Synthesis and biofilm inhibition studies of 2-(2-amino-6-arylpyrimidin-4-yl)quinazolin-4(3H)-ones

Article abstract of DOI:10.1016/j.bmcl.2020.127550

Synthesis of novel 4(3H)-quinazolinonyl aminopyrimidine derivatives has been achieved via quinazolinonyl enones which in turn were obtained from 2-acyl-4(3H)-quinazolinone. They have been assayed for biofilm inhibition against Gram-positive (methicillin-resistant Staphylococcus aureus (MRSA)) and Gram-negative bacteria (Acinetobacter baumannii). The analogues with 2,4,6-trimethoxy phenyl, 4-methylthio phenyl, and 3-bromo phenyl substituents (5h, 5j & 5k) have been shown to inhibit biofilm formation efficiently in MRSA with IC₅₀ values of 20.7–22.4 μM). The analogues 5h and 5j have demonstrated low toxicity in human cells in vitro and can be investigated further as leads.

Full text of DOI:10.1016/j.bmcl.2020.127550

Bioorganic & Medicinal Chemistry Letters 30 (2020) 127550
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biofilm inhibition studies of 2-(2-amino-6-arylpyrimidin-4-yl)
quinazolin-4(3H)-ones
a,⁎
a
a
a
c
Sivappa Rasapalli , Zachary F. Murphy , Vamshikrishna Reddy Sammeta , James A. Golen ,
b
b
b
Alexander W. Weig , Roberta J. Melander , Christian Melander , Prathyushakrishna Macha ,
c
Milana C. Vasudev
b
a
University of Massachusetts Dartmouth, Department of Chemistry and Biochemistry, 285 Old Westport Rd, North Dartmouth, MA 02747, United States
University of Notre Dame, Department of Chemistry and Biochemistry, 252A McCourtney Hall, Notre Dame, IN 46556, United States
c
University of Massachusetts Dartmouth, Department of Biomedical Engineering, 285 Old Westport Rd, North Dartmouth, MA 02747, United States
A R T I C L E I N F O
A B S T R A C T
Dedicated to Dr. Subhash P. Chavan on the
occasion of his retirement from CSIR-NCL,
Pune, India.
Synthesis of novel 4(3H)-quinazolinonyl aminopyrimidine derivatives has been achieved via quinazolinonyl
enones which in turn were obtained from 2-acyl-4(3H)-quinazolinone. They have been assayed for biofilm in-
hibition against Gram-positive (methicillin-resistant Staphylococcus aureus (MRSA)) and Gram-negative bacteria
(
Acinetobacter baumannii). The analogues with 2,4,6-trimethoxy phenyl, 4-methylthio phenyl, and 3-bromo
Keywords:
phenyl substituents (5h, 5j & 5k) have been shown to inhibit biofilm formation efficiently in MRSA with IC50
values of 20.7–22.4 μM). The analogues 5h and 5j have demonstrated low toxicity in human cells in vitro and
can be investigated further as leads.
4
2
(3H)Quinazolinone
-Acetylquinazolin-4(3H)-one
Aminopyrimidine
Anti-biofilm
MRSA biofilm
The incidence of multidrug-resistant (MDR) bacteria is on the rise.1
quinazoline, has been shown to possess antibiofilm activity by several
laboratories (Scheme 1).
This situation has been further complicated by the antibacterial drug
drought for the past 30 years, thus it is important to focus on identifying
Notably, Shaw, et al., showed that N2, N4-disubstituted quinazoline
analogues exhibited potent antibacterial activity against A. baumannii,
displaying single-digit micromolar MICs, as well as eradicating 90% of
cells within a biofilm at or near the minimum inhibitory concentration
2
ways to address the health crisis caused by the same. According to the
Centers for Disease Control and Prevention (CDC), it is estimated that
2
.8 million people are infected by antibiotic-resistant bacteria annually,
8
and of those infected, about 35,000 die. The CDC predicts these num-
bers will only increase if pertinent attention is not brought to these
(MIC). These compounds also exhibited potent antibacterial activity
against methicillin-resistant S. aureus (MRSA), with MIC values around
3
9
persistent bacterial strains. Bacterial species such as Staphylococcus
0.5 μM (Fig. 1). Previous studies have also established that 2-amino-
aureus, and Acinetobacter baumannii, have evolved to evade many cur-
quinazoline derivatives have significant anti-biofilm activity against
1
0
rent antibiotics. A common defense mechanism observed in MDR bac-
Mycobacterium smegmatis (IC50 values around 15 µM). Additionally,
4
teria, and others is the formation of biofilms. In general, biofilms are
multiple studies have shown that 2-aminopyrimidine derivatives pos-
1
1
composed of a surface-attached community of bacteria enclosed in an
extracellular matrix of biomolecules. It is known that bacteria in a
biofilm are at least 1000-fold more resistant to antibiotics. Therefore,
inhibition of biofilm formation is an attractive approach to combat the
sess anti-biofilm properties. Analogues of meridianin D, a 2-amino-
pyrimidine containing natural product show potent biofilm inhibitory
1
2
activity against S. aureus (MRSA) (IC50 values as low as 9 μM) (Fig. 1).
Inspired by these findings, we have designed analogues of 2-(2-amino-
6-arylpyrimidin-4-yl)quinazolin-4(3H)-ones (Fig. 1) by combining the
structural fragments of the leads and tested the role of these analogues
in the inhibition of biofilms. We have developed a facile synthetic route
to access our designed compounds that centered on the condensation of
quinazolinonyl enone intermediates with guanidine. Herein, we report
5
threat of MDR bacteria.
The 4(3H)-quinazolinone, an important pharmacophore with many
6
useful bioactivities found in numerous alkaloids and drugs currently in
the market, has been studied for antibacterial potential but not for the
ability to inhibit biofilm development.7 The close congener, amino-
⁎
Corresponding author.
E-mail address: srasapalli@umassd.edu (S. Rasapalli).
https://doi.org/10.1016/j.bmcl.2020.127550
Received 19 April 2020; Received in revised form 30 August 2020; Accepted 7 September 2020
Available online 12 September 2020
0960-894X/ Published by Elsevier Ltd.

S. Rasapalli, et al.
Bioorganic & Medicinal Chemistry Letters 30 (2020) 127550
Fig. 1. Anti-biofilm activity of analogues containing quinazoline or 2-aminopyrimidine.
the synthesis of the pyrimidinyl quinazolinone derivatives, their anti-
biofilm activity against MRSA, and A. baumannii as well as cytotoxicity
in human colon cells in vitro.
compounds did not prove to exhibit antibiofilm activity against A.
baumannii, with none of the compounds tested inhibiting biofilm for-
mation by more than 25% at 100 µM (Table S1 Supporting
Information). Against MRSA however, 14 of the 22 compounds in-
hibited biofilm formation by more than 50% at 100 µM, and IC50 values
were, therefore, determined (Table 1).
Our synthetic plan was to access the 2-(2-amino-6-arylpyrimidin-4-
yl)quinazolin-4(3H)-ones through conjugate addition and subsequent
condensation between guanidine and various quinazolinonyl enones
(
4a-v). We proposed to access these enones through the aldol con-
The unsubstituted phenyl derivative 5a exhibited moderate activity,
returning an IC50 of 34.41 µM, while the incorporation of a methyl
substituent at either the 2- or 4-position of the phenyl ring (5b and c)
resulted in reduced activity. The monomethoxy derivative 5d, and the
3,4- and 3,5-di-methoxy derivatives 5e and 5f displayed reduced ac-
tivity compared to the parent, however the 2,4-di-methoxy analogue 5g
exhibited comparable activity to the parent. The addition of a second
ortho-methoxy group to this scaffold in compound 5h resulted in in-
creased activity, with this compound exhibiting an IC50 of 21.2 µM,
while the 3,4,5-tri-methoxy isomer 5i displayed considerably reduced
activity. The thioether derivative 5j displayed comparable activity to
5h (IC50 22.4 µM). The most active compound from this series was the
3-bromo derivative 5k which exhibited an IC50 value of 20.7 µM, while
the 4-bromo derivative 5l displayed reduced activity. Other halogen
substituents including 2-chloro (5m), and 2,4-dichloro (5o) were
moderately active while the 4-chloro (5n) and 4-fluoro (5p) derivatives
did not inhibit biofilm formation by more than 50% at 100 µM. Placing
a trifluoromethyl substituent at the 4- position (5q) resulted in reduced
activity compared to the parent. Benzyl derivatives followed the same
trend as the methoxy derivatives in that placement at the 3-position
(5r) reduced activity, while placement at the 4-position (4s) conferred
moderate activity comparable to the parent. Finally, the effect of in-
corporation of aromatic substituents was investigated, with the naph-
thyl and pyridinyl derivatives 5t and 5u effecting less than 50% in-
hibition at 100 µM, and the thiophenyl and furan derivatives 5u and 5v
exhibiting reduced activity compared to the parent.
densation of 2-acetyl-4(3H)quinazolinone (3) with various substituted
aromatic aldehydes.
Accordingly, our synthesis process started with identifying a syn-
thetic route to 2-acetylquinazolin-4(3H)-one (3) amenable to large scale
synthesis. There were only a few reports available in the literature for
1
3
the synthesis of 3 that involved either the use of selenium dioxide or
1
4
triphenylphosphine, reagents problematic for our intended large-
1
5
scale synthesis. The benzodiazepine ring contraction route also failed
to deliver 3 in satisfactory yields in our hands. We then resorted to
1
6
pyruvic acid route utilized by Hart et al., which yielded the desired
compound 3 albeit in poor yield in our hands due to an unknown di-
meric side product. After a great deal of experimentation, we success-
fully modified the preparation of the diamide (2) that resulted in a 61%
yield on a > 100 g reaction scale. The dehydrative cyclization of 2
using mild bases provided compound 3 in 65% yield on a reaction scale
of 10 g. In addition to suppressing the unwanted dimer formation, the
modification of utilizing aq. basic conditions allowed us to run the
subsequent aldol condensation in the same pot to obtain quinazolinonyl
enones (4a-4v) in a single operation at an improved overall yield
(
75–89%), thus rendering the method practical and straightforward to
implement (Scheme 2).
Under basic conditions, the enones (4a-4v) were then refluxed in
ethanol in the presence of guanidine to yield the target pyrimidinyl
quinazolinones (5a-5v, Scheme 2). The amassed collection was assayed
for their anti-biofilm activity against Gram-positive (MRSA) and Gram-
negative (A. baumannii) bacteria.
In general, the structure-activity relationship (SAR) data generated
from this initial library indicates that electron donating groups placed
at the 2- or 4- (or both) positions leads to higher activity, while the
placement of such groups at the 3-position leads to reduced activity (as
seen for compounds 5h and 5j). Incorporation of bromine at the 3-
position confers increased activity (compound 5k) but when a halogen
is placed at the 2- or 4- position a reduction in activity is observed.
We next tested the ability of the library to disperse pre-formed
MRSA biofilms as previously described16 however none of the
Compounds were initially screened for the ability to inhibit biofilm
formation by A. baumannii ATCC 19606 and MRSA ATCC 43300 at a
concentration of 100 µM using a crystal violet reporter assay as pre-
1
7
viously described. All compounds that inhibited biofilm formation by
greater than 50% as compared to an untreated control at this con-
centration were subjected to a dose–response assay to determine the
IC50 value (which we define as the concentration at which the com-
pound effects a 50% reduction in the amount of biofilm). This class of
2

S. Rasapalli, et al.
Bioorganic & Medicinal Chemistry Letters 30 (2020) 127550
Scheme 2. Synthesis of 2-(aryl) aminopyrimidinyl quinazolin-4(3H)-ones.
Table 1
IC50 values for inhibition of MRSA ATCC 43300
biofilm formation.
Compound
IC50 (µM)
Scheme 1. Synthesis of 2-acetylquinazolin-4(3H)-one and aryl quinazolinonyl
enones.
5a
34.4 ± 0.54
84.8 ± 6.63
50.5 ± 5.55
38.4 ± 1.70
21.2 ± 3.33
22.4 ± 3.44
20.7 ± 2.71
47.3 ± 4.50
56.2 ± 2.32
42.7 ± 6.77
86.5 ± 7.08
37.3 ± 2.83
71.0 ± 11.32
56.9 ± 13.60
5
5
d
f
compounds effected more than 25% dispersion (compared to biofilms
treated with fresh media alone) at 100 µM. Finally, MICs of all com-
pounds against MRSA ATCC 433300 were recorded to ensure that the
observed biofilm inhibition activity was not a result of planktonic
toxicity, and all compounds returned MICs of greater than 100 µM
5g
5h
5
5
5
5
j
k
l
m
(
highest concentration tested).
5o
5q
We have also assessed whether the most active compounds (5h, 5j,
5
5
5
s
and 5k) exhibit any toxicity to human cells at the concentrations that
u
v
were used to inhibit biofilm formation. Normal human colon cells
(
CCD-18 Co cells) were cultured in 96-well plates for three days fol-
lowed by the addition of the three active compounds in triplicate (5h,
5
j, and 5k) at three concentrations of each compound, 50 μM, 25 μM
DMSO or the compounds were used to compare the effect of just DMSO
and DMSO in combination with compounds on the cells. This control
was higher in viability indicating damage to the cells in the presence of
DMSO (1 μL and 0.5 μL) though in a low volume. In the case of com-
pound, 5h, the cellular toxicity was minimal even at the highest con-
centration used (50 µM). Whereas in the case of compound 5J, 50 μM
and 12.5 μM. MTT cytotoxicity assay was performed on day 1 following
the addition of the compounds as shown in Fig. 2. The control samples
in this study were DMSO (1 µL) treated cells since the trace amount of
DMSO in the wells also had some cytotoxic effect on the cells. An ad-
ditional positive control of the CC-18 Co cells not exposed to either
3

S. Rasapalli, et al.
Bioorganic & Medicinal Chemistry Letters 30 (2020) 127550
provided, including copies of 1H and C NMR spectra of all new
compounds and HRMS analysis. X-ray data for 2, 3, 4h, 4i, 4s, 4w, 5f
and 5i) to this article can be found online at https://doi.org/10.1016/j.
bmcl.2020.127550.
13
References
1
2
3
. Tanwar J, Das S, Fatima Z, Hameed S. Interdiscip Perspect Infect Dis.
014;2014:541340.
. Mantravadi PK, Kalesh KA, Dobson RCJ, Hudson AO, Parthasarathy A. Antibiotics
2
(
Basel). 2019;8:8.
. Center for Disease and Control Prevention, 2019, retrieved from: https://www.cdc.
gov/drugresistance/about.html.
4
5
. Donlan RM. Emerg Infect Dis. 2002;8:881.
. (a) Welsh MA, Blackwell HE. FEMS Microbiol Rev. 2016;40:774;
(
b) Geske GD, Wezeman RJ, Siegel AP, Blackwell HE. J Am Chem Soc.
Fig. 2. Normalized MTT absorbance indicating the cell viability of CCD-18 Co
cells after 1-day treatment with the three bioactive compounds (5h, 5k, and 5j).
2005;127:12762.
6. (a) Kahn I, Zaib S, Batool S, Abbas N, Ashraf Z, Iqbal J, Saeed A. Bioorg Med Chem.
016;24:2361;
b) Abbas SY, El-Bayouki KAM, Basyouni WM, Mostafa EA. Med Chem Res.
2018;27:571;
c) El-Hashash MAEM, Salem MS, Al-Mabrook SAM. Res Chem Intermed.
018;44:2545;
d) Noolvi MN, Patel HM, Bhardwaj V, Chauhan A. Eur J Med Chem. 2011;46:2327;
(e) Dohle W, Jourdan FL, Menchon G, et al. J Med Chem. 2018;61:1031;
f) Hrast M, Rozman K, Jukic M, Patin D, Gobec S, Sova S. Bioorg Med Chem Lett.
017;27:3529;
2
(
proved to more cytotoxic whereas the lower concentrations were
comparable to the DMSO controls. Among the three compounds used in
this study, 5h demonstrated the lowest cytotoxicity, in comparison with
the DMSO controls whereas compound 5K had the highest toxicity.
Compound 5k had significant toxicity even at the lowest concentration
used (12.5 µM) and comparable to the toxicity at the highest con-
centration used (50 µM).
(
2
(
(
2
(
g) Ighachane H, Sedra MH, Lazrek HBJ. Mater Environ Sci. 2017;8:134;
(h) Abdel-Aziz AA, Abou-Zeid LA, El Tahir KEH, Ayyad RR, Sayed MA, El-Azab AS.
Eur J Med Chem. 2016;121:410;
In conclusion, novel 2-(2-amino-6-arylpyrimidin-4-yl) quinazolin-
(
(
i) Abuelizz HA, El-Dib RA, Marzouk M, Al-Salahi R. Microb Pathog. 2018;117:60;
j) Yang L, Ge S, Huang J, Bao X. Mol Divers. 2018;22:71;
4
(3H)-ones were designed and accessed via guanidine condensation
with the quinazolinonyl aryl enones that were in turn derived from 2-
(k) Dinari M, Gharahi F, Asadi P. J Mol Struct. 2018;1156:43;
(l) Dash B, Dash S, Laloo D, Madhi CJ. Appl Pharm Sci. 2017;7:20;
acetyl 4(3H) quinazolinone. The synthetic collection was assayed for
(
m) Jain N, Jaiswal J, Pathak A, Singour PK. Cent Nerv Syst Agents Med Chem.
the antibiofilm inhibition against
a representative gram-positive
2
018;18:63;
n) Al-Salahi R, Abuelizz HA, Ghabbour HA, El-Dib R, Marzouk M. Chem Cent J.
2016;10:21;
(
MRSA) and representative gram-negative bacteria (A. baumannii). The
(
screening results revealed that some of the analogues (5h, 5j, 5k) in-
hibited biofilm formation efficiently in MRSA (IC50 ~ 20 μM) while
further optimization through additional diversification could poten-
tially augment activity further. The cytotoxicity assay of the active
compounds revealed that compounds 5h and 5j have low toxicity at
lower concentrations whereas compound 5k was highly toxic to the
cells making compounds 5h and 5k as possible leads for further mod-
ification. Efforts are underway in this direction and the results will be
reported.
(
o) Ji Q, Yang D, Wang X, et al. Bioorg Med Chem. 2014;22:3405.
7
. (a) Alagarsamy V, Chitra K, Saravanan G, Solomon VR, Sulthana MT, Narendhar B.
Eur J Med Chem. 2018;151:628;
(b) Zhang L, Chen Q, Li XQ, Wu SQ, Wan JL, Ouyang G. J Heterocycl Chem.
2
018;55:743;
(
(
c) Gatadi S, Lakshmi TV, Nanduri SE. J Med Chem. 2019;170:157;
d) Bouley R, Ding D, Peng Z, et al. J Med Chem. 2016;59:5011;
(e) Jafari E, Khajouei MR, Hassanzadeh F, Hakimelahi GH, Khodarahmi GA. Res
Pharm Sci. 2016;11:1;
(
(
(
f) Zhan X, Xu Y, Qi Q, Wang Y, Shi H, Mao Z. Chem Biodivers. 2018;15;
g) Osarumwense PO, Iyekowa O. Trop J Nat Prod Res. 2017;1:173;
h) Rakesh KP, Kumara HK, Ullas BJ, Shivakumara J, Channe GD. Bioorg Chem.
2
019;90;
Declaration of Competing Interest
(
(
i) Gatadi S, Gour J, Shukla M, et al. Eur J Med Chem. 2019;175:287;
j) Hantsu SH, Gupta V, Narang R. J Pharm Res. 2017;11:1111;
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
(k) Wang HX, Liu HY, Li W, et al. Med Chem Res. 2019;28:203.
8
9
. Fleeman R, Van Horn KS, Barber MM, et al. Antimicrob Agents Chemother.
2
017;61:e00059.
. Van Horn KS, Burda WN, Fleeman R, Shaw LN, Manetsch R. J Med Chem.
2
014;57:3075.
1
1
1
0. Cox KE, Melander C. Medchemcomm. 2019;10:1177.
1. Barot VM, Desai SD. Int J Chem Sc. 2013;11:865.
Acknowledgments
2. Huggins WM, Baker WT, Baker JT, Hahn NA, Melander RJ, Melander C. ACS Med
Chem Lett. 2018;9:702.
This work has been supported by LSMF, and OTCV Phase-I funds of
UMass System and instrumentation grants from the NSF (Grants CHE-
1
1
3. Han X, Peng B, Xiao BB, et al. Eur J Med Chem. 2019;162:586.
4. Alemagna A, Del Buttero P, Licandro E, Maiorana S, Papagni A. Tetrahedron.
1
229339 and CHE-1429086 for funds to purchase of the 400 MHz NMR
1
985;41:3321.
spectrometer and X-ray diffractometer, respectively, used in this re-
search) and NIH/NIDCR grant R01 DE022350 (CM).
15. Heindel ND, Fish VB, Lemke TF. J Org Chem. 1968;33:3997.
1
1
6. Freed JD, Hart DJ, Magomedov NA. J Org Chem. 2001;66:839.
7. Huggins WM, Barker WT, Baker JT, Hahn NA, Melander RJ, Melander C. Med Chem
Lett. 2018;9:702.
Appendix A. Supplementary data
Supplementary data (complete experimental procedures are
4