lable at ScienceDirect

European Journal of Medicinal Chemistry 85 (2014) 371e390

Contents lists avai

European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Mini-review

1,3,5-Triazine-based analogues of purine: From isosteres to privilegedscaffolds in medicinal chemistry

Felicia Phei Lin Lim a, Anton V. Dolzhenko a, b, *

a School of Pharmacy, Curtin Health Innovation Research Institute, Curtin University, GPO Box U1987, Perth, Western Australia 6845, Australiab School of Pharmacy, Monash University Malaysia, Jalan Lagoon Selatan, Bandar Sunway, Selangor Darul Ehsan 47500, Malaysia

a r t i c l e i n f o

Article history:Received 31 December 2013Received in revised form11 April 2014Accepted 31 July 2014Available online 1 August 2014

Keywords:IsosterePurineTriazineEnzyme inhibitorAnticancerAntiviral

* Corresponding author. School of Pharmacy, MonaLagoon Selatan, Bandar Sunway, Selangor Darul Ehsan

E-mail addresses: A.Dolzhenko@curtin.edu.au, AnDolzhenkoAV@gmail.com (A.V. Dolzhenko).

http://dx.doi.org/10.1016/j.ejmech.2014.07.1120223-5234/© 2014 Elsevier Masson SAS. All rights re

a b s t r a c t

Purines can be considered as the most ubiquitous and functional N-heterocyclic compounds in nature.Structural modifications of natural purines, particularly using isosteric ring systems, have been in thefocus of many drug discovery programs. Fusion of 1,3,5-triazine ring with pyrrole, pyrazole, imidazole,1,2,3-triazole or 1,2,4-triazole results in seven bicyclic heterocyclic systems isosteric to purine. Appli-cation of the isosterism concept for the development of new compounds with therapeutic potential inareas involving purinergic regulation or purine metabolism led to significant advances in medicinalchemistry of the azolo[1,3,5]triazines. These 1,3,5-triazine-based purine-like scaffolds significantly in-crease level of molecular diversity and allow covering chemical space in the important areas of medicinalchemistry. Some of these azolo[1,3,5]triazine systems have become privileged scaffolds in the devel-opment of inhibitors of various kinases, phosphodiesterase, xanthine oxidase, and thymidine phos-phorylase, antagonists of adenosine and corticotropin-releasing hormone receptors, anticancer andantiviral agents.

© 2014 Elsevier Masson SAS. All rights reserved.

1. Introduction

Purine, a nitrogen containing heterocycle, is found abundantlyin nature. It is the core structure of adenine and guanine in RNA andDNA [1]. From a biological perspective, attention to purines ismainly driven by the fact that purine nucleotides (ATP, GTP, cAMP,cGMP, NAD, FAD) act as co-factors, substrates, or mediators in thefunctioning of many proteins [1,2]. These proteins are estimated toinclude half of the most druggable targets, primarily enzymes andreceptors. For example, phosphorylation of ATP is carried out byprotein kinases [3] while hydrolysis of cAMP and cGMP is associ-ated with cyclic nucleotide phosphodiesterases [4]. Purine catab-olism involves the key enzyme xanthine oxidase [5] whereas theubiquitous purine nucleoside phosphorylase [6] also plays a vitalrole in the purine salvage pathway. Adenosine receptors constitutea very promising group of G-protein coupled receptors (GPCRs) [7].

Advancements in drug design and development on the basis ofpurine motif inspired parallel development in the chemistry and

sh University Malaysia, Jalan47500, Malaysia.

ton.Dolzhenko@monash.edu,

served.

biology of structurally related heterocyclic system viz. purine iso-steres. Increasingmolecular diversity using various purine isosteresis ideal for the discovery of novel therapeutic agents that targetselectively purine dependent enzymes and receptors.

A number of renowned drugs with purine isostere core struc-ture are available in the market (Fig. 1), including chemothera-peutic temozolomide [8], a pro-drug functioning as a moleculartransporting device to deliver a reactive methylating species toguanine-rich sequences in the major groove of DNA. One of thecornerstones of acute leukemia treatment is antimetabolite 8-azaguanine [9], while an alternative therapeutic agent forodesine[10], a second generation purine nucleoside analogue, is currentlyundergoing clinical trials. Forodesine exhibits highly selective pu-rine nucleoside phosphorylase inhibitory activity in preclinicalstudies with malignant cells and clinical efficacy against T-cellacute lymphoblastic leukemia and cutaneous T-cell lymphoma [10].Another structurally similar purine nucleoside phosphorylase in-hibitor ulodesine (BCX-4208) [11] is also under clinical investiga-tion as the drug for treatment of gout in hope of a synergistictherapy with hypoxanthine bioisostere allopurinol, a drug inhibit-ing xanthine oxidase and therefore preventing formation of uricacid [12]. Well known PDE5 inhibitors, sildenafil and vardenafil [13]have been extensively used for the treatment of erectile dysfunc-tion while their prototype zaprinast was found to demonstrate

Fig. 1. Therapeutic agents based on isosteric purine skeletons.

F.P.L. Lim, A.V. Dolzhenko / European Journal of Medicinal Chemistry 85 (2014) 371e390372

interesting orphan G-protein coupled receptor GPR35 activatingproperties [14]. Reversible purine P2Y12 receptor antagonist tica-grelor with the 8-azapurine scaffold is an oral antiplatelet agentrecently approved for themanagement of acute coronary syndrome[15]. Hypnotic agents zaleplon [16] and indiplon [17] exert theireffects through binding to the GABAA receptors at the benzodiaz-epine recognition site, for which hypoxanthine and inosine arenatural ligands. Also noteworthy, a group of 7-deazapurinestubercidin [18], toyocamycin [19] and sangivamycin [19] whichfunction as antimetabolites and are used as broad spectrumantibiotics.

There are extensive and ongoing research and developmentactivities around purine isosteres and one of the promising di-rections has a focus on using 1,3,5-triazine-based isosteres of pu-rine (Fig. 2). This group of azolo[1,3,5]triazine systems has anitrogen atom in the position 5 of the purine ring and therefore canbe generally categorized as 5-aza-isosteres of purine. The presentreview focuses on the scope of biological activities displayed bycompounds with these scaffolds (IeVII) and their potential appli-cations as therapeutic agents. The research work on chemistry andbiological activity of compounds constructed on the basis of scaf-folds IeVII is not equally distributed between different groups.Thus, pyrazolo[1,5-a][1,3,5]triazines (5-aza-9-deazapurines, II)and 1,2,4-triazolo[1,5-a][1,3,5]triazines (5-azapurines, V) have

undergone more active investigations [20,21], while biologicalproperties of systems I, VI and VII have remained almost unex-plored. Therefore, primary organization of sections in this review isbased on pharmacological effects of the compounds rather thantheir relation to the specific heterocyclic system.

2. Enzymes inhibitors

Almost half of all marketed drugs specifically target enzymes,therefore demonstrating a huge potential for development of newtherapeutic agents modulating enzyme activity [22]. The com-pounds constructed using 1,3,5-triazine-based isosteres of purineaffect enzymes of different groups as discussed below.

2.1. Kinase inhibitors

Kinases are one of the most promising groups of enzymes in thefield of drug discovery. Protein kinases represent about 20% of thedruggable genome. They have been in the focus of intensive in-vestigations resulting in 20 kinase-targeting drugs approved forclinical use over the past decade and hundreds of drug candidatesundergoing clinical trials [23]. It is estimated that protein kinaseinhibitors are main targets in 50e70% of current cancer drug dis-covery programs [23].

Cyclin-dependent kinases (CDKs) are a family of enzymesplaying a key role in cell cycle regulation. Overexpression of CDKs incancer cells makes them an attractive drug target in the fightagainst oncological diseases [24e28]. Development of CDK in-hibitors appears to be a promising strategy in the search for neweffective anticancer agents. A number of CDK inhibitors arecurrently at different stages of clinical trials.

Isosteric to purine system, pyrazolo[1,5-a][1,3,5]triazine scaffoldwas used as a template for new potent CDK2 inhibitors. Variouslydecorated 2,4-diamino substituted pyrazolo[1,5-a][1,3,5]triazines(1e4) demonstrated high CDK2 inhibitory activity (Fig. 3) [29e31].The CDK inhibitory activity was translated into in vitro anti-proliferative activity against prostate (PC3) and colon cancer(HCT116) cell lines [31]. Even though there was a decrease of abouta hundred times in the potency of CDK2 inhibition between com-pounds 2 and 3, macrocyclic compound 3 showed almost ten timesimprovement in the anticancer activity [31]. The macrocyclic lac-tam ring provided a less planar and more three-dimensionalstructure of overall molecule resulting in an increase in mem-brane permeability ultimately improving cellular activity.Enhancement of CDK2 inhibitory potency was successfully ach-ieved by replacing the cyclopropylamino group with a more com-plex substituted arylamino moiety (compound 4) [31]. Thismodification of the structure also improved aqueous solubility of 4which led to an increase in cellular activity against both humanprostate and colon cancer cell lines in in vitro experiments [31].

A purine based CDK inhibitor (R)-roscovitine (5) is a drugcandidate currently undergoing clinical trials as an anticancertherapeutic agent [32e34]. This molecule has been serving as a leadfor the development of new potent compounds targeting CDKs. Anisosteric replacement of purine system of roscovitine (5) with thepyrazolo[1,5-a][1,3,5]triazine core led to the development of apotent CDK2 inhibitor 6 [33]. This isosteric modification providedmore than 5 times improvement in the inhibitory activity. Rosco-vitine (5) and pyrazolo[1,5-a][1,3,5]triazine 6 demonstrated similarconformations and binding modes to CDK2 [33]. A similarity in thepharmacokinetic profile of 6 and roscovitine (5) was also observed[33].

Tested against the National Cancer Institute panel of 60 tumorcell lines, 6 was about 14 times more potent than roscovitine (5)with no preference towards any specific form of tumor [34]. An

Fig. 2. 1,3,5-Triazine-based purine-like scaffolds.

F.P.L. Lim, A.V. Dolzhenko / European Journal of Medicinal Chemistry 85 (2014) 371e390 373

improved in vitro biological activity of compound 6was also provenin the Ewing's sarcoma xenograft mouse model system. Bothroscovitine (5) and 6 showed more than 70% of tumor inhibition,but dose of roscovitine was twice higher (50 mg/kg) than that of 6(25 mg/kg) [34].

Additionally to antiproliferative activity, inhibition of CDKs wasfound to play an important role in the suppression of viral

Fig. 3. CDK in

replication [35]. CDK inhibitors were proposed as prospective drugcandidates for the treatment of HIV-1 infection [35,36]. (R)-Roscovitine (5) inhibited viral transcription by targeting the CDK2/cyclin E complex and binding to ATP pockets that led to apoptosis inHIV-1 infected T-cells, peripheral blood mononuclear cells andmonocytes [37]. More potent viral inhibitionwas achieved by usingroscovitine analogue CR8 (7), which also demonstrated better

hibitors.

F.P.L. Lim, A.V. Dolzhenko / European Journal of Medicinal Chemistry 85 (2014) 371e390374

elimination of HIV-1 infected cells compared to uninfected cells.Using pyrazolo[1,5-a][1,3,5]triazine scaffold instead of the purinesystem led to the development of MRT 3-028 (8). In inducing celldeath, MRT 3-028 (8) was 3 times more selective towards HIV-1infected ACH2 and OM10.1 cells compared to uninfected cells(CEM, JURKAT, and U937). Modification of side chain of 8 resulted inthe development of another interesting compound CR8#13 (9) [35].The unique aspects of CR8#13 (9) manifested in its low cytotoxicityprofile and high downregulation of HIV-1 transcription [35,37]. Inaddition, null effect demonstrated by CR8#13 (9) on the down-stream CDK9 effector genes and cell viability suggested the capa-bility of selective targeting towards HIV-1 transcripts [35].

Recently, studies have been conducted to search for compoundswith selective CDK7 inhibition. CDK7, which is part of the CDK-activating kinase (CAK) complex, plays an essential role in activa-tion of other CDKs through phosphorylation. Hence, the require-ment for CDK7 as a regulator of transcription makes it a viabletarget for antiviral and anticancer treatment. Compounds 10[38,39] and 11 [40,41] were found to possess selective inhibition ofCDK7 in comparisonwith CDK2 and CDK9. This selectivity was 2e3orders higher than that of the ‘state of the art’ CDK inhibitor fla-vopiridol (alvocidib).

Activated Cdc42-associated tyrosine kinase 1 (ACK1) is a non-receptor tyrosine kinase, which has been recently explored as atarget for cancer therapy [42]. Amplification of the ACK1 gene inprimary tumors leads to poor prognosis. Hence, using ACK1 in-hibitors was suggested for the anti-cancer treatment. In the searchfor new inhibitors of ACK1, Jiao et al. identified imidazo[1,2-a][1,3,5]triazine 12 exhibiting inhibition of ACK1 in micromolarconcentrationwhen evaluated in biochemical autophosphorylationassays (Fig. 4) [43].

Casein Kinase II (CK2) is a ubiquitous and highly conservedprotein serine/threonine kinase involved in cell proliferation,transformation, senescence and apoptosis [44,45]. Experimentalevidence confirming the association of an increase expression ofCK2 in human cancers underlines the importance of CK2 as anexcellent target for anti-cancer therapy [44,45]. Hu et al. [46] re-ported that substituted 2,4-diaminopyrazolo[1,5-a][1,3,5]triazine,such as 13a,b effectively targeted CK2 (Fig. 4). Cyano group in po-sition 8 of the pyrazolo[1,5-a][1,3,5]triazine system seems to play acritical role in the activity of the compounds. Even small alterationby changing the cyano group to an ethyl caused almost a 4-orderdrop in the inhibitory potency of the compounds.

2.2. Phosphodiesterase inhibitors

Cyclic nucleotide phosphodiesterases (PDEs) represent a groupof enzymes responsible for the hydrolysis of secondary messengerscAMP and cGMP. Uneven distribution of specific PDE types invarious tissues and cell types prompted the search of novel PDEinhibitors with focus on the selective inhibition of enzyme iso-forms. Using purine and its isosteres as scaffolds in the PDE in-hibitor design has been proved a fruitful avenue with several

Fig. 4. Other kinase inhibitors.

launched blockbuster drugs [47e54]. Variations in isosteric scaf-folds and manipulation with substitution pattern allowed tuningthe selectivity of compounds towards specific PDE types.

Some PDE inhibitory compounds with pyrazolo[1,5-a][1,3,5]triazine scaffold were identified at the beginning of the PDE era(Fig. 5). These compounds, such as 14, were hundreds time morepotent than reference of that time theophylline [55].

Awide range of substituents at the pyrazolo[1,5-a][1,3,5]triazinesystem was tolerated with changing selectivity profile towardsvarious PDE. Compound 15 [56,57] exhibited selective inhibition ofcAMP PDE isolated from the lung, while 16 [56,58,59] demon-strated high level of selectivity on cAMP PDE obtained from thebrain. Another interesting pyrazolo[1,5-a][1,3,5]triazine derivativerevealed at that time was 4-azido-7-phenylpyrazolo[1,5-a][1,3,5]triazine (17) [60]. This compound (17) demonstrated strong irre-versible inhibition of PDE1. Detail analysis of selectivity for thiscompound has not been reported, but earlier data [55] suggestedsome selectivity in the inhibitory activity of 17 to PDE1.

Development of PDE4 inhibitors as potential candidates for thetreatment of autoimmune and inflammatory diseases led to thediscovery of very potent pyrazolo[1,5-a][1,3,5]triazines, e.g. 18a,b[61].The selectivity profile of 18a,b towards PDE4 was analyzed bycomparisonwith PDE1, PDE2, PDE3 and PDE5. The results showed ahigh level of their selectivity towards the PDE4 isoform [61].

Pyrazolo[1,5-a][1,3,5]triazine 19 demonstrated subnanomolarinhibition of PDE10 and should be mentioned among recentlyclaimed inhibitors of this enzyme [62].

In search for compounds displaying PDE5 inhibitory activity,Haning et al. [63] conducted a comparative study, identifying imi-dazo[1,5-a][1,3,5]triazines as a suitable skeleton for the construc-tion of potential PDE inhibitors, e.g. compounds 20 and 21.Adjusting the substitution at both imidazole and triazine ringsallowed successful tuning of selectivity to PDE5. It should be notedthat imidazo[1,5-a][1,3,5]triazine analogue of Sidenafil, compound21 also demonstrated in vivo oral efficacy when tested on a rabbitmodel of erectile dysfunction.

The imidazo[1,5-a][1,3,5]triazine scaffold was successfully usedin the development of selective PDE7 inhibitors [64]. Compound 22and its analogues were identified as potent PDE7 inhibitors with a10 times higher selectivity towards PDE7 when compared to PDE4.

2.3. Xanthine oxidase inhibitors

Xanthine oxidase (XO) is a key enzyme of the purine catabolisminvolved in the oxidation of hypoxanthine to xanthine and finally touric acid. This enzyme is a well-recognized target in therapy ofhyperuricemia and chronic gout [65e67].

A variety of compounds based on isosteric to guanine, hypo-xanthine and xanthine scaffolds were extensively investigated byRobins et al. (Fig. 6) [68]. 5-Aza-hypoxanthine (23), which is also anisostere of the standard anti-gout drug allopurinol, demonstratedrelatively low XO inhibitory activity. 5-Azaxanthine (24a), isostericto oxypurinol, and its thio-analogue 24b were also marginallyactive. S-Methylation of 24b yielded compound 25b, which was 70times more active than the parent molecule (24b) and was alsomore effective than allopurinol. O-Methylation of 5-azaxanthine(24a) had an opposite effect on the activity (25a was even lessactive than parent structure 24a). An isostere of guanine withimidazo[1,2-a][1,3,5]triazine scaffold (26) also demonstrated someXO inhibitory effect [68].

The most interesting results were obtained when pyrazolo[1,5-a][1,3,5]triazine skeleton was used for the construction of XO in-hibitors. The isostere of hypoxanthine and allopurinol based on thisskeleton (27a) possessed lowactivity, similar to that of its structuralanalogue with 1,2,4-triazolo[1,5-a][1,3,5]triazine scaffold (23) [68].

Fig. 5. PDE inhibitors.

F.P.L. Lim, A.V. Dolzhenko / European Journal of Medicinal Chemistry 85 (2014) 371e390 375

Interestingly, introduction of phenyl group to position 7 of thepyrazolo[1,5-a][1,3,5]triazine system led to dramatic increase in theXO inhibitory effect. Compound 27bwas identified as a very potentXO inhibitor with more than three order greater activity in com-parison to unsubstituted 5-aza-9-deazahypoxanthine (27a). It wasdemonstrated that XO inhibitory activity could not tolerate sub-stitutions at position 2 of 27b. Even minor structural modificationat this position (e.g. 27c) led to significant loss of the activity.

A very potent XO inhibitor, BOF 4272 (28) was synthesized by aJapanese research group [69e71]. Promising results from in vitroand in vivo studies [72e74] together with experimental data onhealthy human volunteers [75] demonstrated great potential ofBOF 4272. A significant reduction of the XO activity was achieved bytargeting the liver and small intestine, which are main organs foruric acid production in human. Cell necrosis could also be

Fig. 6. XO in

prevented by BOF 4272 through the decrease in concentration offree radicals generated by XO [76]. Detailed study of themechanismof XO inhibition [72,73] by BOF 4272 showed that the activity ofBOF 4272 was governed by the stereochemistry of the molecule.Both enantiomers were determined to be mixed type inhibitorswith the (S)-(�)-enantiomer having much higher potency than the(R)-(þ)-enantiomer (Fig. 6) [72,73]. The stereoselectivity alsodetermined pharmacokinetic parameters and biotransformation ofBOF 4272 [77e81]. Therefore, further studies were conducted onthe asymmetrical (S)-(�)-BOF 4272 [82e84].

2.4. Thymidine phosphorylase inhibitors

Thymidine phosphorylase (TP), also known as gliostatin orplatelet-derived endothelial cell growth factor, is a key enzyme in

hibitors.

F.P.L. Lim, A.V. Dolzhenko / European Journal of Medicinal Chemistry 85 (2014) 371e390376

the pyrimidine nucleoside salvage pathway. TP is overexpressed invarious solid tumors where it is involved in regulation of cellapoptosis, proliferation and angiogenesis whichmakes this enzymea valid target for anticancer therapy [85,86].

5-Aza-xanthine derivatives and their thio-analogues based oncore structures 24were evaluated as potential TP inhibitors (Fig. 7).Compounds based on 24a scaffold were inactive, while 24binhibited this enzyme with IC50 value of 58.1 mM [87] that led tofurther exploration of this series.

Many substitutions were tested at position 2 of the triazolo[1,5-a][1,3,5]triazine system without any loss of activity. Particularimprovementwas observedwith introduction of an aromatic groupin this position (e.g. compound 29) [87e89]. One of the most activecompound identified in this series was triazolo[1,5-a][1,3,5]triazine30, which inhibited TP via a competitive mixed-type (with respectto thymidine) mechanism with a Ki value of 20.1 mM. The TPinhibitory potency was further improved by introducing a methy-lene spacer between dichlorophenyl moiety and the triazolo[1,5-a][1,3,5]triazine core. The resulted compound 31was also found to bea competitive mixed-type TP inhibitor (Ki ¼ 19.6 mM). Compounds30 and 31 inhibited expression of some angiogenesis markers,namely matrix metallopeptidase 9 (MMP-9) and vascular endo-thelial growth factor (VEGF) in MDA-MB-231 cells [88].

Further exploration of structureeactivity relationship revealedthat changes of the substitution pattern at the 1,3,5-triazine ringcannot be tolerated. The alkylation of a thiocarbonyl group orswapping positions of 5-thiocarbonyl and 7-carbonyl groupsresulted in significant loss of TP inhibitory activity [88,90].

Fig. 7. TP inh

Similar observations were reported for related molecules basedon the pyrazolo[1,5-a][1,3,5]triazine core [91]. Phenyl substitutionat C-7 of the pyrazolo[1,5-a][1,3,5]triazine system also increased TPinhibitory activity (32a), which were further improved by intro-ducing lipophilic substituents to the phenyl ring. The most activecompound in this series was 32b. Changing position of the arylsubstituent at the pyrazole ring significantly improved the TPinhibitory activity (33a). Interestingly, introduction of a penta-fluorosulfanyl group (compound 33b) to the phenyl ring led todramatic increase of the activity. Among pyrazolo[1,5-a][1,3,5]tri-azines, this compound (33b) was the most potent, inhibiting TP inthe non-competitive manner [91].

An attempt to improve TP inhibitory activity of pyrazolo[1,5-a][1,3,5]triazines of both types by the introduction of the chlorouracilmoiety, pharmacophoric for this type of activity led to series ofactive compounds (e.g. 34 and 35), which were usually more potentthan parent structures 32 and 33 (Fig. 7) [92]. However 35b, beingthe most potent compound in this series, was almost one order lessactive than its prototype 33b. Enzyme kinetics study revealed amixed type of TP inhibition for 35b.

In another series of the pyrazolo[1,5-a][1,3,5]triazine based in-hibitors of TPwith the thioureamoiety in position 7, introduction oflipophilic substituent in position 8 was reported to increase theenzyme inhibitory activity [93]. A selection of 8-benzyl substitutedcompounds and their analogues was tested and 4-trifluoromethylbenzyl substituted compound 36b was found to bethe most potent in this series. The introduction of the 4-trifluoromethyl group to the phenyl ring led to almost 8 times

ibitors.

F.P.L. Lim, A.V. Dolzhenko / European Journal of Medicinal Chemistry 85 (2014) 371e390 377

more potent compound than 8-benzyl substituted pyrazolo[1,5-a][1,3,5]triazine 36a. Similarly to other compounds with triazolo[1,5-a][1,3,5]triazine and pyrazolo[1,5-a][1,3,5]triazine scaffolds, 36binhibited TP via a competitive mixed-type (with respect to thymi-dine) mechanism with a Ki value of 1.8 mM. It was also found that36b significantly reduced the VEGF expression in MDA-MB-231cells without affecting the cell growth at concentrations up to100 mM.

2.5. Miscellaneous enzyme inhibitors

Dipeptidyl peptidase IV (DPP-IV), also known as adenosinedeaminase complexing protein 2, is a type II membrane proteinused as a target in the treatment of some metabolic disorders,most notably type 2 diabetes [94]. Compounds with variousisosteric to purine scaffolds, such as 37, 38 and 39 demonstratedselective inhibition of DPP-IV (Fig. 8) [95]. They were claimed toinhibit DPP-IV at the concentrations 50 times lower than thoserequired for inhibition of similar enzyme, fibroblast activationprotein a (FAPa).

Compounds constructed using the pyrazolo[1,5-a][1,3,5]triazinescaffold were reported to have inhibitory activity against variousenzymes. Compounds 40 and 41 (Fig. 8), with the basic alkylaminomoiety linked to position 4 of the pyrazolo[1,5-a][1,3,5]triazineskeleton via long alkyl chain, were tested in form of hydroiodidesand demonstrated inhibition of protein methyl transferase 1 (PRMT1) [96]. Some pyrazolo[1,5-a][1,3,5]triazines were able to inhibitbacterial enzyme, DNA gyrase, a target for the antibacterial therapy[97]. Compound 42 demonstrated similar values (0.5 mg/mL) for themaximal non-effective concentration (MNEC) towards DNA gyraseand the minimal inhibitory concentration against Staphylococcuspyogenes [97]. Similarly to purine-based antiviral agent (S)-9-(2,3-dihydroxypropyl)adenine (S-DHPA), its pyrazolo[1,5-a][1,3,5]triazine analogue 43 was reported [98] to inhibit S-adenosylho-mocysteinase, though being less active than S-DHPA.

Fig. 8. Miscellaneous e

The 1,2,4-triazolo[4,3-a][1,3,5]triazine ring system was used inthe design of 44, which selectively inhibited migration of COS-1cells expressing MMP-9 and did not affect migration of MMP-2and MMP-14 expressing cells (Fig. 8) [99]. This compound (44)also inhibited migration and invasion processes of human invasivecancer cell lines expressing high endogenous level of MMP-9 (HT-1080 andMBD-MB-435). At the same time, 44 did not affect MMP-9expression and demonstrated no proteolytic activity or cytotoxicityproviding therefore solid evidence of MMP-9 inhibition as anexplanation of its effects. It should also be noted that the samecompound (44) was also claimed to be a PDE4 inhibitor [100].

3. Receptor ligands

Receptors are responsive to a plethora of endogenous ligandsand stimuli and hence responsible for the regulation of diversecellular responses. An estimate of 40% clinically approved drugsutilizes the receptor-ligand signaling pathway making themattractive targets for drug discovery [101,102]. The compoundsconstructed using 1,3,5-triazine-based isosteres of purine affectdifferent types of receptors as discussed below.

3.1. Adenosine receptor ligands

Adenosine is an endogenous nucleoside ubiquitous inmammalian cells. It mediates many physiological effects viawidelydistributed specific cell membrane G-protein coupled receptors(GPCR) of four known subtypes viz. A1, A2A, A2B and A3 adenosinereceptors [103,104]. The A2A adenosine receptors are widelyexpressed in the central nervous system and are involved in theregulation of many physiological and pathological processes.Structural similarity to purine drives the extensive exploration ofazolo[1,3,5]triazines as potential A2A receptor ligands. Comparativestudies [105,106] of A2A receptor antagonists with different het-erocyclic skeletons showed that 1,2,4-triazolo[1,5-a][1,3,5]triazines

nzyme inhibitors.

F.P.L. Lim, A.V. Dolzhenko / European Journal of Medicinal Chemistry 85 (2014) 371e390378

(5-azapurines) displayed the highest binding affinity to A2A re-ceptors. The ability of 1,2,4-triazolo[1,5-a][1,3,5]triazines to act asselective A2A receptor antagonists opens an opportunity for them toserve as potential therapeutic agents for the treatment of Parkin-son's disease, depression, anxiety, cerebrovascular disorders andseizures.

The structureeactivity relationship studies of adenosine recep-tor ligands based on the 1,2,4-triazolo[1,5-a][1,3,5]triazine systemrevealed crucial importance of the furyl moiety at position 2 andthe primary amino group at position 7. These structural features areessential for the effective binding affinity towards adenosine re-ceptors (especially A2A subtype), while variations of substituents atposition 5 can be used to further tune affinity, selectivity andbioavailability of the compounds [107]. Introduction of the phe-noxy group at position 5, while maintaining the furyl moiety andprimary amino group at positions 2 and 7 respectively, led to potentA2A adenosine receptor ligand 45, which was also active in theadenyl cyclase functional assay at the A2B receptor subtype(EC50 ¼ 3.42 mM) (Fig. 9) [107,108]. An interesting transformation ofa rather selective A2B receptor antagonist LUF 5451 (46) into a se-lective A1 receptor ligand 47 was carried out through the additionof a second phenyl ring at the a-carbon of the benzylamino moietyat position 5 of 1,2,4-triazolo[1,5-a][1,3,5]triazine core [109].

Another interesting molecule is ZM241385 (48), a well-knownpotent and selective A2A receptor antagonist [110]. X-ray crystal-lography studies showed pattern of key interactions of ZM241385with the A2A adenosine receptors [110]. The hydrophobic

Fig. 9. 5-Azapurines as adeno

interactions between the furan moiety and Trp 246, an importantresidue in the A2A receptor activation, was believed to constrain thereceptor in an inactive state by inhibiting structural rearrange-ments necessary for reactivation. ZM241385 is now widely used asa [3H]-ZM241385 probe in adenosine receptor studies [111]. Re-ports on pharmacological activity of ZM241385 include anticon-vulsant properties when tested against amygdala-kindled seizures[112] and functioning as neuroprotective agents against cerebralischemia [113], Parkinson's disease [113e116] and progressivesupranuclear palsy [115]. Derivatives of ZM241385 (48) were foundto tolerate substitution at the phenolic oxygen [107,117]. A2A re-ceptor binding potential of the ligands was mostly maintainedupon alkylation or acylation at this position. At the same time,alkylation or acylation of the 7-amino group was found to signifi-cantly decrease binding affinity of ligands towards A2A adenosinereceptors [117]. However, the affinity towards human A3 receptorscan be improved by replacement of the 7-amino group by the ureamoiety [107].

Using ZM241385 as a lead, enhancement of oral bioavailabilityof the compounds was achieved via inclusion of a piperazine spacerat position 5 of the 1,2,4-triazolo[1,5-a][1,3,5]triazine scaffold. Thisyielded Biogen 34 (49) [108,118], which demonstrated very goodresults in the radioligand binding assays and showed significantactivity in the animal Parkinson's disease model at 3 mg/kg ( p.o.).Yang et al. conducted further evaluation on Biogen 34 (49) andfound that its affinity to A2A adenosine receptors differed inhumans (Ki ¼ 12.4 nM) and rats (Ki ¼ 1.2 nM) [118].

sine receptor antagonists.

Fig. 11. Adenosine receptor ligands.

F.P.L. Lim, A.V. Dolzhenko / European Journal of Medicinal Chemistry 85 (2014) 371e390 379

Pharmacokinetic results revealed that Biogen 34 (49) had a shorthalf-life and poor brain penetration therefore indicating that thiscompound was more suitable for the in vitro A2A adenosine re-ceptor studies rather than therapeutic use [118].

Interesting fluorinated compounds (e.g. 50 and 51) with apiperazine spacer were identified in radioligand binding assays[119,120]. These compounds (50 and 51) also showed significantactivity in animal Parkinson's disease models at 3 mg/kg (p.o.).

Federico et al. [121] further explored variations at the C-5 po-sition of the 1,2,4-triazolo[1,5-a][1,3,5]triazines with the aim ofsynthesizing A2A receptor antagonists with improved water solu-bility and physico-chemical properties. Among the series of com-pounds evaluated, the two most potent compounds, 52 and 53 (astrifluoroacetates) displayed better water solubility and good affin-ity and selectivity towards A2A adenosine receptors.

In an attempt to design an antiparkinsonian prodrug consistingof two synergistic molecules viz. dopamine (57) and a triazolo-triazine A2A receptor antagonist 52, which are linked by a succinicacid spacer, compound 54 was developed (Fig. 10) [122]. In ratstriatum and hA2A CHO cells, 54 demonstrated high affinity towardsA2A adenosine receptors, but showed no affinity to D2 dopaminereceptors. Degradation of 54 in whole human blood followed firstorder kinetics and suggested the breakdown of the prodrug viahydrolysis providing 52 and inactive N-succinyldopamine (55). Inin vitro testing using rat brain homogenates, it was observed thatcontrolled release during hydrolysis of prodrug 54 may providemoderate dopamine tone at higher interval times in the CNScompared to those of the L-DOPA therapy. This may potentially limitneuronal toxicity and side effects from pulsatile L-DOPA long termtreatments acting as a possible alternative to the DUODOPA sys-tems. These advantageous effects may be supported by the A2Aantagonistic effect of prodrug 54 together with its main hydrolysisproduct (56) which has a better stability in rat brain homogenatescompared to its parent antagonist (52) [122].

The pyrazolo[1,5-a][1,3,5]triazine scaffold was also successfullyused in the design of A2A receptor antagonists. Based on the potentA2A adenosine receptor antagonist, ZM241385 (48), a deaza-analogue 58 was synthesized (Fig. 11) [109]. This compound (58)was found to possess high affinity to adenosine receptors, but itsselectivity towards A2A receptors was lower compare to ZM241385(48).

A number of structural variations were explored for compounds59with the imidazo[1,2-a][1,3,5]triazine scaffold (Fig. 11) [123,124].

Fig. 10. Antiparkinsonian 5-azapurine-based prodrug (53).

The phenyl moiety was preferred at position 2, while position 7could tolerate various groups (including phenyl, e.g. 59a), but smallalkyl groups were preferred. Modifications of the amino group atposition 4 were used to tune selectivity to adenosine receptorsubtypes. Thus, introduction of acetonyl moiety to the amino groupat position 4 led to a very potent and highly selective A1 adenosinereceptor ligand 59b [124]. Similarly to 5-azapurines and othersimilar structures, acylation of the primary amino group increaseaffinity to A3 adenosine receptors and decrease affinity to A2A re-ceptors as exemplified by very potent A3 receptor ligands 59c and59d [123].

3.2. Cannabinoid receptor ligands

Cannabinoid (CB) receptors are known to control downstreamsignaling pathways regulating energy metabolism and processes inthe CNS and immune system. To date, two subtypes of the receptorshave been identified: CB1 receptors, localized predominantly onpresynaptic terminals, and CB2 receptors, widely distributed inperipheral tissues, primarily in immune cells [125]. Other receptors(GPR55 and GPR119) are also sometimes claimed to be CB receptors[126].

The CB1 receptors are connoted to be targets for the treatment ofCNS disorders, inflammation and metabolic diseases [125,127]. Itwas reported that pyrazolo[1,5-a][1,3,5]triazine 60a (CE 178253),used as benzenesulfonate salt, possessed high affinity and selec-tivity towards CB1 receptors (Fig. 12) [128]. Antagonism of com-pound 60a towards CB1 receptors and its synergism withantiparkinsonian action of levodopa was demonstrated using amodel of Parkinsonism in rhesus monkeys thus providing a newdrug combination for treatment of Parkinson's disease [128,129].

It was demonstrated that administration of CE 178253 (60a) ledto sustained weight loss in diet induced obese rats and mice sug-gesting the use of CB1 receptor antagonists for the treatment ofobesity [129]. CE 178253 (60a) showed not only substantialanorectic activity in rodents relative to its CB1-binding affinity butalso an increase in energy expenditure.

Another investigation was carried out using obese cynomolgusmonkeys to explore the relative contribution of CB1 antagonism onweight loss. Treatment using CB1 antagonist PF 095453 (60b)showed significant reduction in food consumption, body weightand body fat in addition to higher adiponectin and lower leptinconcentrations [130]. Adiponectin and leptin are both involve in theregulation of fat metabolism and metabolic processes. Improve-ment in inflammation, insulin sensitivity and vascular reactivity

Fig. 12. CB receptor ligands.

F.P.L. Lim, A.V. Dolzhenko / European Journal of Medicinal Chemistry 85 (2014) 371e390380

due to increased adiponectin levels by CB1 antagonists are yet to beconfirmed.

Even though CB2 receptors are more predominant in immunecells, CB1 antagonists were also found to exhibit anti-inflammatoryproperties. Thus, administration of CE 178253 (60a) using an in vivomodel of LPS induced inflammation led to significant decrease inthe plasma TNF-a level [131]. Prophylactic treatment with 60aeffectively decreased inflammation in the mouse model of collageninduced arthritis [131].

3.3. Corticotrophin-releasing factor receptor ligands

Corticotrophin-releasing factor (CRF) is a neurotransmitter andhormone that regulates stress responses through the endocrineand autonomic nervous system. Receptors for CRF are members ofG-protein coupled receptors divided into type 1 and type 2. CRF1receptor antagonists are believed to be potential candidates for thetreatment of anxiety and mood disorders [132].

DMP-696 (61) was found to exhibit non-competitive antago-nism for CRF1 receptor with high affinity and selectivity (Fig. 13)[133,134]. Additionally, this compound (61) was shown to havesignificant oral bioavailability and desirable pharmacokineticproperties with high anxiolytic and antidepressant activities. DMP-696 (61) has been extensively used as a reference compound forfurther investigations in the role of CRF and its receptors [133,134].

Another compound with CRF1 receptor antagonism (62) wassynthesized and used in the radiolabeled form [76Br] MJL 1-109-2for the autoradiography visualization of CRF1 receptor. To increasepenetration through the blood brain barrier, compound 62 wasmodified by the addition of methoxy groups into the phenyl moietyyielding therefore compounds 63 and 64 [135]. From the results ofthe in vitro autoradiography saturation studies, compound 63demonstrated high subnanomolar affinity binding to both rat andmonkey frontal cortex: Kd ¼ 0.2 nM (rat prefrontal cortex), 0.3 nM(monkey prefrontal cortex). These results for 63 were consistentwith the CRF1 receptor regional distribution while Kd of 64 couldnot be determined due to non-specific binding [135,136]. Thein vivo studies in rats andmice showed the ability of compounds 63and 64 to penetrate the blood brain barrier and remain mostlymetabolized in the rat's brain in 1 h post injection [135]. In thestudies conducted with P-glycoprotein (P-gp) knockout mice, datashowed that compounds 63 and 64 are not P-gp substrates [135].

From a study of 8-(4-methoxyphenyl)pyrazolo[1,5-a][1,3,5]tri-azines, BMS-561388 (65) [137] was found to exhibit potent antag-onism with CRF1 receptor. It showed good pharmacokinetic profile

in dogs in addition to being orally effective when tested in two ratmodels of anxiety. On the basis of its favorable pharmacokineticprofile, compound 65 was advanced to clinical studies in humans.The same research team conducted a study on 8-pyridyl substitutedpyrazolo[1,5-a][1,3,5]triazines, where pexacerfont (BMS-562086,66) [138], another potent orally bioavailable antagonist of CRF1 wasobtained.

Pexacerfont (66) underwent biotransformation into primarymetabolites via O-demethylation, hydroxylation and N-dealkylationmainly by cytochrome P450 [139]. Metabolic clearance involved bothrenal and biliary excretion viamultiple pathways. In vitro testing wasconducted to determine permeability of pexacerfont (66) acrossCaco-2 cell monolayers and protein binding in serum of rat, dog andhuman. Results indicated that pexacerfont demonstrated goodpermeability and high protein binding. Further in vivo evaluationwasconducted in rats, dogs and chimpanzees showing that pexacerfont(66) has rapid absorption and good oral bioavailability. Takentogether, the excellent ADME and pharmacokinetic profile of pex-acerfont (66) favored its advancement into clinical development as apotential therapy to irritable bowel syndrome and generalizedanxiety disorders. Clinical trials of pexacerfont (66) are currentlybeing carried out by Bristol-Myers Squibb [140].

In the light of the extensive research being carried out for noveldevelopment of CRF1 receptor antagonists, Zuev et al. [141] syn-thesized a series of compounds for evaluation as selective CRF1receptor radioligand for positron emission tomography. Discoveryof such compounds could enable assessing of the receptor occu-pancy in clinical trials of CRF1 receptor antagonists in addition tomonitoring the concentrations of CRF in patients. Compound 67,which is an analogue of pexacerfont (66), was found to exhibit agood balance between binding potency and lipophilicity with acalculated log P value of 2.3 and therefore was selected for furtherradiosynthesis and biodistribution studies.

Saito et al. [142] synthesized a series of compounds amongwhich a pyrazolo[1,5-a][1,3,5]triazine analogue, 68 possessedrelatively potent binding affinity to the human CRF1 receptor.Additionally, when examined in a CRF-stimulated adenylate cyclaseassay, good antagonistic activity was observed with the EC50 valueof 26 nM.

Other purine-like systems also have been explored as CRF re-ceptor ligands. Thus compounds with the pyrrolo[1,2-a][1,3,5]tri-azines scaffold i.e. 69 [143], the imidazo[1,3,5]triazine scaffold i.e.70 [144], and the 1,2,3-triazolo[1,5-a][1,3,5]triazine scaffold i.e. 71[145e148] were claimed as CRF receptor antagonists.

3.4. Chemokine receptor ligands

Chemokine receptors are responsible for the regulation throughGPCRs a variety of biological processes, predominantly cell migra-tion and inflammation [149,150]. Involvement of chemokine re-ceptors in angiogenesis, oncogenesis, hematopoiesis and immunemodulation makes them feasible targets in the therapy of inflam-matory diseases, cancers and HIV-1 infections. Pyrazolo[1,5-a][1,3,5]triazine 72 exhibited dual antagonist activity against CeCchemokine receptor type 2 and CeC chemokine receptor type 5together with good bioavailability (Fig. 14) [151]. In vivo study of 72was conducted in a murine model to explore its effects onthioglycollate-induced peritonitis over the duration of two days.Significant inhibition of cellular infiltration (71%) was achieved at adose of 200 mg/kg [151].

3.5. Opioid receptor ligands

The opioid receptors are classified into at least three subtypesviz. m-, d- and k-receptors and further functionally sub-classified

Fig. 13. CRF receptor ligands.

F.P.L. Lim, A.V. Dolzhenko / European Journal of Medicinal Chemistry 85 (2014) 371e390 381

into several pharmacological subtypes, which are distributedwidely in the brain in addition to being expressed in the spinal cordand digestive tract [152].

Fig. 14. Chemokine receptor ligand.

In the search for new antinociceptive agents, imidazo[1,2-a][1,3,5]triazin-2,4-diones were explored as potential opioid m-re-ceptor agonists (Fig. 15) [153e159]. In the pharmacological testing,3,8-diaryl substituted compounds of this type demonstrateddepressive action on the CNS with very low acute toxicity; they alsopossess antinociceptive activity in animal models and decreasedanimal body temperature [153,154]. Compound 73, which exhibi-ted the most promising in vivo results, was also examined for thebinding affinity towards suspected receptors. Results indicated that73 possessed micromolar binding affinity towards opioid m-re-ceptors, at the same time demonstrating no significant effect onserotonin and benzodiazepine receptors [154].

It appears that different substituents at positions 3 and 8 of theimidazo[1,2-a][1,3,5]triazin-2,4-dione system can be tolerated.Replacementof arylwithbenzyl or phenylethyl-based substituents atposition 3 [155,156] and introduction of 2-pyridyl moiety instead of(un)substituted phenyl [157] did not significantly affect pharmaco-logical profile of the compounds (74e76). These molecules (74e76)demonstrated sedation and lowacute toxicity (LD50>2000mg/kg) in

Fig. 15. Opioid receptor ligands.

F.P.L. Lim, A.V. Dolzhenko / European Journal of Medicinal Chemistry 85 (2014) 371e390382

addition to causing hypothermia and significant antinociceptive ac-tivity in the writhing test in mice [155e157]. The antinociceptive ef-fect of 74, 75 and 76was reversible by injection of a small dose of anopioid antagonist naloxone (5 mg/kg, s.c.) therefore indicating theinvolvement of opioid receptors in the activity of these compounds[155e157]. Other types of imidazo[1,2-a][1,3,5]triazin-2,4-dioneswere also explored, e.g. 77 and 78 [158,159]. The CNS pharmacolog-ical profile of 77was similar to the above described analogues [158],however 78 demonstrated no hypothermia and sedation typical foropioid receptor agonists; analgesic effect of 78was also not reversedby naloxone [159].

3.6. Miscellaneous receptor ligands

A number of active compounds with affinity to different re-ceptors were prepared varying the substitution pattern of 5-aza-9-deaza-isosteres of adenine and hypoxanthine (Fig. 16).

Fig. 16. Miscellaneous

A group of purine receptors known as the P2Y1 receptors areactivated mainly by ADP and related diphosphate analogues. Adiphosphate 79, based on the 5-aza-9-deaza-adenine scaffold, wassynthesized as a potential P2Y1 receptor antagonist [160]. Eventhough there were no direct data on the affinity of 79 towards theP2Y1 receptor, this compound demonstrated activity in functionalassays blocking ADP-induced platelet aggregation in vitro(pA2 ¼ 6.5) and in vivo.

Involvement of the neuropeptide Y receptor signaling system inregulation of a number of psychological responses such as feedingand fat storage, anxiolytic behaviors and regulation of the coronarytone makes this system an attractive therapeutic target for thetreatment of metabolic disorders, CNS disorders and cardiovasculardiseases [161]. Pyrazolo[1,5-a][1,3,5]triazines with general struc-ture 80 were prepared and claimed to be modulators of neuro-peptide Y1 receptors [162,163].

receptor ligands.

Fig. 17. Compounds with antiproliferative activity.

F.P.L. Lim, A.V. Dolzhenko / European Journal of Medicinal Chemistry 85 (2014) 371e390 383

5-Hydroxytryptamine (5-HT) receptors are highly diverse. Theyinclude 14 types and subtypes involved in the regulation of manyimportant physiological processes in the CNS and other systems[164]. Pyrazolo[1,5-a][1,3,5]triazines 81 were claimed to be effec-tive ligands for 5-HT6 receptors [165], which are the suitable targetfor the treatment of cognitive dysfunction associated with Alz-heimer's disease, obesity, depression and anxiety [166]. Anothersubtype of 5-HT receptors linked with the potential for the CNSdisorder treatment, namely 5-HT2c receptors, was effectively tar-geted by pyrazolo[1,5-a][1,3,5]triazine 82, which was identified inthe virtual screening and showed submicromolar affinity to 5-HT2creceptors in vitro [167].

The antagonists of androgen receptors (AR) were successfullydeveloped using 9-substituted 5-aza-9-deaza-hypoxanthines 83[168]. These compounds demonstrated nanomolar activity in the3H-mibolorene binding assay.

Pyrazolo[1,5-a][1,3,5]triazine skeleton was used for the con-struction of angiotensin II (AII) receptor antagonists, e.g. 84 [169].This compound (84) showed high affinity to AII receptors in vitroand demonstrated significant decrease in AII hypertensive responsein vivo.

The transient receptor potential ankyrin 1 (TRPA1) ion channelshave been recognized as a target for pain therapy [170]. A series of

compounds bearing the imidazo[1,2-a][1,3,5]triazine scaffold (e.g.85) were synthesized and evaluated as potential TRPA1 antagonists,blocking 90% of TRPA1 response at the 1.0 mM concentration [171].

4. Biological activity with undefined molecular target

For the compounds based on purine isosteres with 1,3,5-triazinering as a part of the molecule skeleton, exact biological targets werenot always identified and mechanisms of their biological activityremain unknown or unclear. This situation is often observed for thecompounds with antiproliferative and antiviral activities.

4.1. Antiproliferative activity

Anticancer evaluation of pyrazolo[1,5-a][1,3,5]triazines yieldedcompounds 86 (Fig. 17) [172]. In antiproliferative screening, 86bdisplayed the activity against colorectal cancer cells (HCT116,SW48, SW480) and CEM lymphoma cell line. This compound (86b)also exhibited excellent cell cycle arrest at the G2/M phase after24 h treatment of colorectal cancer cells. In in vitro testing usingbovine brain purified tubulin, both compounds 86a and 86b wereequally potent in inhibition of tubulin polymerization.

Fig. 18. Compounds with antiviral activity.

F.P.L. Lim, A.V. Dolzhenko / European Journal of Medicinal Chemistry 85 (2014) 371e390384

Antileukemic properties were reported for 5-aza-9-deaza-adenosine (87) and 5-aza-9-deaza-inosine (88), which were oneorder more potent than their isosteres formycins A and B againsttwo cell lines L1210 and P815 [173]. Some modifications at position4 were also explored and 90 showed further improvements in theactivity.

Saczewski et al. [174] synthesized and evaluated in vitro usingtwelve human cancer cell lines a series of imidazo[1,2-a][1,3,5]triazine analogues of 6-mercaptopurine and identified compound92 as a potent antiproliferative agent against urinary bladder (RT4)and breast (MCF-7) cancer cells. This compound (92) displayedhigher antiproliferative activity compared to 6-mercaptopurine onthe RT4 urinary bladder and KYSE-70 esophagus cancer cell lines.

In evaluation of biological activity of a series of fused 1,3,5-triazine derivatives by Bekircan et al. [175], 1,2,4-triazolo[1,5-a][1,3,5]triazines demonstrated promising results in the anti-proliferative and antioxidant assays. Particularly, 2-amino-5,7-diphenyl-1,2,4-triazolo[1,5-a][1,3,5]triazines (93) showed substan-tial growth inhibition against A498 renal cancer cell line in thescreening against 60 human cancer cell lines.

A series of 6,7-dihydro[1,2,4]triazolo[1,5-a][1,3,5]triazines wasdesigned as potential DHFR inhibitors with anticancer properties[176]. The most interesting compound in the series, 94was found to

displayantiproliferativeactivityagainstbreastcancercells (MDA-MB-231). However, none of the compounds in this series showed anysubstantial inhibition of the bovine DHFR activity suggesting there-foreanothermechanismfor theanticancereffectof thesecompounds.

4.2. Antiviral activity

Viruses are the leading cause of human diseases worldwide.Hence, there is an active ongoing search for novel antiviral agents,which may provide highly effective and safe treatment of humanvirus infections. The antiviral properties of purines and their de-rivatives have been well recognized resulting in a number ofpotent drugs in the market. Therefore, interest to the purine iso-steres with 1,3,5-triazine ring in this area of medicinal chemistryis logical. Results of the research in this direction are outlinedbelow.

Compound 35B2 (95), a rather simple derivative of pyrazolo[1,5-a][1,3,5]triazin-4-one, was identified as an active varicella-zostervirus (VZV) inhibitor (Fig. 18) [177]. Antiviral activity of 35B2 (95)against VZV (V-Oka) was determined using plaque reduction assayin GPL and HEL cells. Analysis indicated that 35B2 (95) targetedORF40, VZV major capsid protein (MCP) by inducing MCP aggre-gation and inhibiting capsid assembly [177].

Fig. 19. Compounds with miscellaneous biological activities.

F.P.L. Lim, A.V. Dolzhenko / European Journal of Medicinal Chemistry 85 (2014) 371e390 385

More decorated pyrazolo[1,5-a][1,3,5]triazines 96 and 97showed antiviral activity against herpes simplex virus-1 (HSV-1)with low cytotoxicity [178].

The compounds with the 5-aza-7-deazaguanine core structurewere discussed in literature [179e183] as potential treatment forhosts infected with viruses of Flaviviridae family. The most prom-ising molecule in this category was 5-aza-7-deazaguanosine (98a),encoded as ZX-2401. The in vitro virus yield reduction assaysshowed that at day 2 post initiation ZX-2401 (98a) significantlyreduced virus production (EC90 ¼ 3.3 mg/ml) [179,182]. Furtherantiviral testing of ZX-2401 (98a) against viruses of the Flaviviridaefamily, such as West Nile Virus (WNV), Hepatitis C (HCV), YellowFever Virus (YFV), Dengue Virus (DV), Bovine Viral Diarrhea Virus(BVDV) and Banzi Virus (BV) showed good antiviral activity of thecompound with low cytotoxicity [179,182]. Furthermore, combi-nation of ZX-2401 (98a) with interferon exhibited synergisticantiviral effect [179,182].

ZX-2401 (98a) was also evaluated for the treatment of influenza.In the cytopathic effect (CPE) assay with MDCK cells infected withinfluenza A/Shangdong/09/93 (H3N2), ZX-2401 (98a) demon-strated greater inhibition of influenza A compared to control drugribavirin [184]. When tested against influenza B virus using CPEassay, ZX-2401 (98a) showed clear dose dependent inhibition butwith lower activity compared to the control drug.

Antiviral activity of imidazo[1,5-a][1,3,5]triazines was exploredand compounds 99aed, which had substitution pattern similar to 95,were identified as potential leads for the development of antiviralagent against influenza A or respiratory syncytial virus (RSV) [185].

4.3. Miscellaneous biological activities

The spectrum of biological effects exhibited by the 1,3,5-triazine-based isosteres of purines is very diverse and rangesfrom antimicrobial activity to the effects on CNS.

The antibacterial activity, mainly against Clostridium perfringens,was reported for substituted 5-aza-9-deazaguanine 100 (Fig. 19).The in vitro antibacterial activity spectrum of 100 is limited to grampositive bacteria with no appreciable activity against gram negativebacteria [186].

2,4-Diaminopyrazolo[1,5-a][1,3,5]triazines were studied as po-tential anti-asthmatic agents [187e191]. Dametralast (LA 2851,101)was found to be a potential anti-asthmatic agent exhibiting pro-nounced bronchodilatory [187,188] and antiallergic [187e189] ac-tivities together with low toxicity. Dametralast (101) also possessedanti-inflammatory properties not related to cyclooxygenase inhi-bition or mediator release [190]. The lipoxygenase pathway waspostulated to be responsible for the pharmacological action ofdametralast (101). The inhibition of PDE to a certain extent was also

F.P.L. Lim, A.V. Dolzhenko / European Journal of Medicinal Chemistry 85 (2014) 371e390386

mentioned as the secondary activity of dametralast (101). Thesynthesis and further evaluation of anti-asthmatic properties usinga variety of animal models together with clinical testing in humanswere conducted with dametralast (101), its analogues (102 and103) and derivatives (104) [191]. A series of 1,2,4-triazolo[1,5-a][1,3,5]triazines 105 was also claimed to act as potent bronchodila-tors [192] while another compound with this heterocyclic scaffold(106) was reported as a potential therapy for diseases related toinflammation [193]. Mechanism of action involved in the biologicalactivities of these compounds has not been clearly identified.However, structural similarity together with observed pharmaco-logical profile, especially effects on the cardiovascular system andCNS, suggested antagonism with adenosine receptors as one of thepossible mechanisms.

In the search for new immunosuppressants, the screening of aseries of 1,2,4-triazolo[1,5-a][1,3,5]triazines was carried out usingdifferent models of eosinophilia [194,195]. Among the reportedcompounds, 107 was the most potent in the inhibition of eosino-philia (active at 0.3 mg/kg, p.o.).

1,2,4-Triazolo[1,5-a][1,3,5]triazine 108 was claimed to be a goodcandidate for the treatment of depression [196] and Parkinson'sdisease [197]. The pharmacology of this compound could also belinked to the antagonism with adenosine receptors.

Lisofylline (LSF), a dimethylxanthine compound demonstratedimmediate reduction in the development of type 1 diabeteswhen tested using non-obese diabetic mouse confirming itstherapeutic use for the prevention of type 1 diabetes [198]. Dueto potency and pharmacokinetic disadvantages of LSF, Cui et al.[199] designed and synthesized new LSF analogues as potentialcandidates for the treatment of type 1 diabetes. 5-Aza-7-deazaxanthine analogue of LSF, 109 was identified as a leadcompound. This compound (109) displayed protective effects onthe b-cells from cytokine-induced injury in addition to main-taining insulin secreting capability. The effects of 109 werecomparable with that of LSF. Satisfactory metabolic stability of109 was also observed during in vitro testing with human livermicrosomes at pH 7.4. These promising results led to ongoinginvestigations on the pharmacokinetic profile to determine if thegoal of generating an LSF analogue with improved potency,bioavailability and metabolic stability can be achieved by usingthis 5-aza-7-deazaxanthine scaffold.

5. Conclusion

Continuous inspiration by successes in purine chemistry andpharmacology [1,200,201] led to extensive exploration of theisosteric heterocyclic systems. Interest towards purine-like het-erocycles comprising 1,3,5-triazine ring has been mainly centeredaround two types of the systems: 5-azapurines (1,2,4-triazolo[1,5-a][1,3,5]triazines) and 5-aza-9-deazapurines (pyrazolo[1,5-a][1,3,5]triazines) [20,21]. These heterocyclic systems demonstrated a greatpotential in the development of inhibitors of various kinases, PDE,XO, and TP, antagonists of adenosine, CB and CRF receptors, anti-cancer and antiviral agents, therefore becoming privileged scaffoldsin medicinal chemistry. The success in the development of newbioactive compounds constructed using these heterocyclic systemsis expected to continue growth.

The imidazo-fused 1,3,5-triazines are less explored in medicinalchemistry, but many interesting compounds with potential thera-peutic applications (e.g. PDE inhibitors, adenosine and opioid re-ceptor ligands, and antiviral agents) were identified among them.The medicinal chemistry of the pyrrolo[1,2-a][1,3,5]triazines and1,2,3-triazolo[1,5-a][1,3,5]triazines is in early stages of develop-ment and value of these scaffolds as purine isosteres is still to bediscovered.

References

[1] M. Legraverend, D.S. Grierson, The purines: potent and versatile smallmolecule inhibitors and modulators of key biological targets, Bioorg. Med.Chem. 14 (2006) 3987e4006.

[2] J.M. Murray, D.E. Bussiere, Targeting the purinome, Methods Mol. Biol. 575(2009) 47e92.

[3] G.L. Russo, M. Russo, P. Ungaro, AMP-activated protein kinase: a target forold drugs against diabetes and cancer, Biochem. Pharmacol. 86 (2013)339e350.

[4] M.J. Perry, G.A. Higgs, Chemotherapeutic potential of phosphodiesterase in-hibitors, Curr. Opin. Chem. Biol. 2 (1998) 472e481.

[5] A.K. Werner, C.-P. Witte, The biochemistry of nitrogen mobilization: purinering catabolism, Trends Plant Sci. 16 (2011) 381e387.

[6] A. Bzowska, E. Kulikowska, D. Shugar, Purine nucleoside phosphorylases:properties, functions, and clinical aspects, Pharmacol. Ther. 88 (2000)349e425.

[7] B.B. Fredholm, A.P. IJzerman, K.A. Jacobson, J. Linden, C.E. Müller, Interna-tional Union of Basic and Clinical Pharmacology. LXXXI. Nomenclature andclassification of adenosine receptors e an update, Pharmacol. Rev. 63 (2011)1e34.

[8] B.J. Denny, R.T. Wheelhouse, M.F.G. Stevens, L.L.H. Tsang, J.A. Slack, NMR andmolecular modeling investigation of the mechanism of activation of theantitumor drug temozolomide and its interaction with DNA, Biochemistry 33(1994) 9045e9051.

[9] M. Dourado, A.B. Sarmento, S.V. Pereira, V. Alves, T. Silva, A.M. Pinto,M.S. Rosa, CD26/DPPIV expression and 8-azaguanine response in T-acutelymphoblastic leukaemia cell lines in culture, Pathophysiology 14 (2007)3e10.

[10] P. Robak, T. Robak, Older and new purine nucleoside analogs for patientswith acute leukemias, Cancer Treat. Rev. 39 (2013) 851e861.

[11] S. Bantia, C. Parker, R. Upshaw, A. Cunningham, P. Kotian, J.M. Kilpatrick,P. Morris, P. Chand, Y.S. Babu, Potent orally bioavailable purine nucleosidephosphorylase inhibitor BCX-4208 induces apoptosis in B- and T-lympho-cytes e a novel treatment approach for autoimmune diseases, organ trans-plantation and hematologic malignancies, Int. Immunopharmacol. 10 (2010)784e790.

[12] J. Chao, R. Terkeltaub, A critical reappraisal of allopurinol dosing, safety, andefficacy for hyperuricemia in gout, Curr. Rheumatol. Rep. 11 (2009)135e140.

[13] J.D. Corbin, A. Beasley, M.A. Blount, S.H. Francis, Vardenafil: structural basisfor higher potency over sildenafil in inhibiting cGMP-specific phosphodies-terase-5 (PDE5), Neurochem. Int. 45 (2004) 859e863.

[14] Y. Taniguchi, H. Tonai-Kachi, K. Shinjo, Zaprinast, a well-known cyclic gua-nosine monophosphate-specific phosphodiesterase inhibitor, is an agonistfor GPR35, FEBS Lett. 580 (2006) 5003e5008.

[15] J.W.M. Cheng, Ticagrelor: oral reversible P2Y12 receptor antagonist for themanagement of acute coronary syndromes, Clin. Ther. 34 (2012) 1209e1220.

[16] E. Sanna, F. Busonero, G. Talani, M. Carta, F. Massa, M. Peis, E. Maciocco,G. Biggio, Comparison of the effects of zaleplon, zolpidem, and triazolam atvarious GABAA receptor subtypes, Eur. J. Pharmacol. 451 (2002) 103e110.

[17] J.K. Walsh, A. Moscovitch, J. Burke, R. Farber, T. Roth, Efficacy and tolerabilityof indiplon in older adults with primary insomnia, Sleep Med. 8 (2007)753e759.

[18] A. Bloch, R.J. Leonard, C.A. Nichol, On the mode of action of 7-deaza-aden-osine (tubercidin), Biochim. Biophys. Acta 138 (1967) 10e25.

[19] R.M. McCarty, V. Bandarian, Deciphering deazapurine biosynthesis: pathwayfor pyrrolopyrimidine nucleosides toyocamycin and sangivamycin, Chem.Biol. 15 (2008) 790e798.

[20] A.V. Dolzhenko, A.V. Dolzhenko, W.-K. Chui, 1,2,4-Triazolo[1,5-a][1,3,5]tri-azines (5-azapurines): synthesis and biological activity, Heterocycles 68(2006) 1723e1759.

[21] A.V. Dolzhenko, A.V. Dolzhenko, W.-K. Chui, Pyrazolo[1,5-a][1,3,5]triazines(5-aza-9-deazapurines). Synthesis and biological activity, Heterocycles 75(2008) 1575e1622.

[22] A.L. Hopkins, C.R. Groom, Opinion: the druggable genome, Nat. Rev. DrugDiscov. 1 (2002) 727e730.

[23] P. Cohen, D.R. Alessi, Kinase drug discovery e What's next in the field? ACSChem. Biol. 8 (2013) 96e104.

[24] M. Canavese, L. Santo, N. Raje, Cyclin dependent kinases in cancer: potentialfor therapeutic intervention, Cancer Biol. Ther. 13 (2012) 451e457.

[25] M. Gallorini, A. Cataldi, V. di Giacomo, Cyclin-dependent kinase modulatorsand cancer therapy, BioDrugs 26 (2012) 377e391.

[26] A. Stone, R.L. Sutherland, E.A. Musgrove, Inhibitors of cell cycle kinases:recent advances and future prospects as cancer therapeutics, Crit. Rev.Oncog. 17 (2012) 175e198.

[27] Y. Tian, H. Wan, G. Tan, Cell cycle-related kinase in carcinogenesis (review),Oncol. Lett. 4 (2012) 601e606.

[28] J. Wesierska-Gadek, M. Maurer, Promotion of apoptosis in cancer cells byselective purine-derived pharmacological CDK inhibitors: one outcome,many mechanisms, Curr. Pharm. Des. 17 (2011) 256e271.

[29] T.J. Guzi, K. Paruch, Preparation of pyrazolotriazines as kinase inhibitors fortreating cancer and other diseases associated with a kinase, US Patent2005197219, 2005.

F.P.L. Lim, A.V. Dolzhenko / European Journal of Medicinal Chemistry 85 (2014) 371e390 387

[30] G. Prevost, M.-O. Lonchampt, S. Kim, B. Morgan, G. Ulibarri, C. Thurieau,Pyrazolo[1,5-a]-1,3,5-triazine derivatives with activity as cyclin-dependentkinase (CDK) and glycogen synthase kinase-3 (GSK-3) inhibitors, and theirpreparation, pharmaceutical compositions, and use as, e.g., antiproliferativeagents, WO Patent 2002050079, 2002.

[31] Z. Nie, C. Perretta, P. Erickson, S. Margosiak, J. Lu, A. Averill, R. Almassy,S. Chu, Structure-based design and synthesis of novel macrocyclic pyrazolo[1,5-a][1,3,5]triazine compounds as potent inhibitors of protein kinase CK2and their anticancer activities, Bioorg. Med. Chem. Lett. 18 (2008)619e623.

[32] R. Jorda, K. Paruch, V. Krystof, Cyclin-dependent kinase inhibitors inspired byroscovitine: purine bioisosteres, Curr. Pharm. Des. 18 (2012) 2974e2980.

[33] K. Bettayeb, H. Sallam, Y. Ferandin, F. Popowycz, G. Fournet, M. Hassan,A. Echalier, P. Bernard, J. Endicott, B. Joseph, L. Meijer, N-&-N, a new class ofcell death-inducing kinase inhibitors derived from the purine roscovitine,Mol. Cancer Ther. 7 (2008) 2713e2724.

[34] F. Popowycz, G. Fournet, C. Schneider, K. Bettayeb, Y. Ferandin, C. Lamigeon,O.M. Tirado, S. Mateo-Lozano, V. Notario, P. Colas, P. Bernard, L. Meijer,B. Joseph, Pyrazolo[1,5-a]-1,3,5-triazine as a purine bioisostere: access topotent cyclin-dependent kinase inhibitor (R)-roscovitine analogue, J. Med.Chem. 52 (2009) 655e663.

[35] F. Kashanchi, Modulators of viral transcription, and methods and composi-tions therewith, US Patent 20120149708, 2012.

[36] G. Nemeth, Z. Varga, Z. Greff, G. Bencze, A. Sipos, C. Szantai-Kis, F. Baska,A. Gyuris, K. Kelemenics, Z. Szathmary, J. Minarovits, G. Keri, L. Orfi, Novel,selective CDK9 inhibitors for the treatment of HIV infection, Curr. Med.Chem. 18 (2011) 342e358.

[37] F. Kashanchi, Modulators of viral transcription for treatment of human im-munodeficiency virus (HIV) infection, US Patent 20120083498, 2012.

[38] J. Eickhoff, G. Zischinsky, U. Koch, Preparation of amino-pyrimidinylpyridinone derivatives and analogs for use as serine/threoninekinase inhibitors, EP Patent 2634190, 2013.

[39] J. Eickhoff, G. Zischinsky, U. Koch, Preparation of pyrazolotriazine derivativesfor use as selective cyclin-dependent kinase inhibitors, WO Patent2013128028, 2013.

[40] J. Eickhoff, G. Zischinsky, U. Koch, G. Ruehter, C. Schultz-Fademrecht, P.Nussbaumer, Preparation of pyrazolo-triazine derivatives as selective cyclin-dependent kinase inhibitors, EP Patent 2634189, 2013.

[41] J. Eickhoff, G. Zischinsky, U. Koch, G. Ruehter, C. Schultz-Fademrecht, P.Nussbaumer, Preparation of pyrazolo[1,5-a][1,3,5]triazine derivatives as se-lective cyclin-dependent kinase inhibitors, WO Patent 2013128029, 2013.

[42] K. Mahajan, N.P. Mahajan, ACK1 tyrosine kinase: targeted inhibition to blockcancer cell proliferation, Cancer Lett. 338 (2013) 185e192.

[43] X. Jiao, D.J. Kopecky, J. Liu, J. Liu, J.C. Jaen, M.G. Cardozo, R. Sharma, N. Walker,H. Wesche, S. Li, E. Farrelly, S.-H. Xiao, Z. Wang, F. Kayser, Synthesis andoptimization of substituted furo[2,3-d]-pyrimidin-4-amines and 7H-pyrrolo[2,3-d]pyrimidin-4-amines as ACK1 inhibitors, Bioorg. Med. Chem. Lett. 22(2012) 6212e6217.

[44] M. Montenarh, Cellular regulators of protein kinase CK2, Cell Tissue Res. 342(2010) 139e146.

[45] I.M. Hanif, I.M. Hanif, M.A. Shazib, K.A. Ahmad, S. Pervaiz, Casein kinase II: anattractive target for anti-cancer drug design, Int. J. Biochem. Cell Biol. 42(2010) 1602e1605.

[46] X. Hu, Y. Hu, M. Vogt, D. Stumpfe, J. Bajorath, MMP-Cliffs: systematic iden-tification of activity cliffs on the basis of matched molecular pairs, J. Chem.Inf. Model 52 (2012) 1138e1145.

[47] S.H. Francis, M.D. Houslay, M. Conti, Phosphodiesterase inhibitors: factorsthat influence potency, selectivity, and action, Handb. Exp. Pharmacol. 204(2011) 47e84.

[48] S.H. Francis, K.R. Sekhar, H. Ke, J.D. Corbin, Inhibition of cyclic nucleotidephosphodiesterases by methylxanthines and related compounds, Handb.Exp. Pharmacol. 200 (2011) 93e133.

[49] A. Garcia-Osta, M. Cuadrado-Tejedor, C. Garcia-Barroso, J. Oyarzabal,R. Franco, Phosphodiesterases as therapeutic targets for Alzheimer's disease,ACS Chem. Neurosci. 3 (2012) 832e844.

[50] P. Gresele, S. Momi, E. Falcinelli, Anti-platelet therapy: phosphodiesteraseinhibitors, Br. J. Clin. Pharmacol. 72 (2011) 634e646.

[51] T. Keravis, C. Lugnier, Cyclic nucleotide phosphodiesterase (PDE) isozymes astargets of the intracellular signalling network: benefits of PDE inhibitors invarious diseases and perspectives for future therapeutic developments, Br. J.Pharmacol. 165 (2012) 1288e1305.

[52] C.P. Page, D. Spina, Selective PDE inhibitors as novel treatments for respi-ratory diseases, Curr. Opin. Pharmacol. 12 (2012) 275e286.

[53] C. Schudt, A. Hatzelmann, R. Beume, H. Tenor, Phosphodiesterase inhibitors:history of pharmacology, Handb. Exp. Pharmacol. 204 (2011) 1e46.

[54] R. Sengupta, T. Sun, N.M. Warrington, J.B. Rubin, Treating brain tumors withPDE4 inhibitors, Trends Pharmacol. Sci. 32 (2011) 337e344.

[55] J. Kobe, D.E. O'Brien, R.K. Robins, 2-Aryl-7-substituted pyrazolo[1,5a]-1,3,5-triazines, US Patent 3865824, 1975.

[56] K. Senga, D.E. O'Brien, M.B. Scholten, T. Novinson, J.P. Miller, R.K. Robins,Synthesis and enzymic activity of various substituted pyrazolo[1,5-a]-1,3,5-triazines as adenosine cyclic 3',5'-phosphate phosphodiesterase inhibitors,J. Med. Chem. 25 (1982) 243e249.

[57] J. Kobe, R.H. Springer, D.E. O'Brien, Pyrazolo(1,5-a)1,3,5-triazines, US Patent3846423, 1974.

[58] J.P. Miller, C.C. Sigman, H.L. Johnson, T. Novinson, R.H. Springer, K. Senga,D.E. O'Brien, R.K. Robins, Inhibition of cyclic AMP phosphodiesterases bycyclic nucleotide analogs and nitrogen heterocycles, Adv. Cycl. NucleotideProtein Phosphorylation Res. 16 (1984) 277e290.

[59] D.E. O'Brien, K. Senga, T. Novinson, Pyrazolo(1,5-a)-1,3,5-triazines, US Patent3910907, 1975.

[60] T.A. Sullivan, B.H. Duemler, N.J. Kuttesch, T.M. Keravis, J.N. Wells, Irreversibleinhibition of calmodulin-sensitive cyclic nucleotide phosphodiesterase,J. Cycl. Nucleotide Protein Phosphorylation Res. 11 (1986) 355e364.

[61] P. Raboisson, D. Schultz, C. Muller, J.-M. Reimund, G. Pinna, R. Mathieu,P. Bernard, Q.-T. Do, R.L. DesJarlais, H. Justiano, C. Lugnier, J.-J. Bourguignon,Cyclic nucleotide phosphodiesterase type 4 inhibitors: evaluation of pyr-azolo[1,5-a]-1,3,5-triazine ring system as an adenine bioisostere, Eur. J. Med.Chem. 43 (2008) 816e829.

[62] E. Kawanishi, Y. Tanaka, T. Matsumura, Y. Kado, H. Taniuchi, Fused hetero-cyclic compounds, method for inhibiting PDE10 by them, and pharmaceu-tical compositions containing them, WO Patent 2013027794, 2013.

[63] H. Haning, U. Niewoehner, T. Schenke, T. Lampe, A. Hillisch, E. Bischoff,Comparison of different heterocyclic scaffolds as substrate analog PDE5 in-hibitors, Bioorg. Med. Chem. Lett. 15 (2005) 3900e3907.

[64] H. Inoue, H. Murafuji, Y. Hayashi, Imidazotriazinone derivatives as PDE7(phosphodiesterase 7) inhibitors, their preparation, and pharmaceuticalcompositions containing them, WO Patent 2004111053, 2004.

[65] P. Pacher, A. Nivorozhkin, C. Szab�o, Therapeutic effects of xanthine oxidaseinhibitors: renaissance half a century after the discovery of allopurinol,Pharmacol. Rev. 58 (2006) 87e114.

[66] R. Hille, T. Nishino, Flavoprotein structure and mechanism. 4. Xanthine ox-idase and xanthine dehydrogenase, FASEB J. 9 (1995) 995e1003.

[67] P. Higgins, J. Dawson, K.R. Lees, K. McArthur, T.J. Quinn, M.R. Walters,Xanthine oxidase inhibition for the treatment of cardiovascular disease: asystematic review and meta-analysis, Cardiovasc. Ther. 30 (2012)217e226.

[68] R.K. Robins, G.R. Revankar, D.E. O'Brien, R.H. Springer, T. Novinson, A. Albert,K. Senga, J.P. Miller, D.G. Streeter, Purine analog inhibitors of xanthine oxi-dase e structure activity relationships and proposed binding of the molyb-denum cofactor, J. Heterocycl. Chem. 22 (1985) 601e634.

[69] S. Fujii, H. Kawamura, H. Kiyokawa, S. Yamada, Preparation of pyrazolo-triazines as xanthine oxidase inhibitors, EP Patent 269859, 1988.

[70] S. Sato, K. Tatsumi, T. Nishino, A novel xanthine dehydrogenase inhibitor(BOF-4272), Adv. Exp. Med. Biol. 309A (1991) 135e138.

[71] K. Okamoto, T. Iwamoto, T. Nishino, New tight binding inhibitors of xanthineoxidase, in: Flavins and Flavoproteins 1993, Walter de Gruyter & Co, Berlin,1994, pp. 731e734.

[72] K. Okamoto, New inhibitor of xanthine oxidase, Yokohama Igaku 45 (1994)47e53.

[73] K. Okamoto, T. Nishino, Mechanism of inhibition of xanthine oxidase with anew tight binding inhibitor, J. Biol. Chem. 270 (1995) 7816e7821.

[74] S. Naito, M. Nishimura, Y. Tamao, Evaluation of the pharmacological actionsand pharmacokinetics of BOF-4272, a xanthine oxidase inhibitor, in mouseliver, J. Pharm. Pharmacol. 52 (2000) 173e179.

[75] T. Uematsu, M. Nakashima, Pharmacokinetic and pharmacodynamic prop-erties of a novel xanthine oxidase inhibitor, BOF-4272, in healthy volunteers,J. Pharmacol. Exp. Ther. 270 (1994) 453e459.

[76] H. Suzuki, M. Suematsu, H. Ishii, S. Kato, H. Miki, M. Mori, Y. Ishimura,T. Nishino, M. Tsuchiya, Prostaglandin E1 abrogates early reductive stressand zone-specific paradoxical oxidative injury in hypoperfused rat liver,J. Clin. Investig. 93 (1994) 155e164.

[77] A. Shibukawa, M. Kadohara, J.-Y. He, M. Nishimura, S. Naito, T. Nakagawa,Study of the enantioselective binding between BOF-4272 and serum albu-mins by means of high-performance frontal analysis, J. Chromatogr. A 694(1995) 81e89.

[78] M. Nishimura, K. Yamaoka, S. Naito, T. Nakagawa, Influence of albumin onenantioselective local disposition of BOF-4272, a xanthine oxidase inhibi-tor with chiral sulfoxide, in rat liver, Biol. Pharm. Bull. 20 (1997)1285e1289.

[79] S. Naito, M. Nishimura, Enantioselective uptake of BOF-4272, a xanthineoxidase inhibitor with a chiral sulfoxide, by isolated rat hepatocytes, Yaku-gaku Zasshi 121 (2001) 989e994.

[80] S. Naito, M. Nishimura, In vitro and in vivo studies on the stereoselectivepharmacokinetics and biotransformation of an (S)-(-)- and (R)-(þ)-pyr-azolotriazine sulfoxide in the male rat, Xenobiotica 32 (2002) 491e503.

[81] S. Naito, M. Nishimura, Stereoselective pharmacokinetics of BOF-4272racemate after oral administration to rats and dogs, Biol. Pharm. Bull. 25(2002) 674e677.

[82] K. Hashimoto, M. Inai, Preparation of optically active 4-hydroxy-8-(3-alkoxy-4-phenylsulfinylphenyl)pyrazolo[1,5-a]-1,3,5-triazine salts for treatment ofgout, EP Patent 414200, 1991.

[83] M. Matsugi, K. Hashimoto, M. Inai, N. Fukuda, T. Furuta, J.-i. Minamikawa,S. Otsuka, Asymmetric synthesis of a xanthine dehydrogenase inhibitor (S)-(-)-BOF-4272: utility of chiral alkoxysulfonium salts, Tetrahedron Asymme-try 6 (1995) 2991e3000.

[84] K. Hashimoto, M. Matsugi, N. Fukuda, Y. Kurogi, Asymmetric synthesis ofxanthine dehydrogenase inhibitor (S)-(-)-BOF-4272: mechanism of chiraldiaryl sulfoxide formation, Phosphorus Sulfur Silicon Relat. Elem. 120 & 121(1997) 305e315.

F.P.L. Lim, A.V. Dolzhenko / European Journal of Medicinal Chemistry 85 (2014) 371e390388

[85] Y. Nakajima, R. Madhyastha, M. Maruyama, 2-Deoxy-D-ribose, a down-stream mediator of thymidine phosphorylase, regulates tumor angiogenesisand progression, Anti-Cancer Agents Med. Chem. 9 (2009) 239e245.

[86] I.V. Bijnsdorp, M. de Bruin, A.C. Laan, M. Fukushima, G.J. Peters, The role ofplatelet-derived endothelial cell growth factor/thymidine phosphorylase intumor behavior, Nucleosides Nucleotides Nucleic Acids 27 (2008) 681e691.

[87] H. Bera, W.-K. Chui, S.D. Gupta, A.V. Dolzhenko, L. Sun, Synthesis, in vitroevaluation of thymidine phosphorylase inhibitory activity, and in silico studyof 1,3,5-triazin-2,4-dione and its fused analogues, Med. Chem. Res. 22 (2013)6010e6021.

[88] H. Bera, B.J. Tan, L. Sun, A.V. Dolzhenko, W.-K. Chui, G.N.C. Chiu, A structure-activity relationship study of 1,2,4-triazolo[1,5-a][1,3,5]triazin-5,7-dione andits 5-thioxo analogues on anti-thymidine phosphorylase and associated anti-angiogenic activities, Eur. J. Med. Chem. 67 (2013) 325e334.

[89] H. Bera, A.V. Dolzhenko, L. Sun, S.D. Gupta, W.-K. Chui, Synthesis and in vitroevaluation of 1,2,4-triazolo[1,5-a][1,3,5]triazine derivatives as thymidinephosphorylase inhibitors, Chem. Biol. Drug Des. 82 (2013) 351e360.

[90] H. Bera, M.H. Lee, L. Sun, A.V. Dolzhenko, W.K. Chui, Synthesis, anti-thymidine phosphorylase activity and molecular docking of 5-thioxo-[1,2,4]triazolo[1,5-a][1,3,5]triazin-7-ones, Bioorg. Chem. 50 (2013) 34e40.

[91] L. Sun, H. Bera, W.K. Chui, Synthesis of pyrazolo[1,5-a][1,3,5]triazine de-rivatives as inhibitors of thymidine phosphorylase, Eur. J. Med. Chem. 65(2013) 1e11.

[92] L. Sun, J. Li, H. Bera, A.V. Dolzhenko, G.N.C. Chiu, W.K. Chui, Fragment-basedapproach to the design of 5-chlorouracil-linked-pyrazolo[1,5-a][1,3,5]tri-azines as thymidine phosphorylase inhibitors, Eur. J. Med. Chem. 70 (2013)400e410.

[93] H. Bera, P.K. Ojha, B.J. Tan, L. Sun, A.V. Dolzhenko, W.K. Chui, G.N.C. Chiu,Discovery of mixed type thymidine phosphorylase inhibitors endowed withantiangiogenic properties: synthesis, pharmacological evaluation and mo-lecular docking study of 2-thioxo-pyrazolo[1,5-a][1,3,5]triazin-4-ones. PartII, Eur. J. Med. Chem. 78 (2014) 294e303.

[94] R. Baetta, A. Corsini, Pharmacology of dipeptidyl peptidase-4 inhibitors:similarities and differences, Drugs 71 (2011) 1441e1467.

[95] J. Feng, S.L. Gwaltney, J.A. Stafford, M.B. Wallace, Z. Zhang, Heterocyclic dipep-tidyl peptidase inhibitors for therapeutic use, US Patent 20060135767, 2006.

[96] M. Jung, R. Schuele, E. Metzger, A. Spannhoff, J. Liebscher, M. Paetzel, W.Sippl, Pharmaceutical composition containing inhibiting agents of proteinmethyltransferase I and use thereof to treat tumor diseases, EP Patent2221053, 2010.

[97] T. Lubbers, P. Angehrn, H. Gmunder, S. Herzig, J. Kulhanek, Design, synthesis,and structure-activity relationship studies of ATP analogues as DNA gyraseinhibitors, Bioorg. Med. Chem. Lett. 10 (2000) 821e826.

[98] G.V. Ullas, C.K. Chu, M.K. Ahn, Y. Kosugi, Synthesis of C-nucleoside analog of(S)-9-(2,3-dihydroxypropyl)adenine and related acyclonucleosides, J. Org.Chem. 53 (1988) 2413e2418.

[99] J. Cao, A. Dufour, Ligands for the PEX domain of matrix metalloproteinase 9as inhibitors of dimerization in cancer therapy, US Patent 20120277236,2012.

[100] S.P. Govek, G. Oshiro, S.A. Noble, J.W. Malecha, A.K. Shiau, Heterocyclic in-hibitors of phosphodiesterase 4, WO Patent 2008045664, 2008.

[101] A.P. Russ, S. Lampel, The druggable genome: an update, Drug Discov. Today10 (2005) 1607e1610.

[102] B.C. Heng, D. Aubel, M. Fussenegger, An overview of the diverse roles of G-protein coupled receptors (GPCRs) in the pathophysiology of various humandiseases, Biotechnol. Adv. 31 (2013) 1676e1694.

[103] J.-F. Chen, H.K. Eltzschig, B.B. Fredholm, Adenosine receptors as drug targetse what are the challenges? Nat. Rev. Drug Discov. 12 (2013) 265e286.

[104] C.E. Müller, K.A. Jacobson, Recent developments in adenosine receptor li-gands and their potential as novel drugs, Biochim. Biophys. Acta 1808 (2011)1290e1308.

[105] C.B. Vu, D. Pan, B. Peng, L. Sha, G. Kumaravel, X. Jin, D. Phadke, T. Engber,C. Huang, J. Reilly, S. Tam, R.C. Petter, Studies on adenosine A2a receptorantagonists: comparison of three core heterocycles, Bioorg. Med. Chem. Lett.14 (2004) 4831e4834.

[106] H. Peng, G. Kumaravel, G. Yao, L. Sha, J. Wang, H. Van Vlijmen, T. Bohnert,C. Huang, C.B. Vu, C.L. Ensinger, H. Chang, T.M. Engber, E.T. Whalley,R.C. Petter, Novel bicyclic piperazine derivatives of triazolotriazine and tri-azolopyrimidines as highly potent and selective adenosine A2A receptorantagonists, J. Med. Chem. 47 (2004) 6218e6229.

[107] G. Pastorin, S. Federico, S. Paoletta, M. Corradino, F. Cateni, B. Cacciari, K.-N. Klotz, Z.-G. Gao, K.A. Jacobson, G. Spalluto, S. Moro, Synthesis and phar-macological characterization of a new series of 5,7-disubstituted-[1,2,4]tri-azolo[1,5-a][1,3,5]triazine derivatives as adenosine receptor antagonists: apreliminary inspection of ligand-receptor recognition process, Bioorg. Med.Chem. 18 (2010) 2524e2536.

[108] C.B. Vu, B. Peng, G. Kumaravel, G. Smits, X. Jin, D. Phadke, T. Engber, C. Huang,J. Reilly, S. Tam, D. Grant, G. Hetu, L. Chen, J. Zhang, R.C. Petter, Piperazinederivatives of [1,2,4]triazolo[1,5-a][1,3,5]triazine as potent and selectiveadenosine A2A receptor antagonists, J. Med. Chem. 47 (2004) 4291e4299.

[109] M. De Zwart, R.C. Vollinga, M.W. Beukers, D.F. Sleegers, J.K. Von FrijtagDrabbe Kunzel, M. De Groote, A.P. Ijzerman, Potent antagonists for the hu-man adenosine A2B receptor. Derivatives of the triazolotriazine adenosinereceptor antagonist ZM241385 with high affinity, Drug Dev. Res. 48 (1999)95e103.

[110] V.-P. Jaakola, M.T. Griffith, M.A. Hanson, V. Cherezov, E.Y.T. Chien, J.R. Lane,A.P. Ijzerman, R.C. Stevens, The 2.6 angstrom crystal structure of a human A2aadenosine receptor bound to an antagonist, Science 322 (2008) 1211e1217.

[111] S.P.H. Alexander, A. Mathie, J.A. Peters, Guide to receptors and channels(GRAC), 3rd edition, Br. J. Pharmacol. 153 (Suppl. 2) (2008) S1eS209.

[112] X. Li, H. Kang, X. Liu, Z. Liu, K. Shu, X. Chen, S. Zhu, Effect of adenosine A2Areceptor antagonist ZM241385 on amygdala-kindled seizures and progres-sion of amygdala kindling, J. Huazhong Univ. Sci. Technol. Med. Sci. 32 (2012)257e264.

[113] K. Gotembiowska, A. Dziubina, M. Kowalska, K. Kaminska, Effect of adeno-sine A2A receptor antagonists on L-DOPA-induced hydroxyl radical formationin rat striatum, Neurotox. Res. 15 (2009) 155e166.

[114] K. Golembiowska, A. Dziubina, Effect of adenosine A2A receptor antagonistsand l-DOPA on hydroxyl radical, glutamate and dopamine in the striatum of6-OHDA-treated rats, Neurotox. Res. 21 (2012) 222e230.

[115] V. Belcastro, A. Tozzi, M. Tantucci, C. Costa, M. Di Filippo, A. Autuori,B. Picconi, S. Siliquini, E. Luchetti, F. Borsini, P. Calabresi, A2A adenosine re-ceptor antagonists protect the striatum against rotenone-induced neuro-toxicity, Exp. Neurol. 217 (2009) 231e234.

[116] R. Pepponi, A. Ferrante, R. Ferretti, A. Martire, P. Popoli, Region-specificneuroprotective effect of ZM 241385 towards glutamate uptake inhibition incultured neurons, Eur. J. Pharmacol. 617 (2009) 28e32.

[117] M. J€org, J. Shonberg, F.S. Mak, N.D. Miller, E. Yuriev, P.J. Scammells,B. Capuano, Novel adenosine A2A receptor ligands: a synthetic, functionaland computational investigation of selected literature adenosine A2A re-ceptor antagonists for extending into extracellular space, Bioorg. Med. Chem.Lett. 23 (2013) 3427e3433.

[118] M. Yang, D. Soohoo, S. Soelaiman, R. Kalla, J. Zablocki, N. Chu, K. Leung, L. Yao,I. Diamond, L. Belardinelli, J.C. Shryock, Characterization of the potency,selectivity, and pharmacokinetic profile for six adenosine A2A receptor an-tagonists, Naunyn-Schmiedeb. Arch. Pharmacol. 375 (2007) 133e144.

[119] C.B. Vu, D. Pan, B. Peng, G. Kumaravel, G. Smits, X. Jin, D. Phadke, T. Engber,C. Huang, J. Reilly, S. Tam, D. Grant, G. Hetu, R.C. Petter, Novel diamino de-rivatives of [1,2,4]triazolo[1,5-a][1,3,5]triazine as potent and selectiveadenosine A2A receptor antagonists, J. Med. Chem. 48 (2005) 2009e2018.

[120] C.B. Vu, P. Shields, B. Peng, G. Kumaravel, X. Jin, D. Phadke, J. Wang, T. Engber,E. Ayyub, R.C. Petter, Triamino derivatives of triazolotriazine and tri-azolopyrimidine as adenosine A2A receptor antagonists, Bioorg. Med. Chem.Lett. 14 (2004) 4835e4838.

[121] S. Federico, S. Paoletta, S.-L. Cheong, G. Pastorin, B. Cacciari, S. Stragliotto,K.N. Klotz, J. Siegel, Z.-G. Gao, K.A. Jacobson, S. Moro, G. Spalluto, Synthesisand biological evaluation of a new series of 1,2,4-triazolo[1,5-a]-1,3,5-triazines as human A2A adenosine receptor antagonists with improved wa-ter solubility, J. Med. Chem. 54 (2011) 877e889.

[122] A. Dalpiaz, B. Cacciari, C.B. Vicentini, F. Bortolotti, G. Spalluto, S. Federico,B. Pavan, F. Vincenzi, P.A. Borea, K. Varani, A novel conjugated agent betweendopamine and an A2A adenosine receptor antagonist as a potential anti-parkinson multitarget approach, Mol. Pharm. 9 (2012) 591e604.

[123] F. Da Settimo, G. Primofiore, S. Taliani, C. La Motta, E. Novellino, G. Greco,A. Lavecchia, B. Cosimelli, M. Iadanza, K.-N. Klotz, D. Tuscano, M.L. Trincavelli,C. Martini, A1 adenosine receptor antagonists, 3-aryl[1,2,4]triazino[4,3-a]benzimidazol-4-(10H)-ones (ATBIs) and N-alkyl and N-acyl-(7-substituted-2-phenylimidazo[1,2-a][1,3,5]triazin-4-yl)amines (ITAs): different recogni-tion of bovine and human binding sites, Drug Dev. Res. 63 (2004) 1e7.

[124] E. Novellino, E. Abignente, B. Cosimelli, G. Greco, M. Iadanza, S. Laneri,A. Lavecchia, M.G. Rimoli, F. Da Settimo, G. Primofiore, D. Tuscano,L. Trincavelli, C. Martini, Design, synthesis and biological evaluation of novelN-alkyl- and N-acyl-(7-substituted-2-phenylimidazo[1,2-a][1,3,5]triazin-4-yl)amines (ITAs) as novel A1 adenosine receptor antagonists, J. Med. Chem.45 (2002) 5030e5036.

[125] I. Sví�zensk�a, P. Dubový, A. �Sulcov�a, Cannabinoid receptors 1 and 2 (CB1 andCB2), their distribution, ligands and functional involvement in nervous sys-tem structures e a short review, Pharmacol. Biochem. Behav. 90 (2008)501e511.

[126] A.J. Brown, Novel cannabinoid receptors, Br. J. Pharmacol. 152 (2007)567e575.

[127] B. Bosier, G.G. Muccioli, E. Hermans, D.M. Lambert, Functionally selectivecannabinoid receptor signalling: therapeutic implications and opportunities,Biochem. Pharmacol. 80 (2010) 1e12.

[128] X. Cao, L. Liang, J.R. Hadcock, P.A. Iredale, D.A. Griffith, F.S. Menniti, S. Factor,J.T. Greenamyre, S.M. Papa, Blockade of cannabinoid type 1 receptors aug-ments the antiparkinsonian action of levodopa without affecting dyskinesiasin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated rhesus monkeys,J. Pharmacol. Exp. Ther. 323 (2007) 318e326.

[129] J.R. Hadcock, P.A. Carpino, P.A. Iredale, R.L. Dow, D. Gautreau, L. Thiede,D. Kelly-Sullivan, J.S. Lizano, X. Liu, J. Van Deusen, K.M. Ward, R.E. O'Connor,S.C. Black, D.A. Griffith, D.O. Scott, Quantitative in vitro and in vivo phar-macological profile of CE-178253, a potent and selective cannabinoid type 1(CB1) receptor antagonist, BMC Pharmacol. 10 (2010) 9, http://dx.doi.org/10.1186/1471-2210-10-9.

[130] J.D. Wagner, L. Zhang, K. Kavanagh, G.M. Ward, J.E. Chin, J.R. Hadcock,B.J. Auerbach, H.J. Harwood Jr., A selective cannabinoid-1 receptor antago-nist, PF-95453, reduces body weight and body fat to a greater extent thanpair-fed controls in obese monkeys, J. Pharmacol. Exp. Ther. 335 (2010)103e113.

F.P.L. Lim, A.V. Dolzhenko / European Journal of Medicinal Chemistry 85 (2014) 371e390 389

[131] S.J. Mnich, R.R. Hiebsch, R.M. Huff, S. Muthian, Anti-inflammatory propertiesof CB1-receptor antagonist involves b2 adrenoceptors, J. Pharmacol. Exp.Ther. 333 (2010) 445e453.

[132] R.L. Hauger, D.E. Grigoriadis, M.F. Dallman, P.M. Plotsky, W.W. Vale,F.M. Dautzenberg, International Union of Pharmacology. XXXVI. Currentstatus of the nomenclature for receptors for corticotropin-releasing factorand their ligands, Pharmacol. Rev. 55 (2003) 21e26.

[133] Y.-W. Li, L. Fitzgerald, H. Wong, S. Lelas, G. Zhang, M.D. Lindner, T. Wallace,J. McElroy, N.J. Lodge, P. Gilligan, R. Zaczek, The pharmacology of DMP696and DMP904, non-peptidergic CRF1 receptor antagonists, CNS Drug Rev. 11(2005) 21e52.

[134] C. Chen, Recent advances in small molecule antagonists of the corticotropin-releasing factor type-1 receptor-focus on pharmacology and pharmacoki-netics, Curr. Med. Chem. 13 (2006) 1261e1282.

[135] E.M. Jagoda, L. Lang, K. McCullough, C. Contoreggi, B.M. Kim, Y. Ma,K.C. Rice, L.P. Szajek, W.C. Eckelman, D.O. Kiesewetter, [76Br]BMK-152, anonpeptide analogue, with high affinity and low nonspecific binding forthe corticotropin-releasing factor type 1 receptor, Synapse 65 (2011)910e918.

[136] L. Lang, Y. Ma, B.M. Kim, E.M. Jagoda, K.C. Rice, L.P. Szajek, C. Contoreggi,P.W. Gold, G.P. Chrousos, W.C. Eckelman, D.O. Kiesewetter, [76Br]BMK-I-152, a non-peptide analogue for PET imaging of corticotropin-releasinghormone type 1 receptor (CRHR1, J. Label. Compd. Radiopharm. 52(2009) 394e400.

[137] P.J. Gilligan, L. He, T. Clarke, P. Tivitmahaisoon, S. Lelas, Y.-W. Li, K. Heman,L. Fitzgerald, K. Miller, G. Zhang, A. Marshall, C. Krause, J. McElroy, K. Ward,H. Shen, H. Wong, S. Grossman, G. Nemeth, R. Zaczek, P. Hartig,D.W. Robertson, G. Trainor, 8-(4-Methoxyphenyl)pyrazolo[1,5-a]-1,3,5-triazines: selective and centrally active corticotropin-releasing factorreceptor-1 (CRF1) antagonists, J. Med. Chem. 52 (2009) 3073e3083.

[138] P.J. Gilligan, T. Clarke, L. He, S. Lelas, Y.-W. Li, K. Heman, L. Fitzgerald,K. Miller, G. Zhang, A. Marshall, C. Krause, J.F. McElroy, K. Ward, K. Zeller,H. Wong, S. Bai, J. Saye, S. Grossman, R. Zaczek, P. Hartig, D. Robertson,G. Trainor, Synthesis and structure-activity relationships of 8-(pyrid-3-yl)pyrazolo[1,5-a]-1,3,5-triazines: potent, orally bioavailable corticotropinreleasing factor receptor-1 (CRF1) antagonists, J. Med. Chem. 52 (2009)3084e3092.

[139] L. Zhou, R.C. Dockens, P. Liu-Kreyche, S.J. Grossman, R.A. Iyer, In vitro andin vivo metabolism and pharmacokinetics of BMS-562086, a potent andorally bioavailable corticotropin-releasing factor-1 receptor antagonist, DrugMetab. Dispos. 40 (2012) 1093e1103.

[140] V. Coric, H.H. Feldman, D.A. Oren, A. Shekhar, J. Pultz, R.C. Dockens, X. Wu,K.A. Gentile, S.-P. Huang, E. Emison, T. Delmonte, B.B. D'Souza, D.L. Zimbroff,J.A. Grebb, A.W. Goddard, E.G. Stock, Multicenter, randomized, double-blind,active comparator and placebo-controlled trial of a corticotropin-releasingfactor receptor-1 antagonist in generalized anxiety disorder, Depress. Anx-iety 27 (2010) 417e425.

[141] D. Zuev, R.J. Mattson, H. Huang, G.K. Mattson, L. Zueva, J.M. Nielsen,E.S. Kozlowski, X.S. Huang, D. Wu, Q. Gao, N.J. Lodge, J.J. Bronson, J.E. Macor,Potential CRF1R PET imaging agents: N-Fluoroalkyl-8-(6-methoxy-2-methylpyridin-3-yl)-2,7-dimethyl-N-alkylpyrazolo[1,5-a][1,3,5]triazin-4-amines, Bioorg. Med. Chem. Lett. 21 (2011) 2484e2488.

[142] T. Saito, T. Obitsu, C. Minamoto, T. Sugiura, N. Matsumura, S. Ueno, A. Kishi,S. Katsumata, H. Nakai, M.-A. Toda, Pyrazolo[1,5-a]pyrimidines, triazolo[1,5-a]pyrimidines and their tricyclic derivatives as corticotropin-releasing factor1 (CRF1) receptor antagonists, Bioorg. Med. Chem. 19 (2011) 5955e5966.

[143] A. Nakazato, T. Okubo, D. Nozawa, T. Tamita, L.E.J. Kennis, Preparation ofpyrrolopyrimidines and pyrrolotriazines as CRF antagonists, WO Patent2005066142, 2005.

[144] J.R. McCarthy, Preparation of bicyclic nitrogen-containing heterocycles asCRF receptor antagonists and methods relating thereto, WO Patent 9835967,1998.

[145] A.G. Arvanitis, R.J. Chorvat, Preparation of azolotriazines and -pyrimidines ascorticotropin releasing factor (CRF) antagonists, WO Patent 9803510, 1998.

[146] L. He, P. Gilligan, R. Chorvat, A.G. Arvanitis, Preparation of azolotriazines and-pyrimidines as corticotropin releasing factor (CRF) antagonists, US Patent6313124, 2001.

[147] L. He, P. Gilligan, R. Chorvat, A.G. Arvanitis, Preparation of azolotriazines andpyrimidines as corticotropin releasing factor (CRF) antagonists, US Patent6191131, 2001.

[148] P. Gilligan, R. Chorvat, A.G. Arvanitis, Azolotriazines and pyrimidines usefulas corticotropin releasing factor (CRF) antagonists, US Patent 6060478, 2000.

[149] M. Szpakowska, V. Fievez, K. Arumugan, N. van Nuland, J.-C. Schmit,A. Chevign�e, Function, diversity and therapeutic potential of the N-terminaldomain of human chemokine receptors, Biochem. Pharmacol. 84 (2012)1366e1380.

[150] J. David, F. Mortari, Chemokine receptors: a brief overview, Clin. Appl.Immunol. Rev. 1 (2000) 105e125.

[151] P. Norman, A dual CCR2/CCR5 chemokine antagonist, BMS-813160? ExpertOpin. Ther. Pat. 21 (2011) 1919e1924.

[152] Y. Feng, X. He, Y. Yang, D. Chao, L.H. Lazarus, Y. Xia, Current research onopioid receptor function, Curr. Drug Targets 13 (2012) 230e246.

[153] D. Matosiuk, S. Fidecka, Preparation of 1-phenyl-5,7(1H)-dioxo-2,3-dihydroimidazo[1,2-a][1,3,5]triazines for treating CNS disorders, PL Patent186228, 2003.

[154] D. Matosiuk, S. Fidecka, L. Antkiewicz-Michaluk, J. Lipkowski, I. Dybala,A.E. Koziol, Synthesis and pharmacological activity of new carbonyl de-rivatives of 1-aryl-2-iminoimidazolidine Part 2. Synthesis and pharmaco-logical activity of 1,6-diaryl-5,7(1H)dioxo-2,3-dihydroimidazo[1,2-a][1,3,5]triazines, Eur. J. Med. Chem. 37 (2002) 761e772.

[155] M. Rzadkowska, E. Szaco�n, D. Matosiuk, E. Kedzierska, S. Fidecka, Synthesis ofnew 1-aryl-6-benzylimidazo[1,2-a][1,3,5]triazines with potential pharma-cological activity, Acta Pol. Pharm. Drug Res. 69 (2012) 1270e1275.

[156] M. Rzadkowska, E. Szacon, D. Matosiuk, E. Kedzierska, S. Fidecka, Preparationof new derivatives of 1-aryl-6-arylalkyl-5,7(1H)dioxo-2,3-dihydroimidazo[1,2-a][1,3,5]triazine as analgesics, PL Patent 211865, 2012.

[157] M. Rzadkowska, E. Szacon, D. Matosiuk, E. Kedzierska, S. Fidecka, Preparationof new derivatives of 1-(2-pyridyl)-6-substituted imidazo[1,2-a][1,3,5]triazine, PL Patent 212333, 2012.

[158] M. Rzadkowska, E. Szacon, D. Matosiuk, E. Kedzierska, S. Fidecka, Preparationof new derivatives of 1-aryl-6-substituted imidazo[1,2-a][1,3,5]triazine asCNS agents, PL Patent 211768, 2012.

[159] M. Rzadkowska, E. Szacon, D. Matosiuk, E. Kedzierska, S. Fidecka, Preparationof new derivatives of 1-aryl-6-aminocarbonylmethyl-imidazo[1,2-a][1,3,5]triazine as CNS agents, PL Patent 211769, 2012.

[160] P. Raboisson, A. Baurand, J.-P. Cazenave, C. Gachet, D. Schultz, B. Spiess, J.-J. Bourguignon, A general approach toward the synthesis of C-nucleosidepyrazolo[1,5-a]-1,3,5-triazines and their 3',5'-bisphosphate C-nucleotideanalogues as the first reported in vivo stable P2Y1-receptor antagonists,J. Org. Chem. 67 (2002) 8063e8071.

[161] S.P. Brothers, C. Wahlestedt, Therapeutic potential of neuropeptide Y (NPY)receptor ligands, EMBO Mol. Med. 2 (2010) 429e439.

[162] J.W. Darrow, S. De Lombaert, C. Blum, J. Tran, M. Giangiordano, D.A. Griffith,P.A. Carpino, Preparation of certain alkylene diamino-substituted pyrazolo[1,5-a]-1,5-pyrimidines and pyrazolo[1,5-a]-1,3,5-triazines as selectivemodulators of NPY1 receptors, WO Patent 2001023387, 2001.

[163] J.W. Darrow, S. De Lombaert, C. Blum, J. Tran, M. Giangiordano, D.A. Griffith,P.A. Carpino, Preparation of amino substituted pyrazolo[1,5-a]-pyrimidinesand pyrazolo[1,5-a]-1,3,5-triazines as NPY receptor antagonists, WO Patent2001023388, 2001.

[164] A.B. Pithadia, S.M. Jain, 5-Hydroxytryptamine receptor subtypes and theirmodulators with therapeutic potentials, J. Clin. Med. Res. 1 (2009) 72e80.

[165] M. Boes, C. Riemer, H. Stadler, Preparation of pyrazolopyrimidines andpyrazolotriazines with 5-HT6 receptor affinity, EP Patent 941994, 1999.

[166] H.-M. Yun, H. Rhim, The serotonin-6 receptor as a novel therapeutic target,Exp. Neurobiol. 20 (2011) 159e168.

[167] A. Ahmed, H. Choo, Y.S. Cho, W.-K. Park, A.N. Pae, Identification of novelserotonin 2C receptor ligands by sequential virtual screening, Bioorg. Med.Chem. 17 (2009) 4559e4568.

[168] H. Kiyokawa, S. Yamada, K. Miyajima, K. Edamatsu, K. Tatsumi, T. Yamauchi,K. Kishi, K. Kiyono, Condensed pyrazole derivatives, method ofmanufacturing the same, and androgen inhibitor, EP Patent 594149, 1994.

[169] A.M. Venkatesan, Angiotensin II receptor blocking 2,3-substituted pyrazolo[1,5-a]-1,3,5-triazin-4(3H)-ones, US Patent 5358947, 1994.

[170] E.L. Andrade, F.C. Meotti, J.B. Calixto, TRPA1 antagonists as potential anal-gesic drugs, Pharmacol. Ther. 133 (2012) 189e204.

[171] S.S. Chaudhari, S. Kumar, A. Thomas, N.P. Patil, A.B. Kadam, N.T. Waghmare,N. Khairatkar-Joshi, I. Mukhopadhyay, Preparation of amides of heterocycliccompounds as TRPA1 inhibitors, WO Patent 2011114184, 2011.

[172] F. Popowycz, C. Schneider, S. De Bonis, D.A. Skoufias, F. Kozielski,C.M. Galmarini, B. Joseph, Synthesis and antiproliferative evaluation ofpyrazolo[1,5-a]-1,3,5-triazine myoseverin derivatives, Bioorg. Med. Chem.17 (2009) 3471e3478.

[173] S.Y.K. Tam, R.S. Klein, I. Wempen, J.J. Fox, Nucleosides. 112. Synthesis of somenew pyrazolo[1,5-a]-1,3,5-triazines and their C-nucleosides, J. Org. Chem. 44(1979) 4547e4553.

[174] F. Saczewski, M. Maruszak, P.J. Bednarski, Synthesis and cytotoxic activity ofimidazo[1,2-a]-1,3,5-triazine analogues of 6-mercaptopurine, Arch. Pharm.341 (2008) 121e125.

[175] O. Bekircan, M. Kuxuk, B. Kahveci, S. Kolayli, Convenient synthesis of fusedheterocyclic 1,3,5-triazines from some N-acyl imidates and heterocyclicamines as anticancer and antioxidant agents, Arch. Pharm. 338 (2005)365e372.

[176] A.V. Dolzhenko, B.J. Tan, A.V. Dolzhenko, G.N.C. Chiu, W.K. Chui, Synthesisand biological activity of fluorinated 7-aryl-2-pyridyl-6,7-dihydro[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-amines, J. Fluor. Chem. 129 (2008)429e434.

[177] N. Inoue, M. Matsushita, Y. Fukui, S. Yamada, M. Tsuda, C. Higashi, K. Kaneko,H. Hasegawa, T. Yamaguchi, Identification of a Varicella-Zoster virus repli-cation inhibitor that blocks capsid assembly by interacting with the floordomain of the major capsid protein, J. Virol. 86 (2012) 12198e12207.

[178] K.S. Gudmundsson, B.A. Johns, J. Weatherhead, Pyrazolopyrimidines andpyrazolotriazines with potent activity against herpesviruses, Bioorg. Med.Chem. Lett. 19 (2009) 5689e5692.

[179] J.O. Ojwang, Ribofuranosyl imidazotriazinone compounds as broad-spectrum inhibitors of viruses in the Flaviviridae family, US Patent20060035848, 2006.

[180] G. Gosselin, P. La Colla, F. Seela, R. Storer, D. Dukhan, F. Leroy, Preparation of5-aza-7-deazapurine and C-branched nucleosides as antiviral agents fortreating Flaviviridae, WO Patent 2006000922, 2006.

F.P.L. Lim, A.V. Dolzhenko / European Journal of Medicinal Chemistry 85 (2014) 371e390390

[181] P. La Colla, G. Gosselin, F. Seela, D. Dukhan, F. Leroy, Preparation of 5-aza-7-deazapurine nucleosides as antiviral agents for treating Flaviviridae, WOPatent 2004096197, 2004.

[182] J.O. Ojwang, S. Ali, D.F. Smee, J.D. Morrey, C.D. Shimasaki, R.W. Sidwell,Broad-spectrum inhibitor of viruses in the Flaviviridae family, Antivir. Res.68 (2005) 49e55.

[183] D. Dukhan, F. Leroy, J. Peyronnet, E. Bosc, D. Chaves, M. Durka, R. Storer, P. LaColla, F. Seela, G. Gosselin, Synthesis of 5-aza-7-deazaguanine nucleosidederivatives as potential anti-flavivirus agents, Nucleotides Nucleic Acids 24(2005) 671e674.

[184] J.O. Ojwang, Use of 2-amino-8-(b-D-ribofuranosyl)imidazo[1,2-a]-s-triazin-4-one for the treatment of influenza, WO Patent 2001017518, 2001.

[185] B. Golankiewicz, P. Januszczyk, S. Ikeda, J. Balzarini, E. De Clercq, Synthesisand antiviral activity of benzyl-substituted imidazo[1,5-a]-1,3,5-triazine(5,8-diaza-7,9-dideazapurine) derivatives, J. Med. Chem. 38 (1995)3558e3565.

[186] J.E.G. Guillemont, P.J.-M.B. Raboisson, N. Lounis, Preparation of substitutedtriazinoindazolamines with antibacterial activity against Clostridium, EPPatent 63434, 2012.

[187] J.L. Junien, M. Guillaume, C. Lakatos, J. Sterne, Bronchodilator and antiallergicproperties of LA 2851 (2-4-diamino-7-methylpyrazolo[1,5-a][1,3,5]triazine),Arch. Int. Pharmacodyn. Ther. 252 (1981) 313e326.

[188] M. Guillaume, C. Lakatos, LA 2851, a new bronchodilator and anti-allergiccompound which inhibits slow-reacting substance formation, in: Prosta-glandins Ser, vol. 3, 1983, pp. 332e337.

[189] F. Ruff, A. Floch, D. Chastagnol, M. Blanc, M.C. Santais, Anti-allergic propertiesof 2-4-diamino-7-methyl-pyrazolo(1,5-a)1,3,5-triazine (LA 2851): Possibleinhibition of the leukotrienes, in: Prostaglandins Ser, vol. 3, 1983, pp.205e210.

[190] J.L. Junien, C. Lakatos, J. Brohon, M. Guillaume, J. Sterne, Anti-inflammatoryeffect of LA 2851 and reference drugs on some models of inflammation.Investigation of the mechanism of action, Agents Actions 12 (1982) 459e465.

[191] C. Cohen, Diaminopyrazolo[1,5-a]-s-triazines, DE Patent 2900288, 1979.[192] J.J. Wade, Preparation and testing of substituted 1,2,4-triazolo[1,5-a]triazines

as bronchodilators, US Patent 4734413, 1988.[193] T. Sawai, T. Hirakawa, M. Kozasa, R.S.J. Clark, A. Kimura, K. Harada, K. Chiba,

Preparation and effect of aromatic alkane derivatives as G protein inhibitors,JP Patent 2001011064, 2001.

[194] F. Akahoshi, S. Takeda, T. Okada, M. Kajii, H. Nishimura, M. Sugiura, Y. Inoue,C. Fukaya, Y. Naito, T. Imagawa, N. Nakamura, Synthesis and pharmacologicalactivity of triazolo[1,5-a]triazine derivatives inhibiting eosinophilia, J. Med.Chem. 41 (1998) 2985e2993.

[195] F. Akahoshi, T. Okada, S. Takeda, Y. Naito, C. Fukaya, S. Kuwahara, M. Kajii, H.Nishimura, M. Sugiura, Triazole derivatives and pharmaceutical use thereof,WO Patent 9503286, 1995.

[196] F. Suzuki, N. Koike, J. Shimada, S. Kitamura, S. Ichikawa, J. Nakamura, S.Shiozaki, Depression remedy, WO Patent 9507282, 1995.

[197] F. Suzuki, J. Shimada, N. Koike, S. Ichikawa, J. Nakamura, T. Kanda, S. Kita-mura, Remedy for parkinson's disease, WO Patent 9503806, 1995.

[198] Z. Yang, M. Chen, J.L. Nadler, Lisofylline: a potential lead for the treatment ofdiabetes, Biochem. Pharmacol. 69 (2005) 1e5.

[199] P. Cui, T.L. Macdonald, M. Chen, J.L. Nadler, Synthesis and biological evalu-ation of lisofylline (LSF) analogs as a potential treatment for Type 1 diabetes,Bioorg. Med. Chem. Lett. 16 (2006) 3401e3405.

[200] M. Legraverend, Recent advances in the synthesis of purine derivatives andtheir precursors, Tetrahedron 64 (2008) 8585e8603.

[201] A. Manvar, A. Shah, Microwave-assisted chemistry of purines and xanthines.An overview, Tetrahedron 69 (2013) 8105e8127.