Synthesis and glucose-6-phosphatase inhibitory activity of (thiouriedo)alkanoic acid esters

Article abstract of DOI:10.1016/j.bmcl.2004.02.079

A series of (3-pyridin-2-yl-thiouriedo)alkanoic acid esters (5a-j) have been synthesized by the reaction of pyridin-2-yl-dithiocarbamic acid methyl ester (2) and amino acid esters (4). Most of the synthesized compounds have been evaluated against glucose-6-phosphatase enzyme but only four compounds (5g-j) displayed significant inhibitory activity of the enzyme.

Full text of DOI:10.1016/j.bmcl.2004.02.079

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2571–2574
Synthesis and glucose-6-phosphatase inhibitory activity of
(thiouriedo)alkanoic acid estersq
Farhanullah,a Diptesh Sil,a Brajendra K. Tripathi,b Arvind K. Srivastavab
and Vishnu Ji Rama,*
aDivision of Medicinal Chemistry, Central Drug Research Institute, Lucknow 226001, India
bDivision of Biochemistry, Central Drug Research Institute, Lucknow 226001, India
Received 24 December 2003; accepted 23 February 2004
Abstract—A series of (3-pyridin-2-yl-thiouriedo)alkanoic acid esters (5a–j) have been synthesized by the reaction of pyridin-2-yl-
dithiocarbamic acid methyl ester (2) and amino acid esters (4). Most of the synthesized compounds have been evaluated against
glucose-6-phosphatase enzyme but only four compounds (5g–j) displayed significant inhibitory activity of the enzyme.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
The high intolerance of these compounds necessitated to
modify the structures to obtain efficacious compounds.
The structural modification of I–III led to design and
synthesize (3-pyridin-2-yl-thiouriedo)alkanoic acid ester
IV, which were equipotent and less toxic.
Diabetes mellitus type 2 is a metabolic disorder, char-
acterized by resistance of the peripheral target tissues to
the binding of insulin.1;2 Nearly 90% of the diabetic
population is suffering from type-2 diabetes. The etiol-
ogy of type-2 diabetes is quite complex and in most of
the cases insulin resistance is developed that leads to
hyperinsulinemia. In such a situation b-cells of the
pancreas do not maintain the hyperinsulinemic state for
longer period resulting insulin deficiency leading to
hyperglycemia.
In search of more effective and least toxic antihyper-
glycemic agents, compounds with in built amidine,
thiourea, and amino acid ester pharmacophores were
synthesized as potential inhibitors of glucose-6-phos-
phatase (G-6-pase), an enzyme responsible for main-
taining normal blood glucose level.8
Recently, it has been reported that 3-guanidinoprop-
ionic acid (I), 3-(pyridin-2-yl-amino)-propionic acid (II),
and 3-carbamimidoylsulfanyl-propionic acid (III) pos-
sess both antihyperglycemic and antiobesity activities in
KKAy mouse.3–5 The mode of action of these com-
pounds is obscure but it is believed that probably they
increased the disposal of glucose without affecting glu-
coneogenesis, hepatic glycogen content or intestinal
glucose absorption.5 The antihyperglycemic potential of
lipophilic guanidine derivatives is known since a decade6
but significance of zwitterionic guanidine derivatives in
diabetes was realized from the work of Horlicks et al.7
2. Chemistry
Various (3-pyridin-2-yl-thiouriedo)alkanoic acid esters
have been synthesized in two steps. The first step is the
formation of pyridin-2-yl-dithiocarbamic acid methyl
esters9 (2) from the reaction of 2-aminopyridine (1) and
carbon disulfide in presence of alkali in DMSO followed
Keywords: Thiouriedoalkanoic acid; Glucose-6-phosphatase; Hyper-
insulinemic; Hyperglycemic.
q CDRI Communication no. 6475.
* Corresponding author. Tel.: +91-522-22124116; fax: +91-522-222-
3405; e-mail: vjiram@yahoo.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.079

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Farhanullah et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2571–2574
other chemicals and reagent used were of analytical
grade and were purchased from the local suppliers.
3.2. Partial purification of G-6-pase (D-glucose-6-phos-
phate phosphorylase; EC 3.1.3.9) from rat liver
The liver of male rats of Wistar strain was exercised. A
10% homogenate was prepared in 150 mM KCl using
Potter Elvejhem glass homogenizer fitted with Teflon
pestle. The homogenate was centrifuged at 1000g for
15 min; supernatant was decanted and used as enzyme
source.
Scheme 1. Reagents and conditions: (i) DMSO/KOH; (ii) CS2/MeI/
20 °C; (iii) RNH2/C2H5OH/85 °C; (iv) NH2(CHRn)CO2R0/C2H5OH/
85 °C.
by methylation with methyl iodide. The dithiocarbamic
acid methyl esters (2) thus formed on reaction with
amino acid esters (4) transformed to (3-pyridin-2-yl-
thiouriedo)alkanoic acid esters (5a–j) in good to mod-
erate yields. Further reaction of 2 with ambiphilic
nucleophiles such as hydrazine hydrate and guanidine
separately yielded N-(2-pyridinyl)-1-hydrazinecarbothio-
amide (3a) and 2-[({[amino(imino)methyl]amino}carbo-
thioyl)amino]pyridine (3b). All the synthesized
compounds (Scheme 1) possess amidine, thiourea, ami-
no acid ester, and guanidine pharmacophores in their
molecular make-up, responsible for their antihypergly-
cemic activity. These compounds were properly char-
acterized by spectroscopic and elemental analyses.10
This class of compounds has not been explored earlier
for their glucose-6-phosphatase inhibitory activity.
The effect of test compounds was studied by pre-incu-
bating 100 lg of the compound in 1.0 mL reaction sys-
tem for 10 min and then determining the residual
glucose-6-phosphatase activity according to the method
of Hubscher and West.11 The 1.0 mL assay system
contained 0.3 M citrate buffer (pH 6.0), EDTA 28 mM,
NaF 14 mM, 30 mL water, glucose-6-phosphate
200 mM, and enzyme protein. The mixture was incu-
bated at 37 °C for 30 min after which 1.0 mL of 10%
TCA was added. Estimation of inorganic phosphates
(Pi) in protein free supernatant was done according to
the method of Taussky and Shorr.12 Glucose-6-phos-
phatase activity was defined as micromole Pi released
per minute per milligram protein.
Out of the 12 compounds evaluated for their glucose-6-
phosphatase inhibitory activity, only four compounds
5g–j demonstrated 74.6%, 54.4%, 50%, and 52.2% inhi-
bition, respectively. Among these four active com-
pounds 5g was most active as it inhibited the enzyme
activity by 74.6%. Rest of the compounds either dis-
played moderate or insignificant inhibition (Table 1).
3. Results and discussion
Most of the synthesized compounds were evaluated for
in vitro glucose-6-phosphatase inhibitory activity.
Among the 10 screened compounds, four compounds
(5g–j) demonstrated good inhibitory activity ranging
from 50% to 74.6% tested at 100 lM concentration.
It is evident from the screening results of 5a and 5g that
the presence of halo substituent in pyridine ring
increases inhibitory activity. An increasing pattern of
percent inhibition has been noticed in case of com-
pounds 5e, 5i (29–50%) and 5f, 5j (21.5–52.2%), and 5c,
5h (18–54.4%). Compounds with hydrazino and amid-
ino substituents (3a,b) were also evaluated for their
glucose-6-phosphatase inhibitory activity but they
did not display significant inhibition. Thus, it was
3.1. Glucose-6-phosphatase enzyme assay
Glucose-6-phosphate, EDTA, TCA, and NaF were
purchased from the Sigma Chemicals Co. (USA). All
Table 1. In vitro glucose-6-phosphatase enzyme inhibition results for compounds 3a,b and 5a–j at 100 lM concentration
Compounds 3 and 5
n
R
X
R0
% Inhibitiona
3a
3b
5a
5b
5c
5d
5e
5f
––
––
1
NH2
Amidino
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
––
––
38.5
20
H
CH3
CH3
C2H5
C2H5
CH3
C2H5
CH3
CH3
C2H5
C2H5
CH3
17.7
––
1
2
H
18
––
1
(CH3)2CHCH2
C6H5CH2
Indol-3-yl-methyl
H
1
29
1
21.5
74.6
54.4
50
5g
5h
5i
1
2
H
1
C6H5CH2
Indol-3-yl-methyl
5j
1
52.2
a Values are means of three experiments.

Farhanullah et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2571–2574
2573
Compound 5a. Yield 68%; mp 129–130 °C; MS (FAB) 226
(Mþ+1); IR (KBr) 1739.0 (CO), 3458 cmꢀ1 (NH); 1H
NMR (200 MHz, CDCl3) d 3.81 (s, 3H, OCH3), 4.57 (d,
J ¼ 5:0 Hz, 2H, CH2), 6.78 (d, J ¼ 8:2 Hz, 1H, Py), 6.95–
7.01 (m, 1H, Py), 7.62–7.69 (m, 1H, Py), 8.24 (d,
J ¼ 4:4 Hz, 1H, Py), 8.71 (br s, 1H, NH). Anal. Calcd
for C9H11N3O2S: C, 47.99; H, 4.92; N, 18.65. Found: C,
48.23; H, 4.78; N, 18.78.
concluded that presence of highly basic moieties reduce
the inhibition.
Acknowledgements
The authors are thankful to Dr. Atul Goel for his
valuable suggestions during the preparation of the
manuscript. One of the authors Farhanullah is thankful
to CSIR, New Delhi for senior research fellowship.
Compound 5b. Yield 65%; mp 92–93 °C; MS (FAB) 254
1
(Mþ+1); IR (KBr) 1737 (CO), 3440 cmꢀ1 (NH); H NMR
(200 MHz, CDCl3) d 1.31 (t, J ¼ 7:2 Hz, 3H, CH3), 1.61
(d, J ¼ 7:2 Hz, 3H, CH3), 4.26 (q, J ¼ 7:2 Hz, 2H, OCH2),
5.10–5.17 (m, 1H, CH), 6.69 (d, J ¼ 8:4 Hz, 1H, Py), 6.95–
7.01 (m, 1H, Py), 7.61–7.65 (m, 1H, Py), 8.26 (d,
J ¼ 4:2 Hz, 1H, Py). Anal. Calcd for C11H15N3O2S: C,
52.15; H, 5.97; N, 16.59. Found: C, 52.28; H, 5.82; N,
16.69.
References and notes
1. DeFronze, R. A. Diabetes 1998, 37, 667.
Compound 5c. Yield 62%; mp 102–103 °C; MS (FAB) 254
(Mþ+1); IR (KBr) 1726.0 (CO), 3448 cmꢀ1 (NH); 1H
NMR (200 MHz, CDCl3) d 1.26 (t, J ¼ 7:2 Hz, 3H, CH3),
2.78 (t, J ¼ 6:2 Hz, 2H, CH2), 4.03 (t, J ¼ 6:2 Hz, 2H,
NCH2), 4.16 (q, J ¼ 7:2 Hz, 2H, OCH2), 6.74 (d,
J ¼ 8:4 Hz, 1H, Py), 6.93–6.99 (m, 1H, Py), 7.59–7.68
(m, 1H, Py), 8.20 (d, J ¼ 4:2 Hz, 1H, Py), 8.52 (br s, 1H,
NH). Anal. Calcd for C11H15N3O2S: C, 52.15; H, 5.97; N,
16.59. Found: C, 52.32; H, 5.91; N, 16.75.
Compound 5d. Yield 64%; colorless oil; MS (FAB) 282
(Mþ+1); IR (KBr) 1740.0 (CO), 3440 cmꢀ1 (NH); 1H
NMR (200 MHz, CDCl3) d 0.99 (d, J ¼ 5:2 Hz, 6H,
2CH3), 1.25–1.26 (m, 1H, CH), 1.82–1.87 (m, 2H, CH2),
3.77 (s, 3H, OCH3), 5.13–5.17 (m, 1H, NCH), 6.74 (d,
J ¼ 8:4 Hz, 1H, Py), 6.94–7.00 (m, 1H, Py), 7.60–7.65 (m,
1H, Py), 8.22 (d, J ¼ 4:2 Hz, 1H, Py), 8.60 (br s, 1H, NH).
Anal. Calcd for C13H19N3O2S: C, 55.49; H, 6.81; N, 14.93.
Found: C, 55.62; H, 6.96; N, 14.84.
2. Reaven, G. M. Diabetes Metab. Rev. 1993, 9, 55.
3. Meglasson, M. D. U.S. Patent 5,34,164, July 28, 1992.
4. Meglasson, M. D.; Wilson, J. M.; Yu, J. H. J. Pharmacol.
Exp. Ther. 1993, 266, 1454.
5. Larsen, S. D.; Connell, M. A.; Cudahy, M. M.; Evans, B.
R.; May, P. D.; Meglasson, M. D.; O’sullivan, T. J.;
Schostarez, H. J.; Sin, J. C.; Stevens, F. C.; Tanis, S. P.;
Tegley, C. M.; Tucker, J. A.; Vaillancourt, V. A.; Vidmar,
T.; Watt, W.; Yu, J. H. J. Med. Chem. 2001, 44, 1217.
6. Baily, C. J. Prog. Med. Chem. 1993, 30, 203.
7. (a) Horlicks, Ltd Fr. Patent 5924, 1968; (b) Kaddurah-
Daouk, R.; Teicher, B.A. WO97/13507, 1997.
8. Singh, J.; Nordlie, R. C.; Jorgenson, R. A. Biochim.
Biophys. Acta 1981, 678, 477.
9. Trofimova, B. A.; Sobenina, L. N.; Mekhaleva, A. I.;
Demenev, A. P.; Tarasova, O. A.; Ushkov, I. A.;
Zinchenko, S. V. Tetrahedron 2001, 56, 7325.
10. Synthetic procedures and characterization data for the
prepared compounds:
Compound 5e. Yield 70%; mp 120–121 °C; MS (FAB) 330
(Mþ+1); IR (KBr) 1737.1 (CO), 3440 cmꢀ1 (NH); 1H
NMR (200 MHz, CDCl3) d 1.23 (t, J ¼ 7:2 Hz, 3H, CH3),
3.32 (d, J ¼ 6:0 Hz, 2H, CH2), 4.19 (q, J ¼ 7:2 Hz, 2H,
OCH2), 5.39–5.48 (m, 1H, CH), 6.72 (d, J ¼ 8:4 Hz, 1H,
Py), 6.90–6.94 (m, 1H, Py), 7.19–7.24 (m, 5H, phenyl),
7.58–7.66 (m, 1H, Py), 8.04 (d, J ¼ 4:6 Hz, 1H, Py), 8.56
(br s, 1H, NH). Anal. Calcd for C17H19N3O2S: C, 61.98;
H, 5.81; N, 12.76. Found: C, 62.13; H, 5.93; N, 12.64.
Compound 5f. Yield 64%; mp 170–172 °C; MS (FAB) 355
(Mþ+1); IR (KBr) 1748.0 (CO), 3422 cmꢀ1 (NH); 1H
NMR (200 MHz, CDCl3) d 3.51 (d, J ¼ 5:4 Hz, 2H, CH2),
3.70 (s, 3H, OCH3), 5.49–5.52 (m, 1H, CH), 6.64 (d,
J ¼ 8:4 Hz, 1H, Py), 6.82–6.83 (m, 1H, Py), 7.02–7.10 (m,
4H, indolyl), 7.31–7.35 (m, 1H, indolyl), 7.56–7.61 (m, 1H,
Py), 7.71 (d, J ¼ 4:0 Hz, 1H, Py), 8.10 (br s, 1H, NH), 8.46
(s, 1H, NH, indolyl). Anal. Calcd for C18H18N4O2S: C,
61.00; H, 5.12; N, 15.81. Found: C, 61.13; H, 5.18; N,
15.95.
Procedure (3a). A mixture of pyridin-2-yl-dithiocarbamic
acid methyl ester (2, 2 mmol) and hydrazine hydrate
(2 mmol) was refluxed in alcohol for 5 h. The reaction
mixture was left overnight at room temperature. The
crystals thus formed were filtered and washed with chilled
alcohol. Yield 54%; mp 122–123 °C; MS (FAB) 169
1
(Mþ+1); IR (KBr) 3424 cmꢀ1 (NH); H NMR (200 MHz,
CDCl3) d 6.90–6.96 (m, 1H, Py), 7.09 (d, J ¼ 8:2 Hz, 1H,
Py), 7.57–7.65 (m, 1H, Py), 8.14 (d, J ¼ 4:6 Hz, 1H, Py),
10.30 (br s, 1H, NH). Anal. Calcd for C6H8N4S: C, 42.84;
H, 4.79; N, 33.31. Found: C, 42.77; H, 4.64; N, 33.44.
Procedure (3b). An equimolar mixture of pyridin-2-yl-
dithiocarbamic acid methyl ester (2, 2 mmol) and
guanidine hydrochloride (2 mmol) was stirred at room
temperature for 6 h in acetone in the presence of K2CO3
(2 mmol). Acetone was evaporated; solid product obtained
was washed with water followed by alcohol. Yield 65%,
mp 192–193 °C; MS (FAB) 196 (Mþ+1); IR (KBr)
3425 cmꢀ1 (NH); 1H NMR (200 MHz, CDCl3) d 6.81–
6.87 (m, 1H, Py), 7.43–7.59 (m, 1H, Py), 8.15 (d,
J ¼ 5:2 Hz, 1H, Py), 8.28 (d, J ¼ 7:4 Hz, 1H, Py), 8.38
(br s, 1H, NH). Anal. Calcd for C7H9N5S: C, 43.06; H,
4.65; N, 35.87. Found: C, 43.19; H, 4.77; N, 35.96.
General procedure (5a–j). A mixture of pyridin-2-yl-dithio-
carbamic acid methyl esters (2, 2 mmol) and hydrochloride
salt of the amino acid ester (4, 2.0 mmol) was refluxed in
presence of triethylamine (2.0 mmol) for 5–6 h. Ethanol
was used as a solvent for the reaction of amino acid ethyl
ester while methanol was the solvent of choice for the
synthesis of methyl ester derivatives. Reaction mixture was
left overnight; solid thus obtained was filtered and washed
with alcohol.
Compound 5g. Yield 67%; mp 186–187 °C; MS (FAB) 260
1
(Mþ+1); IR (KBr) 1738 (CO), 3435 cmꢀ1 (NH); H NMR
(200 MHz, CDCl3) d 3.82 (s, 3H, OCH3), 4.55 (d,
J ¼ 5:0 Hz, 2H, CH2), 6.83 (d, J ¼ 8:8 Hz, 1H, Py), 7.61
(dd, J ¼ 8:8, 2.6 Hz, 1H, Py), 8.20 (d, J ¼ 2:4 Hz, 1H, Py),
9.11 (br s, 1H, NH). Anal. Calcd for C9H10ClN3O2S: C,
41.62; H, 3.88; N, 16.18. Found: C, 41.74; H, 3.77; N,
16.25.
Compound 5h. Yield 63%; mp 178–179 °C; MS (FAB) 288
(Mþ+1); IR (KBr) 1740 (CO), 3458 cmꢀ1 (NH); 1.31 (t,
J ¼ 7:2 Hz, 3H, CH3), 2.79 (t, J ¼ 6:2 Hz, 2H, CH2), 4.13
(t, J ¼ 6:2 Hz, 2H, NCH2), 4.27 (q, J ¼ 7:2 Hz, 2H,
OCH2), 6.85 (d, J ¼ 8:8 Hz, 1H, Py), 7.64 (dd, J ¼ 8:8,
2.6 Hz, 1H, Py), 8.24 (d, J ¼ 2:4 Hz, 1H, Py), 9.15 (br s,
1H, NH). Anal. Calcd for C11H14ClN3O2S: C, 45.91; H,

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Farhanullah et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2571–2574
4.90; N, 14.60. Found: C, 46.07; H, 4.83; N, 14.68.
(Mþ+1); IR (KBr) 1740.7 (CO), 3413.6 cmꢀ1 (NH); 1H
NMR (200 MHz, CDCl3) d 3.50 (d, J ¼ 5:4 Hz, 2H, CH2),
3.72 (s, 3H, OCH3), 5.47–5.51 (m, 1H, CH), 6.59 (d,
J ¼ 8:4 Hz, 1H, Py), 7.00–7.04 (m, 2H, Py, indolyl), 7.12–
7.19 (m, 1H, indolyl), 7.35 (d, J ¼ 8:2 Hz, 1H, Py), 7.49–
7.58 (m, 3H, Py, indolyl), 8.10 (br s, 1H, NH), 8.31 (s, 1H,
NH). Anal. Calcd for C18H17ClN4O2S: C, 55.59; H, 4.41;
N, 14.41. Found: C, 55.67; H, 4.47; N, 14.55.
Compound 5i. Yield 50%; mp 190–192 °C; MS (FAB) 350
(Mþ+1); IR (KBr) 1754.0 (CO), 3434 cmꢀ1 (NH); 1H
NMR (200 MHz, CDCl3) d 3.32 (d, J ¼ 5:8 Hz, 2H, CH2),
3.75 (s, 3H, OCH3), 5.41–5.45 (m, 1H, CH), 6.66 (d,
J ¼ 8:8 Hz, 1H, Py), 7.16–7.30 (m, 5H, phenyl), 7.58 (dd,
J ¼ 8:8, 2.6 Hz, 1H, Py), 8.00 (d, J ¼ 2:4 Hz, 1H, Py), 8.41
(br s, 1H, NH). Anal. Calcd for C16H16ClN3O2S: C, 54.93;
H, 4.61; N, 12.01. Found: C, 55.08; H, 4.70; N, 12.11.
Compound 5j. Yield 60%; mp 138–140 °C; MS (FAB) 389
11. Hubscher, G.; West, G. R. Nature 1965, 205, 799.
12. Taussky, H. H.; Shorr, E. J. Biol. Chem. 1953, 202, 675.