European Journal of Medicinal Chemistry 95 (2015) 76e95

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Mini-review

Recent advances in the chemistry and biology of pyridopyrimidines
rour a, M. Akssira b, G. Guillaumet a, S. Routier a, * F. Buron a, J.Y. Me a b

Institut de Chimie Organique et Analytique, Universit
e d’Orl
eans, UMR CNRS 7311, rue de Chartres, BP 6759, 45067 Orl
eans Cedex 2, France
Equipe de Chimie Bioorganique & Analytique, URAC 22, Universit
e Hassan II Mohammedia-Casablanca, BP 146, 28800 Mohammedia, Morocco

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 October 2014 Received in revised form 19 February 2015 Accepted 13 March 2015 Available online 14 March 2015

The interest in pyridopyrimidine cores for pharmaceutical products makes this scaffold a highly useful building block for organic chemistry. These derivatives have found applications in various areas of medicine such as anticancer, CNS, fungicidal, antiviral, anti-inflammatory, antimicrobial, and antibacterial therapies. This review mainly focuses on the progress achieved since 2004 in the chemistry and biological activity of pyridopyrimidines. © 2015 Elsevier Masson SAS. All rights reserved.

Keywords: Heterocycles Pyridopyrimidine Synthesis Pharmacological activities

1. Introduction Pyridopyrimidines are fused 6,6-bicyclic heterocycles consisting of a pyridine ring fused to a pyrimidine. Depending on the position of the nitrogen atom in the pyridine moiety, four isomeric structures of pyridopyrimidines 1e4 are available (Scheme 1). These scaffolds, which are nitrogen bioisosters of quinoxaline, are associated with a diverse range of biological activities such as anticancer, CNS disorders, and antiviral agents. They have been widely used in medicinal chemistry due to their five possible substitution sites and potentially accessible products which cover a large chemical diversity space. In the literature, previous reviews have mainly dealt with the chemistry of pyridopyrimidines, with only a minor interest in their pharmacological properties [1e5] and another review has investigated recent developments in the chemistry of pyridopyrimidines with one bridgehead nitrogen atom [6]. In the present account, new developments over the last ten years in pyridopyrimidine synthesis (excluding pyridopyrimidinone derivatives) with a focus on pharmacological properties will be discussed. These derivatives are considered as a key framework for target interaction and are platforms which orient substituents in a tridimensional manner. During the period covered in this review, few synthetic methods were investigated and, more specifically, most of the chemical developments were devoted to

* Corresponding author. E-mail address: [email protected] (S. Routier). http://dx.doi.org/10.1016/j.ejmech.2015.03.029 0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

the regioselective introduction of various substituents. To give an overview of the potential of pyridopyrimidines, the review is structured as follows. First the useful chemistry leading to the skeletons is depicted. Several building strategies are listed for each regioisomer. The regioselective functionalization of these scaffolds is the presented followed by the biological development of drugs which contain the pyridopyrimidine moiety. 2. Chemistry of pyridopyrimidines The synthesis of pyridopyrimidine derivatives has markedly increased in the literature. The number of references containing the pyridopyrimidine core soared over the period 1975e2014 (Fig. 1). Several methods have been developed and two main cyclization strategies are commonly used to access these scaffolds. The first strategy involves ring closure by formation of the pyrimidine and the second strategy ring closure by formation of the pyridine. 2.1. Synthesis by ring closure 2.1.1. Synthesis by formation of the pyrimidine ring Wu et al. synthesized 8-cyano-7-methoxy pyrido[4,3-d]pyrimidines 8 by pyrimidine ring closure in order to test cytotoxic activity [7]. Formamidate 6, prepared from the reaction of the aminopyridine derivative 5 with triethyl orthoformate, cyclized smoothly in the presence of ammonia or primary amines, generating either the 4-methyl-8-cyano-7-methoxy pyrido[4,3-d]pyrimidine 7 or the 4-methylene-8-cyano-7-methoxy-3,4-dihydropyrido[4,3-d]

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Scheme 1. Structures of pyridopyrimidines.

Scheme 3. Synthesis of pyrido[4,3-d]pyrimidines via an imine. Fig. 1. Number of references containing the pyridopyrimidine core over the period 1923e2014. a Search performed on 20 January 2015 by Scifinder Research Topic using pyridopyrimidine as keyword.

pyrimidine 8 respectively in moderate to good yields (Scheme 2). An amino pyridine framework can be fused to the prebuilt pyrimidine ring. To this end, cyclocondensation with imine derivatives was used by Lobanov et al. for the design of pyrido[4,3-d] pyrimidines. 1-(4-Chloro-2-methylsulfanyl-pyrimidin-5-yl)ethanone 10 reacted with ethyl 3,3-diaminoprop-2-enoate 9 to produce the pyrido[4,3-d]pyrimidine derivative 11 in 71% yield at room temperature in DMF. At elevated temperature (175
C), the same reaction with chloropyrimidine 12 led in a few minutes to the bisamino derivative 13 in good yield (Scheme 3) [8,9]. Recently, a new synthetic approach to 4-aminopyrido[2,3-d] or [3,2-d]pyrimidines was described by Maes et al. [10]. The authors investigated the use of Pd-catalyzed cross coupling reactions between N-(bromopyridyl)amidines 15 and secondary or tertiary isocyanides to efficiently access disubstituted pyridopyrimidines 16 in moderate yields (Scheme 4). Elneairy et al. investigated the use of a Dimroth rearrangement for the synthesis of pyrido[2,3-d]pyrimidine derivatives [11]. Pyridin-2-(1H)-thione 17 reacted with phenyl isothiocyanate in refluxing pyridine to give substituted pyrido[2,3-d]pyrimidine 19 in 59% yield as the result of intermediate 18 rearrangement.

Compound 19 then reacted with various halides such as chloroacetonitrile in DMF in the presence of potassium hydroxide to give thioalkyl derivatives 20. Interestingly, these derivatives were subjected to a second intramolecular thiophenyl ring creation leading to thieno[30 ,50 :5,6]pyrido[2,3-d]pyrimidine derivatives 21 (Scheme 5). 2.1.2. Synthesis by formation of the pyridine ring 2.1.2.1. Pyrido[2,3-d]pyrimidines. Dorokov et al. studied a €nder strategy to access pyrido[2,3-d]pyrimidines [12]. The Friedla reaction of 2,6-disubstituted-5-acetyl-4-aminopyrimidine hydrochloride 22 in refluxing acetylacetone afforded 6-acetyl pyrido[2,3d]pyrimidine 23 in 55% yield. When a phenyl substituent was adjacent to the keto group, the cyclization of the enamine intermediate required the addition of DBU in order to cyclize into compound 25 (53% yield). Using sodium methoxide led to compound 26 with concomitant deacetylation. The use of ethyl acetoacetate instead of acetylacetone led to ethyl pyrido[2,3-d] pyrimidine-6-carboxylate 27 in lower yield (Scheme 6). Using the method of Gangjee [13e15], 2,4,6-triaminopyrimidine 28 reacted with bromomalondialdehyde in acidic conditions. A further protection of the 2,4-diamino-6-bromopyrido[2,3-d]pyrimidine with the pivaloyl group increased solubility and facilitated purification of the final compound. Under these conditions, bis pivaloyl derivative 29 was obtained in a global yield of 39% (Scheme 7). 2.1.2.2. Pyrido[3,2-d]pyrimidines. Recently, Quattropani et al. reported an efficient route to 2,4,8-trichloropyrido[3,2-d]pyrimidine derivatives [16]. Aminouracil 30 and 3-substituted 3-alkoxyacrylic acid ethyl ester were heated at 140
C in isopropanol under microwave irradiation, yielding enamines 31 as a mixture of ethyl and isopropyl esters having E and Z configurations. The mixture was cyclized into the 6-substituted 2,4,8-trihydroxypyrido[3,2-d]pyrimidines at 250
C under microwave irradiation in a mixture of 1,2-dichlorobenzene and dimethylacetamide. Chlorination with excess of POCl3 in N,N-diethylaniline or in neat POCl3 produced the highly valuable platform 32 (Scheme 8).

Scheme 2. Synthesis of pyrido[4,3-d]pyrimidines via imidate.

2.1.2.3. Pyrido[3,4-d]pyrimidines. Malhotra et al. described the only convenient process to prepare pyrido[3,4-d]pyrimidines [17].

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Scheme 4. Synthesis of pyrido[2,3-d] or [3,2-d]pyrimidines by palladium-catalyzed isocyanide insertion.

4-Chloro-8-methoxypyrido[3,4-d]pyrimidine-2-amine 34 was prepared from 33, by reaction of freshly made chloroformamidine hydrochloride in DMSO at 140
C, producing 2-amino-8chloropyrido[3,4-d]pyrimidine-4-ol in 93% yield, which upon treatment with sodium methylate in methanol and a final chlorination with phosphorous oxychloride produced 34 in a global yield of 31% from 33 (Scheme 9). 2.2. Functionalization of pyridopyrimidines

Scheme 5. Synthesis of 4-phenylamino-5-(4-methoxyphenyl)pyrido[2,3-d]pyrimidine-2,7-dithiols.

Most of the previously reported strategies have lead to functionalized pyridopyrimidines through the judicious choice of reagents. Such methods constitute a real breakthrough in large library development. Over the past decade, palladium-catalyzed cross-coupling reactions have been the main method for (C or N)eC bond formation in the pyridopyrimidine series. Additionally halogen leaving groups, which can be discriminated by their nature and position on poly-halopyridopyrimidines, have been extensively studied. 2.2.1. Functionalization by displacement of mono halogenopyridopyrimidines 2.2.1.1. By substitution of mono halogeno pyrido[2,3-d]pyrimidine derivatives. In the work described by Gangjee et al. on the synthesis of novel antifolates, the bromo derivative 24 was unsuccessfully subjected to amination reactions using a standard BuchwaldHartwig coupling protocol (BINAP/Pd(OAc)2 and sodium tert-butoxide as base) [14]. Consequently, the authors decided to find an efficient catalytic system by optimizing all of the reaction parameters (ligand screening, base, palladium catalyst). It was shown that 3 equivalents of LiHMDS prevented deactivation of the catalyst Pd2dba3 and that X-Phos was the best ligand for the coupling reaction. Anilines with electron-donating substituents gave better yields (Scheme 10). Pivaloyl deprotection of 35 was carried out with liquid ammonia at room temperature.

Scheme 6. Synthesis of pyrido[2,3-d]pyrimidines via imine.

Scheme 7. Synthesis of 2,4-di-pivaloylamino-6-bromopyrido[2,3-d]pyrimidine.

2.2.1.2. By substitution of mono halogeno pyrido[3,2-d]pyrimidine derivatives. Our laboratory has described the regioselective dechlorination of 2,4-dichloropyrido[3,2-d]pyrimidine to access 2monosubstituted pyrido[3,2-d]pyrimidines [18]. Mono dechlorination of 36 was regioselectively achieved to produce 2chloropyrido[3,2-d]pyrimidine 37 in presence of tributyltin hydride and Pd(PPh3)4 in toluene in 86% yield. The compound 37 was then reacted with various arylboronic acids in the presence of Pd(PPh3)4 in a mixture of toluene/ethanol to afford the corresponding 2-arylpyrido[3,2-d]pyrimidines 38 in 80e88% yield (Scheme 11). 2.2.1.3. By substitution of mono halogeno pyrido[3,4-d]pyrimidine derivatives. Malhotra et al. studied the displacement of the chlorine in position C-4 of compound 34 in order to design pyridopyrimidines as potential anticancer agents [17]. Direct nucleophilic aromatic substitution (SNAr) on the 4-chloro derivative 34 with

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Scheme 8. Synthesis of pyrido [3,2-d]pyrimidine via enamine 31.

Scheme 9. Synthesis of 2-amino-4-chloro-8-methoxypyrido[3,4-d]pyrimidine.

Scheme 12. Substitution reaction on the pyrimidine ring of pyrido[3,4-d]pyrimidine. Scheme 10. Substitution reaction on the pyridine ring of pyrido[2,3-d]pyrimidine.

amines, thiols, and phenols, promoted either with acid or base, was successfully performed and is illustrated by the synthesis of 39. The palladium-catalyzed reaction with aryl boronic acids (Suzuki reaction) smoothly occurred as seen by the synthesis of 40 (Scheme 12). 2.2.1.4. By substitution of 6-chloropyrido[4,3-d, 3,4-d or 2,3-d]pyrimidine derivatives. Crawford et al. studied the introduction of a halogenophenyl substituent on 4-amino-6-chloropyrimido[3,4d or 4,3-d or 2,3-d]pyrimidines of type 41 to generate potent MAP4K4 inhibitors 42 [19]. These reactions were accomplished using the corresponding arylboronic acids, PdCl2 [(di-tert-butyl(4dimethylaminophenyl)phosphine)]2, and K3PO4 in dioxane/water at 100
C (Scheme 13). 2.2.2. Functionalization by displacement of dihalogenopyridopyrimidines 2.2.2.1. Substitution of 2,4-dichloropyrido[2,3-d]pyrimidine derivatives. A selective bi-functionalization of pyrido[2,3-d]pyrimidines from 2,4-dichloropyrido[2,3-d]pyrimidine 43 was described by Berteina-Raboin et al. [20]. The higher reactivity of position 2 of compound 43 made it possible to obtain mono-substituted pyrido [2,3-d]pyrimidines 44 via a SNAr reaction with an alcohol, thiol or amine as nucleophile. To complete this study, the final functionalization of these derivatives with a Suzuki or Stille cross coupling reaction in position 4 gave 2,4-disubstituted pyrido[2,3-d]pyrimidines in good to excellent yields (Scheme 14). This methodology was extended through two successive

regioselective palladium cross-coupling reactions involving original chlorine discrimination [21]. A first SuzukieMiyaura crosscoupling reaction in C-4 position was performed with classical Suzuki conditions producing derivatives 46 in good yields (Scheme 15). The reactivity of the C-2 position was explored and the second aryl was introduced by C-2 chlorine displacement to give derivatives 47 in 61e86% yields using ethanol as co-solvent to increase reagent solubility. A second pathway was investigated to synthesize 2,4-di(het)arylpyrido[2,3-d]pyrimidines using an isopropylsulfanyl group in position C-2 as a protecting group. The first arylation in C-2 position was performed with arylboronic acids, Na2CO3, 5 mol % of Pd(PPh3)4 in a mixture of DME/water at 75
C to give 49 in good yields. The displacement of the iso-propylsulphanyl group by palladium cross coupling required the presence of copper(I)-thiophene-2-carboxylate as a cofactor, using the conditions of Liebeskind [22,23] in order to access 2,4-di(het)arylpyrido[2,3-d] pyrimidines 50 (71e86% yield, Scheme 15).

2.2.2.2. Substitution of 2,7-dichloropyrido[2,3-d]pyrimidine derivatives. Pursuing this original cross-coupling strategy, our research team developed an efficient and easy access to 2,7edisubstituted pyrido[2,3-d]pyrimidines by two orthogonal palladium-catalyzed cross-coupling reactions via successive C-7 chlorine and C-2 methylsulfur release [24]. Starting from 7-chloro2-methylsulfanylpyrido[2,3-d]pyrimidine 51, the first cross coupling reaction with electron-rich and electron-poor aryl residues or representative heterocycles was performed to give 52 in moderate to good yields. Next, the C-alkylsulfanyl release was

Scheme 11. Substitution reaction on the pyrimidine ring of 2-chloropyrido[3,2-d]pyrimidine.

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Scheme 13. Introduction of halogenophenyl substituent chloropyrimido[3,4-d or 4,3-d or 2,3-d] pyrimidines.

on

4-amino-6-

achieved using a large variety of (het)arylboronic acids with Liebeskind conditions and CuTC as cofactor to yield compounds 53 (Scheme 16).

2.2.2.3. Substitution of 2,4-dichloropyrido[3,2-d]pyrimidine derivatives. At the beginning of our investigations, we developed a new strategy to synthesize 2,4-dissymmetrical pyrido[3,2-d]pyrimidines. 2,4-Dichloropyrido[3,2-d]pyrimidine 36 was regioselectively substituted at position C-4 with various nucleophiles such as propan-2-thiol in the presence of sodium hydride, providing

compound 54 in 88% yield [18]. The second halogen in position C-2 in compound 54 was then substituted by a Suzuki reaction with arylboronic acids (phenyl, (4-methoxy)phenyl, 2-thienyl) [25]. All Suzuki reactions were achieved in 1e3 h in a mixture of DME/water at 75
C and produced the derivatives 55 in excellent yields (73e90%). A second palladium cross-coupling reaction using copper (I) cofactor under Liebeskind conditions was then performed. Thus, 2-thiophene copper(I)carboxylate, Pd(PPh3)4, and arylboronic acids in THF at 50
C or a Stille cross-coupling reaction with aryl stannanes in refluxing DME allowed the synthesis of compounds 56 from 55 in good yields (Scheme 17). Differential substitution of compound 36 can also be achieved via SuzukieMiyaura reactions or Stille reactions. Exclusive monoarylation at position C-4 was observed either with arylboronic acid (1.0 equiv.) in toluene at 100
C for 2 h or in 34e72% yield with Stille cross coupling. A library of 14 products was developed with yields of 72e89%. The second C-2 chloride atom was then displaced with a slight excess of arylboronic acid and Na2CO3 in a mixture of toluene/EtOH at 100
C for 4e6 h in the presence of only 5% Pd(PPh3)4, in excellent yields (89e98%). Alternatively, the same compound 58 was also produced from 57 using classical Stille conditions in moderate to good yields (52e87%). Reactions required a stoichiometric amount of stannanes with 2.8 equiv. of LiCl in DMF at 90
C (Scheme 18) [25]. Finally, the difference in reactivity of the two chlorine atoms allowed a sequential one-pot palladium-catalyzed reactions giving access to dissymmetrical 2,4-di(het)aryl derivatives via a double SuzukieMiyaura reaction. First, compound 36 and the heteroarylboronic acid (1.05 equiv.) was reacted using the catalytic system Pd(OAc)2/triphenylphosphine in refluxing toluene with potassium carbonate, until its disappearance (1e2 h) then a second

Scheme 14. Substitution and Cross coupling reactions of the pyrimidine ring of pyrido[2,3-d]pyrimidine.

Scheme 15. Cross coupling reactions on the pyrimidine ring of pyrido[2,3-d]pyrimidine.

Scheme 16. Cross coupling reactions of the pyrimidine ring of pyrido[2,3-d]pyrimidine.

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Scheme 17. Substitution reaction on the pyrimidine ring of pyrido[3,2-d]pyrimidine.

Scheme 18. Synthesis of 2,4-di(het)aryl pyrido [3,2-d]pyrimidines.

Scheme 21. Synthesis of 2,4-di(aminoaryl) pyrido[3,2-d]pyrimidine. Scheme 19. One-pot synthesis of 2,4-di(het)aryl pyrido[3,2-d]pyrimidines.

heteroarylboronic acid (1.2 equiv.) in ethanol was added and reacted in only a few minutes (10e30 min) to give 59 from 36 in 81% yield (Scheme 19) [26]. A similar sequential substitution of the two halogen atoms was envisioned to prepare arylamino derivatives 61 [26]. It was demonstrated that alkyl amination smoothly occurred regioselectively at C-4 [18] at room temperature and the second amination reaction at position C-2 with heating (Scheme 20). Thus, the 2,4dichloro compound 36 reacted first with aniline derivatives in the presence of a stoichiometric amount of Et3N in THF at room temperature to produce the arylamino-2-chloro pyrido[3,2-d]pyrimidine 60 (i.e. Ar1 ¼ Ph in 87% yield) whereas the introduction of arylamine at position C-2 was achieved in refluxing dioxane to give 61 (i.e. Ar1 ¼ Ph, Ar2 ¼ 4-MeOBn in 92% yield). The authors also demonstrated that a one pot reaction also lead to 61 with the same efficiency for the two-step procedure (78% instead of 80%). With a deactivated amine (6-aminoquinoline) the amination reaction at position C-2 was achieved in 78% yield if Buchwald conditions are used (Xantphos, Pd(OAc)2 reflux, 46 h). Reaction time is decreased to 2 h using microwave irradiation at 140
C (Scheme 21). 2.3. Functionalization by halogen displacement of tri halogenopyridopyrimidines 2.3.1. Substitution of 2,4,7-trichloro pyrido[3,2-d]pyrimidine derivatives 2,4,7-Trichloropyrido[3,2-d]pyrimidine 64 was obtained from 1H,3H-pyrido[3,2-d]pyrimidine-2,4-dione 63 by reacting a mixture of POCl3 and PCl5 at 160
C in a sealed tube under microwave irradiation (Scheme 22) [27]. With this trichloro derivative 64, an

extension of the previous work was proposed and led to a sequential reaction of three chloride atoms. Regioselective arylation at C-4 was first performed using a near stoichiometric amount of phenylboronic acid in the presence of potassium carbonate and Pd(PPh3)4 (5 mol%) in toluene at 100
C. Complete consumption of 64 was observed with the formation of the monophenyl derivative 65 in a good 77% yield (Scheme 22). Arylation at the C-2 position was then achieved in a second reaction without any effect on the chlorine atom at C-7. The best conditions were obtained by adding ethanol as co-solvent and using sodium carbonate as base. The (3methoxyphenyl)boronic acid reacted with 65 in a few hours to give the bis(arylated) compounds 66 in good yields. Full C-2 vs. C-7 regioselectivity was obtained, and no obvious steric effect of the arylboronic acid was observed. The introduction of a third het(aryl) group at C-7 was achieved using standard Suzuki conditions; thus a pyridinyl group was introduced at position C-7 in good yield (10 min reaction time) to produce derivative 67 in 88% yield (Scheme 22). Selective pallado-dehalogenation of 2,4,7-trichloropyrido[3,2-d] pyrimidine 64 was successfully achieved with tributyltin hydride to produce 2,7-dichloropyrido[3,2-d]pyrimidine 68 in 90% yield (Scheme 23) [28]. Discrimination of the reactivity of the two chlorine atoms of 68 was achieved by performing a selective C-2 arylation. The 2, 3 or 4-hydroxyphenylboronic acid isomers reacted in the presence of Pd(PPh3)4 and sodium carbonate to produce the C-2 hydroxyphenyl derivatives 69 in 63e70% yield. If the reaction on 68 was performed under microwave irradiation at 150
C with an excess of hydroxyphenylboronic acid, Pd(PPh3)4, the symmetrical diarylated C-2/C-7 derivatives 70 were obtained in 15 min in good yields (62e79%). The C7eCl bond of the monoarylated derivative 69 (Ar1OH ¼ C6H4(4-OH) could also be reduced to 71 in 54% yield by microwave assisted palladium reduction in the presence of

Scheme 20. Synthesis of 2,4-diaminoaryl pyrido[3,2-d]pyrimidines.

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Scheme 22. Regioselective SuzukieMiyaura reactions.

Scheme 23. Regioselective palladoecatalyst reaction.

formic acid or could react with various het(aryl)boronic acids under microwave irradiation to produce the dissymmetrically diarylated derivatives 72 in 61e73% yield. Tris(amination) of 2,4,7-trichloropyrido[3,2-d]pyrimidine was also achieved stepwise (Scheme 24) [27]. The introduction of benzylamine at C-4 was achieved at room temperature in THF in the presence of Et3N to produce compound 73 from 64 in 90% yield. The second reaction required thermal activation (refluxing dioxane) and a slight excess of amine (ethanolamine) to achieve C-2 chlorine displacement. Compound 74 was thus obtained after 12 h in 92% yield. It was possible to reduce the remaining CeCl bond of 74 using formic acid and palladium diacetate in THF under microwave irradiation, producing 75 in 57% yield. The third amination of 74 was achieved under Buchwald conditions using palladium

acetate and Xantphos in dioxane to give 2-aminopyrimidine 76 in 73% yield. 2.3.2. Substitution of 2,4,8-trichloro pyrido[3,2-d]pyrimidine derivatives Bouscary-Desforges et al. showed that 2,4,8-trichloropyrido[3,2d]pyrimidine-6-carboxylic acid methyl ester 77, if reacted with primary alkyl or aryl amines in the presence of Hunig's base (DIPEA) in acetonitrile, led to a regioselective C-4 amination compound 78 in good yields (73e82%, Scheme 25) [29]. The displacement of the two remaining chlorine atoms via SNAr and metalcatalyzed reactions was performed. Amination was realized with both primary and secondary amines in excess in a solution of DMF/ CH3CN at 90
C. Only one regioisomer 79 was obtained in good

Scheme 24. Regioselective tris(amination) of 2,4,7-trichloropyrido[3,2-d]pyrimidine.

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Scheme 25. Amination of 2,4,8 trichloropyrido[3,2-d]pyrimidine-6-carboxylic acid methyl ester.

yield (77e82%). The use of aniline required refluxing dioxane for 48 h (72% yield) for a reaction to occur. Hydrogenolysis of the CeCl bond of compound 80 with ammonium formate and Pd/C gave 81 in 69% yield. The SNAr reaction was also studied with compound 82 in the presence of thiols. Benzylthiol or p-thiocresol in the presence of Hunig's base or sodium methylthiolate were added to 82 in DMF at 0
C to avoid a double addition as observed at higher temperature. After 2 h of reaction at 0
C two regioisomers (C-8 and C-2) were observed with benzylthiol and p-thiocresol in a C-8/C-2 ratio of 73/23 and 68/32 respectively. With sodium methylthiolate only traces (3%) of the second regioisomer (C-2 substitution) were observed; the C-8 derivative 83 was obtained in 77% yield. By switching to a protic solvent such as iso-propanol, the C-2 addition was quasi exclusive and compound 84 was obtained in 77% yield. The chlorine atom at C-2 position of derivative 83 was easily converted to an aryl group using a Suzuki reaction with an arylboronic acid to afford 74 (79e88% yield). The thiomethyl substituent present at the C-8 position in compounds 85 could be replaced by an aryl group in presence of CuTc, Pd(PPh3)4 to give 86 in 84e88% yield (Scheme 25). The C-2 chlorine atom present in compound 83 was also reacted with n-butylamine, benzylamine or diethyl amine, affording the corresponding C-2,C-4 diamino C-8 methylthio derivatives 87 in 71e74% yield (Scheme 25) [29]. The lability of the chloro substituent at C-4 of the trichloro compounds 88 was investigated by using sodium methylthiolate in THF at 10
C affording compound 89 (84e85%) [16]. Reaction at higher temperatures produced multiple methylsulfanyl addition. Arylation of 89 was then achieved via a Liebeskind cross-coupling reaction of an arylboronic acid, generating the monoaryl

compounds 90 in 77e82% yield (Scheme 26). Amination with benzylamine, butylamine or diethylamine at C-4 of 88 was achieved in acetonitrile under microwave irradiation at 90
C whereas sodium methylthiolate or benzylthiol afforded a mixture of regioisomers. C-2 substitution was largely predominant over that in C-8. Direct arylation of the chloro derivative 91 via metal-catalyzed cross-coupling reactions generated compounds 92 in 80e86% yields (Scheme 26). 3. Design of pyridopyrimidines for biological activity The previously reported synthetic efforts and resulting biological tests have led to the discovery of pyridopyrimidines acting on several proteins. The final highly substituted derivatives have found applications in various areas of medicine such as anticancer, CNS, fungicidal, antiviral, anti-inflammatory, antimicrobial and antibacterial therapies as shown below. 3.1. Anticancer activity The mammalian target of rapamycin (mTOR) is a key target in the development of anti-tumor therapies. This kinase is activated by stimulation of the phosphatidylinositol 3-kinase (PI3K)-AktmTOR pathway and is a central regulator of cell-growth and proliferation [30e32]. Pass et al. reported a potent inhibitor of mTOR kinase (96, Ku 0063794, IC50 PI3Ka ¼ 2.5 nM) which displayed a high level of selectivity against other members of the PIKK isoform family (a, b, g, d; IC50 > 30mM). Compound AZD2014 (97, IC50 ¼ 2.8 nM) possessed the best pharmacokinetic parameters and was selected for clinical development.

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Scheme 26. Reactivity of 2,4,8 trichloropyrido[3,2-d]pyrimidine.

Their synthesis began by first introducing a morpholino group onto 2,4,7-trichloropyrido[2,3-d]pyrimidine 93 at position C-4 to give compound 94 (92%). Cis 2,6-dimethylmorpholine then reacted at 70
C in DMA at position C-2 to give compound 95 (45%). A further Suzuki reaction with [2-methoxy-5-(4,4,5,5-tetramethyl1,2,3-dioxaborolan-2-yl)phenyl]methanol in presence of Pd(PPh3)4 yielded KU0063794 96 in 75% yield. Compound AZD2014 97 was obtained by a two-step reaction of 3S-methyl morpholine with 93 (C-4 substitution with 87% yield at room temperature then C-2 substitution at 70
C in DMA in 71% yield). Introduction of the [3(methylcarbamoyl) phenyl]substituent at position C-7 was performed with several boronic derivatives (Scheme 27) [30,31]. Recently, our group investigated the synthesis of Phosphoinositide-3-Kinase (PI3K)/mammalian target of rapamycin (mTOR) dual inhibitors. In cancer, tumors exhibit a high frequency of activating mutations in the PI3K and mTOR coding genes, leading to an elevated pathway activity and resulting in an extended survival, growth and angiogenesis of tumor cells. Activation of the PI3K/Akt/ mTOR pathway is not vertical and it has been shown that mTOR exerts a negative feedback loop on PI3K. For this reason, inhibition of mTOR or PI3K alone is not sufficient to efficiently block this signaling pathway in cancer cells and a new strategy involved the combined inhibition of both enzymes [32,33]. Starting from 2,4-dichloropyrido[3,2-d]pyrimidine 36, the introduction first of a morpholino substituent at position C-2, then

reaction of 3-hydroxyphenyl or a 4-aminophenylboronic acid in the presence of Pd(PPh3)4 at position C-4 afforded respectively compounds 98 and 99 (60e70% overall yield). The corresponding pyrido[2,3-d]pyrimidine 100 derivative was also synthesized (Scheme 28). Urea derivatives such as 100 were designed to enhance binding. Compound 100 exhibited an IC50 of 58 nM and 5 nM inhibition on PI3K and mTor respectively. Cellular effects of 100 were determined on six cancer lines and compared to those on a healthy diploid cell line for cellular toxicity. Reversing the C-4 morpholino substituent and the C-2 aryl group on the pyrido[3,2-d]pyrimidine scaffold required the synthesis of 101. (3-Methoxymethoxy)phenyltrifluoroborate potassium was used to perform the Suzuki type reaction in toluene at 100
C. Only the C-4 arylated product was observed and isolated in 45% yield. Introduction of the morpholine was achieved in refluxing THF for 12 h in 68% yield whereas a final acidic MOM deprotection at room temperature afforded 101 in 80% yield (Scheme 28). This compound showed an inhibition of PI3Ka (IC50 ¼ 59 nM) which was still 3-fold less efficient than 98. The same general behavior was observed with [2,3-d] pyridopyrimidine compound 103 (Scheme 28). Other kinase targets are Cyclin-dependent kinase CDKs which are located in the nucleus and mainly participate in cell cycle control, transcription and post-transcriptional modification as well as in cell differentiation and cancer cell death. For example, abnormal expression of CDK2 has been observed in breast,

Scheme 27. Synthesis of Ku-0063794.

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Scheme 28. Synthesis of dual PI3K/mTOR inhibitors.

colorectal, ovarian, and prostate cancers. Consequently, many pharmacological inhibitors of CDK2 have been found to display promising antitumor and/or neuroprotective activities. Ibrahim et al. described the design and synthesis of a small library of 4aminopyrido[2,3-d]pyrimidine derivatives as anti-proliferative cyclin-dependent kinase 2 inhibitors [34]. Starting from 104, reaction of thiourea with 3-cyano-2-aminopyridine 105 produced the 4-aminopyrido[2,3-d]pyridine derivative 106 in 72% yield. Introduction of an aminoethylalcohol substituent was followed by cyclization with sulfur in the presence of morpholine in DMF affording 108, which was more active than roscovitine (IC50 CDK2/ A ¼ 90 nM vs roscovotine IC50 ¼ 500 nM, Scheme 29). Font et al. synthesized 2,4-diamino[2,3-d]pyrimidine derivatives which exhibit cytotoxic and pro-apoptotic activities. The compounds were tested in vitro on three tumor cell lines (breast MD-MBA-231, bladder T-24, and colon HT-29) [35] and comparison between the activities of the quinazoline and pyrido[2,3-d]pyrimidine series led to a reduction in LogP values in favor of the pyrido [2,3-d]pyrimidine family for further development (Scheme 30).

Mitogen-activated protein kinase MAP4K4, a seronine threonine kinase, is involved in a variety of signaling pathways. Inhibiting MAP4K4 might be beneficial in pathological processes including cancer. Crawford et al. investigated a fragment-based lead discovery approach to generate potent and selective MAP4K4 inhibitors with pyrido[3,2-d]pyrimidine as scaffold 41. The best inhibitor 111 showed a nanomolar range of activity against MAP4K4, and appeared to be very selective towards other kinases with in vivo pharmacodynamic effects in a human tumor xenograft model (Scheme 31) [19]. Receptors could be targeted by pyridopyrimidine derivatives as described below. The ErbB protein family and epidermal growth factor receptor (EGFR) family are transmembrane receptor tyrosine kinases involved in a wide range of signal transduction and cellular functions, and are associated with many types of cancer. Inhibitors of the growth factor signaling pathway via ErbB2 or EGFR in particular have thus been designed as potential anticancer drugs. Dual ErbB2/EGFR tyrosine kinase inhibitors 112e115 were studied by Rusnak et al. as potential anticancer agents using the pyrido[3,4-

Scheme 29. Synthesis of CDK2 inhibitors.

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Scheme 30. Examples of [2,3-d] with cytotoxic and pro-apoptotic activities.

Scheme 33. ErbB2 Inhibitors. Scheme 31. Example of MAP4K4 inhibitor.

d]pyrimidine scaffold (Scheme 32) [36]. Pilla et al. studied the synthesis of pyrido[3,4-d]pyrimidine as highly selective ErbB2 inhibitors [37]. Despite kinase homology domains between EGFR and ErbB-2, a selective ErbB-2 kinase approach was chosen to avoid unwanted toxicity from inhibiting EGRF and to facilitate the ability to combine a selective erbB2 agent with other selective drugs. The pyrido[3,4-d]pyrimidine ErbB2 inhibitors 116 were found to be 200 fold more selective over EGFR kinase (Scheme 33). The hepatocyte growth factor (HGF) c-Met, a receptor tyrosine kinase, is involved in several cellular processes such as cell proliferation, migration, invasion and embryological development. However, the overexpression of c-Met/HGF has been linked to human cancer and has become a main target in oncology [38]. Zhang et al. synthesized and screened a series of pyrido[2,3-d]pyrimidine derivatives to evaluate their inhibition potency against cMet tyrosine kinase. The 3-nitrobenzyl derivative 119, synthesized from 117, showed an IC50 value of 6.5 nM on c-Met. A co-crystal structure of 119 was investigated to understand the binding mode of the compounds to the c-Met active domain (Scheme 34). When a specific kinase target are unidentified, cell effects can be used in order to evaluate the drug effects. He et al. studied the synthesis and cytoxic effects of pyrido[4,3-d]pyrimidines against KB cells (the drug-sensitive human oral carcinoma), CNE2 cells (human nasopharyngeal carcinoma), and MGC-803 cells (human gastric carcinoma). The most active compound 120 (R ¼ 4MeOPhCH2) exhibited an IC50 value of 0.48, 0.15, and 0.59 mM respectively against these cell lines (Scheme 35) [7]. Cordeu et al. reported a potent anticancer drug. Bis-(4methoxybenzyl)-pyrido[2,3-d]pyrimidine-2,4-diamine 121 was tested in vitro on three tumoral cell lines and showed a good anticancer activity as an apoptosis inducer associated with activating caspase-3 and inducing DNA fragmentation (Scheme 36) [39,40].

Palop et al. investigated sulfur and selenium derivatives of pyrido[2,3-d]pyrimidine for their potential cytotoxicity. The seleno compounds 125 were synthesized in two steps. The first step involved the condensation of 2-amino-cyanopyridine 123 with isoselenocyanates 122 in the presence of pyridine. Alkylation of the selenium atom with various alkyl iodides was performed to give library 125 (Scheme 37). Compound 127 exhibited a marked cytotoxic effect on leukemia CCFF-CEM, colon HT-29, lung HTB-54, breast MCF-7 cell lines and replacement of the sulfur by a selenium atom 126 did not significantly affect biological activity [41]. An increase in Caspase-3 activity accompanied by cell cycle perturbation in a time-dependent manner was demonstrated with the hydroselenite salt 128 (Scheme 38) [42]. Malhotra et al. investigated the synthesis of 4-substituted 2amino pyrido[3,4-d]pyrimidine derivatives 129 as potential anticancer agents. A tumor cell line screening was performed using the NCI 60 cancer cell lines panel, and showed inhibitory effects against the growth of the UO-31 renal cancer cell line, MDA-MB-468 and MCF-7 breast cancer cell lines (Scheme 39) [17].

3.2. Central nervous system, anti-inflammatory activities Enzymes can also be used to solve CNS related disorders. As pyridopyrimidines can be considered toxic for kinase active sites, adenosine kinase (AK) could be targeted by specific pyridopyrimidine derivatives. This target is an intracellular enzyme which regulates intra and extracellular concentrations of adenosine, an endogenous modulator of intercellular signaling that reduces cell excitability during tissue stress and trauma, and has a therapeutic potential as an analgesic and anti-inflammatory agent [43]. Bhagwat et al. reported the synthesis of pyrido[2,3-d]pyrimidines as non-nucleoside adenosine kinase inhibitors. Inhibition of AK was found with compound 131, prepared from the cyclization of 130 with formamide in 63% yield. The C-7 position was used to

Scheme 32. ErbB2 Inhibitors.

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Scheme 34. Synthesis of tyrosine kinase c-Met inhibitors.

Scheme 35. Pyrido[4,3-d]pyrimidine against KB.

Scheme 38. Example of pyridopyrimidines with cytotoxic effects on leukemia CCFFCEM.

Scheme 36. Apoptosis inducer.

introduce diversity. Pharmacomodulation showed that the pyridinyl substituent could be replaced by pyrazine, pyrimidine, thiophenyl, thiazole, oxazole, or imidazole moieties (Scheme 40). The second synthetic strategy, developed by the same authors seeking to use pyridopyrimidines as AK inhibitors, is indicated in Scheme 41 [44]. The synthesis of 6,7-disubstituted-4-aminopyrido [2,3-d]pyrimidines 134 was accomplished by an aza-Cope rearrangement of the intermediate 133 formed by reaction of 132 with an aryl aldehyde in moderate yield. At the C-7 position, a variety of aromatic groups such as substituted phenyl, pyridyl, and furyl were studied. Only the 2-thiophene moiety exhibited a nanomolar range inhibitory activity (R1 ¼ 3-BrC6H4CH2, R2 ¼ 2-thiophenyl, IC50 ¼ 4 nM). These studies led to the emergence of ABT-702 135, a nonnucleoside adenosine kinase inhibitor of pain and inflammation with oral activity in animal models. (IC50 ¼ 2 nM, Scheme 42) [45]. Glutamate, the major excitatory neurotransmitter in the central nervous system, is implicated in a number of neurological conditions through glutamate receptors (ionotropic and metabotropic). Metabotropic glutamate receptors (mGluRs) are members of the G protein-coupled receptors and among these eight mGluR subtypes, mGluR5 plays a key role in a variety of normal brain functions and is also involved in several neuropsychiatric disorders. The modulation of mGluR5 may be useful for the treatment of both peripheral and CNS disorders. Wendt et al. described a novel series of pyrido[2,3-d] pyrimidines mGlu5 receptor antagonists. The pharmacological activity of the lead compound 137 (Ar ¼ 3,5-diClC6H3, IC50 ¼ 0.72 nM)

Scheme 39. Example of pyrido[3,4-d]pyrimidine derivatives as potential anticancer agents.

was reported and showed positive effects on anxiety and pain in rodent models (Scheme 43) [46]. Chemokine receptors are members of the G-protein-coupled receptor group (RCPG) involved in regulating leukocyte trafficking and inflammation in both physiological and pathological conditions. The CCR family comprises 10 receptors (CCR1-10) with their 25 ligands (CCL). Chemokine receptor expression is up-regulated in various inflammatory conditions and CCR4 appears to be a pivotal factor in the development of allergic inflammations. A series of pyrido[2,3-d]pyrimidine ligands was described by Gong et al. For example, compounds 138 and 139 demonstrated high chemotaxis inhibition activities for CCR4 with high selectivity against the other

Scheme 40. Synthesis of AK inhibitors.

Scheme 37. Pyrido[2,3-d]pyrimidines for their potential cytotoxicity.

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Scheme 41. Synthesis of AK inhibitors.

Scheme 42. Example of non-nucleoside adenosine kinase inhibitor.

Scheme 43. Pyrido[2,3-d]pyrimidines as m-Glu-5 receptor antagonists.

related chemokine receptor types (Scheme 44) [47]. The human histamine receptor H4 (hH4R) is mainly expressed in cells of the immune system and modulates the release of various inflammatory mediators. Antagonists are considered as potential drugs for the treatment of allergic asthma. Smits et al. described the synthesis of the pyrido[2,3-d] pyrimidine ligand as a histamine H4 receptor antagonist to reduce hERG binding, with good affinities to the human and rodent histamine receptor H4R [48]. The chiral derivative 142 was prepared from the 4-chloro-7-bromoderivative 141 by substitution of the 4-chloro atom which allowed the introduction of the (R)-3-Bocaminopyrolidine substituent. Compound 142 reacted with isopropenylboronic acid pinacol ester under classical SuzukieMiyaura conditions. A further hydrogenation of the isoprenyl group and cleavage of the acetyl group under acidic conditions gave 143 in moderate yields. Compound 143 showed a pKi (hH4R) ¼ 7.91 with a selectivity of only 12 over hH3R. 3-Boc-4methylaminoazitidine was first reacted with 144, then an acetylamino group was introduced at the C-2 position using palladium catalyzed Buchwald cross coupling reaction and acetamide. Finally cyclopropylpotassium trifluoroborate was reacted, allowing the introduction of a cyclopropyl substituent at position C-7 and

Scheme 44. CCR4 inhibitors.

Scheme 46. DYRK1A inhibitor.

Scheme 45. Synthesis of histamine H4 receptor antagonist.

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Scheme 47. Hepatitis C virus replication inhibitors.

producing compound 146 after protecting group removal. Interestingly, 146 showed a pKi of 8.28 with an enhanced selectivity (up to 100 fold) vs hH3R (Scheme 45). Dual-specificity tyrosine phosphorylation-regulated kinase 1a (DYRK1A) is a member of the dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) family and it's overexpression is implicated in Alzheimer's disease (AD) and Down Syndrome (DS). For these reasons, much research effort has focused on developing inhibitors of this kinase. Our research team recently described the synthesis and optimization of V-shaped derivatives of 2,4-disubstituted pyrido[3,2-d]pyrimidines as DYRK1A inhibitors (Scheme 46) [49,50]. Using previous work in the pyrido[3,2-d]pyrimidine series [28], we explored the potency of this series and developed efficient SAR. Compound 147 inhibited DYRK1A in the submicromolar range (IC50 ¼ 0.06 mM) with excellent selectivity (GSK and CDK5 IC50 > 2 mM).

3.3. Antiviral agents Chronic hepatitis C virus (HCV) infection is a blood-borne pathogen and effective treatment represents a major unmet medical need. HCV is classified into six genotypes (1e6), with subgenotypes 1a and 1b the most prevalent in the United States, Japan, and Western Europe. Lazerwith et al. studied a novel series of HCV replication inhibitors based on a pyrido[3,2-d]pyrimidine core and

Scheme 48. Hepatitis C virus replicon inhibitors.

optimized them for pharmacokinetics in rats (decrease of polar surface area PSA and increase of cLogP). The compound 149 was synthesized in three steps from 148. A 2,2,2-trifluoroethylamine group was first introduced onto 2,4,6-trichloropyrido[2,3-d]pyrimidine 148 at position C-4 at room temperature in 91% yield. Next, the 4-(1H-1,2,4-triazol-1-yl)phenyl)methanamine hydrochloride was reacted at 120
C in NMP at position C-2, followed by a Suzuki reaction with 6-cyclopropoxypyridin-3-ylboronic acid in presence of Pd(PPh3)4 which yielded 149 in 60% yield. Compound 149 exibited an EC50 ¼ 3 nM with a moderate PSA and a calculated cLogP value of 4.7. Finally, the authors concluded that the measurement of compound PSA was not sufficient to correctly evaluate the bioavailability of each HCV replication inhibitor. The weighted combination of hydrogen bond donor count and cLogP was found to be a better predictor of bioavailability for the moderately high PSA compounds (Scheme 47) [51]. The related research area prompted Krueger et al. to investigate the synthesis and SAR of fused pyrimidine derivatives as Hepatitis C virus replicon inhibitors. The most potent isopropylpyrido[2,3-d] pyrimidine analogs 151 were constructed by cyclization of pyridodimethylamidine 150 with the substituted phenylsulfanyl aniline in moderate yield. Compound 151 exhibited a submicromolar potency against both subtype 1a and 1b replicons (EC50(1a) ¼ 170 nM and EC50(1b) ¼ 16 nM) and displayed submicromolar potency in one cell culture assay containing 40% human serum (Scheme 48) [52]. Additionally, DeGoey et al. discovered a novel series of pyrido [2,3-d]pyrimidine compounds which displayed potent inhibition of

Scheme 50. Synthesis of pyridopyrimidine with antimicrobial and anti-oxidant activities.

Scheme 49. Synthesis of Hepatitis C virus replicon inhibitor 155.

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Scheme 51. Examples of DHFR inhibitors.

Scheme 52. Synthesis of DHFR inhibitor.

the HCV genotypes 1a and 1b in the replicon assay. The pyridino [2,3-d]pyrimidine core 154 was synthesized by reaction of cyanoamidine 153 with methyl 4-[4-(tert-butoxycarbonylamino) phenyl]sulfanyl-3-methyl-benzoate 152 in refluxing acetic acid. The introduction of chiral methyl benzyl amine was carried out to show the effects of chirality for biological activities. Compound 155 exhibited an EC50(GT1a) ¼ 0.045 mM (S isomer) vs 0.180 mM (R isomer) and EC50(GT1b) ¼ 0.007 mM (R isomer) vs ¼ 0.10 mM (S isomer) (Scheme 49) [53]. Rao et al. investigated the synthesis and bioassay studies of 7substituted pyrido[2,3-d]pyrimidines. 7-substituted pyrido[2,3-d] pyrimidine 157, easily obtained from 4-aminopyrimidine-5carbaldehyde 156 and various aromatic or heteroaromatic ketones, showed antimicrobial and anti-oxidant activities (Scheme 50) [54]. In the field of antiviral research, a major concern of immunocompromised patients, in particular those with AIDS, is susceptibility to infection caused by opportunistic pathogens. Many examples exist such as the fungus Pneumocystis jirovecii which causes pneumonia, toxoplasmosis caused by the protozoan Toxoplasma gondii, or reinfection caused by the bacterium

Scheme 53. DHFR inhibitor.

Mycobacterium avium. One target to treat infection with these organisms is the inhibition of the dihydrofolate reductase (DHFR) enzyme which converts dihydrofolate into tetrahydrofolate, a cofactor required for the synthesis of purines. DHFR has been successfully targeted in antimicrobial therapy [14,55,56]. Lipophilic, nonclassical antifolates with a pyrimidine or a pyrimidine-fused core are approved agents for the treatment and prophylaxis of opportunistic infections in immunocompromised patients, including M. avium-intracellulare infection. Piritrexim 158, a pyrido[2,3-d]pyrimidine-2,4-diamine-structure, is a nonselective DHFR inhibitor which exhibits an IC50 of 19 nM and 17 nM against Pneumocystis carinii dihydrofolate reductase (DHFR) and T. gondii DHFR respectively [57,58]. Gangjee et al. reported the design, synthesis and molecular modeling of piritrexim analogs, by transposition of the 5-methyl group of PTX to the N9-position in pyrido[2,3-d]pyrimidine analogs. Compound 159 was the most selective inhibitor with excellent potency and selectivity against P. jirovecii DHFR (IC50 ¼ 4.2 nM, 18-fold more selective than PTX, Scheme 51) [14]. Rosowsky et al. described the synthesis of DHFR inhibitors combining the high potency of piritrexim 158 with the high antiparasitic vs mammalian selectivity of trimethoprim 160, using pyrido[2,3-d]pyrimidines as scaffolds (Scheme 52). Formation of the pyrido[2,3-d]pyrimidine scaffold was achieved by reaction of 2,4,6-triaminopyridine 161 and b-ketoester 162, affording 163 in 47% yield. The acetylene derivative 165, obtained from 164 via a Sonogashira reaction, proved its efficiency with a measured IC50 value of 0.65 nM against plasmodium carinii DHFR. This novel derivative was not more potent than PTX 158 but had an excellent potency against P carinii DHFR (IC50 ¼ 1.2 nM) [59]. Other DHFR inhibitors are also under investigation [13,60].

Scheme 54. Example of DHFR inhibitors.

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DHFRs. Structural data has been investigated for complexes of 166 with human DHFR. These results provide useful insight into the role of aminoacid residues in the optimization of highly selective inhibitors of DHFR against the opportunistic pathogen P. jirovecii (Scheme 53) [61]. The same research team also explored the structural data of O(u-carboxyalkoxybenzyl) pyrido[2,3-d]pyrimidine derivatives 167e169 which are potent and selective for P. carinii or human DHFRs for five antifolates compounds167 (Scheme 54) [62]. Scheme 55. Example of FGFR3 inhibitor.

Scheme 56. Example of pyridopyrimidine against Gaucher disease.

Cody et al. studied the kinetic and structural data for 2,4diamino-6-[(2,5-dichloro anilino)methyl]pyrido[2,3-d]pyrimidine 166 which is a potent inhibitor of DHFR from P. jirovecii (Ki ¼ 2.7 nM). This inhibitor showed a 9-fold selectivity compared to its action for human DHFR (Ki ¼ 24.4 nM) against only a 2-fold selectivity for P. carinii DHFR (Ki ¼ 6.3 nM). The variant that P. carinii DHFR forms with K37Q and K37Q/F69N mutations, which makes the enzyme more like the human form, also makes these enzymes more sensitive to the inhibitory activity of 166 with Ki values of 0.26 and 0.71 nM, respectively. A similar gain in sensitivity was also observed for the human DHFR variant, which showed a lower Ki value (0.58 nM) than native human, P. carinii, or P. jirovecii

3.4. Other therapies Legeai-Mallet et al. described a novel tyrosine kinase inhibitor 170 which inhibited constitutive FGFR3 phosphorylation, restored chondrocyte differentiation and promoted bone growth factor in a gain-of-function Fgfr3Y367C/þ mouse model (Scheme 55) [63]. FGFR3 is a tyrosine kinase receptor and constitutive FGFR3 activation impairs endochondral ossification and triggers severe disorganization of the cartilage. Gaucher disease is a lysosomal storage disorder caused by deficiency in the glucocerebrosidase enzyme. Marugan et al. described the biological evaluation of small molecular chaperones of protein without an iminosugar scaffold. Among them, pyrido [2,3-d]or [3,2-d]pyrimidine compounds 171 and 172 show a modest AC50 (corresponding to half the maximum activity concentration, either inhibitory or activating) in the micromolar range for inhibition of the hydrolysis of 4-methylumbelliferone b-D-glucopyranoside in N370S tissue (Scheme 56) [64]. 4. Patents The diversity of biological targets is a good way to measure the medicinal chemistry impact of the 7-pyridopyrimidine core. Numerous compounds have been described in the patent literature, but only a few examples are given in this review Refs. [65e89].

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5. Conclusion This review highlights the medicinal importance of pyridopyrimidine core with an overview of the synthetic methodology used to give polyfunctionalized compounds. These derivatives have found applications in various areas of medicine such as

anticancer, CNS, fungicidal, antiviral, anti-inflammatory, antimicrobial and antibacterial therapies. Large focused libraries can be obtained after selection of the best synthetic pathway. The early development of efficient and versatile methodologies in organic chemistry has again proved its benefits in medicinal chemistry programs.

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Conflicts of interest The authors declare no conflicts of interest. Acknowledgments
gion du The authors thank the INCa, Ligue contre le cancer intere ^ le Grand Ouest CGO and its grand ouest, the Canceropo strand « Valorisation des produits de la mer », the PHC Volubilis
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