Efficient synthesis of (6-deoxy-glycopyranosid-6-yl) sulfone derivatives and their effect on Ca2+-ATPase

Article abstract of DOI:10.1016/j.ejmech.2010.09.069

Synthesis of a series of novel (6-deoxy-glycopyranosid-6-yl) sulfone derivatives has been achieved using a general synthetic strategy. Yields were excellent in every case. The synthetic compounds were evaluated for their biological potential against Ca

Full text of DOI:10.1016/j.ejmech.2010.09.069

European Journal of Medicinal Chemistry 45 (2010) 6012e6019
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Efficient synthesis of (6-deoxy-glycopyranosid-6-yl) sulfone derivatives
and their effect on Ca2þ-ATPase
*
Chinmoy Mukherjee, Swatilekha Ghosh, Pinki Nandi, Parimal C. Sen, Anup Kumar Misra
Division of Molecular Medicine, Bose Institute, A.J.C. Bose Centenery Campus, P-1/12, C. I. T. Scheme VII-M, Kolkata 700054, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of a series of novel (6-deoxy-glycopyranosid-6-yl) sulfone derivatives has been achieved using
a general synthetic strategy. Yields were excellent in every case. The synthetic compounds were eval-
uated for their biological potential against Ca2þ-ATPase, an important enzyme involves in tranporting
Ca2þ across the cell membranes.
Received 26 April 2010
Received in revised form
30 September 2010
Accepted 30 September 2010
Available online 7 October 2010
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Glycosides
Disaccharides
Sulfonyl
Ca2þ-ATPase
Stimulator
1. Introduction
phosphorylationedephosphorylation in the transport cycle [5]
with transient phosphorylation of the enzyme by ATP on an
Variation in the intracellular calcium levels (Ca2þ) is essential
for the normal cellular activities related to the development of cells,
immune response, muscle movement, modulation of neuronal
processes etc. Calcium ATPase (Ca2þ-ATPase) and its isoforms
control the level of Ca2þ ion in cells, which has strong influence on
calcium homeostasis [1]. In animal cells, Ca2þ homeostasis depends
on the function of ATP-driven Ca2þ-pump that transports cytosolic
active aspartyl group being the key step in Ca2þ translocation.
Inhibition of this enzyme activity using vanadate and lanthanum
chloride suggested its similarity with the SERCA family of Ca2þ
-
ATPases [4]. A considerable number of stimulators as well as
inhibitors for Ca2þ-ATPase have been reported and their mecha-
nism of actions has also been well established [6]. Several peptide
toxins have shown strong inhibitory activity on this enzyme [7]. In
the past several carbohydrate derivatives and pseudodisaccharide
derivatives were reported to act as inhibitors for a number of
important enzymes [8]. It was envisaged that the development of
novel sulfone-linked pseudodisaccharide derivatives acting as
stimulators and/or inhibitors of Mg2þ independent Ca2þ-ATPase
would be beneficial in this context. Carbohydrate derivatives could
serve as better stimulators or inhibitors because of their less toxic
nature, hydrophilicity and presence of chirality in the structure.
Replacement of ether linkage between two sugar units with
a sulfuratom or sulfone groupcould provide useful glycomimetics to
study their potential as enzyme inhibitors. A number reports
appeared in the literature for the synthesis of thioether linked
disaccharide derivatives having biological activities [9e13].
Although sulfur-linked pseudodisaccharides are reported earlier
[14,15], preparationof sulfone-linked pseudodisaccharides and their
biological evaluation are rather scarce [16]. In an ongoing program
for the development of carbohydrate derived small molecule stim-
ulators and/or inhibitors of Mg2þ independent Ca2þ-ATPase, we
free calcium into the intracellular storage compartments. Ca2þ
-
ATPase is a vesicular integral membrane protein located in the
sarcoplasmic reticulum (SR), plays significant role for pumping out
of calcium ion from the cytoplasm to lumen. The existence of
a number of Ca2þ-ATPases have been reported earlier whose
activities depend on the presence Mg2þ ion [2]. Earlier, Sen et al.
and others reported a Ca2þ-ATPase from rat testicular membranes
[3a], brain [3b] and goat spermatozoa membranes [4] that could be
activated by Ca2þ ion alone without the requirement of Mg2þ ion in
contrast to the classical Mg2þ dependent Ca2þ-ATPases [2c,d]. The
enzyme belongs to the family of P-type ATPases and couple
ATP hydrolysis with cation transport [4], which is essential in
cellular calcium homeostasis. Like most other Ca2þ-ATPases, it
is involved in ATP-dependent Ca2þ-transport and undergoes
* Corresponding author. Tel.: þ91 33 2569 3240; fax: þ91 33 2355 3886.
E-mail address: akmisra69@gmail.com (A.K. Misra).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.09.069

C. Mukherjee et al. / European Journal of Medicinal Chemistry 45 (2010) 6012e6019
6013
BnO
BnO
3. Biology
I
I
I
I
OBn
O
O
O
BnO
BnO
O
BnO
BnO
BnO
OMe
OMe
BnO
BnO
BnO
In order to examine the bioactivity of the synthetic compounds
(7aeh), their effects were tested against an important enzyme
known as Mg2þ independent Ca2þ-ATPase. Since all compounds
(7aeh) were found to stimulate Ca2þ-ATPase, they were tested in
the presence of a number of calcium channel blockers like verap-
amil, thapsigargin and vanadate to examine their effect. Biological
activities of compounds 7aeh alone and in combination with
known calcium blockers against Mg2þ independent Ca2þ-ATPase
are presented in Table 1. The Mg2þ independent Ca2þ-ATPase
activity assay was performed in the microsomal membranes of goat
spermatozoa according to Sikdar et al. [4]. In the present study, all
compounds (7aeh) have been found to stimulate the enzyme and
30e50% stimulation was observed at an average concentration of
BnO
3d
OMe
OMe
OMe
3a
3c
3b
BnO
BnO
SAc
O
SAc
O
SAc
O
AcS
OBn
BnO
BnO
O
BnO
BnO
BnO
BnO
BnO
BnO
BnO
4d
OMe
OMe
OMe
4a
4c
4b
Fig. 1. Suitably functionalized monosaccharide intermediates.
report herein, efficient synthesis of a series of novel (6-deoxy-gly-
copyranosid-6-yl) sulfone derivatives and their effect on Mg2þ
independent Ca2þ-ATPase.
about 20 mg/L (70 nM) (Fig. 4).
2. Chemistry
4. Results and discussion
Synthesis of
a series of bis-(6-deoxy-glycopyranosid-6-yl)
sulfone derivatives (7aeh) starting from a range of suitably func-
tionalized monosaccharide derivatives was achieved using
The biological experiments using synthetic compounds 7aeh in
the presence of known calcium blockers revealed that the inhibi-
tory effect of Thapsigargin predominates over the stimulating effect
of target compounds 7ae7h on Ca2þ-ATPase, whereas the inhibi-
tory effect of Verapamil on Ca2þ-ATPase was effectively compen-
sated by the presence of disaccharides 7be7d and 7fe7h. In the
case of orthovanadate, a reasonable reduction in the inhibitory
effect on Ca2þ-ATPase was observed in the presence of disaccha-
rides 7b and 7c only (Table 1). These findings suggest that even the
modification of one stereocenter may exert a considerable influ-
ence on the ability of the (6-deoxy-glycopyranosid-6-yl)sulfone
target compounds to compensate the effect of various Ca-ATPase
inhibitors. Since Mg2þ independent Ca2þ-ATPase is involved in
calcium transport, the stimulatory effects on this enzyme by these
synthetic compounds and subsequent competition with some
known calcium blockers support their biological significance.
a general synthetic strategy. Methyl
a-
D-glucopyranoside (1a) was
converted to methyl 2,3,4-tri-O-benzyl-6-deoxy-6-iodo-
a-D-glu-
copyranoside (3a) [17] using a reaction sequence involving selec-
tive tritylation [18] of 6-hydroxy group using trityl chloride in
pyridine, per-O-benzylation [19] using benzyl bromide and sodium
hydroxide and acidic removal [20] of trityl group followed by
treatment with iodine in the presence of triphenylphosphine and
imidazole [21]. Compound 3a [17] was treated with potassium
thioacetate to furnish methyl 2,3,4-tri-O-benzyl-6-deoxy-6-thio-
acetyl-
reaction conditions, compounds 3bed [17] and 4b, 4c [23] and
4d were synthesized from methyl glycosides of -galactose,
-mannose and -allose respectively (Fig. 1, Scheme 1).
Synthesis of (6-deoxy-glycopyranosid-6-yl) sulfide derivatives
a-D-glucopyranoside (4a) [22] in 88% yield. Following similar
D
D
D
was carried out by coupling compounds 3aed [17] with 4aed. After
a series of experimentations, it was observed that treatment of
compound 3a with compound 4a in the presence of solid sodium
hydroxide (2 equiv.) in dimethylformamide at room temperature
furnished compound 5a in 86% yield. Following similar reaction
condition, a series of symmetric and asymmetric (6-deoxy-glyco-
pyranosid-6-yl) sulfide derivatives (5aeh) have been synthesized
in excellent yield (Fig. 2, Scheme 2). Compounds 5aeh were treated
with excess 3-chloroperbenzoic acid (m-CPBA) [24] to give
(6-deoxy-glycopyranosid-6-yl) sulfone derivatives (6aeh) in
excellent yield. Conventional hydrogenolysis [25] of compounds
6aeh over Pearlman’s catalyst furnished symmetric and asym-
metric (6-deoxy-glycopyranosid-6-yl) sulfone (7aeh) in excellent
yield (Fig. 3, Scheme 2).
5. Conclusion
In summary, a series of a new class of symmetric and asymmetric
(6-deoxy-glycopyranosid-6-yl) sulfone have been synthesized by
coupling a set of sugar iodides with sugar thiols in a very straight-
forward and highyielding reaction condition. The reaction condition
can be applied in a scale-up synthesis. The synthetic intermediates
(3aed and 4aed) were prepared from the same starting material.
Furthermore compounds 7aeh are found to be biologically impor-
tant as evident from their stimulatory effects on Mg2þ independent
Ca2þ-ATPase. Further optimization for the development of Ca2þ
ATPase stimulators are currently in progress in our laboratory.
-
SAc
O
I
OH
b
O
c
O
a
Methyl α-D-
(BnO)3
BnO
glycopyranosides
(BnO)3
BnO
(BnO)3
OMe
OMe
OMe
1a-c
4a-c
2a-c
OH
3a-c
I
SAc
O
a
O
b
c
O
BnO
BnO
Methyl β-D-
OMe
OMe
OMe
allopyranoside
BnO
BnO
BnO
BnO
BnO
1d
2d
4d
3d
Sugars: a: D-glucose, b: D-galactose, c: D-mannose.
Scheme 1. Reagents: (a) (i) Trityl chloride, pyridine, 70 ꢀC; (ii) BnBr, NaOH, r. t.; (iii) 80% aq. AcOH, 70 ꢀC (75% for 2a; 72% for 2b; 72% for 2c and 70% for 2d); (b) I2, PPh3, Imidazole,
toluene, 60 ꢀC (84% for 3a; 79% for 3b; 82% for 3c and 79% for 3d); (c) KSAc, DMF, r. t. (88% for 4a; 82% for 4b; 88% for 4c and 85% for 4d).

6014
C. Mukherjee et al. / European Journal of Medicinal Chemistry 45 (2010) 6012e6019
S
OBn
S
S
BnO
O
O
BnO
BnO
BnO
BnO
OMe
5a
O
O
O
BnO
BnO
O
BnO
BnO
BnO
BnO
OMe
BnO
BnO
BnO
BnO
OMe
BnO
BnO
OMe
OMe
OMe
5b
5c
BnO
S
BnO
S
OBn
BnO
S
O
O
O
BnO
BnO
O
BnO
BnO
BnO
BnO
BnO
OMe
O
O
OMe
BnO
BnO
OMe
BnO
BnO
BnO
OMe
5d
BnO
BnO
OMe
OMe
5f
5e
BnO
S
S
O
O
O
O
BnO
BnO
BnO
BnO
OMe
5g
BnO
OMe
OMe
OMe
BnO
BnO
BnO
BnO
BnO
BnO
5h
Fig. 2. Synthesized (6,60)-thiolinked protected pseudodisaccharide derivatives (5aeh).
6. Experimental section
6.1.2. General experimental procedure for the preparation of methyl
6-S-acetyl-2,3,4-tri-O-benzyl-6-deoxy- -glycopyranoside (4aed)
a/b-D
6.1. General remarks
To a solution of compound 3a (1.5 g, 2.6 mmol) in dry DMF
(15 mL) was added potassium thioacetate (600 mg, 5.2 mmol) and
the reaction mixture was stirred at room temperature for 3 h. The
reaction mixture was then concentrated under reduced pressure,
diluted with CH2Cl2 (25 mL) and washed with water (2 ꢁ 10 mL)
and 10% aq. Na2S2O3 (2 ꢁ 10 mL) solution respectively. The organic
layer was dried (Na2SO4), filtered and concentrated under reduced
pressure and the crude product was purified over SiO2 using hex-
aneeEtOAc (6:1) as eluant to give pure product as syrup.
All reactions were monitored by thin layer chromatography over
silica gel coated TLC plates. The spots on TLC were visualized by
warming ceric sulphate (2% Ce(SO4)2 in 2 N H2SO4) sprayed plates on
a hot plate. Silica gel 230e400 mesh was used for column chroma-
tography.1H and 13C NMR, 2D COSY, HMQC spectrawere recorded on
Brucker DRX500 MHzspectrometer usingCDCl3 andD2O assolvents
and TMS as internal reference unless stated otherwise. Chemical
shift values are expressed in ppm. ESI-MS were recorded on
a Micromass Quattro II triple quadrupole mass spectrometer.
Elementary analysis was carried out on Carlo Erba-1108 analyzer.
Optical rotations were measured at 25 ꢀC on a Perkin Elmer 341
polarimeter. Biological experiments were carried out in a Shimadzu
UV-2401PC spectrophotometer. Commercially available grades of
organic solvents of adequate purity are used in all reactions.
6.1.2.1. Methyl 6-S-acetyl-2,3,4-tri-O-benzyl-6-deoxy-
anoside (4a). Yellowish oil (1.2 g, 88%); Rf: 0.7 (hexaneeEtOAc, 2:1);
IR (neat): 2914, 2363, 1695, 1358, 1114, 1071, 1003, 740, 694 cmꢂ1
1H NMR (CDCl3, 300 MHz):
7.29e7.24 (m, 15H, Ar-H), 4.95
a-D-glucopyr-
;
d
(d, J ¼ 10.9 Hz, 1H, PhCH2), 4.86 (d, J ¼ 10.8 Hz, 1H, PhCH2), 4.79 (d,
J ¼ 10.9 Hz, 1H, PhCH2), 4.76 (d, J ¼ 12.1 Hz, 1H, PhCH2), 4.63
(d, J ¼ 12.1 Hz, 1H, PhCH2), 4.60 (d, J ¼ 10.7 Hz, 1H, PhCH2), 4.51 (d,
J ¼ 3.5 Hz, 1H, H-1), 3.94 (t, J ¼ 9.2 Hz each, 1H, H-3), 3.73e3.69
(m, 1H, H-5), 3.47 (dd, J ¼ 9.7, 3.5 Hz, 1H, H-2), 3.41 (dd, J ¼ 13.8,
3.0 Hz, 1H, H-6a), 3.34 (s, 3H, OCH3), 3.26 (t, J ¼ 9.1 Hz each, 1H, H-
4), 2.98 (dd, J ¼ 13.6, 7.7 Hz, 1H, H-6b), 2.31 (s, 3H, COCH3); 13C NMR
6.1.1. General experimental procedure for the preparation of methyl
2,3,4-tri-O-benzyl-6-deoxy-6-iodo-a/b-D-glycopyranoside (3aed)
To a solution of compound 2aed (3 g, 6.5 mmol) in dry toluene
(30 mL) were added triphenylphosphine (3.4 g, 12.9 mmol) and
imidazole (2.2 g, 32.3 mmol) respectively, followed by the addition
of iodine (2.5 g, 9.7 mmol) and the reaction mixture was stirred at
60 ꢀC for 1 h. Methanol (10 mL) was added to the reaction mixture
and the entire solvent was evaporated out. The crude reaction
mixture was then diluted with CH2Cl2 (40 mL) and the organic layer
was then washed with 10% aq. Na2S2O3 solution (2 ꢁ 15 mL) and
brine (2 ꢁ 15 mL) respectively. The organic layer was dried
(Na2SO4), filtered and concentrated under reduced pressure and
the crude product was purified over SiO2 using hexaneeEtOAc (8:1)
as eluant to give pure product as syrup.
(CDCl3, 75 MHz):
d 194.2 (COCH3), 138.6e127.5 (Ar-C), 97.8 (C-1),
81.7, 80.5, 80.0, 75.6, 75.0, 73.2, 69.4, 55.1, 30.8, 30.4; ESI-MS: m/
25
z ¼ 545.3 [M þ Na]þ; [
a]
þ 30.3 (c 1.0, CHCl3); Anal. Calcd. for
D
C30H34O6S (522.21): C, 68.94; H, 6.56. Found: C, 68.75; H, 6.80.
6.1.2.2. Methyl 6-S-acetyl-2,3,4-tri-O-benzyl-6-deoxy-a-D-galacto-
pyranoside (4b). Yellowish oil (1.1 g, 82%). Rf: 0.6 (hexaneeEtOAc,
3:1); IR (neat): 3021, 2357, 1678, 1595, 1425, 1216, 1043, 764,
672 cmꢂ1 1H NMR (CDCl3, 300 MHz):
; d 7.35e7.22 (m, 15H, Ar-H),
4.99 (d, J ¼ 11.3 Hz, 1H, PhCH2), 4.86 (d, J ¼ 11.8 Hz, 1H, PhCH2), 4.80
O
O
S
3a-d
S
O
c
O
a
(BnO)3
OMe
(BnO)3
O
O
+
(HO)3
(HO)3
OMe
OMe
4a-d
OMe
5a-h: X = S
6a-h: X = SO2
b
7a-h
Scheme 2. Reagents: (a) Solid NaOH, DMF, r. t. (86% for 5a, 85% for 5b, 88% for 5c, 86% for 5d, 83% for 5e, 86% for 5f, 88% for 5g and 84% for 5h); (b) m-CPBA, CH2Cl2, 0 ꢀCer. t.
(83% for 6a, 80% for 6b, 84% for 6c, 79% for 6d, 80% for 6e, 83% for 6f, 80% for 6g and 76% for 6h); (c) 20% Pd(OH)2/C, CH3OH-EtOAc (3:1), r. t. (78% for 7a, 75% for 7b, 80% for 7c, 76%
for 7d, 75% for 7e, 76% for 7f, 78% for 7g and 73% for 7h).

C. Mukherjee et al. / European Journal of Medicinal Chemistry 45 (2010) 6012e6019
6015
O
O
O
O
O
O
S
OH
O
OH
S
S
O
O
HO
HO
O
HO
HO
O
HO
HO
O
HO
HO
OMe
HO
HO
HO
HO
HO
HO
OMe
7a
HO
OMe
HO
OMe
7b
OMe
OMe
7c
O
O
O
O
O
O
O
OH
O
S
S
OH
O
OH
HO
OH
O
S
O
HO
HO
HO
HO
HO
HO
HO
OMe
O
OMe
HO
HO
HO
OH
OMe
OH
HO
HO
OMe
OMe
OMe
7f
7d
7e
O
O
O
O
O
S
S
O
O
O
HO
HO
HO
HO
HO
OMe
OMe
OMe
OH
OH
OH
OMe
7g
OH
OH
OH
7h
Fig. 3. Synthesized (6,60)-sulfonyl linked pseudodisaccharides (7aeh).
(d, J ¼ 12.0 Hz, 1H, PhCH2), 4.73 (d, J ¼ 11.8 Hz, 1H, PhCH2), 4.66
(d, J ¼ 12.1 Hz, 1H, PhCH2), 4.62e4.57 (m, 2H, H-1, PhCH2), 3.99 (dd,
J ¼ 9.9, 3.5 Hz, 1H, H-2), 3.89 (dd, J ¼ 10.0, 2.7 Hz, 1H, H-3),
3.87e3.85 (m, 1H, H-4), 3.64 (t, J ¼ 6.6 Hz, 1H, H-5), 3.35 (s, 3H,
OCH3), 3.06e2.94 (m, 2H, H-6ab), 2.30 (s, 3H, COCH3); 13C NMR
J ¼ 11.4 Hz, 1H, PhCH2), 4.74 (d, J ¼ 8.0 Hz, 1H, H-1), 4.58
(d, J ¼ 12.2 Hz, 1H, PhCH2), 4.48 (d, J ¼ 11.5 Hz, 1H, PhCH2), 4.33 (d,
J ¼ 11.5 Hz, 1H, PhCH2), 4.06e3.96 (m, 2H, H-3 and H-5), 3.51 (s, 3H,
OCH3), 3.53e3.46 (m, 1H, H-6a), 3.17e3.12 (m, 2H, H-2 and H-4),
2.98 (dd, J ¼ 13.6, 7.7 Hz, 1H, H-6b), 2.34 (s, 3H, COCH3); 13C NMR
(CDCl3, 75 MHz):
d
194.8 (COCH3), 138.7e127.4 (Ar-C), 98.7 (C-1),
(CDCl3, 75 MHz): d 194.5 (COCH3), 138.8e127.4 (Ar-C), 101.8 (C-1),
79.1, 77.2, 76.3, 76.1, 74.8, 73.5, 69.7, 55.2, 30.4, 29.9; ESI-MS:
79.0, 78.3, 74.5, 74.3, 73.0, 71.4, 71.2, 56.6, 31.1, 30.5; ESI-MS: m/
25
m/z ¼ 545.2 [M þ Na]þ; [
a
]
25 þ 23.4 (c 1.0, CHCl3); Anal. Calcd. for
z ¼ 545.2 [M þ Na]þ; [
a
]
D
þ 32.6 (c 1.0, CHCl3); Anal. Calcd. for
D
C30H34O6S (522.21): C, 68.94; H, 6.56. Found: C, 68.78; H, 6.70.
C30H34O6S (522.21): C, 68.94; H, 6.56. Found: C, 68.72; H, 6.78.
6.1.2.3. Methyl 6-S-acetyl-2,3,4-tri-O-benzyl-6-deoxy-
a-D
-mannopy-
6.1.3. General experimental procedure for the preparation of
ranoside (4c). Yellowish oil (1.2 g, 86%). Rf: 0.6 (hexaneeEtOAc,
(methyl 2,3,4-tri-O-benzyl-6-deoxy-
(methyl 2,3,4-tri-O-benzyl-6-deoxy-
sulfide (5a-h)
a
/
b
b
-
D
-glycopyranosid-6-yl)-
-glycopyranosid-6-yl)
2:1); IR (neat): 3028, 2352, 1676, 1595, 1425, 1216, 1045, 764,
a/
-D
675 cmꢂ1 1H NMR (CDCl3, 300 MHz):
; d 7.86e7.22 (m, 15H, Ar-H),
4.93 (d, J ¼ 10.8 Hz,1H, PhCH2), 4.74 (d, J ¼ 12.4 Hz,1H, PhCH2), 4.69
(d, J ¼ 12.4 Hz, 1H, PhCH2), 4.66 (br s, 1H, H-1), 4.65 (d, J ¼ 10.9 Hz,
1H, PhCH2), 4.60 (br s, 2H, PhCH2), 3.85 (dd, J ¼ 9.1, 3.0 Hz, 1H, H-3),
3.79e3.75 (m, 2H, H-2 and H-4), 3.68 (ddd, J ¼ 10.9, 8.2, 2.7 Hz, 1H,
H-5), 3.57 (dd, J ¼ 13.5, 2.8 Hz, 1H, H-6a), 3.28 (s, 3H, OCH3), 3.07
(dd, J ¼ 13.5, 8.1 Hz, 1H, H-6b), 2.33 (s, 3H, COCH3); 13C NMR (CDCl3,
To a solution of compound 3aed (300 mg, 0.52 mmol) and
compound 4aed (275 mg, 0.52 mmol) in dry DMF (6 mL) was
added crushed NaOH (125 mg, 3.1 mmol) and the resulting reaction
mixture was then stirred at room temperature for 3 h. The reaction
mixture was then concentrated under reduced pressure, diluted
with saturated aq. NH4Cl solution (8 mL) and extracted with CH2Cl2
(3 ꢁ 10 mL). The combined organic layer was dried (Na2SO4),
filtered and concentrated under reduced pressure and the crude
product was purified over SiO2 using hexaneeEtOAc (5:1) as eluant
to give pure product as colorless oil.
75 MHz): d 195.1 (COCH3), 138.3e127.5 (Ar-C), 98.9 (C-1), 80.0, 77.5,
75.2, 74.6, 72.7, 72.1, 70.8, 54.7, 31.2, 30.4; ESI-MS: m/z ¼ 545.3
25
[M þ Na]þ; [
a
]
D
þ 36.9 (c 1.0, CHCl3); Anal. Calcd. for C30H34O6S
(522.21): C, 68.94; H, 6.56. Found: C, 68.75; H, 6.82.
6.1.2.4. Methyl 6-S-acetyl-2,3,4-tri-O-benzyl-6-deoxy-
b
-D
-allopyr-
6.1.3.1. Bis-(methyl 2,3,4-tri-O-benzyl-6-deoxy-a-D-glucopyranosid-
anoside (4d). Yellowish oil (1.15 g, 85%). Rf: 0.6 (hexaneeEtOAc,
3:1); IR (neat): 3273, 2925, 2360, 1651, 1218, 1088, 769, 674 cmꢂ1
1H NMR (CDCl3, 300 MHz):
7.36e7.22 (m, 15H, Ar-H), 4.83
6-yl) sulfide (5a). Yellowish oil (420 mg, 86%). Rf: 0.5 (hex-
aneeEtOAc, 2:1); IR (neat): 3020, 2359, 1595, 1216, 1052, 761,
;
d
670 cmꢂ1
;
1H NMR (CDCl3, 300 MHz):
d
7.30e7.22 (m, 15H, Ar-H),
(d, J ¼ 10.6 Hz, 1H, PhCH2), 4.82 (d, J ¼ 12.1 Hz, 1H, PhCH2), 4.75 (d,
4.94 (d, J ¼ 10.9 Hz, 1H, PhCH2), 4.86 (d, J ¼ 11.1 Hz, 1H, PhCH2), 4.77
Table 1
Effects of pseudosacchrides (7aeh) on Mg2þ independent Ca2þ-ATPase in absence and presence of different calcium blockers.
Concentration of compounds
Control
Compound No.
7a
7b
7c
7d
7e
7f
7g
7h
Thapsigargin (TG) (25 nM)
ꢂ50
NA
NA
NA
NA
þ32
ꢂ38
NA
NA
þ43
ꢂ35
NA
NA
NA
NA
NA
NA
Compounds (20
m
g)
þ36
ꢂ28
NA
þ30
ꢂ31
NA
þ53
ꢂ36
NA
þ49
ꢂ29
NA
þ45
ꢂ27
NA
þ30
ꢂ33
NA
TG (25 mM) þ Compound (20
m
g)
Verapamil (300
Verapamil (300
Orthovanadate (1 mM)
Orthovanadate (1 mM) þ Compound (20
m
m
M)
M) þ Compound (20
ꢂ25
m
g)
NA
ꢂ14
ꢂ5
ꢂ3
ꢂ9
ꢂ21
ꢂ2
ꢂ5
ꢂ8
ꢂ30
NA
NA
NA
NA
NA
NA
NA
NA
NA
m
g)
ꢂ24
ꢂ8
þ4
ꢂ17
ꢂ25
ꢂ27
ꢂ19
ꢂ21
Enzyme activity: percent stimulation (þ) or inhibition (ꢂ). NA: Not applicable. Enzyme activity was measured either in the presence of a fixed concentration of any calcium
blocker, or in the presence of a fixed concentration of a pseudosaccharide alone or in combination of any of specific calcium blocker and pseudodisaccharide. The results shown
are the average of four separate assays.

6016
C. Mukherjee et al. / European Journal of Medicinal Chemistry 45 (2010) 6012e6019
Fig. 4. Effect of different concentrations of pseudosaccharides on Mg2þ independent Ca2þ-ATPase. Activity was assayed in presence of varying concentrations of each of the
compound as described in methods. In plot A: -: compound 7a;*: compound 7b; C: compound 7c and :: compound 7d. In plot B: -: compound 7e; *: compound 7f;
C: compound 7g and :: compound 7h.
(d, J ¼ 10.9 Hz, 1H, PhCH2), 4.71 (d, J ¼ 12.1 Hz, 1H, PhCH2), 4.59 (d,
J ¼ 12.1 Hz, 1H, PhCH2), 4.58 (d, J ¼ 11.2 Hz, 1H, PhCH2), 4.49
(d, J ¼ 3.5 Hz, 1H, H-1), 3.91 (t, J ¼ 9.2 Hz each, 1H, H-3), 3.73
(ddd, J ¼ 9.6, 7.9, 1.7 Hz, 1H, H-5), 3.44 (dd, J ¼ 9.6, 3.5 Hz, 1H, H-2),
3.35 (dd, J ¼ 8.4, 3.5 Hz, 1H, H-4), 3.34 (s, 3H, OCH3), 2.90 (dd,
3.5 Hz, 1H, H-2), 3.34 (s, 3H, OCH3), 3.32e3.29 (m, 1H, H-4), 3.27 (s,
3H, OCH3), 3.02e2.95 (m, 2H, H-6ab0), 2.73e2.59 (m, 2H, H-6ab); 13
NMR (CDCl3, 75 MHz):
138.8e127.5 (Ar-C), 98.8 (C-10), 97.7 (C-1),
C
d
81.9, 80.7, 80.3, 80.2, 77.6, 75.6, 75.0, 74.9, 74.7, 73.2, 72.8, 72.7, 72.1,
71.0, 55.0, 54.6, 35.0, 34.9; ESI-MS: m/z ¼ 949.5 [M þ Na]þ;
25
J ¼ 13.6, 2.2 Hz, 1H, H-6a), 2.60 (dd, J ¼ 13.6, 7.7 Hz, 1H, H-6b); 13
C
[a
]
þ 38.7 (c 1.0, CHCl3); Anal. Calcd. for C56H62O10S (926.41): C,
D
NMR (CDCl3, 75 MHz):
d
138.7e127.4 (Ar-C), 97.6 (C-1), 81.8, 80.4,
72.54; H, 6.74. Found: C, 72.36; H, 6.98.
80.1, 75.5, 74.8, 73.0, 71.0, 54.9, 34.9; The proton integration in the
1H NMR and carbon signals in the 13C NMR spectra appeared as
monomer because of the symmetric nature of the molecule; ESI-
6.1.3.4. (Methyl 2,3,4-tri-O-benzyl-6-deoxy-a-D-glucopyranosid-6-yl)-
(methyl 20,30,40-tri-O-benzyl-6-deoxy-
b-D-allopyranosid-6-yl) sulfide
MS: m/z ¼ 949.6 [M þ Na]þ; [
a]
25 þ 43.4 (c 1.0, CHCl3); Anal. Calcd.
(5d). Yellowish oil (420 mg, 86%). Rf: 0.7 (hexaneeEtOAc, 1:1); IR
D
for C56H62O10S (926.41): C, 72.54; H, 6.74. Found: C, 72.35; H, 6.90.
(neat): 3019, 2359, 1596, 1215, 1080, 760, 670 cmꢂ1; 1H NMR (CDCl3,
300 MHz):
d
7.32e7.19 (m, 30H, Ar-H), 4.93 (d, J ¼ 10.9 Hz, 1H,
6.1.3.2. (Methyl 2,3,4-tri-O-benzyl-6-deoxy-
yl)-(methyl 20,30,40-tri-O-benzyl-6-deoxy-
-galactopyranosid-6-yl)
sulfide (5b). Yellowish oil (415 mg, 85%). Rf: 0.4 (hexaneeEtOAc,
3:1); IR (neat): 3020, 2926, 2360, 1594, 1216, 1049, 760, 671 cmꢂ1
1H NMR (CDCl3, 300 MHz):
7.37e7.19 (m, 30H, Ar-H), 4.96 (d,
a-
D-glucopyranosid-6-
PhCH2), 4.87e4.70 (m, 7H, H-10 and PhCH2), 4.63e4.52 (m, 4H, H-1
and PhCH2), 4.46 (d, J ¼ 11.5 Hz, 1H, PhCH2), 4.32 (d, J ¼ 11.5 Hz, 1H,
PhCH2), 4.08e4.03 (m, 2H, H-30 and H-50), 3.91 (t, J ¼ 9.1 Hz each, 1H,
H-3), 3.78e3.74 (m, 1H, H-5), 3.49e3.44 (m, 1H, H-2), 3.45 (s, 3H,
OCH3), 3.39e3.33 (m, 1H, H-4), 3.36 (s, 3H, OCH3), 3.28 (dd, J ¼ 9.4,
2.2 Hz,1H, H-40), 3.14 (dd, J ¼ 7.9, 2.4 Hz,1H, H-20), 3.02e2.94 (m, 2H,
a-D
;
d
J ¼ 11.4 Hz, 1H, PhCH2), 4.95 (d, J ¼ 10.9 Hz, 1H, PhCH2), 4.86
(d, J ¼ 11.2 Hz, 1H, PhCH2), 4.84 (d, J ¼ 11.8 Hz, 1H, PhCH2), 4.79 (d,
J ¼ 12.1 Hz, 1H, PhCH2), 4.77 (d, J ¼ 10.9 Hz, 1H, PhCH2), 4.74
(d, J ¼ 12.1 Hz, 1H, PhCH2), 4.71 (d, J ¼ 11.8 Hz, 1H, PhCH2), 4.65 (d,
J ¼ 11.9 Hz, 1H, PhCH2), 4.62 (d, J ¼ 12.0 Hz, 1H, PhCH2), 4.59
(d, J ¼ 2.8 Hz, 1H, H-10), 4.57 (d, J ¼ 11.2 Hz, 1H, PhCH2), 4.55 (d,
J ¼ 11.2 Hz, 1H, PhCH2), 4.51 (d, J ¼ 3.5 Hz, 1H, H-1), 4.00e3.85
(m, 4H, H-3, H-20, H-30 and H-40), 3.74e3.68 (m, 2H, H-5 and H-50),
3.45 (dd, J ¼ 9.6, 3.5 Hz, 1H, H-2), 3.38e3.28 (m, 1H, H-4), 3.34 (s,
3H, OCH3), 3.31 (s, 3H, OCH3), 2.84 (dd, J ¼ 13.4, 2.1 Hz, 1H, H-6a),
H-6a and H-6a ), 2.79e2.64 (m, 2H, H-6b and H-6b ); 13C NMR (CDCl3,
0
0
75 MHz):
d
138.9e127.3 (Ar-C), 101.9 (C-10), 97.8 (C-1), 82.0, 80.4,
80.2, 79.2, 78.1, 77.2, 75.6, 74.9, 74.5, 74.4, 73.2, 73.0, 71.3, 70.9, 56.6,
55.0, 35.4, 35.1; ESI-MS: m/z ¼ 949.4 [M þ Na]þ; [
a]
D
25 þ 46.5 (c 1.2,
CHCl3); Anal. Calcd. for C56H62O10S (926.41): C, 72.54; H, 6.74. Found:
C, 72.35; H, 6.95.
6.1.3.5. (Methyl 2,3,4-tri-O-benzyl-6-deoxy-
yl)-(methyl 20,30,40-tri-O-benzyl-6-deoxy-
sulfide (5e). Yellowish oil (405 mg, 83%). Rf: 0.5 (hexaneeEtOAc,
4:1); IR (neat): 3020, 2359, 1596, 1216, 760, 670 cmꢂ1 1H NMR
a-D-galactopyranosid-6-
a-D-mannopyranosid-6-yl)
0
2.73 (dd, J ¼ 13.5, 7.3 Hz, 1H, H-6a ), 2.61e2.44 (m, 2H, H-6b and H-
6b ); 13C NMR (CDCl3, 75 MHz):
d
138.8e127.5 (Ar-C), 98.7 (C-10),
;
0
97.8 (C-1), 81.9, 80.5, 80.2, 79.3, 76.4, 76.0, 75.7, 75.0, 74.8, 73.5,
(CDCl3, 300 MHz): d 7.31e7.19 (m, 30H, Ar-H), 4.93e4.56 (m, 14H,
73.4, 73.3, 71.1, 70.9, 55.2, 55.1, 34.9, 33.9; ESI-MS: m/z ¼ 949.6
H-1, H-10 and PhCH2), 4.02e3.60 (m, 8H, H-2, H-3, H-4, H-5, H-20, H-
30, H-40 and H-50), 3.35 (s, 3H, OCH3), 3.28 (s, 3H, OCH3), 2.95e2.64
[M þ Na]þ; [
a]
þ 23.3 (c 1.2, CHCl3); Anal. Calcd. for C56H62O10
S
25
D
(926.41): C, 72.54; H, 6.74. Found: C, 72.32; H, 7.0.
(m, 4H, H-6ab and H-6ab0); 13C NMR (CDCl3, 75 MHz):
d 138.8e127.3
(Ar-C), 98.8 (C-10), 98.6 (C-1), 80.1, 79.2, 77.5, 76.4, 75.9, 75.0, 74.8,
74.6, 73.3, 73.2, 72.7, 72.6, 71.9, 70.5, 55.1, 54.6, 34.9, 33.6; ESI-MS:
6.1.3.3. (Methyl 2,3,4-tri-O-benzyl-6-deoxy-
a-D-glucopyranosid-6-
yl)-(methyl 20,30,40-tri-O-benzyl-6-deoxy-
a-
D-mannopyranosid-6-yl)
m/z ¼ 949.6 [M þ Na]þ; [
a
]
25 þ 31.8 (c 1.0, CHCl3); Anal. Calcd. for
D
sulfide (5c). Yellowish oil (430 mg, 88%). Rf: 0.5 (hexaneeEtOAc,
C56H62O10S (926.41): C, 72.54; H, 6.74. Found: C, 72.32; H, 7.0.
2:1); IR (neat): 3020, 2361,1593,1216,1052, 761, 670 cmꢂ1; 1H NMR
(CDCl3, 300 MHz):
d
7.33e7.23 (m, 30H, Ar-H), 4.94 (d, J ¼ 10.9 Hz,
6.1.3.6. Bis (methyl 2,3,4-tri-O-benzyl-6-deoxy-a-D-mannopyr-
1H, PhCH2), 4.91 (d, J ¼ 11.1 Hz, 1H, PhCH2), 4.84 (d, J ¼ 11.2 Hz, 1H,
PhCH2), 4.77 (d, J ¼ 11.0 Hz, 1H, PhCH2), 4.72 (d, J ¼ 12.1 Hz,
1H, PhCH2), 4.67 (d, J ¼ 3.7 Hz, 1H, H-10), 4.63e4.56 (m, 7H, PhCH2),
4.51 (d, J ¼ 3.4 Hz, 1H, H-1), 3.92 (t, J ¼ 9.1 Hz each, 1H, H-3),
3.82e3.62 (m, 5H, H-5, H-20, H-30, H-40 and H-50), 3.46 (dd, J ¼ 9.6,
anosid-6-yl) sulfide (5f). Yellowish oil (420 mg, 86%). Rf: 0.6
(hexaneeEtOAc, 3:1); IR (neat): 3020, 2360, 1522, 1216, 762,
670 cmꢂ1; 1H NMR (CDCl3, 300 MHz):
d 7.34e7.20 (m, 15H, Ar-H),
4.90 (d, J ¼ 11.2 Hz, 1H, PhCH2), 4.71e4.56 (m, 6H, H-1, PhCH2),
3.82e3.63 (m, 4H, H-2, H-3, H-4 and H-5), 3.27 (s, 3H, OCH3), 3.06

C. Mukherjee et al. / European Journal of Medicinal Chemistry 45 (2010) 6012e6019
6017
(dd, J ¼ 13.6, 1.6 Hz, 1H, H-6a), 2.72 (dd, J ¼ 13.5, 8.6 Hz, 1H, H-6b);
13C NMR (CDCl3, 75 MHz):
138.7e127.4 (Ar-C), 98.8 (C-1), 80.3,
H-6a), 3.11 (t, J ¼ 9.2 Hz each, 1H, H-4), 2.96e2.87 (m, 1H, H-6b);
13C NMR (CDCl3, 75 MHz):
138.4e127.6 (Ar-C), 97.8 (C-1), 81.6,
d
d
77.8, 75.0, 74.8, 72.8, 72.7, 72.1, 54.6, 34.9; The proton integration
79.7, 79.1, 75.6, 74.5, 73.0, 65.9, 56.3, 55.8; The proton integration
in the 1H NMR and carbon signals in the 13C NMR spectra appeared
in the 1H NMR and carbon signals in the 13C NMR spectra
as monomer because of the symmetric nature of the molecule;
appeared as monomer because of the symmetric nature of the
25
25
ESI-MS: m/z ¼ 949.4 [M þ Na]þ; [
a
]
þ 42.6 (c 1.0, CHCl3); Anal.
molecule; ESI-MS: m/z ¼ 981.5 [M þ Na]þ; [
a
]
þ 76.5 (c 1.0,
D
D
Calcd. for C56H62O10S (926.41): C, 72.54; H, 6.74. Found: C, 72.35;
H, 6.98.
CHCl3); Anal. Calcd. for C56H62O12S (958.40): C, 70.12; H, 6.52.
Found: C, 70.0; H, 6.78.
6.1.3.7. (Methyl 2,3,4-tri-O-benzyl-6-deoxy-
yl)-(methyl
a
-
-
D
-mannopyranosid-6-
-allopyranosid-6-yl)
6.1.4.2. (Methyl 2,3,4-tri-O-benzyl-6-deoxy- -glucopyranosid-6-yl)-
(methyl a-D-galactopyranosid-6-yl)
a
-
D
20,30,40-tri-O-benzyl-6-deoxy-
b
D
20,30,40-tri-O-benzyl-6-deoxy-
sulfide (5g). Yellowish oil (430 mg, 88%). Rf: 0.6 (hexaneeEtOAc,
3:2); IR (neat): 3020, 2363, 1594, 1216, 761, 670 cmꢂ1 1H NMR
(CDCl3, 300 MHz):
7.34e7.21 (m, 30H, Ar-H), 4.90 (d, J ¼ 11.1 Hz,
sulfone (6b). Yellowish oil (290 mg, 80%). Rf: 0.5 (hexaneeEtOAc, 3:1);
;
IR (neat): 3020, 2359, 1726, 1594, 1216, 1046, 760, 671 cmꢂ1; 1H NMR
d
(CDCl3, 300 MHz):
d
7.27e7.18 (m, 30H, Ar-H), 4.98 (d, J ¼ 11.4 Hz, 1H,
1H, PhCH2), 4.84 (d, J ¼ 11.8 Hz, 1H, PhCH2), 4.82 (d, J ¼ 12.2 Hz, 1H,
PhCH2), 4.76e4.55 (m, 9H, H-1, H-10 and PhCH2), 4.46 (d, J ¼ 11.5 Hz,
1H, PhCH2), 4.34 (d, J ¼ 11.5 Hz, 1H, PhCH2), 4.10e4.04 (m, 2H, H-30
and H-50), 3.80e3.66 (m, 4H, H-2, H-3, H-4 and H-5), 3.44 (s, 3H,
OCH3), 3.32e3.28 (m, 1H, H-40), 3.29 (s, 3H, OCH3), 3.16 (dd, J ¼ 7.9,
PhCH2), 4.96 (d, J ¼ 10.8 Hz, 1H, PhCH2), 4.86 (d, J ¼ 11.7 Hz, 1H,
PhCH2), 4.84 (d, J ¼ 11.5 Hz, 1H, PhCH2), 4.78 (d, J ¼ 12.3 Hz, 1H,
PhCH2), 4.77 (d, J ¼ 10.4 Hz, 1H, PhCH2), 4.73 (d, J ¼ 11.9 Hz, 1H,
PhCH2), 4.70 (d, J ¼ 11.6 Hz, 1H, PhCH2), 4.63 (d, J ¼ 12.1 Hz, 1H,
PhCH2), 4.61 (d, J ¼ 12.2 Hz, 1H, PhCH2), 4.52e4.45 (m, 4H, H-1, H-10
and PhCH2), 4.22e4.10 (m, 2H, H-5 and H-50), 3.99e3.88 (m, 3H, H-3,
H-20 and H-30), 3.65e3.56 (m, 2H, H-6a and H-40), 3.45 (dd, J ¼ 9.6,
3.5 Hz, 1H, H-2), 3.36 (s, 3H, OCH3), 3.29e3.20 (m, 4H, H-6b and
0
2.5 Hz, 1H, H-20), 3.11e3.00 (m, 2H, H-6a and H-6a ), 2.85e2.69
(m, 2H, H-6b and H-6b ); 13C NMR (CDCl3, 75 MHz):
d 139.0e127.3
0
(Ar-C), 101.9 (C-10), 98.8 (C-1), 80.3, 79.2, 78.2, 77.6, 75.0, 74.8, 74.6,
0
74.4, 73.1, 73.0, 72.7, 72.6, 72.1, 71.4, 56.5, 54.6, 35.5, 35.1; ESI-MS:
OCH3), 3.12 (t, J ¼ 9.2 Hz each, 1H, H-4), 3.01e2.92 (m, 1H, H-6a ),
m/z ¼ 949.5 [M þ Na]þ; [
a
]
25 þ 35.6 (c 1.2, CHCl3); Anal. Calcd. for
2.55e2.49 (m, 1H, H-6b ); 13C NMR (CDCl3, 75 MHz):
d
138.4e127.6
0
D
C56H62O10S (926.41): C, 72.54; H, 6.74. Found: C, 72.36; H, 7.0.
(Ar-C), 98.8 (C-10), 98.0 (C-1), 81.8, 79.7, 78.9, 78.6, 76.1, 75.7, 74.7, 74.5,
73.8 (2C), 73.4, 73.2, 66.3, 66.0, 56.8, 56.1, 55.9, 55.7; ESI-MS: m/
z ¼ 981.4 [M þ Na]þ; [
a]
D
þ 74.5 (c 1.0, CHCl3); Anal. Calcd. for
25
6.1.3.8. Bis (methyl 2,3,4-tri-O-benzyl-6-deoxy-
b
-
D
-allopyranosid-6-
yl) sulfide (5h). Yellowish oil (410 mg, 84%). Rf: 0.6 (hexaneeEtOAc,
C56H62O12S (958.40): C, 70.12; H, 6.52. Found: C, 69.95; H, 6.80.
2:1); IR (neat): 3021, 2359, 1594,1216,1039, 761, 671 cmꢂ1; 1H NMR
(CDCl3, 300 MHz):
d
7.38e7.24 (m, 15H, Ar-H), 4.88e4.73 (m, 4H, H-
6.1.4.3. (Methyl 2,3,4-tri-O-benzyl-6-deoxy-
yl)-(methyl 20,30,40-tri-O-benzyl-6-deoxy-
-mannopyranosid-6-yl)
sulfone (6c). Yellowish oil (305 mg, 84%). Rf: 0.4 (hexaneeEtOAc,
3:1); IR (neat): 3020, 2363, 1666, 1596, 1216, 1071, 760, 671 cmꢂ1
1H NMR (CDCl3, 300 MHz):
7.33e7.20 (m, 30H, Ar-H), 4.97 (d,
a-D-glucopyranosid-6-
1 and PhCH2), 4.59 (d, J ¼ 12.2 Hz, 1H, PhCH2), 4.48 (d, J ¼ 11.6 Hz,
1H, PhCH2), 4.36 (d, J ¼ 11.6 Hz, 1H, PhCH2), 4.11e4.06 (m, 2H, H-3
and H-2), 3.48 (s, 3H, OCH3), 3.30 (dd, J ¼ 9.5, 2.2 Hz, 1H, H-4), 3.20
(dd, J ¼ 9.8, 7.3 Hz,1H, H-5), 3.09 (dd, J ¼ 13.9, 2.5 Hz,1H, H-6a), 2.77
(dd, J ¼ 13.9, 7.4 Hz, 1H, H-6b); 13C NMR (CDCl3, 75 MHz):
a-D
;
d
J ¼ 10.9 Hz, 1H, PhCH2), 4.92 (d, J ¼ 11.5 Hz, 1H, PhCH2), 4.87 (d,
J ¼ 11.5 Hz, 1H, PhCH2), 4.78e4.47 (m, 11H, H-1, H-10 and PhCH2),
4.18 (t, J ¼ 9.6 Hz each, 1H, H-3), 4.13e4.06 (m, 1H, H-50), 3.97
(t, J ¼ 9.2 Hz each, 1H, H-30), 3.84 (dd, J ¼ 9.3, 3.0 Hz, 1H, H-20),
3.74e3.71 (m, 1H, H-5), 3.62 (t, J ¼ 9.5 Hz each, 1H, H-40), 3.45 (dd,
J ¼ 9.7, 3.5 Hz, 1H, H-2), 3.309e3.28 (m, 5H, H-4, H-6a and OCH3),
d
138.9e127.3 (Ar-C), 101.8 (C-1), 79.1, 78.1, 74.4, 74.3, 73.1, 72.8,
71.3, 56.6, 35.2. The proton integration in the 1H NMR and carbon
signals in the 13C NMR spectra appeared as monomer because of
the symmetric nature of the molecule; ESI-MS: m/z ¼ 949.4
25
[M þ Na]þ; [
a
]
þ 21.6 (c 1.0, CHCl3); Anal. Calcd. for C56H62O10
S
D
0
(926.41): C, 72.54; H, 6.74. Found: C, 72.38; H, 6.98.
3.24e3.10 (m, 5H, H-6b, H-6a and OCH3), 3.05e2.96 (m, 1H, H-6b );
13C NMR (CDCl3, 75 MHz):
d
138.5e127.6 (Ar-C), 99.0 (C-10), 97.8
6.1.4. General experimental procedure for the preparation of (methyl
(C-1), 81.7, 80.1, 79.8, 79.4, 75.9, 75.7, 74.8, 74.6 (2C), 73.1, 73.0, 72.1,
2,3,4-tri-O-benzyl-6-deoxy-
a/
b-
D-glycopyranosid-6-yl)-(methyl 2,3,
67.3, 65.7, 56.6, 56.5, 55.8, 55.3; ESI-MS: m/z ¼ 981.5 [M þ Na]þ;
25
4-tri-O-benzyl-6-deoxy-
a/
b-D
-glycopyranosid-6-yl) sulfone (6aeh)
[a
]
þ 86.6 (c 1.0, CHCl3); Anal. Calcd. for C56H62O12S (958.40): C,
D
To a solution of compound 5aeh (350 mg, 0.38 mmol) in dry
CH2Cl2 (5 mL) was added m-CPBA (325 mg, 1.89 mmol) at 0 ꢀC and
the reaction mixture was then stirred at room temperature for 2 h.
The reaction mixture was then diluted with CH2Cl2 (10 mL) and
washed with 10% aq. Na2S2O3 solution (2 ꢁ 8 mL), saturated aq.
Na2CO3 solution (2 ꢁ 8 mL) and brine (2 ꢁ 8 mL) respectively. The
organic layer was dried (Na2SO4), filtered and concentrated under
reduced pressure and the crude product was purified over SiO2
using hexaneeEtOAc (4:1) as eluant to give pure product as syrup.
70.12; H, 6.52. Found: C, 69.96; H, 6.75.
6.1.4.4. (Methyl 2,3,4-tri-O-benzyl-6-deoxy-a-D-glucopyranosid-6-yl)-
(methyl 20,30,40-tri-O-benzyl-6-deoxy-
b
-
D
-allopyranosid-6-yl) sulfone
(6d). Yellowish oil (285 mg, 79%). Rf: 0.6 (hexaneeEtOAc, 2:1); IR
(neat): 3021, 2360, 1727, 1589, 1216, 1080, 761, 671 cmꢂ1 1H NMR
(CDCl3, 300 MHz):
;
d
7.35e7.15 (m, 30H, Ar-H), 4.95 (d, J ¼ 10.8 Hz, 1H,
PhCH2), 4.88e4.70 (m, 7H, PhCH2), 4.63e4.39 (m, 6H, H-1, H-10 and
PhCH2), 4.22e4.14 (m, 2H, H-30 and H-50), 4.07e4.06 (m, 1H, H-5),
3.97 (t, J ¼ 9.1 Hz each, 1H, H-3), 3.44 (dd, J ¼ 9.6, 3.5 Hz, 1H, H-2),
3.42e3.39 (m, 1H, H-4), 3.29e3.24 (m, 1H, H-6a), 3.17e3.06 (m, 5H,
H-6b, H-20, H-40 and H-6ab0); 13C NMR (CDCl3, 75 MHz):
6.1.4.1. Bis (methyl 2,3,4-tri-O-benzyl-6-deoxy-a-D-glucopyranosid-
6-yl) sulfone (6a). Yellowish oil (300 mg, 83%). Rf: 0.6 (hex-
aneeEtOAc, 2:1); IR (neat): 2908, 2371, 1631, 1453, 1360, 1313,
d
138.5e127.5 (Ar-C), 102.1 (C-10), 97.9 (C-1), 81.7, 79.8, 79.3, 78.6,
1127, 1090, 1037, 739, 696 cmꢂ1
;
1H NMR (CDCl3, 300 MHz):
77.3, 75.7, 74.5 (2C), 73.6, 73.2, 73.0, 70.8, 68.3, 66.1, 57.2, 56.8, 56.5,
55.8; ESI-MS: m/z ¼ 981.6 [M þ Na]þ; [
a]
D
þ 44.3 (c 1.0, CHCl3);
25
d
7.31e7.18 (m, 15H, Ar-H), 4.95 (d, J ¼ 10.9 Hz, 1H, PhCH2), 4.84
(d, J ¼ 11.5 Hz, 1H, PhCH2), 4.76 (d, J ¼ 10.9 Hz, 1H, PhCH2), 4.71
(d, J ¼ 12.1 Hz, 1H, PhCH2), 4.60 (d, J ¼ 12.2 Hz, 1H, PhCH2), 4.51
(d, J ¼ 11.5 Hz, 1H, PhCH2), 4.41 (d, J ¼ 3.4 Hz, 1H, H-1), 4.13 (t,
J ¼ 9.9 Hz each, 1H, H-5), 3.95 (t, J ¼ 9.1 Hz each, 1H, H-3), 3.42
(dd, J ¼ 9.6, 3.5 Hz, 1H, H-2), 3.33 (s, 3H, OCH3), 3.26e3.21 (m, 1H,
Anal. Calcd. for C56H62O12S (958.40): C, 70.12; H, 6.52. Found: C,
69.95; H, 6.80.
6.1.4.5. (Methyl 2,3,4-tri-O-benzyl-6-deoxy- -galactopyranosid-6-
a
-
D
yl)-(methyl 20,30,40-tri-O-benzyl-6-deoxy-
a-D-mannopyranosid-6-yl)

6018
C. Mukherjee et al. / European Journal of Medicinal Chemistry 45 (2010) 6012e6019
sulfone (6e). Yellowish oil (290 mg, 80%). Rf: 0.5 (hexaneeEtOAc,
2:1); IR (neat): 3023, 2365, 1668, 1596, 1216, 1073, 760, 675 cmꢂ1
1H NMR (CDCl3, 300 MHz):
7.39e7.21 (m, 30H, Ar-H), 5.01 (d,
6.1.5. General experimental procedure for the preparation of
(methyl 6-deoxy- -glycopyranosid-6-yl)-(methyl 6-deoxy-
-glycopyranosid-6-yl) sulfone (7aeh)
To the solution of compound 6aeh (200 mg, 0.21 mmol) in
methanol and ethyl acetate (3:1, 12 mL) was added 20% Pd(OH)2/C
(50 mg) and the reaction mixture was allowed to stir at room
temperature under a positive pressure of hydrogen for 24 h. The
reaction mixture was filtered through a CeliteÒ bed and evaporated
to dryness to give compound 7aeh as a white powder, which was
further purified by passing through a column of Sephadex-LH-20
using CH3OH as eluant.
;
a/
b-
D
a/b-
d
D
J ¼ 11.4 Hz, 1H, PhCH2), 4.94 (d, J ¼ 11.3 Hz, 1H, PhCH2), 4.87 (d,
J ¼ 11.8 Hz, 1H, PhCH2), 4.77 (d, J ¼ 12.1 Hz, 1H, PhCH2), 4.72 (d,
J ¼ 11.8 Hz, 1H, PhCH2), 4.67e4.51 (m, 9H, H-1, H-10 and PhCH2),
4.32e4.24 (m, 1H, H-50), 4.18e4.10 (m, 1H, H-3), 3.96 (br s, 2H, H-2
and H-4), 3.87 (dd, 0J ¼ 9.2, 3.0 Hz, 1H, H-30), 3.77e3.58 (m, 4H, H-5,
H-20, H-40 and H-6a ), 3.45e3.38 (m,1H, H-6a), 3.28e3.20 (m, 7H, H-
6b and 2 OCH3), 2.78e2.72 (m, 1H, H-6b); 13C NMR (CDCl3,
75 MHz):
138.4e127.5 (Ar-C), 98.9 (C-10), 98.6 (C-1), 79.9, 78.5,
0
d
77.0, 75.9, 75.8, 74.8, 74.7, 74.4, 73.6, 73.3, 72.8, 71.9, 67.0, 65.9, 56.7,
56.2, 55.7, 55.1; ESI-MS: m/z ¼ 981.4 [M þ Na]þ; [
a
]
25 þ 62.5 (c 1.0,
6.1.5.1. Bis(methyl 6-deoxy-
Glass (85 mg, 78%). Rf: 0.4 (CH2Cl2eCH3OH, 6:1); 1H NMR (D2O,
300 MHz):
4.86 (d, J ¼ 3.6 Hz, 1H), 4.19 (t, J ¼ 9.2 Hz each, 1H),
a-D-glucopyranosid-6-yl) sulfone (7a).
D
CHCl3); Anal. Calcd. for C56H62O12S (958.40): C, 70.12; H, 6.52.
Found: C, 69.92; H, 6.75.
d
3.86e3.68 (m, 3H), 3.64 (dd, J ¼ 9.7, 3.5 Hz, 1H), 3.50 (s, 3H), 3.36 (t,
6.1.4.6. Bis (methyl 2,3,4-tri-O-benzyl-6-deoxy-a-D-mannopyranosid-
J ¼ 9.2 Hz each, 1H); 13C NMR (D2O, 75 MHz):
d 99.4, 72.7, 71.9, 70.9,
6-yl) sulfone (6f). Yellowish oil (300 mg, 83%). Rf: 0.7 (hex-
aneeEtOAc, 1:1); IR (neat): 3021, 2923, 2361, 1664, 1596, 1216, 1124,
66.4, 55.7, 55.6; The proton integration in the 1H NMR and carbon
signals in the 13C NMR spectra appeared as monomer because of
1067, 761, 670 cmꢂ1; 1H NMR (CDCl3, 300 MHz):
d
7.34e7.20 (m,15H,
the symmetric nature of the molecule; ESI-MS: m/z ¼ 441.3
[M þ Na]þ; [
a]
þ 111.4 (c 1.0, H2O); Anal. Calcd. for C14H26O12
S
25
Ar-H), 4.94 (d, J ¼ 11.4 Hz, 1H, PhCH2), 4.65 (br s, 2H, PhCH2),
4.57e4.53 (m, 4H, H-1 and PhCH2), 4.14 (t, J ¼ 9.8 Hz each, 1H, H-5),
3.85 (dd, J ¼ 9.3, 3.0 Hz, 1H, H-3), 3.74e3.72 (m, 1H, H-2), 3.65 (t,
J ¼ 9.5 Hz each, 1H, H-4), 3.46e3.41 (m, 1H, H-6a), 3.29e3.21 (m, 4H,
D
(418.11): C, 40.19; H, 6.26. Found: C, 40.0; H, 6.50.
6.1.5.2. (Methyl 6-deoxy-
-galactopyranosid-6-yl) sulfone (7b). Glass (82 mg, 75%). Rf: 0.5
(CH2Cl2eCH3OH, 6:1); 1H NMR (D2O, 300 MHz):
4.83e4.81 (m,
a-D
-glucopyranosid-6-yl)-(methyl 60-deoxy-
H-6b and OCH3); 13C NMR (CDCl3, 75 MHz):
d
138.1e127.6 (Ar-C),
a-D
98.8 (C-1), 80.1, 76.1, 74.8, 74.6, 72.8, 72.0, 67.1, 56.6, 55.3; The proton
integration in the 1H NMR and carbon signals in the 13C NMR spectra
appeared as monomer because of the symmetric nature of the
d
2H), 4.42 (d, J ¼ 9.8 Hz, 1H), 4.13 (t, J ¼ 9.5 Hz each, 1H), 3.91e3.76
(m, 5H), 3.70e3.55 (m, 4H), 3.46e3.41 (m, 7H); 13C NMR (D2O,
molecule; ESI-MS: m/z ¼ 981.5 [M þ Na]þ; [
a
]
25 þ 64.9 (c 1.0, CHCl3);
75 MHz): d 99.7, 99.3, 72.7, 71.9, 70.9, 70.7, 69.1, 67.6, 66.3, 65.6, 55.7
D
Anal. Calcd. for C56H62O12S (958.40): C, 70.12; H, 6.52. Found: C, 70.0;
H, 6.80.
(2C), 55.6, 55.5; ESI-MS: m/z ¼ 441.3 [M þ Na]þ; [
a
]
25 þ 148.3 (c 1.0,
D
H2O); Anal. Calcd. for C14H26O12S (418.11): C, 40.19; H, 6.26. Found:
C, 39.95; H, 6.45.
6.1.4.7. (Methyl2,3,4-tri-O-benzyl-6-deoxy-
(methyl 20,30,40-tri-O-benzyl-6-deoxy-
-allopyranosid-6-yl) sulfone
(6g). Yellowish oil (290 mg, 80%). Rf: 0.6 (hexaneeEtOAc, 2:1); IR
(neat): 3025, 2363, 1726, 1596, 1216, 1045, 760, 675 cmꢂ1 1H NMR
7.38e7.18 (m, 30H, Ar-H), 4.94e4.43 (m, 14H, H-
a-D-mannopyranosid-6-yl)-
b-D
6.1.5.3. (Methyl 6-deoxy-
-mannopyranosid-6-yl) sulfone (7c). Glass (87 mg, 80%). Rf: 0.4
(CH2Cl2eCH3OH, 6:1); 1H NMR (D2O, 300 MHz):
4.82e4.80 (m,
a-D
-glucopyranosid-6-yl)-(methyl 60-deoxy-
a-D
;
d
(CDCl3, 300 MHz):
d
2H), 4.18e4.07 (m, 2H), 3.97e3.95 (m, 1H), 3.84e3.75 (m, 3H),
1, H-5, H-10 and PhCH2), 4.23 (d, J ¼ 11.6 Hz, 1H, PhCH2), 4.18e4.10
(m, 2H, H-30 and H-50), 3.88 (dd, J ¼ 9.2, 3.0 Hz, 1H, H-3), 3.77e3.75
(m, 1H, H-2), 3.64 (t, J ¼ 9.5 Hz each, 1H, H-4), 3.60e3.55 (m, 1H, H-
3.71e3.53 (m, 5H), 3.46 (s, 3H), 3.44 (s, 3H), 3.35e3.32 (m, 1H); 13C
NMR (D2O, 75 MHz):
d 101.0, 99.4, 72.8, 71.9, 71.0, 70.2, 69.7, 68.7,
67.2, 66.4, 55.8 (2C), 55.6, 55.3; ESI-MS: m/z ¼ 441.2 [M þ Na]þ;
25
0
6a), 3.42 (s, 3H, OCH3), 3.37e3.34 (m, 2H, H-20 and H-6a ), 3.26e3.13
[a
]
þ 90.0 (c 1.0, H2O); Anal. Calcd. for C14H26O12S (418.11): C,
D
(m, 6H, H-6b, H-40, H-6b and OCH3); 13C NMR (CDCl3, 75 MHz):
40.19; H, 6.26. Found: C, 40.0; H, 6.48.
0
d
138.6e127.4 (Ar-C), 101.8 (C-10), 98.7 (C-1), 80.0, 78.5, 75.9, 74.6,
74.5, 74.4, 73.7, 72.9 (3C), 72.0, 70.8, 67.9, 67.4, 56.8 (2C), 56.5, 55.2;
6.1.5.4. (Methyl 6-deoxy-
-allopyranosid-6-yl) sulfone (7d). Glass (83 mg, 76%). Rf: 0.4
(CH2Cl2eCH3OH, 5:1); 1H NMR (D2O, 300 MHz):
4.82e4.80 (m,
a-D
-glucopyranosid-6-yl)-(methyl 60-deoxy-
25
ESI-MS: m/z ¼ 981.5 [M þ Na]þ; [
a]
D
þ 34.3 (c 1.2, CHCl3);
b-D
Anal. Calcd. for C56H62O12S (958.40): C, 70.12; H, 6.52. Found: C,
69.95; H, 6.82.
d
1H), 4.65 (d, J ¼ 8.2 Hz, 1H), 4.26e4.15 (m, 3H), 3.79e3.55 (m, 7H),
3.56 (s, 3H), 3.47 (s, 3H), 3.35e3.28 (m, 2H); 13C NMR (D2O,
6.1.4.8. Bis (methyl 2,3,4-tri-O-benzyl-6-deoxy-
b
-
D
-allopyranosid-
75 MHz): d 101.2, 99.4, 72.9, 72.2, 71.0, 70.7, 70.1, 69.1, 68.5, 66.3,
6-yl) sulfone (6h). Yellowish oil (275 mg, 76%). Rf: 0.5 (hex-
aneeEtOAc, 2:1); IR (neat): 3023, 2357, 1726, 1594, 1216, 1046,
57.4, 56.4, 55.7 (2C); ESI-MS: m/z ¼ 441.2 [M þ Na]þ; [
a
]
25 þ 35.0 (c
D
1.0, H2O); Anal. Calcd. for C14H26O12S (418.11): C, 40.19; H, 6.26.
Found: C, 39.96; H, 6.50.
765, 674 cmꢂ1 1H NMR (CDCl3, 300 MHz):
; d 7.38e7.16 (m, 15H,
Ar-H), 4.86 (d, J ¼ 11.6 Hz, 1H, PhCH2), 4.82 (d, J ¼ 10.7 Hz, 1H,
PhCH2), 4.80 (d, J ¼ 7.9 Hz, 1H, H-1), 4.75 (d, J ¼ 11.9 Hz, 1H,
PhCH2), 4.59 (d, J ¼ 12.2 Hz, 1H, PhCH2), 4.53e4.44 (m, 2H, H-5
and PhCH2), 4.21 (d, J ¼ 11.8 Hz, 1H, PhCH2), 4.09e4.08 (m, 1H, H-
3), 3.48e3.43 (m, 1H, H-6a), 3.37 (s, 3H, OCH3), 3.34e3.25 (m, 1H,
H-6b), 3.17 (dd, J ¼ 7.9, 2.4 Hz, 1H, H-2), 3.12 (dd, J ¼ 9.6, 2.1 Hz, 1H,
6.1.5.5. (Methyl6-deoxy-
-mannopyranosid-6-yl) sulfone (7e). Glass (82 mg, 75%). Rf: 0.5
(CH2Cl2eCH3OH, 5:1); 1H NMR (D2O, 300 MHz):
4.82e4.80 (m, 2H),
4.41 (d, J ¼ 9.9 Hz, 1H), 4.13e4.06 (m, 1H), 3.96e3.50 (m, 10H), 3.43
a-D
-galactopyranosid-6-yl)-(methyl60-deoxy-
a-D
d
(br s, 6H); 13C NMR (D2O, 75 MHz):
d 101.0, 99.7, 70.7, 70.1, 69.7, 69.1,
H-4); 13C NMR (CDCl3, 75 MHz):
d
138.5e127.4 (Ar-C), 102.0 (C-1),
68.7, 67.6, 67.1, 65.6, 55.7 (3C), 55.2; ESI-MS: m/z ¼ 441.3 [M þ Na]þ;
78.6, 77.2, 74.4, 73.4, 73.0, 70.6, 68.3, 57.1, 56.8; The proton
[
a
]
25 þ 122 (c 1.0, H2O); Anal. Calcd. for C14H26O12S (418.11): C, 40.19;
D
integration in the 1H NMR and carbon signals in the 13C NMR
H, 6.26. Found: C, 40.28; H, 6.50.
spectra appeared as monomer because of the symmetric nature of
25
the molecule; ESI-MS: m/z ¼ 981.4 [M þ Na]þ; [
a
]
þ 38 (c 1.0,
6.1.5.6. Bis(methyl 6-deoxy-
Glass (83 mg, 76%). Rf: 0.4 (CH2Cl2eCH3OH, 6:1); 1H NMR (D2O,
300 MHz):
4.76 (d, J ¼ 3.5 Hz, 1H), 4.10 (t, J ¼ 9.3 Hz each, 1H),
a-D-mannopyranosid-6-yl) sulfone (7f).
D
CHCl3); Anal. Calcd. for C56H62O12S (958.40): C, 70.12; H, 6.52.
Found: C, 69.90; H, 6.75.
d

C. Mukherjee et al. / European Journal of Medicinal Chemistry 45 (2010) 6012e6019
6019
3.96e3.94 (m, 1H), 3.84e3.64 (m, 3H), 3.56 (t, J ¼ 9.7 Hz each, 1H),
Appendix. Supplementary data
3.45 (s, 3H); 13C NMR (D2O, 75 MHz):
d 101.1, 70.1, 69.7, 68.7, 67.3,
55.8, 55.4; The proton integration in the 1H NMR and carbon signals
in the 13C NMR spectra appeared as monomer because of the
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2010.09.069.
symmetric nature of the molecule; ESI-MS: m/z ¼ 441.1 [M þ Na]þ.
25
[
a]
þ 56.6 (c 1.0, H2O); Anal. Calcd. for C14H26O12S (418.11): C,
D
References
40.19; H, 6.26. Found: C, 40.0H, 6.50.
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[4] R. Sikdar, U. Ganguly, P. Pal, B. Mazumder, P.C. Sen, Mol. Cell. Biochem. 103
(1991) 121e130.
6.1.5.7. (Methyl 6-deoxy-
deoxy- -allopyranosid-6-yl) sulfone (7g). Glass (85 mg, 78%). Rf:
0.5 (CH2Cl2eCH3OH, 5:1); 1H NMR (D2O, 300 MHz):
4.82e4.81 (m,
a-D
-mannopyranosid-6-yl)-(methyl 60-
b-D
d
1H), 4.68 (d, J ¼ 8.3 Hz, 1H), 4.34e4.27 (m, 1H), 4.23e4.15 (m, 2H),
4.01e3.99 (m, 1H), 3.84e3.68 (m, 5H), 3.66e3.58 (m, 5H),
3.53e3.49 (m, 4H); 13C NMR (D2O, 75 MHz):
d 101.2, 101.0, 70.7,
70.2, 70.1, 69.8, 69.0, 68.8, 68.5, 67.0, 57.4, 56.3, 55.7, 55.3; ESI-MS:
25
m/z ¼ 441.3 [M þ Na]þ; [
a
]
D
þ 20 (c 1.0, H2O); Anal. Calcd. for
C14H26O12S (418.11): C, 40.19; H, 6.26. Found: C, 40.05; H, 6.50.
[5] (a) R. Sikdar, U. Ganguly, S. Chandra, G. Adhikary, P.C. Sen, J. Biosci. 18 (1993)
73e82;
(b) R. Sikdar, K. Roy, A.K. Mandal, P.C. Sen, J. Biosci. 24 (1999) 317e321.
[6] (a) T. Sengupta, S. Ghosal, S.R. Dungdung, G.C. Majumder, P.C. Sen, Mol. Cell.
Biochem. 311 (2008) 93e103;
(b) T. Sengupta,S. Ghosal, P.C. Sen, Comp. Biochem. Physiol. 146 (2006)131e138;
(c) J. Segal, J. Hardiman, S.H. Ingbar, Biochem. J. 261 (1989) 749e754;
(d) B. Mazumder, R. Sikdar, P.C. Sen, Ind. J. Biochem. Bjophys. 28 (1991) 136e139.
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(b) K.J. Baker, J.M. East, A.G. Lee, Biochemistry 34 (1995) 3596e3604;
(c) A. Xu, N. Narayanan, J. Biol. Chem. 275 (2000) 4407e4416;
(d) C.L. Longland, M. Mezna, F.I. Michelangeli, J. Biol. Chem. 274 (1999)
14799e14805.
6.1.5.8. Bis (methyl 6-deoxy-
Glass (80 mg, 73%). Rf: 0.4 (CH2Cl2eCH3OH, 6:1); 1H NMR (D2O,
300 MHz):
4.62 (d, J ¼ 7.8 Hz, 1H), 4.26e4.15 (m, 2H), 3.72e3.33
b-D-allopyranosid-6-yl) sulfone (7h).
d
(m, 7H); 13C NMR (D2O, 75 MHz):
d 101.1, 70.8, 70.1, 69.2, 68.3, 57.4,
56.2; The proton integration in the 1H NMR and carbon signals in
the 13C NMR spectra appeared as monomer because of the
symmetric nature of the molecule; ESI-MS: m/z ¼ 441.2 [M þ Na]þ;
25
[
a]
ꢂ 25.7 (c 1.0, H2O); Anal. Calcd. for C14H26O12S (418.11): C,
D
40.19; H, 6.26. Found: C, 39.96; H, 6.50.
[8] (a) S. Ogawa, S. Yokoi, N. Kimura, Y. Shibata, N. Chida, Carbohydr. Res. 181
(1988) 57e66;
6.2. Assay of ATPase activity
(b) I. Carvalho, A.H. Haines, J. Chem. Soc. Perkin Trans. 1 (1999) 1795e1800;
(c) Y. Blériot, T. Dintinger, N. Guillo, C. Tellier, Tetrahedron Lett. 36 (1995)
5175e5178;
(d) C. Saotome, Y. Kanie, O. Kanie, C.H. Wong, Bioorg. Med. Chem. 8 (2000)
2249e2261.
ATPase activity was assayed in the microsomal membranes iso-
lated from goat spermatozoa which was reported to be enriched
with this enzyme. The microsomal membranes were isolated from
goat spermatozoa following the method reported before by Sikdar
et al. [4]. In a typical experiment, 2 mg of the enzyme preparation and
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C.M. thanks CSIR, New Delhi for providing a Senior Research
Fellowship. This work was supported by DST, New Delhi in the form
of Ramanna Fellowship (AKM); CSIR, New Delhi (37/1088/01/EMR-
II) (PCS) and Bose Institute, Kolkata.
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