Synthesis of montbretin A analogues yields potent competitive inhibitors of human pancreatic α-amylase

Article abstract of DOI:10.1039/c9sc02610j

Simplified analogues of the potent human amylase inhibitor montbretin A were synthesised and shown to bind tightly, K_I = 60 and 70 nM, with improved specificity over medically relevant glycosidases, making them promising candidates for controlling blood glucose. Crystallographic analysis confirmed similar binding modes and identified new active site interactions.

Full text of DOI:10.1039/c9sc02610j

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Synthesis of montbretin A analogues yields potent
competitive inhibitors of human pancreatic a-
amylase†
Cite this: Chem. Sci., 2019, 10, 11073
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
a
b
a
a
Christina R. Tysoe, Sami Caner, Matthew B. Calvert, Anna Win-Mason,
b
a
Gary D. Brayer and Stephen G. Withers
*
Received 29th May 2019
Accepted 16th October 2019
Simplified analogues of the potent human amylase inhibitor montbretin A were synthesised and shown to
bind tightly, K
I
¼ 60 and 70 nM, with improved specificity over medically relevant glycosidases, making them
DOI: 10.1039/c9sc02610j
promising candidates for controlling blood glucose. Crystallographic analysis confirmed similar binding
modes and identified new active site interactions.
rsc.li/chemical-science
The healthcare burden of diabetes mellitus has steadily risen new pharmacophore for a-amylase inhibition involving stacked
over the past century, with an increase from 108 million adult phenolic rings that interact closely with the conserved active
1
cases worldwide in 1980 to 422 million cases in 2014. Strik- site carboxylic acids. The parent molecule, montbretin A (MbA),
2
ingly, type II diabetes accounts for 90–95% of all cases. The is a complex avonol glycoside produced by Crocosmia croc-
inhibition of carbohydrate catabolism has been explored as osmiiora that acts as a potent and selective inhibitor of HPA (K
I
9
a means of mediating post-prandial blood glucose levels for the ¼ 8 nM). This inhibitor has been tested in ZDF (Zucker diabetic
3
treatment of type II diabetes. Targeting of carbohydrate fatty) rats and produced a signicant decrease in blood glucose
catabolism provides two benets: beyond directly modulating plasma levels, highlighting this compound as a promising lead
blood glucose levels it can also promote weight loss in pre- for the therapeutic reduction of post-prandial blood glucose
10
diabetic individuals to prevent the onset of diabetes. Miglitol, levels. However its large-scale extraction and purication from
voglibose and acarbose are three inhibitors of carbohydrate the corms of Crocosmia crocosmiiora is a challenging multistep
catabolism that are currently in medical use. These compounds process and requires a substantial amount of biomass. Conse-
act as inhibitors of the mammalian gut a-glucosidases, thereby quently the design and synthesis of novel inhibitors based upon
4
mediating post-prandial blood glucose levels. While effective in MbA's structure has been proposed as a means of generating
preventing hyperglycemia, the resulting displacement of di- and chemically accessible structures of similar potency and
11
tri-saccharides into the lower gut produces unwanted side specicity.
effects arising from their strong osmotic effects within the large
Montbretin A contains a myricetin avonol core glycosylated
0
intestine and their rapid processing by anaerobic bacteria, at the 3- and 4 -positions (Fig. 1a). The 3-OH is adorned with an
5
resulting in diarrhea, nausea, and abdominal discomfort.
a-linked D-glucopyranosyl-(b-1 / 2)-D-glucopyranosyl-(b-1 /
Human pancreatic a-amylase (HPA) catalyzes the endo- 2)-L-rhamnopyranoside trisaccharide to which a 6-O-caffeic
6
0
ester is attached on the central D-glucosyl group. The 4 -OH of
hydrolysis of ingested starches into maltose and maltotriose
and its activity has been positively correlated with post-prandial myricetin bears a b-linked L-rhamnopyranosyl-(a-1 / 4)-D-
blood glucose levels. Consequently the targeted inhibition of xyloside. X-ray crystallographic analysis of MbA in complex with
this enzyme has been highlighted as a means of managing post- HPA showed notable points of contact occurring between the
prandial blood glucose levels while avoiding side effects asso- enzyme's catalytic residues and MbA's caffeic ester and
7
ciated with general a-glucosidase inhibition. Most designs of
glycosidase inhibitors have been based upon azasugar scaffolds
in which a nitrogen atom replaces O5 or C1 of the sugar ring in
8
the ꢀ1 binding site. In this manuscript we explore a completely
a
Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver
BC, Canada V6T 1Z1. E-mail: withers@chem.ubc.ca
b
Department of Biochemistry and Molecular Biology, University of British Columbia,
2
†
1
350 Health Sciences Mall, Vancouver BC, Canada V6T 1Z3
Electronic supplementary information (ESI) available. See DOI:
0.1039/c9sc02610j
Fig. 1 Structures of montbretin A and mini-MbA.
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11
myricetin A-ring. Meanwhile, minimal interactions were a protecting group strategy. Quercetin-3-O-rutinoside was thus
observed with the myricetin B-ring or any of MbA's carbohy- reacted with benzyl bromide in the presence of K CO , followed
2
3
drate appendages. Step-wise chemoenzymatic degradation of by cleavage of the 3-O-glycoside by treatment with HCl/ethanol to
MbA had yielded a ‘minimum inhibitory structure’ termed yield benzyl quercetin 2, freeing the 3-OH for subsequent modi-
14
mini-MbA (Fig. 1b). This inhibitor contained only the myricetin cation. Analogues with simple linkers were rst synthesized to
avonol and caffeic acid linked through a D-glucopyranosyl-(b-1 test the basic concept of linking the two phenolics (Fig. 2).
2)-L-rhamnopyranose disaccharide, and had a K
¼ 400 nM Optimal linker length and polarity was then explored through
ref. 12) towards HPA (lone myricetin and ethyl caffeate have K a series containing three, ve, seven and eight atoms. Analogues
100 mM and 1.3 mM, respectively ). The retained potency of M01 to M03 contained simple alkyl linkers of three, ve and

/
I
(
¼
I
9
the simplied mini-MbA structure suggested that a suitable seven atoms long while M04 contained a similarly exible eight-
synthetic analogue could achieve equivalent levels of potency, atom triethylene glycol linker. Focus then changed to design of
thus mini-MbA was used as inspiration for our synthesis. It was linkers that incorporate appropriate conformational constraints
not clear however, whether such a compound would retain the while seeking out favourable interactions of the side chains with
required specicity for HPA over the gut alpha-glucosidases active site residues. To that end analogues M05 to M11 were
given the active site similarities.
generated containing an amino acid linked to quercetin's 3-OH
Quercetin was selected to replace myricetin as the avonol through a propyl chain (Scheme 1).
core in our analogue synthesis since it, and its glycosylated
Tri-O-benzyl quercetin 2 was functionalized with 1-bromo-3-
derivatives, are substantially less expensive than other avo- chloropropane,
1-bromo-5-chloropentane,
1-bromo-7-
nols. Quercetin differs from myricetin only in hydroxylation of azidoheptane, or 1-bromo-8-azido-triethylene glycol depending
the B-ring and, as noted earlier, there are very few interactions on the analogue synthesized. Treatment with tetrabutylammo-
13
between HPA and this ring. In mini-MbA, the caffeic acid and nium azide produced a terminal azide, which was reduced with
myricetin groups are connected by a disaccharide, which forms trimethylphosphine. In the cases of analogues M01 to M04, this
a bridge of 7 atoms. This region formed no direct interactions
with the enzyme, though it likely provides some conformational
constraints. As a result, a variety of different chemical structures
could be explored for the linker region. Requirements for linker
length and rigidity could be assessed, as well as the possibility
of incorporating new functionalities to create favorable inter-
actions with the enzyme.
Quercetin-3-O-rutinoside was used as a convenient and inex-
pensive starting material for the synthesis since the pre-existing
sugar at C3 could be used to differentiate the hydroxyls in
Scheme 1 Synthesis of analogues M01 to M04: (a) PFP-TFA (1.2
equiv.), pyr (1.2 equiv.), DMF, 93%; (b) BnBr (5 equiv.), K
DMF, 70 C, 18 h; (c) EtOH/HCl (20 : 1), 70 C, 2 h 70% over two steps;
2
CO
3
(5 equiv.),
ꢁ
ꢁ
(
d) Br(CH
DMF/THF, 61–90% (e) N(C
PMe
equiv.), DCM/THF, 70%; (h) BBr
2
)
n
Cl or Br(C
2
H
4
O)
2
C
2
H
4
N
3
(1.5 equiv.), K
2
CO
3
(1.5 equiv.),
ꢁ
4
H
9
)
4
N
3
(2 equiv.), DMF, 50 C, 90–95%; (f)
3
(3 equiv.), THF/NaOH (0.05 M), 50–80%; (g) 1 (1 equiv.), pyr (1.5
ꢁ
Fig. 2 Structures of synthetic MbA analogues.
3
(7 equiv.), DCM, ꢀ78 C, 7–16%.
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could be coupled directly to caffeic acid through use of activated Table 1 Kinetic analysis of MbA analogues
pentauorophenyl caffeic ester 1. For analogues M05 to M11,
I
K against HPA
(mM)
the propylamine derivative 4 propyl was coupled to one of six
MbA analogue
Linker
chosen Fmoc-L-amino acids (with appropriate side chain pro-
15
tecting groups) (Scheme 2). The Fmoc group could be depro- Quercetin/myricetin
—
ꢂ100
tected with 20% piperidine in dichloromethane followed by M01
Heptylene
Pentylene
Propylene
TEG
4.2 ꢃ 0.8
4.0 ꢃ 0.4
110 ꢃ 10
90 ꢃ 20
M02
a second coupling with the pentauorophenyl caffeic ester 1.
M03
Deprotection of the benzyl ethers and amino acid protecting
M04
3
groups was achieved through treatment with BBr . While this
M05
Gly
60 ꢃ 10
synthesis allowed the construction of a small library of M06
analogues, the nal deprotection step in particular was M07
Pro
Thr
Glu
Phe
Tyr
DOPA
1.0 ꢃ 0.1
50 ꢃ 10
M08
34 ꢃ 5
prohibitively low yielding, posing a signicant barrier to future
M09
applications. The continued study of any promising analogues
M10
2.8 ꢃ 0.4
0.07 ꢃ 0.01
0.06 ꢃ 0.02
required an improved synthesis, as will be explored below.
M11
The eleven analogues were tested as inhibitors of HPA as
shown in Table 1. Analogues M01 and M02 both had a K
mM, demonstrating that joining the avonol and caffeic acid its aromatic side chain likely creates favorable p stacking
I
¼ 4
ꢀ
1
groups with a simple linker could increase potency by 25 times interactions in the active site, contributing 1.8 kcal mol to
9
,13
compared to the lone avonol (K ꢂ 100 mM). However, these binding affinity. Installation of hydroxyl groups onto the phenyl
I
analogues were substantially less potent than mini-MbA, ring, as seen with the tyrosine-based analogue M10 and the
despite having the potential to form all the same interactions DOPA-based analogue M11, produced a further 40 to 47-fold
within the amylase active site. M03 was no more potent than the increase in potency relative to M09, equating to a further
ꢀ
1
lone avonol, suggesting that its three-atom linker was too ꢂ2.5 kcal mol
contribution to the binding affinity. With
short to allow for proper orientation of the phenolic moieties in inhibition constants of 70 and 60 nM, M10 and M11 bind
the active site. M04, which contained an eight-atom triethylene approximately six times more tightly than the mini-MbA that
glycol-based linker (TEG), also failed to offer a signicant inspired their synthesis. Thus not only have we replicated the
increase in potency over the lone avonol. Despite having affinity of the natural product with a simple, synthetically
a linker of similar length and exibility to those of M01 and accessible analogue, but also we have substantially improved
M02, the incorporation of a triethylene glycol-based chain in upon its binding affinity.
place of a simple alkyl chain led to a 21-fold drop in potency,
To gain insights into the binding modes of the best of these
potentially due to the differences in polarity.
analogues, and particularly to understand the greatly improved
While amino acid based analogues M05, M07, and M08 affinity of M10 and M11, X-ray crystallographic analysis of
offered only meagre increases in potency compared to the lone analogues M06 and M10 in complex with HPA was pursued. The

avonol, M06 fared better with a K ¼ 1 mM, perhaps due to the resulting crystal structures (PDBs 6OCN and 6OBX at 1.15 and
I
limited conformational range of its proline pre-organising the 1.30 A˚ resolution) revealed the analogues in non-covalent
ligand for binding.
complex within the enzyme active site. Importantly, an overlay
Introduction of aromatic residues into the linkers also of the M06/HPA and M10/HPA structures with that of mini-
afforded an increase in binding affinity. The phenylalanine- MbA/HPA showed essentially complete overlap of the avonol
based analogue M09 bound 22-fold tighter than the un- and caffeic acyl moieties in each case, simplifying future design
functionalized glycine-based analogue M05, suggesting that (Fig. 3a).
Proline-containing analogue M06 created all of the polar
contacts within the HPA active site that were previously observed
with mini-MbA (Fig. 3b). Hydrogen bonding was observed
between the caffeic acid catechol and R195, D197 and E233 of the
enzyme. The 7-OH of M06's avonol formed hydrogen bonds with
D197 and H201. A hydrogen bond was also observed between the
C4 carbonyl of the avonol and the side chain of T163. Meanwhile,
0
the 3 -OH of the avonol formed a hydrogen bond with the side
chain of H201, as was also observed in the mini-MbA/HPA
complex. This indicates that the more cost-effective quercetin
was sufficient to form the necessary interactions within the active
0
site. The absence of this 4 -O-disaccharide on mini-MbA opens
this position up for hydrogen bonding with K200 in the active site,
an interaction that is also seen with M06. In addition to these
polar contacts, a p–CH stacking interaction appears to form
equiv.), pyr (1 equiv.), DCM, 75–90%; (b) 20% piperidine/DCM, 1 h; (c) 1
Scheme 2 Synthesis of analogues M05 to M11: (a) Fmoc-L-Xaa-pfp (1
(
ꢀ
1 equiv.), pyr (1.5 equiv.), THF/DCM, 50–75%; (d) BBr
78 C, 5–25%.
3
(7 equiv.), DCM, between the proline side chain of M06 and W59 of the enzyme.
ꢁ
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Table 2 Specificity analysis of M10
Enzyme
M10 IC50 (mM)
Roseburia inulivorans amylase A
Butyrivibrio brisolvens amylase B
Homo sapiens maltase-glucoamylase
H. sapiens C-term sucrase-isomaltase
Saccharomyces cerevisiae a-glucosidase
Agrobacterium b-glucosidase
Green coffee bean a-galactosidase
E. coli b-galactosidase
Jack bean a-mannosidase
NI
NI
NI
NI
10.5 ꢃ 0.7
35.4 ꢃ 5.7
NI
NI
NI
intestinal maltase-glucoamylase and sucrase-isomaltase, and
two intestinal bacterial a-amylases (Table 2). The analogue
showed no inhibitory activity towards these a-glucosidases at
I
a concentration over 100-fold higher than its K value for HPA.
M10 did show some inhibitory activity against the non-
relevant S. cerevisiae a-glucosidase and Agrobacterium b-
glucosidase. However, the binding affinity of M10 towards
yeast a-glucosidase is actually lower than that of lone quer-
1
6
cetin (IC50 ¼ 7 mM). Comparison of these results with those of
previous specicity analyses of mini-MbA and MbA indicated
that M10 was in fact more selective for HPA over the intestinal
glucosidases, since mini-MbA and MbA inhibited sucrase-
9
,11
isomaltase and B. brisolvens a-amylase, respectively.
This
bodes well for the use of M10 as a selective amylase inhibitor.
It also implies that this pharmacophore may have restricted
application to other glycosidases.
Inhibitor M10 demonstrates highly promising HPA inhi-
Fig. 3 X-ray crystal structures of M06/HPA and M10/HPA complexes. bition, however the synthetic route that led to its discovery has
(
a) Overlays of M06 (purple) and M10 (pink) with mini-MbA (yellow)
a number of shortcomings that would hamper its development
as a potential pharmaceutical. As such, an alternative
approach was devised (Scheme 3). Selective azide reduction of
show very similar placement of flavonol and catechol groups. (b)
Interactions between M06 (purple) and residues at the HPA active site
(
grey). Hydrogen bonds are shown as dashed lines. Polar interactions
occurred between the flavonol and catechol of M06 and the enzyme. 3 propyl could be carried out in the presence of 6 to give amide
c) Two different views of the interactions between M10 (pink) and 7, which could then be globally deprotected to give amine 8.
(
residues of the HPA active site (grey). In addition to the polar contacts
formed with the analogue flavonol and catechol, polar contacts
occurred between the linker tyrosine and T163 of the enzyme. p-
based interactions also occurred between the enzyme and aromatic
tyrosine side chain.
Treatment of crude amine 8 with 1 then delivered M10 in 49%
yield over four steps, with no purication necessary between
steps. This improved procedure delivers M10 in eight steps
from rutin (24% overall yield), with further optimization
certainly possible.
Despite its high degree of alignment and reproducibility of polar
contacts, M06 is still 2.5 times less potent than mini-MbA, high-
lighting the importance of MbA's glycosidic appendages in stabi-
lization of a pre-stacked conformation.
Analogue M10 also formed all of the same hydrogen bonds
within the amylase active site previously observed for mini-MbA
and M06 (Fig. 3c). In addition, and presumably largely
responsible for its affinity enhancement, the tyrosine of M10's
linker formed hydrogen bonds with the main chain carbonyl
and side chain hydroxyl of T163. These are accompanied by
a number of CH–p–interactions between the tyrosine side chain
and residues W59, Q63, and L165 of the enzyme.
To probe its specicity towards HPA, M10 was tested as an
Scheme 3 Improved synthesis of M10. (a) Lindlar catalyst (60% w/w),
inhibitor of ten other glycosidases, including human H , DMF; (b) pentafluorophenol (4 equiv.), 5% Pd/Al O (30% w/w), H ,
2
2
3
2
3
DMF; (c) 1 (2 equiv.), Et N (2.5 equiv.); 49%.
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P41GM103393). The contents of this publication are solely the
responsibility of the authors and do not necessarily represent
Conclusions
The synthesis and kinetic analysis of MbA analogues M01 to the official views of NIGMS or NIH.
M11 provides new insight into the structural requirements for
HPA inhibition. The fact that most of the analogues bound
more weakly than mini-MbA highlights the importance of References
MbA's constrained carbohydrate linker. Despite forming no
1
2
World Health Organization, Global Report on Diabetes, 2006,
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avonol and caffeic acid moieties for optimal interaction. Initial

exploration of the effect of incorporation of rigidity into the
analogue linkers was achieved with the proline linker of M06.
This increased potency some 60-fold compared to the non-
functionalized glycine linker, with more possibly achievable
by freezing out other motions. Affinity enhancement through
the acquisition of stabilising interactions was explored through
the incorporation of phenolic side chains to recruit some of the
hydrophobic and hydrophilic interactions ordinarily developed
2
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indeed more potent than the mini-MbA that inspired their
development. With further optimization of their synthesis
underway, pharmacokinetic and efficacy studies with these
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Conflicts of interest
The University of British Columbia has applied for patent
protection on the inhibitors described.
11 L. K. Williams, X. Zhang, S. Caner, C. Tysoe, N. T. Nguyen,
J. Wicki, D. E. Williams, J. Coleman, J. H. McNeill,
V. Yuen, R. J. Andersen, S. G. Withers and G. D. Brayer,
Nat. Chem. Biol., 2015, 11(9), 691.
Acknowledgements
We thank Emily Kwan for purication of HPA and Ethan 12 Revised K for mini-MbA (Fig. S49†).
I
Goddard-Borger for useful discussions at the outset. We 13 E. Lo Piparo, H. Scheib, N. Frei, G. Williamson, M. Grigorov
acknowledge funding from the Canadian Glycomics Network/
and C. J. Chou, J. Med. Chem., 2008, 51(12), 3555.
Networks of Centres of Excellence (Project DO-2) DOI: 14 H. Huang, Q. Jia, J. Ma, G. Qin, Y. Chen, Y. Xi, L. Lin, W. Zhu,
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out at the Stanford Synchrotron Radiation Lightsource, SLAC
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Department of Energy, Office of Science, Office of Basic Energy 16 K. Tadera, Y. Minami, K. Takamatsu and T. Matsuoka, J.
Sciences under Contract No. DE-AC02-76SF00515. The SSRL
Nutr. Sci. Vitaminol., 2006, 52(2), 149.
Structural Molecular Biology Program is supported by the DOE 17 G. D. Brayer, G. Sidhu, R. Maurus, E. H. Rydberg, C. Braun,
Office of Biological and Environmental Research, and by the
Y. Wang, N. T. Nguyen, C. M. Overall and S. G. Withers,
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