Synthesis of new pyrimidine-fused derivatives as potent and selective antidiabetic α-glucosidase inhibitors

Article abstract of DOI:10.1016/j.carres.2013.07.008

The synthesis of a set of pyrimidine-fused derivatives (L1-L8), resulting from the incorporation of different fragments on the pyrimidine-fused heterocycle (PFH) of the earlier reported α-glucosidase (α-Gls) inhibitor (C1-C5), allowed the discovery of new ligands with modest and selective inhibitory activity. The PFH core (substructure 2) was proved to play a significant role in their inhibitory properties. Additionally, the substituent on substructures 1 and 3 of the heterocyclic ring was demonstrated to be important in the enzyme inhibitory action of the pyrimidine-fused derivatives. Moreover, these ligands show selective inhibitory properties for α-Gls over porcine pancreatic α-amylase (α-Amy) which is important in terms of their reduced susceptibility for the possible development of intestinal disturbance side effects. Therefore, low to moderate α-Amy inhibition with effective α-Gls inhibitory action may offer a better therapeutic strategy. Overall, these compounds can potentially offer a new opportunity to develop novel antidiabetic drugs with selective inhibitory action against α-Gls.

Full text of DOI:10.1016/j.carres.2013.07.008

Carbohydrate Research 380 (2013) 81–91
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of new pyrimidine-fused derivatives as potent
and selective antidiabetic
a-glucosidase inhibitors
a
b,
Farhad Panahi a,b, Reza Yousefi a, , Mohammad Hossein Mehraban , Ali Khalafi-Nezhad
⇑
⇑
a Protein Chemistry Laboratory (PCL), Department of Biology, College of Sciences, Shiraz University, Shiraz, Iran
b Department of Chemistry, College of Sciences, Shiraz University, Shiraz 71454, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 June 2013
Received in revised form 20 July 2013
Accepted 21 July 2013
Available online 6 August 2013
The synthesis of a set of pyrimidine-fused derivatives (L1–L8), resulting from the incorporation of differ-
ent fragments on the pyrimidine-fused heterocycle (PFH) of the earlier reported -glucosidase ( -Gls)
inhibitor (C1–C5), allowed the discovery of new ligands with modest and selective inhibitory activity.
The PFH core (substructure 2) was proved to play a significant role in their inhibitory properties. Addi-
tionally, the substituent on substructures 1 and 3 of the heterocyclic ring was demonstrated to be impor-
tant in the enzyme inhibitory action of the pyrimidine-fused derivatives. Moreover, these ligands show
a
a
Keywords:
selective inhibitory properties for
in terms of their reduced susceptibility for the possible development of intestinal disturbance side effects.
Therefore, low to moderate -Amy inhibition with effective -Gls inhibitory action may offer a better
therapeutic strategy. Overall, these compounds can potentially offer a new opportunity to develop novel
antidiabetic drugs with selective inhibitory action against -Gls.
Ó 2013 Elsevier Ltd. All rights reserved.
a-Gls over porcine pancreatic a-amylase (a-Amy) which is important
a-Glucosidase
Inhibitors
Antidiabetic
Pyrimidine-fused heterocycles
a
a
a
1. Introduction
importance, as the non-specific inhibition of other Gls especially
pancreatic -Amy may lead to accumulation of non-digested car-
a
a
-Glucosidase (
a
-Gls) has a crucial role in digestion of carbohy-
bohydrates, which in turn results in abdominal cramping, diarrhea,
drates and biosynthesis of glycoproteins.1 Therefore, the inhibition
of this enzyme plays an important role in treatment of degenera-
tive diseases such as type-II diabetes mellitus and human immuno-
deficiency virus (HIV) infection.2 The inhibitors can reduce the
complications of diabetes as they interfere with the enzymatic ac-
and flatulence.3
The pyrimidine ring is an important chemical moiety which can
be seen in the molecular scaffold of a large number of alkaloids,
drugs, antibiotics, agrochemicals, and antimicrobial agents.11
Moreover, simple and biologically active pyrimidine fused hetero-
cycles (PFH) such as purine and pteridine are existed in the chem-
ical structure of many natural compounds.12,13 Also, some of the
PFH derivatives are used as antileukemic drugs14 or potassium-
conserving diuretics.15 A number of fused thieno[3,2-d]pyrimi-
dines serves as anti-allergy drugs and others act as fungicides.11
Additional to inflammatory properties and antioxidant effects,
PFH compounds demonstrate further biological activities such as
anticancer, antimicrobial, and antiviral actions.16,17 However, their
tion of intestinal
a
-Gls.3 Consequently, the liberation of glucose
into bloodstream will be retarded, resulting in delaying glucose
absorption and decreasing postprandial hyperglycemia.4 In addi-
tion,
a-Gls inhibition is a promising strategy for development of
novel anti-HIV agents, because glycosylation of viral envelope pro-
teins is essential for the virus infectivity.5 Therefore, during the re-
cent years
a-Gls inhibitors have been the imperative entities of
various studies in the field of medicinal chemistry.6–8 Moreover,
some
a
-Gls inhibitors such as Acarbose, Voglibose, and Miglitol
inhibitory activity against a-Gls was not investigated yet with the
exception of our recent study which proved for the first time that
have been used as important drugs by diabetic patients.9 The
pseudotetrasaccharide inhibitor Acarbose is a potent pig intestinal
sucrase inhibitor with an IC50 value of 0.5 mM.10 However use of
Acarbose leads to intestinal disturbances in many patients and
there exists a need for development of better and more tolerable
PFH based compounds demonstrate inhibitory action against
a
a
-Gls.17 The chemical structure of the synthesized PFH-based
-Gls inhibitors is shown in Figure 1.
Our studies revealed that some of the synthesized compounds
a-Gls inhibitors. The selectivity of
a-Gls inhibitors is of great
not only revealed inhibitory activity toward type-I (yeast) a-Gls,
but also they demonstrate potent inhibitory action against
type-II (mammalian) enzyme counterpart.17 Also, PFH-based
compounds with aromatic substitution (C3) revealed a strong
⇑
Corresponding authors. Fax: +98 711 2280916 (R.Y.); fax: +98 711 2280926
(A.K.-N).
E-mail addresses: ryousefi@shirazu.ac.ir (R. Yousefi), khalafi@chem.susc.ac.ir
inhibitory activity against both
a-Gls. This compound demon-
strated an improved inhibitory action on the yeast enzyme
(A. Khalafi-Nezhad).
0008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2013.07.008

82
F. Panahi et al. / Carbohydrate Research 380 (2013) 81–91
O2
S
HO
HO
OH
(1)
OH
OH
NH2
C3
NH
C1
C2
HO
N
R =
O
O
(2)
NH
Br
N
HN
10
C5
5
O
N
H
N
N
H
O
C4
R
(3)
Figure 1. The chemical structure of synthetic PTH-based inhibitors of a-Gls.
compared to the widely applied inhibitor drug of acarbose (28-fold
In this protocol barbituric acid was treated with amines and
aldehydes. By changing the amine and aldehyde components a
set of PFHs were synthesized. In the selection of aldehyde compo-
nents, it was decided to use only two aldehydes isovanillin and 3-
indole carboxaldehyde, because they have been used as a chemical
source in synthesis of many biological active compounds.22,23 For
the synthesis of ligands L1–L3, 4-bromo-aniline was used as an
amine component, because this chemical moiety previously
stronger).17 In addition, the aliphatic substitutions (C4 & C5) exhib-
ited no significant inhibition against yeast and mammalian a
-Gls.17
As shown in Figure 1, this scaffold has three available substructures
for further optimization of the inhibitory properties. In design of li-
gands C1–C5, our speculation was that the poly hydroxyl chain has
an important role in the inhibitory action. In this study we revealed
that the PFH (substructure 2) has a significant role in the inhibitory
activity, although the effect of substitution in substructures 1 and 3
was also determinable. Therefore, in the course of searching for the
novel, more specific, and easy accessible a-Gls inhibitors, we exam-
ined PFH derivatives of different aromatic moieties in the substruc-
tures 1 and 3 (Fig. 1). Herein, we report the chemical synthesis and
showed
a-Gls inhibitory activity in the structure of ligand C2
(Fig. 1). In the synthesis of ligands L4–L7 a series of bi-functional-
ized aromatic amines was applied to produce a set of PFH with free
NH2 group in their structures. L4–L7 was synthesized according to
our previous ligands as they have been demonstrated significant
a-Gls inhibitory properties of PFH compounds and discuss the
inhibitory activity against a-Gls. Moreover, it is noteworthy to
enzymatic mechanism of their inhibition. In order to get a better
understanding of their selective inhibition, the effect of these com-
pounds on porcine pancreatic a-amylase (a-Amy) was also exam-
ined. Furthermore, circular dichroism (CD) and fluorescence
spectroscopy were conducted to obtain a deeper insight into the
interaction between the ligands and yeast a-Gls.
mention that the free amino groups in their structures can possibly
improve their ability for hydrogen bonding interaction. In this class
of compounds, one equivalent of amine component was used.
However, in one case in order to synthesize a new derivative based
on ligand L5, 0.5 equiv of amine component was used in the reac-
tion (Scheme 2). This green and efficient reaction was performed in
refluxing EtOH in the presence of catalytic amount of tungstophos-
phoric acid (TPA) as catalyst.24
2. Results and discussion
The synthesized compounds L1–L8 were also characterized,
using different spectroscopic techniques such as 1H NMR, 13C
NMR, infrared (IR), and mass spectroscopy. The data clearly re-
vealed that these molecules were produced successfully. As the
purity of synthetic compounds examined using elemental analysis,
they were highly pure (>97%).
2.1. Synthesis of pyrimidine-fused heterocycle derivatives
From the historical point of view, there are three general syn-
thetic methods for the synthesis of pyrimidine-fused derivatives
in the literature.18,19 In the Bischler’s strategy the synthesis of
PFH is achieved from ortho-acetamidobenzaldehyde deriva-
tives.18 The second method is the Riedel’s synthesis, which PFH
compounds can be obtained via reductive cyclization of bisfor-
mamido derivatives of ortho-nitrobenzaldehydes.19 The so-called
Niementowski’s synthesis involves preparation of fused pyrimi-
dines from the anthranilic acid derivatives.18 In modern organic
synthesis these compounds have been synthesized using multi-
component reactions (MCRs).20 MCRs are attractive synthetic
strategies, as complex products are formed in a single step and
the diversity can be achieved simply by varying the reaction
components.20 Thus, functionally-substituted starting materials
can be used to synthesize the desired biologically active com-
pounds.21 The syntheses of new PFH analogues remain challeng-
ing, especially when optimizing their activities. Hence, MCRs
represent useful procedures for this goal, because by simply
changing the starting material, a large number of pyrimidine-
fused analogues with convenient optimization and range of
biological activities can be synthesized. The following multicom-
ponent reaction was used to synthesis PFH ligands L1–L7
(Scheme 1). It is noteworthy that the products are precipitated
after production and so they are isolated from reaction mixture
by simple filtration with high purity.
2.2. The inhibitory activity of pyrimidine fused compounds
against a-Gls
The inhibitory activity of eight synthetic ligands (pyrimidine-
fused derivatives) against yeast and mouse -Gls was assessed
a
based on the method described in the experimental section. Also
mode of inhibition was determined by Lineweaver–Burk and Cor-
tes plots. To discover the pharmaceutical characteristics of the
promising inhibitors, their corresponding IC50 values were calcu-
lated by Dixon plots. Also Acarbose which is a widely used antidi-
abetic drug with inhibitory action against mammalian
used to serve as the positive control.
a-Gls was
In our previous study, we proved that the introduction of poly
hydroxyl chain in pyrimidine fused heterocycle compound results
in generation of a synthetic compound with significant inhibitory
action against both yeast and mouse
a-Gls. The replacement of
poly hydroxyl chain group with 3-hydroxy-4-methoxy-phenyl
and 1H-indol-3-yl substitutions (in ligand C2) resulted in the pro-
duction of ligands L1 and L2, respectively (Scheme 1). However,
these ligands did not exhibit any inhibitory action. Also, the
replacement of oxo group in L2 with thioxo to produce ligand L3

F. Panahi et al. / Carbohydrate Research 380 (2013) 81–91
83
Ar2
O
O
O
TPA (2 mol%)
NH2
Ar1
HN
CHO
Ar2
HN
NH
+
+
2
EtOH
reflux
X
N
H
O
X
N
H
N
N
H
X
Ar1
X = O, S
L1-L7
OMe
OH
O
NH
NH
O
O
O
O
O
HN
NH
HN
NH
HN
NH
S
N
H
N
N
H
S
O
N
H
N
N
H
O
O
N
H
N
N
H
O
Br
Br
Br
L3
L1
L2
OMe
OMe
OMe
OMe
OH
O
OH
O
OH
O
OH
O
O
O
O
O
HN
NH
HN
NH
HN
NH
HN
NH
O
N
H
N
N
H
O
O
N
H
N
O
N
H
O
O
N
H
N
N
H
O
O
N
H
N
N
H
O
O2S
H2C
C
O
NH2
NH2
NH2
NH2
L4
L7
L5
L6
Scheme 1. The synthetic route for the synthesis of ligands L1–L7.
OMe
O
O
OH
TPA (2 mol%)
HN
+
2
4
+
NH2
EtOH
reflux
H2N
O
N
H
O
O
O
O
O
HN
NH
HN
NH
O
O
N
N
NH
HN
MeO
O
N
H
O
O
N
O
OMe
L8
H
OH
OH
Scheme 2. One-pot synthesis of ligand L8.
did not show any effect, suggesting that the latter has no signifi-
cant effect on the inhibitory action of the synthetic compounds.
According to the results of synthetic compounds applied in our
previous study (Fig. 1), C3 with the substituted moiety as ‘4-(4-
aminophenylsulfonyl) phenyl’ (4-APSP) revealed strong inhibitory
activity with non-competitive and competitive inhibition mode
Scheme 1, L4 was synthesized based on C3 with replacement of
the poly hydroxyl chain with 3-hydroxy-4-methoxy-phenyl (the
solvability of L1 was better than L2). To our surprise, L4 did not
show any inhibitory activity against these enzymes. So here, the
obtained results demonstrate that the activities of compounds
are highly depended on poly hydroxyl chain substitution.
Therefore, L5 was synthesized based on chemical structure of L4
against yeast and mouse
a-Gls, respectively. Thus, as shown in

84
F. Panahi et al. / Carbohydrate Research 380 (2013) 81–91
as following: SO2 group in L4 was replaced with the ether linkage
in L5 (Scheme 1) and as a result of this modification, L5 demon-
against both yeast and mouse enzymes. According to their corre-
sponding IC50 values as reported in Table 1, L8 demonstrates the
inhibitory action 1.9- and 4-fold stronger than L5 on inhibition of
strated inhibitory activity against both yeast and mouse
a-Gls,
with the IC50 values of 148 1 and 159 M, respectively (Ta-
3
l
mouse and yeast a-Gls, respectively. This result also demonstrates
ble 1). In spite of the poor inhibition results obtained herein, they
clearly indicated that in the absence of poly hydroxyl chain moiety,
only by changing of the substitution in substructures 1 and 3
(Fig. 1), one can optimize the inhibitory properties of PFH com-
pounds. It is clear that in the structure of L5, there are both hydro-
gen bonds donating and accepting moieties; acting as a hydrogen
the effect of PFH structure on the inhibitory properties of the syn-
thetic ligands.
The double reciprocal lineweaver–burk plots of inhibition of the
synthetic compounds on yeast and mouse a-Gls are shown in Fig-
ures 4 and 5, respectively. As shown in Figure 4, L5 and L6–L8 in-
hibit, respectively, the yeast enzyme with mixed-uncompetitive
and mixed-competitive modes.
donor/acceptor which is one of the most important features of
a-
Gls inhibitors. The ligands L4 and L5 are different only in the
hinge-like connection (in substructure 3) of the main structure.
Considering this issue, we decided to synthesize L6 and L7 by
changing the connecting group, using corresponding amine com-
ponent in the designed reaction (Scheme 1). Interestingly, L6 with
a 4-(4-aminobenzyl) benzenamine moiety demonstrates the high-
Figure 5 shows the inhibition mechanism of L5 and L8 against
mouse
a-Gls in which both of the ligands demonstrate a mixed-
competitive type of inhibition. The inhibitory parameters of the
synthetic compounds are illustrated in Table 1.
2.3. Pancreatic a-Amy inhibition
est inhibitory activity against yeast
M (Table 1). However, due to the structural difference of
the target enzymes, this ligand did not show any inhibitory activity
against mouse -Gls.
More importantly, L7 similar to L4 demonstrated a weak inhib-
itory activity with an IC50 value of 376 M against yeast -Gls.
Also, comparable to L6, this ligand (L7) did not inhibit mouse -Gls
(Table 1). Comparing the inhibition results of L4–L7, it can be sug-
gested that rigidity of the group connecting two phenyl moieties in
these synthetic ligands is an important factor for their inhibitory
action (Scheme 1 and Table 1). The molecular flexibility index of
an inhibitor is important for the optimal protein–ligand interaction
and enzyme inhibition.25 Therefore, the critical degree of molecu-
lar flexibility which would contribute to the effective enzyme inhi-
bition can be obtained precisely from the modified ligands with
proper connecting group.26 It seems that in ligands L5 and L6 the
level of rigidity is less than ligands L4 and L7, because the created
rigidity by –SO2–27 and –CO–28 groups is higher than –CH2–29 and
–O–30 linkers, so these ligands can easily interact with the corre-
sponding binding residues of protein in the proper connection
(Fig. 2).
a-Gls with an IC50 value of
9
1
l
The effects of synthetic compounds against porcine pancreatic
-Amy activity were examined with a concentration higher than
a
a
their IC50 values for yeast and mouse
a-Gls. As depicted in Figure 6,
a maximum reduction of 20% in activity of
a-Amy was observed in
1
l
a
the presence of L6. Other synthetic ligands demonstrate even low-
er inhibitory action against this enzyme. Hence, in comparison
with Acarbose (70% inhibition), these ligands weakly inhibit the
a
activity of pancreatic
a-Amy.
The specific inhibitors of human pancreatic
a
-Amy have poten-
tial as oral agents for the control of blood glucose levels in the
treatment of diabetes and obesity.31 Nonetheless, it is important
to mention here that
that are acted upon by isomaltases, especially
cose. The presence of potent -Gls inhibitory activity therefore ap-
pears more important in controlling the release of glucose from
disaccharides in the gut than -Amy inhibition. However, moder-
ate -Amy inhibition with potent -Gls inhibitory activity may of-
a-Amy breaks down starch into disaccharides
a
-Gls to release glu-
a
a
a
a
fer better therapeutic strategy that could slowdown the availability
of dietary carbohydrate substrate for glucose production in gut,
reducing the level of possible intestinal disturbance side effects.
Therefore, from these views, the synthetic compounds seem to of-
fer plausible structure–activity relationship.
The optimized molecular structures for the most stable confor-
mation of the synthesized compounds are shown in Figure 3.
As shown, the flexibility of ligands L5 and L6 allows them to ob-
tain different morphologies to the other structures which may af-
fect the interaction of these ligands to their corresponding
binding sites on the protein.
2.4. Fluorescence investigation of the interaction between yeast
a-Gls and the synthetic inhibitors
The selective inhibitory properties of L6 and L7 against yeast
a
-
Fluorescence studies were conducted to obtain a better under-
Gls can be raised from the particular structural constraint of their
target enzyme.
standing of the interaction between these compounds and yeast
-Gls. The Ksv, Ka, n, and thermodynamic values G, H, and
a
D
D
DS
Overall, the results show that for the inhibitory activity of syn-
thetic ligands the PFH ring is essential, but by selecting the appro-
priate fragments in substructures 1 and 3, we can synthesize
are summarized in Table 2. Quenching mechanisms are classified
in two groups as the following: in dynamic quenching the Stern–
Volmer constant is expected to increase with the elevation of tem-
perature; in contrast, as temperature increases the Stern–Volmer
constant decreases in the static quenching.32 The latter was proved
to be the quenching mechanism during the interaction of L6, L7,
potent and selective
a-Gls inhibitors. For instance, accumulation
of another PFH moiety to L5 as shown in Scheme 2, increases sig-
nificantly the inhibitory properties of the obtained compound (L8)
Table 1
The IC50, Ki and inhibition mode of the synthetic compounds.
Entry
IC50
(
l
M)
Kic
Yeast
222 1.5
(
l
M)
Kim
Yeast
37 1.5
(
l
M)
Mode of inhibition
Yeast
Yeast
Mouse
159
–
–
Mouse
Mouse
370
–
–
Mouse
L5
L6
L7
L8
148
9
376
37
1
3
86 2.5
–
–
48 2.5
–
3
Mixed Un.a
Mixed Co.
Mixed Co.
Mixed Co.
Competitive
Mixed Co.b
–
–
Mixed Co.
–
1
1
1
1
8
333
31
1
2
1
7
500
1
2
84
11
2
1
43 1.5
–
167
3
Acarbose
296
–
–
a
Competitive binding modes
Uncompetitive binding modes
b

F. Panahi et al. / Carbohydrate Research 380 (2013) 81–91
85
OMe
OMe
OMe
OMe
OH
O
OH
OH
OH
O
O
O
O
O
O
O
HN
NH
HN
NH
HN
NH
HN
NH
O
N
H
N
X
N
H
O
O
N
H
N
X
N
H
O
O
N
H
N
X
N
H
O
O
N
H
N
X
N
H
O
H2N
X = CO, SO2
NH2
H2N
NH2
X = CH2, O
Figure 2. A perspective of molecular rigidity and flexibility in synthetic ligands L4–L7.
Figure 3. Optimized molecular structures of ligands L4–L8 by AM1 method (using Chembio 3D Ultra 11.0 software).
and L8 with yeast
a
-Gls (Figs. 7 and 8). However, the interaction of
that the hydrogen bonding may be the driven force of the enzyme–
inhibitor complex formation which is in good agreement with our
speculation on their hydrogen donor/acceptor abilities as men-
tioned above. Therefore hydrogen bonding may stimulate the
L5 with yeast -Gls was through dynamic quenching mode. Also,
a
the inhibitor–enzyme complex formed by L6, L7, and L8 was
accompanied by the negative enthalpy changes (Table 2). Accord-
ing to the rules summarized by Ross and Subramanian,33 the neg-
interaction of these inhibitors and yeast
proved inhibition of the enzyme. Furthermore, the negative values
of S in the interaction of L7 and L8 prove that vander waals forces
a-Gls, resulting in the im-
ative
DH values are observed whenever there is hydrogen bonding
in the interaction between ligand and protein. These results prove
D

86
F. Panahi et al. / Carbohydrate Research 380 (2013) 81–91
Figure 4. The lineweaver–Burk plots derived from the inhibition of yeast
of pNPG concentration (0.1–2 mM) in the absence and presence of inhibitors (0–300
different symbols represent the absence (d) and presence (ꢀ, ., j) of these inhibitors in the reaction mixtures.
a
-Gls by the synthetic compounds L5, L6, L7, and L8. The
a-Gls activity was measured as a function
l
M). The experiments performed in 100 mM NaPi buffer, pH 7.0 at 25 °C for 10 min. The
Figure 5. The lineweave–Burk plots derived from the inhibition of mouse
concentration (0.6–3 mM) in the absence and presence of inhibitors (0–200
symbols represent the absence (d) and presence (ꢀ, ., j) of inhibitor in the reaction mixtures.
a
l
-Gls by synthetic compounds L5 and L8. The
M). The experiments performed in 10 mM NaPi buffer, pH 7.0 at 37 °C for 30 min. The different
a-Gls activity was measured as a function of pNPG
may have a minor role in the ligand–inhibitor interaction (Table 2).
Values of H and S of the interaction of L5 and yeast -Gls were
both positive, suggesting contribution of a typical hydrophobic
(Fig. 9). Yeast a-Gls has a content of 11.8% a-helical structures,
D
D
a
46.9% b-sheets, and 41.3% random coils.34 Upon interaction, no
meaningful structural alteration was induced by the synthetic
compounds in this enzyme. Therefore, it is likely that the structural
interaction during binding of this inhibitor to the yeast enzyme
(Table 2). The negative values of
D
G demonstrated the easy forma-
perturbation of yeast a-Gls is not involved in the inhibition mech-
tion of inhibitor–enzyme complex and spontaneity of the interac-
tion. Based on the calculated binding constants, the order of
binding at 25 °C (the same as enzyme inhibition assays) was
L8 > L5 > L6 > L7.
anism of the synthetic pyrimidine-fused derivatives.
3. Conclusion
In conclusion, a set of pyrimidine-fused heterocycles (eight
compounds) with a diversity of molecular structures were syn-
2.5. Secondary structure assessment of yeast
presence of synthetic inhibitors
a-Gls in the
thesized based on the previously reported
synthesis of these compounds, a convenient and efficient method
via three-component condensation reaction of aldehydes,
amines, and barbituric acid was described. This reaction
a
-Gls inhibitors.17 For
We recorded the far-UV CD spectra of yeast
a
-Gls in the absence
a
and presence of different concentrations (20–80
l
M) of the ligands

F. Panahi et al. / Carbohydrate Research 380 (2013) 81–91
87
proceeded smoothly in good to excellent yields and the pure
products were separated from the reaction mixture by simple fil-
tration. Since the inhibition of
a-Gls has become a perfect target
in treatment of diabetes especially type-II and AIDS/HIV control,
the activities of all compounds were investigated, demonstrating
that both PFH ring and the substituents on substructures 1 and 3
of the heterocyclic ring (Fig. 1) play a significant role in the inhib-
itory activity of these ligands. The results of structure–activity
relationships suggest that the rigidity/flexibility of hinge-like con-
nection in substructure 3 of ligands L4–L7 plays an important
role in their
two PFH moiety shows a significant ability for inhibition of
mouse -Gls, suggesting that PFH core plays a significant role
a-Gls inhibitory action. Also, compound L8 with a
a
in the inhibitory properties of the synthetic ligands. Compared
to Acarbose, these ligands weakly inhibit the activity of pancre-
atic
a
-Amy. Overall, these compounds can be considered as a
-Gls inhibitors which may
Figure 6. The inhibitory activity of the synthetic compounds against porcine
pancreatic a-Amy.
foundation for synthesizing novel
a
Table 2
The thermodynamic parameters, calculated binding and Stem–Volmer constant and the number of bound molecules of the ligands per
a-glucosidase molecule (n).
Entry
T
Ksv
KA
n
D
H
D
G
DS
(L mol-1
)
(L mol-1
)
(KJ mol-1
)
(KJ mol-1
)
(J mol-1K-1
)
L5
298
304
310
298
304
310
298
304
310
298
304
310
0.16 ꢀ 106
0.35 ꢀ 106
0.38 ꢀ 106
0.43 ꢀ 106
0.2 ꢀ 106
4 ꢀ 104
12 ꢀ 104
14 ꢀ 104
1.8 ꢀ 104
7 ꢀ 104
1.28
1.33
1.2
1.8
1.18
0.9
1.72
1.55
1.38
1.63
1.50
1.44
71.83
–26.62
–29.57
–30.57
–24.27
–28.19
–29.1
–30.31
–28.59
–25.35
–27.69
–27.56
–26.47
0.331
L6
L7
L8
–95.67
–153.26
–57.84
5.83
–0.411
–0.1
0.06 ꢀ 106
2.99 ꢀ 106
0.78 ꢀ 106
0.09 ꢀ 106
0.9 ꢀ 106
8 ꢀ 104
0.2 ꢀ 104
0.08 ꢀ 104
0.01 ꢀ 104
7 ꢀ 104
0.35 ꢀ 106
0.18 ꢀ 106
5.4 ꢀ 104
2.8 ꢀ 104
Figure 7. The fluorescence emission spectra of ligand/
a-Gls complexes L5, L6, L7, and L8 at 31 °C. A constant concentration of 0.1 mg/mL yeast a-Gls and different
concentrations of ligands (0–50 M) were used. The reaction performed in 100 mM NaPi buffer pH 7.0 at 25, 31, and 37 °C.
l

88
F. Panahi et al. / Carbohydrate Research 380 (2013) 81–91
Figure 8. Stern–Volmer plot for the binding of ligands L5, L6, L7, and L8 with yeast
a
-Gls. Data for 298 K (d), 304 K (j) and 310 K (N) are shown.
Figure 9. Far-UV CD spectra of yeast
a-Gls in the presence of the inhibitors L5, L6, L7, and L8. Protein concentration was 0.2 mg/mL and inhibitor concentrations of 20, 40, and
80 lM were added in 100 mM NaPi buffer pH 7.0.
efficiently and specifically inhibit
they may cause less side effects compared to the conventional
-Gls inhibitors such as Acarbose. Further studies on the struc-
ture–activity relationship of PFHs are in progress in our laborato-
ries to improve bioactivity of these compounds for achieving
a
-Gls and more importantly
was used for mass analysis of products. FTIR spectroscopy
(Shimadzu FT-IR 8300 spectrophotometer) was employed for
the ligand characterization. Melting points were determined in
open capillary tubes in a Barnstead Electrothermal 9100 BZ cir-
culating oil melting point apparatus. The reaction monitoring
was accomplished by TLC on silica gel PolyGram SILG/UV254
plates.
a
more promising
a-Gls inhibitors.
4. Experimental section
4.1. Chemistry
4.1.2. General procedure for preparation of ligands L1–L7
A
mixture of barbituric acid (0.26 g, 2 mmol), aldehyde
(1 mmol), amine (1 mmol), and H3PW12O40 (0.04 g, 2 mol %) in
ethanol (5 mL) at 80 °C was stirred for 12 h. As completion of
the reaction confirmed by TLC (eluent EtOAc/MeOH), the
reaction mixture was cooled to room temperature. Then, the
precipitated product was filtered and washed with
water (2 ꢀ 10 mL) and ethanol (2 ꢀ 5 mL) to afford the pure
product.
4.1.1. General
Chemicals were purchased from Fluka and Aldrich chemical
companies and used without further purification. 1H and 13C
NMR spectra were recorded on a Bruker Avance 250 MHz spec-
trometer in DMSO solution with TMS as an internal standard.
GC/MS: Shimadzu GC/MS-QP 1000-EX apparatus; in m/z (rel%)

F. Panahi et al. / Carbohydrate Research 380 (2013) 81–91
89
4.1.3. 9-(4-Bromo-phenyl)-10-(3-hydroxy-4-methoxy-phenyl)-
9,10-dihydro-1H,8H-1,3,6,8,9-pentaaza-anthracene-2,4,5,7-
tetraone (L1)
4.1.8. 9-[4-(4-Amino-benzyl)-phenyl]-10-(3-hydroxy-4-
methoxy-phenyl)-9,10-dihydro-1H,8H-1,3,6,8,9-pentaaza-
anthracene-2,4,5,7-tetraone (L6)
Yield: 91%; yellow solid; mp >350 °C. IR (KBr): 3315, 3098,
Yield: 85%; yellow solid; mp >350 °C. IR (KBr): 3401, 3118,
1698, 1680 cmꢁ1
.
1H NMR (250 MHz, DMSO-d6/TMS): d = 3.86 (s,
1691, 1674 cmꢁ1 1H NMR (250 MHz, DMSO-d6/TMS): d = 3.72 (s,
.
3H, OCH3), 4.92 (s, 1H, CH), 6.55–6.75 (m, 3H, Ar), 6.80 (d,
J = 7.7 Hz, 1H, Ar), 6.93 (d, J = 6.7 Hz, 1H, Ar), 7.21 (d, J = 7.5 Hz,
2H, Ar), 11.13 (s, 2H, NH), 11.25 (s, 2H, NH). 13C NMR (62.9 MHz,
DMSO-d6/TMS): d = 27.3, 57.6, 80.9, 114.1, 116.6, 118.2, 118.5,
123.9, 132.5, 133.8, 143.8, 146.8, 147.0, 152.1, 162.9, 177.5. MS:
526.03 (46.1%, M+). Anal. Calcd for C22H16BrN5O6 (526.30): C,
50.21; H, 3.06; N, 13.31. Found: C, 50.48; H, 3.15; N, 13.44.
3H, OCH3), 3.86 (s, 2H, CH2), 4.83 (s, 1H, CH), 6.48 (s, 1H, Ar),
6.64 (d, J = 8.2 Hz, 2H, Ar), 6.80 (d, J = 8.5 Hz, 4H, Ar), 7.00–7.06
(m, 4H, Ar), 10.02 (br s, 1H, OH), 11.11 (s, 1H, NH), 11.14 (s, 1H,
NH), 11.26 (s, 2H, NH). 13C NMR (62.9 MHz, DMSO-d6/TMS):
d = 27.5, 55.7, 78.8, 111.2, 114.4, 118.5, 120.3, 125.3, 129.4, 135.2,
145.6, 150.2, 150.6, 167.7. MS: 552.16 (15.9%, M+). Anal. Calcd for
C29H24N6O6 (552.54): C, 63.04; H, 4.38; N, 15.21. Found: C, 63.13;
H, 4.43; N, 15.39.
4.1.4. 9-(4-Bromo-phenyl)-10-(1H-indol-3-yl)-9,10-dihydro-
1H,8H-1,3,6,8,9-pentaaza-anthracene-2,4,5,7-tetraone (L2)
Yield: 90%; yellow solid; mp >350 °C. IR (KBr): 3341, 3112,
4.1.9. 9-[4-(4-Amino-benzoyl)-phenyl]-10-(3-hydroxy-4-
methoxy-phenyl)-9,10-dihydro-1H,8H-1,3,6,8,9-pentaaza-
anthracene-2,4,5,7-tetraone (L7)
1713, 1692 cmꢁ1 1H NMR (250 MHz, DMSO-d6/TMS): d = 4.95 (s,
.
1H, CH), 6.25 (d, J = 10.7 Hz, 2H, Ar), 6.72 (d, J = 6.7 Hz, 2H, Ar),
6.92–711 (m, 5H, Ar), 9.46 (br s, 1H, NH), 10.09 (br s, 1H, NH),
11.13 (s, 1H, NH), 11.26 (s, 1H, NH). 13C NMR (62.9 MHz, DMSO-
d6/TMS): d = 26.3, 80.5, 112.3, 113.5, 114.6, 118.4, 120.5, 121.3,
124.1, 124.8, 132.5, 133.4, 137.3, 146.5, 152.1, 165.6. MS: 519.09
(38.5%, M+). Anal. Calcd for C23H15BrN6O4 (519.31): C, 53.20; H,
2.91; N, 16.18. Found: C, 53.53; H, 2.98; N, 16.32.
Yield: 81%; yellow solid; mp >350 °C. IR (KBr): 3396, 3112,
1692, 1675, 14, 1270 cmꢁ1 1H NMR (250 MHz, DMSO-d6/TMS):
.
d = 3.79 (s, 3H, OCH3), 4.84 (s, 1H, CH), 6.65 (s, 1H, Ar), 6.85 (d,
J = 9.5 Hz, 2H, Ar), 7.02 (d, J = 9.2 Hz, 4H, Ar), 7.83 (d, J = 9.5 Hz,
4H, Ar), 9.43 (br s, 1H, OH), 11.12 (s, 2H, NH), 11.25 (s, 2H, NH).
13C NMR (62.9 MHz, DMSO-d6/TMS): d = 27.1, 57.0, 80.4, 115.8,
116.8, 118.1, 123.5, 127.9, 129.2, 132.1, 143.5, 146.9, 151.8,
152.5, 166.3. MS: 566.08 (27.4%, M+). Anal. Calcd for C29H22N6O7
(566.52): C, 61.48; H, 3.91; N, 14.83. Found: C, 61.58; H, 3.99; N,
14.96.
4.1.5. 9-(4-Bromo-phenyl)-10-(1H-indol-3-yl)-2,7-dithioxo-
2,3,7,8,9,10-hexahydro-1H,6H-1,3,6,8,9-pentaaza-anthracene-
4,5-dione (L3)
Yield: 89%; yellow solid; mp >350 °C. IR (KBr): 3316, 3117, 1673,
1703 cmꢁ1. 1H NMR (250 MHz, DMSO-d6/TMS): d = 4.89 (s, 1H, CH),
6.60–6.63 (m, 2H, Ar), 7.17–7.34 (m, 3H, Ar), 7.56–7.61 (m, 2H, Ar),
7.86–7.90 (m, 2H, Ar), 8.70 (s, 1H, NH), 9.57 (s, 1H, NH), 12.18 (s, 1H,
NH), 12.22 (s, 1H, NH), 12.92 (s, 1H, NH). 13C NMR (62.9 MHz,
DMSO-d6/TMS): d = 29.0, 96.3, 108.7, 111.0, 113.7, 119.2, 122.7,
125.3, 128.8, 137.7, 143.3, 161.0, 170.2, 183.9. MS: 550.95 (26.8%,
M+). Anal. Calcd for C23H15BrN6O2S2 (551.44): C, 50.10; H, 2.74; N,
15.24. Found: C, 50.31; H, 2.82; N, 15.34.
4.1.10. 10-(3-Hydroxy-4-methoxy-phenyl)-9-(4-{4-[10-(3-
hydroxy-4-methoxy-phenyl)-2,4,5,7-tetraoxo-1,3,4,5,6,7,8,10-
octahydro-2H-1,3,8,9-tetraaza-anthracen-9-yl]-10-(3-Hydroxy-
4-methoxy-phenyl)-9-phenyl-9,10-dihydro-1H,8H-1,3,6,8,9-
pentaaza-anthracene-2,4,5,7-tetraone (L8)
For synthesis of this compound, a mixture of barbituric acid
(0.26 g, 2 mmol), isovanillin (1 mmol), amine (0.5 mmol), and
H3PW12O40 (0.04 g, 2 mol %) in ethanol (5 mL) at 80 °C was stirred
for 24 h. As completion of the reaction confirmed by TLC (eluent
EtOAc/MeOH), the reaction mixture was cooled to room tempera-
ture. Then, the precipitated product was filtered and washed with
water (2 ꢀ 10 mL) and ethanol (2 ꢀ 5 mL) to afford the pure prod-
uct. Yield: 86%; yellow solid; mp >350 °C. IR (KBr): 3385, 3115,
4.1.6. 9-[4-(4-Amino-phenoxy)-phenyl]-10-(3-hydroxy-4-
methoxy-phenyl)-9,10-dihydro-1H,8H-1,3,6,8,9-pentaaza-
anthracene-2,4,5,7-tetraone (L4)
Yield: 85%; yellow-orange solid; mp >350 °C. IR (KBr): 3310,
1691, 1701 cmꢁ1 1H NMR (250 MHz, DMSO-d6/TMS): d = 3.86 (s,
.
3101, 1708, 1697 cmꢁ1
.
1H NMR (250 MHz, DMSO-d6/TMS):
6H, OCH3), 4.91 (s, 2H, CH), 6.81–6.87 (m, 6H, Ar), 6.94–6.99 (m,
4H, Ar), 7.02–7.13 (m, 4H, Ar), 10.07 (br s, 6H, NH & OH), 11.14
(s, 2H, NH), 11.27 (s, 2H, NH). 13C NMR (62.9 MHz, DMSO-d6/
TMS): d = 27.7, 57.1, 80.9, 115.5, 116.2, 118.1, 119.0, 123.6, 132.3,
140.5, 143.4, 147.1, 151.9, 152.5, 166.1. MS: 908.08 (4.8%, M+).
Anal. Calcd for C44H32N10O13 (908.78): C, 58.15; H, 3.55; N, 15.41.
Found: C, 58.38; H, 3.63; N, 15.65.
d = 3.87 (s, 3H, OCH3), 4.82 (s, 1H, CH), 6.61-6.71 (m, 5H, Ar),
6.92 (d, J = 6.5 Hz, 1H, Ar), 7.04 (d, J = 7.0 Hz, 2H, Ar), 7.16 (d,
J = 8.7 Hz, 1H, Ar), 7.22 (d, J = 6.5 Hz, 1H, Ar), 9.22 (br s, 2H, NH2),
11.43 (s, 1H, NH), 11.60 (s, 1H, NH), 12.25 (s, 1H, NH), 12.34 (s,
1H, NH). 13C NMR (62.9 MHz, DMSO-d6/TMS): d = 27.3, 55.4, 78.9,
115.2, 116.0, 118.2, 119.8, 123.6, 132.3, 140.1, 143.4, 146.9,
147.3, 151.2, 151.5, 166.5. MS: 554.11 (21.2%, M+). Anal. Calcd for
C28H22N6O7 (554.51): C, 60.65; H, 4.00; N, 15.16. Found: C, 60.78;
4.2. Biological experiments
H, 4.07; N, 15.23.
4.2.1.
Yeast
zyme for investigating the potential inhibitors. The yeast
say was conducted based on the previous spectrophotometric
method with slight changes.35 In this assay, 4-nitrophenyl-b-
glucopyranosiduronic acid (pNPG) was used as substrate and the
liberation of p-nitrophenol (pNP; the colored reagent) from pNPG
was monitored at 410 nm as an indicator of the enzyme activity.
The experiments were performed with a T90+ UV/vis spectropho-
tometer instrument (PG instrument, UK) equipped with Peltier
Temperature Controller (Model PTC-2). In brief, 1 U/mL of the yeast
enzyme was added to different concentrations of pNPG (0.1–
2 mM); then at a constant substrate concentration, the increasing
amounts of each inhibitor were added in order to determine the
a
-Gls inhibitory activity
-Gls was available in pure form and used as a model en-
-Gls as-
4.1.7. 9-[4-(4-Amino-benzenesulfonyl)-phenyl]-10-(3-hydroxy-
4-methoxy-phenyl)-9,10-dihydro-1H,8H-1,3,6,8,9-pentaaza-
anthracene-2,4,5,7-tetraone (L5)
a
a
Yield: 83%; yellow solid; mp >350 °C. IR (KBr): 3374, 3115,
D-
1693, 1677, 935, 807 cmꢁ1
.
1H NMR (250 MHz, DMSO-d6/TMS):
d = 3.86 (s, 3H, OCH3), 4.82 (s, 1H, CH), 6.68 (s, 1H, Ar), 6.85 (d,
J = 9.5 Hz, 2H, Ar), 7.02 (d, J = 9.2 Hz, 4H, Ar), 7.83 (d, J = 9.5 Hz,
4H, Ar), 9.43 (br s, 1H, OH), 11.12 (s, 2H, NH), 11.25 (s, 2H, NH).
13C NMR (62.9 MHz, DMSO-d6/TMS): d = 27.1, 57.0, 80.4, 115.8,
116.8, 118.1, 123.5, 127.9, 129.2, 132.1, 143.5, 146.9, 151.8,
152.5, 166.3. MS: 602.11 (8.9%, M+). Anal. Calcd for C28H22N6O8S
(602.57): C, 55.81; H, 3.68; N, 13.95. Found: C, 55.93; H, 3.73; N,
14.06.

90
F. Panahi et al. / Carbohydrate Research 380 (2013) 81–91
1þA2
FCor ¼ Fobs10A
ð3Þ
inhibition mode by Lineweaver–Burk plots. The IC50 value of each
inhibitor which shows the concentration required for 50% inhibi-
where Fcor and Fobs are the correct and observed intensities how-
tion of a
-Gls was calculated using dixon plots,36 in which the inter-
ever, A1 and A2 are the sum of
a-Gls and the inhibitor absorbance
cepts on the abscissa show the IC50 values. The inhibition constants
(Ki) were calculated using Cortes method in which the slope of a
plot of 1/IC50 versus V0/V (relative velocity) shows the inhibition
constant.36 The results of inhibition of both yeast and mammalian
at the excitation and emission wavelengths, respectively.38
Thermodynamic parameters are the main clue to confirm the
binding force of the interaction namely hydrogen bonds, van der
Waals force, hydrophobic bonds, electrostatic interactions, etc. If
a
a
-Gls are summarized in Table 1. The mouse enzyme is a type II
-Gls which is located in the brush border of intestinal cells. To
the enthalpy change (D
Hh) does not vary significantly over the
studied temperature range, its value and that of entropy (
can be determined by van‘t Hoff equation:
D
Sh)
assay the activity of this enzyme, we used the intestinal acetone
powder and the assay was conducted according to our previous
publication.17 Briefly, 10
lL of the enzyme solution was added to
D
Hh
D
Sh
R
ln KA
¼
þ
ð4Þ
a sample solution containing the synthetic compounds and pNPG,
and the release of p-nitrophenol (pNP) from pNPG monitored at
410 nm for 30 min at 37 °C. The inhibition mode was determined
RT
The free energy change (
lowing equation:
D
Gh) was calculated based on the fol-
by incubating mouse
a-Gls (0.3 units/mL) in the presence of
increasing concentrations of pNPG (0.6–3 mM) and different con-
centrations of each synthetic inhibitor.
D
Gh ¼ ꢁRT ln KA
ð5Þ
where, R is the gas constant 8.314 J molꢁ1 Kꢁ1 and T is the temper-
ature (K).
4.2.2. Pancreatic
The -Amy inhibition was determined based on conventional
methods.3 In this study, type VI-B porcine pancreatic
-Amy was
a-Amy inhibition assay
a
4.2.4. The circular dichroism (CD) experiment
a
used. Based on this assay, 1 mg/mL of the maltose solution (pH
The CD spectra of yeast a-Gls in the presence and absence of the
inhibitors were recorded with a CD spectrophotometer instrument
(Aviv, Model-215, USA). Far-UV CD region (200–260 nm) was se-
lected to investigate the secondary structure contents of the yeast
6.9) can be hydrolyzed by 1 unit of this enzyme for 3 min at
20 °C. A total of 500
l
L of each inhibitor and 500
lL of 20 mM NaPi
buffer (pH 6.9 with 6 mM NaCl), containing
a-Amy solution
(0.5 mg/mL) were incubated at 25 °C for 10 min. The synthetic li-
a-Gls, using 1 mm path cuvettes. The concentration of the enzyme
was kept constant (0.2 mg/mL), while varying the inhibitors’ con-
gands were used with a concentration higher than their IC50 values
for yeast and mouse
a
-Gls (200
l
M). After pre incubation, 500
l
L
centration (20–80 lM). The results were expressed in molar ellip-
ticity [h] (deg cm2/dmol) and molar ellipticity was calculated using
of a 0.5% starch solution in 20 mM NaPi buffer (pH 6.9 with
6 mM NaCl) was added to each tube at timed intervals. The reac-
tion was stopped with 1.0 mL of dinitrosalicylic acid color reagent.
The test tubes were then incubated at 90 °C in a water bath for
10 min and cooled down to the room temperature. The reaction
mixture was then diluted 1:10 with double distilled water, and
absorbance was measured at 540 nm with a T90+ UV/vis spectro-
photometer. The readings were compared with the controls, con-
taining buffer instead of sample extract. The results were
the following equation:39
MRW ꢀ hobs
½hꢂ ¼
ð6Þ
10 ꢀ d ꢀ C
where MRW is the mean amino acid residue weight of the
a
-Gls
(116), hobs is the observed ellipticity in degrees at a given wave-
length, d is the length of the cuvette in cm, and C is the concentra-
tion of the protein in mg/mL. The secondary structure alteration of
the enzyme was predicted using CDNN CD spectra deconvolution
software.
expressed as percentage of a-Amy inhibition.
4.2.3. Fluorescence spectroscopy
Fluorometric studies were carried out on a Cary-Eclipse spec-
trofluorimeter (Model Varian, Sydney, Australia). Solution of yeast
Acknowledgments
a
-Gls (0.1 mg/mL) was prepared in 100 mM NaPi buffer pH 7.0.
We would like to thank financial support of Iran National Sci-
ence Foundation (INSF)-Grant No. 88001894. Also the financial
supports of research councils of Shiraz University are gratefully
acknowledged.
Also stock solution of the inhibitors (5 mM) was made in 0.1 M
NaOH. Lower concentrations of the inhibitors were made from
the above stock by dilution. Samples with constant concentration
of yeast
a-Gls (0.1 mg/mL) and different concentrations
(0–50 M) of each inhibitor were mixed. The fluorescence spectra
l
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