Synthesis and Ribonucleotide reductase inhibitory activity of thiosemicarbazones

Article abstract of DOI:10.1016/j.bmcl.2008.09.097

Ribonucleotide reductase (RR) is an important therapeutic target for anticancer drugs. The structure of human RR features a 1:1 complex of two homodimeric subunits, hRRM1 and hRRM2. Prokaryotically expressed and highly purified recombinant human RR subunits, hRRM1 and hRRM2, were used for holoenzyme-based [³H]CDP reduction in vitro assay. Ten new thiosemicarbazones (7-16) were synthesized and screened for their RR inhibitory activity. Two thiosemicarbazones derived from p-hydroxy benzaldehyde (9 and 10) were found to be active but less potent than the standard, Hydroxyurea (HU). Guided by the activity of compounds 9 and 10, 11 new thiosemicarbazones (17-27) derived from p-hydroxy benzaldehyde were prepared and screened for their RR inhibitory activity. All the 11 compounds were more potent than HU.

Full text of DOI:10.1016/j.bmcl.2008.09.097

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6248–6250
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and Ribonucleotide reductase inhibitory activity
of thiosemicarbazones
a
a
Kesavan Krishnan a, Kumari Prathiba a, Venkatesan Jayaprakash a, , Arijit Basu , Nibha Mishra ,
*
Bingsen Zhou b, Shuya Hu b, Yun Yen b,
*
a Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi, Jharkhand 835 215, India
b Department of Clinical and Molecular Pharmacology, City of Hope National Medical Center, Duarte, CA 91010, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 June 2008
Revised 22 September 2008
Accepted 27 September 2008
Available online 2 October 2008
Ribonucleotide reductase (RR) is an important therapeutic target for anticancer drugs. The structure of
human RR features a 1:1 complex of two homodimeric subunits, hRRM1 and hRRM2. Prokaryotically
expressed and highly purified recombinant human RR subunits, hRRM1 and hRRM2, were used for holo-
enzyme-based [3H]CDP reduction in vitro assay. Ten new thiosemicarbazones (7–16) were synthesized
and screened for their RR inhibitory activity. Two thiosemicarbazones derived from p-hydroxy benzalde-
hyde (9 and 10) were found to be active but less potent than the standard, Hydroxyurea (HU). Guided by
the activity of compounds 9 and 10, 11 new thiosemicarbazones (17–27) derived from p-hydroxy benz-
aldehyde were prepared and screened for their RR inhibitory activity. All the 11 compounds were more
potent than HU.
Keywords:
p-Hydroxybenzaldehyde
thiosemicarbazones
RR inhibitory activity
Human recombinant RR subunit
hRRM1 and hRRM2
Ó 2008 Elsevier Ltd. All rights reserved.
[3H]CDP reduction assay
Ribonucleotide reductase (RR) catalyzes the reduction of ribo-
nucleotides to their corresponding deoxyribonucleotides, which
are the building blocks for DNA in almost all the living cells.1 They
transform ribonucleotides to deoxyribonucleotides by catalyzing
the substitution of the 20-OH group of ribonucleotide with 20-H.
Human RR belongs to Class Ia. Since the reduction of ribonucleo-
tides is the rate-limiting step of DNA synthesis, inactivation of RR
stops DNA synthesis, which inhibits cell proliferation. This along
with the fact that RR has low activity in resting cells, high activity
in rapidly growing normal cells, and very high activity in cancer
cells has made it an important target for cancer therapy.2 Also
studies have indicated the critical role of RR in tumor promotion.
Therefore, RR is considered as a relevant molecular target for the
design and development of antitumor agents.3
gested to interact with the iron in the R2 subunit of RR.6 Iron
chelating property and carcinostatic activity of acylhydrazones
and thiosemicarbazones derived from salicylaldehyde and 1-naph-
thol-2-carboxaldehyde (Fig. 1) have been well documented, but
their inhibitory activity of RR has to be explored. In this paper,
we report the synthesis and RR inhibitory activity of 21 new aro-
matic/heteroaromatic aldehyde thiosemicarbazones. These com-
pounds were designed and synthesized to test the concept of
iron chelation. In this work, those compounds which satisfy the
functional group requirements for iron chelation and also those
that do not satisfy these requirements but are structurally similar-
ity to trimidox and didox were synthesized in order establish a
structure–activity relationship (Scheme 1).
The synthesis of thiosemicarbazones was carried out by follow-
ing the procedures reported earlier.7,8 Potassium hydrazine
carbodithioate was prepared by the reaction of hydrazine hydrate
Several potentially useful class of RR inhibitors were reported
which includes: (1) Free-radical scavengers: Hydroxy urea, Trimi-
dox, Didox, etc., (2) Iron chelators: Triapine, PIH, 311, etc., and
2
N
(3) Substrate analogs (Gemcitabin, Azido CDP, etc.). A class of
a
-(N)-Heterocyclic carboxaldehyde thiosemicarbazones (HCT)
N
OH
4
HO
O
NH
HO
O
were reported to be carcinostatic possibly due to their iron che-
2
lation property.5 HCTs were reported to inhibit RR and are sug-
N
N
N
N
N
NH
H N
NH
NH
2
S
* Corresponding authors. Tel.: +91 6512276247 (V.J.); +1 626 359 8111ꢀ65707
TRIAPINE
PIH
311
(Y.Y.).
E-mail addresses: venkatesanj@bitmesra.ac.in (V. Jayaprakash), yyen@coh.org (Y.
Yen).
Figure 1. Iron chelators with RR inhibitory activity and carcinostatic property.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.097

K. Krishnan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6248–6250
6249
ii
RR inhibitory activity was carried out using recombinant pro-
teins, that is, prepared by following the procedure reported ear-
lier.9–11 The coding sequences of hRRM2 and hRRM1 were
obtained from human oropharyngeal carcinoma KB cells and
cloned in-frame with an N-terminal 6ꢀHis-tag into the prokaryotic
expression vector pET28 (Novagen, Madison, WI). The proteins
were expressed in BL21 (DE3) bacteria (Stratagene, La Jolla, CA)
and purified using Ni(II) affinity chromatography. In vitro RR inhi-
bition assays were based on Steeper and Stuart CDP reductase
activity method.12 Each compound was dissolved in neat DMSO
and diluted with 50 mM Tris–HCl (pH 7.5). The final concentration
of DMSO in the reaction mixtures was 1% v/v. To perform the assay
to test the potency of RR inhibitors using purified recombinant pro-
teins, the method was modified and standardized as follows:
Step 1: A mixture of purified hRRM1 and hRRM2 was incubated
at room temperature for 30 min with various concentrations of
each compound. HU 20 mM was used as a positive control. Step
i
NH
NH
S
2
H N
2
CH
3
H N
2
S
1
iii
NH NH
S
NH
S
N
S
N
CH
3
S
1
R
R
O
R
2-4
7-12
17-27
O
S
5
13-14
6
15-16
Scheme 1. Synthetic route for thiosemicarbazones. Reagents and conditions: (i)
CS2, KOH, <10 °C, stirring, 15–30 min; CH3I, <10 °C, stirring, 30–45 min, (ii) R–C6H4–
CHO, i-PrOH, rt, stirring, 30 min, (iii) R1–C6H4–NH2, EtOH, reflux, 8–12 h.
2: To initiate enzymatic reduction, a reaction buffer (0.125 lM
(85%), carbon disulfide and potassium hydroxide below 10 °C with
constant stirring for a period of 15–30 min. This was then converted
in to methylhydrazine carbodithioate (1) by the action of methyl io-
dide, added dropwise with stirring maintaining the temperature be-
low 10 °C for a period of 30–45 min. Substituted benzaldehyde
hydrazones of methylhydrazine carbodithioate (2–6) were prepared
by the reaction of 1 with the respective substituted benzaldehydes
in isopropanol, stirring at room temperature for a period of 30–
45 min. Ten thiosemicarbazones (7–16) were prepared by the reac-
tion of 2–6 with their respective aniline derivative in ethanol and by
refluxing for a period of 8–12 h till the evolution of methyl mercap-
tan ceased.. Later another 11 thiosemicarbazones (17–27) were pre-
pared by the reaction of and 3, with respective anilines in a similar
manner described above. All the intermediates were characterized
by IR spectroscopy and by elemental analysis for CHNS. In the ele-
mental analysis, the percentage variations between observed and
calculated values were within 0.4%. Final compounds were charac-
terized by 1H NMR and FAB-MS (Table 3, Supplemental material).
The structure and physico-chemical characterization data of com-
pounds 7–27 were presented in Table 1.
[3H]CDP, 50 mM HEPES (pH 7.2), 6 mM DTT, 4 mM MgOAc, 2 mM
ATP, 0.05 mM CDP, 100 mM KCl, and 0.24 mM NADPH) was added
to the protein/inhibitor mixture from Step 1 up to a final volume of
100 ll. The reaction mixture was incubated at 37 °C for 30 min.
The enzyme substrate [3H]CDP and the resulting product [3H]dCDP
in the reaction mixture were dephosphorylated by phosphodies-
terase. Step 3: The [3H]cytidine and [3H]deoxycytidine in the reac-
tion mixture were separated by HPLC using a C18 reversed phase
column connected to a Model 2 b-RAM Radio Flow-Through detec-
tor (IN/US Systems, Tampa, FL). Negative control samples, which
were run with each experiment, contained only 1% v/v DMSO.
The inhibition of RR was expressed as percent of the negative con-
trol (relative activity). The relative enzyme activity dependence on
inhibitor concentration was fitted using a non-linear regression
equation (f(x) = (a ꢁ d)/[1 + (x/c)b] + d, where a = asymptotic maxi-
mum, b = slope parameter, c = value at the inflection point, and
d = asymptotic minimum). The IC50 values, namely the compound’s
concentration that produces 50% inhibition, were calculated by set-
ting f(x) = 50. The inhibitory potency is reported as the mean of
three separate tests, each performed in duplicate.
The first 10 thiosemicarbazones (7–16, Table 1) synthesized
were screened for RR inhibitory activity. Interestingly those thio-
semicarbazones that can complex iron (7 and 8) were inactive.
Table 1
Structure and physico-chemical characterization of compounds 7–27
NH NH
N
Table 2
S
1
Ribonucleotide reductase inhibitory activity of compounds 7–27
R
R
a
Mpa (°C)
Yieldb (%)
Compound
IC50
(l
M)
Compound
R
R1
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
>500
>500
7
8
9
2-OH
2-OH
4-OH
4-OH
3-OMe-4-OH
3-OMe-4-OH
2-Furyl
2-Furyl
2-Thiophenyl
2-Thiophenyl
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
4-OH
–H
4-Cl
–H
4-Cl
–H
4-Cl
–H
170–171
180–183
188–190
165–166
158–160
178–181
170–175
168–170
180–182
190–192
132–133
120–122
141–143
92–93
75
70
70
62
55
50
76
70
80
85
60
70
74
61
57
54
49
62
57
47
65
242.0
287.0
>500
>500
>500
>500
>500
>500
46.8
29.3
36.5
30.4
14.2
41.6
39.3
45.1
29.3
28.6
43.3
148.0
2.25
10
11
12
13*
14*
15*
16*
17
18
19
20
21
22
23
24
25
26
27
4-Cl
–H
4-Cl
2-Cl
3-Cl
2-Me
3-Me
4-Me
2-OMe
2-NO2
4-NO2
2-OH
3-OH
4-OH
73–75
138–141
106–108
166–169
195–198
132–135
158–161
23
24
25
26
27
HU
4-OH
4-OH
*
Furyl and thiophene ring replaces phenyl ring (R–C6H4–).
Melting point determined by capillary method and are uncorrected.
Percentage yield of final step.
3-AP (Triapine)
a
a
b
Values are means of three experiments.

6250
K. Krishnan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6248–6250
Surprisingly, 4-hydroxybenzaldehyde derived thiosemicarbazones
(9 and 10) were found to be active but around 100- and 2-fold less
potent than the standard drugs used in the study, Triapine and
Hydroxyurea, respectively (Table 2).
tral data. Kesavan Krishnan and Kumari Prathiba are thankful
to the University Grants Commission for the Junior Research
Fellowship.
Guided by the activity of 4-hydroxybenzaldehyde thiosemicar-
bazones, we prepared 11 analogs (17–27) of these active com-
pounds (9 and 10) with different substitution on the N4-phenyl
ring (Table 1.). The compounds 17–27 were potent than com-
pounds 9 and 10, and were more potent than standard drug
hydroxyurea (Table 2). Also these compounds are found to be more
potent than most of the N-hydroxy semicarbazones of aromatic
aldehydes reported earlier.10
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.09.097.
References and notes
Within this small set of active molecules, a concrete SAR discus-
sion may not be feasible. But few guiding principles can be derived
to proceed further in designing this class of molecules as RR inhib-
itors. Rational selection of substitution for N1- and N4-phenyl rings
guided by the activity of compounds 17–27 and synthesizing the
new analogs may be considered as a next suitable approach to-
wards reaching a more potent molecule. Present work suggests
p-hydroxy substitution in N1-phenyl ring and m-hydroxy or p-
methyl substitution in N4-phenyl ring are favorable for RR inhibi-
tory activity. Guided by this principle, synthesis of analogs of com-
pounds 21 and 26 are currently underway.
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We are grateful to the Head, Sophisticated Analytical Instru-
ment Facility (SAIF), CDRI, Lucknow, India, for providing spec-