DABCO–PEG ionic liquid-based synthesis of acridine analogous and its inhibitory activity on alkaline phosphatase

Article abstract of DOI:10.1080/00397911.2017.1409898

In world, many people struggle with viral, parasitic, bacterial, cancer, and other diseases. Therefore, numerous chemists seek to develop less toxic, more selective, and effective medicines. Most therapeutic medicines are based on inhibition of specific e

Full text of DOI:10.1080/00397911.2017.1409898

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: http://www.tandfonline.com/loi/lsyc20
DABCO–PEG ionic liquid-based synthesis of
acridine analogous and its inhibitory activity on
alkaline phosphatase
Muhammad Faisal, Shereena Shahid, Sarfaraz Ali Ghumro, Aamer Saeed,
Fayaz Ali Larik, Zeenat Shaheen, Pervaiz Ali Channar, Tanzeela Abdul Fattah,
Samina Rasheed & Parvez Ali Mahesar
To cite this article: Muhammad Faisal, Shereena Shahid, Sarfaraz Ali Ghumro, Aamer
Saeed, Fayaz Ali Larik, Zeenat Shaheen, Pervaiz Ali Channar, Tanzeela Abdul Fattah, Samina
Rasheed & Parvez Ali Mahesar (2018): DABCO–PEG ionic liquid-based synthesis of acridine
analogous and its inhibitory activity on alkaline phosphatase, Synthetic Communications, DOI:
10.1080/00397911.2017.1409898
To link to this article: https://doi.org/10.1080/00397911.2017.1409898
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SYNTHETIC COMMUNICATIONS®
https://doi.org/10.1080/00397911.2017.1409898
DABCO–PEG ionic liquid-based synthesis of acridine
analogous and its inhibitory activity on alkaline phosphatase
a
b
b
a
Muhammad Faisal
, Shereena Shahid , Sarfaraz Ali Ghumro , Aamer Saeed
,
a
a
a
a
Fayaz Ali Larik
, Zeenat Shaheen , Pervaiz Ali Channar , Tanzeela Abdul Fattah ,
Samina Rasheed , and Parvez Ali Mahesar
a
a
a
b
Department of Chemistry, Quaid-i-Azam University, Islamabad, Pakistan; H.E.J. Research Institute of
Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan
ABSTRACT
ARTICLE HISTORY
Received 14 October 2017
In world, many people struggle with viral, parasitic, bacterial, cancer,
and other diseases. Therefore, numerous chemists seek to develop less
toxic, more selective, and effective medicines. Most therapeutic
medicines are based on inhibition of specific enzymes. Acridines are
interesting heterocyclic structures that are much sought after targets
attributed to their wide biological activities and feature to display
potent enzymes inhibitory effects. Their approach of action is owing
to DNA interaction and subsequent effects on the biological functions
linked to DNA and associated enzymes. In this regards, we synthesized
acridine analogous through 1,4-diazabicyclo[2.2.2]octane (DABCO)–
polyethylene glycol-400 (PEG-400) mediated ionic liquid approach.
DABCO–PEG-400-mediated IL was prepared through the DABCO
alkylation using 1-bromopentane followed by mixing with PEG-400.
The synthesized analogous were investigated as inhibitors of alkaline
phosphatase, which is a nonspecific phosphomonoester hydrolase
that catalyzes the hydrolysis of broad spectrum of organic monopho-
sphates. Analogue viz. 3,3,6,6-tetramethyl-9-(4-nitrophenyl)-
KEYWORDS
Acridines; alkaline
phosphatase; DABCO;
enzyme inhibitor; ionic
liquid
2
,3,4,5,6,7,9,10-octahydroacridine-1,8-dione was found to be potent
alkaline phosphatase inhibitor.
GRAPHICAL ABSTRACT
CONTACT Aamer Saeed
aamersaeed@yahoo.com; Fayaz Ali Larik
fayazali@chem.qau.edu.pk
Department of
Chemistry, Quaid-i-Azam University, 45320 Islamabad, Pakistan.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lsyc.
Supplemental data (full experimental detail and proton and carbon-13 NMR spectra) can be accessed on the
publisher’s website.
©
2018 Taylor & Francis

2
M. FAISAL ET AL.
Introduction
Acridine analogous are significant type of nitrogen-mediated heterocycles attributed to
their wide spectrum of pharmaceutical activities. Almost 10,000 acridines analogous have
been disclosed, which contains biological, therapeutic and photochemical properties.[1]
Acridine analogous are characterized through exclusive chemical and physical characteris-
tics, industrial applications and biological potencies. In 1870, first time separation of
acridine from high boiling fraction of coal tar was done by Heinrich Caro and Carl Grabe
[
2]
in Germany. In the middle of 19th century, acridine analogous were already industrially
[
3]
applied as dyes and pigments. More critical to the medicinal industry, acridine analogous
have displayed potent biopotencies, e.g., anti-inflammatory 1, antimicrobial 2 and 3,
anticancer 3 and 4, antimalarial 5 and 6, antibacteria 7, antiviral 8, antitubercular 9, anti-
[
1]
leishmanial 10, antiparasitic 11, analgesic 12, and fungicidal 13 activity (Fig. 1).
One of the most attractive feature of acridine derivatives are that these entities displayed
potent enzymes inhibitory effects. The unique conjugated planar structure of acridine ring
allows them to interact with DNA intercalators and to inhibit terminal transferase or
[
4]
enzymes. A library of acridine analogous have been prepared, for instance, amsacrine
(
m-AMSA) 14, triazoloacridone (C-1305) 15, and DACA 16 have entered medical
[
1]
investigations (Fig. 2). Among them, amsacrine 14 was the first synthetic medicine dis-
playing medical potency as inhibitor of topoisomerase. Furthermore, through the acridine
Figure 1. Structure of potent bioactive acridine derivatives.

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Figure 2. Structure of potent acridine-based inhibitors.
structure modification, acridines can bind selectively to specific DNA configurations, for
instance, BRACO-19 17 can specifically bind to G-4 DNA with poor efficacy to double
[
5]
DNA, thus inhibiting the potency of terminal transferase. In the recent years, acridine
analogous have been applied for inhibiting permeability glycoprotein and various
[
6]
protein-kinases. Other examples in which acridine analogous shows potent inhibitory
effect are WFU001a 18 (displays potent potency against NCI-H460 cell with IC nearly
50
2
.81 nM), 9-aminoacridine derivatives, for example, 19, carboxylic group-mediated
acridine 20 (intercalate DNA of tumor cells), bisacridines, for example, D11B 21 (potent
affinity against S295 human glioblastoma multiforme cell, i.e., IC ¼ 177 nM) and human
50
colorectal cancer cell, i.e., IC ¼ 167 nM), SN 28049 22 (displayed strong activity toward
5
0
p388 leukemia cell, i.e., IC ¼ 2.1 nM) and LDN-192960 23 (potent haspin kinase, i.e.,
5
0
IC < 49 nM and DYRK (dual specificity tyrosine regulated kinase) protein, i.e., IC < 49 nM
5
0
50
[1]
inhibitor) (Fig. 2).
Alkaline phosphatase (AP) is a class of phosphatidylinositol-mediated membrane
proteins. The AP family proteins are zinc metalloenzymes that hydrolyze phosphate
containing substrates at alkaline pH.[ It comprises a family of membrane bound enzymes
found across diverse species including bacteria, mammals, reptiles, fungi, plants and
7]
[
8]
amphibians. AP is extensively dispersed in nature, including higher eukaryotes and
[
9]
prokaryotes. The wide distribution of this family of isozymes suggests its involvement
[
10]
in significant biological function, but their role in vivo remains unclear.
In humans,
several distinct classes of AP have been identified, each encoded by a separate gene. AP
is found in different tissues and also in the circulation. Membrane-bound alkaline phos-
[
9]
phatase is a homodimer, which consists of two identical 66-kDa glycosylated subunits.
In human serum, AP predominantly circulates as a hydrophilic homodimer, which may
be released from the membrane by phospholipase C.[9] In vitro the APs are known to

4
M. FAISAL ET AL.
remove phosphates form phosphorylated molecules at alkaline pH between 8 and 10.5,
hinting at possible in vivo roles in cell–cell signaling, transmembrane signaling, shuttling,
and mineralization. Two distinct forms of mammalian APs exist tissue nonspecific AP and
tissue-specific APs (TSAPs). The TSAPs may be farther subdivided depending on tempera-
ture stability, pH optimum, inhibitor sensitivities, and expression patterns into forms with
names, for example, human placental AP, intestinal AP, mouse embryonic AP, and human
germ cell alkaline phosphatases.[11]
The above discussion provides strong evidence that the level of AP is linked to
numerous diseases, such as prostatic cancer, hepatitis, a dynamic bone disease,[12] etc.
Controlling AP through enzyme inhibitors have received extensive attraction in recent
year. Owing to potent properties of acridine analogous as enzymes inhibits, we decided
to investigate them as inhibitors for alkaline phosphatase. The objective of this study
was to synthesize novel acridine analogous through highly advanced and efficient 1,4-
diazabicyclo[2.2.2]octane (DABCO)–polyethylene glycol-400 (PEG-400) based ionic liquid
approach and examine their effect on the catalytic activity of AP. The ionic liquid approach
for synthesis of acridine analogous are safer, nonvolatile, environmentally benign, thermal
[
13]
stable, efficient recyclable, and nonflammable.
The application of PEG polymer-based
deep eutectic solvent (DES) as benign reaction media for organic syntheses offers exciting
opportunities to be explored in the realm of green synthesis.
Results and discussion
Chemistry
For the acridine analogous synthesis, DABCO–PEG-400 mediated ionic liquid approach
was utilized. DABCO-mediated ionic liquid (IL) was prepared through the DABCO 24
alkylation using 1-bromopentane (Scheme 1). The synthesized DABCO-based ionic salt
2
5 was mixed and heated with PEG-400 as hydrogen bond donor (HBD) in the molar ratio
[
13]
of 1:2 to prepare new DES in 86% yield.
These DABCO–PEG-based DES were success-
fully used for the acridine analogous synthesis (Fig. 3). The three-component cyclo-
condensation Hantzsch reaction of dimedone 27 and substituted aromatic aldehydes 26
in the presence of ammonium acetate at temperature 115–116 °C for 12–14 h in
DABCO–PEG-based DES conditions afforded acridine derivatives in excellent yields
(
Scheme 2). In this reaction, ammonium acetate provides source of nitrogen. In alternative
to ammonium acetate, other nitrogen source such as urea, ammonium bicarbonate,
ammonium acetate on basic alumina, ammonium hydroxide, hydroxylamine, methyla-
[
14]
mine, or substituted anilines were also utilized in literature. In recent literature, acridine
derivatives were also synthesized in the presence of triethylbenzylammonium chloride,
Scheme 1. Synthesis of DABCO–PEG-400 mediated ionic liquid. Note: DABCO, 1,4-diazabicyclo[2.2.2]
octane; PEG, polyethylene glycol.

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Figure 3. Synthesis of acridine analogous.
p-dodecylbenzenesulfonic acid, proline, amberlyst 15, and ammonium chloride or
[
14]
Zn(OAc) · 2H O.
While the classical approaches for the synthesis of acridine deriva-
2
2
tives are Ullmann synthesis, Bernthesen synthesis, Friedlander synthesis and C-acylated
[
14]
diphenylamines.
Low volatility, high thermal stability, efficient recyclability, high
catalytic ability, and nonflammability are the main reasons for using ionic liquid approach
for synthesizing acridine derivatives while all other approaches lack these features. The
high catalytic ability of DABCO is due to two active nitrogen atoms (instead of one
nitrogen) on DABCO for catalytic action. In alternative to PEG-400, alcohols can also
be utilized but it is investigated that PEG-400 provides a much more safe, nonvolatile,
and environmentally benign reaction medium for synthetic reactions. The recyclability
of DABCO–PEG-based DES is similar to those of ILs.[15] The filtrate (which is obtained
after filtering the reaction mixture of acridine analogous) can be reused for at least four
reactions with no significant loss of yield.
The proposed reaction mechanism is displayed in Figure 4. The HBD capability of the
ionic liquid may increase the enol 27b content in the keto-enol equilibrium. The C=O of
the aldehyde was polarized by the development of hydrogen bonds with the carboxyl group
on the cation of the ionic liquid, which was then attacked by 27b to afford precursor 28 and
then dehydrated to 29. Ammonium acetate was decomposed to ammonia and acetic acid,
and ammonia attacked enol 27b to furnish precursor 30. The precursor 30 reacted with 29
to deliver the product 28 (Fig. 4).
Scheme 2. Synthesis of acridine analogous.

6
M. FAISAL ET AL.
Figure 4. The purposed reaction mechanism for the IL-catalyzed Hantzsch reaction.
Calf intestinal alkaline phosphatase inhibition assay
All the synthesized entities 28a–28j were investigated for their potential to calf intestinal
alkaline phosphatase (CIAP). In Table 1, the results of CIAP are summarized. In this
investigation, four electron donating groups (EDGs), i.e., OMe, OH, OEt, and Me and five
electron withdrawing groups (EWDs), i.e., Br, Cl, F, NO , and CN were used on phenyl
2
ring of acridine to examine inhibitory potency for alkaline phosphatase. The inhibitory
potencies of acridines were compared to standard monopotassium phosphate (4.317 �
0
.201 µM). The inhibitory potency of fluorinated analogue of acridine 28f is better than
chlorinated analogue of acridine 28e. In the case of substituent on 4th position of phenyl
ring of acridine, methyl inhibitor 28g displayed more potential then fluorine and chlorine
inhibitors (28f and 28e) while NO -based inhibitor 28h demonstrate better potency than
2
methyl-based inhibitor 28g. Entities which contains EWDs at fourth position are the most
better inhibitors than entities contain EDG at fourth position. The present inquiry suggests
that the EDGs and EWDs have serious role on activity of inhibitor. Among all, the
analogous 28b (OCH at fourth position and Br at third position) and 28d (OH at fourth
3
position and OEt at third position) showed minimum potential against CIAP. NO2
Table 1. Alkaline phosphatase inhibitory activity of acridine
derivatives 28a–28j.
Inhibitors
Alkaline phosphatase IC50 (µM)
2
2
2
2
2
2
2
2
2
2
8a
8b
8c
8d
8e
8f
0.2996 � 0.013
0.6579 � 0.032
0.0882 � 0.003
0.3361 � 0.016
0.1541 � 0.007
0.137 � 0.006
0.0685 � 0.003
0.0102 � 0.0005
0.0593 � 0.0029
0.0768 � 0.0037
4.317 � 0.25
8g
8h
8i
8j
KH
2
PO
4
Values are presented as mean � standard error of mean.

SYNTHETIC COMMUNICATIONS®
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−
1
Figure 5. Free radical percentage scavenging potency of synthesized entities at 100 µg mL
concentration.
containing analogue 28h exhibited highest potential in the series with IC 0.0102 � 0.0005
50
(
standard KH PO 4.317 � 0.201 µM). These characteristics of acridine analogous are
2
4
owing to their unique conjugated planar heterocyclic structure, which significantly interca-
late with alkaline phosphatase. The possible reason for excellency of 28h is that the NO2
group is the most powerful deactivating group.
Free radical scavenging
The synthesized library of acridines based inhibitors were screened for their radical
scavenging potency. In comparison to reference drug vitamin C, entities 28b, 28d, and
2
8g displays outstanding radical scavenging potency. Other units did not illustrate
−1
excellent radical scavenging potency even at 100 µg mL concentration (Fig. 5).
Conclusion
The present work describes the DABCO–PEG ionic liquid-based synthesis and enzyme
inhibitory activity of acridine analogous. The excellent yield of synthesized acridine anal-
ogous suggest that ionic liquid approach for the synthesis of acridines is the most suitable
and convenient methodology. Introduction of IL as an environment-friendly medium for
the acridines synthesis has been a major step in this direction. The use of PEG-400 as HBD
in DES proved to be a safer medium for reaction and opens new avenue in this area for
further research. Demonstration of potent inhibitory activity by acridine analogous suggest
that acridines have high potential to inhibit alkaline phosphatase. This potent inhibitory
potential of acridine analogous are owing to their unique conjugated planar heterocyclic
structure, which significantly intercalate with alkaline phosphatase. Analogue 28h
exhibited highest potential in the series with IC50 0.0102 � 0.0005 (standard KH PO
2
4
4
.317 � 0.201 µM). However, understanding of structure–activity relation is required to
construct more potent analogous of acridines. This work delivers a strong platform from
which to conduct further research in this field and investigate additional hypotheses.
Further, to extend the application of acridine analogous in the field of drug synthesis,

8
M. FAISAL ET AL.
modification of the nature and position of the groups on the acridine nucleus is required.
Moreover, concentration to construct of aza-mediated acridines or other heteromoiety-
based acridines is also required. Well-organized approaches for optimization and
identification of acridine analogous to localize (produce effect) at the disease sites still
needs to be more established. We hope the result of our work offers a strong motivation
for further research progress.
Experimental section
Materials and general methods
In this research work all reagents and other chemicals were obtained from Fluka, Merck,
and Sigma-Aldrich. All reagents and other chemicals were used without additional purifi-
cation while all the solvents were purified through distillation before used. Bruker model
AM-300, which functions at 300 MHz for proton NMR and 75 MHz for carbon-13
NMR, was used to recorded proton and carbon-13 NMR. TMS as internal standard and
DMSO as solvent are used. Chemical shifts are expressed in ppm unit. All synthesized
compounds were fully characterized after purification. Melting points of compounds were
−1
recorded with apparatus model 12000 PLDSC at heating rate of 5 °C min . Using KBr
disks, IR spectra were measure on a Bruker Penssor 27 spectrophotometer. Only most
significant and strongest peaks were listed. The reactions progress was monitored through
precoated silica gel-60 TCL plates.
Synthesis
1
,4-Dipentyl-1,4-diazabicyclo[2.2.2]octane-1,4-diium Bromide (25)
In a typical reaction, DABCO 24 (1.0 g, 8.92 mmol, and 1 equiv.) was added to the
oven-dried round-bottom flask along with the solvents toluene/DCM (3:5). To the result-
ing solution, the corresponding 1-pentylbromide (2.94 g, 19.6 mmol, and 2.2 equiv.) was
added at room temperature and heated at reflux (120–130 °C) until no starting material
was observed on the TLC analysis (10% n-hexane in EtOAc). On cooling, the resulting
ionic salt was separated and washed further with cold toluene and dried in vacuo. The
obtained material was further washed with cold toluene (10 mL ꢀ 3) to get yellow
thick oil (3.15 g, 86%). (This compound was previously reported by Ali Ghumro
[
13]
−1
et al. ) R : 0.45 [n-hexane:EtOAc (1:9)]; IR (cm ): 3425, 2960, 2870, 2511, 2057, 1630,
f
1
1
468, 1391, 1320, 1210, 1118, 1058, 856; H NMR (300 MHz, DMSO): δ H 3.91 (12H,
H
s, CH ꢀ 6), 3.61–3.58 (4H, m, J ¼ 8.5 Hz, CH ꢀ 2), 1.72–1.66 (4H, m, CH ꢀ 2), 1.37–
2
2
2
13
1
6
.25 (8H, m, CH ꢀ 4), 0.89 (6H, t, J ¼ 7 Hz, Me ꢀ 2); C NMR (75 MHz, DMSO): δc
2
3.5 (CH ꢀ 2), 50.4 (CH ꢀ 6), 28 (CH ꢀ 2), 22 (CH ꢀ 2), 21 (Me ꢀ 2), 14 (Me).
2
2
2
2
General procedure for acridine analogous synthesis (28a–28j)
In a general procedure, an oven-dried round-bottom flask was charged with DABCO-IL 25
and HBD (PEG-300) in a ratio of 1:2, respectively. The reaction content was heated at 90 °C
to homogenize it, and then to this mixture aromatic aldehyde 26 (2 mmol), dimedone 27
(
4 mmol), NH OAc (2.4 mmol) were added. Next, reaction content was stirred and heated
4
at reflux (78–80 °C) for 10–12 h. The reaction progress was observed through TLC by
comparison with the starting materials (20% EtOAc in n-hexane). Upon starting material
complete consumption, the reaction content was cooled to RT and then crashed ice was

SYNTHETIC COMMUNICATIONS®
9
added to the reaction flask. The resulting mixture was stirred to get precipitates that were
filtered carefully and washed with cold distilled H O to get the desired product in crude
2
form (28a–28j). Silica gel column chromatography with the gradient eluent mixture of
EtOAc and hexane (1:9 to 9:1) was used to purify the product.
General procedure for alkaline phosphatase assay
Through spectrophotometric assay procedure as previously reported by Iqbal,[16] potency
of CIAP was determined. The reaction mixture consists of 50.0 mM Tris-hydrochloride
buffer (5.0 mM magnesium chloride, 0.1 mM zinc chloride pH 9.0) and the entity
−1
(
0.10 mM with final DMSO 1% (v/v)) and by adding 5 µL of CIAP (0.025 U mL , the
mixture was preincubated for 20 min). Then, 10.0 µL of substrate 0.5 mM p-NPP (viz.
p-nitrophenylphosphate disodium salt) was added to start the reaction process and for
3
0–40 min at 40 °C, the assay mixture was incubated again. The absorbance change of
released 4-nitrophenolate was observed at 405 nm, through a 96-well microplate reader
(
ARRIS OM4100 Opti Max). In a triplicate manner, all the experiments were repeated
three times. Monopotassium phosphate was used as the reference inhibitor of CIAP.
General procedure for free radical scavenging assay
Through modifying already reported method of Zhang et al.,[17] radical scavenging potency
was determined through 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazino (DPPH) assay.
The assay solution comprised of 100.0 µL of DPPH (150.0 µM), 20.0 µL of increasing
concentration of compounds (which was subjected under investigation) and the volume
was adjusted to 200.0 µL in each well with CH OH. For 40 min at room temperature,
3
the reaction mixture was then incubated. L-Ascorbic acid (vitamin C) was used as a
reference inhibitor. The measurements of assay were observed at 517 nm through micro
plate reader (ARRIS OM4100 Opti-Max). The rates of reaction were compared and the
[
18]
percentage inhibition caused by the existence of examined inhibitors was estimated.
In a triplicate manner, all the experiments were repeated three times.
ORCID
Muhammad Faisal
http://orcid.org/0000-0001-9181-9262
Aamer Saeed
http://orcid.org/0000-0002-7112-9296
http://orcid.org/0000-0002-8491-5229
Fayaz Ali Larik
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