Structure-based design and optimization of pyrimidine- and 1,2,4-triazolo[4,3-a]pyrimidine-based matrix metalloproteinase-10/13 inhibitors via Dimroth rearrangement towards targeted polypharmacology

Article abstract of DOI:10.1016/j.bioorg.2020.103616

Recently, interest in matrix metalloproteinases (MMPs) -10 and -13 has been revitalized with the growing knowledge on their relevance within the MMPs network and significance of their inhibition for treatment of various diseases like arthritis, cancer, atherosclerosis and Alzheimer. Within this approach, dual MMP-10/13 inhibition was disclosed as new approach for targeted polypharmacology. While several efficient MMP-13 inhibitors are known, very few potent and selective MMP-10 inhibitors were reported. This study describes the design, synthesis and optimization of novel MMP-10/13 inhibitors with enhanced MMP-10 potency and selectivity towards polypharmacology. Starting with a lead fused pyrimidine-based MMP-13 inhibitor with weak MMP-10 inhibition, a structure-based design of pyrimidine and fused pyrimidine scaffolds was rationalized to enhance activity against MMP-10 in parallel with MMP-13. Firstly, a series of 6-methyl pyrimidin-4-one hydrazones 6–10 was synthesized via conventional and ultrasonic-assisted methods, then evaluated for MMP-10/13 inhibition. The most active derivative 9 exhibited acceptable dual potency with 7-fold selectivity for MMP-10 (IC₅₀ = 53 nM) over MMP-13. Such hydrazones were then cyclized to the corresponding isomeric 1,2,4-triazolo[4,3-a]pyrimidines 12–19. Their MMP-10/13 inhibition assay revealed, in most cases, superior dual activities with general MMP-10 selectivity compared to the corresponding precursors 6–10. In addition, a clear structure activity relationship trend was deduced within the identified regioisomers, where the 5-oxo-1,2,4-triazolo[4,3-a]pyrimidine derivatives 15 and 16 were far more active against MMP-10/13 than their regioisomers 12 and 13. Remarkably, the p-bromophenyl derivative 16 exhibited the highest MMP-10 inhibition (IC₅₀ = 24 nM), whereas the p-methoxy derivative 18 was the most potent MMP-13 inhibitor (IC₅₀ = 294 nM). Moreover, 16 exhibited 19-fold selectivity for MMP-10 over MMP-13, 10-fold over MMP-9, and 29-fold over MMP-7. Docking studies were performed to provide reasonable explanation for structure-activity relationships and isoform selectivity. 16 and 18 were then evaluated for their anticancer activities against three human cancers to assess their therapeutic potential at cellular level via MTT assay. Both compounds exhibited superior anticancer activities compared to quercetin. Their in silico ligand efficiency metrics, physicochemical properties and ADME parameters were drug-like. Guided by such findings that point to 16 as the most promising compound in this study, further structure optimization was carried out via photoirradiation-mediated Dimroth rearrangement of the inactive triazolopyrimidine 13 to its potent regioisomer 16.

Full text of DOI:10.1016/j.bioorg.2020.103616

Journal Pre-proofs
Structure-based design and optimization of pyrimidine- and 1,2,4-triazo-
lo[4,3-a]pyrimidine-based matrix metalloproteinase-10/13 inhibitors via Dim-
roth rearrangement towards targeted polypharmacology
El Sayed Helmy El Ashry, Laila Fathy Awad, Mohamed Teleb, Nihal Ahmed
Ibrahim, Marwa M. Abu-Serie, Mohamed Nabil Abd Al Moaty
PII:
S0045-2068(19)31828-0
DOI:
https://doi.org/10.1016/j.bioorg.2020.103616
YBIOO 103616
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
28 October 2019
20 January 2020
22 January 2020
Please cite this article as: E. Sayed Helmy El Ashry, L. Fathy Awad, M. Teleb, N. Ahmed Ibrahim, M.M. Abu-
Serie, M. Nabil Abd Al Moaty, Structure-based design and optimization of pyrimidine- and 1,2,4-triazolo[4,3-
a]pyrimidine-based matrix metalloproteinase-10/13 inhibitors via Dimroth rearrangement towards targeted
polypharmacology, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103616
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Structure-based design and optimization of pyrimidine- and 1,2,4-triazolo[4,3-a]pyrimidine-
based matrix metalloproteinase-10/13 inhibitors via Dimroth rearrangement towards targeted
polypharmacology
El Sayed Helmy El Ashry a*, Laila Fathy Awad a, Mohamed Teleb b, Nihal Ahmed Ibrahim a,
Marwa M. Abu-Serie c and Mohamed Nabil Abd Al Moaty a*
a Chemistry Department, Faculty of Science, Alexandria University, Alexandria 21321, Egypt.
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University, Alexandria 21521, Egypt.
c Medical Biotechnology Department, Genetic Engineering and Biotechnology Research Institute, City of Scientific
Research and Technological Applications (SRTA-City), Egypt.
* Corresponding authors; Email addresses: eelashry60@hotmail.com ORCID ID 0000-0001-5480-239X,
mohamednabil_sc_chem@yahoo.com
ABSTRACT
Recently, interest in matrix metalloproteinases (MMPs) -10 and -13 has been revitalized with the
growing knowledge on their relevance within the MMPs network and significance of their inhibition
for treatment of various diseases like arthritis, cancer, atherosclerosis and Alzheimer. Within this
approach, dual MMP-10/13 inhibition was disclosed as new approach for targeted polypharmacology.
While several efficient MMP-13 inhibitors are known, very few potent and selective MMP-10
inhibitors were reported. This study describes the design, synthesis and optimization of novel MMP-
10/13 inhibitors with enhanced MMP-10 potency and selectivity towards polypharmacology. Starting
with a lead fused pyrimidine-based MMP-13 inhibitor with weak MMP-10 inhibition, a structure-
based design of pyrimidine and fused pyrimidine scaffolds was rationalized to enhance activity against
MMP-10 in parallel with MMP-13. Firstly, a series of 6-methyl pyrimidin-4-one hydrazones 6-10 was
synthesized via conventional and ultrasonic-assisted methods, then evaluated for MMP-10/13
inhibition. The most active derivative 9 exhibited acceptable dual potency with 7-fold selectivity for
MMP-10 (IC50 = 53 nM) over MMP-13. Such hydrazones were then cyclized to the corresponding
isomeric 1,2,4-triazolo[4,3-a]pyrimidines 12-19. Their MMP-10/13 inhibition assay revealed, in most
cases, superior dual activities with general MMP-10 selectivity compared to the corresponding
precursors 6-10. In addition, a clear structure activity relationship trend was deduced within the
identified regioisomers, where the 5-oxo-1,2,4-triazolo[4,3-a]pyrimidine derivatives 15 and 16 were
far more active against MMP-10/13 than their regioisomers 12 and 13. Remarkably, the p-
bromophenyl derivative 16 exhibited the highest MMP-10 inhibition (IC50 = 24 nM), whereas the p-
methoxy derivative 18 was the most potent MMP-13 inhibitor (IC50 = 294 nM). Moreover, 16
exhibited 19-fold selectivity for MMP-10 over MMP-13, 10-fold over MMP-9, and 29-fold over
MMP-7. Docking studies were performed to provide reasonable explanation for structure-activity
relationships and isoform selectivity. 16 and 18 were then evaluated for their anticancer activities
against three human cancers to assess their therapeutic potential at cellular level via MTT assay. Both
compounds exhibited superior anticancer activities compared to quercetin. Their in silico ligand
efficiency metrics, physicochemical properties and ADME parameters were drug-like. Guided by such
findings that point to 16 as the most promising compound in this study, further structure optimization
was carried out via photoirradiation-mediated Dimroth rearrangement of the inactive
triazolopyrimidine 13 to its potent regioisomer 16.
Keywords:
MMP-10/13; Polypharmacology; Pyrimidine; Triazolopyrimidine; Dimroth rearrangement;
Anticancer.

1. Introduction
Matrix metalloproteinases (MMPs) are family of zinc dependent structurally and functionally
related endopeptidases. They are majorly implicated in the extracellular matrix degradation and
remodeling [1-3]. Hence, a plethora of vital biological processes rely on their activities, such as cell
adhesion and proliferation, angiogenesis, tissue remodeling and immune responses mediation. To date,
twenty-three human MMPs were identified and classified as collagenases (MMP-1, -8, -13, and -18),
gelatinases (MMP-2, and -9), stromelysins (MMP-3, and-10), matrilysins (MMP-7, and -26),
membrane-type MMPs (MMP-14, -15, -16, -17, -24, and -25), and others (MMP-12, -19, -20, -21, -
23, -27, -28) [4-7]. All MMPs share nearly the same catalytic domain that is constituted by about 170
amino acid residues, an active-site zinc together with another zinc ion and one to three calcium ions
[8]. The domain is divided into C-terminal and N-terminal subdomains by a cleft of six binding pockets
(S1, S2, S3, S1´, S2´ and S3´), of which S1´ shows variations in composition of its residues among
different MMPs, thus viewed as the selectivity binding pocket [9]. MMPs Dysfunction leads to a broad
field of devastating pathological conditions ranging from osteoarthritis, multiple sclerosis,
autoimmune diseases to cancer [5,10]. The relevance of MMP inhibition for treating such diseases has
been recently revitalized and validated by knockout experiments [11]. Within this approach, MMP-10
and -13 have gained considerable interest as attractive druggable targets, where both MMPs are
involved in different types of cancer [12-16], arthritis [17-19], atherosclerosis [20] and most recently
Alzheimer's disease [21]. Considering the negative clinical outcomes delivered by potent unspecific
MMP inhibitors, extensive efforts have been exerted to design and synthesize selective inhibitors. For
MMP-10, potent selective inhibitors are rarely reported in literature [22], whereas several selective
MMP-13 with nanomolar or even sub-nanomolar affinity were widely introduced [23-29]. Literature
review revealed that selective MMP-13 inhibitors are generally classified into zinc binding and non-
zinc binding inhibitors which are subdivided into various groups based on their structural scaffolds.
As the selectivity of zinc binding inhibitors is hard and rather challenging to be optimized, non-zinc
binding inhibitors are viewed as the most promising selective MMP-13 inhibitors. An important class
of non-zinc binding inhibitors is mainly based on pyrimidine (compounds I [30] and IV [22]) or fused
pyrimidine (compounds II-IV [23,24]) core linked to aromatic ring via, in most cases, amide backbone
which forms hydrogen bond interactions with the catalytic site of the enzyme [23,24,29,30] (Figure
1). A prime member of this class is a selective quinazoline-2-carboxamide II (Figure 1) MMP-13
inhibitor (IC50: 12 nM) that was identified by High-throughput screening [23] and utilized for further
optimization studies [24,31]. Optimization strategies were mainly focused on (i) modification of the
carboxamide linker; (ii) modification of the core via substitution, opening or replacement of the ring
A with various heterocyclic moieties [24] (Figure 1).
In a recent study, a potent pyrimidine-based dual MMP-10/13 inhibitor VI [22] (Figure 1) with
outstanding MMP-10 affinity (IC50 MMP-10: 31 nM, IC50 MMP-13: 5 nM) and clean inhibition profile
was synthesized and identified as a promising starting point for developing therapeutic agents based
on the principle of targeted polypharmacology (i.e. targeting multiple receptors) with less drug
interactions than mixtures of inhibitors [22,32]. The benefit of such dual activity relies on the reported

disease relevance of both MMP-10 and -13, in addition to the network organization of MMPs, where
MMP-13 is known to be super activated by MMP-10 [18].
Ring "A"
O
O
N
N
NH
NH
H
H
H
H
N
N
N
N
N
O
N
O
O
O
O
O
(II)
(I)
(III)
(IC50 MMP-13: 72 nM) [30]
(IC50 MMP-10: 3400 nM)
(IC50 MMP-13: 12 nM) [23, 24]
(IC50 MMP-13: 12 nM) [24]
O
O
N
H
N
O
NH
H
O
NH
O
F
N
S
S
N
N
O
N
H
N
O
(IV)
(V)
(VI)
O
Cl
(IC50 MMP-13: 1.1 nM) [24]
O
(IC50 MMP-13: 3.4 M) [33]

(IC50 MMP-10: 31 nM)
(IC50 MMP-13: 5 nM) [22]
Figure 1. Reported lead MMP-13 and MMP-10/13 inhibitors.
Motivated by such approach, we set out to design novel pyrimidine- and triazolopyrimidine- based
(fused pyrimidine) scaffolds as potential dual MMP-10/13 inhibitors with possible enhanced MMP-
10 inhibitory activity starting with a lead quinazolin-4-one MMP-13 inhibitor II (IC50: 12 nM)
exhibiting a rather poor MMP-10 inhibitory activity (IC50: 3400 nM) [23]. Herein, the design strategy
(Figure 2) focused on both modifying the linker as well as ring A opening (2-(substituted
benzylidenehydrazinyl)-6-methylpyrimidin-4-one 6-10), besides replacement of the quinoline core
with triazolopyrimidine moiety (1,2,4-triazolo[4,3-a]pyrimidine derivatives 12-19) via linker
cyclization. It is worth mentioning that the triazolyl moiety is known to confer high potency against
MMPs [34]. Molecular alignment studies (Figure 3) and docking simulations (Figure 4) were
performed to assess such design at the molecular level. The designed compounds (6-19) (Figure 2)
were synthesized via conventional method as well as ultrasound irradiation, then in vitro screened for
possible MMP-10/13 dual inhibitory activities. Guided by the observed structure-activity relationship
data especially within the possible triazolopyrimidine regioisomers (Figure 2), further structure
optimization was performed to maximize the activity against MMP-10 in parallel with MMP-13.
Within this approach, photoirradiation-mediated Dimroth rearrangement was successfully performed
to convert the inactive triazolopyrimidines to the corresponding potent regioisomers. Dimroth
rearrangement has been recognized as a general phenomenon in heterocyclic chemistry where a
translocation of two hetero atoms in the heterocyclic system takes place with or without changing the
ring structure [35-37]. The Dimroth rearranged products are obtained as a result of ring opening ring
closing processes which depend not only on the pH of the solution [38], but also on the number of
atoms or bulkiness of the substituents. The rearrangement is generally performed under heat, light,
acidic or basic conditions [39]. Furthermore, with the goal of exploring the potencies of the most
active dual MMP-10/13 inhibitors against other members of the MMPs family to evaluate their
selectivity profiles, their IC50 values were determined on MMP-7 and-9. Docking simulations were

performed to allow better understanding of the observed structure-activity relationships and isoform
selectivity. The most promising MMP inhibitors were then evaluated for their anticancer activities
against three human cancers namely; triple negative breast cancer (MDA-MB 231), liver cancer (HepG-
2), and colon cancer (Caco-2) compared to quercetin, an anticancer MMP inhibitor, as a reference via
MTT assay to assess their therapeutic potentials at the cellular level [40]. It is worth mentioning that
these selected cancers are among the most common and lethal human cancers according to the global
cancer statistics in 2018 [41]. Also, they are characterized by central role and /or overexpression of
MMP-10 and -13 [42,43]. Finally, ligand efficiency metrics, drug-likeness data, physicochemical
properties and ADME parameters of the promising compounds were in silico computed.
2. Results and Discussion
2.1. Design rationale
Firstly, the target 2-hydrazonyl-6-methylpyrimidin-4-ones 6-10 were designed to mimic the
thematic structural features of the lead MMP-13 inhibitor [23] (II) while modifying the three-atom
carboxamide linker to a hydrazone one (Figure 2) with approximately similar length (4.72 and 4.47
Å, respectively) and comparable charge distribution. (Figure 3A and B).
O
NH
R
Linker modification
Ring “A” opening
N
Ring "A"
N
N
H
O
R = Cl, Br, NO , OMe, -OH
p- p-
p-
p-
o
6-10;
2
NH
H
N
N
O
O
(II)
O
N
Linker modification
and cyclization
Ring “A” opening
R
O
N
N
Linker
N
Ac
Ac
N
N
N
N
R
Ac
Ac
R = Cl, Br, NO , OMe, -OAc
p- p- p- p-
o
12-19;
2
Figure 2. Design strategy of the target MMP-10/13 inhibitors.
In attempt to gain considerable theoretical evidence into such design at the molecular level,
molecule alignment study between the designed 2-[2-(4-methoxybenzylidene)hydrazinyl]-6-

methylpyrimidin-4-one 9, as a representative of the series, and the lead quinazolin-4-one MMP-13
inhibitor II (Figure 3C), was performed employing Molecular Operating Environment (MOE)
software version 2015.10 [44] (Figure 3D). As seen, both the lead MMP-13 inhibitor II and the
designed hydrazone derivative 9 aligned well with a similarity measure of alignment configuration (F
score) equals -137.047 and a grand alignment score (S score) equals -145.575.
A
B
4.72 Å
4.47 Å
C
Figure 3. (A) Electrostatic potential map of the lead MMP-13 inhibitor II, (B) Electrostatic potential
map of the designed 2-[2-(4-methoxybenzylidene)hydrazinyl]-6-methylpyrimidin-4-one 9, and (C)
Alignment of II (magenta) and 9 (cyan).
With the aim of exploring the influence of the hydrazone linker on possible interactions with
MMP-10, we performed docking simulations of the complexes between the designed pyrimidinone
derivative 9 and MMP-10 catalytic domain (PDB: 1Q3A [8]). Interestingly, the hydrazone moiety had
the potential to bind the catalytic zinc and form hydrogen bond with the backbone Ala181 (Figure
4A). Besides, the hydrazone as amenable chemical entity allowed expanding the chemical diversity of
the designed compounds and enriching the structure activity relationship studies through cyclization
reactions to the corresponding triazolopyrimidinone derivatives 12-19 with possible regioisomerism
(Figure 2). Such modification that allowed modifying the core of the lead MMP-13 inhibitor II was
also studied computationally to estimate the binding propensities of the designed triazolopyrimidine
derivatives to MMP-10 and -13. Among the possible regioisomers of the triazolopyrimidine
derivatives, the 3-(4-methoxyphenyl)-5-oxo-1,2,4-triazolo[4,3-a]pyrimidine derivative 18 was

selected as a representative of the series and docked to the active site of MMP-10 catalytic domain
(PDB: 1Q3A [8]). Interestingly, such cyclization allowed more favorable accommodation of the
triazolopyrimidine core in the S1´ selectivity pocket through hydrogen bond interaction with Tyr239
(Figure 4B and C). Besides, 18 fitted well in the MMP-13 active site (PDB: 456C [45]) via binding
catalytic zinc, H-π interactions with the zinc chelating residue His232, as well as His222 and hydrogen
bonding with backbone Leu185 flanking the catalytic site (Figure 4D).
A
B
C
D
Figure 4. (A) 3D binding mode of 9 (cyan sticks) in MMP-10 active site (PDB: 1Q3A [8]), (B) 3D
binding mode of 18 (yellow sticks) in MMP-10 active site (PDB: 1Q3A [8]), (C) Overlay of 9 (cyan
sticks) on 18 (yellow sticks) in MMP-10 active site (PDB: 1Q3A [8]), and (D) 3D binding mode of 18
(yellow sticks) MMP-13 active site (PDB: 456C [45]). The zinc ions appear as light blue spheres.
2.2. Chemistry

6-Methyl-2-thiouracil 1 has been selected as a precursor for the synthesis of the two scaffolds;
ethyl-2[(6-methyl-4-oxo-3,4-dihydropyrimidin-2-yl)thio]acetate and 2-hydrazinyl-6-methyl
3
pyrimidin-4-one 4. The ethyl-2[(6-methyl-4-oxo-3,4-dihydropyrimidin-2-yl)thio]acetate 3 [46] was
synthesized under conventional method in 83 % yield by stirring 6-methyl-2-thiouracil 1 with ethyl
bromoacetate in acetone and in the presence of triethylamine for 24 h. A better yield (85 %) within a
shorter reaction time (2 h) at room temperature can be obtained under ultrasound irradiation (Scheme
1).
Reaction of the thioacetate derivative 3 with hydrazine hydrate in ethanol or even methanol for 9
h under reflux afforded 2-hydrazinyl-6-methylpyrimidin-4-one 4 rather than 2-[(4-methyl-6-oxo-1,6-
dihydropyrimidin-2-yl)thio]acetohydrazide 5. Alternatively, compound 4 can be obtained by refluxing
the 6-methyl-2-methylthio-4-pyrimidinone 2 with hydrazine hydrate in 2-propanol [47]. Attempted
synthesis of 4 by reaction of 1 with hydrazine hydrate in ethanol-water mixture under reflux was
unsuccessful. However, using higher boiling butanol [48] instead of ethanol afforded the desired
product.
O
O
CH3I
Et3N / Acetone
CM or US
NH
NH
N
2
S
N
1
SH
NH2NH2.H2O
BrCH2COOEt
Et3N/Acetone
CM or US
Reflux
BuOH
NH2NH2.H2O
2-propanol, reflux
O
O
NH2NH2.H2O
EtOH, reflux
NH
NH
N
4
NHNH2
N
3
SCH2COOEt
O
NH
N
5
SCH2CONHNH2
CM = Conventional method
US = Ultrasound irradiation
Scheme 1. Synthesis of 2-hydrazinyl-6-methylpyrimidin-4-one 4.
Condensation of the 2-hydrazinyl-6-methylpyrimidin-4-one 4 with aromatic aldehydes in ethanol
and in the presence of a catalytic amount of glacial acetic acid under reflux afforded the respective
hydrazones 6-10. Due to the possibility of amide-iminol tautomerism in the pyrimidine ring, the latter
hydrazones were found to exist in solutions of DMSO-d6 as equilibrated mixture of the amide 6-10A
1
and the iminol 6-10B forms in a ratio of 9:1 as deduced from their H-NMR spectra. At the most
downfield region, a broad D2O exchangeable peak of 1.9 proton intensity at δH 11.22-11.41 ppm could
be assigned to the hydrazonyl NH and pyrimidine N-3 protons. The presence of the iminol tautomer

was shown from the signal corresponding to the OH proton assigned at δH 10.15-10.40 ppm with 0.1
intensity. Moreover, the pyrimidine proton H-5 resonated as two singlets at δH 5.50-5.52 ppm and
5.25-5.34 ppm, respectively, whereas their carbon signals resonated at δC 101.5-108.5 ppm, and this
may be due to the amide-iminol tautomerism (9:1) rather than the E/Z isomerism. Accordingly, the
amide tautomer signals showed the NH, and H-5 at higher frequency compared with that of the iminol
tautomer (OH, H-5). However, the methyl protons of the amide tautomers 6-10A were assigned at
lower frequency at δH 2.03-2.09 ppm, whereas those of the iminol tautomers 6-10B were assigned at
higher frequency at δH 2.15-2.21 ppm. Their respective carbons showed different manner where that
of the amide tautomers 6-10A resonated at δC 22.9-23.9 ppm and those of the iminol isomers were
assigned at δC 19.0-21.1 ppm, respectively (Scheme 2).
O
O
OH
ArCHO
EtOH/ AcOH
Reflux
NH
NH
N
N
Ar
N
Ar
N
N
N
4
NHNH2
N
N
H
H
H
H
A;
E
B;
E
O
6 Ar = -ClC H
p
6
4
7 Ar = -BrC H
4
p
NH
6
8 Ar = -NO C H
4
p
2
6
N
H
9 Ar = -OMeC H
p
6
4
N
N
H
10 Ar = -OHC H
o
6
4
Ar
isomer
Z
Scheme 2. Synthesis of 2-[2-(substitutedbenzylidene)hydrazinyl]-6-methylpyrimidin-4-one 6-10.
The azomethine proton showed two singlet signals at δH 7.99-8.35 and 8.12-8.42 ppm. The 9:1
ratio of these signals may be due to the tautomerism in the pyrimidine ring of the more stable E
configurational isomer. As a module, calculating the minimized energy of 6 showed that the structure
of the E configurational structure of the amide tautomer has lower minimized energy than the
corresponding Z isomer. In addition, the iminol tautomer of the E configurational structure showed
almost similar minimized energy as the corresponding Z isomer (Figure 5). Therefore, the NMR
spectra as well as the minimized energy calculations assigned the presence of the amide tautomer of
the E configurational structure and the corresponding iminol tautomer in 9:1 ratio. The aryl protons of
compounds 6, 7, 9 and 10 also resonated as two sets of signals in the same ratio 9:1, whereas those of
the nitro derivative 8 were assigned from the multiplet signal at δH 8.18-8.26 ppm.

E isomer, iminol tautomer
E isomer, amide tautomer
E = 16.5358 Kcal/mol
E = 3.3728 Kcal/mol
Z isomer, iminol tautomer
Z isomer, amide tautomer
E = 29.5952 Kcal/mol
E = 15.8503 Kcal/mol
Figure 5. Minimized energy structure for the E and Z configurational isomers of 6 for amide and
iminol tautomers using Chem 3D Pro12.0.
As proposed in the adopted design strategy, 1,2,4-triazolo[4,3-a]pyrimidine derivatives were
synthesized. Treatment of the hydrazones 6-10 with acetic anhydride under reflux was expected to
afford either a mixture of the 5-oxo-1,2,4-triazolo[4,3-a]pyrimidine and 7-oxo-1,2,4-triazolo[4,3-
a]pyrimidine regioisomers or only one regioisomer. Herein, cyclization of the hydrazones 6 or 7 with
acetic anhydride under reflux for 9 hours afforded a mixture of two isomeric products that could be
separated by column chromatography. Structure elucidation of the 7-oxo-1,2,4-triazolo[4,3-
a]pyrimidines 12 and 13 as well as the 5-oxo-1,2,4-triazolo[4,3-a]pyrimidines 15 and 16 was
1
13
confirmed by H and C-NMR spectra which proved that the kinetically controlled products N,N'-
diacetyl-3-aryl-5-methyl-7-oxo-1,2,4-triazolo[4,3-a]pyrimidine 12 and 13 as well as the

thermodynamically products N,N'-diacetyl-3-aryl-7-methyl-5-oxo-1,2,4-triazolo[4,3-a]pyrimidine 15
and 16, respectively were obtained (Scheme 3).
O
N
O
N
O
OAc
H
N
NH
N
Ac2O
-AcOH
N
Ar
Reflux
Ac
Ac
Ac
N
N
H
N
N
N
H
H
H
N
Ar
Ac
6; Ar = -ClC H
p
6
4
Ar
7; Ar = -BrC H
p
6
4
12; Ar = -ClC H
13; Ar = -BrC H
14; Ar = -NO C H
p
p
p
6
4
8; Ar = -NO C H
4
p
11
2
6
6
4
9; Ar = -OMeC H
p
6
4
2
6
4
10;Ar = -OHC H
o
6
4
Dimroth
rearrangement
H+
O
O
N
Ar
H
N
N
Ac
N
Ac
N
N
H
N
Ac
N
Ar
Ac
15; Ar = -ClC H
p
6
4
16; Ar = -BrC H
p
6
4
17; Ar = -NO C H
4
p
2
6
18; Ar = -OMeC H
p
6
4
19; Ar = -OAcC H
o
6
4
Scheme 3. Synthesis of 1,2,4-triazolo[4,3-a]pyrimidine derivatives 12-19 and the postulated
Dimroth rearrangement mechanism.
1H-NMR spectrum of 12 and 13 as well as 15 and 16 showed the presence of two N-acetyl protons
signals at δH 1.27-2.70 ppm which were assigned for both isomers. They confirmed the acetylation of
both nitrogens of 1,2,4-triazole moiety, whereas, the methyl protons at position-5(7) resonated at δH
1
13
1.97-2.34 ppm. The H and C-NMR spectra of 15 and 16 showed the H-3 protons resonating as
singlet signals at δH 7.06 ppm, while their carbons resonated at δC 74.0 and 74.1 ppm, respectively. At
lower frequency, a singlet signal corresponding to H-6 proton was observed at δH 5.15 and 5.19 ppm,
respectively, whereas their corresponding carbons C-6 resonated at δC 97.8 and 98.1 ppm, respectively.
On the other hand, the kinetically less stable separated products 12 and 13, showed some differences
throughout their NMR elucidation than the thermodynamic isomers 15 and 16. Downfield shifted H-
3 of the triazole moiety as well as H-6 of the pyrimidine ring were observed at δH 7.46-7.48 and 6.14

ppm, respectively. Alternatively, their C-6 of the pyrimidine moiety resonated at δC 109.3 ppm while
the asymmetric C-3 carbon showed upfield shift and resonated at δC 70.9 ppm in both compounds.
This postulated structure elucidation was in accordance with that reported in literature describing
identification of isomeric 1,2,4-triazolopyrimidinones[49] based on chemical shifts of their H-6, C-6
and carbonyl carbon of the pyrimidine moiety [50-52].
In the current study, cyclization regioselectivity of the triazolopyrimidine series was also
evidenced by HMBC spectra of the regioisomers 12 and 15 as representatives (Figure 6). Obviously,
H-3 of the 7-oxo-1,2,4-triazolo[4,3-a]pyrimidine 12, resonating at δH 7.48 ppm showed correlation
with C-5 at δC 147.2 ppm, while correlation with C-7 (δC 165.1 ppm) was missed. On the other hand,
H-3 of the 5-oxo derivative 15 resonated at δH 7.07 ppm, correlated to C-5 at δC 146.8 ppm and didn’t
display a correlation with C-7 (δC 151.2 ppm). Accordingly, these detected connectivities confirmed
the two isomeric structures.
B
A
O
Cl
N
O
H
Ac
N
N
N
N
H
N
Ac
Ac
N
N
Ac
15
Cl
12
Figure 6. (A) HMBC spectrum of
N,N'-diacetyl-3-(4-chlorophenyl)-5-methyl-7-oxo-1,2,4-
triazolo[4,3-a]pyrimidine 12, and (B) HMBC spectrum of N,N'-diacetyl-3-(4-chlorophenyl)-7-methyl-
5-oxo-1,2,4-triazolo[4,3-a]pyrimidine 15.

Moreover, extra signals corresponding to the thermodynamic regioisomers 15 and 16 were
assigned in a 1:9 ratio compared to the corresponding kinetic isomers 12 and 13 at almost the same
chemical shift found of the NMR of the pure separated thermodynamic isomers 15 and 16 although
both isomers were well separated and purified by column chromatography. This gave considerable
indication that the kinetically controlled isomers separated chromatographically underwent Dimroth
rearrangement to the more thermodynamically stable isomers 15 and 16, respectively.
The cyclization process can be attributed to the formation of a di-N-acetyl intermediate 11 due to
the presence of acetic anhydride. Under these reaction conditions, the di-N-acetyl intermediate 11
losses a molecule of acetic acid with subsequent cyclization because of the nucleophilic attack of N-1
of pyrimidine moiety to the azomethine carbon forming the kinetically controlled isomer 12 or 13 as
the major product. This regioisomer was then rearranged by the aid of the acid present in the medium
to the more thermodynamically stable Dimroth product 15 or 16 as a minor one. Thus, under the acidic
medium of the reaction, Dimroth rearrangement [53] has taken place according to the postulated
mechanism (Scheme 3).
From our observations, the chromatographically separated derivatives 12 and 13 were rearranged
by time in presence of light (photoirradiation) to the thermodynamically more stable products 15 and
16, respectively. Therefore, exposure of a solution of 12 or 13 in ethanol to light for 56-63 h afforded
the rearranged pure products 15 or 16, respectively, which were confirmed by TLC monitoring
followed by NMR spectroscopy. However, no changes were observed when 12 and 13 were irradiated
using ultrasound in dark within time ranges from 5 to 120 minutes at room temperature in the presence
of polar or non-polar solvent such as ethanol and dichloromethane, respectively. Under these
circumstances, Dimroth rearrangement could take place by photoirradiation of the triazolopyrimidine
derivatives 12 and 13 to give their respective thermodynamically stable regioisomers 15 and 16,
respectively (Scheme 4).
O
N
O
N
O
N
Ar
N
N
Light
N
Ac
N
Dimroth
rearrangement
Ac
N
Ac
N
N
Ac
N
N
Ar
Ar
Ac
Ac
12; Ar = -ClC H
p
15; Ar = -ClC H
p
6 4
6
4
13; Ar = -BrC H
p
16; Ar = -BrC H
p
6 4
6
4
14; Ar = -NO C H
p
17; Ar = -NO C H
p
2 6 4
2
6
4
Scheme 4. Postulated mechanism of Dimroth rearrangement in presence of light for 7-oxo-1,2,4-
triazolo[4,3-a]pyrimidine.
The nitro derivative 8 was treated similarly with acetic anhydride for 12 h, to afford a mixture of
the two isomers (14 and 17) in a ratio 9:1 as that confirmed from their 1H and 13C-NMR spectra. The

1H-NMR spectrum of 14 showed two singlet peaks at δH 7.58 and 7.15 ppm corresponding to the H-3
of the kinetically regioisomer 14 and the thermodynamically regioisomer 17, respectively in 9:1 ratio,
13
while the H-6 proton resonated at δH 6.17 and 5.21 ppm, respectively in the same ratio. C-NMR
spectrum also confirms the presence of both isomers where two singlet signals corresponding to C-3
resonated at δC 70.3 and 73.6 ppm, respectively while C-6 resonated at δC 109.3 and 98.4 ppm,
respectively.
Most remarkable, when the nitro derivative 14 was exposed to light for 63 h, a mixture of both
isomers was obtained in ratio 2:3 (kinetic isomer: thermodynamic isomer). 14 didn’t rearranged
completely to the thermodynamic isomer 17 although it was the same time required for the chloro and
bromo derivatives to be rearranged completely to the more stable thermodynamic isomer via Dimroth
rearrangement.
In contrast, under the same reaction conditions refluxing 9 or 10 with acetic anhydride afforded
only the thermodynamically stable N,N'-diacetyl-3-aryl-7-methyl-5-oxo-1,2,4-triazolo[4,3-
a]pyrimidine derivatives 18 or 19 rather than 7-oxo-1,2,4-triazolo[4,3-a]pyrimidine analogues. The
1
structures of these compounds were confirmed by NMR spectra. Where the H-NMR spectra of 18
and 19 showed the presence of a singlet signal corresponding to the H-3 proton resonating at δH 6.97
and 7.11 ppm, respectively, whereas their corresponding carbons resonated at δC 74.3 and 72.4 ppm,
respectively. At upfield region, the H-6 pyrimidine proton resonated at δH 5.08 and 5.15 5.52 ppm,
respectively, while their carbons resonated at δC 98.4 and 97.9 ppm, respectively. The methyl group at
position 7 was assigned as a singlet signal at δH 2.04 and 2.19 ppm for 18 and 19, respectively.
Moreover, all these compounds showed two N-acetyl protons signals at δH 2.00-2.31 ppm. Compound
19 showed an extra signal at δH 2.32 ppm because of acetylation of the phenolic OH group.
From the previous results, it was concluded that cyclization via acetic anhydride of 2-hydrazonyl
pyrimidine containing electron donating group as the methoxy group in 9 or the hydroxyl group as in
10 gave only the pure more stable 5-oxo-1,2,4-triazolo[4,3-a]pyrimidine regioisomers 18 and 19. On
the other hand, electron withdrawing groups as p-chloro, p-bromo or p-nitro group facilitates the
formation of the 7-oxo-1,2,4-triazolo[4,3-a]pyrimidine analogue as a major product in addition to
the thermodynamically stable 5-oxo-1,2,4-triazolo[4,3-a]pyrimidine isomer.
2.3. MMPs inhibitory activity and ligand efficiency metrics
IC50 values of all the newly synthesized pyrimidine derivatives were determined on MMP-10
and -13 in comparison to N-Isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid (NNGH)
as a reference MMP inhibitor (Table 1). Interestingly, all compounds exhibited higher MMP-10
inhibitory activities (i.e. selectivity) in comparison to MMP-13, except for compound 6 displaying
similar potencies against both enzymes. The most potent MMP-10 inhibitor was compound 16 (IC50
= 24 nM), followed by 9 (IC50 = 53 nM), 4 (IC50 = 62 nM) and 15 (IC50 = 93 nM). The four
compounds were more active than NNGH against MMP-10. 8 and 18 were nearly equipotent (IC50
≈ 130 nM), while other compounds displayed moderate to weak MMP-10 inhibition with IC50 values
ranging from 223 to 1633 nM. On the other hand, the most potent MMP-13 inhibition was detected

for compound 18 (IC50 = 294 nM) which was also more potent than the reference NNGH.
Interestingly, 9 was comparable to NNGH and slightly less active than 18 (IC50 = 354 nM), followed
by 19 and 16 exhibiting almost similar IC50 values (IC50 = 441 and 460 nM, respectively), while the
remainder compounds were relatively weak MMP-13 inhibitors (IC50 = 538-7777 nM).
With the goal of further exploring the potencies of the most active MMP-10 and -13 inhibitors
against other members of the MMPs family, their IC50 values were determined on MMP-7 and -9.
Obviously, both 16 and 18 were more selective against MMP-9 compared to MMP-7, with 18 (IC50
= 146 and 397 nM, respectively) exhibiting higher inhibitory activities than 16 (IC50 = 251 and 711
nM, respectively). Considering the obtained results, compound 16 exhibited more than 19-fold
selectivity for MMP-10 over MMP-13, 10-fold selectivity over MMP-9 and 29-fold selectivity over
MMP-7.
Table 1: In vitro MMPs inhibition and ligand efficiency metrics of the synthesized compounds
MMP-13
LEb
MMP-10
LE
MMP-9
LE
MMP-7
LE
Cpd.
No.
IC50
(nM)
538
IC50
(nM)
62
IC50
(nM)
IC50
(nM)
a
pIC5
LLEc
LELPd
pIC50
LLE
LELP
pIC50
LLE
LELP
pIC50
LLE
LELP
4
6
7
8
9
6.269
5.878
5.866
6.092
6.450
5.920
5.734
5.109
6.085
6.337
6.120
6.531
6.355
-
0.858
0.447
0.446
0.417
0.465
0.450
0.327
0.291
0.347
0.361
0.322
0.357
0.322
-
6.809
3.688
3.546
4.622
4.880
4.470
4.504
3.749
4.855
4.977
5.610
5.931
6.305
-
-0.617
4.899
5.201
3.525
3.376
3.222
3.761
4.673
3.544
3.767
1.583
1.686
0.155
-
7.207
5.869
6.197
6.866
7.275
5.933
6.425
5.787
7.031
7.619
6.308
6.886
6.651
-
0.986
0.446
0.478
0.470
0.524
0.451
0.366
0.330
0.401
0.434
0.332
0.377
0.337
-
7.747
3.679
3.877
5.396
5.705
4.483
5.195
4.427
5.801
6.259
5.798
6.286
6.601
-
-0.547
4.190
4.853
3.127
2.996
3.215
3.360
4.121
3.067
3.133
1.536
1.591
0.148
-
-
-
-
-
-
-
-
-
-
-
1323
1360
809
1349
634
136
53
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
354
-
-
-
-
-
-
-
-
-
-
1201
1845
7770
822
1166
375
1633
93
-
-
-
-
-
-
-
-
-
-
10
12
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
13
15
-
-
-
-
-
-
-
-
-
-
16
460
24
251
-
6.600
0.376
5.24
3.617
711
-
6.148
0.350
4.788
3.885
17
757
492
130
223
101
-
-
-
-
-
-
-
-
18
294
146
-
6.835
0.374
6.253
1.604
397
-
6.401
0.350
5.801
1.714
19
441
-
-
-
-
-
-
-
-
NNGH
360
73
-
-
-
-
240
-
-
-
-
apIC50: negative logarithm of half-maximal inhibitory concentration (in molar concentration).
bLE: ligand efficiency.
cLLE: lipophilic ligand efficiency.
dLELP: ligand efficiency-dependent lipophilicity index.
Moreover, various ligand efficiency metrics were computed for comparing, prioritizing and
assessing the drug likeness of the synthesized compounds. Firstly, ligand efficiency (LE) was
calculated for each compound as reported [54]. This metric represents the balance between potency
and size as it reflects the average binding affinity per heavy atom instead of the whole molecule
[54,55]. All the evaluated compounds succeeded to pass the acceptable limit (0.3) [56] against all the
tested MMPs, except for compound 13 which was slightly beyond it in case of MMP-13. While
comparing molecules, LE normally prioritizes small compounds with relatively lower potencies rather
than larger higher potency ones [57]. It comes as no surprise then that compound 4 recorded the highest
LE values, even superior to the most active compounds, due to its relatively lower molecular size.
Secondly, a broader metric termed liphophilic ligand efficiency (LLE) was evaluated to provide a
straightforward link between potency and lipophilicity through reflecting the price paid in lipophilicity

on expense of potency [57,58]. LLE is defined as the difference of log P and the negative logarithm
of potency measure (pIC50). Applying the reported equation [57], again all the studied compounds
fulfilled the optimum LLE value for lead molecules (LLE ≥ 3) [56] against all the evaluated MMPs.
Moreover, compounds 4, 17, 18 and 19 could be considered as drug-like candidates (LLE ≥ 5) [56]
against MMP-13. Results of MMP-10 evaluation were more promising, where most compounds (4, 8,
9, 12, 15, 16, 17, 18 and 19) recorded drug-like LLE values.
Although LE and LLE are widely employed ligand efficiency metrics, both have been subjected
to criticism, where LE fails to take lipophilicity into account and LLE is not size adjusted [57]. A new
descriptor combining molecular size, lipophilicity and potency into one composite metric was thus
introduced as the ligand efficiency-dependent lipophilicity index (LELP) and defined as the ratio of
log P and LE [56]. Studies showed that drug candidates with suboptimal LELP fail to proceed in the
development pipeline [59]. As reported the optimal LELP range is -10<LELP<10 [56]. Accordingly,
all the synthesized compounds displayed acceptable LELP values against MMP-10 and -13.
2.4. Anticancer evaluation
In light of the aforementioned MMP-10/13 inhibition results, the most active MMP inhibitors 16
and 18 were evaluated for their anticancer potential to assess their biological activities at cellular level.
Herein, the compounds under investigation were tested for cytotoxic activities against three human
cancers namely; triple negative breast cancer (MDA-MB 231), liver cancer (HepG-2), and colon cancer
cell (Caco-2) compared to quercetin as a reference anticancer MMP inhibitor utilizing MTT assay [40]
(Table 2).
Table 2: Cytotoxicity of the most active MMP-10/13 inhibitors.
IC50 (µM)
Cpd. No.
MDA-MB231
HepG-2
Caco-2
16
18
0.256±0.012
0.290±0.014
0.283±0.001
0.240±0.0004 0.235±0.002
0.262±0.002 0.256±0.0008
0.278±0.001 0.277±0.00002
Quercetin
Both 16 and 18 displayed potent anticancer activities superior or comparable to that of quercetin
against the three cell lines. 16 was slightly more active than 18 against all the screened cells. Caco-2
cells may be considered as the most sensitive cell line to the tested compounds. Interestingly, these
results enrich the therapeutic potential of the newly synthesized compounds.
MDA-MB231, HepG-2 and Caco-2 cells were also examined morphologically after treatment with
16 and 18 in comparison with quercetin-treated cells and the untreated cancer cells (Figure 7). The
cancer cells treated with 16 and 18 lost their characteristic shape. Additionally, their shrinkage
reflected the apoptotic potential of the tested compounds in comparison to quercetin.

Figure 7. Morphological alterations of the most active derivatives-treated cancer cell lines.
2.5. In silico physicochemical properties, drug-likeness and ADME
Recently drug discovery programs involve computational prediction of physicochemical and
ADME properties as useful lead identification and optimization tool. In this study, the
physicochemical properties formulating Lipinski’s rule [60] were firstly computed for the most active
MMP 10/13 inhibitors utilizing Molinspiration software [61] (Table 3). According to Lipinski, oral
bioavailability is possible if the following rules are obeyed, log P ≤ 5, molecular weight ≤ 500 Da,
hydrogen bond acceptors ≤ 10 and hydrogen bond donors ≤ 5. Interestingly, the selected compounds
16 and 18 were in full accordance to Lipinski’s parameters.
Molinspiration [61] was also employed to calculate topological polar surface area (TPSA) as
additional bioavailability descriptor [62] that is utilized to calculate the estimated % absorption
[63,64]. Herein, compounds 16 and 18 displayed drug-like TPSA values. (<140–150 A2) [65,66] and
reasonable % ABS (around 80 %), predicting promising oral bioavailability.
Table 3: In silico physicochemical properties, drug-likeness and ADME data of the
most active compounds
Cpd.
No.
Log
Pa
Lipinski’s
Violation
Volumes
(A)3
Sg
(mg/L)
Drug-
likeness
CYP3A4
Inhibition
CYP2D6
inhibition
M.Wtb
HBAc
HBDd
TPSAe
%ABSf
Caco2h
MDCKi
HIAj
BBBk
PPBl

model
score
0.22
1.36
0.60
391.23
342.36
7
8
0
0
0
0
75.52
84.75
82.94
79.76
292.74
300.40
1146.62
7788.34
19.473
25.855
0.492
2.171
98.579
98.044
0.356
0.176
80.494
72.069
Non
Non
Non
Non
16
18
0.09
aLog P: logarithm of compound partition coefficient between n-octanol and water.
bM.Wt: molecular weight.
cHBA: number of hydrogen bond acceptors.
dHBD: number of hydrogen bond donors.
eTPSA: polar surface area. Drug-like TPSA < 140–150 A2 [65,66].
f%ABS: percentage of absorption.
gS: aqueous solubility.
hCaco2: permeability through cells derived from human colon adenocarcinoma. PCaco2 values < 4 nm/sec
(low permeability), values ≈ 4 - 70 nm/sec (medium permeability) and values > 70 nm/sec (high
permeability) [67-69].
iMDCK: permeability through Madin-Darby Canin kidney cells. PMDCK values < 25 nm/sec (low
permeability), values ≈ 25 – 500 nm/sec (medium permeability) and values > 500 nm/sec (high
permeability) [68].
jHIA: human intestinal absorption. HIA values < 20% (poorly absorbed), values ≈ 20-70% (moderately
absorbed) and values > 70% (well bound) [70].
kBBB: blood-brain barrier penetration. BBB values < 0.1 (low CNS absorption), values ≈ 0.1-2
(medium CNS absorption) and values > 2 (high CNS absorption) [71].
lPPB: plasma protein binding. PPB values < 90% (poorly bound) and values > 90% (strongly bound)
[72].
Aqueous solubility of the investigated compounds 16 and 18 as well as their drug-likeness scores
(Table 3) were predicted utilizing Molsoft software [73]. Herein, both compounds recorded excellent
predicted solubility (1.1 and 7.7 g/L, respectively) and drug-likeness model scores, indicating their
promising drug-like potential.
Furthermore, Pre-ADMET software [72] was used for ADME prediction of the selected
compounds 16 and 18. Accordingly, CaCo2 and MDCK cell permeability coefficients, human
intestinal absorption (HIA), blood-brain barrier penetration (BBB), plasma protein binding (PPB), as
well as inhibition to cytochromes P450 2D6 (CYP2D6) and P450 3A4 (CYP3A4) were computed and
listed in Table 3. Both 16 and 18 displayed medium CaCo2 cell model permeability values (19.47 and
25.85 nm/sec, respectively), and low MDCK cell model ones (0.49 and 2.17 nm/sec, respectively).
Their HIA (> 98%) and BBB (0.35 and 0.17, repectively) values demostrated excellent predicted
intestinal absorption and acceptable CNS bioavailability of both compounds. Additionally, both 16
and 18 were predicted to be devoid of the undesirable CYP3A4 and CYP2D6 inhibition actvities,
hence potential drug-drug interactions are most probably excluded.
2.6. Structure-activity relationship
The general MMP-10/13 inhibition pattern revealed that the designed pyrimidine and triazolo[4,3-
a]pyrimidine scaffolds conserved the intrinsic activity of the parent lead inhibitors (Figure 1).

However, the inhibition profile regarding potency and selectivity was found to be a function of
substitution pattern and regioisomerism (Figure 8). As expected, the p-methoxy substituted hydrazone
9 showed acceptable MMP-13 inhibitory activity and was evaluated as the most potent derivative
among its series. Fortunately, it exhibited excellent superior MMP-10 inhibition (7 folds) and was also
the most active MMP-10 inhibitor among the synthesized hydrazones. Other hydrazones didn’t exhibit
considerable MMP-10/13 inhibition activities, whereas the hydrazinyl precursor 4 displayed
promising MMP-10 inhibition while maintaining moderate activity against MMP-13.
With the goal of expanding the chemical diversity and mimicking the fused pyrimidine lead MMP-
10/13 inhibitors, hydrazones were cyclized to afford the corresponding triazolopyrimidine derivatives.
Interestingly, such modification, in most cases, enhanced MMPs inhibitory activities, where the most
active compounds in this study were detected among the triazolopyrimidine series. The
methoxyphenyltriazolopyrimidine derivative 18 was the most potent MMP-13 inhibitor, while the
bromophenyl-5-oxo-1,2,4-triazolo[4,3-a]pyrimidine 16 exhibited the most potent MMP-10 inhibitory
activity. Seemingly, all the promising compounds share enhanced MMP-10 selectivity over MMP-13.
Furthermore, a clear structure activity relationship trend was observed within the identified 1,2,4-
triazolo[4,3-a]pyrimidine regioisomers, where the triazolopyrimidine derivatives 15 and 16 were far
more active more than their regiosiomers 12 and 13 against MMP-10 and -13. Such variation in
potency was better disclosed when comparing the MMP-10 inhibition of the p-bromophenyl
regioisomers 13 and 16 rather than that of the p-chloro isomers. As obvious, the bromo derivative 16
was 68-fold more active than its regioisomer 13, whereas the p-chloro derivative 15 was only 4-fold
more active than its regioisomer 12. Similar comparisons regarding MMP-13 activities revealed lower
activity differences. Hence, this observation could point to possible correlation between the substituent
size and the overall potency especially against MMP-10, where the bromo substitution conferred
higher MMP inhibition than the chloro substitution. Within the triazolopyrimidine series, the p-
bromophenyl derivative 16 exhibited the highest MMP-10 inhibitory activity, followed by the p-choro
15, p-methoxy 18, o-acetoxy 19 and p-nitro 17 derivatives, respectively. On the other hand, different
trend was observed against MMP-13, where the p-methoxy derivative 18 was the most potent
inhibitor, followed by the o-acetoxy 19, p-bromo 16, p-nitro 17 and p-choro 15 derivatives,
respectively.
R
O
N
Ac
N
N
N
Ac

15-19
MMP-10 inhibition:
R= p-Br> p-Cl>p-OCH3>o-OAc>p-NO2
MMP-13 inhibition:
R=p-OCH3>o-OAc>p-Br>p-NO2>p-Cl
Activity↑↑
6-10
MMP-10 inhibition:
R=p-OCH3>p-NO2>>p-Br>o-OH>p-Cl
MMP-13 inhibition:
R=p-OCH3>>p-NO2>o-OH>p-Cl>p-Br
121,21, 313
MMP-10 inhibition:
R= p-Cl > p-Br
MMP-13 inhibition:
R= p-Cl > p-Br
Figure 8. Schematic representation of the general structure-activity relationship data.
2.7. Docking
Docking studies were performed by MOE 2015.10 [44] to provide reasonable explanation for the
structure-activity relationships observed among the triazolopyrimidine regioisomers, to explore the
possible binding modes of the active isomers, and to possibly justify MMP-10 selectivity over MMP-
13.

The coordinates of MMP-10 and -13 catalytic domains were obtained from their crystal structures;
PDB ID: 1Q3A [8] and PDB ID: 456C [45], respectively. The optimized domain consisted of a MMP-
10 monomer, two zinc and three calcium ions (Figure 9). Docking was validated and conducted
several times to select the best interactions and binding energies. The most potent MMP-10 inhibitor,
compound 16, chelated the catalytic zinc ion via the triazolopyrimidine core C=N (N-8) and the nearby
acetyl oxygen. Additionally, the core carbonyl oxygen exhibited hydrogen bonding with Ser179
residue that is known to bind one of the calcium ions at the catalytic site. Probably, these interactions
allowed possible contact between the bromophenyl moiety and the S1´ hydrophobic selectivity pocket,
adjacent to the catalytic site, through H-π interactions with Tyr239. Regarding the relative orientation
of the corresponding inactive regioisomer 13 in the active site of MMP-10 with respect to 16, it is
clearly obvious that 13 failed to occupy similar spatial area. This may be due to lacking interactions
with the active sites except for single interaction with the zinc ion. A postulated explanation for such
observation may be based on the relative position of the core carbonyl group with respect to the p-
bromophenyl moiety. As seen in Figure 9, proximity of the core carbonyl group to the p-bromophenyl
moiety in compound 16 allowed favorable accommodation of triazolopyrimidine core into the active
site. On the other hand, the triazolopyrimidine core of compound 13 was not properly positioned when
the carbonyl group is located relatively far from the p-bromophenyl group.
Similar trend was observed when comparing docking simulations of the two regioisomers 16 and
13 complexed in the binding site of MMP-13 (PDB: 456C) [45], but with relatively less interactions
than that observed with MMP-10. Herein, 16 displayed only H-π interactions with Thr 245, whereas
13 didn’t show considerable interactions. With these observations, it seems that MMP-10 selectivity
may be attributed to more favorable interactions, especially those posed by its S1´ selectivity pocket,
with the active derivative 16 compared to the interactions offered by MMP-13. Interestingly, docking
simulations were consistent with MMP-10/13 inhibition assays and may reasonably explain the
questionable structure-activity relationship data.

B
A
C
E
D
F
Figure 9. (A) 3D binding mode of 16 (green sticks) in MMP-10 active site (PDB: 1Q3A [8]), (B) 2D
binding mode of 16, (C) 3D binding mode of 13 (orange sticks) in MMP-10 active site (PDB: 1Q3A
[8]), (D) 2D binding mode of 16, (E) 3D binding mode of 16 (green sticks) in MMP-13 active site
(PDB: 456C [45]), and (F) 2D binding mode of 16. The zinc ions are shown as light blue spheres.

3. Conclusion
The current work portrays a structure optimization study of pyrimidine- and fused pyrimidine-
based MMP-10/13 inhibitors towards targeted polypharmacology. Starting with a lead quinazolin-4-
one MMP-13 inhibitor with poor activity against MMP-10, we set up a structure-based design strategy
to possibly enhance activity against MMP-10 in parallel with MMP-13. Accordingly, a series of 6-
methylpyrimidin-4-one hydrazones 6-10 was synthesized utilizing conventional method and
ultrasound irradiation, then evaluated for potential MMP-10/13 inhibition. The p-methoxy substituted
hydrazone 9 was the most active dual MMP-10/13 inhibitor among its series showing acceptable
MMP-13 inhibition (IC50 = 354 nM) and excellent superior (7 folds) MMP-10 inhibition (IC50 = 53
nM). With the aim of gaining access to fused pyrimidine derivatives, modifying the core, expanding
the chemical diversity and enriching the structure-activity relationship study, the synthesized
hydrazones were cyclized to the corresponding isomeric 1,2,4-triazolo[4,3-a]pyrimidines 12-19.
MMP-10/13 inhibition assay showed promising results where most triazolopyrimidines showed
superior dual inhibitory activities with observed MMP-10 selectivity compared to their hydrazone
precursors. Interestingly, a clear structure activity relationship trend was observed within the identified
1,2,4-triazolo[4,3-a]pyrimidine regioisomers, where the 5-oxo-1,2,4-triazolo[4,3-a]pyrimidine
derivatives 15 and 16 were far more active than their regioisomers 12 and 13 against MMP-10 and -
13. Docking simulations displayed the possible orientations of different regioisomers (13 and 16) in
the active sites of MMP-10 and -13, thus providing possible explanation of the observed structure-
activity relationships at the receptor level. Such finding guided our research protocol to further
structure optimization via photoirradiation-mediated Dimroth rearrangement of the inactive
triazolopyrimidines 12 and 13 to their potent regioisomers 15 and 16. Based on the observed results,
the most potent MMP-10 inhibitor was the bromophenyl-5-oxo-1,2,4-triazolo[4,3-a]pyrimidine 16
(IC50 = 24 nM), whereas the p-methoxy derivative 18 was the most potent MMP-13 inhibitor (IC50 =
294 nM) among the synthesized compounds. With the goal of exploring their selectivity profiles
against other members of the MMPs family, namely MMP-7 and-9, compound 16 exhibited more than
19-fold selectivity for MMP-10 over MMP-13, 10-fold selectivity over MMP-9, and 29-fold
selectivity over MMP-7. MMP-10 selectivity over MMP-13 was also explained computationally via
postulated docking simulations. Furthermore, the most promising MMP inhibitors 16 and 18 exhibited
superior anticancer activities against MDA-MB 231, HepG-2, and Caco-2 cell lines compared to
quercetin. Finally, there in silico predicted ligand efficiency metrics, drug-likeness data,
physicochemical properties and ADME parameters were remarkably good. With this data, it could be
concluded that 4-bromophenyl-5-oxo-1,2,4-triazolo[4,3-a]pyrimidine 16 represents a promising
starting point for developing dual MMP-10/13 inhibitors with enhanced MMP-10 activity towards
targeted polypharmacology. Chemical modifications may be directed to provide additional
interactions with the selectivity pocket of MMP-10 to possibly enhance isoform selectivity. Also,
introducing various zinc-binding groups [74] to the designed scaffolds will possibly improve potency.
However, balancing selectivity and potency must be carefully considered [75].
4. Experimental

4.1. Chemistry
General Methods and Instruments
All Melting points were determined using Mel-Temp apparatus and are uncorrected. Thin Layer
Chromatography (TLC) was conducted on silica gel (Kiesel gel G, Merk) and spots were detected
under UV light at 254 nm. Irradiation under ultrasound was established using Ultrasonic Cleaner
model UD50SH-2LQ. IR spectra were recorded for the compounds in a KBr matrix with a Bruker
Tensor 37 FTIR spectrophotometer. 1H- NMR and 13C-NMR spectra were recorded by Bruker DRX
400, Faculty of Pharmacy, Cairo University, Egypt and JEOL ECA500, NMR unit, Mansoura
University, Egypt. HMBC spectra were recorded by Bruker DRX 400, Faculty of Pharmacy,
Mansoura University, Egypt. Chemical shifts (δ) are given in ppm using tetramethyl silane as a
reference, the data are described as; s = singlet, bs = broad singlet, d = doublet, t = triplet, q =
quartet, dd = doublet of doublet, m = multiplet. Microanalysis was performed at Konstanz
University, Germany and at the Chemistry Department, Faculty of Science, El Azhar University,
Egypt.
6-Methyl-2-methylthio-4-pyrimidinone 2.
A mixture of 2-mercapto-6-methylpyrimidin-4-one 1 (0.142 g, 1 mmol) and triethylamine (0.2 mL) in
acetone (20 mL) was stirred for 15 minutes; methyl iodide (0.09 mL, 1.5 mmol) was added on the
reaction mixture and continue stirring for 24 h at room temperature or by using ultrasonic irradiation
at room temperature in 15 minutes. The precipitate formed was filtered off, washed with water and
dried, then recrystallized from absolute ethanol, it was obtained as colorless crystals in 83 % yield; m.
p. 230-231 °C (Lit. [76] m. p. 228 °C), RF = 0.43 (Ethyl acetate/ n-Hexane 2:1).
Ethyl 2-[(6-methyl-4-oxo-3,4-dihydropyrimidin-2-yl)thio]acetate 3.
A mixture of 4-hydroxy-2-mercapto-6-methyl pyrimidine 1 (2.24 g, 15.7 mmol) and triethylamine
(2.4 mL, 17.2 mmol) in acetone (50 mL) was stirred for 15 minutes; ethyl bromoacetate (1.9 mL, 17.1
mmol) was added on the reaction mixture and continue stirring for 24 h at room temperature or by
using ultrasonic irradiation at room temperature in 120 minutes. The white precipitate formed was
filtered off, washed with water and dried, then recrystallized from absolute ethanol, it was obtained as
colorless crystals in 83.4 % yield; m. p. 142 °C (Lit. [46] m. p. 140 °C), RF = 0.58 (Ethyl acetate/ n-
1
Hexane 2:1). IR (KBr) υ (cm-1): 3452 (NH), 1739 (COOEt), 1654 (CONH). H-NMR (CDCl3, 400
MHz) δH ppm: 1.29 (t, 3 H, J = 7.2 Hz, CH3CH2), 2.25 (s, 3 H, CH3), 3.96 (s, 2 H, SCH2), 4.23 (q, 2
H, J = 7.2 Hz, CH2CH3), 6.09 (s, 1 H, H-5 pyrimidine), 10.96 (bs, 1 H, D2O exchangeable, NH). 13C-
NMR (CDCl3, 100 MHz) δC ppm: 14.1 (CH3CH2), 23.8 (CH3), 32.8 (SCH2), 61.9 (OCH2), 108.4 (C-
5 pyrimidine), 159.2 (C-6), 165.5 (C-2), 165.6 (C-4), 168.2 (C2H5OC=O).
2-Hydrazinyl-6-methylpyrimidin-4-one 4.

A mixture of compound 3 (0.9 g, 3.9 mmol) and hydrazine hydrate (0.24 mL, 5 mmol) in ethanol (30
mL) was refluxed for 9 h in presence of a catalytic amount of glacial acetic acid. The white precipitate
formed was filtered off, washed with ether and dried. Recrystallized from ethanol/ H2O. It was
obtained as a white precipitate in 73 % yield; m. p. 250 °C (decomp.) (Lit. [47] m. p. > 235 °C
(decomp.)), RF = 0.73 (Acetone/ H2O 11.5:0.5). IR (KBr) υ (cm-1): 3336 (NH), 1634 (CONH). 1H-
NMR (DMSO-d6, 400 MHz) δH ppm: 2.01 (s, 3 H, CH3), 2.03 (s, 2 H, D2O exchangeable, NH2), 5.38
(s, 1 H, H-5 pyrimidine), 9.06 (bs, 2 H, D2O exchangeable, 2 × NH). 13C-NMR (DMSO-d6, 100 MHz)
δC ppm: 23.8 (CH3), 100.3 (C-5), 157.5 (C-2, C-6), 163.0 (C-4).
General procedure for the preparation 2-[2-(4- or 2- substituted benzylidene)hydrazinyl]-6-
methylpyrimidin-4-one 6-10.
Method A
A mixture of compound 4 (1 mmol) and aryl aldehyde (1 mmol) was refluxed in ethanol (30 mL) for
8-12 h in the presence of a catalytic amount of glacial acetic acid to afford the corresponding aromatic
hydrazones 6-10 which were filtered off, washed with ethanol and dried, then recrystallized from
absolute ethanol.
Method B
A mixture of compound 4 (1 mmol) and aryl aldehyde (1 mmol) was heated in ethanol (30 mL) at 70-
75 °C under ultrasound irradiation for 30-45 minutes in the presence of a catalytic amount of glacial
acetic acid to afford the corresponding aromatic hydrazones 6-10 which were filtered off, washed with
ethanol and dried, then recrystallized from absolute ethanol.
E-2-[2-(4-Chlorobenzylidene)hydrazinyl]-6-methylpyrimidin-4-one 6.
Yield 84 % (method A) and 87 % (method B), m. p. 248-251 °C (Lit. [46] m. p. 280 °C), RF = 0.61
(Ethyl acetate/ n-Hexane 4:1).IR (KBr) υ (cm-1): 3127, 3037 (2 X NH), 1659 (CONH), 1579
(CH=N).1H-NMR (DMSO-d6,400 MHz) δH ppm: 2.03, 2.07 (2 s, 2.7 H, CH3 6A), 2.18 (s, 0.3 H, CH3
6B), 5.29 (s, 0.1 H, H-5 6B), 5.52 (s, 0.9 H, H-5 6A), 7.46 (d, 2 H, J = 8.8 Hz, Ar-H, 6A), 7.94 (d, 0.2
H, Ar-H, 6B), 8.00 (d, 1.8 H, J = 8.4 Hz, Ar-H, 6A), 8.04 (s, 0.9 H, CH=N, 6A), 8.18 (s, 0.1 H, CH=N,
6B), 10.25 (s, 0.1 H, D2O exchangeable, OH 6B), 11.30 (bs, 1.9 H, D2O exchangeable, NH's 6A and
NH 6B). 13C-NMR (DMSO-d6,100 MHz) δC ppm: 19.2 (CH3 6B), 22.9 (CH3 6A), 101.9 (C-5), 128.9,
129.6, 133.9, 134.3 (Ar-C), 143.6 (CH=N), 152.9 (C-2, C-6), 162.9 (C-4). Anal. Cal. For
C12H11ClN4O: C, 54.87, H, 4.22, N, 21.33. Found: C, 55.19, H, 4.45, N, 21.08.
E-2-[2-(4-Bromobenzylidene)hydrazinyl]-6-methylpyrimidin-4-one 7.

Yield 75 % (method A) and 77 % (method B), m. p. 247-251 °C, RF = 0.4 (CH2Cl2/ CH3OH 9.5:0.5).
IR (KBr) υ (cm-1): 3128, 3037 (2 X NH), 1659 (CONH), 1602 (CH=N).1H-NMR (DMSO-d6, 400
MHz) δH ppm: 2.03, 2.07 (2 s, 2.7 H, CH3 7A), 2.18 (s, 0.3 H, CH3 7B), 5.29 (s, 0.1 H, H-5 7B), 5.52
(s, 0.9 H, H-5 7A), 7.59 (d, 2 H, J = 8.4 Hz, Ar-H 7A), 7.86 (d, 0.2 H, Ar-H 7B), 7.93 (d, 1.8 H, J =
8.4 Hz, Ar-H 7A), 8.02 (s, 0.9 H, CH=N, 7A), 8.16 (s, 0.1 H, CH=N, 7B), 10.25 (s, 0.1 H, D2O
exchangeable, OH 7B), 11.29 (bs, 1.9 H,D2O exchangeable, NH's 7A and NH 7B). 13C-NMR (DMSO-
d6, 100 MHz) δC ppm: 19.2 (CH3 7B), 22.9 (CH3 7A), 102.0 (C-5), 123.1, 129.8, 131.8, 134.3 (Ar-C),
143.7 (CH=N), 152.9 (C-2, C-6), 162.9 (C-4). Anal. Cal. For C12H11BrN4O: C, 46.93, H, 3.61, N,
18.24. Found: C, 47.21, H, 3.80, N, 18.49.
E-2-[2-(4-Nitrobenzylidene)hydrazinyl]-6-methylpyrimidin-4-one 8.
Yield 72 % (method A) and 75 % (method B), m. p. 282-284 °C, RF = 0.46 (CH2Cl2/ CH3OH 9.5:0.5).
1
IR (KBr) υ (cm-1): 3358, 3126 (2 X NH), 1662 (CONH), 1570 (CH=N). H-NMR (DMSO-d6, 400
MHz) δH ppm: 2.03, 2.09 (2 s, 2.7 H, CH3 8A), 2.21 (s, 0.3 H, CH3 8B), 5.34 (s, 0.1 H, H-5 8B), 5.55
(s, 0.9 H, H-5 8A), 8.16 (s, 1 H, CH=N 8A), 8.21-8.26 (m, 4 H, Ar-H 8A, 8B), 10.40 (s, 0.1 H, D2O
exchangeable, OH 8B), 11.41 (bs, 1.9 H, D2O exchangeable, NH's 8A and NH 8B). 13C-NMR (DMSO-
d6, 100 MHz) δC ppm: 21.1 (CH3 8B), 23.9 (CH3 8A), 108.5 (C-5), 124.0, 128.7, 128.8, 129.3, 134.7
(Ar-C), 147.8 (CH=N), 158.7 (C-6), 162.93, 162.96 (C-2, C-4). Anal. Calc. for C12H11N5O3: C, 52.75,
H, 4.06, N, 25.63. Found: C, 52.53, H, 4.24, N, 25.87.
E-2-[2-(4-Methoxybenzylidene)hydrazinyl]-6-methylpyrimidin-4-one 9.
Yield 80 % (method A) and 79 % (method B), m. p. 216-218 °C (Lit. [46] m. p. 220 °C), RF = 0.55
1
(CH2Cl2/ CH3OH 9:1). IR (KBr) υ (cm-1): 3388, (NH), 1663 (CONH), 1511 (CH=N). H-NMR
(DMSO-d6, 400 MHz) δH ppm: 2.07 (s, 2.7 H, CH3 9A), 2.17 (s, 0.3 H, CH3 9B), 3.80 (s, 3 H, OCH3),
5.25 (s, 0.1 H, H-5 9B), 5.50 (s, 0.9 H, H-5 9A), 6.96 (d, 2 H, J = 8.8 Hz, Ar-H 9A), 7.84 (d, 0.2 H,
Ar-H, 9B), 7.89 (d, 1.8 H, J = 8.4 Hz, Ar-H, 9A), 7.99 (s, 0.9 H, CH=N, 9A), 8.12 (s, 0.1 H, CH=N,
9B), 10.15 (s, 0.1 H, D2O exchangeable, OH 9B), 11.23 (bs, 1.9 H, D2O exchangeable, NH's 9A and
NH 9B). 13C-NMR (DMSO-d6, 100 MHz) δC ppm: 19.1 (CH3 9B), 23.1 (CH3 9A), 55.7 (OCH3), 101.6
(C-5), 114.4, 127.5, 129.5 (Ar-C), 144.7 (CH=N), 152.8 (C-6) 160.9 (C-2), 162.9 (C-4).
E-2-[2-(2-Hydroxybenzylidene)hydrazinyl]-6-methylpyrimidin-4-one 10.
Yield 81 % (method A, method B), m. p. 245-247 °C, RF = 0.32 (CH2Cl2/ CH3OH 9.5:0.5). IR (KBr)
1
υ (cm-1): 3429 (OH), 1671 (CONH), 1531 (CH=N). H-NMR (DMSO-d6, 400 MHz) δH ppm: 2.03,
2.07 (2 s, 2.7 H, CH3 10A), 2.15 (s, 0.3 H, CH3 10B), 5.29 (s, 0.1 H, H-5 10B), 5.52 (s, 0.9 H, H-5
10A), 6.83 (t, 1 H, Ar-H), 6.88 (d, 1 H, Ar-H), 7.21 (t, 1 H, J = 7.2 Hz, Ar-H), 7.82 (d, 0.1 H, Ar-H,
10B), 8.05 (d, 0.9 H, J = 7.6 Hz, Ar-H, 10A), 8.35 (s, 0.9 H, CH=N, 10A), 8.42 (s, 0.1 H, CH=N,
10B), 9.97 (bs, 1 H, D2O exchangeable, OH), 10.28 (s, 0.1 H, D2O exchangeable, OH 10B), 11.22 (bs,
13
1.9 H, D2O exchangeable, NH's 10A and NH 10B). C-NMR(DMSO-d6,100 MHz) δC ppm: 19.0
(CH310B), 23.0 (CH310A), 101.5 (C-5), 116.4, 119.6, 120.8, 128.4, 131.2 (Ar-C), 142.9 (CH=N),
152.8 (C-6), 156.5 (C-2), 163.1 (C-4). Anal. Calc. for C12H12N4O2: C, 59.01, H, 4.95, N, 22.94. Found:
C, 59.27, H, 5.12, N, 23.21.

General procedures for the preparation of N,N'-diacetyl-3-(4-chloro or bromophenyl)-5-methyl-
7-oxo-1,2,4-triazolo[4,3-a]pyrimidine 12, 13 and N,N'-diacetyl-3-(4-chloro or bromophenyl)-7-
methyl-5-oxo-1,2,4-triazolo[4,3-a]pyrimidine 15, 16.
Method A
A mixture of compound 6 or 7 (0.3 mmol) and acetic anhydride (2 ml) was refluxed; the clear yellow
solution obtained was monitored by TLC using ethyl acetate: n-hexane (2:1) as an eluent, completion
of the reaction required 9 h and two products were observed on TLC. The reaction mixture was poured
onto crushed ice and the white precipitate obtained was filtered off, washed with water and dried. The
afforded products were separated through column chromatography (Ethyl acetate/ n-Hexane 2:1); the
fractions collected were evaporated under diminished pressure to give compounds 12-13 and 15-16.
Method B
A mixture of compound 6 or 7 (0.3 mmol) and acetic anhydride (2 ml) was heated under ultrasonic
irradiation at 70-75 °C. The reaction required 5 h to be ended. The reaction mixture was poured onto
crushed ice and the precipitate obtained was filtered off and dried, then separated through column
chromatography to obtain 12-13 and 15-16.
Method C
A solution of 12, 13 or 14 (50 mg) in ethanol was placed in a sample tube and exposed to sunlight for
56-63 h to afford compound 15, 16 or 17, respectively, in 99 % yield, the reaction was monitored by
TLC using ethyl acetate: n-hexane eluent (2:1), the solvent was evaporated under reduced pressure
and recrystallized from absolute ethanol.
N,N'-Diacetyl-3-(4-chlorophenyl)-5-methyl-7-oxo-1,2,4-triazolo[4,3-a]pyrimidine 12.
Compound 12 was separated by column chromatography using ethyl acetate/ n-hexane (2:1) in 41 %
(method A) and 45 % (method B) yield as colorless crystals; m. p. 168-171 °C, RF = 0.51 (Ethyl
acetate/n-Hexane 1:1). IR (KBr) υ (cm-1): 1714, 1689 (COCH3), 834, 807 (C-Cl). 1H-NMR (CDCl3,
400 MHz) δH ppm: 2.08 (s, 0.36 H, CH3 15), 2.12 (s, 0.1 H, NAc 15), 2.18 (s, 0.36 H, NAc 15), 2.25
(s, 2.7 H, NAc 12), 2.33 (s, 3 H, CH3 12), 2.68 (s, 3 H, NAc 12), 5.16 (s, 0.1 H, H-6 15), 6.13 (s, 0.9
H, H-6 12), 7.07 (s, 0.1 H, H-3 15), 7.36 (d, 2 H, J = 8.4 Hz, Ar-H 12 and 15), 7.47 (d, 2 H, Ar-H 12
and 15), 7.48 (s, 0.9 H, H-3 12). 13C-NMR (CDCl3, 100 MHz) δC ppm: 19.0, 20.6, 20.8, 20.9, 22.7,
23.9 (CH3 and 2 X CH3CO 12 and 15), 70.8 (C-3 12), 73.9 (C-3 15), 98.1 (C-6 15), 109.3 (C-6 12),
127.7, 128.4, 128.8, 129.3, 132.2, 134.6, 135.3, 136.0 (Ar-C 12 and 15), 147.2 (C-5 12), 148.0 (C-5
15), 151.5 (C-7 15), 158.0 (C-8a 12), 158.7 (C-8a 15), 165.1 (C-7 12), 166.7, 176.1 (2 X CH3CO).
Anal. Calc. for C16H15ClN4O3: C, 55.42, H, 4.36, N, 16.16. Found: C, 55.22, H, 4.54, N, 16.11.
N,N'-Diacetyl-3-(4-chlorophenyl)-7-methyl-5-oxo-1,2,4-triazolo[4,3-a]pyrimidine 15.
Compound 15 was separated by column chromatography using ethyl acetate/ n-hexane (2:1) in 10 %
(method A) and 13 % (method B) yield; m. p. decomp. > 260 °C, RF = 0.35 (Ethyl acetate). IR (KBr)

υ (cm-1): 1694 (COCH3), 826 (C-Cl). 1H-NMR (CDCl3, 400 MHz) δH ppm: 1.27 (s, 3 H, NAc), 1.97
(s, 3 H, CH3), 2.26 (s, 3 H, NAc), 5.15 (s, 1 H, H-6), 7.07 (s, 1 H, H-3), 7.34 (d, 2 H, J = 8.4 Hz, Ar-
H), 7.47 (d, 2 H, Ar-H). 13C-NMR (CDCl3, 100 MHz) δC ppm: 18.9 (CH3), 20.8 (CH3CO), 29.6
(CH3CO), 74.0 (C-3), 97.8 (C-6), 128.4, 128.7, 134.8, 135.3 (Ar-C), 146.8 (C-5), 151.8 (C-7), 158.3
(C-8a), 166.9 (COCH3). Anal. Calc. for C16H15ClN4O3: C, 55.42, H, 4.36, N, 16.16. Found: C, 55.65,
H, 4.42, N, 16.39.
N,N'-Diacetyl-3-(4-bromophenyl)-5-methyl-7-oxo-1,2,4-triazolo[4,3-a]pyrimidine 13.
Compound 13 was separated by column chromatography using ethyl acetate/ n-hexane (2:1) in 60 %
(method A, B) yield as colorless crystals; m. p. 174-178 °C, RF = 0.55 (Ethyl acetate/ n-Hexane 1:1).
IR (KBr) υ (cm-1): 1714, 1688 (COCH3), 838, 804 (C-Br). 1H-NMR (CDCl3, 400 MHz) δH ppm: 2.07
(s, 0.49 H, CH3 16), 2.12 (s, 0.18 H, NAc 16), 2.18 (s, 0.43 H, NAc 16), 2.24 (s, 2.70 H, NAc13), 2.33
(s, 3 H, CH3 13), 2.68 (s, 3 H, NAc 13), 5.16 (s, 0.1 H, H-6 16), 6.14 (s, 0.9 H, H-6 13), 7.06 (s, 0.16
H, H-3 16), 7.41 (d, 2 H, J = 8.4 Hz, Ar-H 13 and 16), 7.46 (s, 1 H,H-3 13), 7.52 (d, 2 H, Ar-H 13 and
16). 13C-NMR (CDCl3, 100 MHz) δC ppm: 19.1, 20.6, 20.9, 22.7, 23.9, 29.7 (CH3, 2 X CH3CO 13 and
16), 70.9 (C-3 13), 74.1 (C-3 16), 98.2 (C-6 16), 109.3 (C-6 13), 123.5, 124.2, 127.9, 128.7, 131.7,
132.3, 132.8, 135.3 (Ar-C 13 and 16), 148.0 (C-5 13), 150.7 (C-7 16), 157.9, 158.5 (C-8a 13 and 16),
165.0 (C-7 13), 167.7, 173.9 (2 X CH3CO). Anal. Calc. for C16H15BrN4O3: C, 49.12; H, 3.86; N, 14.32.
Found: C, 49.17, H, 3.89, N, 14.04.
N,N'-Diacetyl-3-(4-bromophenyl)-7-methyl-5-oxo-1,2,4-triazolo[4,3-a]pyrimidine 16.
Compound 16 was separated by column chromatography using ethyl acetate/ n-hexane (2:1) in 5 %
(method A) and 12 % (method B) yield; m. p. 150-154 °C, RF = 0.28 (Ethyl acetate). 1H-NMR (CDCl3,
400 MHz) δH ppm: 1.27 (s, 3 H, NAc), 2.07 (s, 3 H, CH3), 2.24 (s, 3 H, NAc), 5.19 (s,1 H, H-6), 7.06
(s, 1 H, H-3), 7.42 (d, 2 H, J = 6.4 Hz, Ar-H), 7.51 (d, 2 H, Ar-H). 13C-NMR (CDCl3, 100 MHz) δC
ppm: 19.1 (CH3), 20.8 (CH3CO), 29.7 (CH3CO), 74.1 (C-3), 98.1 (C-6), 123.5, 128.7, 131.7, 135.3
(Ar-C), 146.6 (C-5), 151.3 (C-7), 158.0 (C-8a), 166.9 (COCH3). Anal. Calc. for C16H15BrN4O3: C,
49.12, H, 3.86, N, 14.32. Found: C, 49.38, H, 3.97, N, 14.50.
N,N'-Diacetyl-3-(4-nitrophenyl)-5-methyl-7-oxo-1,2,4-triazolo[4,3-a]pyrimidine 14.
A mixture of compound 8 (0.37 mmol) and acetic anhydride (3 ml) was dissolved, the clear yellow
solution obtained was refluxed. The reaction was monitored by TLC and required 11-12 h to be ended.
The reaction mixture was poured onto crushed ice and the white precipitate obtained was filtered off,
washed with water and dried. Recrystallized from absolute ethanol in 60 % yield as colorless crystals;
m. p. decomp. 214 °C, RF = 0.50 (Ethyl acetate/ n-Hexane 1:1). IR (KBr) υ (cm-1): 1712 (COCH3).
1H-NMR (CDCl3, 400 MHz) δH ppm: 2.11 (s, 0.7 H, NAc 17), 2.24 (s, 0.3 H, CH3 17), 2.26 (s, 2.3 H,
NAc 14), 2.34 (s, 2.70 H, CH3 14), 2.67, 2.70 (2 s, 3 H, NAc 14 and 17), 5.21 (s, 0.1 H, H-6 17), 6.17
(s, 0.9 H, H-6 14), 7.15 (s, 0.1 H, H-3 17), 7.58 (s, 0.9 H, H-3 14), 7.76 (d, 2 H, J = 8.8 Hz, Ar-H 14
and 17), 8.23 (d, 2 H, Ar-H 14 and 17). 13C-NMR (CDCl3, 100 MHz) δC ppm: 19.2, 20.6, 20.8, 22.1,
22.7, 24.0 (CH3, 2 X CH3CO 14 and 17), 70.3 (C-3 14), 73.6 (C-3 17), 98.4 (C-6 17), 109.3 (C-6 14),
123.9, 124.2, 127.6, 128.2, 140.3, 142.4 (Ar-C 14 and 17), 147.8 (C-5 14), 148.7 (C-5 17), 151.0 (C-7

17), 157.7 (C-8a 17), 158.5 (C-8a 14), 165.2 (C-7 14), 166.7, 167.7, 174.2, 175.4 (2 X CH3CO). 1H-
NMR of 14:17 (2:3) method C (CDCl3, 400 MHz) δH ppm: 2.14, 2.36 (2 s, 3 H, NAc 14 and 17),
2.21, 2.28 (2 s, 3 H, CH3 14 and 17), 2.68, 2.71 (2 s, 3 H, NAc 14 and 17), 5.21, 6.18 (2 s, 1 H, H-6
14 and 17), 7.16, 7.60 (2 s, 1 H, H-3 14 and 17), 7.72-7.77 (m, 2 H, Ar-H 14 and 17), 8.24-8.25 (m, 2
H, Ar-H 14 and 17). Anal. Calc. for C16H15N5O5: C, 53.78, H, 4.23, N, 19.60. Found: C, 54.01, H,
4.39, N, 19.48.
N,N'-Diacetyl-3-(4-methoxyphenyl)-7-methyl-5-oxo-1,2,4-triazolo[4,3-a]pyrimidine 18.
A mixture of compound 9 (0.77 mmol) and acetic anhydride (2 ml) was dissolved, the clear yellow
solution obtained was refluxed. The reaction was monitored by TLC and required 9 h to be ended. The
reaction mixture was poured onto crushed ice and the precipitate obtained was filtered off, washed
with water and dried. Compound 18 was purified by column chromatography using ethyl acetate in 38
% yield as yellow crystals; m. p. decomposition 214 °C, RF = 0.23 (Ethyl acetate). IR (KBr) υ (cm-1):
1704, 1662 (COCH3). 1H-NMR (CDCl3, 400 MHz) δH ppm: 2.01, 2.04, 2.25 (3 s, 9 H, CH3 and 2 X
NAc), 3.69, 3.78 (2 s, 3 H, OCH3), 5.08, 5.65 (2 s, 1 H, H-6), 6.95, 6.97 (2 s, 1 H, H-3), 6.78, 6.86,
7.36, 7.41, 7.63, 7.78 (6 d, 4 H, J = 8.4 and 8.8 Hz, Ar-H). 13C-NMR (CDCl3, 100 MHz) δH ppm: 19.1,
20.8, 21.0 (CH3 and 2 X CH3CO), 55.2, 55.5 (OCH3), 74.3 (C-3), 98.4 (C-6), 113.9, 114.5, 128.2,
128.6, 129.9, 132.0 (Ar-C), 147.0 (C-5), 150.9 (C-7), 160.2 (C-8a), 166.4, 175.8 (2 X COCH3). Anal.
Calc. for C17H18N4O4: C, 59.64, H, 5.30, N, 16.37. Found: C, 59.88, H, 5.46, N, 16.52.
N,N'-Diacetyl-3-(2-acetoxyphenyl)-7-methyl-5-oxo-1,2,4-triazolo[4,3-a]pyrimidine 19.
A mixture of compound 10 (0.2 g, 0.8 mmol) and acetic anhydride (2 mL) was dissolved, the clear
solution obtained was refluxed for 11 h where the reaction was monitored by TLC till its end. The
reaction mixture was poured onto crushed ice and the white precipitate obtained was filtered off,
washed with water, dried and purified by column chromatography using ethyl acetate in 33 % yield;
1
m. p. > 214 °C (decomp.), RF = 0.23 (Ethyl acetate). IR (KBr) υ (cm-1): 1742, 1695 (COCH3). H-
NMR (CDCl3, 400 MHz) δH ppm: 2.00 (s, 3 H, NAc), 2.19 (s, 3 H, CH3), 2.31, 2.32 (2 s, 6 H, NAc,
OAc), 5.15 (s, 1 H, H-6), 7.11 (bs, 2 H, H-3, Ar-H), 7.25, 7.38 (2 bs, 2 H, Ar-H), 7.58 (d, 1 H, J = 7.2
Hz, Ar-H). 13C-NMR (CDCl3, 100 MHz) δC ppm: 18.9, 20.5, 20.7, 21.1 (CH3 and 3 X CH3CO), 72.4
(C-3), 97.9 (C-6), 123.6, 125.8, 127.8, 130.4, 147.3 (Ar-C), 148.6 (C-5), 151.8 (C-7), 158.3 (C-8a),
166.7, 168.9, 175.4 (3 X COCH3). Anal. Calc. for C18H18N4O5: C, 58.37, H, 4.90, N, 15.13. Found:
C, 58.60, H, 4.78, N, 15.27.
4.2. MMPs inhibitory activity
The synthesized compounds were evaluated for in vitro MMPs -13,-10,-9 and -7 inhibitory
activities utilizing Abcam® MMP13 Inhibitor Screening Assay Kit (ab139451),SensoLyte®520 MMP-
10 Assay Kit, Abcam® MMP9 Inhibitor Screening Assay Kit (ab139448), and Abcam® MMP7
Inhibitor Screening Assay Kit (ab139445), respectively, following the manufacturers’ instructions.
4.3. Cytotoxicity screening

Cytotoxicity was performed via MTT assay as previously described [77] and detailed in the
supplementary data.
4.4. Molecular modeling
4.4.1. Docking
The compounds under investigation were built in silico and energy minimized utilizing the
MMFF94x force field at a gradient of 0.01 RMSD. Coordinates for MMP-10 and -13 catalytic domains
were retrieved from RCSB PDB (PDB ID: 1Q3A [8] and PDB ID: 456C [45], respectively), handled
with MOE 2015.10. Unwanted chains, solvents, and ligands were eliminated. The preparation was
conducted employing “Structure Preparation” module with the default settings. The optimized domain
consisted of MMP monomer, zinc, and calcium ions. The active site was selected based on the co-
crystallized ligand interactions taking into consideration the reported binding sites [8,45]. Docking
simulations were conducted utilizing ‘Triangle Matcher’ as the placement method and ‘London dG’
scoring for calculating Gibbs energy for binding.
4.4.2. In silico physicochemical properties and drug-likeness scores
Physicochemical properties were predicted by Molinspiration online property calculation toolkit.
Drug-likeness scores and solubility were calculated by MolSoft software. ADME profiling was
performed by PreADMET calculator.
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