Synthesis of 6-[4-(4-Propoxyphenyl)piperazin-1-yl]-9H-purine Derivatives as Antimycobacterial and Antifungal Agents: In Vitro Evaluation and In Silico Study

Article abstract of DOI:10.1002/cbdv.202000053

A series of novel alkyl substituted purines were synthesized. 6-[4-(4-Propoxyphenyl)piperazin-1-yl]-9H-purine was used as the key starting material, which was synthesized via a multistep protocol and finally subjected for N-alkylation with various alkyl halides with an aim to get prospective antimicrobial agents. The structures of the novel compounds were established by substantiating them through spectral techniques like ¹H-NMR, ¹³C-NMR, FT-IR and EI-MS. They were explored for antitubercular activity against Mycobacterium tuberculosis H37RV. Furthermore, they were checked for their antimicrobial activity concerning bacterial and fungal strains. The title compounds exhibited considerable antimicrobial activity without any significant toxicity. In silico studies depicted their good binding profile against Mycobacterium tuberculosis enoyl reductase (InhA; PDB ID: 4TZK) and Candida albicans dihydrofolate reductase (PDB ID: 1AI9). The title compounds obeyed Lipinski's parameters and have exhibited good drug-like properties.

Full text of DOI:10.1002/cbdv.202000053

Title: Synthesis of 6-(4-(4-propoxyphenyl) piperazin-1-yl)-9H-purine
derivatives as antimycobacterial and antifungal agents: In-vitro
evaluation and in-silico study
Authors: Afra Quasar Nadaf, Mahesh S Najare, Manjunatha
Garbhagudi, Shivaraj Mantur, Manjunath G. Sunagar, Supreet
Gaonkar, Shrinivas Joshi, and Imtiyaz Ahmed M. Khazi
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Biodiversity 10.1002/cbdv.202000053
Link to VoR: http://dx.doi.org/10.1002/cbdv.202000053

10.1002/cbdv.202000053
Chemistry & Biodiversity
Synthesis of 6-(4-(4-propoxyphenyl) piperazin-1-yl)-9H-purine derivatives as
antimycobacterial and antifungal agents: In-vitro evaluation and in-silico
study
AfraQuasar A. Nadafa, Mahesh S. Najarea, Manjunatha Garbhagudia, Shivaraj Mantura,
Manjunath G. Sunagara, Supreet Gaonkara, Shrinivas Joshib and Imtiyaz Ahmed M. Khazi a*
aDepartment of Chemistry, Karnatak University, Dharwad 580003, Karnataka, India
bNovel Drug Design and Discovery Laboratory, Department of Pharmaceutical Chemistry, S.E.T.’s College of
Pharmacy, Sangolli Rayanna Nagar, Dharwad,
580 002, Karnataka, India
Corresponding author mail: drimkorgchem@gmail.com
Abstract
A series of novel alkyl substituted purines were synthesized. 6-(4-(4-propoxyphenyl) piperazin-1-
yl)-9H-purine was used as the key starting material, which was synthesized via a multistep
protocol and finally subjected for N-alkylation with various alkyl halides with an aim to get
prospective antimicrobial agents. The structures of the novel compounds were established by
substantiating them through spectral techniques like 1H NMR, 13C NMR, FT-IR and EI-MS. They
were explored for antitubercular activity against Mycobacterium tuberculosis H37RV.
Furthermore, they were checked for their antimicrobial activity concerning bacterial and fungal
strains. The title compounds exhibited considerable antimicrobial activity without any significant
toxicity. In-silico studies depicted their good binding profile against Mycobacterium tuberculosis
enoyl reductase (InhA) (PDB ID: 4TZK) and Candida albicans dihydrofolate reductase (PDB ID:
1AI9). The title compounds obeyed Lipinski’s parameters and have exhibited good drug-like
properties.
Key words: Propoxyphenyl piperazin-1-yl; Antitubercular activity; Antimicrobial;
Mycobacterium tuberculosis H37RV; Docking Study.
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Chemistry & Biodiversity
Graphical Abstract
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10.1002/cbdv.202000053
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1. Introduction
There is a formidable urge for the synthesis of novel leads in order to develop potent drugs for the
treatment of life-threatening as well as infectious diseases. Tuberculosis is one of the most
pervasive communicable disease accounting for millions of death globally. The appearance of
unfamiliar virulent strains of TB like the multi drug resistance TB (MDR-TB), extremely drug
resistance TB (XDR-TB) and their association with human immuno deficiency virus (HIV)
aggravates the situation.[1-3] Also, the advent of multi-drug resistance strains of bacteria and fungi
have constituted a challenge for the treatment of infectious diseases.[4-6] An indispensable
component of drug designing programs is the search of new leads by synthesis of low molecular
weight compounds, which are novel but still resemble well known pharmacologically active
fragments having imperative structural components.[7-8]
It has been entrenched that heterocyclic scaffolds play a vital role in designing new
structural motifs with medicinal applications. Amongst them, purine nuclei have contributed
immensely for augmentation of the antimicrobial activity comparatively to the other hetero
moieties. Being one of the most ubiquitous nitrogen-containing heterocycles, purine occurs
abundantly in nature and also forms a core moiety of nucleic acids, which are of prime importance
for genetic and proper functioning of most of the metabolic processes in living organisms.[9]
Purines are involved with multiple number of enzymes and receptors which exhibit crucial roles in
different phases of the cell cycles, in cell signaling and other fundamental reactions in the
[10-11]
biological system.
Concisely, purines and their substituted analogues have grabbed
considerable attention of research in medicinal chemistry.
Literature reports have shown that naturally occurring purine as well as their synthetic
counterparts exhibit a wide range of biological activity.[12-16] Di, tri and tetra substituted purines
have been accounted for possessing antiviral and antitubercular properties. They also modulate
CRH-R1 and act as inhibitors of Hsp90, sulfotransferase, leukotriene A4 hydrolase, and kinases
phosphodiesterases.[17] Furthermore, there are reports available regarding their role in enzyme
inhibition,[18-19] as antihyperglycemic,[20] antifungal and antibacterial agents.[21-24] According to a
report; WHO stresses for research on purines as potential anti-mycobacterials.[25] As a result, a
broad range of bioactive moieties with purine skeletal have been designed and synthesized which
have added to the launch of several powerful drugs with varied applications.[26] The rationale for
the display of biological activities is conferred to the diversity of substituents that can be combined
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10.1002/cbdv.202000053
Chemistry & Biodiversity
on the C-2, C-6, C-8 and N-9 centers of purine nucleus whereas C-6 and N-9 positions are the
most anchored ones.[27] The explanation of the selectivity and efficacy of derivatives of purine is
attained principally by the cell surface transporters which mediate access to the cell and later the
enzymes of the metabolic pathways modify the prodrug into an active metabolite.[28]
Piperazine and its derivatives are reported for inhibiting tumor metastasis in most of the
cancer cell lines. Mechanistic studies of the analogues containing piperazine have showed that this
property is accomplished by impeding synthesis of microtubules, progression of the cell cycle,
angiogenesis and obliteration of the tumor cells through the initiation of apoptosis.[29-30] Further,
literature proves that incorporation of piperazine into the first generation quinolines had
significantly improved the bioavailability of the second generation fluoroquinolones. This
indicates that piperazine skeleton might aid to regulate the physiochemical properties and enhance
their pharmacokinetic and pharmacodynamic behavior. SQ786 and LL-3858 from Lupin Ltd., with
a piperazine moiety in their framework showed promising antitubercular activity.[31] In addition,
biologically active compounds with varying alkyl/acyl groups have realized to regulate their
lipophilicity and permeability[32-33] whereas the alkenyl and alkynl purines are often cytotoxic to
the human cells.[34]
With this outlook and in extension of our work with purines; we emphasized on the design
and development of potent molecules with purine as the core moiety, hybridized with phenyl
piperazine at its C-6 position and exploring its N-9 position with various alkyl groups. Hence we
attempted the synthesis of alkyl-substituted purines by simple and efficient multi-step protocol.
The title compounds were explored for their in-vitro and in-silico antimicrobial and antitubercular
properties. Also, their physiochemical properties have been investigated.
2. Results and Discussion
2.1. Chemistry
N-9 substituted alkyl derivatives of purine (PPC01-PPC10) are synthesized as shown in Scheme
1 as per the reported procedures.[35] N-protected 4-hydroxyphenyl piperazine (PP1) and propyl
bromide which are commercially available were refluxed using triethyl amine to get the O-
alkylated product (PP2). This (PP2) was now subjected to de-acetylation with HCl (4 N, 20 mL)
to obtain the first precursor 4-propoxyphenyl piperazine (PP3). The key intermediate (PP4) was
obtained by nucleophilic substitution of PP3 with 6-chloro purine. Finally, the reaction of this
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10.1002/cbdv.202000053
Chemistry & Biodiversity
key intermediate (PP4) with different alkyl halides accorded the target compounds i.e., N-9 alkyl
substituted purines with yields about 74-85 %. The chemical structures of the final compounds
1
were confirmed by spectroscopic techniques like H and 13C NMR, FT-IR and EI-MS. The
characterization data of the final molecules PPC01-PPC10 are in accordance with the
synthesized compounds and are specified in the experimental section.
1
In PPC01, the H NMR spectrum showed two singlet’s at δ 8.38 and δ 7.78 ppm,
accounting for the C-H proton of pyrimidine and imidazole of the purine scaffold respectively.
The characteristic quartet-triplet pattern of the ethyl chain appeared at δ 4.25 and δ 1.52 ppm
whereas the propyl chain of propoxy phenyl piperazine moiety resonated as triplet multiplet
triplet pattern at δ 3.88, δ 1.78 and δ 1.02 ppm respectively. It was further established by
recording 13C NMR spectrum. The signals present in the spectrum interpreted for all the C atoms
present in PPC01. The EI-MS of PPC01 revealed a molecular ion peak at 366[M+]. This
coincides with its respective molecular formula C20H26N6O.
The 1H NMR spectrum of PPC04 showed a signal at δ 8.36 ppm as singlet relating to the
C-H of pyrimidine moiety whereas the C-H proton of imidazole in the purine scaffold appeared
as singlet at δ 7.73 ppm. The butyl substituent appeared as a triplet corresponding to 2H linked to
nitrogen at δ 3.86 ppm, multiplet corresponding to 4H of 2 CH2 at δ 1.79 ppm and triplet
corresponding to 3H of CH3 at δ 1.00 ppm. The EI-MS spectrum showed a molecular ion peak at
394[M+] which further supported the formation of compound PPC04. The characterization
details of the remaining molecules are in accordance with their expected chemical structures.
2.2. Biological Evaluation
All the synthesized title compounds were evaluated for their antimycobacterial activity against
Mycobacterium tuberculosis by making use of microplate Alamar Blue assay (MABA). The title
compounds were also tested for antibacterial activity against Gram-positive Staphylococcus
aureus and Bacillus subtilis, Gram-negative Escherichia coli and Pseudomonas aeruginosa;
antifungal activity against Aspergillus flavus, Trichoderma harzianum, Penicillium chrysogenum
and yeast Candida albicans by macrodilution broth method.
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OH
O
O
N
b
a
N
N
N
N
N
H
O
O
PP3
82 % yield
PP1
PP2
80 % yield
O
O
O
N
N
N
N
N
Cl
N
N
d
N
c
N
N
H
N
N
N
N
H
N
H
N
R
N
N
PP4
85 % yield
PPC01-PPC10
74-85 % yield
PP3
Eth-1-yl, Isoprop-1-yl, Prop-1-yl, But-1-yl, Pent-1-yl, Hex-1-yl, Hept-1-yl, Oct-1-yl, Non-1-yl, Dec-1-yl
R=
Scheme 1:
Synthesis of PPC01-PPC10
Reagents and conditions:
(a) Propyl bromide (1.2 eq); anhyd. K2CO3(2 eq), DMF, 120 0C, 12 h; (b) 4 N HCl (20 mL), 80 0C, 3 h.
(c) triethyl amine (2.7 eq ), n-butanol, 110 0C, 12 h; (d) R–Br (1 eq), K2CO3(1.2 eq), DMF, RT, 10 h.
Scheme 1: Synthetic route for PPC01-PPC10
2.3. Antimycobacterial Activity
PPC01-PPC10 were assessed for antimycobacterial activity against M. tuberculosis H37Rv by
employing microplate Alamar Blue assay (MABA).[36] Pyrazinamide, Ciprofloxacin and
Streptomycin were used as positive controls. Among the tested compounds PPC01- PPC04
having ethyl, isopropyl, propyl and butyl groups have shown good results whereas rest of the
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10.1002/cbdv.202000053
Chemistry & Biodiversity
compounds have appeared moderate. PPCO2 and PPC03 have shown good activity with MIC
3.125 μg/mL similar to the two positive standards; pyrazinamide and ciprofloxacin, whereas
PPC01 and PPC04 exhibited MIC 6.25 μg/mL which is the same as that of streptomycin
standard. The Screening results are briefed in Table 1.
Table 1: Anti-mycobacterial activity of PPC01-PPC10
COMPOUNDS
PPC01
PPC02
PPC03
PPC04
PPC05
MIC μg/mL
6.25
STANDARDS
Ciprofloxacin
Pyrazinamide
Streptomycin
MIC μg/mL
3.125
3.125
3.125
6.25
3.125
6.25
12.5
PPC06
12.5
PPC07
12.5
PPC08
12.5
PPC09
12.5
PPC10
12.5
2.4. Antibacterial studies
The antibacterial screening of PPC01-PPC10 against human pathogenic bacterial strains by
[37]
macrodilution broth method
have shown moderate to good activity. Tetracycline was the
positive control. Screening results are encapsulated in Table 2. PPCO1-PPC06 have shown
good activity in comparison with the standard whereas rest of them have exhibited moderate
activity against all the strains.
2.5. Antifungal Studies
PPC01-PPC10 were explored for their antifungal activity against four fungal strains[37] using
nystatin and fluconazole as standards. Among the tested compounds PPCO1-PPC06 have
emerged active against all the tested fungal strains, whereas rest of them have shown good
activity. The results are tabulated in Table 3.
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Table 2: Antibacterial Activity of PPC01-PPC10 (minimum inhibitory concentration, μg/mL)
Test organism
Staphylococcus
aureus
Bacillus
subtilis
Escherichia
coli
Pseudomonas
aeruginosa
Compounds
PPC01
PPC02
PPC03
PPC04
PPC05
PPC06
PPC07
4
4
2
2
2
2
4
4
4
2
2
2
4
2
4
4
4
2
4
2
2
2
4
2
16
16
64
32
4
32
8
32
16
32
64
2
32
16
64
32
4
PPC08
PPC09
32
64
2
PPC10
Tetracycline
2.6.Cytotoxicity of the synthesized compounds
The synthesized compounds were screened for their toxicity against normal Human Embryonic
kidney cell line (HEK293). As depicted in Table 4, the compounds have exhibited moderate to
low levels of cytotoxicity with IC50 values in the range of 580.8-753.6 μg/mL. None of the
compounds have exhibited any considerable cytotoxic effects, recommending good potential for
their further use.
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Table 3: Antifungal Activity of PPC01-PPC10 (minimum inhibitory concentration, μg/mL)
Test organism
Compounds
Aspergillus
flavus
Trichoderma
harzianum
Penicillium
chrysogenum
Candida
albicans
PPC01
PPC02
PPC03
PPC04
PPC05
PPC06
PPC07
4
2
4
2
2
4
2
2
2
2
4
2
1
2
4
4
8
16
16
32
32
32
64
2
8
16
8
8
16
16
64
64
64
2
16
32
64
64
2
32
16
64
64
4
PPC08
PPC09
PPC10
Nystatin
Fluconazole
2
4
2
2
Table 4: Cytotoxicity of PPC01-PPC10 against normal human embryonic kidney cells using
MTT assay
Compound Name
PPC01
IC50 (µg/mL)
750.4
Compound Name
PPC06
IC50 (µg/mL)
695.4
PPC02
659.8
PPC07
710.1
PPC03
646.1
PPC08
686.6
PPC04
753.6
PPC09
702.4
PPC05
624.4
PPC10
580.8
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2.7. Prediction of Physio-chemical properties of PPC01-PPC10
The correlation between physio-chemical properties of the chemical moieties and the drug
pharmacokinetics in the human body is calculated by the Lipinski rule of five.[38-39] As stated by
this rule a chemical moiety is more likely to exhibit poor absorption or permeation if
i. The molecular weight of the moiety is greater than 500
ii. The measure of lipophilicity i.e., Log P (Octanol/Water partition coefficient) is more than 5
iii. The number of H-bond donors exceeds 5,
iv. The number of H-bond acceptors i.e., the sum of Nitrogen and Oxygen atoms is over 10.
Despite of some exceptions, it is widely accepted method to screen all patterns of drugs.
The results deduced using molinspiration server are summarized in Table 5. The data disclosed
that the molecular weights of compounds are below 500 and range between 366.47- 478.69. The
Log P values lie in the limits of 2.97- 6.83. All the compounds have shown 4 hydrogen bond
acceptors. Six compounds have shown no violations of Lipinski.
2.8. Structure-activity relationship study
The SAR studies have interpreted the role of the length of alkyl chains in deciding the biological
activities of the synthesized pharmacophores. In case of anti-mycobacterial activity, it has been
seen that PPCO1-PPCO4 have shown good activity with MIC of 3.125 to 6.25 µg/mL equivalent
to the positive control whereas the remaining compounds have shown moderate activity with
MIC of 12.5 µg/mL. PPC01- PPC04 bears smaller length alkyl substituents namely ethyl,
isopropyl, propyl and butyl. The molecular docking studies against InhA was carried out and all
of the molecules exhibited higher C-score values than the standard which suggested that the all
the compounds should have good activity but a look into their Lipinski’s parameters showed that
the compounds of chain length 7 to 10 violated the rules in the parameter of lipophilicity. This
may be the reason for the decrease in the in-vitro activity.
The antimicrobial activity results showed that PPC01 and PPC03 have appeared active
against E. coli with MIC 2 µg/mL, whereas PPC02 is active against B. subtilis with MIC 2
µg/mL which is as same as the standard tetracycline. PPC04 is active towards S. aureus having
MIC 4 µg/mL. The remaining compounds have exhibited good antibacterial activity towards all
the four strains. Molecules with smaller chain alkyl groups are more active than their longer
counterparts.
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Table 5: Predicted properties of PPC01-PPC10
Lipinski’s Parameters
Bioactivity score
TPSA Molar %ABS Drug GPCR Ion Kinase Nuclear Protease
Compounds Log P MW HDA HBD
Violations
volume
Likeliness ligand channel inhibitorreceptorinhibitor
(A3)
modulator
ligand
PPC01
PPC02
PPC03
PPC04
PPC05
PPC06
PPC07
PPC08
PPC09
PPC10
2.97 366.47
3.32 380.50
3.45 380.50
3.93 394.52
4.42 408.55
4.90 422.58
5.38 436.60
5.86 450.63
6.34 466.46
6.83 478.69
4
4
4
4
4
4
4
4
4
4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
59.32 346.79 88.53
0.16
0.40
0.16
0.32
0.35
0.35
0.35
0.35
0.34
0.33
0.32
0.31
0.30
0.14
0.41
0.48
0.39
0.37
0.36
0.35
0.34
0.33
0.32
0.31
-0.58
-0.22
-0.28
-0.18
-0.15
-0.14
-0.13
-0.13
-0.12
-0.12
-0.12
59.32 363.37 88.53
59.32 363.59 88.53
0.14
0.17
0.17
0.17
0.16
0.16
0.15
0.15
0.13
-0.51
-0.55
-0.53
-0.51
-0.49
-0.48
-0.46
-0.45
-0.43
59.32 380.39 88.53 -0.03
59.32 397.19 88.53 -0.07
59.32 413.99 88.53 -0.07
59.32 430.80 88.53 -0.07
59.32 447.60 88.53 -0.07
59.32 460.40 88.53 -0.07
59.32 481.20 88.53 -0.07
Calculated from online server [38] http://www.molinspiration.com/cgi-bin/properties
[39] http://www.molsoft.com/mprop/
HDA-Number of hydrogen bond acceptors (n-ON)
HBD-Number of hydrogen bond donors (n-OHNH)
MW-Molecular weight
LogP-Logarithm of partition coefficient between n-octanol and water (miLogP)
TPSA-Topological polar surface area
%ABS-Percent of absorption; GPCR-G-Protein-Coupled Receptor
A molecule having bioactivity score more than 0.00 is likely to exhibit considerable biological activity, while value -0.50 to 0.00 are expected to be
moderately active and if the score is less than -0.50 it is presumed to be inactive
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In case of antifungal activity PPC01 to PPC04 have appeared active against all the fungal
strains with MIC ranging between 1-4 µg/mL. PPC05 and PPC06 also possess good activity with
MIC of 8-16 µg/mL whereas the rest of the compounds are moderate with MIC ranging between
16-64 µg/mL. PPC02 and PPC03 have appeared active against C. albicans, A. flavus and T.
harzianum whereas PPC01 exhibited good activity towards P. chrysogenum. However, PPC04
appeared active towards A. flavus. All of the compounds discussed above have shown MIC
comparable to the standards.
The C-score values also show that the molecules with alkyl chain between 1-4 chain length
have higher values than the standard and hence possess good binding interactions. The substituents
with 5 and 6 chain length are having lower C-score values; hence there is a decrease in the in-vitro
activity. On the other hand, the compounds with the 7 to 10 chain length also exhibit high C-score
values but due to violations of lipophilicity parameter, the in-vitro activity decreases in comparison
to the smaller alkyl chains. This explanation also holds accounts for the antibacterial studies.
It has been analyzed that the compounds containing smaller alkyl chains (i.e., chain length
1-4) have exhibited better activities which is also supported by their in-silico as well as
physicochemical studies. It has also been noticed that with increase in the length of an alkyl chain,
the activity of the compounds decreases.
2.9. In-Silico Evaluation
Among the enzymes involved in FAS-II, the NADH-dependent enoyl-ACP reductase encoded by
Mycobacterium gene InhA is the key catalyst in mycolic acid biosynthesis. Studies in the past have
established that InhA is the primary molecular target for INH which has been the frontline drug for
over 40 years used in the treatment of TB. The INH-NADH adduct functions as a potent inhibitor
[40-41]
[42]
of InhA and the well-known non-selective InhA inhibitors diazoborines
and triclosan,
InhA direct inhibitors, pyrrole[43] derivatives and piperazines[44] have all been reported to exhibit
in vivo as well as in vitro antitubercular activities.
On the other hand, Dihydro folate reductase (DHFR) is an important enzyme in both
mammals and microorganism including Mycobacterium tuberculosis (Mtb). It catalyzes folic acid
conversion to dihydro and tetrahydro folic acid which is a crucial step in the folate pathway.
Inhibition of the folate pathway leads to the interruption of thymidine supply resulting in cell
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Chemistry & Biodiversity
death. Hence molecular docking was performed on these two targets in order to find active
molecules.
Molecular modelling was employed to comprehend the effectiveness of the title molecules
by examining their interactions which also reinforced the in-vitro results. Molecular modelling was
done on Mycobacterium tuberculosis enoyl reductase (InhA) coordinated with 1-cyclohexyl-N-
(3,5-dichlorophenyl)-5-oxopyrrolidine-3-carboxamide (PDB ID: 4TZK) from M. tuberculosis and
Candida albicans DHFR (PDB ID: 1AI9) from C. albicans. This was also utilized to explicate the
binding approach of the title molecules. The docking study was carried out using Surflex-Dock
programme of Sybyl-X software extracting proteins from the Protein Data Bank (PDB).[45-46]
2.9.1 Docking studies of PPC01-PPC10 with Mycobacterium tuberculosis enoyl reductase (InhA)
coordinated with 1-cyclohexyl-N-(3,5-dichlorophenyl)-5-oxopyrrolidine-3-carboxamide
(PDB ID: 4TZK) from M. tuberculosis
The docking investigation unfolded that all the title molecules have shown reasonably good
docking score for InhA (Table 6). Fig.1 represents the docked view of PPC01-PPC10 in the
active pocket of the enzyme PDB ID: 4TZK.
Fig.2 illustrates that compound PPC02 makes one H-bonding interaction at the active
pocket of the enzyme (PDB ID: 4TZK). Oxygen of the propoxy group makes an interaction with
hydrogen atom of TYR158 (O------H-TYR158, 2.14 Å). The pyrrolidine carboxamide (4TZK
ligand) exhibited two H-bonding interactions through its carbonyl oxygen with Tyr158 and co-
factor NAD+ as portrayed in Fig.3
PPC10 makes three hydrogen bonding connections on active pocket of the enzyme (PDB
ID: 4TZK), amongst those two interactions are from nitrogen atom of the first position of purine
with hydrogen of NAD500 and TYR158 (N------H-NAD500, 2.26 Å; N------H-TYR158, 2.41 Å)
and the last one raised from the nitrogen of the third position of purine with hydrogen atom of
TYR158 (N------H-TYR158, 2.58 Å). (Supplementary Fig.1 & 2)
Fig.4 (A and B) illustrate the hydrophobic and hydrophilic amino acids encircling PPC10
& PPC02. Docking studies interpreted that most of the title compounds occupied analogous
binding sites as that of pyrrolidine carboxamide. The compounds have exhibited C score between
the range 10.42-6.21, signifying the outline of all the forces of interaction between PPC01-PPC10
and InhA and indicate that molecules competitively bind to InhA than 4TZK reference ligand.
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Table 6: Surflex Docking score (kcal/mol) of PPC01-PPC10 for M. tuberculosis enoyl reductase
(InhA) (PDB ID: 4TZK)
C
Crash
Polar
PMF
Chem
Compounds
D Score[d]
G Score[f]
-285.29
Score[a]
Score[b]
Score[c]
Score[e]
Score[g]
6.13
6.87
6.95
6.59
6.21
6.67
8.80
8.85
8.74
10.42
9.08
-1.39
-2.82
-2.03
-1.57
-2.30
-3.46
-1.83
-2.49
-2.21
-2.40
-6.41
1.18
0.00
1.04
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.78
-168.11
-144.964
-131.628
-135.489
-149.141
-165.361
-161.880
-173.274
-180.275
-195.518
-219.898
-49.19
-43.371
-47.238
-53.734
-48.685
-30.275
-70.888
-38.750
-73.876
-76.801
-45.133
-37.47
4TZK
PPC01
PPC02
PPC03
PPC04
PPC05
PPC06
PPC07
PPC08
PPC09
PPC10
-298.556 -30.210
-248.902
-249.958
-279.305
-335.324
-309.389
-335.916
-331.317
-396.089
-460.212
-28.037
-22.968
-31.068
-32.075
-29.905
-35.675
-32.648
-35.680
-39.608
[a] C Score (Consensus Score) integrates a number of popular scoring functions for ranking the affinity of PPC01-
PPC10 bound to an active pocket of a receptor and reports output of total score.
[b] Crash-score discloses the inappropriate penetration into the binding pocket. The scores close to 0 are favorable
whereas Negative numbers indicate penetration.
[c] Polar score indicates the contribution of polar interactions to the total score. It may be useful for excluding docking
results that make no H-bonds.
[d] D-score depicts charge and Vander Waals interactions between protein and ligand.
[e] PMF-score specifies the Helmholtz free energies of interactions for protein-ligand atom pairs (Potential of Mean
Force, PMF).
[f] G-score shows hydrogen bonding, complex (ligand-protein) and internal (ligand-ligand) energies.
[g] Chem-score points for H-bonding, lipophilic contact and rotational entropy including an intercept term.
Fig.1 Docked view of PPC01-PPC10 in the active pocket of the enzyme (PDB ID: 4TZK)
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H
TYR158
O
N
N
N
N
N
N
Fig.2 Docked view of PPC02 in the active pocket of the enzyme (PDB: 4TZK)
Tyr158
H
O
NAD+
Cl
Cl
O
H
N
NH
Fig.3 Interaction of (InhA) at the binding site of the enzyme (PDB ID: 4TZK)
A
B
Fig.4 A) Hydrophobic amino acids encircling PPC02 (cyan colour) and PPC10 (green colour)
B) Hydrophilic amino acids encircling PPC02 (cyan colour) and PPC10 (green colour)
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2.9.2. Docking studies of PPC01-PPC10 with Candida albicans DHFR (PDB ID: 1AI9) from C.
albicans
The studied compounds have shown good docking scores against the enzyme dihydrofolate
reductase of Candida albicans as revealed by the docking study. (Table 7) summarizes the results.
Fig.5 represents the docked view of PPC01-PPC10 in the active pocket of the enzyme PDB ID:
1AI9.
PPC03 has three H-bonding interactions in the active pocket of the enzyme (PDB ID:
1AI9). The first interaction is through the nitrogen of 7th position of purine with hydrogen of
ARG56 (N------H-ARG56; 2.72 Å), the second one through the nitrogen of 1st position on purine
ring with hydrogen atom of LYS57 (N------H-LYS57; 2.08 Å). The third interaction is raised
through the nitrogen of 3rd position of purine with hydrogen atom of ARG56 (N------H-ARG56;
2.72 Å) as depicted in Fig.6.
PPC08 has three H-bonding interactions in the active pocket of the enzyme (PDB ID:
1AI9). Amongst that an interaction is from the nitrogen atom present on the 7th position of purine
with hydrogen atom of LYS57 (N------H-LYS57; 1.96 Å), nitrogen of 1st position of purine binds
with hydrogen atom of LYS57 (N------H-LYS57; 2.09 Å). The last one is from nitrogen of 3rd
position of purine with hydrogen atom of GLY114 (N------H-GLY114; 2.86 Å). (Supplementary
Fig.3 and 4)
As depicted in fig.7 fluconazole, shows three H-bonding interaction at the active pocket of
Candida albicans DHFR (PDB ID: 1AI9) and all of these are from only one nitrogen of triazole
with hydrogen of SER78 (-N----H-SER78), LYS57 (-N----H-LYS57) and ARG56 (-N----H-
ARG56).
Fig.8 illustrates the hydrophobic and hydrophilic amino acids encircling PPC03 & PPC08.
The synthesized molecules have shown binding interactions with LYS57 and ARG56 as similar to
that of fluconazole. Thus, from these studies, the experimental findings can be substantiated,
wherein the purine derivatives inhibit DHFR.
As DFHR is a very universal enzyme across all living organisms from bacteria to mammals
including humans, docking studies with human dihydrofolate reductase complexed with NADPH
and 2,4-diamino-5-((7,8-dicarbaundecaboran-7-yl)methyl)-6-methylpyrimidine, a novel boron-
containing, non classical antifolate (PDB ID: 2C2T) was explored as shown in Fig. 9 (A and B).
This was done to check whether the synthesized compounds are selective towards DHFR of C.
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10.1002/cbdv.202000053
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albicans or also interact with other DHFR’s. The study showed that compounds did not show any
interactions with the human DHFR and are selective towards dihydrofolate reductase (DFHR)
enzyme of C. albicans which also supports the toxicity study on the normal cell line.
Table 7: Surflex Docking score (kcal/mol) of PPC01-PPC10 for Candida albicans dihydrofolate
reductase (PDB ID: 1AI9)
C
Score[a]
6.12
6.35
7.03
6.23
4.78
5.13
5.79
6.05
6.22
7.81
6.78
Crash
Score[b]
-2.43
-0.91
-3.39
-1.76
-7.45
-3.51
-4.66
-5.46
-6.18
-1.44
-2.35
Polar
Score[c]
4.76
1.19
0.03
0.01
0.21
0.01
0.00
0.02
1.77
0.00
0.00
PMF
Score[e]
-38.035
-6.412
27.538
-43.415
45.507
-12.721
26.196
30.734
4.711
Chem
Score[g]
Compounds
D Score[d]
G Score[f]
-25.709
-101.680
-151.299
-106.269
-175.798
-137.091
-158.458
-169.192
-171.586
-133.758
-147.730
-201.77
-187.612
-330.283
-257.610
-345.851
-273.537
-306.378
-339.918
-312.986
-274.778
-314.429
-11.08
Flucanazole
PPC01
PPC02
PPC03
PPC04
PPC05
PPC06
PPC07
PPC08
PPC09
PPC10
-17.642
-22.817
-19.191
-21.530
-16.253
-22.519
-26.555
-22.591
-17.349
-21.645
2.210
11.838
Fig.5 Docked view of PPC01-PPC10 in the active site of the enzyme (PDB ID: 1AI9)
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O
N
N
A
R
G
56
H
A
R
G
5
6
H
N
N
N
N
H
L
YS
57
Fig.6 Docked view of PPC03 in the active pocket of the enzyme (PDB: 1AI9)
SER78
H
ARG56
N
H
LYS57
N
N
H
N
N
N
F
OH
F
Fig.7 Interaction of Fluconazole at the binding pocket of the enzyme (PDB ID: 1AI9)
A
B
Fig.8 A) Hydrophobic amino acids encircling PPC03 (green colour) and PPC08 (cyan colour)
B) Hydrophilic amino acids encircling PPC03 (green colour) and PPC08 (cyan colour)
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B
A
Fig.9 A) Docked view of PPC01-PPC10 in the active site of the enzyme (PDB ID: 2C2T)
B) Interaction of PPC01- PPC10 at the binding pocket of the enzyme (PDB ID: 2C2T)
3. Conclusion
An efficient and simple synthetic protocol was chosen to synthesize novel N-9 alkyl-substituted
purines employing mild reaction conditions. The title compounds obtained were characterized by
1H NMR, 13C NMR, FT-IR and EI-MS. A dominant display of antimicrobial activity including
antitubercular activity has been shown depending on the length of the alkyl chain. In general,
expression of activity is manifested for the compounds with alkyl substituent of 2 to 7 chain
length. The title compounds exhibited strong antitubercular activity with MIC similar to the
standards used. PPC01-PPC04 appeared better with MIC (3.125-6.25µg/mL) whereas the rest
compounds were moderate (MIC 12.5 µg/mL).
The studied compounds showed pronounced antimicrobial activity expressed against
bacterial and fungal strains with MIC values lower than the standards used. The molecules with
smaller alkyl chain length showed better activity with MIC’s lower than the standards while their
longer chain counterparts exhibited moderate activity.
The docking study with M. tuberculosis enoyl reductase (InhA) depicted that all of the
compounds possessed better docking scores, indicating that the molecules occupy similar binding
sites as that of pyrrolidine carboxamide and preferentially bind to InhA.
The docking study of the molecules with Candida albicans dihydrofolate reductase shows
that the synthesized molecules have shown binding interactions with LYS57 and ARG56 as similar
to that of fluconazole. Thus, from these studies, the experimental findings can be substantiated,
wherein the purine derivatives inhibit DHFR of Candida albicans selectively.
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The promising activities shown by the title molecules established them as crucial
pharmacophores that can be further explored and used as leads to design further novel derivatives
with enhanced activity.
4. Experimental Section
4.1. Chemistry
Chemicals and solvents of reagent grade from Sigma Aldrich were used without further
purification. The open capillary method was used to record melting points and they are
uncorrected. Perkin Elmer FT-IR spectrometer was used for recording FT-IR spectra were
1
recorded on. H and 13C NMR data were collected by using JEOL 400-MHz FT NMR
spectrometer. CDCl3 was the solvent used. Chemical shifts (δ) are represented in parts per million
(ppm) with respect to Tetramethylsilane (TMS) as an internal standard. Mass spectra were
obtained by using QP2010S SHIMADZU instrument.
4.2. General procedure for the synthesis of PP2
N-protected 4-hydroxyphenyl piperazine (PP1) (4.0 g, 18.1 mmol) and propyl bromide (2.66 g,
21.72 mmol) were reacted with anhyd. K2CO3 (5.0 g, 36.2 mmol). Dry DMF was used as solvent.
0
It was refluxed at 120 C at a time period of 12 h. TLC was used to monitor the progress of the
reaction. After the completion, the reaction mixture was cooled to room temperature, the unreacted
K2CO3 was filtered off and the residue was washed with a small volume of solvent. The filtrate
was concentrated under reduced pressure to obtain crude solid which was washed with cold water
to yield the pure product as white colored solid (PP2) with 80 % yield.
4.3. General procedure for the synthesis of PP3
PP2 obtained in the earlier step (3.5 g, 13.3 mmol) was taken in a round bottom flask containing
distilled water (15 mL) and stirred for 5-10 min. To this, 20 mL of HCl (4 N) was added slowly.
0
This reaction mixture was refluxed at 80 C for 3 h and the progress of reaction was examined by
thin-layer chromatography. After the completion of reaction, the reaction mixture was cooled to 5
0C and neutralized using aq. NaOH (25%) to get crude solid. Ethyl acetate was used to
recrystallize the crude compound to afford pure light brown colored solid as (4-propoxyphenyl)
piperazine (PP3) with 82 % yield.
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4.4. General procedure for the synthesis of PP4
(4-propoxyphenyl)piperazine (PP3) (4.0 g, 18.17 mmol) and 6-chloro purine (2.8 g, 18.17 mmol)
were taken in n-butanol. The reaction mixture was stirred for 5 minutes followed by drop wise
addition of triethylamine (5.83 g, 57.64 mmol). On completion of addition, the reaction mixture
was refluxed at 120 0C for 8 h. After the completion of reaction, solvent was evaporated using rota
evaporator to obtain the crude solid. This upon recrystallization using ethyl alcohol to get pure C-6
substituted purine (PP4) with 85 % yields.
4.5. General procedure for the synthesis of PPC01-PPC10
C-6 substituted purine (PP4) (500 mg, 1.47 mmol) was taken in dry DMF and anhyd. K2CO3 (250
mg, 1.77 mmol) was added. This reaction mixture was stirred at room temperature for 10-15 min
and substituted alkyl bromides (160 mg, 1.47 mmol) were added and maintained at room
temperature for about 12 h. The completion of reaction was monitored by TLC and the reaction
mixture was quenched with ice to obtain crude solid. This was filtered and washed with water to
obtain the crude products which were purified by eluting through hexane and ethyl acetate in 80-
90% yields.
4.5.1. 9-ethyl-6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine (PPC01)
o
Obtained as colorless solid; Yield: 80%; m.p: 98-100 C; FT-IR (cm-1): 3110 (C-H), 2927 (C-C-
H),1580 (C=N), 1250 (C-O), 1154 (C-N); 1H NMR (400 MHz, CDCl3) δ: 8.38 (s, 1 H), 7.78 (s, 1
H), 6.94 (d, J 6.8 Hz, 2 H), 6.86 (d, J 9.1 Hz, 2 H), 4.47 (s, 4 H), 4.25 (q, J 7.3 Hz, 2 H), 3.88 (t, J
6.6 Hz, 2 H), 3.19 (s, 4 H), 1.74-1.83 (m, 2 H), 1.52 (t, J 7.3 Hz, 3 H), 1.02 (t, J 7.4 Hz, 3 H); 13C
NMR (100 MHz, CDCl3) δ: 153.96, 153.82, 152.40, 150.98, 145.51, 138.65, 120.17, 118.81,
115.26, 69.83, 51.26, 45.44, 22.87, 14.35, 10.69; EI-MS: m/z calcd for C20H26N6O 366.46, found
366 (M+).
4.5.2. 9-isopropyl-6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine(PPC02)
o
Obtained as colorless solid; Yield: 85%; m.p: 96-98 C; FT-IR (cm-1): 3104 (C-H), 2924 (C-C-
H),1592 (C=N), 1255 (C-O), 1198 (C-N); 1H NMR (400 MHz, CDCl3) δ: 8.37 (s, 1 H), 7.74 (s, 1
H), 6.93 (d, J 9.0 Hz, 2 H), 6.84 (d, J 9.0 Hz, 2 H), 4.46 (s, 4 H), 4.15 (t, J 7.2 Hz, 2 H), 3.19 (s, 4
H), 2.86-2.97 (m, 1H) 1.85-1.94 (m, 2 H), 1.10 (d, J 6.5 Hz, 6H), 0.95 (t, J 7.4 Hz, 3 H); 13C NMR
(100 MHz, CDCl3) δ: 153.85, 153.72, 152.02, 150.65, 145.22, 137.95, 119.32, 117.61, 114.34,
69.86, 51.02, 22.68, 21.47, 10.62; EI-MS: m/z calcd for C21H28N6O 380.49, found 380 (M+).
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4.5.3. 6-(4-(4-propoxyphenyl)piperazin-1-yl)-9-propyl-9H-purine (PPC03)
o
Obtained as colorless solid; Yield: 82%; m.p: 92-94 C; FT-IR (cm-1): 3104 (C-H), 2935 (C-C-
H),1615 (C=N), 1265 (C-O), 1158 (C-N); 1H NMR (400 MHz, CDCl3) δ: 8.37 (s, 1 H), 7.74 (s, 1
H), 6.93 (d, J 9.0 Hz, 2 H), 6.84 (d, J 9.0 Hz, 2 H), 4.46 (s, 4 H), 4.15 (t, J 7.2 Hz, 2 H), 3.87 (t, J
6.6 Hz, 2 H), 3.19 (s, 4 H), 1.81-1.95 (m 2 H), 1.73-1.80 (m, 2 H), 1.01 (t, J 7.4 Hz, 3 H), 0.95 (t, J
7.4 Hz, 3 H); 13C NMR (100 MHz, CDCl3) δ: 153.93, 153.84, 152.17, 150.99, 145.40, 138.28,
120.10, 118.81, 115.30, 69.97, 51.15, 43.71, 26.76, 22.68, 14.06, 10.77; EI-MS: m/z calcd for
C21H28N6O 380.49, found 380 (M+).
4.5.4. 9-butyl-6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine (PPC04)
o
Obtained as colorless solid; Yield: 78%; m.p: 90-92 C; FT-IR (cm-1): 3100 (C-H), 2926 (C-C-
1
H),1585 (C=N), 1270 (C-O), 1152 (C-N); H NMR (400 MHz, CDCl3) δ: 8.36 (s, 1 H), 7.73 (s, 1
H), 6.93 (d, J 9.0 Hz, 2 H), 6.84 (d, J 9.1 Hz, 2 H), 4.45 (s, 4 H), 4.16 (t, J 7.2 Hz, 2 H), 3.86 (t, J
6.6 Hz, 2 H), 3.18 (s, 4 H), 1.74-1.87 (m, 6 H), 1.00 (t, J 7.4 Hz, 3 H), 0.86 (t, J 7.6 Hz, 3 H); 13C
NMR (100 MHz, CDCl3) δ: 153.93, 153.84, 152.19, 150.81, 145.47, 139.13, 120.33, 118.55,
115.00, 70.07, 50.77, 44.04, 31.98, 26.13, 22.57, 14.06, 10.01; EI-MS: m/z calcd for C22H30N6O
394.51, found 394 (M+).
4.5.5. 9-pentyl-6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine (PPC05)
Obtained as colorless solid; Yield: 80%; m.p: 90-92oC; FT-IR (cm-1): 3105 (C-H), 2929 (C-C-H),
1592 (C=N), 1260 (C-O), 1154 (C-N); 1H NMR (400 MHz, CDCl3) δ: 8.38 (s, 1 H), 7.74 (s, 1 H),
6.94 (d, J 9.1, 2 H), 6.86 (d, J 9.1 Hz, 2 H), 4.47 (s, 4 H), 4.19 (t, J 7.2 Hz, 2 H), 3.88 (t, J 6.6 Hz,
2H), 3.20 (s, 4 H), 1.81-1.89 (m, 2 H), 1.76-1.79 (m, 2 H), 1.29-1.42 (m, 4 H), 1.02 (t, J 7.4 Hz, 3
H), 0.95 (t, J 7.4 Hz, 3 H); 13C NMR (100 MHz, CDCl3) δ: 153.94, 153.84, 152.40, 150.86,
145.52, 138.06, 120.21, 118.83, 115.26, 69.87, 51.37, 38.99, 31.35, 29.26, 22.48, 20.27,15.45,
10.38; EI-MS: m/z calcd for C23H32N6O 408.54, found 408 (M+).
4.5.6. 9-hexyl-6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine (PPC06)
Obtained as colorless solid; Yield: 85%; m.p: 88-90oC; FT-IR (cm-1): 3070 (C-H), 2925 (C-C-H),
1582 (C=N), 1249 (C-O), 1154 (C-N); 1H NMR (400 MHz, CDCl3) δ: 8.38 (s, 1 H), 7.74 (s, 1 H),
6.94 (d, J 9.1 Hz, 2 H), 6.86 (d, J 9.1 Hz, 2 H), 4.47 (s, 4 H), 4.19 (t, J 7.2 Hz, 2 H), 3.88 (t, J 6.6
Hz, 2 H), 3.20 (s, 4 H), 1.84-1.88 (m, 2 H), 1.74-1.79 (m, 2 H), 1.29-1.40 (m, 4 H), 1.20-1.23 (m, 2
H), 1.02 (t, J 7.4 Hz, 3 H), 0.95 (t, J 7.4 Hz, 3 H); 13C NMR (100 MHz, CDCl3) δ: 153.87, 153.77,
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152.32, 151.03, 145.37, 138.30, 120.08, 118.50, 115.10, 69.93, 50.84, 44.91, 43.36, 32.02, 29.66,
22.52, 22.02, 13.50, 10.38; EI-MS: m/z calcd for C23H32N6O 422.57, found 422(M+).
4.5.7. 9-heptyl-6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine (PPC07)
Obtained as colorless solid; Yield: 76%; m.p: 84-86oC; FT-IR (cm-1): 3115 (C-H), 2923 (C-C-H),
1
1592 (C=N), 1262 (C-O), 1157 (C-N); H NMR (400 MHz, CDCl3) δ: 8.36 (s, 1 H), 7.73 (s, 1 H),
6.92 (d, J 9.1 Hz, 2 H), 6.84 (d, J 9.1 Hz, 2 H), 4.45 (s, 4 H), 4.16 (t, J 7.2 Hz, 2 H), 3.86 (t, J 6.6
Hz, 2 H), 3.18 (s, 4 H), 1.86-1.90 (m, 2 H), 1.74-1.80 (m, 2 H), 1.28-1.34 (m, 8H), 1.00 (t, J 7.4
Hz, 3 H), 0.87 (t, J 7.0 Hz, 3 H); 13C NMR (100 MHz, CDCl3) δ: 153.99, 153.87, 152.24, 151.24,
145.38, 138.57, 120.05, 118.76, 115.24, 70.12, 51.12, 44.01, 31.98, 31.62, 29.82, 29.78, 28.81,
22.70, 22.22, 13.99, 10.63; EI-MS: m/z calcd for C25H36N6O 436.59, found 436 (M+).
4.5.8. 9-octyl-6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine (PPC08)
Obtained as colorless solid; Yield: 80%; m.p: 80-82oC; FT-IR (cm-1): 3100 (C-H), 2916 (C-C-
H),1588 (C=N), 1242 (C-O), 1182 (C-N); 1H NMR (400 MHz, CDCl3) δ: 8.36 (s, 1 H), 7.73 (s, 1
H), 6.92 (d, J 9.1 Hz, 2 H), 6.84 (d, J 9.1 Hz, 2 H), 4.45 (s, 4 H), 4.16 (t, J 7.2 Hz, 2 H), 3.86 (t, J
6.6 Hz, 2 H), 3.18 (s, 4 H), 1.86-1.92 (m, 2 H), 1.74-1.80 (m, 2 H), 1.42-1.48 (m, 2 H), 1.30-1.36
13
(m, 8 H), 1.00 (t, J 7.4 Hz, 3 H), 0.87 (t, J 7.0 Hz, 3 H); C NMR (100 MHz, CDCl3) δ: 153.99,
153.84, 152.24, 151.24, 145.38, 138.57, 120.05, 118.76, 115.24, 70.12, 51.12, 44.01, 31.98, 31.62,
29.82, 29.78, 28.81, 22.70, 22.22, 13.99, 10.63; EI-MS: m/z calcd for C26H38N6O 450.62, found
450 (M+).
4.5.9. 9-nonyl-6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine (PPC09)
Obtained as colorless solid; Yield: 75%; m.p: 82-84oC; FT-IR (cm-1): 3115 (C-H), 2920 (C-C-H),
1580 (C=N), 1252 (C-O), 1152 (C-N); 1H NMR (400 MHz, CDCl3) δ: 8.36 (s, 1 H), 7.73 (s, 1 H),
6.92 (d, J 9.1 Hz, 2 H), 6.84 (d, J 9.1 Hz, 2 H), 4.45 (s, 4 H), 4.16 (t, J 7.2 Hz, 2 H), 3.86 (t, J 6.6
Hz, 2 H), 3.18 (s, 4 H), 1.88-1.94 (m, 2 H), 1.74-1.80 (m, 2 H), 1.34-1.42 (m, 10 H), 1.25-1.31(m,
2 H), 1.00 (t, J 7.4 Hz, 3 H), 0.87 (t, J 7.0 Hz, 3 H); 13C NMR (100 MHz, CDCl3) δ: 153.89,
153.75, 152.24, 151.24, 145.38, 138.57, 120.05, 118.76, 115.24, 70.12, 51.12, 44.01, 31.98, 31.62,
29.82, 29.78, 28.81, 22.70, 22.22, 21.34, 13.99, 10.63; EI-MS: m/z calcd for C27H40N6O 464.65,
found 464 (M+).
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4.5.10. 9-decyl-6-(4-(4-propoxyphenyl)piperazin-1-yl)-9H-purine (PPC10)
Obtained as colorless solid; Yield: 74%; m.p: 80-82oC; FT-IR (cm-1): 3125 (C-H), 2910 (C-C-H),
1588 (C=N), 1256 (C-O), 1162 (C-N); 1H NMR (400 MHz, CDCl3) δ: 8.36 (s, 1 H), 7.73 (s, 1 H),
6.92 (d, J 9.1 Hz, 2 H), 6.84 (d, J 9.1 Hz, 2 H), 4.45 (s, 4 H), 4.16 (t, J 7.2 Hz, 2 H), 3.86 (t, J 6.6
Hz, 2 H), 3.18 (s, 4 H), 1.82-1.90 (m, 2 H), 1.74-1.82 (m, 2 H), 1.43-1.49 (m, 2 H),1.31-1.37 (m,
12 H), 1.00 (t, J 7.4 Hz, 3 H), 0.87 (t, J 7.0, 3 H); 13C NMR (100 MHz, CDCl3) δ: 153.84, 153.79,
152.24, 151.24, 145.38, 138.57, 120.05, 118.76, 115.24, 70.12, 51.12, 44.01, 31.98, 31.62, 29.82,
29.78, 28.81, 22.70, 22.22, 13.99, 10.63; EI-MS: m/z calcd for C28H42N6O 478.67, found 478
(M+).
4.6. Biological Activity
4.6.1. Anti-mycobacterial activity
The synthesized compounds were assessed for their anti-mycobacterial activity against
Mycobacterium tuberculosis employing microplate Alamar Blue assay (MABA). This
methodology is a parallel method to BACTEC radiometric method and is non-toxic. To all outer
perimeter wells of sterile 96 wells plate, 200μL of sterile de-ionized water was added to reduce
evaporation of media during incubation. 100 μL of the middlebrook 7H9 broth was added to the 96
wells plate and serial dilution of synthesized compounds PPC01-PPC10 was done on the plate. The
final concentrations of the title molecules tested were 100 to 0.2 μg/mL. Parafilm was used to
cover the plates and the plates were incubated for five days at 37 ºC. After 5 days, 1:1 mixture of
Almar Blue reagent and 10% tween 80 were poured onto the plates (25μL, freshly prepared) and
incubated for 24 h. The colour of the well was used to interpret the bacterial growth. A blue colour
in the well was interpreted as no bacterial growth, and pink colour was scored as growth. The
lowest drug concentration at which the color change from blue to pink is prevented is termed as
MIC. The Standard Strain used: Mycobacterium tuberculosis (Vaccine strain, H37RV strain):
ATCC No- 27294.
4.6.2. Antibacterial activity
The title compounds PPC01-PPC10 were screened for antibacterial activity against four bacterial
strains by macro dilution broth method. The synthesized compounds in concentrations of 0.125 -
256 μg/mL respectively were poured into the labeled sterile Mueller Hinton Broth (MHB) medium
tubes. 1 mL lag phase cultures of the Gram-positive bacteria Staphylococcus aureus and Bacillus
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10.1002/cbdv.202000053
Chemistry & Biodiversity
subtilis; Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa were inoculated in
the MHB tubes. These were incubated for 24 h at 37 ºC followed by which the inhibition of growth
of the bacteria was observed in the tubes. The lowest concentration of the synthesized compounds
containing tubes which did not show any visible growth of the test bacteria is termed as MIC.
4.6.3. Antifungal activity
PPC01-PPC10 were screened for antifungal activity by macro dilution broth method. The
synthesized compounds in concentrations of 0.125 - 256 μg/mL respectively were added to labeled
sterile saboraud dextrose broth (SDB) tubes. The lag phase cultures of the fungi Aspergillus flavus,
Trichoderma harzianum, Penicillium chrysogenum and yeast Candida albicans were inoculated in
the SDB tubes and incubated for 48 h at 27 ºC. The inhibition of the growth of test fungi in the
tubes was observed after 48 h. The lowest concentration of the synthesized compounds containing
tubes which did not show any visible growth of the test fungi is termed as MIC.
4.6.4. Cytotoxicity
To evaluate the effects of PPC01-PPC10 on cell viability, Human Embryonic kidney Cells
(HEK293) were employed to perform the cytotoxic assay by MTT method. The measurement in
the reduction of yellow colour of 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) by mitochondrial succinate dehydrogenase is measured through this colorimetric assay. 96-
well flat-bottom micro plate were seeded with HEK293 cells at a density of about 5×103 cells/well
and maintained in 95% humidity and 5% CO2 overnight at 37 °C. The synthesized compounds in
six concentrations (600, 300, 150, 75, 37.5, 18.75 μg/mL) were seeded in the cells and incubated
for 48 h. After this time, the cells in the well were washed with phosphate buffer solution twice
and MTT staining solution (20 μL) was added to each well. The plate was incubated at 37 °C. 100
μL of Dimethyl sulfoxide (DMSO) was poured in each well after 4 h to dissolve the formazan
crystals formed and using micro plate reader the absorbance at 570 nm was recorded.
4.6.5. Molecular docking studies
Information regarding 3D arrangement of the target protein was extracted from Protein Data Bank
(PDB). Surflex Dock program was employed to examine the complete intermolecular interactions
between the ligand and the target protein. A Gasteiger-Huckel charge was used to add polar
hydrogen atoms in the proteins for docking and the water molecules were removed. 3D structure
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10.1002/cbdv.202000053
Chemistry & Biodiversity
was generated using SKETCH module present in the SYBYL program and Tripos force field was
used to obtain its energy-minimized conformation using Gasteiger-Huckel charges. Surflex-Dock
program interfaced with Sybyl- X 2.0. was utilized for performing the molecular docking. The
default values of the miscellaneous parameters were assigned by the software.
Acknowledgement
We
are
thankful
to
the
University
Scientific
Instruments
Centre (USIC and SAIF ), Karnatak University, Dharwad for providing the spectral data, DST-
INSPIRE, New Delhi, India, for providing financial support under DST/INSPIRE
Fellowship/2016/IF160657 and DST-PURSE PHASE-II program for providing necessary
facilities. One of the authors Mahesh S. Najare is thankful to UGC-SRF for financial support.
Author Contribution Statement
AfraQuasar A. Nadaf performed the experiments, analyzed the data and wrote the first draft of the
manuscript. Mahesh S. Najare, Manjunatha Garbhagudi and Shivaraj Mantur helped with the
interpretation of spectral data of the synthesized compounds. Manjunath G. Sunagar Supreet
Gaonkar helped in designing the experiment. Shrinivas Joshi carried out molecular docking work
and Imtiyaz Ahmed M. Khazi supervised the whole work along with drafting it. All the authors
studied and approved the final draft of the manuscript.
Conflict of interest
Authors declare that there are no conflicts of interest.
Appendix A. Supplementary data
Representative spectral data for the synthesized compounds are provided in supporting
information.
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