Synthesis and antiproliferative evaluation of 5-oxo and 5-thio derivatives of 1,4-diaryl tetrazoles

Article abstract of DOI:10.1016/j.bmcl.2010.05.012

A series of 1,4-diaryl tetrazol-5-ones were synthesized by copper mediated N-arylation of 1-phenyl-1H-tetrazol-5(4H)-one with aryl boronic acids, o-R ₁C₆H₄B(OH)₂ where R₁ = H, OMe, Cl, CF₃, Br, CCH. The 1,4-diaryl tetrazol-5-ones substituted with OMe, Cl, CF₃, Br underwent thionation with Lawesson's reagent to yield the corresponding 5-thio derivatives. The 1-(2-bromophenyl)-4-phenyl-1H- tetrazole-5(4H)-thione so obtained was subjected to lithiation/protonation and Sonogashira coupling to produce 1,4-diphenyl-1H-tetrazole-5(4H)-thione and 1-(2-ethynylphenyl)-4-phenyl tetrazole-5-thione, respectively. The title compounds were found to be stable to strong Lewis acid conditions. Three of these novel compounds were found to inhibit L1210 leukemia cell proliferation and SK-BR-3 breast cancer cell growth over several days in culture in vitro. Shorter tetrazole derivative treatments also reduced the expression of the Ki-67 marker of cell proliferation in SK-BR-3 cells and the rate of DNA synthesis in L1210 cells.

Full text of DOI:10.1016/j.bmcl.2010.05.012

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3920–3924
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antiproliferative evaluation of 5-oxo and 5-thio derivatives
of 1,4-diaryl tetrazoles
Aditya S. Gundugola a, Kusum Lata Chandra a, Elisabeth M. Perchellet b, Andrew M. Waters b,
Jean-Pierre H. Perchellet b, Sundeep Rayat a,
*
a Department of Chemistry, Kansas State University, 213 CBC Building, Manhattan, KS 66506-0401, USA
b Anti-Cancer Drug Laboratory, Kansas State University, Division of Biology, Ackert Hall, Manhattan, KS 66506-4901, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 1,4-diaryl tetrazol-5-ones were synthesized by copper mediated N-arylation of 1-phenyl-1H-
tetrazol-5(4H)-one with aryl boronic acids, o-R1C6H4B(OH)2 where R1 = H, OMe, Cl, CF3, Br, C„CH. The
1,4-diaryl tetrazol-5-ones substituted with OMe, Cl, CF3, Br underwent thionation with Lawesson’s
reagent to yield the corresponding 5-thio derivatives. The 1-(2-bromophenyl)-4-phenyl-1H-tetrazole-
5(4H)-thione so obtained was subjected to lithiation/protonation and Sonogashira coupling to produce
1,4-diphenyl-1H-tetrazole-5(4H)-thione and 1-(2-ethynylphenyl)-4-phenyl tetrazole-5-thione, respec-
tively. The title compounds were found to be stable to strong Lewis acid conditions. Three of these novel
compounds were found to inhibit L1210 leukemia cell proliferation and SK-BR-3 breast cancer cell
growth over several days in culture in vitro. Shorter tetrazole derivative treatments also reduced the
expression of the Ki-67 marker of cell proliferation in SK-BR-3 cells and the rate of DNA synthesis in
L1210 cells.
Received 7 April 2010
Revised 4 May 2010
Accepted 7 May 2010
Available online 12 May 2010
Keywords:
Copper catalyzed N-arylation
Lawesson’s reagent
Tetrazol-5-one
Tetrazole-5-thione
Antiproliferative activity
Ó 2010 Elsevier Ltd. All rights reserved.
Among the various heterocycles reported to date, tetrazole and
its derivatives have received much attention in recent years due to
their widespread applications in biology. For instance, the 5-oxo
and 5-thio derivatives of tetrazole form an important structural
framework of many pesticides1,2 and herbicides.3,4 These ring sys-
tems are present in drugs patented for the treatment of central
nervous system disorders,5 HIV,6 sexual dysfunction7–9 asthma10
obesity and diabetes,11,12 as well as for injuries caused by exposure
to chemical warfare agents.13 Tetrazol-5-one and tetrazole-5-thi-
one rings are also featured in many antiviral,14 antibacterial,14,15
treatments with synthesized tetrazole derivatives would inhibit
the expression of human Ki-67 nuclear protein, which is an excel-
lent marker of tumor cell proliferation.27
Although, there are several reports in the literature that discuss
the preparation of 1,4-phenyl alkyl substituted tetrazol-5-ones,28–31
the synthesis ofthe corresponding1,4-diarylderivatives has not been
directly carried out. For instance, Quast and Nahr carried out the
N-alkylationof1-phenyl-1H-tetrazol-5(4H)-onewith3-bromocyclo-
hexene which was followed by the dehydrogenation of the resulting
compound with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone to ob-
tain the 1,4-diaryl tetrazol-5-one.32 The latter have also been re-
ported as side products in the reaction of triarylbismuth diazides
with aryl isocyanates to yield arylcarbamoyl azides.33 There are no
previous reports on the synthesis of 1,4-diaryl tetrazole-5-thiones.
Our approach to synthesize the title compounds is described below.
In recent years, copper mediated C(aryl)–N bond formation
through the cross coupling of aryl boronic acids with nitrogen
nucleophiles has emerged as a powerful strategy for the synthesis
analgesic,16,17 anesthetic,18,19 antihistaminic20
,
antimicrobial,21
antiinflammatory22,23 and anticancer drugs.24 Our interest in these
heterocycles stems from their aforementioned bioactivities. In par-
ticular, we are interested in the search of new anticancer com-
pounds based on tetrazol-5-one and tetrazole-5-thione scaffold.
Herein, we report the facile synthesis of a series of 1,4-diarylated
derivatives of these ring systems 1a–g and 2a–g (Scheme 1) and
evaluation of their antiproliferative activity in rapidly-growing
suspension cultures of L1210 leukemia cells and slow-growing
monolayer cultures of SK-BR-3 mammary tumor cells, using tests
of metabolic activity and DNA synthesis in vitro.25,26 Since the frac-
tion of Ki-67-positive tumor cells is often correlated with the clin-
ical course of cancer, it was also of interest to determine whether
* Corresponding author. Tel.: +1 785 532 6660; fax: +1 785 532 6666.
E-mail address: sundeep@ksu.edu (S. Rayat).
Scheme 1. 1,4-Diaryl tetrazol-5-ones 1a–g and tetrazole-5-thiones 2a–g.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.012

A. S. Gundugola et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3920–3924
3921
of a variety of compounds.34 This reaction has been successfully
used for the N-arylation of a wide range of functional groups such
as amines, amides, imides and sulfonamides.35 Lam et al. have also
reported the C(aryl)–N bond formation during the reaction of aryl
boronic acids with 5-phenyl-2H-tetrazole in the presence of copper
acetate.36 We successfully synthesized 1,4-diaryl tetrazol-5-one
derivatives 1a–f by the copper mediated coupling of a series of
ortho substituted boronic acids 4a–f (R1 = H, OMe, Cl, CF3, Br,
C„CH) with 1-phenyl-1H-tetrazol-5(4H)-one (3)37 in the presence
of pyridine and molecular sieves (Scheme 2). The boronic acids 4a–
e were commercially available while 4f was prepared from phenyl
acetylene by a reported method38 and used without further purifi-
cation due to its low yields.
is an excellent reagent to introduce acetylene group to an aromatic
ring,44–47 we attempted to synthesize 2f via the Sonogashira cou-
pling of 2e with ethynyltrimethylsilane followed by deprotection.
However, our coupling reaction did not go to completion as more
than 50% of 2e remained unreacted after 60 h. Also, the reaction
produced two products that eluted closely with unreacted 2e on
silica gel column and thus, were inseparable and could not be iden-
tified. We also attempted to synthesize 2f through the Sonogashira
coupling of 1e with ethynyltrimethylsilane followed by deprotec-
tion and thionation. Again, the Sonogashira coupling reaction did
not undergo completion in 48 h and resulted in an extremely low
yield of the desired product. Failure of this reaction to give desired
product in useful quantities for synthesis to proceed, prompted us
to search for other reagents to introduce the ethynyl group into or-
ganic structures via Sonogashira coupling. Sabourin and co-work-
Treatment of 1b–e with Lawesson’s reagent 539 yielded the cor-
responding 1,4-diaryl tetrazole-5-thiones 2b–e (Scheme 3). All the
attempts to thionate 1a to 2a with 5 failed. The lack of reactivity of
1a toward 5 may be attributed to the following reason: the mech-
anism of thionation with 5 is believed to involve a dissociative
equilibrium involving the formation of a highly reactive dithio-
phosphine ylide which reacts with carbonyl compounds via the
formation of a Wittig-type intermediate.39 The lone pairs present
on the ortho substituents in 1b–e may provide a coordination site
for electrophilic phosphorous of the ylide40 and thus, facilitate the
attack at nearby carbonyl group. Several other thionation condi-
ers have reported 3-methyl-2-butynol as
a
useful and
inexpensive alternative to ethynyltrimethylsilane in palladium cat-
alyzed cross coupling reaction with aryl halides.48,49 Treatment of
the resulting arylated methylbutynol with alkali-metal hydroxide
yields the corresponding arylacetylene and acetone.50 To our de-
light, the Sonogashira coupling of 1-(2-bromophenyl)-4-phenyl
tetrazole-5-thione 2e with 3-methyl-2-butynol yielded 6 which
upon treatment with sodium hydroxide in toluene gave the desired
1-(2-ethynylphenyl)-4-phenyl tetrazole-5-thione 2f (Scheme 4).
To explore a separate reaction chemistry from the synthesized
tetrazol-5-ones and tetrazole-5-thiones, we subjected 1b and 2b
to treatment with boron tribromide51 to produce 1-(2-hydroxy-
phenyl)-4-phenyl tetrazol-5-one 1g and its thio derivative 2g
(Scheme 5). This reaction demonstrated the stability of tetrazol-
5-ones and tetrazole-5-thiones to withstand strong Lewis acid con-
ditions such as boron tribromide.
tions were tried to induce this transformation, such as P4S10
/
Al2O3,41 PSCl3/H2O/Et3N at 63 °C42 and PSCl3/H2O/Et3N under
microwave conditions,43 however, in vain.
Compound 2a was ultimately prepared by the lithiation of 2e
followed by protonation in ethanol (Scheme 4). Similarly, thiona-
tion of 1f with 5 to yield 2f resulted in the formation of several
products that could not be identified. Since ethynyltrimethylsilane
Compounds 1f, 1g and 2g were most effective in inhibiting the
mitochondrial ability of L1210 leukemia cells to metabolize the 3-
(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium (MTS) reagent in the presence of
phenazine methosulfate (PMS) at days 2 and 4 (Table 1, IC50 values
in the 2.5–16.1
inactive and did not significantly alter the control rate of L1210 tu-
mor cell growth even at the highest concentrations (156.25 M)
tested. All other compounds showed moderate antiproliferative
activities (IC50 values in the 20–50 M range, Table 1s Supplemen-
lM range), whereas compounds 3 and 2a were
Scheme 2. Copper catalyzed N-arylation of 1-phenyl-1H-tetrazol-5(4H)-one (3) to
1a–f.
l
l
tary data). Full concentration-response curves indicated that the
Scheme 3. Thionation of tetrazol-5-ones 1b–e to tetrazole-5-thiones 2b–e.
Scheme 5. Lewis acid catalyzed conversion of 1b and 2b to 1g and 2g, respectively.
Table 1
Antiproliferative activity of the most potent tetrazole derivatives in L1210 tumor cells
in vitroa
Compound
IC50 valuesb
(lM)
Day 2
Day 4
1f
1g
2g
16.1 0.4
12.0 0.3
3.8 0.1
12.9 0.3
7.1 0.2
2.5 0.1
a
Concentrations of 1f, 1g and 2g required to inhibit by 50% (IC50 values) the
metabolic activity of L1210 tumor cells, using the MTS:PMS assay after 2 or 4 days
of culture in vitro. IC50 values were calculated from linear regression of the slopes of
the log-transformed concentration-survival curves.
Scheme 4. Synthesis of tetrazole-5-thiones 2a and 2f from 1-(2-bromophenyl)-4-
phenyl-1H-tetrazole-5(4H)-thione 2e.
b
Means SD (n = 3).

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A. S. Gundugola et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3920–3924
Figure 1. Comparison of the abilities of serial concentrations (plotted on a logarithmic scale) of 1f (blue, 4), 1g (black, }) and 2g (red, h) to inhibit the metabolic activity of
L1210 tumor cells at days 2 (left) and 4 (right) in vitro. Cell proliferation results were expressed as % of the net absorbance of MTS/formazan after bioreduction by vehicle-
treated control cells after 2 (A490 nm = 1.171 0.044, 100 3.8%) and 4 (A490 nm = 1.188 0.055, 100 4.6%) days in culture. The blank values (A490 nm = 0.447 at day 2 and
0.471 at day 4) for cell-free culture medium supplemented with MTS:PMS reagent were subtracted from the results. Bars: means SD (n = 3). aNot different from respective
controls; bP <0.01 and cP <0.005, smaller than respective controls.
inhibitions of L1210 tumor cell growth by the two most effective
compounds 1g and 2g, began around 1.6–4 M and became maxi-
mal or near maximal around 25–62.5 M (Fig. 1). All other com-
Compounds 1f, 1g and 2g also inhibit the proliferation of mono-
layer cultures of human SK-BR-3 breast adenocarcinoma cells at
days 2 and 4 (Fig. 2), however higher micromolar concentrations
of these compounds must be used to achieve inhibitory effects
l
l
pounds 1a–e and 2a–f induced concentration-dependent
inhibitory effects that were able to block L1210 tumor cell prolifer-
similar to those observed in L1210 cells. For instance, 156.25 lM
ation by 90–100% at 62.5–156.25
l
M (data not shown). Moreover,
2g inhibits SK-BR-3 cell proliferation by 60.6% at day 2 and 79.3%
the magnitudes of the antiproliferative effects of the tested com-
pounds were generally more pronounced after 4 than 2 days in cul-
ture (Table 1 and Fig. 1), suggesting that the effectiveness of these
bioactive compounds against L1210 tumor cell growth is a combi-
nation of drug concentration and duration of action.
The inactivity of 3 suggests that the presence of a second aro-
matic ring may be critical to reveal the antitumor effect of the tet-
razole derivatives. In general, the related tetrazole-5-ones and
tetrazole-5-thiones have similar antiproliferative activities but
there were notable exceptions as 2a is totally inactive while 1a
shows bioactivity, and also 2f is much weaker than its tetrazole-
5-one counterpart 1f. The presence of a hydroxyl group in 1g and
2g appears to be an asset in enhancing the bioactivity of this
framework. This may be attributed to the fact that an –OH group
is an excellent hydrogen bond donor which may result into a stron-
ger bonding with its biological target,52 thereby, increasing the
antiproliferative activity of 1g and 2g.
at day 4 (Fig. 2) but these magnitudes of antiproliferation are
achieved by about 25 lM 2g in L1210 cells (Fig. 1), suggesting that
this tetrazole-5-thione is at least 6.25 times more effective against
leukemic than breast cancer cell lines. The fact that the concentra-
tions of 1f, 1g and 2g required to inhibit the mitochondrial ability
of tumor cells to metabolize the MTS:PMS reagent at days 2 and 4
are somewhat higher in SK-BR-3 than L1210 cells suggests that the
antiproliferative action of these drugs is generally greater against
unsynchronized populations of rapidly-growing suspensions of
leukemic cells that are frequently turning through the cell cycle
than against unsynchronized populations of relatively slow-grow-
ing adherent monolayers of solid tumor cells that have smaller
growth fractions.
The antitumor activity of 2g is substantiated by the finding
that this compound inhibits the expression of the human Ki-67
marker of cell proliferation in SK-BR-3 mammary tumor cells at
24 h (Fig. 3, left). The Ki-67 nuclear protein, which is absent from
resting cells (G0), is exclusively detected within the nuclei of cells
progressing through all active phases of the cell cycle (G1, S, G2)
and relocates to the surface of the chromosomes during mitosis.27
Since Ki-67 expression may be absolutely required to maintain
cell proliferation, it is an excellent marker for determining the
growth fraction of tumor cell populations and the fraction of Ki-
67-positive tumor cells is often correlated with the clinical course
of cancer.27 As compared to the level of Ki-67 protein detected by
immunolabeling in untreated SK-BR-3 control tumor cells, the
ability of 62.5–156.25 lM 2g to inhibit Ki-67 expression by
36.5–52.4% at 24 h suggests that this novel antiproliferative com-
pound maintains surviving tumor cells in the resting stage and
prevents them from re-entering the cell cycle to divide (Fig. 3,
left). After antiproliferative 2g treatment, therefore, there are few-
er tumor cells and they fail to express Ki-67, indicating that the
growth fraction of tumor cells progressing through the cell cycle
has been reduced.
A 90-min treatment with 2g is sufficient to inhibit the incorpo-
ration of [3H]thymidine into DNA used to assess the rate of DNA
synthesis over a 30-min period of pulse-labeling in L1210 tumor
cells in vitro (Fig. 3, right). Although it may be somewhat mislead-
ing to compare biological responses measured at very different
times, the concentration-dependent inhibition of DNA synthesis
by 2g suggests that the ability of this compound to prevent tumor
cells from synthesizing DNA at 2 h (Fig. 3, right) may play a role in
Figure 2. Comparison of the abilities of 156.25 lM concentrations of 1f, 1g or 2g to
inhibit the metabolic activity of SK-BR-3 tumor cells at days 2 (black columns) and
4 (grey columns) in vitro. SK-BR-3 cell proliferation results were expressed as % of
the net absorbance of MTS/formazan after bioreduction by vehicle-treated control
(C) cells after 2 (A490 nm = 1.396 0.034, 100 2.4%) and 4 (A490 nm = 1.291 0.061,
100 4.7%) days in culture. The blank values (A490 nm = 0.230 at day 2 and 0.249 at
day 4) for cell-free culture medium supplemented with MTS:PMS reagent were
subtracted from the results. Bars: means SD (n = 3). aP <0.025 and bP <0.005,
smaller than respective controls.

A. S. Gundugola et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3920–3924
3923
Figure 3. Left: Whole cell immunodetection of Ki-67 protein level in vitro. Comparison of the abilities of 25, 62.5 or 156.25
lM concentrations of 2g to inhibit the Ki-67
marker of cell proliferation in SK-BR-3 cells at 24 h. The results were expressed as % of the ratio of Ki-67 protein level (relative luminescence intensity of the anti-Ki-67
primary antibody-antigen immune complex bound to horseradish peroxidase-linked secondary antibody):cell number (relative fluorescence intensity of the Hoechst reagent-
DNA complex) in vehicle-treated control SK-BR-3 tumor cells at 24 h (C: 1.6248 0.1771, 100 10.9%). Bars: means SD (n = 3). aP <0.05, smaller than control. Right:
Comparison of the abilities of 25, 62.5 or 156.25
l
M concentrations of 2g to inhibit the rate of incorporation of [3H]thymidine into DNA measured in L1210 cells over 30 min
following a 90-min period of incubation at 37 °C in vitro. DNA synthesis in vehicle-treated control (C) cells at 37 °C was 11,314 769 cpm (100 6.9%). The blank value
(1282 112 cpm) for control cells incubated and pulse-labeled at 2 °C with 1
bP <0.005, smaller than control.
l
Ci of [3H]thymidine has been subtracted from the results. Bars: means SD (n = 3). aP <0.05 and
its inhibition of Ki-67 expression at 24 h (Fig. 3, left) and antipro-
liferative activity at days 2 and 4 (Table 1 and Fig. 1).
Biology Research and Instruction Enhancement Fund Program).
The authors acknowledge Dr. Ruth Welti and Ms. Pamela Tamura
at the Kansas Lipidomics Research Center (KLRC) for providing
the mass spectra on our compounds.
Concentrations of 2g somewhat higher than those sufficient to
maximally inhibit tumor cell proliferation must be used to partially
inhibit Ki-67 expression and DNA synthesis. Such apparent dis-
crepancy may simply be due to different experimental conditions
and cellular responses to various periods of drug exposure: the rate
of DNA synthesis over 30 min is inhibited in cells treated for only
2 h with 2g and the level of Ki-67 protein is reduced in cells incu-
bated for 24 h in the presence of this antitumor compound,
whereas the more spectacular inhibitions of L1210 and SK-BR-3 tu-
mor cell proliferations are the result of 2- and 4-day long drug
treatments.
Supplementary data
Supplementary data (description of compound syntheses and
their spectral characterization; biological assay methods and
Table 1s showing the IC50 values of 1a – e, 2a – f and 6) associated
with this article can be found, in the online version, at doi:10.1016/
j.bmcl.2010.05.012.
In conclusion, copper mediated coupling of aryl boronic acids
with 1-phenyl-1H-tetrazol-5(4H)-one is a versatile reaction to syn-
thesize a series of 5-oxo derivatives of 1,4-diaryl tetrazoles. The
substituents with lone pairs (such as –OMe, –Cl, –CF3, –Br) on
the aromatic ring of the 1,4-diaryl tetrazole-5-ones promote their
thionation to corresponding tetrazole-5-thiones. Compound 1-(2-
bromophenyl)-4-phenyl-1H-tetrazole-5(4H)-thione thus obtained
is an excellent precursor to introduce other type of substituents
on tetrazole-5-thione through metalation followed by electrophilic
quenching, or via palladium catalyzed cross coupling reactions. The
synthesized tetrazole derivatives are also stable to strong Lewis
acid conditions. These compounds may have interesting bioactivity
but more compounds based on 5-oxo and 5-thio 1,4-diaryl tetra-
zole scaffolds must be synthesized to elucidate structure–activity
relationships, identify more potent antitumor lead compounds,
and investigate their molecular targets and mechanism of action.
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