Convenient syntheses of novel 1-isothiocyano-alkylphosphonate diphenyl ester derivatives with potential biological activity

Article abstract of DOI:10.1016/j.tetlet.2012.08.037

Herein, we describe a convenient method for the syntheses of novel 1-isothiocyano-alkylphosphonate diaryl ester derivatives and their antiproliferative activity. The syntheses are based on dithiocarbamates obtained in situ with the use of carbon disulfide

Full text of DOI:10.1016/j.tetlet.2012.08.037

Tetrahedron Letters 53 (2012) 5845–5847
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Convenient syntheses of novel 1-isothiocyano-alkylphosphonate diphenyl
ester derivatives with potential biological activity
Mateusz Psurski a,b, Marta Piguła a, Jarosław Ciekot b, Łukasz Winiarski a, Joanna Wietrzyk b,c
,
Józef Oleksyszyn a,
⇑
a Division of Medicinal Chemistry and Microbiology, Wroclaw University of Technology, Faculty of Chemistry, Division of Medicinal Chemistry and Microbiology, Coast of Stanislaw
Wyspianski St. 27, 50-370 Wroclaw, Poland
b Department of Experimental Oncology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolf Weigl St. 12, 50-314 Wroclaw, Poland
c Institute of Chemistry, Environmental Protection and Biotechnology, Jan Dlugosz University, National Army Avenue 13, 42-218 Czestochowa, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein, we describe a convenient method for the syntheses of novel 1-isothiocyano-alkylphosphonate
diaryl ester derivatives and their antiproliferative activity. The syntheses are based on dithiocarbamates
obtained in situ with the use of carbon disulfide under basic conditions, and their desulfurization using
several different reagents, of which hydrogen peroxide proved to be the most efficient. The compounds
synthesized demonstrated high antiproliferative activity against several cancer cell lines in vitro, and also
showed some activity as serine protease inhibitors.
Received 5 June 2012
Revised 25 July 2012
Accepted 9 August 2012
Available online 20 August 2012
Keywords:
Isothiocyanates
Ó 2012 Elsevier Ltd. All rights reserved.
Phosphonate diaryl esters
Antiproliferative activity
Serine protease inhibition
The isothiocyanate moiety is a useful functional group in organ-
ic syntheses, acting as a strong electrophile with the carbon atom
as an electrophilic center. It has proved to be useful in a wide vari-
ety of synthetic applications, for example in pseudo-peptide thio-
urea syntheses1 and in heterocyclic chemistry.2 Isothiocyanates
(ITC) are also found in plants in large amounts and have a high de-
gree of structural diversity.3 In numerous in vitro and in vivo stud-
ies these naturally occurring isothiocyanates (such as benzyl
isothiocyanate and allyl isothiocyanate) have shown high antipro-
liferative and anticancer activity (see the review by Zhang et al.4),
and have negligible toxicity. Such advantages make them very
promising natural compounds for cancer treatment. A key phase
responsible for the above activities is fast cellular accumulation
via passive diffusion followed by the cellular glutathione-isothio-
cyanate reaction and/or protein modification resulting in cell cycle
arrest and apoptosis. As a result, the intracellular concentration of
isothiocyanates can reach high levels (e.g., after 30 min of treat-
ment, intracellular concentrations can reach 9.4 mM in Hepa
1c1c7 cells5). We believe that such a phenomenon could be utilized
in order to transport additional biologically active moieties present
in the same structure. In such an approach, even moieties with
lower biological activity could be useful due to their accumulation
inside the cells, which is especially crucial for compounds such as
enzyme inhibitors for example.
The mechanism of action of natural and synthetic isothiocya-
nates is not clear. These electrophilic species could simply deplete
cellular glutathione and/or react with some of the very important
cellular–SH proteins. In the first case, there is no clear structure
activity–relationship, therefore any new synthetic isothiocyanate
could provide additional evidence for such a mechanism of action.
Here, we describe a series of new isothiocyanates containing a
phosphonate diaryl ester moiety. We have found that these com-
pounds are potent antiproliferative agents, but also, like other dia-
ryl phosphonate esters,6 they are able to inhibit irreversibly serine
proteases.
Among several methods for the syntheses of isothiocyanates,
those based on primary amine conversion using thiophosgene,
di-2-pyridyl thionocarbonate, or carbon disulfide are among the
most extensively used. Since thiophosgene is extremely toxic and
di-2-pyridyl thionocarbonate is relatively expensive, we chose
carbon disulfide to convert primary amine hydrobromides into
the corresponding isothiocyanates. In this method, the amine
moiety is first converted into the corresponding dithiocarbamate
by reaction with carbon disulfide in the presence of a base in a
water miscible organic solvent, followed by subsequent desulfur-
ization with an appropriate reagent. Three different desulfurization
agents were tested: O-benzotriazole-N,N,N1,N1-tetramethyluronium
⇑
Corresponding author. Tel.: +48 (071) 3204027; fax: +48 (071) 3202425.
E-mail address: jozef.oleksyszyn@pwr.wroc.pl (J. Oleksyszyn).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.08.037

5846
M. Psurski et al. / Tetrahedron Letters 53 (2012) 5845–5847
O
O
R'
HBF4/MeCN
O
O
P
O
O
P
O
O
NH2
O
N
H
R'
H
3
33% HBr/AcOH
CS2/DIPEA/HBTU/DMF
or
R'
R'
O
P
O
O
P
O
CS2/Et3N/H2O2/DMF
O
O
SCN
H2N
HBr
Scheme 1.
Table 1
Chemical structure and yield of the obtained isothiocyanates
Product
Yield (%) HBTU/H2O2
Product
Yield (%) HBTU/H2O2
O
O
P
O
O
P
1
78/84
6
79/75
NCS
O
NCS
NCS
O
O
P
O
O
P
O
O
O
O
O
2
3
72/86
84/92
7
8
57/55
67/64
NCS
NO2
O
P
O
O
O
P
O
NCS
NCS
NCS
Cl
O
P
O
O
P
O
4
90/93
9
86/77
O
O
NCS
NCS
O
O
P
O
O
O
P
O
5
77/90
10
77/85
O
NCS
hexafluorophosphate (HBTU), tosyl chloride (TsCl), and hydrogen
peroxide (H2O2). In all the methods, 1-amine-alkylphosphonate
products were always present along with the target compound,
thus purification via silica gel column chromatography was neces-
sary. The use of tosyl chloride as the desulfurization agent did not
improve the yields or the purities of the final isothiocyanates,
therefore this reagent was not tested further. The conversion of
the amine hydrobromide into isothiocyanate with the use of
hydrogen peroxide, previously applied to hydroxyalkyl isothiocya-
nate synthesis,8 allowed us to obtain the target compounds in high
yields, and in higher purities in comparison to the HBTU-based
diaryl ester hydrobromides, obtained using a modified a-amidoal-
kylation reaction,6 followed by a standard Cbz-deprotection, were
used as the substrates (see Scheme 1).
The method based on HBTU as the desulfurization agent, as pre-
viously reported by Boas7 and used in the solid-phase syntheses of
thioureas, provided us with the target compounds in moderate to
high yields and with satisfactory purity. However, several side

M. Psurski et al. / Tetrahedron Letters 53 (2012) 5845–5847
5847
Table 2
and examined in order to evaluate the possible correlation be-
tween the chemical structure of the obtained isothiocyanates, their
biological activity and their potential as anticancer agents. Addi-
tionally, the inhibitory potency of the synthesized compounds
against human neutrophil elastase and chymotrypsin was mea-
sured using fluorescent substrates. Some of them inhibited these
The antiproliferative activity of the synthesized compounds against several cancer
cell lines in vitro
ITC
IC50
LoVoDX
[lM] SD
LoVo
A549
MCF7
1
2
3
4
5
6
7
8
9
10
10
10
12
70
16
13
13
69
19
7
1
11
10
54
71
17
21
27
68
12
8
1
36
2
29
33
60
79
30
34
33
80
40
20
1
1
2
1
1
2
2
3
2
1
serine proteases with k2/Kinact in the range of 80–870 MÀ1 sÀ1
.
1
1
1
2
1
1
2
1
1
1
2
3
1
1
1
2
1
1
47
68
86
34
17
18
66
59
31
2
2
2
2
1
1
3
2
2
However, due to the presence of two electrophilic centers, the
mechanism of inhibition is not clear and investigations of this issue
are underway.
In summary, a rapid and convenient method for the syntheses
of novel 1-isothiocyano-alkylphosphonate diaryl esters, as building
blocks in organic chemistry and as anticancer agents, has been de-
scribed. Their in vitro antiproliferative activity against cancer cells
is in the range of natural isothiocyanates despite the significant dif-
ferences in their structures. This seems to suggest that depletion of
glutathione is the main mechanism for the antiproliferative poten-
tial of isothiocyanates, when –NCS reactivity with small molecules
(such as GSH, glutathione sulfide) is not influenced by the size and
shape of other regions of molecules (excluding electron-withdraw-
ing effects). Additionally, the obtained compounds possess serine
protease inhibitory activity. In our opinion such compounds are a
good starting point to develop a new class of biologically active
agents as potential drugs or as tools in cancer studies.
method. Nevertheless, only compounds 1-3 and 6 (see Table 1)
were obtained as satisfactorily pure products, and for all the other
compounds the crude purity was 65–80%. However, the impurities
could easily be removed using flash chromatography. It should be
noted that in this method the temperature of the reaction had to be
kept in the range of 0–15 °C in order to maintain the yield and pur-
ity at high levels. Allowing the reaction mixture to warm up during
H2O2 addition led to the formation of several side products, and in
most cases, separation of the target compound was difficult.
In both of the methods described, the base employed was tri-
ethylamine (Et3N); N,N-diisopropylethylamine (DIPEA) can be used
with no detrimental influence on the yield and purity. Considering
the fact that the isothiocyanate moiety readily reacts with a pri-
mary amine under basic conditions resulting in thiourea derivative
formation, the pH of the reaction should be kept below 8 to avoid
such a side reaction, and/or an excess of CS2 should be added in or-
der to ensure full conversion of the amine substrate into the dithio-
carbamate salt. For most of the compounds the best yields and
purities were achieved when 4–6 equiv of DIPEA and 10–15 equiv
of CS2 were used along with 1.5 equiv of HBTU as the desulfuriza-
tion agent; 1.1-1.5 equiv of Et3N, and 10–15 equiv of CS2 when 3–
4 equiv of hydrogen peroxide are used. However, in the case of
compounds 7 and 8, the conversions, regardless of the method
used, decreased significantly. For example, from 57% for 7 and
67% for 8 to 11% and 9%, respectively, when four molar equivalents
of DIPEA were used in the HBTU-based method. The most probable
explanation for this is the instability of the corresponding hydro-
bromides under basic conditions. For the other compounds de-
scribed in this Letter, such decomposition was not observed.
All of the compounds synthesized were tested for their antipro-
liferative activity against breast (MCF7), lung (A549), and both
doxorubicin-sensitive (LoVo) and doxorubicin-resistant (LoVoDX)
colon cancer cell lines. The results are presented in Table 2. For
the most active compounds, (1, 2 and 10) the antiproliferative
effects were comparable to those of the naturally occurring
isothiocyanates.9 Clearly, more compounds need to be synthesized
Acknowledgments
This work was supported by a grant of the National Science
Center, grant no. 2011/01/N/NZ4/03361. J.C. thanks the European
Social Fund for support. The authors thank Prof. Tadeusz Gajda
and Dr Waldemar Goldeman for useful discussions.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2012.08.
037. These data include MOL files and InChiKeys of the most
important compounds described in this article.
References and notes
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3. Fahey, J. W.; Zalcmann, A. T.; Talalay, P. Phytochem. 2001, 56, 5.
4. Zhang, Y. Carcinogenesis 2012, 33, 2.
5. Zhang, Y. Carcinogenesis 2000, 21, 1175.
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149, 556785.
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