Design and synthesis of 1,4-substituted 1H-1,2,3-triazolo-quinazolin-4(3H)-ones by Huisgen 1,3-dipolar cycloaddition with PI3Kγ isoform selective activity

Article abstract of DOI:10.1016/j.bmcl.2018.02.032

A strategy for construction of medicinally important 1,4-substituted 1H-1,2,3-triazolo-quinazolin-4(3H)-ones has been devised and presented here. The compounds have been synthesized using one-pot multicomponent strategy under microwave assisted conditions. Triazolyl-quinazolinone based D-ring modified analogs are designed based on IC87114 scaffold, which is first known isoform selective inhibitor of PI3Kδ. Herein, we identified two triazolyl-quinazolinone compounds (5a and 5l) based on same scaffold with PI3Kγ specific inhibitory potential, the selectivity towards this isoform is well supported by in silico results, wherein, these compounds show better interaction and affinity and inhibitory activity for PI3Kγ rather than PI3Kδ. This repositioning of scaffold from PI3Kδ to PI3Kγ isoform can be very useful from medicinal chemistry and drug discovery perspective to unravel molecular interactions of this new scaffold in different cellular pathways.

Full text of DOI:10.1016/j.bmcl.2018.02.032

Accepted Manuscript
Design and synthesis of 1,4-substituted 1H-1,2,3-triazolo-quinazolin-4(3H)-
ones by Huisgen 1,3-dipolar cycloaddition with PI3Kγ isoform selective activity
M. Srinivas, Anup Singh Pathania, Priya Mahajan, Praveen K. Verma, Santosh
S. Chobe, Fayaz A. Malik, Amit Nargotra, Ram A. Vishwakarma, Sanghapal
D. Sawant
PII:
S0960-894X(18)30125-2
https://doi.org/10.1016/j.bmcl.2018.02.032
BMCL 25628
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
9 November 2017
5 February 2018
15 February 2018
Please cite this article as: Srinivas, M., Singh Pathania, A., Mahajan, P., Verma, P.K., Chobe, S.S., Malik, F.A.,
Nargotra, A., Vishwakarma, R.A., Sawant, S.D., Design and synthesis of 1,4-substituted 1H-1,2,3-triazolo-
quinazolin-4(3H)-ones by Huisgen 1,3-dipolar cycloaddition with PI3Kγ isoform selective activity, Bioorganic &
Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.02.032
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Design and synthesis of 1,4-substituted 1H-1,2,3-triazolo-quinazolin-4(3H)-
ones by Huisgen 1,3-dipolar cycloaddition with PI3Kγ isoform selective
activity
a,d
b,d
c,d
a
e
M. Srinivas, Anup Singh Pathania, Priya Mahajan, Praveen K. Verma, Santosh S. Chobe,
b
c
a
a
Fayaz A. Malik, Amit Nargotra, Ram A. Vishwakarma, Sanghapal D. Sawant *
a
Medicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road,
Jammu-180 001, India
b
Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road,
Jammu-180 001, India
c
Discovery Informatics Division, CSIR-Indian Institute of Integrative Medicine, Canal Road,
Jammu-180 001, India
d
Academy of Scientific and Innovative Research, Anusandhan Bhawan, 2 Rafi Marg, New Delhi-
1
10 001, India
e
Department of Chemistry, Loknete Vyankatrao Hiray Arts, Science and Commerce College,
Nashik (M.S.), India
E-mail: sdsawant@iiim.ac.in; sawant.rrl@gmail.com; sdsawant@iiim.res.in
Tel: +91-191-2585222; Fax: +91-1912586333

A strategy for construction of medicinally important 1,4-substituted 1H-1,2,3-triazolo-
quinazolin-4(3H)-ones has been devised and presented here. The compounds have been
synthesized using one-pot multicomponent strategy under microwave assisted conditions.
Triazolyl-quinazolinone based D-ring modified analogs are designed based on IC87114 scaffold,
which is first known isofom selective inhibitor of PI3Kδ. Herein, we identified two triazolyl-
quinazolinone compounds (5a and 5l) based on same scaffold with PI3Kγ specific inhibitory
potential, the selectivity towards this isoform is well supported by in silico results, wherein, these
compounds show better interaction and affinity and inhibitory activity for PI3Kγ rather than
PI3Kδ. This repositioning of scaffold from PI3Kδ to PI3Kγ isoform can be very useful from
medicinal chemistry and drug discovery perspective to unravel molecular interactions of this new
scaffold in different cellular pathways.
Keywords: 1H-1,2,3-triazolo-quinazolin-4(3H)-ones, Huisgen 1,3-dipolar cycloaddition, PI3K-γ
inhibitors, multicomponent synthesis

1
-7
1
,2,3-triazoles are widely used in different ways, like peptide bond mimetics and they can act
as an option for double bond in the chemical structures, which is an important feature from the
8
-9
perspective of medicinal chemistry and drug discovery. Moreover, triazoles can also be used
1
0
as furan substitutes as well. Therefore, they are of great importance from structural point of
view and provide fast access to construct five-membered heterocyclic ring. They attribute to
1
1-14
important and wide range of applications in biological and pharmacological activities.
Triazole based structures are exhaustively exploited in many applications in organic,
1
5
organometallic and material chemistry. The synthesis of 1,4-substituted 1,2,3-triazoles from
halides, azides, and acetylenes in the presence of copper (I) salt in one pot is well known
1
6-22
reaction.
Mostly, these 1,2,3-triazole moieties are constructed using click chemistry approach
1
5,23-24
by using Huisgen 1,3-dipolar cycloadditions of azides and alkynes.
Microwave assisted
The increased interest of
25-28
methods for the synthesis of 1,2,3-triazoles are also reported.
chemist in the 1,2,3-triazole remains continued due to their non-toxic properties, benign nature
and their structural rigidity as well as high stability. Triazoles are particularly interesting for
medicinal use because of their water solubility compared to other hetero-aromatic compounds,
2
9
and they are stable in biological systems.
We recently published a protocol for the synthesis of purine-quinazolinone and some other
scaffolds that have been designed on the basis of their biological importance in PI3K isoform
3
0-32
selective inhibitory activity for cancer.
In one of our ongoing study, we aimed to design
analogs based on quinazolinone scaffold by medicinal chemistry of clinical candidates for
developing anticancer agents and isoform specific inhibitors of PI3K-δ. In this direction, we
chose to work on medicinal chemistry of purine quinazolinone IC87114 molecule, the first
3
3-36
isoform-selective PI3K-δ inhibitor reported and patented by ICOS in 2001.
This compound
exhibited nanomolar inhibition against PI3K-δ isoform (IC50= 130 nM) and a 100- to 1000-fold
selectivity against the other class I PI3K’s. Subsequently, several molecules based on
quinazolinone scaffold started appearing in preclinical and clinical investigation stages and one
of the most promising PI3K-δ isoform-selective inhibitors to date is GS1101/CAL-101 by Gilead
with IC50 of 2.5 nM named as Idelalisib, which has been approved recently by US-FDA as
monotherapy for B-cell malignancies and in combination with rituximab as second-line drug for
3
7-40
chronic lymphocytic leukemia (CLL) cases.

PI3K-δ and PI3K-γ isoforms are preferentially expressed in leukocytes, where they have distinct
4
1-43
and non-overlapping roles in immune cell function.
Class IA PI3Ks contain, p110α, p110β
and p110δ as catalytic subunits, and these are activated in tyrosine kinase receptor signaling.
Class IB PI3Ks contain only p110γ as a catalytic subunit, which is mostly activated by seven-
transmembrane G-protein-coupled receptors (GPCRs) via its regulatory subunit p101 and G-
protein γ subunits. While PI3Kα and PI3Kβ are ubiquitously expressed, however, PI3Kγ and
4
4
PI3Kδ expression is mainly restricted to the hematopoietic system. The p110α and p110β
4
5
isoforms are known to stimulate cell proliferation and invasive cell growth, p110δ controls
4
6
47
proliferation in acute myeloid leukemia and migration of breast cancer cells, whereas, a role
4
8
has been suggested for p110γ in tumor angiogenesis and drug resistance of chronic myeloid
4
9
leukemia cells.
In continuation of our interest in medicinal chemistry of quinazolinone scaffold for discovery of
30-32
PI3K isoform specific inhibitors,
we now designed a new triazole based quinazolinone
structures (Fig. 1); as triazole based scaffold, in particular 1,2,4-triazoles are also reported for
5
0
PI3K inhibitory activity. In this endeavor, we report a protocol for the synthesis of 1,4-
substituted 1H-1,2,3-triazolo-quinazolin-4(3H)-one scaffold under microwave assisted
conditions using one-pot multicomponent strategy along with their PI3Kγ specific activity.
Fig. 1. Structural features and similarities in IC87114 and the designer 1H-1,2,3-triazolo-
quinazolin-4(3H)-one scaffold.

As an extension of our earlier work, we initiated our studies to generate functionalized
derivatives and analogs of quinazolinone based structure as PI3K inhibitors. In an effort to
optimize reaction conditions for the synthesis of 1,4-substituted 1H-1,2,3-triazolo-quinazolin-
4
(3H)-ones, we opted to use a procedure similar to one, which has been reported by our group
3
0
recently for purine quinazolinones synthesis. In this study, similar procedure was used to
synthesize and obtain intermediate 3. Further, we reacted, intermediate 3 with alkyne, in this we
used one-pot strategy and typically phenylacetylene (4) was taken for optimization under
microwave irradiation using established conditions of purine quinazolinone, and the reaction was
carried in neat conditions. This has not given any product; we switched to conduct reactions
using solvents to observe the solvent effect. Different solvents were screened as mentioned in
table 1. Here, in case of dimethyl formamide (DMF), the product formation was observed,
reaction in DMF has given highest about 80% yields and reaction in dimethyl sufoxide,
acetonitrile, methanol and acetone gave 5%, 10%, 65% and 70% yield of product 5a
respectively. In case of reaction in water, tetrahydrofuran and dichloromethane, the reaction did
not proceed. Therefore, all the future optimization reactions were performed using the condition
mentioned in entry 8 of table 1.
Table 1
Solvent optimization for the synthesis of 1,4-substituted 1H-1,2,3-triazolo-quinazolin-4(3H)-
ones.
a
Isolated yield.
The intermediate 3 was prepared using reported method, this was used for synthesizing 1,4-
substituted 1H-1,2,3-triazolo-quinazolin-4(3H)-one analogs (Scheme 1).

Scheme
1.
Synthesis
of
5-methyl-2-((4-phenyl-1H-1,2,3-triazol-1-yl)methyl)-3-(O-
tolyl)quinazolin-4(3H)-one using cyclo-addition reaction approach.
The reaction conditions for microwave protocol was optimized as mentioned in table 2, where
o
the best conversion was observed using 100 Watts microwave power at 100 C temperature for 3
minutes irradiation (entry 2, table 2) in DMF. Therefore, all the reactions were conducted using
this optimized condition.
Table 2
Optimization of microwave irradiation conditions for the synthesis of 1,4-substituted 1H-1,2,3-
triazolo-quinazolin-4(3H)-ones.
Entry
Microwave
Power (Watts)
Temp.
( C)
Time
(Min.)
Yield of 5a
o
a
(%)
1
2
80
100
100
3
3
75
80
100
a
Isolated yields.Series of 1,4-substituted 1H-1,2,3-triazolo-quinazolin-4(3H)-one compounds
were prepared using optimized protocol (Table 3). Variety of alkynes viz. substituted aryl
acetylenes; -N and O-propargylated substituted aryls, and also substituted N-propargyl
arylsulphonamides were used to bring the diversity in the scaffold. Different kind of substitutions
were chosen in acetylenes; electron donating as well as electron withdrawing groups have been
accommodated for preparation of diverse triazole analogs of quinazolinone. Moreover, various
substitutions on aryl rings of quinazolinone and N-aryl compounds were also synthesized. The
observed yields were in the range of 70-80% using optimized conditions.
The introduction of a triazole substituent at C-2 position of quinazolinone structure was achieved
using optimized conditions. The triazole substituted compounds were synthesized by
cycloaddition of in situ generated organic-azides with terminal alkynes, which gave expected

1
,4-substituted triazolyl-quinazolinone. 5-Methyl-2-((4-phenyl-1H-1,2,3-triazol-1-yl)methyl)-3-
(
o-tolyl)quinazolin-4(3H)-one (5a) was synthesized from 2-(chloromethyl)-5-methyl-3-(o-
tolyl)quinazolin-4(3H)-one (3) (1.0 equiv.), sodium azide (1.3 equiv.) and phenylacetylene 4 (1.0
equiv.) in presence of CuI (10 mol %) in DMF solvent. In order to bring the diversity in the
scaffold, 26 compounds were prepared from substituted phenylacetylenes, wherein, different
sites on quinazolinone or at the C-5″ position of triazole ring was used for modifications. All the
products were obtained in good yields. For the preparation of triazolyl- or substituted triazolyl-
quinazolinone analogs, we followed scheme 1 and a list of synthesized compounds (5a-z), is
presented in table 3.

Table 3
Synthesis of diverse analogs of 1H-1,2,3-triazolo-quinazolin-4(3H)-one scaffold by
modifications at C-5″ position of triazole moiety.
a
Isolated yields
Having synthesized compounds (5a-5z), we performed in vitro cytotoxicity evaluation against
four human cancer cell lines viz. HL-60 (Leukemia cancer cell line), Colo-205, HCT-116 (colon
cancer cell line), MCF-7 (breast adenocarcinoma cell line), and A549 (lung adenocarcinoma

epithelial cell line), were used for in vitro assay (Table S1, supporting information). Compounds
a and 5l were found to be active against HL-60 cells, these compounds showed lowest IC50
5
values (1 µM) among all.
Further, we checked the specificity of compounds 5a and 5l against different isoforms of
PI3Kinases by western blotting assay (Fig. 2). HL-60 cells were treated with both compounds for
short time intervals at 1 µM concentration and expression of different isoforms were analyzed.
p110γ isoform was significantly down regulated in HL-60 cells treated with compounds 5a and
5
l. Densitometry analysis (Fig. 3) of p110γ western blots revealed that at 2h, more than 50%
expression was down regulated by these compounds. Both compounds have not shown any effect
on p110α, p110β and p110δ expression in HL-60 cells.
Fig. 2. Western blot analysis of compound 5a and 5l at 1 µM against all isoforms of PI3K.

Fig. 3. Densitometry analysis. HL-60 cells were treated with compounds 5a and 5l (1 µM
concentration both) at indicated time intervals. After incubation, cell lysates were prepared and
5
0-70 µg of protein was run on SDS–PAGE gel for western blot analysis. Densitometry was
performed by using image analysis software from Thermo Scientific.
The IC50 of these active compounds-5a and 5l were calculated and they were found to be
supportive to western blot results. These compounds showed selectivity towards PI3Kү isoform
with IC50 of 1 and 3µM respectively (Table 4).
Table 4.
The IC50 of compound-5a and 5l showing selectivity for PI3K isoforms.
Compound
IC50
PI3Kү
PI3Kα
PI3Kβ
PI3Kδ
Compound 5a
Compound 5l
IC87114
250 μM
430 μM
1 μM
5 μM
290 μM
255 μM
355 μM
200 μM
3 μM
10 μM
70 μM
160 nM
Compound 5a and 5l are found to be equipotent in cell based screening against different cancer
cell lines having IC50 1 µM for HL60 (leukemia) cell line. However, in biochemical assay (cell
free) it has been observed that the IC50 1 and 3 µM was noted for 5a and 5l with respect to 70 µM
for PI3K ү isoform of reference compound IC87114. And for PI3Kδ the IC50 values of 5a, 5l and
are 5, 10 µM respectively with respect to reference compound IC87114 showing IC50160 nM.
Thus, the compounds 5a and 5l are more selective towards PI3K ү isoform than δ (approx. 5a is
5
fold and 5l around 3 fold more selective for PI3Kү).
The molecular docking results for series of the designer molecules showed to have proficiency
for hydrophobic π-π interactions and also showed more H-bond interactions in two of series,
th
compound 5a, shows PI3Kγ interaction with the phenyl ring at 4 position of triazole is forming
rd
a π-π stacking with Tyr867 at distance of 3.46A⁰ and o-tolyl at 3 position of quinazoline forms
π-cation interaction with Lys890 at a distance of 3.55A⁰ whereas the benzene ring of indole
moiety of Trp812 is forming bidentate π-π stacking with the quinazolines aromatic rings (4.51A⁰
and 3.67A⁰ distance respectively) and the pyrrole ring of this indole moiety is forming an
additional π-π stacking with the benzene ring (4.45A⁰) to form a stable complex. In case of 5a

rd
with PI3Kδ protein Lys755 forms a H-bonding with nitrogen atom at 3 position of triazole ring
at a distance of 2.31A⁰ and π-cation interaction with phenyl ring at 2.93A⁰ distance and Tyr813
forms π-π with quinazolinones moiety (3.28A⁰) (Fig. 4).
IC87114 with PI3K γ
IC87114 with PI3K δ
Quinazoline-triazole -5a with PI3K δ
Quinazoline-triazole - 5a with PI3K γ
Quinazoline-triazole -5l with PI3K γ
Quinazoline-triazole -5l with PI3K δ

Fig. 4. H-bond interaction and π-π interaction of compound 5a and 5l with PI3K-delta and
gamma protein using in silico docking approach. The reference compound used for studies is
51
IC87114 (PDB ID 2X38)
In triazole-compound 5l with PI3Kγ protein bromophenyl and triazole rings are forming π-cation
interaction with Lys802 (5.41A⁰) and Lys890 (4.69 A⁰). The benzene ring of indole moiety of
Trp812 is forming bidentate π-π stacking with the quinazolinone aromatic rings (3.93 A⁰ and
3
.64A⁰ distance respectively) and the pyrrole ring of this indole moiety is forming an additional
π-π stacking with benzene ring (4.29A⁰) to provide a stable conformation to the molecule. In
th
case of 5l with PI3K δ the Lys755 forms π-cation interaction with bromophenyl at 4 position of
rd
triazole at a distance of 3.25A⁰, Trp760 forms π-π stacking with o-tolyl at 3 position of
quinazolinone at a distance of 3.73A⁰ and Tyr813 forms π-π stacking with quinazolinone
moiety(3.17A⁰).
On comparing the interactions of triazole-compounds 5a and 5l with PI3Kγ and δ protein, it was
identified that in case of PI3Kγ the indole moiety of Tyr812 is forming three (benzene ring of
indole moiety of Tyr is forming bidentate π-π stacking with the quinazolinone aromatic rings and
the pyrrole ring of this indole moiety forms third π-π stacking with the aromatic benzene ring) π-
π stacking with the quinazolinone aromatic rings, whereas in case of PI3Kδ Tyr813 is forming
only one π-π stacking with this quinazolinone moiety. Thus this can be the reason due to which,
the triazole-compounds are showing better affinity and activity for PI3Kγ rather than expected
PI3Kδ. However, on analyzing the interaction of protein in complex with quinazolinone-triazole-
5
a and 5l, it has been observed that the H-bonding interaction with the Glu880 and Val882 has
been interuptted. Where, in PI3K δ, these interactions are retained in IC87114. So, this could be
the reason of selectivity of 5a and 5l towards PI3Kγ isoform.
In conclusion, we have developed a method for the synthesis of 1,4-substituted 1H-1,2,3-
triazolo-quinazolin-4(3H)-ones and evaluation for their cytotoxicity. These compounds are
prepared by microwave assisted conditions using one-pot multicomponent strategy. Compound
5
a and 5l exhibited highest cytotoxicity against HL-60 human cancer cell line and specifically
inhibited PI3Kγ isoform in western blotting analysis. In silico analysis of these two compounds
show better affinity towards the PI3Kγ.

Acknowledgements
MS, ASP thank CSIR/UGC for the award of fellowship. The funding support to carry out this
th
work from BSC-0108 ‘MEDCHEM-CSIR 12 FYP Project’ is gratefully acknowledged. SDS
thanks CSIR for the award of Raman Research Fellowship. PKV is thankful to DST (SERB) for
providing Fasttrack Young Scientist Grant.
1
13
o
Supplementary data: Supplementary data ( H/ C NMR, DEPT at 135 , and mass for all the
compounds).
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Graphical Abstract
Design and synthesis of 1,4-substituted 1H-
Leave this area blank for abstract info.
1
,2,3-triazolo-quinazolin-4(3H)-ones by
Huisgen 1,3-dipolar cycloaddition with
PI3Kγ isoform selective activity
M. Srinivas, Anup Singh Pathania, Priya Mahajan, Praveen K. Verma, Santosh S. Chobe, Fayaz A.
Malik, Amit Nargotra, Ram A. Vishwakarma, Sanghapal D. Sawant*