Med Chem Res MEDICINALCHEMISTRY
DOI 10.1007/s00044-014-1295-3 RESEARCH
REVIEW ARTICLE
A comprehensive review on bioactive fused heterocycles
as purine-utilizing enzymes inhibitors
Monika Chauhan • Raj Kumar
Received: 30 May 2014 / Accepted: 3 November 2014
 Springer Science+Business Media New York 2014
Abstract Purine-utilizing enzymes (PUEs) are involved in Introduction
the control of biological actions of nitrogen-containing
bases, purines and pyrimidines, by participating in their Purine-utilizing enzymes (PUEs) are validated purine and
catabolism, and this has made them a topic of considerate pyrimidine druggable targets for many diseases ranging
interest. The heterocyclics, as purine-utilizing enzyme from inflammation to cancer (Robak and Robak, 2013;
inhibitors (PUEIs), play a vital role in a number of diseases, Bobrovnikova-Marjon and Hurov, 2014; D’Angiolella,
e.g., malaria, cancer, rheumatoid arthritis, inflammation, 2014). These are broadly categorized into six classes
tissue rejection, and autoimmune disorders. The present (Fig. 1) based on their mode of action, basic structures,
review is first of its kind covering the literature up to 2014 on selectivity, and their target sites.
the advances in broad-spectrum medicinal activities exhib- They are represented as adenosine and guanine deam-
ited by heterocycles as PUEIs. The drug designing of the inases, DNA topoisomerases, xanthine oxidase, purine
purine and pyrimidine antimetabolites is based on the nucleoside phosphorylase (Guillen et al., 2014), and
structural mimicking of the existing compounds. The basic hypoxanthine–guanine phosphoribosyltransferase (Robak
consideration during the designing of this class is the intro- and Robak, 2013; Newcombe, 2013). PUEs are responsible
duction of small structural changes without the alteration of for the transformation of some important key steps
basic skeleton of pharmacophore. The balance between the involved in the de novo purine biosynthesis of cell
existing empirical approach and rational approach is yet to be metabolism (Fig. 2).
maintained during the design and synthesis of new PUEIs by The heterocycles exhibit diverse properties including
combining in vivo, in vitro, and in silico methods. The data electrophilic and nucleophilic action, oxidizing and
compiled in the present manuscript on SARs, IC50s, Kis, Km, reducing properties, and acidic and basic attributes which
in silico studies, and their reported X-ray co-crystal struc- are associated with the electronic arrangements in their
tures with PUEs will offer the researchers the rational chemical structures (Pozharskii et al., 2011; Lister, 2009;
approaches for the design and development of selective and Lehman, 2012; Pozharskii et al., 2012; DeVita and
specific PUEIs devoid of adverse effects. Rosenberg, 2012; Chauhan et al., 2005; Bertino and Johns,
1967; Pitt et al., 2009). Because of their similarities with
Keywords Heterocycles  Purine-utilizing enzymes  the purines and pyrimidines, they act as purine-utilizing
Inhibitor  Purines  Pyrimidines enzyme inhibitors (PUEIs) and find their applications in
treatment and management of diseases such as malaria,
cancer, rheumatoid arthritis, inflammation, tissue rejection,
Raj Kumar—CUPB Library Communication Number: P116. T-cell leukemia (Zavialov et al., 2010a; la Marca et al.,
2014), and other autoimmune disorders (e.g., HIV)
M. Chauhan  R. Kumar (&) (Niedzwicki et al., 1991).
Laboratory for Drug Design and Synthesis, Centre for Chemical
The present review is first of its kind detailing medicinal
and Pharmaceutical Sciences, School of Basic and Applied
Sciences, Central University of Punjab, Bathinda 151 001, India applications, structure-activity relationships, and confor-
e-mail: raj.khunger@gmail.com; rajcps@cup.ac.in mational aspects of heterocycles as PUEIs reported till
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Fig. 1 Classes of purine-utilizing enzymes
date. We will discuss the various functions of PUEs, the Hosmane, 1997; Reayi and Hosmane, 2004b; Chauhan and
mechanistic studies existing on several members of their Kumar, 2013).
superfamily, their common structural characteristics, and In 2009, Motta et al. presented the pyrazolo[3,4-d]pyrim-
various PUEIs of heterocyclic skeletons at clinical and idin-4-one ring system as potent adenosine deaminase
preclinical levels. inhibitor. The position-2 of the pyrazolo[3,4-d]pyrimidin-4-
one nucleus was substituted with various alkyl and aryl/alkyl
Adenosine deaminase inhibitors groups. The series of compounds was synthesized and studied
for their structure-activity relationships. The potent com-
Adenosine deaminase (AD; EC 3.5.4.4) is involved in pound 5 of the series (Fig. 4) was evaluated in animal model
metabolism of extracellular adenosine. AD catalyzes the of experimental colitis. The amelioration of both systemic and
deamination of adenosine to inosine. It binds to A1, A2A, intestinal inflammatory alterations was seen. Urea derivatives
A2B, and A3 adenosine deaminase receptors by responding (Fig. 5) with alike substitution as carboxamide derivatives
through G-protein coupled receptor (Hirschhorn and Ra- were found to have less potency. Triflouromethyl group at the
tech, 1980; Reayi and Hosmane, 2004a). It has two kinet- para position of phenyl ring disclosed threefold escalation in
ically distinguishable isoenzymes known as ADA1 potency as compared to carboxamide derivative (La Motta
(adenosine deaminase 1) and ADA2 (adenosine deaminase et al., 2009). Electron-withdrawing substituent increases the
2). ADA1 is a 30- to 40-kDa monomeric protein present in potency and further increase in size increases the activity in
erythrocytes, kidney, lymphocytes, macrophages, liver, and carboxamide derivatives.
intestine (Daddona and Kelley, 1977). It is present in In 2009, Lianmei et al. explored the effect of nimodi-
extracellular fluid and required for the intracellular deple- pine on proliferation induced by rhTGF-a and mRNA
tion of adenosine. ADA2 is a symmetrical homodimer expression of RNA-dependent ADA1 in human laryngeal
possessing catalytic domain, disulfide bond, a signal pep- cancer Hep-2 cell lines. Nimodipine (8; Fig. 6) was found
tide, and two ADGF/ADA2-specific domains. It regulates to block calcium channel of Hep-2 cell and decrease the
the level of adenosine which acts by binding to the aden- expression of ADARI mRNA (Lianmei et al., 2009).
osine deaminase receptors and serves as a key factor in In 2010, Tite et al. designed and synthesized new C-
ordinance of various cellular activities (Zavialov et al., nucleosides as potential ADA inhibitors (Fig. 7). They
2010b). Its levels are increased in HIV infection, AIDS, performed molecular docking study and predicted the high-
autoimmune abnormalities, and tuberculosis (Andreasyan ADA binding affinity of the compounds. The important
et al., 2005; Nosaka et al., 1996; Mediero and Cronstein, interactions seen were hydrogen bond formation of amine
2013; Kuno et al., 2006; Saevels et al., 1996; Antonioli group of the compounds with the glutamate carboxylate
et al., 2012). and hydrogen bonding of N-1 hydrogen with carboxylate of
Azapinomycin-based coformycin and 2-deoxycoformy- Asp296. However, the compounds were found to be poor
cin are natural, and pyrazolo[3,4-d]pyrimidines are syn- inhibitors of ADA due to the poor covalent hydrate sta-
thetic adenosine deaminase inhibitors (Fig. 3) (Hong and bility which emphasized the importance of hydration and
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Fig. 2 Biosynthetic pathways of purine metabolism
Fig. 3 Chemical structures of coformycin, 2-deoxycoformycin, and pyrazolo[3,4-d]pyrimidines
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its decisive function in stability and activity against ADA
(Tite et al., 2010).
In 2010, Zavialov et al. envisioned 6-hydroxy-7,8-di-
hydropurine nucleoside (Fig. 8) as selective inhibitor of
ADA1. The team further discovered ADGF (adenosine
deaminase growth factors)/ADA2-specific domains in
ADA2 protein (Zavialov et al., 2010b).
In 2011, Gillerman and Fischer investigated the molec-
ular recognition of several ADA inhibitors (Fig. 9). The
Fig. 4 Carboxamide derivatives (4 and 5) as adenosine deaminase
inhibitors important interactions of the compounds for the binding and
stabilization with ADA were found to be hydrogen bonding
of Gly184 with N-3 nitrogen of adenosine, hydrogen
bonding between the adenosine N-1 and N-6 with Glu217,
and hydrogen bond formation of Asp296 with the adenosine
N-7. Nebularine compounds having substitution at 2-posi-
tion (MeS or NH2) showed potent inhibitory activity as
compared to substitution at 8-position which were poor
inhibitors of ADA exhibiting Ki values greater than
100 lM. 2-SBu-(14) and 2-SHex-(16) substituted com-
pounds showed tight binding to the lipophilic site explaining
Fig. 5 Urea derivatives (6 and 7) as adenosine deaminase inhibitors their good potency. Further, C-2 substituent was found to be
important for activity (Gillerman and Fischer, 2010).
Fig. 6 Chemical structure of In 2013, Ajlooa et al. studied the effect of two imi-
nimodipine (8) dazolium-based ionic liquids (Fig. 10) on ADA activity. It
was observed that the ionic liquids have a potential to inhibit
the enzyme which is directly associated with their hydro-
phobicity (Ajloo et al., 2013). These act via competitive or
non-competitive inhibition leading to condensed intermo-
lecular hydrogen bond with the tertiary structure of ADA.
Fig. 7 C-nucleosides as
adenosine deaminase inhibitors
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DNA topoisomerase inhibitors both strands of phosphate backbone of the DNA and needs
ATP for complete activity (Baechler et al., 2012). The
DNA topoisomerases have been convoluted in DNA rep- catalytic mechanism of topoisomerases occurs via cleavage
lication, transcription, and recombination as essential and relegation of DNA. The cleavage involves a covalent
enzymes in governing and aligning the topologic states of bond formation by nucleophilic attack on phosphate group
DNA (Pastor et al., 2012; Sperling et al., 2011; Gellert, of DNA phosphodiester bond by the hydroxyl group of
1981; Nitiss, 2009). They are classified into two major catalytic tyrosine residue forming tyrosine phosphate (Dinh
types depending on their mechanisms: DNA topoisomerase et al., 2014; Champoux 2001; Stewart et al., 1998; Corbett
I and DNA topoisomerase II (Vos et al., 2014; Schneider and Berger, 2004).The resealing of DNA strand occurs by
et al., 1990). DNA topoisomerase I asseverates topological the attack on tyrosine phosphate by deoxyribose 50
stress by breaking the single strand of DNA, whereas DNA hydroxyl group. Cancer compels speedy replication and
topoisomerase II relaxes the supercoiled DNA by breaking topoisomerase inhibitors breach DNA strands, causing cell
Fig. 8 Chemical structure of
6-hydroxy-7,8–dihydropurine
nucleoside (13)
Fig. 10 Ionic liquids as adenosine deaminase inhibitors
Fig. 9 Chemical structures of adenosine deaminase inhibitors
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Fig. 11 Structures of camptothecin, 10-hydroxycamptothecin, and 7-ethyl-10-hydroxycamptothecin as topoisomerase I inhibitors
Fig. 12 SAR of 2,5-
disubstituted benzimidazoles
Fig. 13 Purine-based catalytic
topoisomerase II inhibitors
cycle arrest and thus leading to programmed cell death inhibitors (Oksuzoglu et al., 2008). The compound 25 was
(Guerrant et al., 2013). the most potent DNA topoisomerase I inhibitor having IC50
Camptothecin, 10-hydroxycamptothecin, and 7-ethyl- value of 14.1 mM. Other compounds with significant
10-hydroxycamptothecin are some of the topoisomerase I activity against topoisomerase I were 5-amino-2-(p-fluo-
inhibitors (Fig. 11). Maja and team swayed a review on rophenyl)benzoxazole, 5-amino-2-(p-bromophenyl)benz-
explaining the topoisomerase degradation in cancer cell oxazole, and 5-nitro-2-phenoxymethyl-benzimidazole having
resistance linking to camptothecin-like topoisomerase I IC50 values of 132.3, 134.1, and 258 mM, respectively.
inhibitors (Tomicic and Kaina, 2012). Further 2-(p-nitrobenzyl)benzoxazole has shown maximum
In 2008, Oksuzoglu et al. manifested benzoxazole and inhibitory potential against DNA Top-II having IC50 of
benzimidazole derivatives as potent topoisomerase I and II 17.4 mM (Oksuzoglu et al., 2008). Some of the compounds
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Fig. 14 Novel
indenoisoquinolines as
topoisomerase I inhibitors
Fig. 15 Dual-acting histone
deacetylase-topoisomerase I
inhibitors
with the structure-activity relationships are presented in system was insignificant for the inhibition of HeLa cell
Fig. 12. nuclear extract HDACs. The mechanism of topoisomerase
In 2009, Furet et al. designed new class of catalytic inhibition was investigated based on hydrogen bond for-
topoisomerase II inhibitors based on purine motif (Fig. 13) mation of 10-hydroxy moiety of compounds with water
which competed with ATP-binding site (Furet et al., 2009). molecule present in Topoisomerase I, which was further
In 2012, Zhang et al. envisioned novel indenoisoquinolines supported by X-ray crystallographic data.
as topoisomerase I inhibitors. They evaluated the series of In 2012, Luniewski et al. synthesized 11-methyl-6-[2-
compounds in human cancer cell lines A549, HepG2, and (dimethylamino)ethyl]-6H-indolo[2,3-b]quinoline derivatives
HCT-116 and found out the compounds 34 and 35 to be potent as DNA topoisomerase II inhibitors (Fig. 16). The compounds
against HepG2 and HCT-116 (IC50 of 0.019 and 0.093 lM, were tested on different cancer cell lines and analogs with either
respectively) cancer cell lines (Fig. 14) (Zhang et al., 2012). amine or amide linkers were found to exhibit maximum anti-
The nitro group was optimum for the activity. Further meth- proliferative activity (Luniewski et al., 2012).
oxy and fluorine at R showed maximum potency. In 2013, Xu et al. synthesized indenoisoquinoline
In 2013, Guerrant et al. synthesized dual-acting histone derivatives as topoisomerase I inhibitors that suppressed
deacetylase-topoisomerase I inhibitors (Fig. 15) leading angiogenesis by affecting the HIF signaling pathway (Xu
to cell arrest in cell-free and whole-cell studies. The et al., 2013).
compounds were designed on the camptothecin template
having a triazole ring which was joined with hydroxamic HGPRT inhibitors
moiety via a linker (Guerrant et al., 2013). The linker
length had been concluded to be an important factor for the Hypoxanthine–guanine phosphoribosyltransferase (HGPRT;
activity. The ethyl group at the C-7 of camptothecin ring EC 2.4.2.8) is an enzyme belonging to the class of
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Fig. 16 Chemical structures of synthesized indolo[2,3-b]quinoline derivatives as topoisomerase II inhibitors
phosphoribosyl transferases inscribed by HPRT1 gene (Eads In 2009, Keough et al. synthesized neutral 6-oxopurine
et al., 1994; Sculley et al., 1992). The enzyme is tortuous in acyclic nucleoside phosphonates, which showed inhibitory
transition of hypoxanthine and guanine to inosine mono- activity against recombinant PfHGXPRT (Fig. 19) leading
phosphate and guanine monophosphate, respectively to antimalarial effect in vitro. The docking studies revealed
(Fig. 17). It functions via shifting the 5-phosphoribosyl to the the formation of hydrogen bond by three oxygen atoms of
purine from 5-phosphoribosyl 1-pyrophosphate through sal- phosphate/phosphonate group with main-chain amides of
vage pathway (Finette et al., 2002; Torres and Puig, 2007). Gly139 and Asp137 and a water molecule, amide of
The transfer occurs by the binding of 5-phosphoribosyl Thr141, the hydroxyl atom of Thr141 and a water mole-
1-pyrophosphate with free enzyme and successive formation cule, side-chain oxygen, and main-chain amide atom of
of bond between the C-1 of 5-phosphoribosyl 1-pyrophos- Thr138 (Keough et al., 2009). Further in 2013, the same
phate and the N-9 of the purine ring of hypoxanthine or research group revealed that the lipophilic groups linked by
guanine (Keough et al., 2005). Atkinson et al. contemplated phosphoramidate bonds of these compounds escalate the
the HGPRT inhibitory activity of 6-mercaptopurine (Fig. 18) activity.
and structurally related compounds through its competitive In 2012, Krecmerova et al. synthesized purine N9-[2-
inhibition. hydroxy-3-O-(phosphonomethoxy)propyl] derivatives and
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O
2-
HN N
OPO3
O
R N N OPO 2-3PO3
O
HO HOOH Hypoxanthine-guanine phosphoribosyltransferase OHO
P OH
HO 5-phospho-α-D-ribosyl-
O 1-pyrophosphate, Mg2+
Nucleotide
HGPRT, Nucleotide Hypoxanthine-guanine phospho
ribosyltransferase,5-phospho-α-
D-ribosyl-1-pyrophosphate, Mg2+
Mg2+ Pyrophosphate
O
Nucleotide H
HN N
R N N
HGPRT HGPRT Guanine or Hypoxanthine
Guanine or 5-phospho-α-D-ribosyl-
Pyrophosphate, Mg2+ Hypoxanthine 1-pyrophosphate, Mg2+
R=H or for hypoxanthine, R= NH2 for guanine
Fig. 17 Catalytic pathway for hypoxanthine–guanine phosphoribosyl transferase
Fig. 18 Chemical structure of studies revealed that guanine-based compounds were less
6-mercaptopurine (44) effective.
Hockovaa et al. in 2012 synthesized novel N-branched
acyclic nucleoside phosphonates (ANPs) as potent and
selective inhibitors of human, P. falciparum, and P. vivax
6-oxopurine phosphoribosyl transferases (Fig. 21). Guan-
ine emerged to be more potent base for human HGPRT and
their side-chain modified analogs as potential antimalarial bound more tightly than hypoxanthine via formation of
agents (Fig. 20). The synthesized acyclic nucleoside extra hydrogen bond between the 2-amino group and the
phosphonates exhibited very weak inhibition of human active site amino acid groups. Further two-carbon atoms
HGPRT and good inhibition of Plasmodium falciparum as linked between the tri-substituted nitrogen and the N-9
well as Plasmodium vivax enzymes. The Ki values were 2 atom had been found to be optimum for the HGPRT
and 5 lM for P. falciparum HGPRT and P. vivax HGPRT, inhibitory activity. The substituents like COOMe, COOH,
respectively (Krecmerova et al., 2012). The docking and OH were tolerable for inhibitory activity against
Fig. 19 Acyclic nucleoside
phosphonates as a new class of
HGPRT inhibitors
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Fig. 20 N9-[2-hydroxy-3-O-
(phosphonomethoxy)propyl]
derivatives as HGPRT
inhibitors
Fig. 21 N-branched acyclic nucleoside phosphonates as human and plasmodium HGPRT inhibitors
PfHGXPRT, whereas CN was preferably optimum for The linker and the purine base were crucial and found to
PvHGPRT inhibition (Hockovaa et al., 2012). play an important role in the binding of the ANPs to these
In 2012, Hazleton et al. gave acyclic immucillin phos- three enzymes. The compounds with shorter linker did not
phonates as inhibitors of P. falciparum Hypoxanthine– show binding to the enzymes because if the base binds first,
Guanine–Xanthine Phosphoribosyltransferase (Fig. 22). the phosphonate group is unable to reach into the 5-phos-
The phosphonate group of the acyclic immucillin phos- phate binding pocket or on the contrary; if the phosphonate
phonates offered resistance to phosphohydrolases. The group binds to active site first, the base is in a farther
compounds were based on immucillin scaffold but having distance from its binding site (Cesnek et al., 2012).
enhanced stability, specificity, and binding affinity than In 2013, Baszczynski et al. demonstrated the effect of
neutral 6-oxopurine acyclic immucillin phosphonates. The novel [3-fluoro-(2-phosphonoethoxy)propyl]purines on the
important interactions observed were hydrogen bonding inhibition of P. falciparum, P. vivax, and human HGPRT
with Glu144, Asp145, and water molecule coordination by (Fig. 24). The fluorinated ANPs were highly potent as
Mg2? ion. The cationic reaction center of these compounds compared to non-fluorinated ANPs. Nature and location of
had complimentary interactions leading to increased the purine base were critical for the activity. The phos-
affinity for PfHGXPRT (Hazleton et al., 2012). phorous and oxygen interactions with Asp137 and Thr141
In 2012, Ansari et al. carried out comparative modeling amino acids were found to be indispensable and essential
of HGPRT enzyme of L. donovani and studied binding for the tight binding of the acyclic nucleoside phosphonates
affinities of different analogs of GMP. Lys66, Asp74, (Baszczynski et al., 2013).
Arg77, Asp81, Val88, Tyr182, Arg192, and Arg194 are the
important amino acids involved in binding of HGPRT. It
was found out that HGPRT inhibitors like acyclovir or Guanine deaminase inhibitors
pentamidine in combination might have the potential
against HIV and visceral leishmaniasis (Ansari et al., Guanase or guanine deaminase (GDA; EC 3.3.4.3) is a zinc
2012). metalloenzyme involved in catalysis of first step in purine
Cesnek et al. synthesized and evaluated 9-phospho- metabolism and converts guanine to xanthine by the
noalkyl and 9-phosphonoalkoxyalkyl purines as inhibitors hydrolytic deamination (Fernandez et al., 2010). Guanine
of P. falciparum, P. vivax, and human HGPRT (Fig. 23). deaminase consists of two families. The families differ in
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Fig. 22 Chemical structures and inhibitory activities of acyclic immucillin phosphonates (52 and 53)
Fig. 23 Chemical structures of 9-phosphonoalkyl and 9-phosphonoalkoxyalkyl purines
number of amino acids present in them. One of them lipophilicity in its surrounding. These groups escalated the
contains 160 amino acids and other consists of greater than interactions with enzyme. The 5-carbonyl group in along
400 amino acids for e.g., Bacillus subtilis guanase and with 6-hydroxy group was proposed to interact with Zn2?
Escherichia coli guanase, respectively. offering stability to the transition state (Chakraborty et al.,
Wang and Hosmane reported ring-expanded acyclic 2011).
nucleosides, 4,6-diamino-8H-1-hydroxyethoxymethyl-8- In 2012, the same research group synthesized and
iminoimidazo[4,5-e][1,3]diazepines (Fig. 25) as dual evaluated the structural analogs of azepinomycin against
inhibitors of ADA and GDA. The compounds were found GDA (Fig. 27) for determining structure–activity rela-
to act by competent inhibition (Wang and Hosmane, 2001). tionships (Chakraborty et al., 2012). However, the com-
In 2011, Chakraborty et al. proposed the mechanism of pounds were not found to be as potent as natural analog.
GDA inhibitors as transition-state analog inhibitor Further, Tantravedi et al.. (2013) reported synthesis,
(Fig. 26). The compound possessed geminal carbon at biochemical screening, and structure–activity correlations
position-6 and a benzyl group at position-3 which provided of various selectively substituted imidazo[4,5-
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Fig. 24 [3-Fluoro-(2-phosphonoethoxy)propyl]purines as HGPRT inhibitors
Fig. 25 4,6-Diamino-8H-1-hydroxyethoxymethyl-8-iminoimidazo[4,
5-e][1,3]diazepine as dual inhibitor of adenosine and guanine
deaminases
Fig. 27 Synthetic analogs of azepinomycin
Fig. 28 Potential transition-
state analog inhibitor of guanase
Fig. 26 Novel transition-state analog inhibitor of guanase
e][1,4]diazepines as potential transition-state analog azepinomycin exhibited Ki of 2.5 lM against GDA (Tan-
inhibitors of guanase (Fig. 28). It was observed that the travedi et al., 2013).
lipophilicity near O-5 was escalated, and if the hydropho-
bic character of N-3 was shifted to N-1, the activity Xanthine oxidase inhibitors
decreases. Further, N-3 or N-4 hydrophobic character was
directly proportional to inhibitory activity. The N-3 lipo- Xanthine oxidase (XO; EC 1.1.3.22) (Oliveira-Campos
phobic character was crucial for inhibitory activity. The et al., 2008) is a metallo-flavoenzyme which is oxidized
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Fig. 29 Xanthine oxidase catalyzed biotransformation
Fig. 30 Chemical structure of O In 2010, Ali et al. carried out the docking studies based
allopurinol (74) on PMF scoring performances and SAR of 2-substituted
NH
N pyrazolotriazolopyrimidines and 4-substituted pyrazolo-
N
H N pyrimidines for the design of XO inhibitors (Fig. 31). The
active site of the bovine milk xanthine dehydrogenase and
two scoring functions AutoDock 3.05 and the CAChe
form of xanthine oxidase reductase and present in brain, 6.1.10 were used. The oxo group of pyrazolopyrimidine
kidney, heart, liver, gut, lung, etc. (Oliveira-Campos et al., derivatives was found to be crucial for its activity. Further
2008; Kumar et al., 2011; Nepali et al., 2011b). XO is also bicyclic pyrazolopyrimidine derivatives were less potent
acquitted by plasma and some leukocytes for the genera- than the tricyclic derivatives. Docking score of allopurinol
tion of hydrogen peroxide and other forms of reactive was -88.04 (Ali et al., 2010).
oxygen species during inflammatory response (Kayyali In 2011, our research group rationally designed and
et al., 2003; Kim et al., 2013). XO comprises two subunits synthesized 1-acetyl-3,5-diaryl-4,5-dihydro(1H)pyrazoles
(Nepali et al., 2011a) and contains two iron-sulfur centers, as a new class of potential non-purine XO inhibitors
molybdenum-site, and flavin-adenine dinucleotide. It (Fig. 32). 1-Naphthyl group at ring A showed good XO
converts the bicyclic nitrogenous bases to uric acid inhibitory activity (Fig. 32). Replacement of 1-naphthyl at
i.e., hypoxanthine to xanthine and xanthine to uric acid ring A with 2-furyl further increased XO inhibition.
(Fig. 29) (Bhushan et al., 2003). The enzyme generates 4-Pyridyl at ring B (78) exhibited maximum potency.
superoxide anions and hydrogen peroxide which are further Electron-withdrawing groups such as nitro amplified the
converted into hydroxyl radical in the presence of chelated activity, whereas electron-donating groups had the opposite
iron (Beauchamp and Fridovich, 1970; Fridovich, 1970; effect. N-acetyl group was found out to be critical for the
Kumar et al., 1995). activity (Nepali et al., 2011b).
Allopurinol (Fig. 30) was the first XO inhibitor clini- Hu et al. discovered novel xanthone derivatives as XO
cally approved for the treatment of gout (Borges et al., inhibitors (Fig. 33). Cyano group at para position showed
2002). It is a competitive inhibitor of XO and arrests the noteworthy inhibition of XO. Para substituted benzyl
conversion of hypoxanthine and xanthine to xanthine and groups exhibited more XO inhibition as compared to sub-
uric acid, respectively (Massey et al., 1970). stitutes at ortho or meta position. The docked compounds
Fig. 31 Pyrazolopyrimidine
and pyrazolotriazolopyri-
midines as xanthine oxidase
inhibitors
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O2N
O N N
N
77 O
XO inhibitory activity (IC50, μM)= 14.2 O N N
78 O
O2N XO inhibitory activity (IC50, μM)= 5.3
NO2
Ring B O N N
N 79 O O N
N
Ring A N
O XO inhibitory activity (IC O50, μM)= 13.1 80
XO inhibitory activity (IC50, μM)= 12.4
Cl
Cl
Cl
O N N
O N N 82 O
81 O XO inhibitory activity (IC50, μM)= 15.2
XO inhibitory activity (IC50, μM)=19.2
Fig. 32 1-Acetyl-3,5-diaryl-4,5-dihydro(1H) pyrazoles as non-purine xanthine oxidase inhibitors
Fig. 33 Xanthone derivatives as XO inhibitors
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Fig. 34 Thiazolo-pyrazolyl derivatives as XO inhibitors
R
B A
R1
HN N N
R5 R1
N-(1,3-Diaryl-3-oxopropyl)amides N N R C
R4 R2
R2 O R3
N-Acetyl pyrazolines R2 = H
R2 = any alkyl or aryl group
92; R = H, R1 = OH, R2, R4 = Cl, R3, R5 = H, IC50 = 14 µM
93; R = H, R2 = NO2, R1, R3, R4, R5 = H, IC50 = 24 µM
Allopurinol; IC50 = 32 µM
Fig. 35 5,6-Dihydropyrazolo/pyrazolo[1,5-c]quinazoline derivatives as xanthine oxidase inhibitors
were found to interact with molybdenum-protein active In 2012, Beedkar et al. synthesized novel thiazolo-pyr-
site, Phe798, Gln1194, and Gln112 (provides stabilization) azolyl derivatives and evaluated them as xanthine oxidase
and hydrogen bonding with Gln1040, Ser1082, Gln1261, inhibitors and free radical scavengers (Fig. 34). In vitro XO
Gly797, Gln767, and Cys150 (Hu et al., 2011). assay and in silico study evidenced that the hydroxylation
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Fig. 36 9-Deazapurine
ribonucleotide and its
derivatives as PNPase inhibitors
Fig. 37 Four generations of transition-state analogs as PNPase inhibitors
is furnished at the aromatic rings conjoined by a central
fused pyrazole rings near molybdenum metal (Beedkar
et al., 2012).
In 2014, our research group discovered 5,6-dihy-
dropyrazolo/pyrazolo[1,5-c]quinazoline derivatives as XO
inhibitors (Kumar et al., 2014) (Fig. 35). The compounds
were also found to be free radical scavengers. The docking
studies of compounds highlighted the role of important
amino acids of XO in binding interactions.
PNPase inhibitors
Purine nucleoside phosphorylase (PNPase; E.C. 2.4.2.1) is
an omnipresent enzyme involved in purine salvage path-
way and also known as inosine phosphorylase. It catalyzes Fig. 38 Purine nucleoside phosphorylase inhibitors
the conversion of inosine to hypoxanthine, adenosine to
adenine, and guanosine into guanine (Bzowska et al., 2000;
Kline and Schramm, 1993). PNPase has dual role in purine disorders (Furihata et al., 2014; Giblett et al., 1975; Gelf-
metabolism i.e., in synthesis as well as metabolism (Kline and et al., 1978; Borgers et al., 1977; Somech et al., 2013;
and Schramm, 1995). PNPase involves nucleoside seg- Crittenden and Pillinger, 2013; Stoeckler et al., 1986;
mentation resulting in a nucleobase and ribose-1 phosphate Shewach et al., 1986).
by ribose phosphorylation. T-cell immunodeficiency dur- Stoeckler et al. described 9-deazapurine ribonucleotide
ing PNPase deficiency led to the discovery of PNPase and its derivatives as PNPase inhibitors. These compounds
inhibitors against rheumatoid arthritis, tissue rejection, were found to act via competitive inhibition of PNPase. It
chemotherapy for T-cell leukemias, and other autoimmune was observed that the 9-deazapurine ribonucleotides have
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Med Chem Res
Fig. 39 Chemical structures of S S S
new inhibitors of Schistosoma Br Cl
mansoni purine nucleoside NH NH NH
phosphorylase S S SCl HO
O O O
IC (SmPNP) = 12 ± 1 μM IC (SmPNP) = 38 ± 2 μM IC (SmPNP)50 50 50 = 18 ± 2 μM
103 104 105
Fig. 40 SmPNPase inhibitors
much higher affinity for the enzyme than corresponding inhibition. The ortho and para pyridine-substitutions
formycin B series (Fig. 36) (Stoeckler et al., 1986). increased the selectivity of compounds but when substi-
In 2010, Ho et al. established and generated the four tuted with bulkier groups, the selectivity was lost. Further
generations of transition-state analogs for human PNPase meta-substitutions of pyridine produced converse results.
(Fig. 37).The X-ray crystal structures of human PNPase The important interaction seen was the hydrogen bonding
featured that there is formation of an ion-pair between to Tyr202 (Castilho et al., 2010). The compounds are
bound phosphate and cation inhibitor. The phosphate group represented in Fig. 38.
interacts with hydroxyl groups whereas leaving group In 2010, Postigo et al. discovered new inhibitors of S.
interacts to N-1, O-6 and N-7 of 9-deazahypoxanthine. mansoni purine nucleoside phosphorylase (SmPNP) by
Other important interaction was of His257 with the 50- pharmacophore-based virtual screening (Fig. 39). They
hydroxyl group. It was concluded that third and fourth performed the docking studies and the important interactions
generation PNP inhibitors better occupy the binding site in seen were the meta-bromophenyl substituent with the
comparison to first generation inhibitors (Ho et al., 2010). hydrophobic side chain of Ala118, Val262, His259, Met221,
In 2010, Castilho et al. studied crystallographic, kinetic, and Phe161, T-shaped p-stacking of phenyl ring with the side
and structural bases of purine-based compounds for their chain of Phe161 and Vander Waals interaction of the bro-
selective inhibition of PNPase obtained from Schistosoma mine atom with the side chain of Ala 118 and Val262. In the
mansoni. Electronic, hydrogen bonding, volume, and para position, the hydroxyphenyl was best suited as com-
lipophilic properties were correlated with the extent of pared to fluorophenyl or nitrophenyl. Further meta-
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Med Chem Res
S
CH2CH3
N N
N N N N
N N
R O OH
O OH N N OH
OH
OH 111
N N OH
IC50 (μM)= 0.72 112
IC50 (μM)= 0.09 O OH
OH
OH
N N
N
N N N
N N
O OH
O OH
OH
OH OH
OH
IC50 (μM)=1
IC50 (μM)= 0.62
CCRF-CEM cells (American Type Culture Collection)
113 114
Fig. 41 Inhibition of CEM cell growth by C-6-substituted purine nucleosides
Fig. 42 Chemical Structure of O 0
immucillin-H (115) H the 7 N–H group of the purine ring system. The p–p
HN N interactions were seen between base and the side chain of
Tyr202. The bulkier groups were found to play a crucial
N
OH role in lipophilicity of compounds and their interactions.
HN The compounds with aromatic ring (phenyl, pyridine, and
OH thiophene) were less potent as compared to non-aromatic
HO compounds. Further substitutions on phenyl ring on ortho
or meta positions with hydroxyl or chlorine have shown
substituted analogs were less potent than para-substituted escalation in potency. Most potent compounds were gen-
analogs due to the steric hindrance caused by Met221 and erated via substitution at para position (Postigo et al.,
Ala118. The mono-substituted phenyl thioxothiazolidinone 2011).
derivatives were most potent followed by fair potency of Hassan et al. synthesized C-6 alkyl, cycloalkyl, and
disubstituted derivatives and least potent tri-substituted aryl-9-(b-d-ribofuranosyl)purine analogs and evaluated
phenyl thioxothiazolidinone derivatives (Postigo et al., their potential against E. coli PNPase (Fig. 41) (Hassan
2010). et al., 2012).
In 2011, Postigo et al. further demonstrated enzyme In 2012, Wielgus-Kutrowska et al. explored binding of
kinetics, structural analysis, and molecular modeling immucillin-based compounds in the transition state of tri-
studies on a series of SmPNP (Fig. 40). The catalytic res- meric PNPase. It was concluded that one-third-of-the-sites
idue of Asn245 interacts with carbonyl oxygen by donating binding does not occur for trimeric PNP (Wielgus-Ku-
a hydrogen bond whereas it takes a hydrogen bond from trowska et al., 2012).
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Med Chem Res
Table 1 Mechanisms, uses, side effects, and routes of administration of US-FDA-approved purine-utilizing enzyme inhibitors
Drug Proprietary Dosage Route Mechanism of action Uses Side effects
name form
Febuxostat Uloric Injection IV Xanthine oxidase inhibitor Treat hyperuricemia in gout Hepatic abnormalities, nausea,
(Oliveira-Campos et al., patients (Beara-Lasic arthralgia (Becker et al., 2005a, b)
2008; Becker et al., 2010; et al., 2010)
Takano et al., 2005)
Pentostatin Nipent Injection IV Adenosine deaminase Hairy cell leukemia (Spiers Cough, chills, lower back pain,
inhibitor et al., 1987; Kane et al., pain, painful urination, weakness
1991) (Poi et al., 2013; Dighiero, 1996)
Thioguanine Lanvis Tablet Oral Inhibition of HGPRT Treatment of acute Leukopenia, thrombocytopenia,
(Nelson et al., 1975) lymphoblastic leukemia hepatic toxicity, loss of appetite
in children (Nelson et al., etc. (Gearry et al., 2004)
1975)
Mercaptopurine Purinethol Tablet Oral Inhibition of HGPRT Leukemia, non-hodgkin’s Myelosuppression, bone marrow
(Weinshilboum and lymphoma toxicity etc. (Weinshilboum and
Sladek, 1980) (Weinshilboum and Sladek, 1980)
Sladek, 1980)
Allopurinol Zyloprim Tablet Oral Xanthine oxidase inhibitor Treatment of joint Sore throat, headache, peeling,
(Moorhouse et al., 1987) destruction, uric acid redness of skin, upper stomach
lithiasis, leukemia, and pain, itching, anorexia, and brown
lymphoma (Hershfield stools etc. (Hande et al., 1984)
et al., 2013)
Further in the same year, Deves et al. gave immucillin- mutant purine nucleoside phosphorylase (tm-PNP)
H (Fig. 42) as transition-state analog inhibitor of PNP enzymes were exploited as novel compositions for inhibi-
which arrested bone loss in rat periodontal disease models tion of cancer cell (Parker and Sorscher, 2014).
(Deves et al., 2013).
In 2013, Ducati et al. generalized that immucillin-H has no
lethal effect on attenuation of growth in Staphylococcus FDA-approved PUEIs
aureus. Thus, the cell viability is not affected with PNPase in
methicillin-resistant S. aureus (Stratton and Schramm, 2013). 6-Mercaptopurine was the first approved antimetabolite
Ducati et al. swayed a review on transition-state inhib- followed by allopurinol in 1956 as xanthine oxidase
itors of purine salvage and other prospective enzymes that inhibitor. Table 1 includes some of the US-FDA-approved
target malaria (Ducati et al., 2013). drugs which signify the importance of purine-utilizing
In 2014, Donaldson et al. determined structural charac- enzyme inhibitors in various diseases ranging from cancer
teristics of P. falciparum purine nucleoside phosphorylase to autoimmune disorders.
(PfPNP) responsible for the efficiency and specificity of
catalysis. Through site-directed mutagenesis, molecular
simulations, and circular dichroism analysis, the significance Summary and conclusions
of various residues which are vital for PfPNP activity was
investigated. It was also explored that mutation in PfPNP In general, PUEIs require presence of a purine-like het-
was responsible for the loss of 50-methylthio activity while erocyclic structure(s) as a prerequisite in their chemical
retaining inosine activity. Tyr160 is a conventional architecture which is fully reflected in case of naturally
replacement for the Phe residue present in human PNP, occurring or synthetic heterocyclics. Most of these inhibi-
whereas Val66, Val73, and Tyr160 residues are responsible tors act either through competitive or non-competitive
for the catalytic efficiency of PfPNP 50-methylthio activity. inhibition and a few are allosteric inhibitors (Fig. 43). The
50-methylthio group was used for the optimization and as the conformational aspects of the compounds have revealed
rational target against malaria (Donaldson et al., 2014). the crystal structure of the required binding site and various
Parker and Sorscher demonstrated an approach targeting interactions responsible for potency. DNA topoisomerase
wild-type Trichomonas vaginalis purine nucleoside phos- inhibitors include isoindenoquinoline derivatives, novel
phorylase (Tv-PNP) enzyme cancer cell progression. This benzoxazole, benzimidazole derivatives, etc. These inhib-
involved exposure of the enzyme to a substrate cleaved by itors find their applications as anticancer, antiangiogenic,
the enzyme resulting in a cytotoxic purine analog. Tailed and antiproliferative agents. Novel transition-state analog
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Med Chem Res
Fig. 43 Summary of purine-utilizing enzymes and their inhibitors
inhibitors of guanase were based on azepinomycin ring autoimmune disorders. Recently disclosed X-ray crystal
structure, whereas acyclic nucleoside phosphonates were structures of PUEs with endogenous substrates or inhibitors
found to be inhibitor of HGPRT. The inhibitors of HGPRT of heterocyclic nature along with biochemical, biophysical
are mostly used as antimalarials. Non-purine analogs, studies, and medicinal attributes have opened up a new era
thiazolo-pyrazolyl compounds, pyrazoles, substituted pyr- of rational drug designing which will surely offer scope for
azolotriazolopyrimidines derivatives, xanthone derivatives, the development of better and safer drugs.
and substituted pyrazolopyrimidines were established as
xanthine oxidase inhibitors. These have been used as free Acknowledgments Authors are thankful to DST, New Delhi for
providing the financial aid (DST project Grant No. SR/FT/CS-71/2011)
radical scavengers and antioxidants as well. PNP inhibitors
to carry out the present work. The financial assistance received from
find their applications in rheumatoid arthritis, tissue Research Seed Money (RSM), CUPB (GP-25) Grant is also
rejection, chemotherapy for T-cell leukemias, and other
123
Med Chem Res
acknowledged. Authors are also thankful to Dean Academic Affairs, HMX under anaerobic conditions. Biochem Biophys Res
Central University of Punjab, Bathinda for his constant encouragement. Commun 306(2):509–515
Bobrovnikova-Marjon E, Hurov JB (2014) Targeting metabolic
changes in cancer: novel therapeutic approaches. Annu Rev
Med 65:157–170
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