon Fraser University for
NMR services.
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