Design, synthesis and biological evaluation of theophylline containing variant acetylene derivatives as α-amylase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2019.103120

A novel pharmacophore with theophylline and acetylene moieties was constructed by using a fragment-based drug design and a series of twenty theophylline containing acetylene conjugates were designed and synthesized, and all the compounds were evaluated by enzyme-based in vitro α-amylase inhibition activity. The in vitro evaluation revealed that most of the compounds displayed good inhibitory activities, and among them nine analogs 13–15, 20, 21 and 24–27 were exhibited more or nearly as equipotent inhibitory activity with IC₅₀ values 1.11 ± 0.07, 1.14 ± 0.17, 1.07 ± 0.01 and 1.21 ± 0.03, 1.33 ± 0.09, 1.17 ± 0.01, 1.05 ± 0.02, 1.61 ± 0.04, 1.02 ± 0.03 μM respectively, as compared with standard, acarbose 1.37 ± 0.26 μM. Further, molecular docking simulation studies were done to identify the interactions and binding mode of synthesized analogs at binding site of α-amylase enzyme (PBD ID: 4GQR). Among the synthesized analogs, two compounds 25 and 27 were selected on the basis of α-amylase inhibition activity and evaluated for in vivo anti-diabetic activity by High Fat Diet-Streptozotocin (HFD-STZ) model in normal rats. At the dose of 10 mg/kg, bw, po these compounds have significantly reduced Plasma Glucose level in rats as compared to pioglitazone. The anti-diabetic activity results showed that the animal treated with the compounds 25 and 27 could better reverse and control the progression of the disease compared to the standard.

Full text of DOI:10.1016/j.bioorg.2019.103120

Accepted Manuscript
Design, synthesis and biological evaluation of theophylline containing variant
acetylene derivatives as α-amylase inhibitors
Radhakrishnam Raju. Ruddarraju, Gangarapu. Kiran, Adharvana Chari.
Murugulla, Ravichandar. Maroju, Devarakonda Krishna Prasad, Boyina
Hemanth Kumar, Nukala Shravya Reddy
PII:
S0045-2068(19)30113-0
https://doi.org/10.1016/j.bioorg.2019.103120
103120
DOI:
Article Number:
Reference:
YBIOO 103120
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 January 2019
28 June 2019
11 July 2019
Please cite this article as: R. Raju. Ruddarraju, Gangarapu. Kiran, A. Chari. Murugulla, Ravichandar. Maroju, D.
Krishna Prasad, B. Hemanth Kumar, N. Shravya Reddy, Design, synthesis and biological evaluation of theophylline
containing variant acetylene derivatives as α-amylase inhibitors, Bioorganic Chemistry (2019), doi: https://doi.org/
10.1016/j.bioorg.2019.103120
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Design, synthesis and biological evaluation of theophylline
containing variant acetylene derivatives as -amylase
inhibitors
Radhakrishnam Raju. Ruddarrajua*, Gangarapu. Kiranb, Adharvana Chari. Murugullaa, Ravichandar.
Marojuc, Devarakonda Krishna Prasadb, Boyina Hemanth Kumarb, Nukala Shravya Reddyb
a
Dr.MACS Bio-Pharma Pvt. Ltd, Factory: Plot-79/B&C, Pashamylaram, Patancheru, Medak (Dist)-
502307, Telangana, India
bDepartment of Pharmacy, Anurag Group of Institutions, School of Pharmacy, Venkatapur, Ghatkesar,
Medchal, Telangana, India-500 088
cMahatma Gandhi Institute of Technology, Gandipet, Hyderabad-500 075, Telangana, India.
Corresponding Author: rk.raju4u@gmail.com
Abstract: A novel pharmacophore with theophylline and acetylene moieties was constructed by
using a fragment-based drug design and a series of twenty theophylline containing acetylene
conjugates were designed and synthesized, and all the compounds were evaluated by enzyme-
based in vitro α-amylase inhibition activity. The in vitro evaluation revealed that most of the
compounds displayed good inhibitory activities, and among them nine analogs 13–15, 20, 21 and
24–27 were exhibited more or nearly as equipotent inhibitory activity with IC50 values
1.11±0.07, 1.14±0.17, 1.07±0.01 and 1.21±0.03, 1.33±0.09, 1.17±0.01, 1.05±0.02, 1.61±0.04,
1.02±0.03 M respectively, as compared with standard, acarbose 1.37±0.26 M. Further,
molecular docking simulation studies were done to identify the interactions and binding mode of
synthesized analogs at binding site of -amylase enzyme (PBD ID: 4GQR). Among the
synthesized analogs, two compounds 25 and 27 were selected on the basis of -amylase
inhibition activity and evaluated for in vivo anti-diabetic activity by High Fat Diet-Streptozotocin
(HFD-STZ) model in normal rats. At the dose of 10 mg/kg, bw, po these compounds have
significantly reduced Plasma Glucose level in rats as compared to pioglitazone. The anti-diabetic
activity results showed that the animal treated with the compounds 25 and 27 could better
reverse and control the progression of the disease compared to the standard.
Keywords: Theophylline, Acetylenes, α-Amylase, Molecular Docking, Anti-diabetic,
High fat diet, Streptozotocin

1. Introduction
Diabetes being a severe disease is the most raising problem in the world. Estimated data
was collected in 2010 which stated that 285 million peoples were affected by this disease which
may increase to 439 million by 2030[1,2]. Almost 95% of diabetes cases are characterized by
varying blood glucose level because of insulin resistance[3]. This leads to cardiovascular
diseases, high blood pressure, stroke, blindness, and kidney failure[4]. Amylases are the group of
enzymes namely, α-amylase, glucoamylase and β-amylase which hydrolyze α-1,4- and α-1,6-
glucosidic bonds present in starch and glycogen[5]. Inhibition of α-amylase enzyme in the
digestive system is one of the best options to maintain the postprandial glucose level[6,7]. One
therapeutic approach for treating diabetes is to decrease post-prandial hyperglycemia which can
be achieved by delaying the absorption of glucose and also by the inhibition of carbohydrate
hydrolyzing enzyme α-amylase[8,9]. α-Amylase inhibitors significantly decrease the post-
prandial hyperglycemia in the treatment of type-II diabetes[7],[10]. Examples of such inhibitors
which are in clinical use are Acarbose, Miglitol, and Voglibose. The WHO recommends a search
of safe, potent and less toxic natural anti-diabetic agent[11].
Unfortunately, most natural products themselves are not suitable for administration as drugs.
But natural products served as an effective source of drug lead throughout the history[12–15]. In
view of this background, theophylline was selected as lead which belongs to the class called
xanthine family, mainly found in the beans of cocoa and it is highly present in criollo cocoa
beans to an extent of 3.7 mg/kg and is also found in brewed tea[16]. It is reported to have a wide
range of various biological activities such as treatment of respiratory diseases like asthma,
chronic obstructive pulmonary disease (COPD)[17,18], and other airway diseases for more than
75 years. Theophylline containing acetylene derivatives have been reported as DPP-IV inhibitor
with GPR119 agonist particularly in the treatment of type 2 diabetes mellitus[19–21]. Other
biological activities such as anticancer[22], anti-microbial[23] and antidepressant agents[24].
On the other hand, in medicinal chemistry and organic synthesis, acetylenes are a valuable
and important class of compounds. The metabolites of acetylenes belong to a class of molecules
having the triple bonds and they are found in fungi, plants, marine invertebrates and
microorganisms[25–27]. Some of the acetylene, theophylline, and theophylline containing
acetylene bioactive molecules are illustrated (Figure-1)

So, in the present study, new Linagliptin based α-amylase inhibitors were designed by
making suitable chemical modification in the theophylline nucleus. Thus, based on the above
points it was thought worthwhile to prepare new theophylline containing acetylenes with an
objective to obtain pharmacological active, safer, and less adverse effects anti-diabetic agents.
O
N
O
O
H
7
6
3
1
N
N
N
8
HN
N
N
4
2
N
N
N
9
O
O
N
O
N
Caffeine
Theophylline
(CNS stimulant)
(Asthma and COPD)
Theobromine
(vasodilator and blood
pressure)
OH
HO
HN
O
O
N
OH
N
N
N
N
O
N
N
N
N
N
N
N
O
N
O
NH2
O
N
N
Linagaliptin
(anti-diabetic)
Theodrenaline
(cardiac stimulant)
DMPX
(A2-adenosine receptors)
H
N
O
N
O
Cl
F
F F
Efavirenz
(antiretroviral)
Terbinafine
(antifungal)
Figure-1. Multiple bioactive theophylline, acetylene, and theophylline containing acetylene
moieties.

O
N
N
N
N
N
N
N
O
NH2
Linagaliptin
(anti-diabetic)
O
H
N
N
O
N
N
1
Series-B
Series-A
O
O
N
R2
R5
O
O
N
R1
O
N
O
R3
R6
N
N
N
N
N
N
N
N
O
O
O
N
4
, R1 = CH2
8-21 & 23-27,
constructed with amino acid unit
1
R5 = H, R6 = Different Chain length
5
, R1 = CH2CH2CH2
6,7 & 9,
R2 = H, R3 = Different Chain length
10,
R2 = CH3, R3 = Chain length
22,
R5 = CH3, R6 = Chain length
constructed with amino acid unit
O
O
N
N
R4
H
N
N
N
O
8,13,14 & 15,
R4 = Different Chain
length constructed with ester or amide
unit
Figure-2. Design strategy of the novel theophylline series A and B.
2 Results and Discussion
2.1 Chemistry
The synthesis of key intermediate theophyllinoic acid compounds (3a-b) was described first
in Scheme-1 and later, all novel theophylline containing acetylene derivatives were constructed
as in Scheme-2, 3 and 4. Commercially available theophylline 1 was used as the starting material
to synthesize intermediate compounds (3a-b) and final analogs (4-10) by following literature
procedure[28].

O
N
O
N
O
R1
R1
O
O
O
N
O
OH
a) i)
ii)
H
O
O
N
N
N
b
Br
N
N
N
O
N
O
N
O
O
N
3a
3b
2a
2b
1
OH
2a-3a : R1 = H
2b-3b : R1 = CH3
Scheme-1 Synthesis of theophylline acid compounds (3a–3b). Reagents and conditions: (a) (i)
K2CO3, DMF, 85°C, 12 h & (ii) TPP, DIAD, THF, rt, 5 h; (b) LiOH.H2O, THF:H2O, rt, 2 h.
O
N
O
N
O
N
H
N
N
N
N
N
Br
N
Br
a
N
N
N
N
O
O
b
O
4
5
1
O
O
O
N
O
O
N
N
H2N
H
N
N
Br
N
O
N
N
O
N
c
e
O
O
O
N
OH
6
8
N
N
O
N
O
O
N
O
O
N
O
3a
N
N
N
N
f
d
N
O
O
Br
OH
9
7
O
N
O
O
OH
O
O
N
N
N
OH
N
N
g
O
N
O
N
3b
10
Scheme-2 Synthesis of theophylline containing acetylene compounds (4-10). Reagents and
conditions: (a) K2CO3, DMF, 85°C, 2 h; (b) K2CO3, DMF, 85°C, 10 h; (c) K2CO3, DMF, rt, 10 h;
(d) K2CO3, DMF, 80°C, 12 h; (e) HATU, DIPEA, DMF, rt, 12 h; (f & g) DCC, DMAP, DCM, rt,
16h.

Now in Scheme-3, compound 3a was coupled with methyl 3-amino-2,2-
dimethylpropanoate hydrochloride by using HATU and DIPEA at room temperature for 16 hours
gave compound 11 and then followed by hydrolysis with LiOH.H2O in THF and H2O (1:1) gave
compound 12. In a similar way, compound 12 was treated with propargyl bromide in the
presence of K2CO3 in DMF at room temperature to obtain compound 13 (60%) and treated with
5-chloro-1-pentyne at 80°C for 12 hours to get compound 14 (50%). But, compound 12 was
coupled with propargylamine by using reagents T3P and triethylamine (TEA) in DMF at room
temperature to get compound 15 with the yield 36%.
O
O
O
N
N
N
H
H
N
N
N
O
15
H2N
O
e
O
O
O
N
O
N
O
O
N
N
OH
O
N
N
O
O
N
OH
H
H
ClH.H2N
O
N
N
N
N
N
N
N
b
a
O
O
O
12
3a
11
c
Cl
d
Br
O
O
O
O
N
O
N
N
O
O
N
N
O
H
H
N
N
N
N
N
O
O
13
14
Scheme-3 Synthesis of theophylline containing acetylene compounds (13-15). Reagents and
conditions: (a) HATU, DIPEA, DCM, rt, 16 h; (b) LiOH·H2O, THF:H2O, rt, 4 h; (c) K2CO3,
DMF, rt, 10 h; (d) K2CO3, DMF, 80°C, 12 h; (e) T3P, TEA, DMF, rt, 16 h.
In the synthetic Scheme-4, compounds (3a-b) were coupled with various amino acids has
been described in this section. Compound 3a was coupled with glycine methyl ester
hydrochloride (a), alanine methyl ester hydrochloride (b), L-valanine ethyl ester hydrochloride
(c), L-phenyl alanine benzyl ester hydrochloride (d) by using reagents HATU and DIPEA in
dichloromethane at room temperature for 16 hours which gave compounds 16a-d, while

compound 3b was coupled with only alanine methyl ester hydrochloride (b) by using reagents
HATU and DIPEA in dichloromethane at room temperature for 16 hours gave compound 16e.
The ester compounds 16a-c and 16e hydrolysis with LiOH.H2O in THF and H2O at room
temperature for 4 h gave compounds 17a–c and 17e. Compound 16d hydrogenated with Pd/C in
methanol at 60 Psi for 8 h to get compound 17d. Further, compounds 17a-b and 17e were
coupled with propargyl alcohol by using DCC and DMAP in dichloromethane at room
temperature for 16 hours gave compounds 18, 19 (86% and 79%) and 22 with the yield 74%.
Compounds 17c-d were treated with propargyl bromide in the presence of K2CO3 in DMF at
room temperature to obtain compound 20 (54%), 21 (91%). But, compounds 17b-d were treated
with 5-chloro-1-pentyne at 80°C for 12 hours to get compounds 23 (33%), 24 (72%) and 25
(47%), respectively. Compounds 17c-d were coupled with propargylamine by using reagents T3P
and triethylamine (TEA) in DMF at room temperature to get compounds 26 and 27 with yields
63%, 91% respectively.

O
N
R2
H
N
R1
O
N
N
26
27
c :
d :
R1 = H, R2 =
R1 = H, R2 =
H
N
O
N
O
26-27
H2N
vi
R2
O
N
O
N
R2
O
N
R2
R1
R1
R1
O
OH
O
ClH.H2N
OH
O
N
O
O
N
H
H
O
ii (a, b)
N
N
O
N
O
(R2 = a,b,c & d)
N
iv
N
N
i
O
N
O
N
O
N
17a-e
3a-b
16a-e
iii
v
HO
R1
Cl
Br
O
R2
O
R2
O
R1
O
O
N
H
O
N
N
H
N
O
N
O
N
N
N
O
N
O
N
18-22
23-25
R1 = H, R2 = H
a :
b :
c :
18
19
20
b : R1 = H, R2 = CH3
23
24
R1 = H, R2 = CH3
R1 = H, R2 =
c :
R1 = H, R2 =
d : R1 = H, R2 =
R1 = H, R2 =
25
d :
e :
21
22
R1 = CH3, R2 = CH3,
Scheme-4 Synthesis of theophylline containing amino acid acetylene compounds (18-27).
Reagents and conditions: (i) HATU, DIPEA, CH2Cl2, rt, 16 h; (ii) (a) LiOH.H2O, THF:H2O, rt,
4h & (b) Pd/C, MeOH, H2, 60 Psi, rt, 8 h; (iii) DCC, DMAP, CH2Cl2, rt, 16 h; (iv) K2CO3, DMF,
rt, 10 h; (v) K2CO3, DMF, 80°C, 12 h; (vi) T3P, TEA, DMF, rt, 16 h.
2.2 Anti diabetic activity
2.2.1 In vitro a-amylase inhibitory activity
This work is aimed at the biological evaluation of two novel series of theophylline derivatives as
shown in Figure-2. Our plan was based on using theophylline 1 and the simple acetylene as
scaffolds (4-10 and 13-15). Although 4 and 5 have shown low activity, it can be used as a

starting point for the optimization of its biological activity by modifying its added carbonyl unit
and length of chain. The simple acetylenes of series A (4-10 and 13-15) were planned by keeping
the carbonyl scaffold of 1 and substituting it with different acetylenes in order to verify the
importance of such subunits in the α-amylase activity. In the series B (18-27), the planned
derivatives present the theophylline moiety protected by carbonyl and amino acid moieties
aiming to observe the influence of these groups on the activity of the theophylline portion, as
well as to verify the importance of the inclusion of the different amino acid units.
Importance from synthesized a novel series A and B of theophylline containing acetylene
scaffolds (4-10, 13-15 and 18-27) and were evaluated against the in vitro α-amylase inhibitory
activity. All the evaluated analogs were found to be active and shown good inhibitory potentials
with IC50 values ranging between 14.8±0.15 to 1.02±0.03 M and compared with standard
acarbose having IC50 value 1.37±0.26 M as shown in Table 1. Three analogs in series A: 13, 14
and 15 shown excellent inhibitory potential with IC50 values 1.11±0.07, 1.14±0.17 and
1.07±0.01, and also six analogs in the series B: 20, 21, 24, 25, 26, and 27 shown excellent
inhibitory potential with IC50 values 1.21±0.03, 1.33±0.09, 1.17±0.01, 1.05±0.02, 1.61±0.04 and
1.02±0.03 M respectively, when compared with the standard acarbose with IC50 value
1.37±0.26 M. The remaining all other analogs showed good to moderate inhibitory potential. A
limited structure-activity relationship could be established by analyzing at the different
substituents on the theophylline ring part on 7th position.
Table 1. α-Amylase activity of synthesized theophylline acetylene analogs
No
Structure
IC50 (µM)
No
Structure
IC50 (µM)
O
O
O
O
N
N
H
N
N
N
N
O
O
N
N
4
12.9±0.17
18
2.84±0.13
N
O
N
N
N
O
O
O
O
N
O
O
N
N
H
5
14.8±0.15
19
2.62±0.02
N
O
N
N

O
O
N
O
O
N
O
O
N
N
H
N
O
N
O
N
N
6
7
-
20
21
1.21±0.03
N
O
O
N
O
O
N
O
N
O
3.27±0.04
1.33±0.09
O
N
H
N
O
N
O
N
N
O
N
O
N
O
N
O
O
O
N
N
H
H
N
N
O
N
8
4.14±0.09
22
N
2.09±0.11
N
O
N
O
O
O
O
O
N
H
O
N
O
N
O
N
N
N
9
4.01±0.14
4.32±0.04
23
24
2.27±0.09
N
N
O
N
O
O
O
O
O
O
O
N
H
N
O
N
10
1.17±0.01
N
N
N
O
N
N
O
N
O
N
O
O
N
O
N
O
H
N
O
N
13
14
1.11±0.07
25
1.05±0.02
1.61±0.04
O
N
N
H
N
O
O
N
N
O
O
O
N
O
H
O
N
O
N
H
O
N
N
H
N
N
N
O
1.14±0.17
1.07±0.01
26
27
N
O
N
N
O
O
O
O
O
N
N
H
N
H
N
H
N
N
15
1.02±0.03
O
N
N
H
N
O
N
O
N
N
O
Acarbose
1.37±0.26 Acarbose
1.37±0.26
*All the values are expressed as mean ± SD, (n=3);

The inactivity of the derivatives from series A compounds 4 and 5 highlights the importance
of the theophylline moiety for the α-amylase activity IC50 values 12.9±0.17 and 14.8±0.15 M
such as introduced simple alkyl chain at 7th position. Then later, we introduced carbonyl unit
along with different hydroxyl alkyl acetylene chains such as ester analogs 10, 9 and 7 and shown
increasing activity with IC50 values 4.32±0.04, 4.01±0.14 and 3.27±0.04 M having 4-fold
potent than the 4 and 5 and although they are less active than standard drug. Next in that way,
introduced carbonyl unit along with different amine alkyl acetylene chains such as amide analogs
8, 14, 13 and 15 shown excellent activity with IC50 values 4.14±0.09, 1.14±0.17, 1.11±0.07 and
1.07±0.01 M, respectively, having more potent than the all compounds in this series-A but
except compound 8 less active than standard. Finally, in this series we observed, carbonyl having
oxygen or nitrogen introduced alkyl chains shown increasing activity and also here the length of
the chain also plays a key role against the inhibitory activity.
Considering in the series B, the results for compounds 18-27 were proposed in order to
evaluate the importance of the amino acid group in activity by adding it with a carbonyl group at
the 7th position of theophylline moiety. Moreover, the conjugation of a carbonyl with the amino
acid unit could change the conformation of these molecules, in which relative planarity could be
achieved. In this series B, introduced amino acid unit by liking carbonyl is fixed at amine group
and different acetylene chains at carboxyl group, and also shown variant groups at α-carbon.
Firstly, introduced different hydroxyl alkyl acetylene chains having simple neutral non polar
amino acids such as ester analogs glycine 18 (2.84±0.13 M), alanine 19, 22 and 23 (2.62±0.02,
2.09±0.11 and 2.27±0.09 M), valine 20 and 24 (1.21±0.03 and 1.17±0.01 M), and
phenylalanine 21 and 25 (1.33±0.09 and 1.05±0.02 M), respectively in which some of the
analogs 20, 21, 24 and 25 shown excellent activity than the standard (1.37±0.26 M) and the
other analogs 18, 19, 22 and 24 shown less active than standard. In contrast, amide analogs such
as amino acid having different amine alkyl acetylene chains compounds 26 and 27 (1.61±0.04
and 1.02±0.03 M) in which analog 27 shown highest activity and analog 26 shown good
activity as compared with the standard. From these results, it is clearly suggesting that the -
amylase inhibition activity depends on the substitution of α-carbon and with an increasing order
C-H<C-CH3<C-(CH3)2<C-CH2C6H5.

The substitutions of the carbonyl, amino acid unit and length of the acetylene chain are beneficial
to the α-amylase activity, in this case, with the highest potency analogs are observed for 13-15
and 20, 21 and 24-27 are more active than standard.
2.2.2. Molecular docking
The molecular docking studies were carried out of these synthesized compounds against α-
amylase enzyme (PDB ID: 4GQR) in order to predict the possible binding interactions between
the ligands and enzymes. Compound 25 (docking score = -6.49) the most potent compound in
the series-B, purine ring of 5 and 6-rings showed pi-pi interactions with Trp59 (3.13Ao) and
formed H-bond interactions between the carbon atom and carboxylate oxygen atom of Asp300
(3.13Ao) and side chain carbon atom and oxygen atom of Glu233 (3.2Ao). Whereas, other
residues Trp58, His101, Thr163, Leu165, Asp197, and Ala307 showed hydrophobic interactions.
The acetylene chain in compound 25 showed Vander Waals clashes with Ile235 and Glu233
residues.
In series-B, compound 27 (docking score = -6.46) showed good binding interactions with
the active site of 4GQR of -amylase protein. The compound shows strong H-bond interactions
as a donor with His305 with distance 2.68 Ao between the carbon atom of acetylene side chain
with the oxygen atom of Histidine residue. Trp59 formed H-pi interactions with N-CH3 of purine
ring and pi-H interactions with Leu165, Ala307, and Gly308 residues. The hydrophobic
interactions are shown with Asp300, Tyr62, and Trp58 residues.
Compound 14 (docking score -6.86) in the series-A showed good binding interactions with
the active site of amino acid residues. The compound forms pi-H interactions with 5-ring of
purine with Trp59 residue. It also forms H-donor interactions with Tyr62 (3.15) and H-acceptor
interactions with Gln63. The hydrophobic interactions are formed with residues Asp197,
Leu165, Ala198, Trp58 and Ala307. In case of compound 21 also showed similar pi-pi
interaction with Trp59 with distance 3.71 but additional hydrogen bond interaction with NH of
the ligand with ASP300 with distance 2.78 and exhibits hydrophobic interactions with Trp58,
Leu162, Leu165, and Ala198. In addition, compound 21 has dock score and binding energy
values are -6.51 and -3.6 kcal/mol. The acetylene group shows Vander Waals clashes with
Arg195 and Glu233 residues.
Similarly, all the compounds have exhibited binding interactions with the active site
residues. From the docking studies, it could be revealed that acetylene group has formed Vander

Waals interactions, side chain containing amide or carboxylate groups -NH and C=O forms H-
bond interactions and purine containing 6 and 5 membered rings shows pi-H or pi-pi
interactions.
The co-crystal ligand, Myricetin assumed to occupy the α-amylase and established
hydrogen bonding interactions with Gln63, Asp197, and Asp300. All the docked compounds
superimposed well with the cocrystal ligand Myricetin and all common interactions (Gln63,
Asp197, Asp300 and Leu162, Leu165, Trp59, and Trp58) were consistent with the ones in the
crystal complexes of ligand binding domain (LBD) of α-amylase, as shown in Figure-3. The
ligand-protein interactions were illustrated in Table-2.

Compound 14
Compound-27

Compound-25
Compound-21
Figure-3: Binding interactions of compounds with -amylase enzyme (PDB ID: 4GQR)
Table-2: Docking score and Binding interactions of compounds with -amylase enzyme
into the active site of 4GQR
Interactions report
Comp.
Docking score
Ligand
Receptor
Interaction Distance
C 13 OE2 GLU 233
C 14 OE1 GLU 233
H-donor
H-donor
H-acceptor
H-pi
3.15
3.30
2.79
4.41
O 11
C 13
NE2 HIS 299
5-ring HIS 299
04
05
-3.99
-4.59
C 18 OE1 GLU 233
H-donor
3.56

O 15
NE2 HIS 101
H-acceptor
2.85
C 13 OE1 GLU 233
H-donor
H-acceptor
H-pi
3.35
2.90
4.15
4.21
3.29
2.75
3.86
3.06
3.55
3.93
3.32
2.66
4.41
3.63
4.20
3.30
3.15
3.47
4.47
3.69
4.15
3.30
3.15
3.47
4.47
3.69
4.15
3.82
3.94
3.07
3.28
06
07
-4.54
-4.64
O 10
C 19
C 19
NE2 HIS 101
6-ring TRP 59
6-ring TRP 59
H-pi
C 13 OE2 GLU 233
C 14 OD2 ASP 197
H-donor
H-donor
pi-cation
H-donor
H-acceptor
pi-H
H-donor
H-donor
pi-H
H-pi
pi-H
H-donor
H-donor
H-acceptor
H-pi
08
09
-5.51
-4.93
5-ring
C 13
O 10
6-ring
C 8
NE2 HIS 101
O
TYR 62
NE2 HIS 101
CB TYR 62
OD2 ASP 197
C 12
O
TYR 62
10
13
-4.90
-6.01
6-ring
CD1 LEU 165
6-ring TRP 59
CB TRP 59
C
38
5-ring
C
C
N
C
C
5
O
O
TYR 62
TYR 62
39
41
44
44
NE2 GLN 63
5-ring TRP 59
6-ring TRP 59
CB TRP 59
H-pi
pi-H
H-donor
H-donor
H-acceptor
H-pi
14
-6.86
5-ring
C
C
5
O
O
TYR 62
TYR 62
39
41
44
44
N
C
C
5-ring
6-ring
6-ring
NE2 GLN 63
5-ring TRP 59
6-ring TRP 59
CB TRP 59
5-ring TRP 59
5-ring TRP 59
NE2 HIS 201
NE2 HIS 201
H-pi
pi-H
pi-pi
pi-pi
15
18
-5.88
-5.55
O
O
12
31
H-acceptor
H-acceptor
19
20
-5.15
-4.97
O 11
N 13
6-ring
5-ring
6-ring
5-ring
NE2 HIS 201
H-acceptor
2.89
OD2 ASP 300
5-ring TRP 59
CD1 LEU 162
CD1 LEU 162
CD1 LEU 162
H-donor
Pi-pi
pi-H
pi-H
pi-H
2.78
3.71
4.21
4.08
4.25
21
-6.12
22
23
24
-5.05
-5.13
-5.58
6-ring
NE2 HIS 201
pi-cation
4.58
C 18
5-ring TRP 59
OD1 ASP 300
OE1 GLU 233
5-ring TRP 59
5-ring TRP 59
6-ring TRP 59
6-ring TRP 59
H-pi
H-donor
H-donor.
pi-pi
pi-pi
H-pi
H-pi
H-donor
H-pi
pi-H
pi-H
4.21
3.13
3.20
3.54
3.94
4.38
4.38
2.68
4.36
4.51
4.33
4.51
C
C
17
36
5-ring
6-ring
C 27
C 27
25
26
-6.49
-5.54
C
C
38
25
O
HIS 305
6-ring TRP 59
CD1 LEU 165
CA ALA 307
6-ring
6-ring
6-ring
27
-6.46
N
GLY 308
pi-H

O 23
OD2 ASP 300
H-donor
H-donor
H-donor
H-acceptor
pi-H
3.19
3.15
2.78
3.19
4.40
O
O
O
24 OD2 ASP 197
26 OD2 ASP 197
21 NE2 GLN 63
MYC
-14.1194
6-ring
CD1 LEU 165
2.2.3 In vivo anti-diabetic activity
2.2.3.1. HFD-STZ induced diabetic model[30]
The in vitro α-amylase inhibitory activity and docking studies have revealed that the
compounds 25 and 27 showed potential inhibition activity against the enzyme. Hence, these two
compounds 25 and 27 were evaluated for their in vivo anti-diabetic effect using HFD-STZ-
induced diabetic model. HFD along with the low dose of STZ produced impairment of insulin
secretion which was an important characteristic of human insulin-deficient type 2 Diabetes
Mellitus (T2D). Figure-4 showed the significant increase in plasma glucose level (PGL) in
HFD-STZ-Control treated group compared to the NDC group. The pioglitazone and compounds
25 and 27 were administrated at the dose of 10 mg/kg, b.w p.o in 1% Sod CMC in HFD-STZ
rats. The PGL levels on the 7th day were significantly decreased in pioglitazone-treated rats when
compared to HFD-STZ-Control group (P < 0.01) interestingly, compound 25 has shown
moderate significance (P < 0.05) and whereas compound 27 has shown any significant effect
when compared to diabetic control. However, the PGL levels recorded on 14th day showed that
treatment with pioglitazone and compound 25 has significant (P <0.001; P < 0.01) effect evident
by the subsequent decrease in the PGL levels compared to HFD-STZ-Control group. But
compound 27 has shown a mild significant effect (P < 0.05) when compared to diabetic control
on the 14th day. Nevertheless, the antidiabetic activity of selected compound 25 and 27 have
shown moderate activity when compared with known standard drug pioglitazone. Thus,
administration of selected compounds 25 and 27 have shown significant improvement in the
PGL levels.

500
400
300
200
100
0
NDC
HFD-STZ-Control
Pioglitazone
Compound 25
Compound 27
*
*
**
**
***
y
y
y
y
y
y
y
y
a
y
a
y
y
y
a
y
y
y
a
a
a
a
a
a
a
a
a
a
a
a
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
-
-
-
-
-
-
-
-
-
4
-
-
-
4
-
-
-
4
4
4
0
7
0
7
0
7
0
7
0
7
1
1
1
1
1
Figure-4. In vivo anti-diabetic activity of compounds 25, 27 and standard Pioglitazone in HFD-
STZ induced diabetic rats. All Data were expressed as Mean ± SEM (n=6) using One-Way
*
ANOVA followed by Dunnett’s multiple comparison test. p<0.05, **p<0.01 and ***p<0.001 vs
HFD-STZ-Control.
NDC: Normal rats with normal diet; HFD-STZ-Control is Diabetic Control.
2.2.4. Histological analysis of pancreas and liver samples
The haematoxylin and eosin staining were used to analyze the histological aberrations in
pancreas and liver samples. The islets of the pancreas were observed for the appearance of acinar
cells, apoptosis, and degeneration of β-cells in control, diabetic control, standard, compound 25
and 27 groups respectively (Figure 5A-5D). The photomicrographs of pancreas tissue samples
of diseased animals showed degeneration and reduced number of pancreatic islet cells. In non-
diabetic control and pioglitazone group showed normal pancreatic islet cells compared with
HFD-STZ-control group (shown in Figure 5C). Treatment with compound 25 has shown normal
histology and proliferation of pancreatic islet cells is seen interestingly, compound 27 treated rat
pancreas showed recovery and moderate degenerative changes in pancreatic islets compared with
HFD-STZ-Control group (shown in Figure 5D and 5E).
In the diabetic liver histopathology multifocal necrosis in the central lobular region along
with peribiliary inflammation with infiltration of inflammatory cells was noticed in diabetic

control. In non-diabetic control and standard groups, proper arrangement and normal structure of
hepatic cells were noticed. After treatment with test compounds 25 and 27, the inflammation
hepatocytes were significantly alleviated, and liver histology gradually returns to the normal
state, especially in standard and compound 25 groups (Figure 6A-6E).
C
A
B
100 µm
100 µm
100 µm
E
D
100 µm
100 µm
Figure-5. Histopathological changes observed in pancreas tissue samples of HFD-STZ induced
diabetic rats treated with compound-25 and compound-27.
(A) Normal diabetic control group-arrows shows normal proliferation of β‐cells and acinar cells;
(B) Diabetic control group–arrows indicates degeneration of pancreas islet cells; (C)
Pioglitazone treated group–arrow shows recovered pancreas islet cells; (D) Compound-25 treated
group-arrows indicates recovered pancreas islet cells without any degeneration; (E) Compound-
27 treated group–arrows indicates showing partly recovered pancreas islet cells with moderate
degeneration.

C
B
A
100 µm
100 µm
D
E
100 µm
100 µm
Figure-6. Histopathological changes observed in liver tissue samples of HFD-STZ induced
diabetic rats treated with compound-25 and compound-27.
(A) Normal diabetic control–arrows indicating normal histological structure liver cells; (B)
Diabetic control group–arrows showing Multi focal necrosis in centrilobular region along with
peribiliary inflammation with infiltration of inflammatory cells; (C) Pioglitazone treated group–
arrows indicate mild infiltration of inflammatory cells noticed in peribillary region; (D)
Compound-27 treated group–arrows indicating the improved structure with mild periportal
infiltration of inflammatory cells; (E) Compound-25 treated group-arrows showing moderate
periportal infiltration of inflammatory cells.
From these observations, it indicates that the administration compound 25 and 27 showed
partial restoration in the liver and pancreatic cells. Collectively, these results suggest that the
investigation of the anti-hyperglycemic mechanisms of compound 25 and 27 compared to
standard drug and ameliorated the degree of disease and decreased proliferation is seen in
pancreas and liver tissues.
This study has demonstrated that in the combination of HFD and the low dose of STZ is
sufficient to induce type-2 diabetic in rats. The present work is the comprehensively study the
effect of theophylline containing acetylene derivatives on T2DM. The experimental study has
demonstrated that the theophylline containing acetylene derivatives have anti-hyperglycemic
property and further optimization of these leads could discover a novel anti-diabetic agent.

3. Conclusion
In conclusion, two series of novel theophylline containing acetylene compounds were
synthesized and evaluated for their in vitro α-amylase inhibitory activity and in vivo anti-diabetic
activity by HFD-STZ induced diabetic model. Molecular docking simulation study of the
compounds has been done to study interactions and binding mode of synthesized compounds on
-amylase enzyme (PDB ID: 4GQR). Compounds 13–15, 20, 21, and 24–27 have shown
excellent α-amylase inhibitory activity. Structure-activity relationships showed that the enzyme
-amylase inhibition of compounds was dependent on residues on α-carbon and exhibited with
increasing order of activity C-H<C-CH3<C-(CH3)2<C-CH2C6H5. Further in vivo HFD-STZ-
induced diabetic models showed that compound 25 showed significant anti-diabetic activity and
compared with a standard drug. Thus, based on in silico molecular docking and experimental
studies, it can be suggested that theophylline containing acetylenes should be further explored for
the anti-hyperglycemic mechanism.
4. Experimental section
4.1 Chemistry and chemical methods
Starting materials were obtained from commercial suppliers and used without further
1
purification. The H NMR and 13C NMR spectra were taken on a VNMRS 400 MHz
spectrometer using the solvent (CDCl3 7.26 ppm and 77.0 ppm, DMSO-d6 2.49 ppm 39.7 ppm)
and TMS used as an internal standard. Chemical shifts are given in δ ppm and coupling constant
(J) is given in Hz. IR spectra were recorded on a Perkine Elmer FTIR 1600 spectrometer for
samples in KBr discs. The mass spectra were recorded on QSTAR XL hybrid MS/MS system
(Applied Biosystems, USA) under ESI. All compounds were purified by flash chromatography
(FC) was performed using on silica gel (100-200 mesh) and all the reactions were monitored by
Thin-Layer Chromatography (TLC) on Silica Gel 60 F254 plates (VWR, Darmstadt);
visualization by UV detection at 254 nm.
4.2 Synthesis
The synthesis of intermediate compounds (2a-b), (3a-b) and final analogues (4-10) reported by
the following literature[28].

4.2.1 methyl 3-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamido)-2,2-
dimethylpropanoate (11). To a stirred solution of compound 3a (3 g, 12.60 mmol) and 3-
amino-2,2-dimethylpropanoate hydrochloride (3.1 g, 18.90 mmol) in dichloromethane (40 mL),
HATU (7.18 g, 18.90 mmol) and DIPEA (6.5 mL, 37.81 mmol) were added at room temperature.
The reaction mixture was stirred for 16 h. On completion of the reaction as monitored by TLC,
the reaction mixture was poured into water and extracted with dichloromethane. The organic
extracts was washed with aq. NaHCO3 solution and aq. KHSO4 solution, dried over anhydrous
Na2SO4, filtered and evaporated under reduced pressure to afford 11 (3.0 g, 68%) as an off-
white solid. 1H NMR (400 MHz, CDCl3): δ 7.69 (s, 1H), 7.14 (brs, 1H), 4.87 (s, 2H), 3.66 (s,
3H), 3.60 (s, 3H), 3.41 (s, 3H), 3.37 (d, J = 6.4 Hz, 2H), 1.14 (s, 6H); m/z (ES)+: 352.2 [M+H]+.
4.2.2
3-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamido)-2,2-
dimethylpropanoic acid (12). LiOH.H2O (717 mg, 17.09 mmol) was added portion wise to a
stirred solution of ester compound 11 (3 g, 8.547 mmol) in THF (15 mL, 5 vol) and H2O (15 mL,
5 vol) at room temperature and the reaction mixture was stirred for 4 h. After completion, from
the reaction mixture THF was concentrated, diluted with water and acidified with aqueous
KHSO4 solution, extracted with 10% methanol in dichloromethane (2 x 50 mL). The combined
organic extracts were dried over anhydrous Na2SO4, filtered and evaporated under reduced
pressure to afford 12 (2.0 g, 69%) as an off-white solid. 1H NMR (400 MHz, DMSO-d6): δ 12.33
(s, 1H), 8.13 (t, J = 6.02 Hz, 1H), 8.00 (s, 1H), 5.0 (s, 2H), 3.43 (s, 3H), 3.24 (d, J = 6.27 Hz,
2H), 3.19 (s, 3H), 1.06 (s, 1H); m/z (ES)+: 338.16 [M+H]+.
4.2.3
prop-2-yn-1-yl
3-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-
yl)acetamido)-2,2-dimethylpropanoate (13). To a stirred solution of compound 12 (150 mg,
0.44 mmol) in 5 mL of DMF, was added K2CO3 (79 mg, 0.57 mmol) at room temperature. After
20 min, propargyl bromide (0.76 mL, 0.89 mmol) was added to the reaction mixture at room
temperature and was stirred for 10 h. As monitored by TLC, to the reaction mixture water was
added and a solid precipitate was formed that was filtered and dried to afford 13 (166 mg, 60%)
as an off-white solid. Mp: 145–148oC; IR (KBr): υmax/ cm-1 = 3306, 3233, 3111, 2945, 2123,
1722, 1697, 1676, 1649, 1573, 1550, 1472, 1433, 1373, 1261, 1230, 1134, 1029, 979, 747, 698;
1H NMR (400 MHz, DMSO-d6): δ 8.19 (t, J = 6.0 Hz, 1H), 7.97 (s, 1H), 4.97 (s, 2H), 4.65 (d, J

= 2.4 Hz, 2H), 3.52 (s, 1H), 3.41 (s, 3H), 3.25 (d, J = 6.0 Hz, 2H), 3.17 (s, 3H), 1.08 (s, 6H); 13C
NMR (400 MHz, DMSO-d6): δ 175.39, 167.02, 154.82, 151.47, 148.34, 144.11, 106.88, 78.93,
78.09, 52.57, 48.50, 46.65, 43.54, 29.88, 27.90, 23.02; HRMS (ESI): calcd for C17H22N5O5
[M+H]+: 376.1621, found: 376.1631; Anal. calcd for C17H21N5O5: C, 54.39; H, 5.64; N, 18.66%;
Found: C, 54.40; H, 5.68; N, 18.67%; m/z (ES)+: 376.14 [M+H]+.
4.2.4
pent-4-yn-1-yl
3-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-
yl)acetamido)-2,2-dimethylpropanoate (14). To a stirred solution of compound 12 (150 mg,
0.44 mmol) in 5 mL of DMF, K2CO3 (79 mg, 0.57 mmol) was added at room temperature. After
20 min, 5-chloropent-1-yne (0.094 mL, 0.89 mmol) was added to the reaction mixture at room
temperature and was stirred at 80°C for 12 h. As monitored by TLC, reaction mass was cooled to
0°C, diluted with water and extracted with ethyl acetate, dried over anhydrous Na2SO4, filtered
and evaporated under reduced pressure. The crude compound was triturated with DCM and n-
pentane to afford 14 (90 mg, 50%) as an off-white solid. Mp: 78–80oC; IR (KBr): υmax/ cm-1 =
3298, 3226, 3093, 2971, 2946, 2116, 1732, 1700, 1655, 1552, 1474, 1432, 1370, 1231, 1144,
1
1029, 976, 754, 709; H NMR (400 MHz, DMSO-d6): δ 8.14 (t, J = 6.0 Hz, 1H), 7.97 (s, 1H),
4.97 (s, 2H), 4.04 (t, J = 6.0 Hz, 2H), 3.41 (s, 3H), 3.25 (d, J = 6.0 Hz, 2H), 3.17 (s, 3H), 2.78 (s,
1H), 2.20 (t, J = 6.8 Hz, 1H), 1.75–1.71 (m, 2H), 1.07 (s, 6H); 13C NMR (400 MHz, DMSO-d6):
δ 176.10, 166.96, 154.80, 151.45, 148.31, 144.10, 106.85, 83.92, 72.0, 63.40, 48.51, 46.75,
43.54, 40.60, 29.86, 27.87, 27.49, 23.20, 14.94; HRMS (ESI): calcd for C19H26N5O5 [M+H]+:
404.1934, found: 404.1938; Anal. calcd for C19H25N5O5: C, 56.57; H, 6.25; N, 17.36%; Found:
C, 56.59; H, 6.28; N, 17.38%; m/z (ES)+: 404.34 [M+H]+.
4.2.5 3-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamido)-2,2-dimethyl-
N-(prop-2-yn-1-yl)propanamide (15). To a stirred solution of compound 12 (150 mg, 0.44
mmol) in DMF, T3P (0.26 mL, 0.89 mmol) and TEA (0.18 mL, 1.33 mmol) were added at room
temperature. Propargyl amine (0.056 mL, 0.089 mmol) was added to the reaction mixture and
stirred at room temperature for 16 h. On completion of the reaction as monitored by TLC, diluted
with water and extracted with ethyl acetate, dried over anhydrous Na2SO4, filtered and
evaporated under reduced pressure. The crude compound was purified by silica gel (100-200
mesh) eluted in 2% MeOH in dichloromethane to afford 15 (60 mg, 36%) as an off-white solid.

Mp: 157–160oC; IR (KBr): υmax/ cm-1 = 3321, 3066, 2969, 2123, 1740, 1704, 1654, 1558, 1530
1
1476, 1418, 1369, 1229, 1031, 977, 755, 668; H NMR (400 MHz, DMSO-d6): δ 8.06–8.0 (m,
3H), 4.99 (s, 2H), 3.85–3.83 (m, 2H), 3.43 (s, 3H), 3.22–3.19 (m, 5H), 3.06 (t, J = 2.25 Hz, 1H),
1.05 (s, 6H); 13C NMR (400 MHz, DMSO-d6): δ 175.51, 166.49, 154.32, 150.97, 147.84,
143.61, 106.39, 81.52, 72.50, 48.11, 46.48, 42.71, 29.38, 28.21, 27.41, 22.90; HRMS (ESI):
calcd for C17H23N6O4 [M+H]+: 375.1781, found: 375.1791; Anal. calcd for C17H22N6O4: C,
54.54; H, 5.92; N, 22.45%; Found: C, 54.56; H, 5.98; N, 22.47%; m/z (ES)+: 375.12 [M+H]+.
4.3 General procedure for the synthesis of compounds (16a–e)
To a stirred solution of acid compounds 3a–b (1.0 equiv) and their corresponding glycine
methyl ester hydrochloride (a), D,L-alanine methyl ester hydrochloride (b), L-valanine ethyl
ester hydrochloride (c) and L-phenyl alanine benzyl ester hydrochlorid (d) (1.1 to 1.2 equiv) in
dichloromethane was added HATU (1.2 to 1.5 equiv), DIPEA (3.0 equiv) at room temperature.
The reaction mixture was stirred at room temperature for 16 h. On completion of the reaction as
monitored by TLC, the reaction mixture was poured into water and extracted with
dichloromethane. The organic extracts was washed with aq. NaHCO3 solution and aq. KHSO4
solution, dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure to afford
the desired products (16a-e) as off-white solids.
4.3.1
Methyl
2-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamido)
acetate (16a). Compound 3a (2 g, 8.40 mmol) and glycine methyl ester hydrochloride (1.26 g,
10.081 mmol), HATU (4.78 g, 12.60 mmol) and DIPEA (4.4 mL, 25.21 mmol) in
1
dichloromethane (40 mL) were taken to afford 2.1 g (81%) of 16a as a white solid. H NMR
(400 MHz, DMSO-d6): δ 8.69 (t, J = 5.2 Hz, 1H), 8.03 (s, 1H), 5.06 (s, 2H), 3.91 (d, J = 5.6 Hz,
2H), 3.63 (s, 3H), 3.44 (s, 3H), 3.20 (s, 3H); m/z (ES)+: 310.03 [M+H]+.
4.3.2
methyl
2-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-
yl)acetamido)propanoate (16b). Compound 3a (3 g, 12.60 mmol) and D,L-alanine methyl
ester hydrochloride (2.11 g, 15.12 mmol), HATU (7.18 g, 18.90 mmol) and DIPEA (6.6 mL,
1
37.81 mmol) in dichloromethane (45 mL) gave 3.96 g (97%) of 16b as an off-white solid. H
NMR (400 MHz, DMSO-d6): δ 8.74 (d, J = 7.6 Hz, 1H), 8.01 (s, 1H), 5.03 (s, 2H), 4.33–4.29

(m, 1H), 3.62 (d, 3H), 3.43 (s, 3H), 3.20 (s, 3H), 1.30 (d, J = 7.6 Hz, 3H); m/z (ES)+: 324.21
[M+H]+.
4.3.3 (S)-ethyl 2-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamido)-3-
methylbutanoate (16c). Compound 3a (1.7 g, 7.14 mmol) and L-valanine ethyl ester
hydrochloride (1.42 g, 7.85 mmol), HATU (3.2 g, 8.57 mmol) and DIPEA (2.5 mL, 14.28 mmol)
1
in dichloromethane (42 mL) to afford 2.4 g (92%) of 16c as an off-white solid. H NMR (400
MHz, DMSO-d6): δ 8.64 (d, J = 8.28 Hz, 1H), 8.01 (s, 1H), 5.08 (s, 2H), 4.20–4.04 (m, 3H), 3.43
(s, 3H), 3.19 (s, 3H), 2.09–2.01 (m, 1H), 1.19 (t, J = 7.27 Hz, 3H), 0.90 (t, J = 7.02 Hz, 6H); m/z
(ES)+: 366.12 [M+H]+.
4.3.4 (S)-benzyl-2-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamido)-3-
phenylpropanoate (16d). Compound 3a (2.0 g, 8.40 mmol) and L-phenyl alanine bezyl ester
hydrochloride (2.6 g, 9.24 mmol), HATU (3.8 g, 10.08 mmol) and DIPEA (2.9 mL, 16.80 mmol)
1
in dichloromethane (60 mL) to afford 3.8 g (95%) of 16d as an off-white solid. H NMR (400
MHz, DMSO-d6): δ 8.87 (d, J = 7.52 Hz, 1H), 7.97 (s, 1H), 7.33–7.19 (m, 10H), 5.10–4.98 (m,
4H), 4.55 (q, J = 7.78 Hz, 1H), 3.43 (s, 3H), 3.18 (s, 3H), 3.06–2.95 (m, 2H); m/z (ES)+: 476.20
[M+H]+.
4.3.5
Methyl-2-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)propanamido)
propanoate (16e). Compound 3b (2 g, 7.933 mmol) and D,L-alanine methyl ester hydrochloride
(1.32 g, 9.520 mmol), HATU (4.52 g, 11.90 mmol) and DIPEA (4.1 mL, 23.80 mmol) in
1
dichloromethane (40 mL) gave 1.82 g (68%) of 16e as a white solid. H NMR (400 MHz,
DMSO-d6): δ 8.75 (d, J = 5.2 Hz, 1H), 8.25–8.19 (m, 1H), 5.62–5.57 (m, 1H), 4.29–4.23 (m,
1H), 3.61 (d, J = 12.8 Hz, 3H), 3.43 (d, J = 2.0 Hz, 3H), 3.21 (d, J = 6.4 Hz, 3H), 1.72–1.67 (m,
3H), 1.29 (dd, J = 1.2 Hz, 7.2 Hz, 3H); m/z (ES)+: 338.4 [M+H]+.
4.4 General procedure for the synthesis of compounds (17a–c & 17e)
LiOH.H2O (1.2 to 1.5 equiv) was added portion wise to a stirred solution of ester compounds
16a-c and 16e (1.0 equiv) in THF (10 vol) and H2O (10 vol) at room temperature and the
reaction mixture was stirred for 2-4 h. After completion, from the reaction mixture THF was

concentrated and acidified with aqueous KHSO4 solution and extracted with 10% methanol in
dichloromethane. The combined organic extracts were dried over anhydrous Na2SO4, filtered and
evaporated under reduced pressure to afford the desired products.
4.4.1 2-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl) acetamido) acetic acid
(17a). Compound 16a (2.1 g, 6.79 mmol) and LiOH.H2O (342 mg, 8.15 mmol) in 42 mL of
THF:H2O (1:1) to afford 1.6 g (80%) of 17a as a white solid. 1H NMR (400 MHz, DMSO-d6): δ
12.59 (brs, 1H), 8.57 (t, J = 5.2 Hz, 1H), 8.03 (s, 1H), 5.05 (s, 2H), 3.81 (d, J = 5.6 Hz, 2H), 3.44
(s, 3H), 3.20 (s, 3H); m/z (ES)+: 296.10 [M+H]+.
4.4.2
2-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamido)propanoic
acid (17b). Compound 16b (3.96 g, 12.25 mmol) and LiOH.H2O (610 mg, 14.70 mmol) in 78
1
mL of THF:H2O (1:1) to afford 2.6 g (68%) of 17b as a white solid. H NMR (400 MHz,
DMSO-d6): δ 12.64 (brs, 1H), 8.60 (d, J = 7.6 Hz, 1H), 8.01 (s, 1H), 5.02 (d, J = 2.8 Hz, 2H),
4.26-4.19 (m, 1H), 3.43 (s, 3H), 3.20 (s, 3H), 1.29 (d, J = 7.6 Hz, 3H); m/z (ES)+: 310.03
[M+H]+.
4.4.3
(S)-2-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamido)-3-
methylbutanoic acid (17c). Compound 16c (2.4 g, 6.57 mmol) and LiOH.H2O (610 mg, 14.70
mmol) in 40 mL of THF:H2O (1:1) to afford 1.8 g (81%) of 17c as a white solid. 1H NMR (400
MHz, DMSO-d6): δ 8.51 (d, J = 8.8 Hz, 1H), 8.02 (s, 1H), 5.08 (s, 2H), 4.19–4.16 (m, 1H), 3.43
(s, 3H), 3.19 (s, 3H), 2.11–2.02 (m, 1H), 0.91-0.88 (m, 6H); m/z (ES)+: 338.14 [M+H]+.
4.4.4 2-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)propanamido)propanoic
acid (17e). Compound 16e (1.4 g, 4.152 mmol) and LiOH.H2O (200 mg, 4.983 mmol) in 28 mL
of THF:H2O (1:1) to afford 1 g (74%) of 17e as a white solid. 1H NMR (400 MHz, DMSO-d6): δ
12.59 (brs, 1H), 8.65–8.60 (m, 1H), 8.22 (d, J = 17.2 Hz, 1H), 5.64–5.55 (m, 1H), 4.24–4.14 (m,
1H), 3.43 (s, 3H), 3.22 (s, 3H), 1.71–1.66 (m, 3H), 1.28 (d, J = 7.2 Hz, 3H); m/z (ES)+: 324.06
[M+H]+.
4.5 General procedure for the synthesis of compounds (17d)

4.5.1
(S)-2-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)acetamido)-3-
phenylpropanoic acid (17d). Pd/C (500 mg) was added portion wise to a stirred solution of
compound 16d (2 g, 4.20 mmol) in EtOH (100 mL) under H2 atmosphere at 60 Psi for 8 h. After
completion, reaction mixture was filtered on a celite bed and filtrate was evaporated under
reduced pressure to afford 1.5 g (92%) of 17d as an off-white solid. 1H NMR (400 MHz, DMSO-
d6): δ 8.65 (d, J = 7.78 Hz, 1H), 7.97 (s, 1H), 7.31–7.19 (m, 5H), 5.0 (d, J = 6.02 Hz, 2H), 4.44
(q, J = 8.03 Hz, 1H), 3.43 (s, 3H), 3.20 (s, 3H), 3.06–3.01 (m, 1H), 2.94-2.88 (m, 1H); m/z
(ES)+: 386.20 [M+H]+.
4.6 General procedure for the synthesis of compounds (18, 19 & 22)
DCC (1.5 equiv) was added to a stirred and cooled (0°C) solution of acid compounds 17a-b and
17e (1.0 equiv) and propargyl alcohol (2.0 equiv) in dichloromethane (30 vol). After 5 min,
DMAP (0.4 equiv) was added and the solution was allowed to stirred at room temperature for 16
h. After completion, the reaction mixture was added to water and extracted with 10% methanol
in dichloromethane. The combined organic extracts was dried with anhydrous Na2SO4, filtered
and evaporated under reduced pressure. The crude product was triturated with acetone and
diethyl ether, filtered to afford the desired products.
4.6.1
Prop-2-yn-1-yl-2-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)
acetamido) acetate (18). Compound 17a (1.6 g, 5.42 mmol), propargyl alcohol (0.62 mL, 10.84
mmol), DCC (1.67 g, 8.13 mmol) and DMAP (264 mg, 2.168 mmol) in 42 mL of
dichloromethane to afford 1.56 g (86%) of 18 as a white solid. IR (KBr): υmax/ cm-1 = 3281,
3111, 2953, 2130, 1754, 1708, 1659, 1548, 1474, 1457, 1384, 1376, 1217, 1190, 1029, 947, 763,
748; 1H NMR (400 MHz, DMSO-d6): δ 8.73 (t, J = 5.6 Hz, 1H), 8.03 (s, 1H), 5.07 (s, 2H), 4.73
(d, J = 2.4 Hz, 2H), 3.95 (d, J =6.0 Hz, 2H), 3.57 (t, J = 2.4 Hz, 1H), 3.44 (s, 3H), 3.20 (s, 3H);
13C NMR (400 MHz, DMSO-d6): δ 169.37, 167.27, 154.80, 151.44, 148.33, 144.14, 106.79,
78.57, 78.47, 52.66, 48.26, 33.79, 29.88, 27.90; HRMS (ESI): calcd for C14H16N5O5 [M+H]+:
334.1151, found: 334.1157; Anal. calcd for C14H15N5O5: C, 50.45; H, 4.54; N, 21.01%; Found:
C, 50.48; H, 4.56; N, 21.02%; m/z (ES)+: 334.11 [M+H]+.

4.6.2
prop-2-yn-1-yl
2-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-
yl)acetamido)propanoate (19). Compound 17b (1.8 g, 5.82 mmol), propargyl alcohol (0.67 mL,
11.64 mmol), DCC (1.80 g, 8.73 mmol) and DMAP (284 mg, 2.33 mmol) in 54 mL of
dichloromethane to afford 1.6 g (79%) of 19 as a white solid. Mp: 150–156oC; IR (KBr): υmax
/
cm-1 = 3289, 3119, 2949, 2138, 1750, 1702, 1649, 1542, 1476, 1467, 1376, 1352, 1214, 1192,
1023, 942,748; 1H NMR (400 MHz, DMSO-d6): δ 8.79 (d, J = 6.8 Hz, 1H), 8.01 (s, 1H), 5.04 (d,
J = 3.6 Hz, 2H), 4.71 (d, J = 2.8 Hz, 2H), 4.35-4.32 (m, 1H), 3.56 (s, 1H), 3.43 (s, 3H), 3.20 (s,
3H), 1.31 (d, J = 7.2 Hz, 3H); Anal. calcd for C15H17N5O5: C, 51.87; H, 4.93; N, 20.16%; Found:
C, 51.89; H, 4.98; N, 20.15%; m/z (ES)+: 348.3 [M+H]+.
4.6.3
Prop-2-yn-1-yl-2-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)
propanamido) propanoate (22). Compound 17e (1 g, 3.094 mmol), propargyl alcohol (0.35
mL, 6.189 mmol), DCC (0.95 g, 4.64 mmol) and DMAP (151 mg, 1.237 mmol) in 30 mL of
dichloromethane to afford 0.82 g (74%) of 22 as a white solid. Mp: 155–160oC; IR (KBr): υmax
/
cm-1 = 3324, 2928, 2850, 1746, 1705, 1666, 1625, 1573, 1473, 1449, 1435, 1311, 1242, 1213,
1153, 1087, 994, 748; 1H NMR (400 MHz, DMSO-d6): δ 8.80 (d, J = 6.8 Hz, 1H), 8.25–8.19 (m,
1H), 5.61–5.58 (m, 1H), 4.72–4.68 (m, 2H), 4.31–4.27 (m, 1H), 3.58–3.53 (m, 1H), 3.43 (s, 3H),
3.22 (s, 3H), 1.72–1.67 (m, 3H), 1.31 (d, J = 7.6 Hz, 3H); 13C NMR (400 MHz, DMSO-d6): δ
171.83, 169.85, 154.94, 151.34, 148.54, 142.24, 106.36, 78.42, 78.34, 54.92, 52.74, 48.23, 29.87,
28.01, 18.50, 16.95; HRMS (ESI): calcd for C16H19N5O5Na [M+Na]+: 384.1284, found:
384.1283; Anal. calcd for C16H19N5O5: C, 53.18; H, 5.30; N, 19.38%; Found: C, 53.20; H, 5.37;
N, 19.39%; m/z (ES)+: 362.4 [M+H]+.
4.7 General procedure for the synthesis of compounds (20 & 21)
To a stirred solution of compounds 17c-d (1.0 eq) in DMF, K2CO3 (1.2 to 1.3 eq) was added at
room temperature. After 20 min, propargyl bromide (2.0 eq) was added to the reaction mixture at
room temperature and stirring was continued at room temperature for 12 h. As monitored by
TLC, the reaction mass was added to water and a solid precipitate was formed that was filtered
and dried to afford the desired solid compounds.

4.7.1
(S)-prop-2-yn-1-yl 2-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-
yl)acetamido)-3-methylbutanoate(20). Compound 17c (150 mg, 0.44 mmol), propargyl
bromide (0.076 mL, 0.89 mmol), K2CO3 (71 mg, 0.57 mmol) in 5 mL of DMF to afford 70 mg
(54%) of 20 as an off-white solid. Mp: 165–170oC; IR (KBr): υmax/ cm-1 = 3284, 3246, 3117,
1
2964, 2127, 1741, 1649, 1553, 1466, 1372, 1224, 1200, 1142, 1027, 964, 746, 691; H NMR
(400 MHz, DMSO-d6): δ 8.72 (d, J = 6.72 Hz, 1H), 8.01 (s, 1H), 5.09 (s, 2H), 4.76 (s, 2H), 4.26–
13
4.23 (m, 1H), 3.59 (s, 1H), 3.43 (s, 3H), 3.19 (s, 3H), 2.08 (brs, 1H), 0.92 (brs, 6H); C NMR
(400 MHz, DMSO-d6): δ 170.52, 166.53, 154.30, 150.96, 147.79, 143.67, 106.36, 77.84, 57.27,
52.08, 47.75, 30.34, 29.37, 27.40, 18.72, 17.99; HRMS (ESI): calcd for C17H22N5O5 [M+H]+:
376.1621, found: 376.1636; Anal. calcd for C17H21N5O5: C, 54.39; H, 5.64; N, 18.66%; Found:
C, 54.42; H, 5.68; N, 18.70%; m/z (ES)+: 376.42 [M+H]+.
4.7.2
(S)-prop-2-yn-1-yl 2-(2-(1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-
yl)acetamido)-3-phenylpropanoate(21). Compound 17d (200 mg, 0.51 mmol), propargyl
bromide (0.093 mL, 1.03 mmol), K2CO3 (93 mg, 0.61 mmol) in 5 mL of DMF to afford 200 mg
(91%) of 21 as an off-white solid. Mp: 178–180oC; IR (KBr): υmax/ cm-1 = 3310, 3227, 3104,
1
2951, 2127, 1739, 1706, 1650, 1546, 1471, 1368, 1208, 1230, 1032, 969, 719, 698; H NMR
(400 MHz, DMSO-d6): δ 8.81 (d, J = 7.2 Hz, 1H), 7.94 (s, 1H), 7.27–7.19 (m, 5H), 5.0 (d, J =
6.0 Hz, 2H), 4.68 (s, 2H), 4.49 (q, J = 6.0 Hz, 1H), 3.55 (t, J = 2.0 Hz, 1H), 3.41 (s, 3H), 3.18 (s,
3H), 3.04–3.01 (m, 1H), 2.99–2.94 (m, 1H); 13C NMR (400 MHz, DMSO-d6): δ 170.76, 166.74,
154.75, 151.44, 148.27, 144.08, 137.0, 129.64, 128.72, 127.09, 106.76, 78.50, 78.40, 54.11,
52.84, 48.27, 37.10, 29.86, 27.89; HRMS (ESI): calcd for C21H22N5O5 [M+H]+: 424.1621, found:
424.1629; Anal. calcd for C21H21N5O5: C, 59.57; H, 5.00; N, 16.54%; Found: C, 59.62; H, 5.08;
N, 16.58%; m/z (ES)+: 424.16 [M+H]+.
4.8 General procedure for the synthesis of compounds (23, 24 & 25).
To a stirred solution of compounds 17b-d (1.0 eq) in DMF, K2CO3 (1.3 eq) was added at room
temperature. After 20 min, 5-chloro-1-pentyne (2.0 eq) was added to the reaction mixture at
room temperature and was heated to stirred at 80°C for 12 h. As monitored by TLC, reaction
mass was cooled to 0°C, water was added and a solid precipitate was formed that was filtered
and dried to get desired products (or) the reaction mass was added to water and extracted with