Probing the active-site requirements of human intestinal N-terminal maltase glucoamylase: The effect of replacing the sulfate moiety by a methyl ether in ponkoranol, a naturally occurring α-glucosidase inhibitor

Article abstract of DOI:10.1016/j.bmcl.2010.08.020

Ponkoranol is a naturally occurring glucosidase inhibitor isolated from the plant Salacia reticulata. The compound comprises a sulfonium ion with an internal sulfate counter ion. We report here an efficient synthetic route to 3′-O-methyl ponkoranol to test the hypothesis that occupation of a hydrophobic pocket by a methyl group instead of the polar sulfate ion within the active site of human N-terminal maltase glucoamylase would be beneficial. The synthetic strategy relies on the nucleophilic attack of 2,3,5-tri-O-benzyl-1,4- anhydro-4-thio-d-arabinitol at the C-6 position of benzyl 6-O-p-toluenesulfonyl β-d-glucopyranoside, followed by deprotection using boron trichloride and reduction with sodium borohydride. The target compound inhibited the N-terminal catalytic domain of intestinal human maltase glucoamylase (ntMGAM) with a K _i value of 0.50 ± 0.04 μM, higher than those of de-O-sulfonated ponkoranol (K_i = 43 ± 3 nM), or its 5′-stereoisomer (K_i = 15 ± 1 nM). We conclude that the interaction of the methyl group with hydrophobic residues in the active site is not as beneficial to inhibition of ntMGAM as the other interactions of the polyhydroxylated chain with active-site residues.

Full text of DOI:10.1016/j.bmcl.2010.08.020

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5686–5689
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Probing the active-site requirements of human intestinal N-terminal
maltase glucoamylase: The effect of replacing the sulfate moiety by
a methyl ether in ponkoranol, a naturally occurring
a-glucosidase inhibitor
Razieh Eskandari a, Kyra Jones b, David R. Rose b, B. Mario Pinto a,
⇑
a Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6
b Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1
a r t i c l e i n f o
a b s t r a c t
Article history:
Ponkoranol is a naturally occurring glucosidase inhibitor isolated from the plant Salacia reticulata. The
compound comprises a sulfonium ion with an internal sulfate counter ion. We report here an efficient
synthetic route to 30-O-methyl ponkoranol to test the hypothesis that occupation of a hydrophobic pocket
by a methyl group instead of the polar sulfate ion within the active site of human N-terminal maltase
glucoamylase would be beneficial. The synthetic strategy relies on the nucleophilic attack of 2,3,5-tri-
Received 14 July 2010
Revised 3 August 2010
Accepted 4 August 2010
Available online 11 August 2010
O-benzyl-1,4-anhydro-4-thio-D-arabinitol at the C-6 position of benzyl 6-O-p-toluenesulfonyl b-D-gluco-
pyranoside, followed by deprotection using boron trichloride and reduction with sodium borohydride.
The target compound inhibited the N-terminal catalytic domain of intestinal human maltase glucoamy-
Keywords:
a
-Glucosidase inhibitors
30-O-Methylponkoranol
Maltase glucoamylase
Thiosugar
lase (ntMGAM) with a Ki value of 0.50 0.04 lM, higher than those of de-O-sulfonated ponkoranol
(Ki = 43 3 nM), or its 50-stereoisomer (Ki = 15 1 nM). We conclude that the interaction of the methyl
group with hydrophobic residues in the active site is not as beneficial to inhibition of ntMGAM as the
other interactions of the polyhydroxylated chain with active-site residues.
Ó 2010 Elsevier Ltd. All rights reserved.
Sulfonium ion
Glycosidase inhibitors have many potential therapeutic applica-
tions because glycosidase enzyme-catalyzed hydrolysis of complex
carbohydrates is biologically widespread, and has been implicated
in several disease states.1,2 For example, inhibition of starch-hydro-
anol (4) and the corresponding sulfated stereoisomers, respectively,
revealed that the de-O-sulfonated analogs were more potent inhib-
itors than the parent compounds.15,16 Furthermore, we have shown
recently that de-O-sulfonated ponkoranol (7) (Ki = 0.043 0.01
and its 50-stereoisomer (8) (Ki = 0.015 0.01
M) are more potent
inhibitors of ntMGAM than ponkoranol (3) itself (Ki = 0.17
0.03
M) (Fig. 2).17
lM)
lyzing enzymes such as pancreatic
a
-amylase and intestinal
l
a-glucosidases that leads to a delay in digestion of ingested carbohy-
drates is one of the therapeutic approaches for the treatment of type
2 diabetes.3,4 Bioactive components isolated from medicinal plants
often provide the lead structures for drug development programs.5,6
Forexample, a relativelynewand interestingclass ofinhibitorsis the
sulfonium ion containing inhibitors, which were first isolated from
Salacia reticulata, a plant, that is, used in traditional Ayurvedic med-
icine in Sri Lanka and South India for treating type 2 diabetes.7–9 The
active components in S. reticulata were found to include salaprinol
(1),10 salacinol (2),11 ponkoranol (3),10 kotalanol (4),12 de-O-sulfo-
nated kotalanol (5),13 and de-O-sulfonated salacinol (6)14 (Fig. 1),
l
Our previous X-ray crystallographic studies of ntMGAM in com-
plex with kotalanol (4) and de-O-sulfonated kotalanol (5) had indi-
cated that removal of the sulfate group affects the conformation of
the rest of the polyhydroxylated chain.18 We concluded that
although the stereoconfiguration at C30 does not affect inhibitory
activity, the proximity of the sulfate group to the large hydropho-
bic groups (Y299, W406, and F575) likely restricts its conforma-
tional freedom. Therefore, by removing the sulfate group, the
positional constraint imposed by the bulky hydrophobic residues
surrounding the C30 group is relieved, allowing the rest of the poly-
hydroxylated chain to make optimal contacts with the ntMGAM
active site (Fig. 3).18
In view of these findings, it was of interest to question whether
replacing the sulfate group with a hydrophobic methyl ether in
ponkoranol (9) ( Fig. 4) would increase its inhibitory properties
through hydrophobic interactions in the site compared to de-O-
sulfonated ponkoranol 7.
whose structures comprise a 1,4-anhydro-4-thio-D-arabinitol core
and polyhydroxylated acyclic chain (Fig. 1).
Comparison of the inhibitory activities against the human N-ter-
minal catalytic domain of maltase glucoamylase (ntMGAM) of de-O-
sulfonated kotalanol (5) and some of its stereoisomers versus kotal-
⇑
Corresponding author. Tel.: +1 (778) 782 4152; fax: +1 (778) 782 4860.
E-mail address: bpinto@sfu.ca (B.M. Pinto).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.020

R. Eskandari et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5686–5689
5687
OH OH
OH
OH
OH OH
S
S
OSO3
OSO3
S
OH
OSO3
HO
HO
HO
HO
HO
OH
OH
HO
OH
Salaprinol (1)
Ponkoranol (3)
Salacinol (2)
OH OH
OH
OH OH
OH
OH
OH OH
OH
OH
S
OH
HCO2
S
S
OH
CH3OSO3
OH
OSO3
OH
HO
HO
HO
HO
OH
HO
OH
HO
De-O-sulfonated Kotalanol (5)
De-O-sulfonated Salacinol (6)
Kotalanol (4)
Figure 1. Components isolated from Salacia species.
We report here an efficient synthetic route to 30-O-methyl pon-
koranol (9). Our synthetic strategy involved the alkylation of an
appropriate protected anhydrothioarabinitol B at the ring sulfur
atom with agent C. Agent C could be obtained by methylation of
OH
OH OH
3' 5'
OH
2'
OH OH
5'
3'
1'
S
1'
S
6'
4'
2'
4'
6'
Cl
Cl
OH
OH
OH
OH
HO
HO
protected
Thus, benzyl 2,3-di-O-benzyl-4-O-methyl-6-O-tosyl-b-
D
-glucose at C-40 (Scheme 1).
HO
OH
D-gluco-
HO
OH
pyranoside (11)wasobtainedfrom1019 bytreatmentwitha mixture
of methyl iodide in aqueous sodium hydroxide and dimethyl sulfox-
ide (50% w/w)20 to afford 11. Our initial attempt at the coupling
reaction employed 11 with the anhydrothioarabinitol (12)21 in
1,1,1,3,3,3-hexafluoroisopropyl alcohol (HFIP) at 70 °C, as in our pre-
vious work (Scheme 2).22 No product formation and decomposition
of the starting material were observed by TLC; the coupling reaction
7
8
Figure 2. De-O-sulfonated ponkoranol and its 50-stereoisomer.
of benzyl 4-O-methyl-6-O-tosyl-b-D-glucopyranoside (13) with the
anhydrothioarabinitol (12) was also unsuccessful, the same result
being observed (Scheme 2).
Next, a trifluoromethanesulfonyl (OTf) group was chosen as a
leaving group to ensure milder conditions for the coupling reac-
tion. Thus, the primary hydroxyl group in the diol 1419 was pro-
tected as its TBDMS ether followed by sequential methylation of
the secondary hydroxyl group to give 15 in 53% over two steps
(Scheme 3). Removal of the TBDMS group using tetrabutylammo-
nium fluoride (TBAF) gave the alcohol which was treated with tri-
fluoromethanesulfonyl anhydride to yield the glycoside 16 in 76%
yield over two steps. The coupling reaction of the OBn-protected
thioether (12) with benzyl 2,3-di-O-benzyl-4-O-methyl-6-O-tri-
fluoromethanesulfonyl-a-D-glucopyranoside 16 was carried out
in dry CH2Cl2 at room temperature to give the corresponding pro-
tected sulfonium ion 17 as a 4:1 mixture of diastereomers at the
stereogenic sulfur center. However, attempts to separate the mix-
ture of diastereomers, even after deprotection and reduction, were
unsuccessful.
Figure 3. Effect of removing the sulfate group. Superposition of kotalanol (4)
(orange) and de-O-sulfonated kotalanol (5) (purple) structures. Double-headed
arrows show the proximities of the sulfate group to the surrounding hydrophobic
residues Y299, W406, and F575. (Reproduced with permission from Biochemistry,
2010, 49, 443. Copyright American Chemical Society.)
OH OH
O
OP
OP
OH
S
L
L
PO
S
OMe OH
+
OH OH OH
HO
MeO
OP
PO
5'
1'
S
3'
OP
2'
OH
4'
6'
HO
Cl
B
C
A
OH
OMe
HO
O
OP
OP
L
L = leaving group
P = protecting group
HO
OH
HO
9
OP
Figure 4. 30-O-Methyl ponkoranol.
Scheme 1. Retrosynthetic analysis.

5688
R. Eskandari et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5686–5689
S
BnO
OBn
OBn
O
OBn
OBn
O
TsO
TsO
HO
BnO
OBn
MeI, NaOH,
DMSO, rt.
85%
12
Starting material decompositon
MeO
HFIP, 70 oC
OBn
11
S
OBn
10
O
OBn
OH
BnO
BnO
TsO
OBn
12
Starting material decompositon
MeO
HFIP, 70 oC
OH
13
Scheme 2. First attempted syntheses of 9.
O
OBn
OBn
O
OBn
OBn
O
OBn
1. TBAF, THF
2. TfO2, Pyridine,
TfO
MeO
1.TBDMSCl,Imid,
2.MeI, NaOH
TBSO
MeO
HO
HO
CH2Cl2, 76%
OBn DMSO, rt, 53%
OBn
OBn
OBn
15
16
14
OH OH
5'
6'
4'
OMe OH
6'
3'
S
1'
2'
S
5'
1'
O
3'
OBn
2'
OBn
OH
Cl
OTf
S
BnO
BnO
1. BCl3, CH2Cl2,
-78 oC, 6 h
4'
HO
12
OBn
BnO
BnO
MeO
OBn
OBn
2. Amberlyst A-26,
H2O, 3 h
3. NaBH4, H2O, 3 h
OH
HO
CH2Cl2, rt, 90%
9
17
57%
Scheme 3. Second attempted synthesis of 9.
In another attempt, the tosylate 18, containing a butane diacetal
(BDA) protecting group, was chosen as the coupling partner. As
shown in Scheme 4, the tosylate 18 was synthesized in two steps
ester 18 were carried out in HFIP to give the protected sulfonium
ion 21 as a single diastereomer in 70% yield (Scheme 4).
Deprotection of the coupled product 21 was carried out in a
two-step procedure. The benzyl group was first removed by treat-
ment with boron trichloride at ꢀ78 °C in CH2Cl2, followed by
sequential BDA deprotection with 80% TFA. During the course of
benzyl group deprotection with BCl3, the p-toluenesulfonate coun-
terion was partially exchanged with chloride ion. Similar results
were observed in our previous work.18 Hence, after deprotection,
the product was treated with Amberlyst A-26 resin (chloride form)
to completely exchange the p-toluenesulfonate counterion with
from benzyl 2,3-O-[(2R,3R)-2,3-dimethoxybutane-2,3-diyl]-b-D-
glucopyranoside 19 which was, in turn, prepared from D-glucose
according to literature procedures.23 Thus, compound 19 was
treated with p-toluenesulfonyl chloride to afford the p-toluenesul-
fonyl ester 20 in 75%, which was methylated with methyl iodide to
give 18 in 91% yield. The coupling reactions of the OBn-protected
1,4-anhydro-4-thio-D-arabinitol (12) with the p-toluenesulfonyl
O
OBn
TsO
O
OBn
O
OBn
TsO
HO
HO
TsCl, Pyridine
75%
MeI, NaOH,
DMSO, 91%
MeO
O
OMe
HO
O
O
O
OMe
OMe
O
O
MeO
MeO
MeO
18
20
19
BnO
1'
OMe
2'
O
O
OH OH
5'
6'
4'
OMe OH
5'
4'
S
3'
6'
O
1'
3'
OH
OH
2'
1. BCl3, CH2Cl2,
-78 oC, 6 h
2. 80% TFA
OMe
Cl
BnO
BnO
HFIP, 70 oC, 70%
OTs
S
S
OBn
HO
BnO
BnO
3. Amberlyst A-26,
4. NaBH4, H2O, 3 h
OBn
OH
HO
21
9
51%
Scheme 4. Synthesis of compound 9.

R. Eskandari et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5686–5689
5689
Supplementary data
OH
OH
3'
H
1'
5'
Supplementary data (experimental details and 1H and 13C NMR
spectra of compounds) associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.08.020.
OH
2'
6'
4'
Cl
S
OMe OH
H4
HO
OH
HO
9
References and notes
Figure 5. 1D-NOESY correlations of selected protons in compound 9.
1. Lillelund, V. H.; Jensen, H. H.; Liang, X.; Bols, M. Chem. Rev. 2002, 102, 515.
2. Gloster, T. M.; Davies, G. J. Org. Biomol. Chem. 2010, 8, 305.
3. Holman, R. R.; Cull, C. A.; Turner, R. C. Diabetes Care 1999, 22, 960.
4. Jacob, G. S. Curr. Opin. Struct. Biol. 1995, 5, 605.
5. Harvey, A. L. Drug Discovery Today 2008, 13, 894.
6. Butler, M. S. Nat. Prod. Rep. 2008, 25, 475.
7. Chandrasena, J. P. C. The Chemistry and Pharmacology of Ceylon and Indian
Medicinal Plants; H&C Press: Colombo, Sri Lanka, 1935.
8. Jayaweera, D. M. A. Medicinal Plants Used in Ceylon-Part 1; National Science
Council of Sri Lanka: Colombo, 1981.
9. Matsuda, H.; Yoshikawa, M.; Murakami, T.; Tanabe, G.; Muraoka, O. J. Trad. Med.
2005, 22, 145.
10. Yoshikawa, M.; Xu, F. M.; Nakamura, S.; Wang, T.; Matsuda, H.; Tanabe, G.;
Muraoka, O. Heterocycles 2008, 75, 1397.
11. Yoshikawa, M.; Murakami, T.; Shimada, H.; Matsuda, H.; Yamahara, J.; Tanabe,
G.; Muraoka, O. Tetrahedron Lett. 1997, 38, 8367.
12. Matsuda, H.; Li, Y. H.; Murakami, T.; Matsumura, N.; Yamahara, J.; Yoshikawa,
M. Chem. Pharm. Bull. 1998, 46, 1399.
13. Muraoka, O.; Xie, W. J.; Tanabe, G.; Amer, M. F. A.; Minematsu, T.; Yoshikawa,
M. Tetrahedron Lett. 2008, 49, 7315.
14. Minami, Y.; Kurlyarna, C.; Ikeda, K.; Kato, A.; Takebayashi, K.; Adachi, I.;
Fleet, G. W. J.; Kettawan, A.; Karnoto, T.; Asano, N. Bioorg. Med. Chem.
2008, 16, 2734.
15. Jayakanthan, K.; Mohan, S.; Pinto, B. M. J. Am. Chem. Soc. 2009, 131, 5621.
16. Mohan, S.; Jayakanthan, K.; Nasi, R.; Kuntz, D. A.; Rose, D. R.; Pinto, B. M. Org.
Lett. 2010, 12, 1088.
17. Eskandari, R.; Kuntz, D. A.; Rose, D. R.; Pinto, B. M. Org. Lett. 2010, 12, 1632.
18. Sim, L.; Jayakanthan, K.; Mohan, S.; Nasi, R.; Johnston, B. D.; Pinto, B. M.; Rose,
D. R. Biochemistry 2010, 49, 443.
19. Shinkiti, K.; Kusunoki, A.; Hirooka, M. Bull. Chem. Soc. Jpn. 2000, 73, 967.
20. Wang, H.; Sun, L.; Glazebnik, S.; Zhao, K. Tetrahedron Lett. 1995, 36, 2953.
21. Satoh, H.; Yoshimura, Y.; Sakata, S.; Miura, S.; Machida, H.; Matsuda, A. Bioorg.
Med. Chem. Lett. 1998, 8, 989.
chloride ion. Finally, the crude product was reduced with NaBH4 to
provide the desired 30-O-methyl ponkoranol 9 in 51% yield over
three steps (Scheme 4).
The absolute stereochemistry at the stereogenic sulfur center in
9 was established by means of 1D-NOESY experiments (Fig. 5). Cor-
relation between H-10 and H-4 and also a correlation between H-20
and H-4 confirmed the anti relationship between the alkyl side
chain and the C-4 substituent on the anhydroarabinitol moiety in
compound 9.
Finally, the inhibitory activity of compound 9 was examined
against the N-terminal catalytic domain of recombinant human
maltase glucoamylase (ntMGAM), a critical intestinal glucosidase
for processing starch-derived oligosaccharides into glucose. The
30-O-methyl ponkoranol 9 inhibited ntMGAM with a Ki value of
0.50 0.04 lM. By comparison, de-O-sulfonated ponkoranol 7
and its 50-stereoisomer 8 inhibited ntMGAM with Ki values of
43 3 and 15 1 nM, respectively.17 We conclude, therefore, that
the hydrophobic interactions between the methyl group and the
hydrophobic residues Y299, W406, and F575 in the active site
are not as optimal as the interactions of the latter groups with
the rest of polyhydroxylated chain in the absence of the methyl
ether; a similar situation is also observed in the binding of the sul-
fated compound, ponkoranol (3) (Ki = 0.17 0.03 lM).
22. Ghavami, A.; Sadalapure, K. S.; Johnston, B. D.; Lobera, M.; Snider, B. B.; Pinto, B.
M. Synlett 2003, 1259.
23. Liu, H.; Nasi, R.; Jayakanthan, K.; Sim, L.; Heipel, H.; Rose, D. R.; Pinto, B. M. J.
Org. Chem. 2007, 72, 6562.
Acknowledgment
We are grateful to the Canadian Institutes for Health Research
and the Heart and Stroke Foundation of Ontario Grant # NA-
6305 for financial support of this work.