Discovery of pyridoindole derivatives as potential inhibitors for phosphodiesterase 5A: in silico and in?vivo studies

Article abstract of DOI:10.1080/14786419.2021.1925274

The aim of this work was to synthesise derivatives from identified plant based pyridoindole lead scaffold, and to assess phosphodiesterase 5A inhibitory potential by in silico and in?vivo. Pyridoindole derivatives were synthesised by using six-stage reactor. In silico screening was carried out by grip-based docking methodology. In step-I, tryptophan as a starting material was reacted with different aldehydes and ketones to obtain 11 molecules. In step-II, obtained molecules were reacted with ethanol and benzyl alcohols to obtain D1 to D22 derivatives. In silico investigation resulted in best three molecules D12, D4 and D8 with promising BE score. Oral acute toxicity study of selected molecules resulted in LD50 value 500 mg/kg in rats. The result of in?vivo antihypertensive study shown that molecule D12 was found to be the best antihypertensive lead molecule. This study could be a best platform to tailor novel biomolecules for inhibiting phosphodiesterase 5A enzyme in hypertension management.

Full text of DOI:10.1080/14786419.2021.1925274

Natural Product Research
Formerly Natural Product Letters
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Discovery of pyridoindole derivatives as potential
inhibitors for phosphodiesterase 5A: in silico and
in vivo studies
Dipak P. Mali, Dinanath T. Gaikwad, Manish S. Bhatia & Neela M. Bhatia
To cite this article: Dipak P. Mali, Dinanath T. Gaikwad, Manish S. Bhatia & Neela M. Bhatia
(2021): Discovery of pyridoindole derivatives as potential inhibitors for phosphodiesterase 5A: in
silico and in vivo studies, Natural Product Research, DOI: 10.1080/14786419.2021.1925274
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2021.1925274
Discovery of pyridoindole derivatives as potential
inhibitors for phosphodiesterase 5A: in silico and
in vivo studies
Dipak P. Malia , Dinanath T. Gaikwadb , Manish S. Bhatiac and
Neela M. Bhatiaa
aDepartment of Pharmaceutical Quality Assurance, Bharati Vidyapeeth College of Pharmacy,
b
Kolhapur, Maharashtra, India; Department of Pharmaceutics, Bharati Vidyapeeth College of
c
Pharmacy, Kolhapur, Maharashtra, India; Department of Pharmaceutical Chemistry, Bharati
Vidyapeeth College of Pharmacy, Kolhapur, Maharashtra, India
ABSTRACT
ARTICLE HISTORY
Received 12 February 2021
Accepted 26 April 2021
The aim of this work was to synthesise derivatives from identified
plant based pyridoindole lead scaffold, and to assess phospho-
diesterase 5A inhibitory potential by in silico and in vivo.
Pyridoindole derivatives were synthesised by using six-stage
reactor. In silico screening was carried out by grip-based docking
methodology. In step-I, tryptophan as a starting material was
reacted with different aldehydes and ketones to obtain 11 mole-
cules. In step-II, obtained molecules were reacted with ethanol
and benzyl alcohols to obtain D1 to D22 derivatives. In silico
investigation resulted in best three molecules D12, D4 and D8
with promising BE score. Oral acute toxicity study of selected mol-
ecules resulted in LD50 value 500mg/kg in rats. The result of
in vivo antihypertensive study shown that molecule D12 was
found to be the best antihypertensive lead molecule. This study
could be a best platform to tailor novel biomolecules for inhibit-
ing phosphodiesterase 5A enzyme in hypertension management.
KEYWORDS
Biomolecules; heart
disorder; natural product;
phosphodiesterase 5A
inhibitor; pyridoin-
dole scaffold
CONTACT Dipak P. Mali
dipakpmali@rediffmail.com
Supplemental data for this article can be accessed online at https://doi.org/10.1080/14786419.2021.1925274.
ß 2021 Informa UK Limited, trading as Taylor & Francis Group

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D. P. MALI ET AL.
1. Introduction
Natural products are one of the best sources for obtaining a variety of therapeutic
agents in drug discovery (Sircar 1982). Natural products contribute as a foundation of
therapeutic agents and have shown valuable uses. Natural products are a rich source
of biologically active compounds. Isolation, characterisation and pharmacological
screening of natural products are the major challenging task in drug discovery as far
as designing new leads from natural bioactives are concerned (Tariq and
Siddiqi 1985).
Hypertension, a cardiovascular disease (CVD), is a global public health issue and the
leading cause of death in the entire world (WHO 2017). Longstanding high blood pres-
sure entertains major threat like coronary artery disease, heart failure, atrial fibrillation,
dementia peripheral vascular disease, vision loss and kidney disorders (Lackland and
Weber 2015).
Phosphodiesterase 5A (PDE5A) is an enzyme found in lung, cerebellum, heart, brain,
platelets, cardiac myocytes, vascular myocytes, gastrointestinal tissues and penis.
PDE5A inhibitors have mild systemic vasodilatory effects and could, therefore, repre-
sent a treatment for hypertension (Konstantinos and Petros 2009). The PDE5A causes
vasoconstriction by preferentially hydrolysing the cyclic guanosine monophosphate
(cGMP) to inactive GMP. PDE5A inhibition fosters intracellular accumulation of cGMP
which inhibits calcium entry into the cell (Schellack and Agoro 2014; Abusnina and
Lugnier 2017). This causes a decrease in intracellular calcium level and consequently
results in relaxation of pulmonary and vascular smooth muscles (Yu et al. 1995;
Chen 2010).
Recent research reported that nitrogen-containing 1H-Pyrazolo[4,3-d]pyrimidine and
pyrazolopyrimidinones scaffolds could be used in PDE5 inhibition (Tollefson et al.
2010). A study reported that pyrrolo[2,3-d]pyrimidine scaffolds had antihypertensive
application (Katouah and Gaffer 2019). Another nitrogen and oxygen containing scaf-
fold, methoxyl-phenyl-pyrrol-thiophen have also been reported as a PDE5 inhibitor
(Shang et al. 2014). Oxygen-containing benzofuran, naphthofuran, xanthene and chro-
mene chemical backbones as well as nitrogen and oxygen containing furo quinoline,
pyrano quinolizine and dioxalo isoquinolone backbones were also identified as lead
scaffolds inhibiting PDE5A (Mali and Bhatia 2021).
Applying modern tools like computational technology offers an exclusive opportun-
ity to screen a variety of natural products in discovering new drugs (Sircar 1982). The
target-specific complex model was successfully applied for phytochemicals by using
Grip-based docking methodology (Gaikwad and Jadhav 2019). In analysing receptor–-
protein or drug–ligand interactions, molecular docking assists by getting the most
excellent geometry of ligand/receptor composite, identifying the appropriate active
sites in protein and calculating the binding energy for various molecules to design
and optimise more useful ligands (Guedes et al. 2014). Similarity features for drug like-
liness properties have been successfully described by using the pharmacophore mod-
elling approach (Gaikwad and Jadhav 2021).
The potential of natural compounds in providing lead molecules have been
accepted by researchers but it has certain limitations like appropriate screening meth-
odology for lead constituents, optimisation of leads and its target suitability.

NATURAL PRODUCT RESEARCH
3
Moreover, identification of lead scaffolds and their optimisation have less tried till
date. This demands the need for exploration of phyto-lead scaffolds for
drug discovery.
Considering these facts; the present study was attempted to focus on exploration
of nitrogen-containing pyridoindole lead scaffold which was identified by in silico
methodology from natural database (Mali and Bhatia 2019). The objective of the pre-
sent study was to synthesise pyridoindole derivatives from amino acid tryptophan as a
starting material, in silico screening of derivatives for PDE5A inhibition, assessment of
oral acute toxicity and in vivo antihypertensive potential of selected molecules.
2. Results and discussion
2.1. Lead scaffold identification
Docking result exhibited varying patterns in H-bonding, ionic, p-stacking and van der
Waals interactions of ligands with protein (Hansen et al. 1996; Yun et al. 2014).
Fourteen nitrogen-containing tricyclic molecules having three different basic chemical
backbones as lead scaffolds namely pyridoindole, tetrahydro-pyridonaphthyridine and
dihydropyridoquinazoline were identified (Mali and Bhatia 2019).
2.2. Synthesis of derivatives of pyridoindole
Amongst identified tricyclic leads, nitrogen-containing lead scaffold have preferred for
lead scaffold optimisation because standard PDE5A inhibitor (sildenafil) (Wilkens et al.
2001) contains nitrogen heteroatom in its basic skeleton. Critical interpretation of the
docking interaction progressed in the identification of vital structural functionalities
whose optimisation may lead to improved potency of the selected scaffold. The
importance of aromatic hetero ring, substitution at R1 and R2 is evident from the
docking studies of molecules bearing it. Considering the results of virtual screening,
substitutions were proposed on the pyridoindole ring system at R1, R2 and –RCOOH
positions using the protocol described in Section 3. This led to the synthesis of 22 pyr-
idoindole derivatives (D1 to D22).
For synthesis, tryptophan was used as a starting material having indole ring in it,
which makes the structure electron-rich in nature. Initially, various aldehyde and
ketones were used for ring-closing reaction. The optimum yield was obtained as the
reaction was refluxed to 80 ꢀC in acetic acid. Moderate reaction temperature helps in
achieving pure products with minimum by-products and could be separated with suc-
tion filtration and washing. After drying, the product was used for the second step
namely esterification. The carboxyl functionality on the third position of the intermedi-
ate compound was esterified with the use of either ethanol or benzyl alcohol in SOCl2
to obtain designed novel ethyl/benzyl ester.
1
A number of signals (neglecting splitting) in H NMR were observed for confirm-
ation of the presence of different hydrogen environments. The number of signals in
the 13C NMR spectrum along with intensity were observed for deciding the carbon
number in the proposed structure. The physical, chromatographic and spectral charac-
terisation information of D12, D4 and D8 as given in Table S1.

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D. P. MALI ET AL.
Figure 1. Structures of pyridoindole scaffold, D12, D4 and D8.
2.3. In silico study
The developed three feature pharmacophore model disclosed that the molecular
assembly of sildenafil fundamentally encompasses one aromatic centre, one aliphatic
centre and one centre of hydrogen bond acceptor at specific distance (Figure S1). The
novel derivatives of pyridoindole were designed considering pharmacophoric features
required to be present in them and were synthesised. Those derivatives which have all
three features, and showing good interactions and binding energy (BE) score, are
ranked at top whereas those with less than three features are ranked at bottom in
Table S2. In silico docking studies revealed that, within 22 synthesised pyridoindole
derivatives, few show satisfactory BE score as compared to standard. Considering this
post-synthesis docking, all molecules were ranked according to their BE score and
selected top three molecules D12, D4 and D8 with promising BE score ꢁ75.5608,
ꢁ74.4264 and ꢁ70.6706, respectively (Table S2). Although the remaining molecules
were showing marginally poor BE score, we have restricted our pharmacological study
to these three molecules only and these molecules were shortlisted for screening of
their acute oral toxicity and antihypertensive potential.
Amongst all synthesised compounds, the chemical structures of D12, D4 and D8
which have shown promising docking interactions with PDE5A are depicted in
Figure 1.
2.4. Acute oral toxicity studies
After giving 300 mg/kg of D12, one animal of the group was dead, whereas after giv-
ing 300 mg/kg of D4 and D8, all animals of group were alive. In the next step, dosing

NATURAL PRODUCT RESEARCH
5
of three additional animals at 2000 mg/kg dose level was carried out where all animals
for all three compounds were dead. For all three synthesised drugs (D4, D8 and D12)
the LD50 value was found to be 500 mg/kg.
2.5. Ex vivo vasodilatory study pertaining to PDE5A inhibition
Results of % of relaxation of aortic endothelium showed that amongst D4, D8 and
D12, the D12 was having maximum activity 60.26 3.15% and 81.34 2.10% as com-
pared with standard sildenafil 63.45 4.95% and 83.22 3.83% with lower (0.25 mg/mL)
and higher (0.5 mg/mL) concentration, respectively (Table S3).
2.6. In vivo antihypertensive activity
The antihypertensive potential of D4, D8 and D12 were estimated on rats, in which
the hypertension was induced with the help of methyl prednisolone acetate, and was
compared with the antihypertensive effect of standard sildenafil drug (Table S4).
The systolic blood pressure of group of untreated rats was checked initially and
was found to be in the range of 113 to 119 mm Hg. Also, the rats in which hyperten-
sion was induced the systolic blood pressure was found to be in the range of 176 to
183 mm Hg. The oral administration of D4, D8 and D12 suspension (50 mg/kg) mini-
mised the hypertension up to 120, 122 and 119 mm Hg, respectively, within 6 h, and
then reached 171, 178 and 153 mm Hg, respectively, at 12 h (Figure S2). Compared
with the antihypertensive effect of standard sildenafil (50 mg/kg), oral suspension of
D4 and D8 have shown a gradual decrease in blood pressure up to 6 h nonetheless its
action reversed and blood pressure raised to above the regular value at 12 h. The
effect shown by D12 was found to be best because it controlled the blood pressure
to normal (119 mm Hg) at 6 h and its antihypertensive effect remains moderately
active at 12 h (153 mm Hg) as compared to the effect shown by sildenafil.
The above result suggested that the antihypertensive potential of optimised lead
compound D12 was better than the sildenafil; whereas other optimised leads D4 and
D8 have moderate potential. Structurally, it was observed that the benzyl
(O–CH2–C6H6) ester derivatives are more active than ethyl (O–C2H5) esters. Results sug-
gest that if the benzene ring was attached on pyridine then the activity increases than
that if alkyl substituents were attached. It could also be concluded that if the electron
donating group was present on the benzene ring which was attached to pyridine
then antihypertensive potential increases (inhibit PDE5A). The presence of two oxygen
atoms at the ester group acts as hydrogen bond acceptor whereas two heterocyclic
nitrogen moieties acts as hydrogen bond donor are important parts for the thera-
peutic activity. Also, the aromatic ring in the pyridoindole scaffold is essential for the
activity of these derivatives. The results of in vivo pharmacological studies validate the
results obtained in in silico studies. It was also observed that the synthesised lead
compound D12 hold promises for the managing of hypertension that requires to be
further authenticated by the clinical trial. The screening of remaining derivatives for
their antihypertensive potential will be the future scope of this study.

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D. P. MALI ET AL.
3. Experimental
3.1. Lead scaffolds identification
The pyridoindole lead scaffold was identified from selected antihypertensive phyto-
chemicals as per our previous in silico work (Mali and Bhatia 2019).
3.2. Docking parameters
GRIP type of docking was performed in which the ligand was kept nonflexible, rota-
tion angle was set at 10ꢀ, a number of placements were 30 and ligand wise 10 results
(poses) were selected. PDB ID: 1TBF, was selected as a docking template, which was in
complex with sildenafil, a reference PDE5A inhibitor was used. Docking was carried
out using vLife MDS 4.4 software (vLife Sciences Technologies Pvt. Ltd., Pune, India).
Docking analysis of the enzyme/ligand complex, was based on the parameters such as
hydrogen bond interactions, p–p interactions, BE and orientation of the docked com-
pound within the active site.
3.3. Synthesis of derivatives of pyridoindole
To develop novel derivatives of pyridoindole scaffold synthesis scheme was designed
(Figure 2). Tryptophan as a starting material having an indole ring was used.
Bischler–Napieralski or Pictet–Spengler reactions method with some modifications was
designed with step I and II (Milen et al. 2005). In step I, DL-Tryptophan was reacted
with required aldehyde or ketone in glacial acetic acid with stirring and heated till
reflux. Different aldehydes and ketones were used and it includes acetaldehyde, ben-
zaldehyde, p-nitrobenzaldehyde, p-cholorobenzaldehyde, p-methoxybenzaldehyde, 2-
hydroxybenzaldehyde (salicylaldehyde), ethyl methyl ketone, acetone, diethyl ketone,
isopropyl methyl ketone (3-methyl-2-butanone) and propionaldehyde. The reaction
mixture was then cooled, was filtered by suction filtration. Residual acetic acid was
removed by using water and then the product of the first step was dried to give 11
products in step 1. At step 2, ethanol and benzyl alcohols were reacted with step 1
products in the ice salt bath with the reaction mixture containing SOCl2 for 20 min
with stirring. After that the ice bath was taken aside. The mixture was then slowly
heated to reflux for 4.0 h. The mixture was cooled and stirred into 500 mL of ice water.
NaOH solution was used to adjust the pH to neutral and the white precipitate was
formed. Water was used to wash the precipitate, then filtered, and dried using the
vacuum to obtain respective novel ethyl/benzyl ester. To check the reaction develop-
ment, the TLC technique was used. The proposed structures of new synthesised ana-
logues were confirmed by taking their melting point, IR and NMR spectral information.
3.4. Acute oral toxicity studies
Institutional animal ethics committee permission was obtained as per CPCSEA guide-
lines (Approval No: BVCPK/CPCSEA/IAEC/01/23) for carrying out the study on animals
(OECD 2001).

NATURAL PRODUCT RESEARCH
7
Figure 2. Scheme for synthesis.

8
D. P. MALI ET AL.
3.5. Ex vivo vasodilatory study pertaining to PDE5A inhibition
The goat aortic strips were brought from slaughterhouse. Aortic rings were tied to
stainless steel hooks placed in Krebs–Henseleit physiological solution maintained at
37 ꢀC. About 2 g weight was given to all tissues for the creation of basal tension and
changes in basal tension were recorded (Biopac Systems). The contraction of each pre-
pared aorta was maximised by administration of KCl (120 mM) and recorded. The tis-
sues were pre-incubated with D4, D8 and D12 (0.25 and 0.5 mg/mL). Sildenafil was
used as standard (0.25 and 0.5 mg/mL). Relaxation was expressed as a percentage
change from KCl contracted levels, i.e. by comparison between maximum vascular
contraction before and after addition of samples and calculated using the following
formula:
% Relaxation ¼ Tc ꢁ Tt=Tc x 100
where Tc stands for tension due to contracting agents and Tt stands for tension after
adding compounds.
3.6. In vivo antihypertensive activity
A total of 15 rats, aged 3- to 5-month-old of both sex and weighing between 180 and
200 g were used in the course of this study. Five groups with three rats in each group
were used for the testing of optimised leads after molecular modelling studies. The
rats were housed in stainless steel cages with sterilised bedding which were changed
every day. Slight movement and aggression by the animal create the difference in BP
reading and to avoid this training to the animals was given for staying in the
restrainer. Group I was kept as control. To the animals of other four groups (Groups II
to V), hypertension was developed by subcutaneous injection of 20 mg/kg/week of
methylprednisolone acetate for 2 weeks as per the reported method described by Aqil
et al. (2006). Before the drug administration on the day of the actual experiment,
hypertensive rats from Group II to V were observed and BP was recorded. Group II
was treated with standard drug sildenafil. Group III, IV and V were given an oral sus-
pension (50 mg/kg) of test drug D4, D8 and D12, respectively, with a feeding needle.
Suspension of the synthesised compounds was made in 1% w/v sodium CMC and
administered at a lower and higher dose per body weight. The rat was then placed in
the restrainer where the movement of animals was restricted. The dirty matter on the
tail was removed with moist cotton and surface of tail was made smooth by spraying
the talcum powder. The pulse transducer and tail cuff were attached around the tail
and the blood pressure from the tail was recorded by CODA non-invasive BP system
(a tail-cuff method, Kent Scientific Corporation) at predetermined time intervals up to
12 h. The systolic, diastolic and mean blood pressure of the rats were monitored.
4. Conclusion
Nitrogen-containing pyridoindole lead scaffold was successfully explored to discover
potent derivatives by in silico methodology from natural database. Furthermore, in sil-
ico investigation of 22 compounds resulted in best three molecules D12, D4 and D8

NATURAL PRODUCT RESEARCH
9
with promising BE score. Oral acute toxicity study of D12, D4 and D8 showed the
LD50 value 500 mg/kg. Ex vivo and in vivo study results show that molecule D12 was
found to be the best antihypertensive lead molecule. This study would provide a
novel platform for drug discovery of antihypertensive leads from the natural resource.
Acknowledgement
The authors are thankful to Dr. H. N. More, Principal, Bharati Vidyapeeth College of Pharmacy,
Kolhapur, Maharashtra, India for providing facilities for the work.
Disclosure statement
No potential conflict of interest was reported by the author(s).
ORCID
Dipak P. Mali
http://orcid.org/0000-0001-6916-2946
Dinanath T. Gaikwad
http://orcid.org/0000-0002-2780-8216
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