Diversifying the xanthine scaffold for potential phosphodiesterase 9A inhibitors: synthesis and validation

MEDICINAL

CHEMISTRY

RESEARCH

Medicinal Chemistry Research

https://doi.org/10.1007/s00044-021-02722-9

ORIGINAL RESEARCH

Diversifying the xanthine scaffold for potential phosphodiesterase

9A inhibitors: synthesis and validation

Nivedita Singh¹Akhtar Hussain Malik²Parameswar Krishnan Iyer²Sanjukta Patra¹

●

Received: 9 November 2020 / Accepted: 15 March 2021

Abstract

Xanthine and its derivatives have been a great area of interest for the development of potent bioactive agents. In this study,

two synthesis routes have been developed for 1,3,8-tri substituted xanthine derivatives. The synthesis routes exploits

“xanthine” as precursor molecule as it represents maximum unsubstituted sites for maximum possible substitutions. This

study divulged the reactivity pattern of three –NH groups at N₁, N₃and N₇position of xanthine in the order of N₇> N₃> N₁,

which helped in carrying out regio-selective N-alkylation reaction at different –NH sites of xanthine. Selective protection

and selective deprotection at N₃and/or N₇sites of xanthine were the key strategies for developing two synthesis schemes.

Eight newly synthesized compounds C1-8 were evaluated for their biological activity against Phosphodiesterase 9A. All the

compounds were found to be promising inhibitors. To gain further insight for mode of interaction with Phosphodiesterase

9A, these compounds were subjected to docking studies. The present study provides insight into the potential of ‘xanthine’

to contribute to the structural diversiﬁcation of xanthine derivatives in the drug development process. Xanthine based

chemical synthesis is shown to be a cost effective, fast, and highly productive method. This work can be extrapolated to ﬁnd

various selective xanthine based inhibitors targeting other enzymes.

Graphical Abstract

●

Keywords PDE9A cGMP IBMX Inhibition Xanthine Scaffold Scheme SAR

Abbreviations

PDE

cGMP

IBMX

phosphodiesterase

cyclic guanosine mono phosphate

1-methyl 3-isobutyl xanthine

Introduction

Xanthine (1H-purine-2,6 (3H,7H)-diones) is a purine based

alkaloid. It has been a platform for various compounds with

diverse medicinal properties [1–7]. In worldwide popular

beverages (tea, coffee, cocoa, etc), the presence of xanthine

based therapeutic components (such as caffeine, theophylline,

Supplementary information The online version contains

supplementary material available at https://doi.org/10.1007/s00044-

021-02722-9.

* Parameswar Krishnan Iyer

Guwahati, Assam 781039, India

pki@iitg.ernet.in

2

Akhtar Hussain Malik and Parameswar Krishnan Iyer, Department

of Chemistry, Indian Institute of Technology Guwahati,

Guwahati, Assam 781039, India

* Sanjukta Patra

sanjukta@iitg.ernet.in

1

Nivedita Singh and Sanjukta Patra, Department of Biosciences and

Bioengineering, Indian Institute of Technology Guwahati,

Medicinal Chemistry Research

theobromine, etc.) have attracted the attention of medicinal

chemists to further explore the potential of xanthine as a

scaffold for developing its synthetic derivatives [7, 8, 9] In

recent years, substitutions at xanthine scaffold have been

reported for generating important classes of pharmaceutically

active compounds with well-established biological activities

such as adenosine receptor antagonist, phosphodiesterase

(PDE) inhibitor, Topo II inhibitor, dipeptidyl peptidase 4

inhibition, acetylcholinesterase (AChE) inhibitor, inducers of

histone deacetylase activity, etc. Abdulrahman et al. [10–14].

In addition, the gamut of clinical applications of these xan-

thine derivatives has continued to widen the scope in ther-

apeutics as they have been reported as anticonvulsants, anti-

allergic, bronchodilators, and in treatment of various neuro-

degenerative diseases (Ching et al. 2006; [10])

further screened for their inhibitory activity against Phos-

phodiesterase 9A (PDE9A). PDE9A is one of the most

important regulatory enzymes of signal transduction path-

way in brain, kidney, liver, lungs and heart [20–22]. It is a

cyclic guanosine mono phosphate (cGMP) speciﬁc PDE

[23–27] playing pivotal role in signal transduction pathway.

Researchers and pharmaceutical companies have been put-

ting enormous efforts to develop PDE9A inhibitors.

BAY73-6691, a pyrazolopyrimidinone based compound

has been reported as the ﬁrst potent PDE9A inhibitor [28].

Thereafter, other potent PDE9A inhibitors such as PF-

04447943, PF-4181366, and compound 28 s were reported

[26, 29]. Most of the reported PDE9A inhibitors have

shown moderate inhibitory activity mainly because they are

constructed over “pyrazolopyrimidinone” scaffold, a com-

mon scaffold for inhibitors targeting other members of PDE

superfamily [21, 28–32]. Herein, we wish to add some new

“xanthine” based compounds to the repertoire of PDE9A

inhibitors to achieve molecular diversiﬁcation using an

improved strategy for the synthesis of xanthine derivatives.

In our earlier studies, we developed a library of new

xanthine derivatives based on in silico studies [33]. In the

present study, eight compounds were selected for chemical

synthesis (Fig. 1). Herein, the compounds selected were

bestowed with C₈substitution together with substitution at N₁

and N₃sites of xanthine. For synthesis of these derivatives

two new routes were developed using xanthine as scaffold.

Though synthetically derived xanthine derivatives are

pharmaceutically active but their complex synthesis meth-

odologies has been a bottleneck in their drug development

journey ([6, 11, 15]; Cai et al. 2013). This is mainly because

of the limited availability of standard methods for synthe-

sizing diverse xanthine derivatives. The currently available

methods are ring closure synthetic and classical condensa-

tion routes for synthesis of the xanthine derivatives e.g.,

istradefylline (KW-6002), MSX-2 and KW-3902, para-

xanthine, isoparaxanthine, MX-2 etc. ([6, 7, 11, 12, 15]; Cai

et al. 2013;). These schemes suffer from numerous bottle-

necks. Multiple synthesis steps, inaccessible chemicals

required for the process, use of toxic and expensive che-

micals and/or catalysts, use of acid/base or external oxidant,

separation of imine intermediate, high temperature, use of

hazardous solvents, lengthened reaction time, tiresome

workup, formation of by-products, low product yield are

some of these [12, 16–18] These drawbacks, make these

available methods non-viable for expansion of xanthine

derivatives library. Another approach for synthesis of xan-

thine derivatives has been the use of existing xanthine based

molecules such as Theophylline, Caffeine, Theobromine,

etc, as precursor molecule for synthesis [14, 19]. However,

major issue in using these compounds as a starting material

is their limited available sites for substitution. For instance,

in case of Theophylline, N₁and N₃sites are occupied

already by methyl groups. Thus, only N₇and C₈positions

are available for substitution [12]. Similarly, in Caffeine,

only C₈site is available for substitution. A viable alternative

synthesis method is an urgent need to address existing

issues. Abundance of xanthine both biologically and syn-

thetically makes it an ideal choice as a “starting material”

for synthesizing xanthine derivatives. It also has beneﬁts as

ability to generate a plethora of diverse derivatives using

maximum available modiﬁcation sites. The present study

attempts to use xanthine as an economical starting material

for developing synthesis schemes for biologically active

xanthine derivatives. The synthesized compounds were

Results and discussions

Synthesis of target compounds C1–C8

New series of xanthine derivatives were achieved through

efﬁcient and versatile synthesis routes (Scheme-I and

Scheme-II). These routes were developed using xanthine as

starting material as it possesses maximum available

unsubstituted positions. Thus, rather than going through

most commonly used ring closure synthesis methodologies,

present study attempted to develop new synthesis routes

using xanthine as precursor molecule. Figure 2 depicts the

combined form of two new synthesis routes where both

schemes share two common initial steps and one common

ﬁnal step. Three intermediate steps (c, d and e) in scheme-I

and ﬁve intermediate steps (g, h, I, j and k) in scheme-II are

exclusive (Fig. 2).

Bromination of xanthine (X) at its C₈position resulted

into formation of Compound 1 (step a) [13]. Brominated C₈

position was essential for C₈arylation reaction to be carried

out in the 5th step of scheme-I (step e) and scheme-II (step

i) as original xanthine has only one hydrogen atom at C₈

position. It was carried out as the ﬁrst step because selective

bromination at C₈position was not possible after protection

Medicinal Chemistry Research

Fig. 1 Chemical structure of

newly designed xanthine

derivatives

Fig. 2 Combined steps of scheme-I and scheme-II (sufﬁx ‘a’ denotes

scheme - I and ‘b’ denotes scheme – II). Reagents and Conditions: (a)

Br₂, H₂O, 100 °C, 3 h; (b) benzyl chloride, Anhydrous K₂CO₃,

Anhydrous DMF, 2.5 h, 70 °C; (c) Alkyl iodide, Anhydrous K₂CO₃,

Anhydrous DMF, 6 h, 70 °C; (d) Alkyl iodide, Anhydrous K₂CO₃,

Anhydrous DMF, 12 h, 70 °C; (e) Pd (PPh₃)₄, (f) Anhydrous K₂CO₃,

DMF, 48 h, 110 °C, inert argon atmosphere; (g) 4-methoxy benzyl

chloride, Anhydrous K₂CO₃, Anhydrous DMF, 2.5 h, 70 °C; (h) Alkyl

iodide, Anhydrous K₂CO₃, Anhydrous DMF, 12 h, 70 °C; (i) Pd

(PPh₃)₄, Anhydrous K₂CO₃, DMF, 48 h, 110 °C, inert argon atmo-

sphere; (j) TFA, conc H₂SO₄, reﬂux, 22 h. (k) Alkyl iodide, Anhy-

drous K₂CO₃, Anhydrous DMF, 12 h. (l) H₂, 10% Pd/H, Methanol,

48 h, rt

with benzyl group at N₇and/or N₃position due to the

presence of methylene group (-CH₂-) which also present

two hydrogen atom capable of interfering in C₈arylation.

Bromination reaction was followed by substitution reaction

at –NH position. In this study, substitution at N₁, N₃and C₈

positions were selected for synthesis of xanthine deriva-

tives. But the presence of three –NH groups at N₁, N₃and

N₇positions of xanthine was the most challenging part in

Medicinal Chemistry Research

the living system [34]. In the living system, natural trans-

methylation of xanthine takes place in the sequence of

xanthine→7-methylxanthine→3,7-dimethylxanthine (theo-

bromine)→1,3,7-trimethylxanthine (caffeine). This indi-

cates that the reactivity pattern of the three –NH groups

follows the sequence of N₇> N₃> N₁[34]. Thus, indubi-

tably, our substitution sequence analysis conﬁrms the

similarity of the reaction pattern of –NH groups on xanthine

both in synthetic medium and in the living system. The

present study divulges that in chemical synthesis also sub-

stitution follows the same sequential pattern as it is found in

natural system i.e., N₇> N₃> N₁substitution.

Fig. 3 Reactivity pattern of –NH groups at N₁, N₃and N₇positions of

xanthine

the selective substitution endeavor. To develop xanthine led

synthesis routes, it was essential to understand the ‘reac-

tivity pattern’ of three –NH positions of xanthine. The three

–NH sites at xanthine showed different reactivity because of

their different atomic environment. In the present study, by

concentration optimization of reactants (substituents) it was

found that –NH group at N₇position was the most reactive

site as it faced less ‘steric hindrance’ than other –NH

groups. Thus, to make the selective substitution at N₃and

N₁positions, it was essential to protect N₇position ﬁrst.

Benzyl group was introduced at N₇as leaving group or

protecting group. N₇position was selectively protected in

step b, using nucleophyllic substitution reaction which

follow SN²mechanism where concentration of both the

reactants (8-bromoxanthine (1) and benzyl chloride) have

equal role in determining the product formation. Con-

centration of benzyl chloride was optimized to 0.5 equiva-

lents of 8-bromoxanthine for selective protection at N₇

position. Selective protection was necessary to achieve

greater yield in shortest possible time avoiding the tedious

workup and costly solvents for column chromatography. It

was interesting to observe that when concentration of ben-

zyl chloride was higher than the required concentration for

selective protection at N₇position, benzyl chloride attacked

the N₃position. This indicated that –NH at N₃position

possess second reactivity rank among three –NH groups.

Likewise, after occupying all N₃positions by respective

substituent, –NH group at N₁position was attacked by alkyl

reactant. N₁position showed least reactivity as it faced

comparatively higher steric hindrance among the three –NH

groups due to positioning of its reaction center which is

surrounded by carbonyl groups at C₂and C₆positions. The

above analysis showed that reactivity pattern of –NH

groups on the xanthine scaffold followed the order of N₇>

N₃> N₁substitution, as shown in Fig. 3. However, this

sequence is contradictory to the earlier report where N₃

position was considered as the most reactive on xanthine

structure because of the highest acidity of –NH group at N₃

position among all –NH groups and therefore it was

assumed that substitution should follow the sequence of N₃

> N₇> N₁positions [34]. Surprisingly, their assumption was

contradictory to the sequence of substitution occurring in

Thus, after N₇protection, both Scheme-I and Scheme-II

follow two different routes. In Scheme-I, protection at N₇

position was followed by three independent steps while in

Scheme-II it was followed by ﬁve independent steps; ﬁnally

both schemes merged and shared the last common step (Fig. 2).

In Scheme-I (shown in Fig 4), N₇protection step was

followed by selective sequential substitution reactions - ﬁrst

at N₃position (step c) and then at N₁position (step d) of

xanthine using reported alkylation reaction with the opti-

mized concentration of the respective alkyl halides [19].

Alkylation reaction both at N₃and N₁positions followed the

2

S_Nreaction mechanism. Alkyl groups like methyl, ethyl,

propyl, n-butyl and isobutyl groups were selected for sub-

stitution reaction because of their positive biological impli-

cations such as inotropic effect, higher blood-brain barrier

permeability level and plasma protein binding efﬁciency [35].

After substitution at N₃and N₁positions, the intermediate

products obtained were subjected to Suzuki coupling reaction

for C₈arylation (step e) to install phenyl ring (with various

functional groups and side chains) at C₈position. Suzuki

coupling reaction was carried out with respective aryl-

boronic acid in presence of palladium catalyst to give aryl

substituted xanthine derivatives, 5a1–5a7. Finally, after sub-

stitution at all selected positions (N₁, N₃and C₈positions)

deprotection of N₇position was carried out by reported cat-

alytic hydrogenation reaction methodology. The ﬁnal syn-

thesized compounds obtained using scheme-I were 6a1–6a7.

Reaction mechanism for scheme-I is shown in Fig. 5.

Scheme-II (Fig. 6) is an alternate scheme designed for the

synthesis of analogous xanthine derivatives. In this scheme

N₇protection with benzyl group was followed by another

protection with 4-methoxy benzyl chloride at N₃position in

step (g) using regio-selective N-alkylation reaction. The

sequential double protection strategies at N₇and N₃position

of 8-bromoxanthine was followed by alkylation reaction at

N₁position (step h) which was then followed by Suzuki

coupling reaction for arylation reaction (step i) at C₈position.

The sequential alkylation at N₁position and arylation at C₈

position were followed by deprotection of N₃position

selectively by reported acid catalyzed method (step j) to make

N₃site available for various substituent [11]. After selective

Medicinal Chemistry Research

Fig. 4 Scheme-I Synthesis of compound 6a1–6a7 (Reagents and

Conditions: (a) Br₂, H₂O, 100 °C, 3 h, 71% (b) benzyl chloride,

Anhydrous K₂CO₃, Anhydrous DMF, 2.5 h, 70 °C, 67% (c) Alkyl

iodide, Anhydrous K₂CO₃, Anhydrous DMF, 6 h, 70 °C, 78–92%; (d)

Alkyl iodide, Anhydrous K₂CO₃, Anhydrous DMF, 12 h, 70 °C,

90–95%; (e) Pd (PPh₃)₄, Anhydrous K₂CO₃, DMF, 48 h, 110 °C, inert

argon atmosphere, 63–99%; (f) H₂, 10% Pd/H, Methanol, 48 h, rt,

50–93%)

deprotection at N₃position, alkylation reactions (step k) were

carried out for N₃substitutions. In scheme-II, after substitu-

tion at N₁, N₃and C₈positions of xanthine, another depro-

tection reaction (step l) was carried out to deprotect the N₇

position. This was a similar catalytic deprotection method

which was employed in scheme-I (step f). Thus, compound

8b1–8b2 were synthesized using scheme-II. Compound 8b1

(C5) was same compound as 6a5 (C5) obtained from

scheme-I. Compound 8b2 (C6) was synthesized by exclu-

sively using scheme-II.

Interestingly, the basis for the designing and develop-

ment of the above two synthesis routes was their selective

protection and selective deprotection strategies which were

employed at N₃and N₇sites of xanthine. In both scheme-I

and scheme-II, the ﬁnal step was the deprotection of benzyl

group at N₇position, while in scheme-II one more depro-

tection carried out at N₃position. For the deprotection at

both N₁and N₃positions_,different deprotection methods

were attempted. Both acid deprotection method and cata-

lytic hydrogenation method were applied for deprotection at

N₃and N₇positions, but these methods worked differently

at N₃and N₇positions of xanthine. Acid deprotection

method did not work for deprotection of benzyl group at N₇

position of xanthine derivatives. In both scheme-I and

scheme-II, the catalytic hydrogenation method proved as

the best method for selective deprotection at N₇position of

the xanthine derivatives when benzyl group was used as

protecting group. The selectivity of benzyl deprotection at

N₇position was conﬁrmed by testing the catalytic hydro-

genation deprotection method with compound having both

N₇and N₃position protected with benzyl group (3,7-

dibenzyl xanthine derivatives). Acid deprotection method

was another approach applied for deprotection of 3,7-

dibenzyl xanthine derivatives, where it was observed that

deprotection was selectively occurring at N₃position.

However, the yield of the deprotected compound was very

low (6%). To facilitate the selective deprotection at N₃

position with good yield more appropriate protecting group

(p-methoxy benzyl group) was used to protect the N₃

position. The presence of methoxy (an electron releasing)

group spurred the deprotection selectively at N₃position

with good yield (73%). Thus, by obtaining control on the

selective deprotection at both N₃and N₇sites of xanthine,

scheme-II was developed. In scheme-II, two sequential

selective protections were required (ﬁrst protection at N₇

position with benzyl group and second at the N₃position

with 4-methoxy benzyl group) to deprotect selectively at N₃

position in ‘step j’ selectively with acid deprotection while

catalytic deprotection method employed for selective

deprotection at N₇position in ‘step l’.

Thus, the whole synthesis work was divided into two

schemes - Scheme-I and Scheme-II, which simpliﬁed the

Medicinal Chemistry Research

Fig. 5 Reaction mechanism for

Scheme-I

reactivity and substitution pattern of different –NH groups

at xanthine scaffold. Scheme-I can be applied for those

compounds having common N₃substitutions with diverse

N₁and C₈substitutions. Likewise, scheme-II can be applied

for the synthesis of compounds having common N₁and C₈

substituent with different N₃substitutions. It was apparent

from the synthesis of two compounds 8b1 and 8b2 using

one single synthesis pathway of scheme-II. These two

compounds varied only at N₃position. Compound 8b1

caries iso-butyl group at N₃position whereas compound

8b2 carries n-butyl group. Thus, the two separate schemes

are designed to construct diverse library of xanthine deri-

vatives. Comparing with the existing methods, xanthine

based synthesis mechanism has numerous advantages such

as mild reaction conditions, cost-effectiveness and readily

available reagents, use of non-hazardous chemicals, no need

for chromatographic clean up in most of the steps, shorter

reaction time, multiple compounds generation using single

scheme and better product yields. Due to these advantages,

the above schemes are better alternatives as compared to

Medicinal Chemistry Research

Fig. 6 Scheme-II Synthesis of

compound 8b1–8b2 (Reagents

and Conditions: (a) Br₂, H₂O,

100 °C, 3 h, 71%; (b) benzyl

chloride, Anhydrous K₂CO₃,

Anhydrous DMF, 2.5 h, 70 °C,

67 %; (g) 4-methoxy benzyl

chloride, Anhydrous K₂CO₃,

Anhydrous DMF, 2.5 h, 70 °C,

70%; (h) Alkyl iodide,

Anhydrous K₂CO₃, Anhydrous

DMF, 12 h, 70 °C, 88%; (i) Pd

(PPh₃)₄, Anhydrous K₂CO₃,

DMF, 48 h, 110 °C, inert argon

atmosphere; 72%; (j) TFA, conc

H₂SO₄, reﬂux, 22 h, 73%; (k)

Alkyl iodide, Anhydrous

K₂CO₃, Anhydrous DMF, 12 h,

84–95%; (l) H₂, 10% Pd/H,

Methanol, 48 h, rt, 71–77%)

existing methods which have been in use worldwide so far

for the synthesis of xanthine derivatives.

38.55 µM) with ethyl group. Compounds having modiﬁca-

tions with isomeric fragments showed very little difference

in the inhibitory afﬁnity for PDE9A. Interestingly, IC₅₀

values of compound C6 and compound C5 are essentially

the same indicating that nature of the bond length (linear or

branched) at N₃position does not inﬂuence the inhibition

activity. Substitution at C₈position with aryl fragment

created signiﬁcant impact on inhibitory effect of com-

pounds. 1-methyl 3-isobutyl xanthine (IBMX, a synthetic

xanthine derivative) has been reported as non-inhibitor for

PDE9A [38]. The addition of aryl fragment (phenyl ring

with aliphatic side chain/functional group at meta position)

at C₈position of xanthine ring showed a signiﬁcant impact

in increasing the inhibitory afﬁnity of compounds. For

instance, the presence of isopropyl phenyl ring at C₈posi-

tion generates inhibition potential in C₈substituted IBMX

i.e., compound C4 (IC₅₀= 76.19 µM). Furthermore, repla-

cing methyl group with functional group (such as ﬂoro

group in compound C8) at meta-position of phenyl sub-

stituent at C₈position increases the potency of compound

towards PDE9A. However, increase in alkyl chain length

had more impact in enhancing the inhibitory afﬁnity of

compound than using phenyl ring with functional group at

C₈position of xanthine derivatives. Thus, the presence of

phenyl substituent had signiﬁcant role in generating the

inhibition potential in the designed compounds.

Biological activity

Inhibition studies of synthesized compounds

Bio-active nature of newly synthesized compounds was

conﬁrmed by inhibition studies based structure activity rela-

tionship (SAR) analysis. All the synthesized compounds

were tested to check their capability of inhibition towards the

catalytic action of PDE9A using standard in vitro malachite

green spectrophotometric enzymatic assay [36, 37]. The

results are summarized in Table 1. Figure S98 (in supple-

mentary ﬁle) represents the inhibition response plot of eight

newly synthesized compounds towards PDE9A (Fig. 7).

As seen from data of Table 1, C6 shows best inhibition

(IC₅₀= 38.27 µM) among all synthesized compounds. IC₅₀

of synthesized compound suggested that increasing the

chain length at N₁, N₃and meta position of phenyl sub-

stituent at C₈positions of xanthine has immense potentiality

to increase the inhibition afﬁnity of these compounds

towards PDE9A. Increasing alkyl chain length at N₁posi-

tion showed relatively better inhibition afﬁnity than

increasing alkyl chain length at N₃position. For instance, in

compound C1, substitution of methyl group at N₁position

showed nearly two fold less inhibition afﬁnity (IC₅₀

=

91.83 µM) than ethyl group substituted compound C2 (IC₅₀

= 50.46 µM) at N₁position. Similarly, compound C4 (IC₅₀

= 76.19) with methyl group at N₁position showed two fold

Thermal shift assay of synthesized compounds

Thermal stability of the enzyme-inhibitor complex is one of

less inhibition strength than compound C5 (IC₅₀

=

the parameters to examine the stability of inhibitor–enzyme

Medicinal Chemistry Research

Table 1 Chemical structure of

inhibitors (C1–C8) and their

SAR analysis (IC₅₀) with

PDE9A (amino acid 181–506)

Entry Substituted positions at the xanthine IC₅₀of PDE9A (in µM) Afﬁnity (kcal mol⁻¹) AutoDock 4.2

scaffold

R₁

R₂

R₃

C1

C2

–CH₃–C₃H₇

–C₂H₅–C₃H₇

91.83 1.98

50.46 2.21

−8.92

−9.16

C3

C4

–C₂H₅–C₃H₇

86.86 2.43

76.19 1.60

−8.40

−9.16

–CH₃–CH₂CH(CH₃)₂

C5

C6

–C₂H₅–CH₂CH(CH₃)₂

–C₂H₅–C₄H₁₀

38.55 1.90

38.28 1.63

−9.40

−9.21

C7

C8

–C₂H₅–C₄H₁₀

62.60 2.6

−8.72

−8.30

51.82 2.25

Table 2 Melting temperature of substrate and inhibitor bound PDE9A

Compounds cGMP C1 C2 C3 C4 C5 C6 C7 C8

ΔT_m(°C)

53.35 53.75 53.75 52.7 53.35 54.35 53.35 52.75 52.7

was 53.35 °C. The magnitude of T_mshift was more or less

similar for all the synthesized compounds. However, these

values do not always reﬂect the relative binding afﬁnities

[39]. This might be reﬂected by the dependence on con-

tribution of enthalpy and entropy of the compounds [39].

Among all synthesized compounds, C5 showed highest T_mof

54.35 °C while compound C4 and C6 showed T_mvalue equal

to the T_mof substrate-protein complex. From this analysis it

was concluded that the synthesized compounds were bound

and could stabilize the protein in similar manner as was

analyzed in the case of substrate (cGMP) bound protein.

Fig. 7 Thermograph of PDE9A protein bound with chemically syn-

thesized inhibitors (C1–C8)

interaction [39]. In this study, the stability of the PDE9A in

complex with synthesized compounds was determined by

Differential scanning ﬂuorimetry (DSF). Figure 7 illustrates

the thermograph of all synthesized compounds in PDE9A

bound form. Table 2 represents the calculated melting tem-

perature of substrate bound PDE9A and inhibitor bound

PDE9A. T_mof substrate (cGMP) bound PDE9A was used as

a reference point to analyze the stability of protein in inhibitor

bound state. The calculated T_mof cGMP-PDE9A complex

Comparative study of chemically synthesized compounds

(C1–C8) and reported virtual screened compound

ZINC62579975 with PDE9A

The comparative structure activity relationship analysis of

best performing chemically synthesized compounds (C5

Medicinal Chemistry Research

and C6) and reported virtual screened compound

ZINC62579975 (3-butyl-7-methyl-8-[[4-(2-phenoxyethyl)

piperazin-1-yl]methyl] purine 2,6-dione) [40] were carried

out which gave insight over the modiﬁcation required for

improving potency in compound for PDE9A inhibition.

Best performing synthesized compounds (C5 (38.55 µM)

and C6 (38.27 µM)) showed better potency than reported

ZINC compound (46.96 µM) [40]. The probable reason for

this is discussed in the molecular docking section. Fur-

thermore, to understand the stability of synthesized com-

pound over reported ZINC compound in protein active

site, thermal shift assay was performed. ZINC62579975

(47.75 °C) showed comparatively lower Tm than Tm of the

synthesized compounds. This result ascertained that syn-

thesized compounds showed better stability in PDE9A than

ZINC62579975. The higher Tm shift of synthesized com-

pounds was due to more entropically driven binding. The

entropically driven binding was mainly hydrophobic bind-

ing. This was because of the presence of phenyl substituent

with alkyl groups at C₈position. The stability of protein in

inhibitor bound form was important to ensure the stability

of interaction between protein and ligand. Figure 8 repre-

sents the comparative thermograph of compound C6 and

ZINC62579975.

Fig. 8 Thermograph of PDE9A in complex with compound C6 and

ZINC62579975

Table 3 Docking result of eight newly synthesized inhibitors

with PDE9A

Entry Lowest

free energy

Interacting residues Conformations in H-bond

largest cluster

of binding

(kcal/mol)

C1

C2

C3

C4

C5

C6

C7

C8

−8.92

−9.16

−8.40

−9.16

−9.40

−9.21

−8.72

−8.30

GLN453, PHE456 100

GLN453, PHE456 99

GLN453, PHE456 100

2

GLN453, PHE456

95

88

79

93

Molecular docking details

To reveal nature of differences of synthesized compounds in

terms of interaction pattern, orientation of compound,

binding afﬁnity towards the target protein, etc., molecular

docking studies were carried out using AutoDock 4.2

(Table 3). In this study, 2HD1 structure was selected from

protein data bank taking into account its quality in terms of

resolution and similarity with the co-crystallized compound

with studied ones [38]. All synthesized compounds showed

similar interaction pattern in the active site pocket of

PDE9A by forming two hydrogen bonds with invariant

GLN453. The presence of unsubstituted N₇position and

carbonyl group (C = O) at C₆position of new xanthine

derivatives built strong double hydrogen bond interaction

with the side chains of GLN453 of PDE9A. However, they

varied in their binding scores because of their varied sub-

stituent. Increasing chain length at N₁and N₃positions

increased the binding score of compounds towards the tar-

get. Among all synthesized compounds, compound C5

showed highest binding afﬁnity towards PDE9A with

lowest free energy of binding of −9.40 kcal/mol. In this

study, enhancement in the activity of newly developed

inhibitors towards PDE9A was compared with IBMX

(Fig. 9) and ZINC62579975 (Fig. 10).

GLN453, PHE456 100

PDE9A to show inhibitory effect. Verhoest et al. [31] With

increasing the size of synthesized compounds, binding

afﬁnity towards target protein enhanced. In fact, in bound

state of PDE9A, IBMX and synthesized inhibitors showed

similar interaction pattern with invariant GLN453, a key

residue in substrate/inhibitor selectivity. However, in terms

of the binding scores based on lowest free energy of bind-

ing, synthesized compounds (For example, compound C5

(−9.40 kcal/mol)) showed better binding score than IBMX

(−5.71 kcal/mol) towards PDE9A. The addition of C₈

substitution in newly synthesized compounds signiﬁcantly

increased the binding afﬁnity of compounds towards

PDE9A. It was because phenyl substituent at C₈position

was placed towards the hydrophobic region in active site

pocket of PDE9A and thus established strong hydrophobic-

hydrophobic interaction between ligand and protein. Resi-

dues present at hydrophobic region of PDE9A active site

were LEU420, LEU421, PHE441, VAL447 and ALA452

(Fig. 9). With increasing the chain length at meta position of

phenyl substituent, the hydrophobic-hydrophobic interac-

tion between ligand and protein became strong further.

Additionally, with aryl ring substitution at C₈position, the

IBMX has N₁and N₃substitutions with methyl and

isobutyl groups respectively; yet the size of the compound

was relatively small to occupy the active site pocket of

Medicinal Chemistry Research

Fig. 9 Interaction studies of

compound C5 and IBMX with

PDE9A as seen in PyMol

Fig. 10 Interaction studies of

compound C6 and

ZINC62579975 with PDE9A

compounds acquired bigger size to occupy larger area of the

active site pocket and established strong interaction with

protein. This led to more appropriate ﬁtting of the com-

pound in the active site pocket of PDE9A. Thus, both

in vitro and in silico studies showed relative positive impact

of increased size of xanthine derivatives in enhancing the

inhibition potential of compounds towards PDE9A.

than alkylated aryl substituent in ZINC62579975. Phenyl

substituent in synthesized compounds established

hydrophobic-hydrophobic interaction in protein-ligand

complex which was lacking in case of ZINC62579975.

This was apparent from the comparative interaction pattern

analysis of compound C6 and ZINC62579975 in PDE9A

active site as shown in Fig. 10.

In ZINC62579975, unsubstituted –NH at N1 position and

carbonyl group at C6 position together were forming strong

Hydrogen bond with GLN453. It was different from the

positioning of groups forming hydrogen bond interaction

with GLN453 in case of synthesized compounds where

unsubstituted –NH group at N7 position and carbonyl group

at C6 position together interacting with GLN453. As inter-

action with GLN453 was important for generating potency

towards PDE9A, hence, from this analysis it can be con-

cluded that availability of unsubstituted NH group is

required either at N₁or N₃together with carbonyl group at

C₆. As evident from in vitro studies, the presence of alkyl

group at N₃position has important role in generating binding

strength in xanthine derivatives. Both ZINC62579975 and

compound C₆have butyl group at N₃position, so reactivity

of these compounds not differed due to N₃position. C₈

position was occupied in both compounds but by different

types of substituent. The presence of phenyl substituent at C₈

position in synthesized compound showed better interaction

In depth, the structure activity relationship analysis by

biological studies and molecular docking studies suggest that

“xanthine” core with N₁, N₃and C₈substituents present

potential platform for designing compounds C1–C8

(Fig. 11). In conclusion, we observed that together N₇and

C = O at C₆position determine the ‘binding afﬁnity’ of

compounds. N₁position is essential to determine the selec-

tivity of compound towards PDEs. N₃determines the

strength of binding of interms of binding energy inside the

active site pocket of PDEs. Phenyl substituent bound at C₈

ensures the orientation towards the hydrophobic site of

the PDE.

Drug-likeness details

As ﬁnal validation, drug-likeness properties, ADMET

properties and toxicity prediction of all synthesized com-

pounds were performed using PreADMET online server

(https://preadmet.bmdrc.kr/). All the synthesized compounds

Medicinal Chemistry Research

Fig. 11 Structure activity relationship of designed scaffold of xanthine

derivatives

(C1–C8) fulﬁlled the Lipinsiki rule of ﬁve and other drug-

likeness properties such as MDDR rule, WDI rule, CMC

like rule etc. PDE9A has abundance in brain, in physiolo-

gical condition to inhibit PDE9A; the compound should

have predicted blood-brain barrier (BBB) permeability of

greater than 2. Among the synthesized compounds, com-

pound C5 (4.25033) and compound C6 (4.52008) possess

comparatively higher blood-brain barrier (BBB) perme-

ability. The higher BBB permeability supported the suit-

ability of these compounds to target PDE9A in brain to treat

various neurodegenerative diseases. While ZINC62579975

(0.210766) showed relatively very poor BBB permeability

[36]. Hence, all pharmacokinetic properties of synthesized

compounds are come within the reasonable range deﬁned for

human use. Table 4 shows the pharmaceutical properties of

the eight synthesized compounds.

Conclusion

In summary, this investigation has provided vital con-

tribution in xanthine based drug development by developing

two new schemes using xanthine as a precursor molecule.

As the presence of three –NH groups was the most chal-

lenging part in the synthesis initiated with xanthine, the

selective protection and selective deprotection strategy was

adopted. The present study suggested the reactivity of –NH

groups of xanthine follows the order of N₇> N₃> N₁. This

gave us reason to use xanthine directly for synthesis of its

derivatives rather than going for tedious conventional ring

closure and classical condensation methods. Two schemes

were developed to avoid the need to repeat the reaction

every time for new combination of substitutions at N₁, N₃

and C₈positions. The strategy used in the present study

enables the construction of a diverse library of drug-like

xanthine derivatives using all possible substitution sites

without disturbing the basic scaffold of xanthine. The

bioactive nature of synthesized compounds was further

conﬁrmed by using structure activity relationship analysis

Medicinal Chemistry Research

and thermal shift assay. The in silico structure activity

relationship studies were supported with biological eva-

luation and in most of cases SAR explained the nature and

electron effect of substituent on the xanthine ring and pre-

sent prospect for future drug development. Thus, the present

study strongly proposes the newly developed schemes

methodology along with the moiety of xathine scaffold

(Fig. 10) have immense potential to deliver biologically

active compounds in the shortest possible time in a cost

effective manner.

was vacuum dried. White powderd product (2) was

obtained with 67% yield (0.28 g). ¹H NMR (600 MHz,

DMSO-d₆) δ: 11.804 (s, 1H), 11.061 (s, 1H), 7.35 (t, J =

7.41, 7.41 Hz, 2H), 7.30 (t, J = 7.22, 7.22 Hz, 1H), 7.23 (d,

J = 7.77 Hz, 2H), 5.442 (s, 2H); ¹³C NMR (150 MHz,

DMSO-d₆) δ: 155.2, 151.2, 149.1, 136.1, 129.1, 128.8,

128.3, 127.4, 108.9, 49.6; HRMS (ES⁺) m/z: calculated for

C₁₂H₉BrN₄O₂321.9888, found 321.94, 322.94 (MH⁺).

Synthesis of 7-benzyl-8-bromo-3-propyl-1H-purine-2, 6 (3H,

7H)-dione) (3a1)

Materials and methods

To a mixture of compound 2 (7-benzyl-8-bromo-1H-purine-

2, 6 (3H,7H)-dione) (307 mg, 0.956 mmol) and anhydrous

K₂CO₃(132 mg, 0.956 mmol) in a 2 mL anhydrous DMF,

47.36 µL propyl iodide (0.487 mmol) was added and then

reaction mixture was allowed to stir at 70 °C for 6 h. After

the completion of reaction, the reaction ﬂask was kept on

ice for 10 min. 10% diluted HCl was added drop-wise to

neutralize the product mixture. After that water was added

in the neutralized solution. With addition of water white

color precipitate formed which was ﬁltered and washed 3-4

times with water followed by drying in oven at 40 °C. The

resultant solid powder was the mixture of two compounds-

7-benzyl-8-bromo-3-propyl-1H-purine-2, 6 (3H,7H)-dione

and 7-benzyl-8-bromo-1,3-dipropyl-1H-purine-2,6(3H,7H)-

dione. The products were puriﬁed by column chromato-

graphy. The major product was 7-benzyl-8-bromo-3-pro-

pyl-1H-purine-2, 6 (3H,7H)-dione (Compound 3a1) in

white powdered form. The ﬁnal yield of the compound 3a1

(7-benzyl 8-bromo 3-propyl xanthine) was 78 % (270 mg).

¹H NMR (600 MHz, DMSO-d₆)δ: 11.33(s, 1H), 7.36 (t,

J = 7.57, 7.57 Hz, 2H), 7.31(t, J = 7.31, 7.31 Hz, 1H), 7.26

(d, J = 7.17, 1H), 5.48 (s, 2H), 3.83 (t, 2H), 1.65 (sext, 2H),

0.874 (t, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ: 154.1,

150.3, 149.1, 135.6, 128.8, 128.5, 127.9, 127.2, 108.8, 49.3,

43.4, 20.8, 10.9; HRMS (ES⁺) m/z: calculated for

C₁₅H₁₅BrN₄O₂363.2092, found 363.0453, 364.0453 (MH⁺).

Chemistry

¹H NMR and ¹³C NMR spectra were recorded using

600 MHz NMR spectrophotometer in DMSO-d₆using Tetra

Methyl Silane (TMS) as internal standard. Chemical shifts

are denoted in ppm (δ) relative to TMS (¹H). Routine mass

spectra (HRMS) were performed on Agilent Mass Spec-

troscopy using ESI as positive mode.

General procedure for synthesis of 8-bromo-1H-purine-2, 6

(3H, 7H)-dione) (1)

To a mixture of Xanthine (1 g, 657 mmol) in 6.5 mL water

in a glass tube, 711 µL (13.7 mmol) of concentrated bro-

mine (Br₂) solution was added and capped tightly. The

reaction was allowed to stir at 100 °C for 2 h. After com-

pletion of the reaction, product mixture was allowed to cool

at room temperature and ﬁltered. The yellowish solid was

washed with water 2-3 times which followed washing with

diethyl ether (Et₂O) under vacuum. The product was dried

under oven at 40 °C. Light yellowish powdered compound

1

(1) was obtained with yield of 71 % (1.08 g). H NMR

(600 MHz, DMSO-d₆) δ: 14.00 (s, 1H), 11.66 (s, 1H), 10.92

(s, 1H); ¹³C NMR (150 MHz, DMSO-d₆) δ: 154.5, 151.1,

148.8, 124.0, 109.5; HRMS (ES⁺) m/z: calculated for

C₅H₃BrN₄O₂231.0069, found 231.01, 232.0058 (MH⁺).

Synthesis of 7-benzyl-8-bromo-3-isobutyl-1H-purine-2, 6

(3H, 7H)-dione) (3a2)

Synthesis of 7-benzyl-8-bromo-1H-purine-2,6(3H,7H)-dione

(2)

To a mixture of Compound 2 (385 mg, 0.12 mmol) and

anhydrous K₂CO₃(0.33 mg, 0.239 mmol) in a 1 mL anhy-

drous DMF, 137.96 µL isobutyl iodide (0.12 mmol) was

added. The reaction mixture was then stirred at 70 °C for

6 h. The post-reaction processing followed the same pro-

cedure used for 3a1. The resultant powder was the mixture

of two compounds- 7-benzyl-8-bromo-3-isobutyl-1H-pur-

ine-2,6 (3H,7H)-dione and 7-benzyl-8-bromo-1,3-diisobu-

tyl-1H-purine-2,6(3H,7H)-dione. Column chromatography

was carried out to separate two products. 7-benzyl-8-

bromo-3-isobutyl-1H-purine-2,6 (3H,7H)-dione (compound

To a mixture of 8-Bromo xanthine (8-bromo-1H-purine-2, 6

(3H,7H)-dione) (0.3 g, 1.298 mmol) in 3 mL anhydrous

DMF, 1.298 mmol K₂CO₃and 80.6 µL benzyl chloride

(0.7 mmol) were added in a 25 mL round bottom ﬂask.

Reaction was allowed to run at 70 °C for 2.5 h on silica

bath. After the completion of reaction, product mixture was

kept on ice for 10 min. 10% HCl was added to neutralize the

product mixture. White color precipitate was formed which

was ﬁltered and washed 2-3 times with water. The product

Medicinal Chemistry Research

3a2) was the major product as white powdered form with

ﬁnal yield of 83% (373 mg). H NMR (600 MHz, DMSO-

2H), 7.31(t, J = 1.81, 9.03, 1H), 7.26 (m, 2H,), 5.48 (s, J =

3.76, 2H), 3.9 (dd, J = 6.80, 7.91, 2H), 3.22 (s, J = 3.03,

3H), 1.67 (sext, 2H), 0.88 (t, J = 7.45, 7.45 Hz, 3H); ¹³C

NMR (150 MHz, DMSO-d₆) δ: 153.7, 150.3, 147.5, 135.5,

128.7, 128.2, 127.9, 127.1, 108.4, 49.4, 44.4, 27.8, 10.9;

HRMS (ES⁺) m/z: calculated for C₁₆H₁₇BrN₄O₂377.0657,

found 377.0603.

1

d₆) δ: 11.33(s, 1H), 7.36 (t, J = 7.42,7.42 Hz, 2H), 7.31(dd,

J = 5.44, 9.10 Hz, 1H), 7.26 (d, J = 7.34 Hz, 2H,), 5.47 (s,

2H), 3.70 (d, J = 7.48 Hz, 2H), 2.15 (m, 1H), 0.87(d, 6H);

¹³C NMR (150 MHz, DMSO-d₆) δ: 154.1, 150.3, 149.3,

128.7, 127.9, 127.1, 108.8, 49.5, 48.8, 26.7, 19.7; HRMS

(ES⁺) m/z: calculated for C₁₆H₁₇BrN₄O₂377.0657, found

377.0609.

Synthesis of 7-benzyl-8-bromo-1-ethyl-3-propyl-1H-purine-

2,6 (3H,7H)-dione)(4a2)

Synthesis of 7-benzyl-8-bromo-3-butyl-1H-purine-2, 6

(3H,7H)-dione) (3a3)

To a mixture of compound 3a1 (500 mg, 1.38 mmol) and

anhydrous K₂CO₃(381 mg, 2.75 mmol) in 5 mL anhydrous

DMF, 220 µL ethyl iodide (2.75 mmol) was added. The

reaction was allowed to stir at 70 °C for 12 h. After com-

pletion of the reaction, it was allowed to cool on ice for

10 min. The product mixture was neutralized with 10%

HCl. As white precipitate began to appear, water was added

to the neutralized mixture. After addition of water the

product was accumulated in a white precipitated form. The

whitish precipitate was ﬁltered and washed with water 3-4

time. Product was allowed to dry at 40 °C in a hot air oven

to obtain white powder (compound 4a2). The ﬁnal yield of

the compound 4a2 (7-benzyl-8-bromo-1-ethyl-3-propyl-

To a mixture of 7-benzyl 8-bromo xanthine (7-benzyl-8-

bromo-1H-purine-2, 6 (3H,7H)-dione) (90 mg, 0.28 mmol)

and anhydrous K₂CO₃(0.39 mg, 0.280 mmol) in 1 mL

anhydrous DMF, 22.6 µL propyl iodide (0.199 mmol) was

added. The reaction then stirred at 70 °C for 6 h. The post-

reaction processing and work-up followed the same proce-

dure used for 3a1. The powder obtained was a mixture of

two compounds- 7-benzyl-8-bromo-3-butyl-1H-purine-2, 6

(3H,7H)-dione and 7-benzyl-8-bromo-1,3-dibutyl-1H-pur-

ine-2,6(3H,7H)-dione. The product mixture was puriﬁed by

column chromatography. 7-benzyl-8-bromo-3-butyl-1H-

purine-2, 6 (3H,7H)-dione (compound 3a3) was the major

product as white powdered form with a ﬁnal yield of 85 %

1

1H-purine-2,6(3H,7H)-dione) was 95% (510 mg). H NMR

(600 MHz, DMSO-d₆) δ: 7.35 (t, J = 7.40, 7.40, 2H), 7.29

(t, J = 7.35, 7.35, 1H), 7.24 (d, J = 7.31, 2H), 5.55 (s, 2H),

3.89 (q, J = 6.99, 6.99, 6.81, 4H), 1.66 (m, 2H), 1.09 (t,

J = 7.00, 7.00, 3H), 0.87 (t, J = 7.44, 7.44, 3H); ¹³C NMR

(150 MHz, DMSO-d₆) δ: 153.3, 149.9, 147.6, 135.6, 128.3,

127.9, 127.0,1 108.4, 49.3, 44.3, 35.7, 20.7, 12.9, 10.9;

HRMS (ES⁺) m/z: calculated for C₁₇H₁₉BrN₄O₂391.2623,

found 391.0778.

1

(96 mg). H NMR (600 MHz, DMSO-d₆) δ: 11.32(s, 1H),

7.34 (t, J = 7.36, 7.36 Hz, 2H), 7.29 (t, J = 7.05, 7.05 Hz,

1H), 7.23 (d, J = 7.87, 2H), 5.46 (s, 2H), 3.85 (t, J = 7.30,

2H), 1.59 (quint, 2H), 1.28 (sext, 2H), 0.88 (t, J = 7.4,

7.4 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ: 154.0,

150.2, 149.0, 135.5, 128.8, 128.1, 127.9, 127.2, 108.8,

49.3, 41.7, 29.6, 19.4, 13.6; HRMS (ES⁺) m/z: calculated

for C₁₆H₁₇BrN₄O₂377.0657, found 377.0629

Synthesis of 7-benzyl-8-bromo-3-isobutyl-1-methyl-1H-

purine-2,6 (3H,7H)-dione) (4a3)

Synthesis of 7-benzyl-8-bromo-1-methyl-3-propyl-1H-

purine-2, 6(3H,7H)-dione)(4a1)

To a mixture of compound 3a2 (812 mg, 2.15 mmol) and

anhydrous K₂CO₃(595 mg, 4.3 mmol) in 10 mL anhy-

drous DMF, 268 µL methyl iodide (3.47 mmol) was added.

The reaction was then stirred at 70 °C for 12 h. After

completion of the reaction, ﬂask was allowed to cool at

room temperature. The product mixture was diluted with

ethyl acetate and transferred to a separating funnel. The

organic part was extracted from the aqueous part by

washing with water and brine alternatively and dried with

sodium sulfate. The product mixture was vacuum dried in

a rotary evaporator. Dark yellowish product mixture was

obtained. The product mixture was puriﬁed by column

chromatography using ethyl acetate-hexane (10:90). The

white powder (compound 4a3) was obtained. The ﬁnal

yield of the product was 93% (780 mg). ¹H NMR

(600 MHz, DMSO-d₆) δ: 7.36 (t, J = 7.41, 7.41 Hz, 2H),

To a mixture of compound 3a1 (630 mg, 1.73 mmol) and

anhydrous K₂CO₃(480 mg, 3.47 mmol) in 8 mL anhydrous

DMF, 216 µL methyl iodide (3.47 mmol) was added. The

reaction mixture was then stirred at 70 °C for 12 h. After

completion of the reaction, ﬂask was allowed to cool at

room temperature. The product mixture was diluted with

ethyl acetate. Organic part was separated from the aqueous

part by using water and brine alternatively. At the end of

extraction, the organic part was isolated and dried with

sodium sulfate. The yellowish solution obtained was

vacuum dried in rotary evaporator. The yellowish mixture

obtained was puriﬁed by column chromatography with

ethyl-hexane (10:90) solvent. Puriﬁed whitish powder pro-

1

duct (4a1) was obtained with a yield of 92% (604 mg). H

NMR (600 MHz, DMSO-d₆) δ: 7.36 (dd, J = 4.57, 10.13,

Medicinal Chemistry Research

7.31(t, J = 7.31, 7.31, 1H), 7.26 (d, J = 7.31 Hz, 2H), 5.52

(s, 2H), 3.85 (d, J = 7.48 Hz, 2H), 3.23 (s, 3H), 2.17 (sept,

1H), 0.88 (d, 6H); ¹³C NMR (150 MHz, DMSO-d₆) δ:

153.3, 149.9, 147.6, 135.5, 128.3, 127.9, 127.1, 108.4,

49.3, 44.3, 35.7, 20.7, 12.9, 10.9; MS (+ESI) m/z: cal-

culated for C₁₇H₁₉BrN₄O₂391.2623, found 391.07.

Synthesis of 7-benzyl-8-(3-isopropylphenyl)-1-methyl-3-

propyl-1H-purine-2,6(3H,7H)–dione (5a1)

A mixture of compound 4a1 (226 mg, 0.599 mmol), 3-

isopropyl phenyl boronic acid (196 mg, 1.2 mmol), anhy-

drous K₂CO₃(166 mg, 1.2 mmol) was taken in 25 mL

round bottom ﬂask. Then tertakis (triphenylphosphine)

palladium (36 mg, 0.03 mmol) was added under argon

atmosphere in the ﬂask. The reaction was then stirred at

110 °C for 2 days. After completion of the reaction, product

mixture was allowed to cool at room temperature. 10 mL

water was added to the product mixture and stirred for

10 min. Upon cooling, the reaction mixture darkened and

black emulsion appeared on the upper layer of the solution.

The reaction mixture was then diluted with ethyl acetate and

transferred to a separating funnel. Two layers were formed,

the organic layer was extracted with ethyl acetate. Organic

extract was washed with 5% sodium carbonate solution and

brine sequentially. After extraction, the organic phase was

transferred to a 250 mL Erlenmeyer ﬂask equipped with a

magnetic stir bar. Activated charcoal (0.50 g) and sodium

sulfate (2 g) were added to the ﬂask. This mixture was

stirred for 10 min. The solution was then ﬁltered through

1 cm celite bed. The resulting pale yellow solution was

concentrated under reduced pressure to yield the crude

product in oil form. The product was puriﬁed by column

chromatography (10% ethyl acetate: 90% Hexane). The

product 5a1 formed was yellowish oily in nature with yield

of 88 % (220 mg). ¹H NMR (600 MHz, DMSO-d₆): 7.45 (d,

J = 5.71 Hz, 1H), 7.42 (d, J = 7.74 Hz, 1H), 7.35 (t, J =

7.69, 7.69 Hz, 2H), 7.30 (s, 1H), 7.26 (d, J = 7.33 Hz, 2H),

7.01 (d, J = 7.50 Hz, 1H), 5.63 (s, 2H), 4.02 (t, J = 7.18,

7.18 Hz, 2H), 3.24 (s, 3H), 2.85 (dt, J = 6.30, 6.30,

12.43 Hz, 1H), 1.75 (m, 2H), 1.09 (s, 3H), 1.09 (s, 3H), 0.92

(t, J = 7.39, 7.39 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆)

δ: 154.5, 151.9, 150.7, 148.9, 147.7, 137.2, 128.9, 128.8,

127.8, 126.7, 126.4, 125.9, 107.4, 48.8, 44.3, 33.2, 27.6,

23.5, 20.9, 11.1. calculated for C₂₅H₂₈N₄O₂416.5154,

found 416.5114.

Synthesis of 7-benzyl-8-bromo-1-ethyl-3-isobutyl-1H-

purine-2,6(3H,7H)-dione (4a4)

To a mixture of compound 3a2 (0.224 g, 0.646 mmol) in

3 mL anhydrous DMF, 0.178 mmol anhydrous K₂CO₃and

104 µL (0.129 mmol) of ethyl iodide was added. Reaction

was allowed to run at 70 °C for 12 h on silica bath. After

completion of the reaction, product was extracted with

ethyl acetate and water sequentially. The extracted organic

part was dried with sodium sulfate. Yellowish reaction

mixture was obtained after vacuum evaporation. Reaction

mixture was puriﬁed by column chromatography. White

powdered compound 4a4 was obtained with ﬁnal yield of

90 %. ¹H NMR (600 MHz, DMSO-d₆) δ: 7.36 (t, J = 7.41,

2H), 7.31(t, J = 7.31, 1H), 7.26 (d, J = 7.31, 2H), 5.51 (s,

2H), 3.90 (q, 2H), 3.77 (d, 2H), 3.23 (s, 3H), 2.15 (sept,

1H), 0.86 (d, 6H); ¹³C NMR (150 MHz, DMSO-d₆) δ:

153.4, 150.2, 147.8, 135.6, 128.8, 128.5, 127.9, 127.1,

108.4, 49.4, 42.2, 37.7, 35.8, 26.8, 19.8, 12.9. HRMS

(ES⁺) m/z: calculated for C₁₈H₂₁BrN₄O₂405.0889, found

405. 0931.

Synthesis of 7-benzyl-8-bromo-3-butyl-1-ethyl-1H-purine-

2,6 (3H,7H)-dione) (4a5)

To a mixture of compound 3a3 (525 mg, 1.39 mmol) and

anhydrous K₂CO₃(385 mg, 2.78 mmol) in 6 mL anhydrous

DMF, 224 µL ethyl iodide (2.78 mmol) was added. The

reaction was allowed to stir at 70 °C for 12 h. After com-

pletion, reaction mixture was allowed to cool on ice for

10 min. The product mixture was neutralized with 10%

HCl. Then water was added to the neutralized mixture.

After addition of water the product was accumulated in

white precipitated form. The precipitated product was ﬁl-

tered and washed 3-4 times with water. Product was then

allowed to dry at 40 °C in the hot air oven overnight. White

powdered compound 4a5 was obtained with ﬁnal yield of

92.6% (522 mg). ¹H NMR (600 MHz, DMSO-d₆) δ: 7.36 (t,

J = 7.43, 2H), 7.30 (t, J = 7.31, 1H), 7.23 (d, J = 7.33, 2H),

5.52 (s, 2H), 3.94 (dd, J = 7.08, 14.45 Hz, 2H) 3.89 (t, J =

7.01, 7.01 Hz, 2H), 1.62 (quint, 2H,), 1.28 (sext, 2H), 1.106

(t, J = 6.99, 6.99 Hz, 3H), 0.899 (t, J = 7.36, 7.36 Hz, 3H);

¹³C NMR (150 MHz, DMSO-d₆) δ: 153.9, 150.4, 148.1,

136.1, 129.3, 128.9, 128.4, 127.6, 108.9, 49.8, 42.9, 36.3,

30.1, 19.9, 14.1, 13.5; HRMS (ES⁺) m/z: calculated for

C₁₈H₂₁BrN₄O₂405.0889, found 405. 0941.

Synthesis of 7-benzyl-1-ethyl-8-(3-isopropylphenyl)-3-

propyl-1H-purine-2,6(3H,7H)-dione (5a2)

A mixture of compound 4a2 (283 mg, 0.7233 mmol), 3-

isopropyl phenyl boronic acid (237 mg, 1.45 mmol), anhy-

drous K₂CO₃(200 mg, 1.45 mmol) were taken in 25 mL

round bottom ﬂask. Then, Tertakis (triphenylphosphine)

palladium (45 mg) was added under argon atmosphere in

the ﬂask. The reaction was carried out in 5 mL DMF. The

reaction was stirred at 110 °C for 2 days. The post-reaction

processing for obtaining pure product followed the same

procedure used for 5a1. The product formed was whitish

semisolid in nature (compound 5a2) with a yield of 85 %

Medicinal Chemistry Research

1

(265 mg). H NMR (600 MHz, DMSO-d₆): 7.44 (t, J =

152.1, 151.7, 150.9, 149.0, 148.0, 137.1, 128.9, 128.8, 127.5,

126.8, 126.4, 125.9, 107.4, 49.8, 48.8, 33.2, 27.7, 26.9, 23.5,

19.9, 19.2.; HRMS (ES⁺) m/z: calculated for C₂₆H₃₀N₄O₂

431.2402, found 431.2465.

6.74, 6.74 Hz, 1H), 7.42 (d, J = 8.29 Hz, 1H), 7.34 (s, 1H),

7.30 (t, J = 7.31, 7.31 Hz, 2H), 7.25 (t, J = 7.24 Hz, 1H),

7.01 (d, 2H), 5.63 (s, 2H), 4.02 (t, J = 7.18 Hz, 2H), 3.92 (q,

J = 6.83 Hz, 2H), 2.85 (sept, 1H), 1.75 (sext, 2H), 1.14 (s,

3H),1.09 (d, J = 6.89 Hz, 6H), 0.91 (t, J = 7.42,7.42 Hz,

3H); ¹³C NMR (150 MHz, DMSO-d₆) δ: 154.1, 151.7,

150.4, 148.8, 147.7, 137.1, 128.9, 128.7, 128.6, 127.5,

126.7, 126.4, 125.9, 107.4, 48.8, 44.2, 35.6, 33.2, 23.5,

20.9, 13.1, 11.1; HRMS (ES⁺) m/z:. calculated for

C₂₆H₃₀N₄O₂431.2402, found 431. 2457.

Synthesis of 7-benzyl-1-ethyl-3-isobutyl-8-(3-

isopropylphenyl)-1H-purine-2,6(3H,7H)-dione (5a5)

A mixture of compound 4a4 (315 mg, 0.778 mmol), 3-

isopropyl phenyl boronic acid (268 mg, 1.63 mmol), anhy-

drous K₂CO₃(225 mg, 1.63 mmol) were taken in 50 mL

round bottom ﬂask. Tertakis (triphenylphosphine) palla-

dium (60 mg, 0.005 mmol) was added under argon atmo-

sphere in the ﬂask. The reaction was carried out in 6 mL

DMF at 110 °C for 48 h. The post-reaction processing for

obtaining pure product followed the same procedure used

for 5a1. The product formed was semisolid and yellowish in

color (compound 5a5) with a yield of 71%. ¹H NMR

(600 MHz, DMSO-d₆) δ: 7.54 (s, 1H), 7.43 (m, 2H), 7.39 (t,

J = 7.31 Hz, 1H), 7.32 (s, 1H), 7.29 (t, J = 7.40 Hz, 1H),

6.99 (d, J = 7.31 Hz, 2H), 5.63 (s, 2H), 3.92 (q, J = 7.10,

7.10, 6.99 Hz, 2H), 3.88 (d, J = 7.44 Hz, 2H), 2.76 (hept,

1H), 2.24 (ddq, 1H), 1.15 (d, 6H), 1.11(t, 3H), 0.90 (d, 6H);

¹³C NMR (150 MHz, DMSO-d₆) δ: 154.7, 152.5, 151.1,

150.4, 149.6, 148.7, 137.8, 129.8, 129.6, 129.4, 128.4,

128.1, 128.1, 127.4, 127.0, 126.6, 107.8, 50.2, 49.4, 36.3,

34.0, 27.6, 24.5, 24.5, 24.2, 20.6, 13.8.; HRMS (ES⁺) m/z:

calculated for C₂₇H₃₂N₄O₂445.2558, found 445.2631.

Synthesis of 7-benzyl-1-ethyl-3-propyl-8-m-tolyl-1H-purine-

2,6(3H,7H)-dione (5a3)

A mixture of compound 4a2 (218 mg, 0.557 mmol), 3-

methyl phenyl boronic acid (151 mg, 1.14 mmol), anhydrous

K₂CO₃(73.5 mg, 1.14 mmol) were taken in 25 mL round

bottom ﬂask. Tertakis (triphenylphosphine) palladium

(34 mg, 0.003 mmol) was added in the presence of argon

atmosphere. The reaction was carried out in 5 mL DMF. The

reaction was stirred at 110 °C for 2 days. The post-reaction

processing for obtaining pure product followed the same

procedure used for 5a1. The product 5a3 obtained was

1

semisolid and light yellow with yield of 68% (152 mg). H

NMR (600 MHz, DMSO-d₆) δ: 7.35 (d, J = 6.92 Hz, 2H),

7.29 (m, 2H), 7.24 (d, J = 8.55 Hz, 2H), 7.06 (d, J = 5.79 Hz,

1H), 7.00 (m, 2H), 5.63 (s, 2H), 4.02 (t,

J = 7.18 Hz, 2H), 3.92 (q, J = 6.83 Hz, 2H) 2.85 (sept, 1H),

2.29 (s, 3H), 1.75 (sext, 2H), 1.14 (s, 3H); ¹³C NMR

(150 MHz, DMSO-d₆) δ: 154.6, 152.1, 151.7, 150.9, 149.0,

148.0, 137.1, 128.9, 128.8, 127.5, 126.7, 126.4, 125.9, 107.4,

49.8, 48.8, 33.2, 27.7, 26.9, 23.5, 19.9, 19.2; HRMS (ES⁺)

m/z: calculated for C₂₄H₂₆N₄O₂403.2089, found 403.2153.

Synthesis of 7-benzyl-3-butyl-1-ethyl-8-m-tolyl-1H-purine-

2,6 (3H,7H)-dione (5a6)

To a mixture of compound 4a5 (183 mg, 0.452 mmol), 3-

methyl phenyl boronic acid (129 mg, 0.903 mmol), anhy-

drous K₂CO₃(125 mg, 0.903 mmol) was taken in 25 mL

round bottom ﬂask, Tertakis (triphenylphosphine) palladium

(35 mg, 0.003 mmol) was added under argon atmosphere in

the ﬂask. The reaction was carried out in 4 mL DMF at

110 °C for 48 h. The post-reaction processing for obtaining

pure product followed the same procedure used for 5a1. The

product formed was semisolid in nature and light yellow in

nature (compound 5a6) with yield of 85% (159 mg).¹H

NMR (600 MHz, DMSO-d₆) δ: 7.40 (s, 1H), 7.37 (m, 2H),

7.34 (t, J = 7.53, 7.53 Hz, 1H), 7.27 (t, J = 7.43, 7.43 Hz,

2H), 7.23 (t, J = 7.31, 7.31 Hz, 1H), 5.60 (s, 2H), 4.03 (t,

2H) 3.89 (q, J = 6.93, 6.93, 6.99, 2H), 2.30 (s, 3H), 2.15

(quint, 2H), 1.30 (dq, J = 7.32, 7.32, 7.19, 14.63 Hz, 2H),

1.10 (t, J = 6.98 Hz, 3H), 0.90 (t, J = 7.36 Hz, 3H); ¹³C

NMR (150 MHz, DMSO-d₆) δ: 157.1, 154.4, 151.8, 150.1,

147.5, 138.3, 129.6, 129.4, 128.9, 128.7, 128.3, 126.1,

125.9, 126.1, 125.9, 107.4, 48.1, 42.1, 35.6, 29.6, 20.8,

19.5, 13.4, 13.4, 12.9; HRMS (ES⁺) m/z: calculated for

C₂₅H₂₈N₄O₂417.2245, found 417.2288.

Synthesis of 7-benzyl-3-isobutyl-8-(3-isopropylphenyl)-1-

methyl-1H-purine-2,6(3H,7H)-dione (5a4)

A mixture of compound 4a3 (417 mg, 1.07 mmol), 3-

isopropyl phenyl boronic acid (350 mg, 2.13 mmol) and

anhydrous K₂CO₃(295 mg, 2.13 mmol) were taken in 50 mL

round bottom ﬂask. Tertakis (triphenylphosphine) palladium

(64 mg, 0.006 mmol) was added under argon atmosphere in

the ﬂask. The reaction was carried out in 10 mL DMF at

110 °C for 48 h. The post-reaction processing for obtaining

pure product followed the same procedure used for 5a1. The

product formed was semisolid and yellowish in color (com-

pound 5a4) with yield of 66 % (301 mg). ¹H NMR

(600 MHz, DMSO-d₆): 7.538(s, 1H), 7.43 (m, 2H), 7.39 (d,

J = 2.18, 1H), 7.319 (s, 1H), 7.29 (t, J = 7.40, 7.40, 2H),

7.239 (t, J = 7.25,7.25, 1H), 6.997 (d, 2H), 5.62 (s, 2H), 3.8

(d, J = 7.48, 2H), 3.23 (s, 3H), 2.88 (hept, 1H), 2.23 (ddq,

1H), 0.90 (d, 6H).; ¹³C NMR (150 MHz, DMSO-d₆): 154.6,

Medicinal Chemistry Research

Synthesis of 7-benzyl-3-butyl-1-ethyl-8-(3-ﬂuorophenyl)-

1H-purine-2,6(3H,7H)-dione (5a7)

HRMS (ES⁺) m/z: calculated for C₁₈H₂₂N₄O₂327.1776,

found 327.1834; Melting Point: 220 °C.

Compounds 6a2–6a7 were prepared by catalytic depro-

tection as described above.

A mixture of compound 4a5 (130 mg, 0.321 mmol), 3-ﬂoro

phenyl boronic acid (94 mg, 0.674 mmol), anhydrous

K₂CO₃(93 mg, 0.674 mmol) were taken in 25 mL round

bottom ﬂask. Tertakis (triphenylphosphine) palladium

(25 mg, 0.002 mmol) was added under argon atmosphere in

the ﬂask. The reaction was carried out in 3 mL DMF at

110 °C for 48 h. The post-reaction processing for obtaining

pure product followed the same procedure used for 5a1.The

product 5a7 formed was semisolid in nature and light green

in color with yield of 75% (100 mg).¹H NMR (600 MHz,

DMSO-d₆) δ: 7.54 (dd, J = 8.06, 13.95, 1H), 7.44 (dd, 4.74,

13.85 Hz, 2H), 7.38 (td, J = 2.39, 8.75, 8.75 Hz, 1H), 7.27

(t, J = 7.37, 7.37 Hz, 2H), 7.22 (t, J = 7.26, 7.26 Hz, 1H),

6.98 (d, J = 7.35, 2H), 5.69 (s, 2H), 4.03 (t, J = 7.26, 7.26,

2H), 3.901 (q, J = 6.96, 6.96, 6.98, 2H), 1.69 (m, 2H), 1.32

(m, 2H), 1.10 (t, J = 7.00, 7.00 Hz, 3H), 0.91 (t, J = 7.36,

7.36, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ: 163.3, 161.7,

154.6, 150.8, 149.0, 137.4, 131.9, 131.0, 129.4, 128.3,

126.8, 125.8, 118.2, 116.5, 107.7, 49.3, 43.1, 36.4, 30.3,

20.1, 14.3, 13.8; HRMS (ES⁺) m/z: calculated for

C₂₄H₂₅FN₄O₂421.1995, found 421.2130.

1-ethyl-8-(3-isopropylphenyl)-3-propyl-1H-purine-2,6

(3H,7H)-dione(6a2 or C2)

1

Yield, 76%, H NMR (600 MHz, DMSO-d₆) δ:13.82 (s,

1H), 8.04 (s, 1H), 7.90 (, d, J = 7.56 Hz, 1H), 7.42 (t, J =

7.67 Hz, 1H), 7.37 (d, J = 7.64, 1H), 4.03 (t, J = 7.18 Hz,

2H), 2.96 (m, 1H), 1.75 (sext, 2H), 1.26 (d, J = 6.70 Hz,

6H), 1.14 (t, 3H) 0.91 (t, 3H); ¹³C NMR (150 MHz, DMSO-

d₆) δ: 154.5, 151.1, 149.8, 148.6, 129.6, 129.3, 129.1,

124.9, 124.8, 108.2, 45.0, 38.7, 36.4, 34.1, 24.4, 21.4, 21.5,

13.9, 11.7; HRMS (ES⁺) m/z: calculated for C₁₉H₂₄N₄O₂

341.1938, found 341.2002; Melting Point: 237 °C.

1-ethyl-3-propyl-8-m-tolyl-1H-purine-2,6(3H,7H)-dione (6a3

or C3)

1

Yield, 77%, H NMR (600 MHz, DMSO-d₆) δ: 13.76 (s,

1H), 7.945 (d, J = 5.12 Hz, 1H), 7.89 (d, J = 7.6 Hz, 1H),

7.37 (t, J = 7.66, 7.66 Hz, 1H), 7.28 (d, J = 7.52, 1H), 4.00

(m, 2H), 3.93(q, J = 7.00, 7.00, 7.02 Hz, 2H), 2.36 (s, 3H),

1.727 (m, 2H), 1.12 (td, J = 2.39, 6.95, 7.02, 3H), 0.89 (t,

J = 7.43,7.43 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ:

154.5, 151.1, 150.8, 150.6, 148.9, 138.9, 131.5, 129.5,

129.3, 127.6, 124.2, 108.3, 45.1, 36.4, 21.6, 21.5, 13.9,

13.9, 11.7; HRMS (ES⁺) m/z: calculated for C₁₇H₂₀N₄O₂

313.1619, found 313.1684; Melting Point: 248 °C.

Synthesis of 8-(3-isopropylphenyl)-1-methyl-3-propyl-1H-

purine-2,6(3H,7H)-dione (6a1 or C1)

Deprotection of 7-benzyl-8-(3-isopropylphenyl)-1-methyl-

3-propyl-1H-purine-2,6(3H,7H)–dione (220 mg) was car-

ried out in a 25 mL ﬂask equipped with magnetic bead.

3 mL methanol was added into the ﬂask. Then the solution

was degassed and backﬁlled with argon alternatively for

three times. 10% Pd/H (100 mg) was taken into 2nd round

bottom ﬂask and dissolved with 5 mL methanol. The ﬂask

mixture was degassed three times by evacuation and

backﬁlled with argon. The content of 1st ﬂask was poured

into the 2nd ﬂask while stirring via syringe. The 2nd ﬂask

was then purged with H₂gas. Then the reaction was allowed

to stir at room temperature for 48 h. The resultant product

mixture was passed through celite (1 cm). The solution

obtained was concentrated under reduced pressure. Product

formed was yellowish powder which was washed with

1 mL diethyl ether and ﬁltered. The ﬁnal product 6a1

formed was pure white in color with yield of 93% (160 mg).

¹H NMR (600 MHz, DMSO-d₆):13.78 (s, 1H), 8.033 (s,

1H), 7.92 (d, J = 7.69 Hz, 1H), 7.39 (m, 1H), 7.35 (d, J =

7.68 Hz, 1H), 4.02 (m, 2H), 3.26 (s, 1H), 2.94 (ddd, J =

5.19, 8.58, 10.31 Hz, 1H), 1.74 (m, 2H), 1.24 (d, J =

3.57 Hz, 3H), 1.23 (d, J = 3.34 Hz, 3H), 0.90 (t, J = 7.43,

7.43 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ: 154.9,

151.5, 150.7, 149.8, 148.9, 129.6, 129.3, 129.1, 128.4,

128.0, 124.8, 108.3, 45.1, 34.1, 28.4, 24.4, 21.5, 11.7;

3-isobutyl-8-(3-isopropylphenyl)-1-methyl-1H-purine-2,6

(3H,7H)-dione(6a4 or C4)

Yield, 81%, ¹H NMR (600 MHz, DMSO-d₆) δ: 8.05 (s, 1H),

7.9 (d, J = 2.64, 1H), 3.89 (d, J = 7.37 Hz, 2H), 3.27 (s, 3H),

2.95 (dt, J = 6.87, 6.87, 13.78 Hz, 1H), 2.23 (dt,

J = 7.01, 7.01, 13.71 Hz, 1H), 1.25 (d, J = 6.89 Hz, 6H),

0.91 (d, J = 6.66 Hz, 6H). Note [A broader peak came it may

probably submerged NH peak, two aryl peaks]; ¹³C NMR

(150 MHz, DMSO-d₆) δ: 154.8, 151.8, 150.7, 149.8, 149.1,

129.6, 129.4, 129.1, 124.9, 108.2, 50.6, 34.1, 28.5, 27.5,

24.4, 20.6; HRMS (ES⁺) m/z: calculated for C₁₉H₂₄N₄O₂

341.1938, found 341.1999; Melting Point: 249 °C.

Synthesis of 1-ethyl-3-isobutyl-8-(3-isopropylphenyl)-1H-

purine-2,6(3H,7H)-dione (6a5 or C5)

1

Yield, 76 %, H NMR (600 MHz, DMSO-d₆) δ: 13.80 (s,

1H), 8.02 (s, 1H), 7.91 (d, J = 7.62, 1H), 7.41 (t, J = 7.68,

7.68, 1H), 7.35 (d, J = 7.67, 1H), 3.94 (q, J = 6.95, 6.95,

6.97, 2H), 3.87 (d, J = 7.45, 2H), 2.94 (m, 1H), 2.244 (dp,

Medicinal Chemistry Research

J = 6.88, 6.88, 6.87, 6.87, 13.76 Hz, 1H), 1.24 (d, J = 6.92,

6H), 1.12 (t, J = 7.00, 7,00 Hz, 3H), 0.89 (d, J = 6.71, 6H);

¹³C NMR (150 MHz, DMSO-d₆) δ: 154.5, 151.3, 150.7,

149.8, 149.2, 129.6, 129.1, 128.2, 128.1, 124.9, 108.2,

50.5, 36.4, 34.1, 27.5, 24.4, 20.5, 13.8; HRMS (ES⁺) m/z:

calculated for C₂₀H₂₆N₄O₂354.2055, found 354.2182;

Melting Point: 272 °C.

7.26 Hz, 2H), 7.29 (d, J = 6.19 Hz, 1H), 7.26 (t, J = 8.26,

8.26 Hz, 4H), 6.86 (d, J = 8.43 Hz, 2H), 5.45 (s, 2H), 4.98

(s, 2H), 3.69 (s, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ:

159.4, 154.2, 150.9, 149.5, 136.2, 129.8, 129.4, 128.6,

127.9, 114.5, 109.6, 55.7, 50.1, 45.1; HRMS (ES⁺) m/z:

calculated for C₂₀H₁₇BrN₄O₃441.2779, found 441.0397

Synthesis of 3-(4-methoxybenzyl)-7-benzyl-8-bromo-1-

ethyl-1H-purine-2,6(3H,7H)-dione) (4b1)

3-butyl-1-ethyl-8-m-tolyl-1H-purine-2,6(3H,7H)-dione (6a6

or C7)

To a mixture of compound 3b1 (0.98 g, 2.22 mmol) and

anhydrous K₂CO₃(0.644 g, 4.66 mmol) in 10 mL anhy-

drous DMF, 0.3749 µL ethyl iodide (4.66 mmol) was

added. The reaction then stirred at 70 °C for 12 h. After

completion of the reaction, it was allowed to cool at room

temperature. The product mixture was diluted with ethyl

acetate. The organic part was separated from aqueous part

by washing with water and brine alternatively. After

extraction, the organic part was isolated and dried with

sodium sulfate. The yellowish solution obtained was dried

under vacuum in a rotary evaporator. Whitish product

mixture was obtained. The product mixture was puriﬁed by

column chromatography with ethyl-hexane (10:90) solvent.

Product 4b1 was obtained in the form of white powder with

Yield, 50 %, ¹H NMR (600 MHz, DMSO-d₆): 7.93 (s, 1H),

7.89 (d, J = 7.65 Hz, 1H), 7.34 (t, J = 7.57, 7.57 Hz, 1H),

7.22 (d, J = 7.33 Hz, 1H), 4.03 (t, J = 6.95, 6.95 Hz, 2H),

3.93 (q, J = 6.58, 6.58, 6.62 Hz, 2H), 3.15 (s, 1H), 2.35 (s,

3H), 1.68 (m, 2H), 1.31 (dq, J = 7.22, 7.22, 7.46, 14.29 Hz,

2H), 1.11 (t, J = 6.91, 6.91 Hz, 3H), 0.91 (t, J = 7.29,

7.29 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆):154.2,

151.1, 149.9, 148.2, 138.8, 131.5, 129.5, 127.6, 124.3,

108.2, 43.2, 36.4, 30.3, 21.6, 20.0, 14.3, 13.8. HRMS (ES⁺)

m/z: calculated for C₁₈H₂₂N₄O₂327.1776, found 327.1841;

Melting Point: 220 °C.

3-butyl-1-ethyl-8-(3-ﬂuorophenyl)-1H-purine-2,6(3H,7H)-

dione (6a7 or C8)

1

ﬁnal yield of 88 % (915 mg). H NMR (600 MHz, DMSO-

d₆) δ: 7.34 (t, J = 7.40, 7.40 Hz, 2H), 7.29 (d, J = 7.286 Hz,

3H), 7.25 (d, J = 7.53 Hz, 2H), 6.86 (d, J = 8.45 Hz, 2H),

5.49 (s, 2H), 5.04 (s, 2H), 3.88 (q, J = 6.77, 6.77, 6.86 Hz,

3H) (s, 2H), 3.69 (s, 3H Ar), 1.09 (t, J = 6.94, 6.94 Hz,

3H).; ¹³C NMR (150 MHz, DMSO-d₆) δ: 159.3, 153.9,

150.7, 148.1, 136.2, 136.2, 129.9, 129.4, 129.1, 129.0,

128.6, 127.8, 114.5, 114.3, 109.1, 55.7, 50.1, 46.1, 36.6,

13.7; HRMS (ES⁺) m/z: calculated for C₂₂H₂₁BrN₄O₃

469.0811, found 469.0894

Yield, 56%, ¹H NMR (600 MHz, DMSO-d₆) δ: 7.98 (d, J =

7.75 Hz, 1H), 7.92 (d, J = 10.07 Hz, 1H), 7.57 (dd, J = 7.70,

14.21 Hz, 1H), 7.33 (t, J = 8.30, 8.30 Hz, 1H), 4.05 (t, J =

7.02, 7.02 Hz, 2H), 3.94 (q, J = 6.83, 6.83, 6.84 Hz, 2H),

1.69 (m, 2H), 1.324 (m, 2H), 1.13 (, t, J = 6.93, 6.93, 3H),

0.93 (t, J = 7.32, 7.32 Hz, 3H); ¹³C NMR (150 MHz, DMSO-

d₆) δ: 163.8, 162.2, 154.6, 151.0, 131.9, 131.8, 127.1, 123.2,

117.6, 117.5, 113.6, 113.5, 43.3, 36.5, 30.3, 20.0, 14.4, 14.4,

14.3, 13.8; HRMS (ES⁺) m/z: calculated for C₁₇H₁₉FN₄O₂

331.1525, found 331.1575; Melting Point: 246 °C.

Synthesis of 3-(4-methoxybenzyl)-7-benzyl-1-ethyl-8-(3-

isopropylphenyl)-1H-purine-2,6 (3H,7H)-dione (5b1)

Synthesis of 3-(4-methoxybenzyl)-7-benzyl-8-bromo-1H-

purine-2,6(3H,7H)-dione (3b1)

To the mixture of compound 4b1 (1.66 g, 3.55 mmol), 3-

isopropyl phenyl boronic acid (1.22 g, 7.45 mmol), anhy-

drous K₂CO₃(1.03 g, 7.45 mmol) were taken in 50 mL

round bottom ﬂask. Tertakis (triphenylphosphine) palla-

dium (260 mg, 0.2 mmol) was added under argon atmo-

sphere in the ﬂask. The reaction was carried out in 10 mL

DMF at 110 °C for 48 h. After completion of the reaction,

product mixture was allowed to cool at room temperature.

10 mL water was added to the product mixture and stirred

for 10 min. Upon cooling, the reaction mixture darkened

and black emulsion appeared on the upper layer of the

solution. The reaction mixture was then diluted with ethyl

acetate and transferred to a separating funnel. Two layers

were formed; the organic layer was re-extracted with ethyl

acetate. Organic extract was washed with 5% sodium

To a mixture of 7-benzyl 8-Bromo xanthine (7-benzyl-8-

bromo-1H-purine-2,6 (3H,7H)-dione) (1.07 g, 3.33 mmol)

in 10 mL anhydrous DMF, 6.66 mmol of K₂CO₃and

40.65 µL of 4-methoxybenzyl chloride (0.9 mmol) were

added in a 50 mL round bottom ﬂask. Reaction was allowed

to run at 70 °C for 2.5 h on silica bath. After the completion

of the reaction, the product mixture was kept on ice for

10 min. 10% HCl was added drop-wise to neutralize the

product mixture. Light white colored precipitate was

formed. Product was ﬁltered and washed 2-3 times with

water. The product was vacuum dried. White powder pro-

1

duct 3b1 was formed with 70 % (1.03 g) yield. H NMR

(600 MHz, DMSO-d₆) δ: 11.42 (s, 1H), 7.34 (t, J = 7.26,

Medicinal Chemistry Research

carbonate solution and brine sequentially. After extraction

organic phase was transferred to a 250 mL Erlenmeyer ﬂask

equipped with a magnetic stir bar. Activated charcoal

(0.50 g) and sodium sulfate (2 g) were added to the ﬂask.

This mixture was stirred for 10 min. The solution was then

ﬁltered through 1 cm celite bed. The resulting pale yellow

solution was concentrated under reduced pressure to yield

the crude product in oil form. The product was puriﬁed by

column chromatography (10% ethyl acetate: 90% Hexane).

The product 5b1 formed was light yellow and semisolid in

anhydrous DMF, 59 µL isobutyl iodide (0.515 mmol) was

added. The reaction was then stirred at 70 °C for 12 h.

After completion of the reaction, product mixture was

allowed to cool at room temperature. The reaction mix-

ture was then diluted with ethyl acetate and transferred to

a separating funnel. Two layers were formed the organic

layer was re-extracted with ethyl acetate. The organic

extract was washed with 5% sodium carbonate solution

and brine sequentially. After extraction organic phase

was transferred to a 250 mL Erlenmeyer ﬂask equipped

with a magnetic stir bar. The resulting pale yellow

solution was concentrated under reduced pressure to yield

the crude product as oil. The product was then puriﬁed by

column chromatography (10% ethyl acetate: 90% Hex-

ane). The puriﬁed compound 7b1 was obtained in yel-

1

nature with yield of 72 % (1.3 g). H NMR (600 MHz,

DMSO-d₆) δ: 7.45 (t, J = 8.58, 8.58 Hz, 1H), 7.41 (dd, J =

5.98 Hz, 13.53, 2H), 7.37 (d, J = 8.35 Hz, 2H), 7.34 (s, 1H),

7.29 (t, J = 7.33, 7.33 Hz, 2H), 7.24 (d, J = 7.26 Hz, 1H),

7.01 (d, J = 7.30 Hz, 2H), 6.87 (d, J = 8.13 Hz, 2H), 5.62

(s, 1H), 5.15 (s, 1H), 3.89 (dd, J = 6.69, 13.63 Hz, 2H),

3.69 (s, 1H), 2.834 (dt, J = 6.72, 6.72, 13.55 Hz, 1H), 1.09

(t, J = 6.55, 6.55 Hz, 3H); ¹³C NMR (150 MHz, DMSO-d₆)

δ: 159.3, 154.7, 152.5, 150.9, 149.6, 148.2, 137.8, 130.2,

129.6, 129.4, 127.3, 126.6, 114.5, 107.4, 60.4, 55.7, 49.2,

46.2, 36.4, 33.8, 24.2, 21.3, 14.7, 13.8.; HRMS (ES⁺) m/z:

calculated for C₃₁H₃₂N₄O₃509.2508, found 509.2611.

1

lowish oil form with yield of 84% (193 mg); H NMR

(600 MHz, DMSO-d₆) δ: 7.54 (s, 1H), 7.43 (m, 2H), 7.39

(t, J = 7.31 Hz, 1H), 7.32 (s, 1H), 7.29(t, J = 7.40 Hz,

1H), 6.99 (d, J = 7.31 Hz, 2H), 5.63 (s, 2H), 3.92 (q, J =

7.10, 7.10, 6.99 Hz, 2H), 3.88 (d, J = 7.44 Hz, 2H), 2.99

(hept, 1H), 2.24 (ddq, 1H), 1.15 (d, 6H), 1.11(t, 3H), 1.08

(d, 6H); ¹³C NMR (150 MHz, DMSO-d₆) δ: 154.7, 152.5,

151.1, 150.4, 149.6, 148.7, 137.8, 129.8, 129.6, 129.4,

128.4, 128.1, 128.1, 127.4, 127.0, 126.6, 107.8, 50.2,

49.4, 36.3, 34.0, 27.6, 24.5, 24.5, 24.2, 20.6, 13.8.;

HRMS (ES⁺) m/z: calculated for C₂₇H₃₂N₄O₂445.2658,

found 445.2631.

Synthesis of 7-benzyl-1-ethyl-8-(3-isopropylphenyl)-1H-

purine-2,6(3H,7H)-dione (6b1)

A

solution of Compound 5b1 (R₁= Et) (1.257 g,

2.47 mmol), concentrated sulfuric acid (10 drop), anisol

(375 µL, 3.4 mmol) in TFA (5 mL) was taken in 25 mL ﬂask

equipped with magnetic bead. The ﬂask was degassed and

backﬁlled with argon alternatively for three times. The

reaction mixture was reﬂuxed for 22 h. Oily residue was

formed which was diluted with water and isopropyl ether.

This was followed by neutralization with 20% NaOH till pH

5 was obtained. The resultant precipitate was ﬁltered,

washed with water and isopropyl ether, and dried. The

product 6b1 was obtained in greenish powdered form with

Compound 7b2 was prepared by similar alkylation

reaction as described above.

7-benzyl-3-butyl-1-ethyl-8-(3-isopropylphenyl)-1H-purine-

2,6(3H,7H)-dione (7b2)

Yield of 98 %, ¹H NMR (600 MHz, DMSO-d₆) δ: 7.35 (, d,

J = 7.18 Hz, 2H), 7.34 (m, 2H), 7.33 (d, J = 7.82 Hz, 1H),

7.28 (m, J = 7.67 Hz, 1H), 7.16 (s, J = 1.79 Hz, 1H), 7.13

(s, J = 7.67 Hz, 1H), 7.08 (d, J = 8.05 Hz, 1H), 7.03 (t, J =

7.77 Hz, 2H), 5.41 (s, 2H), 3.90 (q, 2H) 3.82 (t, J =

7.14 Hz, 2H), 2.88 (hept, J = 7.00, 6.99, 13.81 Hz, 1H),

2.75 (quint, 2H), 1.60 (sext, 2H), 1.13 (d, 6H), 1.10 (t, J =

7.05 Hz, 3H), 0.81 (t, J = 7.36 Hz, 3H); ¹³C NMR

(150 MHz, DMSO-d₆) δ: 154.6, 152.1, 151.7, 150.9, 149.0,

148.0, 137.1, 128.9, 128.8, 127.5, 126.7, 126.4, 125.9,

107.4, 49.8, 48.8, 33.2, 27.7, 26.9, 23.9, 23.5, 19.9, 19.9,

19.2; HRMS (ES⁺) m/z: calculated for C₂₇H₃₂N₄O₂

445.2658, found 445.2622.

1

yield of 73 % (700 mg). H NMR (600 MHz, DMSO-d₆):

12.03 (s, 1H), 7.39 (d, J = 5.12 Hz, 1H), 7.36 (d, J = 6.38,

6.38 Hz, 3H), 7.28 (t, J = 7.30, 7.30 Hz, 2H), 7.23 (m, 1H),

6.99 (d, J = 7.27 Hz, 2H), 5.604 (s, 1H), 3.84 (q, J = 6.69,

6.69, 6.70 Hz, 2H), 2.83 (dt, J = 6.76, 6.76 Hz, 13.58, 1H),

1.08 (d, J = 7.01 Hz, 6H); ¹³C NMR (150 MHz, DMSO-d₆)

δ: 154.9, 152.1, 150.6, 149.0, 147.3, 137.3, 128.9, 128.8,

128.6, 127.5, 126.4, 126.4, 125.9, 107.4, 48.6, 23.8, 23.6,

13.2; HRMS (ES⁺) m/z: calculated for C₂₃H₂₄N₄O₂

389.1928, found 389.1990.

Synthesis of 1-ethyl-3-isobutyl-8-(3-isopropylphenyl)-1H-

purine-2,6(3H,7H)-dione (8b1 or C5)

Synthesis of 7-benzyl-1-ethyl-3-isobutyl-8-(3-

isopropylphenyl)-1H-purine-2,6(3H,7H)-dione (7b1)

A mixture of compound 7b1 (315 mg, 0.778 mmol), 3-

isopropyl phenyl boronic acid (268 mg, 1.63 mmol),

anhydrous K₂CO₃(225 mg, 1.63 mmol) were taken in

To a mixture of compound 6b1 (0.2 g, 0.515 mmol)

and anhydrous K₂CO₃(0.142 g, 1.028 mmol) in 2 mL

Medicinal Chemistry Research

50 mL round bottom ﬂask. Tertakis (triphenylphosphine)

palladium (60 mg, 0.005 mmol) was added under argon

atmosphere in the ﬂask. The reaction was carried out in

6 mL DMF at 110 °C for 48 h. After completion of the

reaction, product mixture was allowed to cool at room

temperature. 10 mL of water was added to the product

mixture and stirred for 10 min. Upon cooling, the reaction

mixture darkened and black emulsion appeared on the

upper layer of the solution. The reaction mixture was then

diluted with ethyl acetate and transferred to a separating

funnel. Two layers were formed, the organic layer was re-

extracted with ethyl acetate. Organic layer was washed

with 5% sodium carbonate solution and brine sequen-

tially. After extraction organic phase was transferred to a

250 mL Erlenmeyer ﬂask equipped with a magnetic stir

bar. Activated charcoal (0.50 g) and sodium sulfate (2 g)

were added to the ﬂask. This mixture was stirred for

10 min. The solution was then ﬁltered through 1 cm celite

bed. The resulting pale yellow solution was concentrated

under reduced pressure to yield the crude product in oil

form. The product was then puriﬁed by column chroma-

tography (10% ethyl acetate: 90% Hexane). The product

formed was semisolid and yellowish in color (compound

8b1) with a yield of 71%. ¹H NMR (600 MHz, DMSO-d₆)

δ: 13.80 (s, 1H), 8.02 (s, 1H), 7.91 (d, J = 7.62 Hz, 1H),

7.41 (t, J = 7.68, 7.68 Hz, 1H Ar-H), 7.35 (d, J = 7.67 Hz,

1H), 3.94 (q, J = 6.95, 6.95, 6.97 Hz, 2H), 3.88 (d, J =

7.45 Hz, 2H), 2.94 (m, 1H), 2.24 (dp, J = 6.88, 6.88, 6.87,

6.87, 13.76 Hz, 1H), 1.24 (d, J = 6.92 Hz, 6H), 1.12 (t,

J = 7.00, 7,00 Hz, 3H), 0.89 (d, J = 6.71, 6H); ¹³C NMR

(150 MHz, DMSO-d₆)δ: 154.5, 151.3, 150.7, 149.8,

149.2, 129.6, 129.1, 128.2, 128.1, 124.9, 108.2, 50.5,

36.4, 34.1, 27.5, 24.4, 20.6, 13.8; HRMS (ES⁺) m/z:

calculated for C₂₀H₂₆N₄O₂354.2055, found 354.2182;

Melting Point: 272 °C.

Biological evaluation

Cloning, expression and puriﬁcation of PDE9A coding

domain

The Full length cDNA clone of PDE9A [IMAGE ID

3874635, Gene bank Accession no. BC009047] was pur-

chased from Source Bioscience, USA. The catalytic domain

of PDE9A (181-506) was ampliﬁed with the help of a pair of

synthesized oligonucleotide primers. Both ampliﬁed coding

domain of PDE9A and pET15b expression vector were

digested with restriction enzymes NdeI and XhoI at 37 °C. It

was followed by ligation of digested coding domain of

PDE9A and digested expression vector. The resultant

recombinant plasmid PDE9A-pET15b clone was trans-

formed into E. coli BL21 strain for expression of PDE9A

protein. The recombinant plasmid carried BL21 strain was

propagated in LB medium under 37 °C temperature till the

absorption reached at A₆₀₀= 0.7. Induction was carried out

with addition of 0.1 mM β-D-thiogalactopyranoside (IPTG)

and further growth was allowed at 15 °C for 20 h. The

protein was puriﬁed with Ni-NTA afﬁnity puriﬁcation. The

puriﬁed PDE9A protein was conﬁrmed by SDS-PAGE.

Concentration yield of PDE9A protein after puriﬁcation was

1 mg/mL or 20 µM.

Bioassay

The enzyme activity assay of PDE9A were performed by

using malachite green (MLG) assay- a spectrometric assay

[5, 40]. It is a coupled end point assay which depends on the

combined action of two enzymes- PDE9A and calf intestinal

phosphatase (CIAP). The assay buffer was 20 mM Tris-HCl

at pH7.5, 10 mM MgCl₂, 0.10 mM EDTA. The substrate

speciﬁcity and inhibitor potency towards PDE9A were

determined by quantiﬁcation of the inorganic phosphate that

released from the test substrate (cGMP) by combined action

of PDE and CIAP. Inhibition study was a key analysis to

determine the structure-activity relationship of newly syn-

thesized compounds. For PDE9A inhibition study, the

reaction mixture at 30 °C in 1.35 mL contained 0.90 mL

reaction buffer plus an indicated PDE inhibitor, 0.15 mL

solution of CIAP properly diluted in 28% glycerol, 0.20 mL

solution of a PDE9A, 0.10 mL aqueous cGMP for the ﬁnal

concentration at 16 µM (as optimized in separate reaction).

Enzyme reactions were initiated at 30 °C with the addition of

cGMP and were terminated with the addition of 0.25 mL

HClO₄(40%) after an indicated reaction duration. After

centrifugation at 2000 × g for 10 min to remove denaturated

proteins, 0.70 ml of the acidiﬁed supernatant was withdrawn

to quantify inorganic phosphate release from the combined

action of PDE and CIAP. Thereafter, 70 µL molybdate

solutions (50.0 mM ammonium molybdate in 3.4 M sulfuric

Compound 8b2 obtained from the deprotection of com-

pound 7b2 using similar protocol as described above.

3-butyl-1-ethyl-8-(3-isopropylphenyl)-1H-purine-2,6

(3H,7H)-dione (8b2 or C6)

1

Yield, 77%; H NMR (600 MHz, DMSO-d₆) δ: 8.02 (s,

1H), 7.91 (d, J = 7.63 Hz, 1H), 7.38 (t, J = 7.64, 7.64 Hz,

1H), 7.31 (d, J = 7.55 Hz, 1H), 3.94 (t, J = 7.12, 7.12 Hz,

2H), 3.93 (q, J = 6.89, 6.89, 6.92 Hz, 2H), 2.93 (dp, J =

6.72, 6.72, 6.57, 6.57, 13.42 Hz, 1H), 1.69 (m, 2H), 1.31

(dq, J = 7.41, 7.41, 7.46, 14.88 Hz, 2H), 1.24 (d, J = 6.90,

6H), 1.12 (t, J = 6.96, 6.96 Hz, 3H), 0.91 (t, 3H); ¹³C NMR

(150 MHz, DMSO-d₆) δ: 154.8, 151.2, 151.1, 149.7, 149.1,

130.0, 129.5, 128.7, 124.7, 124.7, 109.1, 43.2, 36.3, 34.1,

30.3, 24.4, 20.0, 14.2, 13.9; HRMS (ES⁺) m/z: calculated

for C₂₀H₂₆N₄O₂355.2089, found 355.2142; Melting Point:

232 °C.

Medicinal Chemistry Research

acid) was added to the acidiﬁed reaction solutions of phos-

phate at ﬁrst and then 130 µL MLG reagent (1.0 mM MLG,

0.16% PVA, 6.0 mM sulfuric acid) was added after a lag

time of 2 min. The incubation time for the dye binding kept

30 min at 30 °C before the absorbance at 630 nm was mea-

sured. Phosphate standard was prepared to quantify the ﬁnal

concentration of inorganic phosphate obtained after the

coupling reaction and absorption was limited to 1.0. The

buffer used for the preparation of potassium phosphate

standard contained 10.0 mM MgCl₂and 0.10 mM EDTA.

The estimation of CIAP concentration required for releasing

inorganic phosphate from intermediate GMP (produced by

the catalytic action of PDE9A), was carried out through

separate spectrophotometric assay. The IC₅₀graph was

plotted to determine the percentages inhibition of PDE9A

activities against logarithmic concentrations of a candidate

inhibitor. The linear part of such plots was used for regres-

sion analysis which gave IC₅₀of the candidate inhibitor.

Results were represented as mean standard deviation.

preparation consisted of 90 × 90 × 90 points in x, y and z

direction with equal spacing of 0.253 Å. PDE9A catalytic

domain was used as rigid model with ﬂexible side chains.

Flexible ligand model was used for docking and further

optimization. Parameters used for docking of synthesized

compounds with PDE9A was 100 GA run, 300 population

sizes, 27,000 maximum numbers of generation and

25,000,000 maximum numbers of evaluations. Clustering

of docked complexes was created with 2 Å root-mean-

square deviation tolerance. Molecular docking was carried

out in CentOS Linux system. Docking poses were ana-

lyzed with the help of ADT, PyMol, and Discovery Studio

Visualizer which provided information about hydrogen

bond interactions and π- π interaction. Hasegawa et al.

[19, 22, 7, 41, 42].

Acknowledgements Nivedita Singh acknowledges IIT Guwahati for

providing infrastructure facilities for carrying out her doctoral work

and research fellowship. This work was ﬁnancially supported by

Department of Biotechnology, Government of India under the project

number BTPRI15211/MED/29/282/2011, DeitY, India Project no. 5

(9)/2012-NANO (Vol. II), DST, India (No. DST/SERB/EMR/2014/

000034), DST-Max Planck Society, Germany (IGSTC/MPG/PG(PKI)/

2011A/48).

Thermal shift assay of synthesized compounds

The reaction buffer used for this assay was 20 mM Tris-HCl

at pH 7.5, 10 mM MgCl₂, 0.10 mM EDTA, 10% Glycerol.

The reaction was carried out in presence of 200X SYPRO

Orange (Invitrogen). The excitation and emission spectra of

the dye was performed at 492 nm and 610 nm respectively.

Concentration of protein and inhibitor used for this assay

were 20 and 200 µM (1:10) respectively. DSF assay was

performed in Real Time PCR instruments (Agilent

Mx3005P QPCR System). For this assay, the reaction

buffer was taken in a series of PCR tubes. Reactions were

carried out in triplicates. For this study Lysozyme was used

as standard whereas no protein buffer with dye was used as

control. In this study, a simple ﬁtting procedure was used

for quick calculation of melting temperature (T_m). The

temperatures (T_m) of the transitions were calculated for

melting curves from the midpoint of transition [28].

Compliance with ethical standards

Conﬂict of interest The authors declare no competing interest.

Publisher’s note Springer Nature remains neutral with regard to

jurisdictional claims in published maps and institutional afﬁliations.

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Article Doi

Diversifying the xanthine scaffold for potential phosphodiesterase 9A inhibitors: synthesis and validation

DOI: 10.1007/s00044-021-02722-9

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