Synthesis and SAR study of novel tricyclic pyrazoles as potent phosphodiesterase 10A inhibitors.

European Journal of Medicinal Chemistry 84 (2014) 181e193

Contents lists available at ScienceDirect

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

Original article

Synthesis and SAR study of novel tricyclic pyrazoles as potent phosphodiesterase 10A inhibitors Antonio Dore a, Battistina Asproni a, *, Alessia Scampuddu a, Gerard Aime Pinna a, Claus Tornby Christoffersen b, Morten Langgård c, Jan Kehler c di Sassari, Via F. Muroni 23/A, 07100 Sassari, Italy Dipartimento di Chimica e Farmacia, Universita H. Lundbeck A/S, Department of Molecular Pharmacology, 9 Ottiliavej, DK-2500 Valby, Denmark c H. Lundbeck A/S, Discovery Chemistry and DMPK, 9 Ottiliavej, DK-2500 Valby, Denmark a

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 September 2013 Received in revised form 17 June 2014 Accepted 6 July 2014 Available online 7 July 2014

Novel pyrazolo[5,1-f][1,6]naphthyridines, pyrazolo[5,1-a][2,6]naphthyridines, pyrazolo[5,1-a][2,7]naphthyridines and pyrazolo[5,1-a]isoquinolines phenylimidazole/benzimidazole ethylene-linked were designed and synthesized for PDE10A interaction. An AgOTf and proline-cocatalyzed multicomponent methodology based on use of o-alkynylaldehydes, tosylhydrazide and ketones was developed and proved to be a convenient route for assembly of most of the novel tricyclic pyrazoles synthesized. Pyrazolo[5,1-f] [1,6]naphthyridine 43 and 59, pyrazolo[5,1-a][2,6]naphthyridine 66, and pyrazolo[5,1-a][2,7]naphthyridine 42 showed the highest affinity for PDE10A enzyme (IC50 ¼ 40, 42, 40, 55 nM, respectively). © 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Tricyclic pyrazoles PDE10A inhibition Structureeactivity relationships

1. Introduction Phosphodiesterases (PDEs) are a class of key enzymes in cellular signalling pathways. They are bimetallic hydrolases that degrade the intracellular second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) by catalytic hydrolysis of the 30 -50 phosphodiester bond, forming the inactive 50 -monophosphates AMP and GMP, respectively. Thereby, they play a crucial role in regulation of the intracellular levels of these ubiquitous second messengers. PDEs are a superfamily of enzymes encoded by 21 genes and consist of 11 families (PDE1PDE11) with over 60 isoforms based on structural and functional properties. They can be classified by the substrate specificity: the cAMP-specific PDEs, including PDE4, PDE7, and PDE8; the cGMPselective enzymes PDE5, PDE6 and PDE9; the dual-substrate PDEs, PDE1, PDE2, PDE3, PDE10 and PDE11 [1,2]. PDE10A is a relatively newly identified phosphodiesterase [3] and among the 11 PDE families, it has the most restricted distribution, with mRNA being highly expressed in human brain [4]. In the brain, the enzyme is predominantly expressed in medium spiny neurons of the striatum [5], making it an intriguing target for the

* Corresponding author. E-mail address: [email protected] (B. Asproni). http://dx.doi.org/10.1016/j.ejmech.2014.07.020 0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

treatment of central nervous system disorders associated to dysfunction in the basal ganglia circuit like e.g. Parkinson's disease, Huntington's disease, schizophrenia, addiction and obsessive compulsive disorder [6]. During the last decade, an increasing number of pharmaceutical companies were involved in identifying a huge diversity of chemical scaffolds and molecular architectures for PDE10A interaction and to evaluate their therapeutic potential [7]. In this context, Pfizer has been a leading company having disclosed the natural alkaloid papaverine as the first relatively selective PDE10A inhibitor, evaluating its in vivo activity in animal models predictive of antipsychotic activity [8] and presented the first highly selective PDE10A inhibitor clinical candidate, namely MP-10 (PF-2545920), for the treatment of schizophrenia (Fig. 1) [9]. Preclinical data generated by the investigation of MP-10 [10] and its close N-trifluoroethyl analogue TP-10 [11] (Fig. 1) indicated that a PDE10A inhibitor exhibited antipsychotic activity in several animal models, spanning from positive, cognitive and negative symptoms of schizophrenia. Nevertheless, in spite of the strong preclinical evidences for antipsychotic activity of PDE10A inhibitors, the development of PF2545920 as clinical candidate for the treatment of schizophrenia was recently halted following the observation that the efficacy of MP-10 in acute exacerbation of schizophrenia was not significantly different from placebo [12]. On the other hand, several emerging preclinical data indicated the potential of PDE10A inhibitors for

182

A. Dore et al. / European Journal of Medicinal Chemistry 84 (2014) 181e193

Fig. 1. PDE10A inhibitors Papaverine, MP-10, TP-10, 1,2,4-triazolo[1,5-c]quinazoline I, pyrazolo[1,5-c]quinazoline II and the novel pyrazolonaphthyridines 1, 2, 3 and pyrazoloisoquinoline 4.

treatment of patients with Huntington's disease. Recent reports described the activity of TP-10 in reducing striatal and cortical cell loss in different animal models of Huntington's disease [13]. MP-10 and TP-10 are representative terms of a series of pyrazolyl-phenoxymethyl-quinolines [9] endowed with subnanomolar PDE10A affinity and excellent selectivity (>3000-fold) versus other PDEs [11]. X-ray data of the complex formed between MP-10 and the catalytic domain of PDE10A [9] revealed a binding mode which does not involve the conserved glutamine residue, but

an alternative interaction involving a hydrogen bond between the quinoline nitrogen and a tyrosine residue at the “selectivity pocket” which is in proximity to the catalytic site of the PDE10A enzyme. Occupation of this pocket by the ligand has been reported to provide nearly complete selectivity versus other PDEs [7f]. In a recent disclosure [14] we have developed a series of phenylimidazole-pyrazolo[1,5-c]quinazolines exemplified by compound II, Fig. 1, as potent PDE10A inhibitors (IC50 y 12e400 nM), endowed with high selectivity against the other PDE isoforms


(IC50 > 10000 nM). The reported research was based on design considerations derived from an X-ray analyses of the benzimidazole-triazolo[1,5-c]quinazoline I bound to the catalytic domain of PDE10A [15]. Our structureeactivity relationship (SAR) investigations prompted us to assume that the planar pyrazoloquinazoline ring system might occupy the hydrophobic clamp of the PDE10A catalytic domain. The ethylene group could act as a linker reaching into the well defined selectivity pocket, where the phenylimidazole nitrogen is involved in a hydrogen bond interaction with the tyrosine residue. The concomitant intervention of above mentioned binding interactions presumably contribute to the high potency and especially the selectivity of the investigated phenylimidazole-pyrazolo[1,5-c]quinazolines. As part of our ongoing efforts to generate SARs around II, we envisioned that bioisosteric modification of the pyrazoloquinazoline core might offer new templates that could provide better interactions with the PDE10A enzyme. Thus, four different tricyclic scaffolds including pyrazolo[5,1-f][1,6]naphthyridine 1, pyrazolo [5,1-a][2,6]naphthyridine 2, pyrazolo[5,1-a][2,7]naphthyridine 3 and pyrazolo[5,1-a]isoquinoline 4 have been designed (Fig. 1). We planned also to make use of the phenylimidazole/benzimidazole fragments as useful hydrogen bond acceptors for the selectivity pocket, and evaluate the effect of different substituents such as CH3, COOC2H5, CF3, CH2OCH3, at the pyrazole portion of the tricyclic system. In this paper we report the synthesis and the biological evaluation of novel tricyclic pyrazoles whose structures are reported in Table 1.

183

Table 1 Structure and PDE10A potency for pyrazolonaphthyridines and pyrazoloisoquinolines compounds. Entry

Compound

PDE10A IC50a (nM)

1

40

2

170

3

220

4

1300

5

190

6

42

7

40

8

55

9

480

10

900

2. Results and discussion 2.1. Chemistry The chemistry employed to prepare the pyrazolonaphthyridines- and pyrazoloisoquinoline-based compounds (Fig. 1 and Table 1) is outlined in Schemes 1e4. Schemes 1 and 2 depict the synthetic routes used to synthesize most of the title compounds presented in this paper, and consist firstly in the preparation of methoxymethyl-based tricyclic pyrazoles 18e22 (Scheme 1), whose functionalization reactions (Scheme 2) provided the desired pyrazolonaphthyridines- and pyrazoloisoquinoline-based molecules. The synthetic procedure developed to assemble the pyrazolonaphthyridine and pyrazoloisoquinoline cores (Scheme 1) make use of a multicomponent methodology (step c) which is based on a “dual-activation concept” of the electrophiles and nucleophiles, through one-pot combination of metal and organocatalysis. One-pot combination of AgOTf and proline catalysis was applied successfully by Wu et al. for the synthesis of 1,2dihydroisoquinoline derivatives in multicomponent reactions, starting from appropriate o-alkynylbenzaldehydes, amines, and ketones [16]. More recently, Wu et al. described the unexpected formation of the substituted pyrazolo[5,1-a]isoquinoline core by AgOTf-catalyzed tandem reactions of o-alkynylbenzylidene-tosylhydrazones with alkynes or silyl enolates in the presence of a base [17,18]. Taking into account these results, we reasoned that the pyrazolonaphthyridine and pyrazoloisoquinoline products 18e22 (Scheme 1) could be synthesized by one-pot combination of AgOTf and proline catalysis by reacting the tosylhydrazones 14e17 and appropriate ketones, followed by treatment with a base. The synthetic sequence commenced with o-haloaldehydes 6e9 which were allowed to react with methyl propargyl ether under the conventional Sonogashira conditions to give the corresponding oalkynylaldehydes 10e13. The o-Br-aldehydes 6, 7 and 9 were

(continued on next page)

184


more methine carbon. The structure of novel tricyclic pyrazoles was also confirmed with an alternative six-steps synthetic approach applied for the preparation of pyrazolo[5,1-f][1,6]naphthyridine 18 as representative term of the series (vide infra and Supplementary data, Scheme 5). Methoxymethyl-based tricyclic pyrazoles 18, 19, 21 and 22 (Scheme 2) were converted into the corresponding alcohols 23e26 by treatment with BBr3. SOCl2 chlorination of 23e26 afforded the chloromethyl-based tricyclic pyrazole intermediates 27e30 which were reacted with triphenylphosphine to give the phosphonium salts 31e34, whereas the alcohols 23 and 26 were converted in high yields into the corresponding aldehydes 35 and 36 using DessMartin periodinane (DMP). Wittig reaction between the phosphonium salts 32, 33 and 1-methyl-4-phenyl-1H-imidazole-2carbaldehyde and between the phosphonium salts 31e34 and 1methyl-1H-benzo[d]imidazole-2-carbaldehyde afforded alkenes 37, 38 and 45e48, respectively. Wittig reaction between the aldehydes 35, 36 and ((1-methyl-4-phenyl-1H-imidazol-2-yl)methyl) triphenylphosphonium chloride furnished alkenes 39 and 40, respectively. All alkenes were isolated in general with high yields as E/Z mixtures. Final reduction of alkenes with PTSH allowed to obtain the title compounds 41e44 and 49e52 in satisfactory yields. Alternatively, Scheme 3 depicts a further application of above mentioned multicomponent methodology starting from the oalkynylaldehyde intermediate 56 which incorporates in its structure the necessary ethyl-phenylimidazole portion. Compound 56 was reacted in situ with PTSH to give the corresponding tosylhydrazone in the presence of AgOTf and DL-proline, which after sequential treatment with ethyl pyruvate or trifluoroacetone and Na2CO3 afforded target compounds 41 and 57 in high yields, respectively. Compound 56 was synthesized from 2-(but-3-yn-1yl)-1-methyl-4-phenyl-1H-imidazole (55) which was submitted to Sonogashira reaction with 2-bromonicotinaldehyde (6) to give the desired o-alkynylaldehyde 56 in good yields. This two-step straight synthetic approach used for the synthesis of 41 and 57 make use of the alkyne 55, synthesized starting from 4-pentynoic acid (53), which after sequential treatment with Cs2CO3 and 2bromoacetophenone formed the keto-ester 53′. The addition of NH4OAc to a xylene solution of 53′ at reflux, afforded in high yield the phenylimidazole 54. Alkylation of 54 with CH3I in the presence of NaH gave the desired alkyne 55. Reduction of the ester function of 41 with NaBH4 afforded the alcohol 58 which was methylated to compound 59. Looking at the yields (30e75%) of tricyclic pyrazoles obtained by the multicomponent reaction described in Schemes 1 and 3, the

Table 1 (continued ) Entry

PDE10A IC50a (nM)

Compound

11

1000

12

50

a IC50 values are means of at least two experiments, and a typical standard deviation was ±30%.

commercially available, whereas the o-I-aldehyde 8 was obtained by making use of a trans-halogenation reaction by reacting 4chloronicotinaldehyde (5) with NaI in the presence of acetyl chloride [19]. Compounds 10e13 were condensed in situ with p-toluenesulfonyl hydrazide (PTSH) at room temperature in ethanol and in the presence of AgOTf (10 mol %), DL-proline (10 mol %) to give tosylhydrazones 14e17 (not isolated), which were converted into the desired tricyclic pyrazoles 18e22 by sequential treatment with acetone or ethyl pyruvate, heating the reaction mixture by microwave irradiation to 50e60 C, and finally with Na2CO3 at room temperature. The formation of 18e22 can be explained by the initial formation of 1,2-dihydroisoquinoline intermediate 14′-17′ [16] which undergoes intramolecular dehydrative condensation with the sulfonohydrazide portion of the molecule to afford the tricyclic intermediate 1400 -1700. Base mediated elimination of the tosyl group and aromatization afford the desired tricyclic compounds 18e22. The use of pure tosylhydrazones did not improve the reaction yields, and therefore isolation and purification of these intermediates was omitted. All tricyclic pyrazoles were characterized using 1H, 13C-APT NMR, and mass spectrometry. The 1H NMR spectra of 18e22 revealed the presence of two distinct singlet peaks (1H) at dH 6.85, 7.31 (18), 7.55, 7.63 (19), 6.95, 7.13 (20), 6.95, 7.03 (21), 6.82, 7.05 (22), indicating HeC1/HeC6 for 18, 19, 20, 22 and HeC10/HeC5 for 21 [14,17]. The 13C-APT NMR spectra revealed the presence of at least 10 carbons, five quaternary carbons and five methine carbons, indicating the above described di-substituted pyrazolonaphthyridine or pyrazoloisoquinoline skeleton with the last one with one

N

O

H N

Z

a

Y

Cl 5

O

Z

b

Y

Halo

X

O

Y

X

6: X = N; Y,Z = CH; Halo = Br 7: X,Z = CH; Y = N; Halo = Br 8: X,Y = CH; Z = N; Halo = I 9: X,Y,Z = CH; Halo = Br

N

Z

c (1) O

O X

O 14: 15: 16: 17:

10: X = N; Y,Z = CH 11: X,Z = CH; Y = N 12: X,Y = CH; Z = N 13: X,Y,Z = CH

O S

X = N; Y,Z = CH X,Z = CH; Y = N X,Y = CH; Z = N X,Y,Z = CH

O R R

H

H N c (2)

N

Z Y

X 14'-17'

Ts

Z

O

Y

N N

R

Ts O

X 14"-17"

N c (3)

Z Y

N X

O

18: X = N; Y,Z = CH; R = CH3 19: X = N; Y,Z = CH; R = COOEt 20: X,Z = CH; Y = N; R = CH3 21: X,Y = CH; Z = N; R = CH3 22: X,Y,Z = CH; R = CH3

(75%) (70%) (9%) (58%) (30%)

Scheme 1. Reagents and conditions: (a) NaI, AcCl, dry MeCN, 0 C / r.t., 1 h; (b) methyl propargyl ether, Pd(PPh3)2Cl2, CuI, Et3N, dry DMF, r.t., 1 h; (c) (1) PTSH, AgOTf, DL-Proline, dry EtOH, r.t., 20 min; (2) acetone (for 18, 20e22) or ethyl pyruvate (for 19), MW, 50e60 C, 30 min; (3) Na2CO3, r.t., 12 h.


185

R

R

N Y

b R

R

N Y X

Y

a O

18: X = N; Y = CH; R = CH3 19: X = N; Y = CH; R = COOEt 21: X = CH; Y = N; R = CH3 22: X,Y = CH; R = CH3

Cl

OH

N X


N X

Y

c

X

N

N

N

N

PPh3Cl


R


N Y

d

N O X

35: X = N; Y = CH; R = CH3 36: X,Y = CH; R = CH3

R 32, 33

R

e N Y

N

N

g N

X 35, 36

f

Y

N N

X

N

N

37: X = N; Y = CH; R = COOEt 38: X = CH; Y = N; R = CH3 39: X = N; Y = CH; R = CH3 40: X,Y = CH; R = CH3

41: X = N; Y = CH; R = COOEt 42: X = CH; Y = N; R = CH3 43: X = N; Y = CH; R = CH3 44: X,Y = CH; R = CH3

R

R

N 31-34

h

Y

N

N

g N

X N


Y

N N

X N


Scheme 2. Reagents and conditions: (a) BBr3 (1 M in DCM), dry DCM, 0 C /r.t., N2, 1.5 h; (b) SOCl2, dry DCM, 0 C / r.t., 12 h; (c) PPh3, MeCN, reflux, 12 h; (d) DMP, dry DCE, N2, r.t., 12 h; (e) NaH (60% in mineral oil), 1-methyl-4-phenyl-1H-imidazole- 2-carbaldehyde, dry DMF, 0 C / r.t., 5 h; (f) NaH (60% in mineral oil), ((1-methyl-4-phenyl-1H-imidazol-2- yl) methyl)triphenylphosphonium chloride, dry DMF, 0 C / r.t., 5 h; (g) PTSH, dry DMF, N2, 120 C, 4e6 h; (h) NaH (60% in mineral oil), 1-methyl-1H-benzo[d]imidazole-2carbaldehyde, dry DMF, 0 C / r.t., 5 h.

reaction works well both with acetone, ethyl pyruvate, and trifluoroacetone, and appear to be a general methodology for the assembly of pyrazolo[5,1-f][1,6]naphthyridines 18, 19, 41, 57 (70e75% yield), pyrazolo[5,1-a][2,7]naphthyridine 21 (58% yield), and pyrazolo[5,1-a]isoquinoline 22 (30% yield), even if the last one was obtained with lower yields. Nevertheless, this methodology was found to be not convenient with o-alkynylaldehyde 11 (Scheme 1), since the pyrazolo[5,1-a][2,6]naphthyridine-based compound 20 was obtained only in poor yields (9% yield). This finding prompted us to explore an alternative synthetic route for pyrazolo[5,1-a][2,6] naphthyridine tricyclic ring system that is depicted in Scheme 4 for the synthesis of final compound 66. The key step involves the final heterocyclization of the ketoxime 65 into the pyrazolo[5,1-a][2,6] naphthyridine 66 via catalytic rearrangement of the azirine intermediate 65′ [20]. The synthetic sequence started with 3bromoisonicotinaldehyde (7), which was submitted to Sonogashira reaction with alkyne 55 to afford the o-alkynylaldehyde 60 that in turn was converted into the aldoxime 61 by a conventional procedure. Treatment of 61 with K2CO3 in ethanol at room temperature afforded the 2,6-naphthyridine-2-oxide 62 in high yield [21]. Compound 62 gave satisfactory 1H NMR data, and the dH values of the 2,6naphthyridine 2-oxide system is in agreement with literature data [21]. POCl3 chlorination of 62 gave the 1-chloro-2,6-naphthyridine

63 which upon palladium-catalyzed cross-coupling reaction with isopropenyl acetate in the presence of tributyl(methoxy)stannane, 2-dicyclohexylphosphino-20 -(N,N-dimethylamino)biphenyl (DavePhos) [22] furnished in almost quantitative yield the a-2,6naphthyridine acetone-based derivative 64. Treatment of 64 with hydroxylamine gave the desired ketoxime 65 which treated with trifluoroacetic anhydride (TFAA) and Et3N to give the azirine intermediate 65′ (step f1) [20]. Final rearrangement was achieved by heating the azirine intermediate (not isolated) in the presence of FeCl2 to give the desired final compound 66 in 8% of yield. This procedure works well with 2-(3-methoxyprop-1-yn-yl)nicotinaldehyde (10) which was submitted to the above described sequence reactions to give the corresponding ketoxime which was converted into the pyrazolo[5,1-f][1,6]naphthyridine 18 in 41% of yield (Scheme 5, Supplementary data). 2.2. Inhibition of PDE10A activity and SAR study The ability of pyrazolonaphthyridine and pyrazoloisoquinoline derivatives to inhibit the PDE10A mediated hydrolysis of cAMP was measured using 3H-labelled cAMP in an yttrium silicate SPA bead assay making use of the fact that cAMP has low affinity for yttrium silicate SPA beads, whereas hydrolyzed 50 -AMP has high affinity for

186


OH

O

a

O

O

N

b HN

N

O

53'

53

N

c

54

55

CF3 N d

O

N

e

N

N

N N 56

N 57 (75%)

N e

COOEt

CH2OH

N

CH2OCH3

N

N N

N

N

N

f

N

c N

N

N

N

N

N

41 (71%)

N

58

59

Scheme 3. Reagents and conditions: (a) (1) Cs2CO3, EtOH/H2O 1:1, r.t., 1 h; (2) 2-bromoacetophenone, dry DMF, r.t., 15 min; (b) NH4OAc, xylene, reflux, 12 h; (c) NaH (60% in mineral oil), dry THF, 0 C, 30 min, then MeI, 0 C to r.t., 12 h; (d) 2-bromonicotinaldehyde (6), Pd(PPh3)2Cl2, CuI, Et3N, dry DMF, MW, 50 C, 20 min; (e) (1) PTSH, AgOTf, DL-Proline, dry EtOH, r.t., 20 min; (2) ethyl pyruvate (for 41) or 1,1,1-trifluoroacetone (for 57), MW, 50e60 C, 30 min; (3) Na2CO3, r.t., 12 h; (f) NaBH4, EtOH, reflux, 12 h.

O N

O

a

Br

N

b

N

OH

60

N

N 61

N

N 62

N

N

OH

Cl N

d

N

N 64

N

OH

N

b

N

N 63

N

e

N

O

N

N 7

N

c

N

N

N 65

N

N N N

f (1) N

N 65'

N

f (2) N

N

N 66

N

Scheme 4. Reagents and conditions: (a) 2-(but-3-yn-1-yl)-1-methyl-4-phenyl-1H-imidazole (55), Pd(PPh3)2Cl2, CuI, Et3N, dry DMF, MW, 70 C, 2 h; (b) NH2OH,HCl, AcONa,3H2O, EtOH, r.t., 4 h; (c) dry K2CO3, dry EtOH, r.t., 6 h; (d) POCl3, MeCN, reflux, 2 h; (e) isopropenyl acetate, DavePhos, nBu3SnOMe, Pd2(dba)3, dry toluene, N2, 100 C, 12 h; (f) (1) TFAA, Et3N, dry DME, N2, 0 C / r.t.; (2) FeCl2, 75 C, 12 h.

yttrium silicate SPA beads [14,15]. The potency in inhibiting PDE10A was calculated and the IC50 values are shown in Table 1. For comparison, the IC50 value of the lead compound 2-methyl-5-(2-(1methyl-4-phenyl-1H-imidazol-2-yl)ethyl)pyrazolo[1,5-c]quinazoline (II) has been reported. Compound 43 (Entry 1), incorporating the pyrazolo[5,1-f][1,6]naphthyridine core, exhibited high potency in inhibiting PDE10A activity, with an IC50 value very similar to that of the reference compound II (Entry 12), indicating that the shifting of nitrogen from the central ring to the side one does not significantly influence the affinity for the enzyme. The replacement of the phenylimidazole motif in 43 with the benzimidazole to give compound 49 (Entry 2) weakened the affinity about 4-fold. Interestingly, the trend of the phenylimidazole being slightly more potent than the corresponding benzimidazole appear to be a constant in all tested compounds (see 43 versus 49, 41 versus 50, 42 versus 51

and 44 versus 52). The carboxyethyl analogues 41 and 50 exhibited 5- and 7-fold decreased affinity when compared to compounds 43 and 49, respectively. A similar trend was observed with the trifluoromethyl analogue 57 (Entry 5) which exhibited 5-fold decreased affinity with respect to 43. Considering that the methyl and trifluoromethyl groups are comparable in size, the observed decreased affinity in 57 could be speculated to be influenced by the electron-withdrawing properties of the CF3 group. To further investigate the interaction mode of compounds 43 and 57 in the catalytic domain of PDE10A, molecular docking study was performed using the Glide XP protocol [23]. The receptor docking site was generated from the PDE10A X-ray structure in complex with the close analogue triazoloquinazoline ligand I, which was obtained from the Protein Data Bank (PDB code 2Y0J). As showed in Fig. 2, the docking study of 43 and 57 in the PDE10A catalytic


domain suggests that the pyrazole nitrogen of tricyclic system interacts with the conserved glutamine residue through hydrogenbond interaction, and the strength of the H-bond might be weakened by an electron-withdrawing group. This result seems to be consistent with the binding mode depicted from the X-ray structure of triazoloquinazoline I bound to PDE10A catalytic site [14,15], where the triazolo N-3 nitrogen interact with the glutamine residue of PDE10A through hydrogen-bond interaction. However, although a similar argument could be made to explain the weaker affinity of the carboxyethyl analogue 41, the docking study suggest an alternative binding mode (Fig. 2, C), where the N-7 of the pyrazolo[5,1-f][1,6]naphthyridine interacts with the conserved glutamine residue of PDE10A. The unfavourable effect was not exerted by the methoxymethyl group in compound 59 (Entry 6), which was equipotent with 43 in inhibiting the PDE10A. Compounds 66, and 42 (Entry 7 and 8), incorporating the pyrazolo[5,1-a][2,6]naphthyridine and pyrazolo[5,1-a][2,7]naphthyridine core respectively, showed PDE10A potency almost similar to that of 43. In contrast, compound 44 (Entry 10), incorporating the pyrazolo[5,1-a]isoquinoline core, displayed marked decrease of potency with respect to the aza-analogues 43, 66 and 42, highlighting the same versatility of all pyrazolonaphthyridines to inhibit the PDE10A enzyme activity with a potency lying in a similar range. The new compounds were all screened in a panel of nine other PDEs representing members from the other PDE families and data are shown in Table 2 [14,15]. It can be noted, that all compounds demonstrated a high selectivity towards PDE1C, PDE2A, PDE3A, PDE4D6, PDE5A, PDE7B, PDE8A, PDE9 and PDE11, as might also have been expected based upon an assumption of a binding mode, where the phenylimidazole and the benzimidazole are occupying the selectivity pocket. For five of the compounds the in vitro permeability using MDCKMDR1 cells [24] was assessed showing that the new compounds possessed a range of permeabilities, ranging from low passive permeability (44: PAPP-A-B ¼ 3.1; 52: PAPP-A-B ¼ 1.1; 57: PAPP-AB ¼ 3.1) to medium passive permeability (66: PAPP-A-B ¼ 11.7; 59: PAPP-A-B ¼ 17.8 e comparable to MP-10) and importantly, none of them appeared to be pgp-substrates PAPP-A-B(PAPP-A-B/PAPP-BA-ratio < 0.62). For six of the compounds (41e43 and 49e51) the respective metabolic stability was assessed by measuring the intrinsic clearance in human and rat liver microsomes (HLM Cl, int and RLM Cl, int) and all of the compounds appeared to possess high clearance in both species (>14 L/min in HLM and >170 L/min in RLM). 3. Conclusions In summary, a new series of pyrazolo[5,1-f][1,6]naphthyridines, pyrazolo[5,1-a][2,6]naphthyridines, pyrazolo[5,1-a][2,7]naphthyridines and pyrazolo[5,1-a]isoquinolines phenylimidazole/benzimidazole ethylene-linked were designed and synthesized. An AgOTf and

187

proline-cocatalyzed multicomponent methodology based on use of oalkynylaldehydes, PTSH and ketones was developed and proved to be a convenient route for assembly of most of the novel tricyclic pyrazoles synthesized. Our in vitro studies highlighted the same ability of all pyrazolonaphthyridines to affect PDE10A activity with a potency in the near nM range, close to that of the lead compound II. Within the series, a clear trend of the phenylimidazole better than benzimidazole and methyl, methoxymethyl better than trifluoromethyl, carboxyethyl was observed. All compounds were profiled for PDE isoform selectivity and showed high selectivity for PDE10A. Even if the aim to obtain new templates for PDE10A interaction was achieved, the high microsomal intrinsic clearance of most of the novel compounds presented in this paper hampered their further development. 4. Experimental section 4.1. Chemistry: general procedures €fler melting point appaMelting points were obtained on a Ko ratus and are uncorrected. 1H NMR spectra were taken on a Varian Unity 200 NMR spectrometer with 1H being observed at 200 MHz. 13 C NMR spectra were taken on a Bruker 400 Avance III NanoBay spectrometer with 13C being observed at 100.6 MHz. Chemical shifts for 1H and 13C NMR spectra were reported in d (ppm) downfield from tetramethylsilane, and coupling constants (J) were expressed in Hertz. Multiplicities are recorded as s (singlet), br s (broad singlet), d (doublet), t (triplet), dd (doublet of doublets), m (multiplet). Atmospheric Pressure Ionization Electrospray (API-ES) mass spectra were obtained on an Agilent 1100 series LC/MSD spectrometer. Elemental analyses were performed with a PerkineElmer 2400 analyzer, and results were within ±0.40% of the calculated values. Microwave experiments were carried out by a Biotage Iniziator-8-microwave system (max pressure 20 bar, IR temperature sensor). TLC was performed on Merck silica gel 60 TLC plates F254 and visualized using UV. Flash chromatography (FC) was performed using Merck silica gel 60 (230e400 mesh ASTM). The o-haloaldehydes 5e7 and 9 were purchased by Livchem GmbH. 1-Methyl-1H-benzo[d]imidazole-2-carbaldehyde and 4pentynoic acid (53) were purchased by Lancaster. 1-Methyl-4phenyl-1H-imidazole-2-carbaldehyde [25] was obtained according to reported procedure. 4.1.1. Synthesis of 4-iodonicotinaldehyde (8) NaI (20 eq, 56 mmol) was added under nitrogen atmosphere to a solution of 4-chloronicotinaldehyde (5) (1 eq, 2.80 mmol) in dry MeCN (40 mL), the whole cooled to 0 C, and acetyl chloride (3 eq, 8.40 mmol) was then added. The resulting reaction mixture was maintained at room temperature for about 1 h and then quenched with ice and neutralized with saturated aqueous NaHCO3 solution. The crude product was extracted with DCM. The combined organic layers were washed with saturated aqueous Na2S2O3 solution,

Fig. 2. Proposed binding mode of pyrazolo[5,1-f][1,6]naphthyridines 43 (A), 57 (B), and 41 (C) in the PDE10A catalytic site.

188


Table 2 Selectivity of final compounds towards PDE isoforms. Entry (Compd.)

% Inhibition @ 10 mM PDE1C

1 (43) 2 (49) 3 (41) 4 (50) 5 (57) 6 (59) 7 (66) 8 (42) 9 (51) 10 (44) 11 (52) a b

15 28 20 27 3 7 24 11 10 1 7

PDE2A 28 55 44 20 13 69 17 20 54 14 8

PDE3A 5 16 13 1 28 51 49 13 30 20 15

PDE4D 38 23 45 24 44 58 68 25 15 12 11

PDE5A 35 67 13 38 33 38 59 15 39 22a 20

PDE7B 18 27 4 17 41 47 53 41 3 13 11

PDE8A 16 13 46 6 31 29 37 25 46 17 36

PDE9A 5 21 20 2 8 15 12 53 15 NDb 10

PDE10A a

102 93a 88a 73a 101 106 102 100a 79a 72 77

PDE11A 34 43 7 10 25a 70a 37a 7 1 13a 12a

% inhibition @ 2 mM. ND is not determined.

water, brine, dried over Na2SO4, and evaporated to afford 8 as yellow solid (g 0.489, 75%) used in the following step with no further purification. Rf 0.47 (petroleum ether/AcOEt 6:4); mp 163e165 C; 1H NMR (CDCl3) 10.09 (s, 1H), 8.90 (s, 1H), 8.34 (d, J ¼ 4.8 Hz, 1H), 7.92 (d, J ¼ 4.8 Hz, 1H). API-ES m/z: [MþH]þ calcd for C6H5INO: 233.9, found 233.7. 4.1.2. General procedure I. Synthesis of o-alkynylaldehydes (10e13) A mixture of the appropriate o-haloaldehyde (1 eq, 2.60 mmol), methyl propargyl ether (1.2 eq, 3.12 mmol), Pd(PPh3)2Cl2 (0.04 eq, 0.10 mmol), CuI (0.075 eq, 0.19 mmol), Et3N (1.5 eq, 3.90 mmol), in dry DMF (12 mL), was stirred under nitrogen atmosphere, at room temperature for 1 h. The mixture was quenched with H2O and the product was extracted with Et2O. The combined organic layers were washed with water, brine, dried over Na2SO4, and evaporated. The crude product was purified by FC (petroleum ether/AcOEt 6:4).

4.1.2.1. 2-(3-Methoxyprop-1-yn-yl)nicotinaldehyde (10). Synthesized from the o-bromoaldehyde 6. Cream solid (0.414 g, 91%); Rf 0.23 (petroleum ether/AcOEt 6:4); mp 53e55 C; 1H NMR (CDCl3) 10.53 (s, 1H), 8.78 (dd, Jm ¼ 1.8 Hz, Jo ¼ 4.8 Hz, 1H), 8.18 (dd, Jm ¼ 1.8 Hz, Jo ¼ 8.0 Hz, 1H), 7.41 (dd, J ¼ 4.8 Hz, 8.0 Hz, 1H), 4.42 (s, 2H), 3.48 (s, 3H). API-ES m/z: [MþH]þ calcd for C10H10NO2: 176.1, found: 176.0.

4.1.2.2. 3-(3-Methoxyprop-1-yn-1-yl)isonicotinaldehyde (11). Synthesized from the o-bromoaldehyde 7. Cream solid (0.373 g, 82%); Rf 0.24 (petroleum ether/AcOEt 6:4); mp 56e57 C; 1H NMR (CDCl3) 10.51 (s, 1H), 8.90 (s, 1H), 8.74 (d, J ¼ 5.0 Hz, 1H), 7.70 (d, J ¼ 5.0 Hz, 1H), 4.42 (s, 2H), 3.49 (s, 3H). API-ES m/z: [MþH]þ calcd for C10H10NO2: 176.1, found: 176.0. 4.1.2.3. 4-(3-Methoxyprop-1-yn-yl)nicotinaldehyde (12). Synthesized from the o-iodoaldehyde 8. Yellow solid (0.291 g, 64%); Rf 0.34 (petroleum ether/AcOEt 6:4); mp 58e60 C; 1H NMR (CDCl3) 10.51 (s, 1H), 9.10 (s, 1H), 8.76 (d, J ¼ 5.2 Hz, 1H), 7.44 (d, J ¼ 5.2 Hz, 1H), 4.42 (s, 2H), 3.49 (s, 3H). API-ES m/z: [MþH]þ calcd for C10H10NO2: 176.1, found: 176.0. 4.1.2.4. 2-(3-Methoxyprop-1-yn-1-yl)benzaldehyde (13) [26]. Synthesized from the o-bromoaldehyde 9. Obtained heating the reaction mixture using microwave irradiation to 70 C for 2 h. The crude product was purified by FC (petroleum ether/AcOEt 8:2). Yellow oil (0.411 g, 91%). Rf 0.61 (petroleum ether/AcOEt 8:2).

4.1.3. General procedure II. Synthesis of methoxymethyl-based tricyclic pyrazoles (18e22) A mixture of the appropriate o-alkynylaldehyde (1 eq, 1.88 mmol), PTSH (1.05 eq, 1.97 mmol), AgOTf (0.1 eq, 0.19 mmol), DL-Proline (0.1 eq, 0.19 mmol), in dry ethanol (15 mL) was stirred at room temperature for 20 min. The appropriate ketone (10 eq, 18.80 mmol) was added, and the whole heated by microwave irradiation to 60 C for 30 min. The reaction mixture was cooled to room temperature; Na2CO3 (6 eq, 11.28 mmol) was added and the mixture stirred at room temperature for 12 h. The solvent was evaporated to afford a crude residue which was purified by FC (petroleum ether/AcOEt 3:7). 4.1.3.1. 5-(Methoxymethyl)-2-methylpyrazolo[5,1-f][1,6]naphthyridine (18). Synthesized from the o-alkynylaldehyde 10 and acetone as ketone. Pale yellow solid (0.320 g, 75%); Rf 0.53 (petroleum ether/AcOEt 3:7); mp 134e135 C; 1H NMR (CDCl3) 8.82 (dd, Jm ¼ 1.6 Hz, Jo ¼ 4.8 Hz, 1H), 8.30 (d, J ¼ 8.0 Hz, 1H), 7.44 (dd, J ¼ 4.8 Hz, 8.0 Hz, 1H), 7.31 (s, 1H), 6.85 (s, 1H), 4.99 (s, 2H), 3.62 (s, 3H), 2.55 (s, 3H); 13C NMR (CDCl3) 151.5, 150.4, 146.2, 139.1, 138.8, 131.2, 121.5, 119.0, 109.4, 98.6, 69.0, 59.4, 14.2; API-ES m/z: [MþH]þ calcd for C13H14N3O: 228.1, found: 227.9. 4.1.3.2. Ethyl 5-(methoxymethyl)pyrazolo[5,1-f][1,6]naphthyridine-2carboxylate (19). Synthesized from the o-alkynylaldehyde 10 and ethyl pyruvate as ketone. Grey solid (0.375 g, 70%); Rf 0.61 (petroleum ether/AcOEt 2:8); mp 130e132 C; 1H NMR (CDCl3) 8.93 (dd, Jm ¼ 1.6 Hz, Jo ¼ 4.4 Hz, 1H), 8.41 (d, J ¼ 7.6 Hz, 1H), 7.63 (s, 1H), 7.55 (s, 1H), 7.53e7.49 (m, 1H), 5.10 (s, 2H), 4.51 (q, J ¼ 7.0 Hz, 2H), 3.66 (s, 3H), 1.47 (t, J ¼ 7.0 Hz, 3H); 13C NMR (CDCl3) 162.5, 151.1, 146.0, 145.2, 139.8, 138.9, 131.4, 122.2, 119.5, 112.5, 101.8, 68.6, 61.5, 59.6, 14.4; API-ES m/z: [MþH]þ calcd for C15H16N3O3: 286.1, found: 286.0. 4.1.3.3. 5-(Methoxymethyl)-2-methylpyrazolo[5,1-a][2,6]naphthyridine (20). Synthesized from the o-alkynylaldehyde 11 and acetone as ketone. Pale yellow solid (0.038 g, 9%); Rf 0.55 (petroleum ether/ AcOEt 3:7); mp 104e106 C; 1H NMR (CDCl3) 9.05 (s, 1H), 8.63 (d, J ¼ 5.6 Hz, 1H), 7.83 (d, J ¼ 5.4 Hz, 1H), 7.13 (s, 1H), 6.95 (s, 1H), 4.98 (s, 2H), 3.64 (s, 3H), 2.56 (s, 3H); 13C NMR (CDCl3) 151.3, 149.8, 145.7, 137.5, 137.0, 127.9, 123.8, 116.5, 105.3, 99.5, 69.0, 59.4, 14.1; API-ES m/z: [MþH]þ calcd for C13H14N3O: 228.1, found: 227.9. 4.1.3.4. 6-(Methoxymethyl)-9-methylpyrazolo[5,1-a][2,7]naphthyridine (21). Synthesized from the o-alkynylaldehyde 12 and acetone as ketone. Obtained by using 0.2 eq of AgOTf. White solid (0.247 g, 58%); Rf 0.53 (petroleum ether/AcOEt 3:7); mp


112e113 C; 1H NMR (CDCl3) 9.40 (s, 1H) 8.64 (d, J ¼ 5.6 Hz, 1H), 7.55 (d, J ¼ 5.6 Hz, 1H), 7.03 (s, 1H), 6.95 (s, 1H), 4.99 (s, 2H), 3.65 (s, 3H), 2.56 (s, 3H); 13C NMR (CDCl3) 151.9, 146.6, 146.5, 140.0, 137.8, 133.7, 119.9, 118.9, 105.3, 97.7, 68.9, 59.5, 14.1; API-ES m/z: [MþH]þ calcd for C13H14N3O: 228.1, found: 228.0. 4.1.3.5. 5-(Methoxymethyl)-2-methylpyrazolo[5,1-a]isoquinoline (22). Synthesized from the o-alkynylaldehyde 13 and acetone as ketone. The crude product was purified by FC (petroleum ether/ AcOEt 8:2). Pale yellow solid (0.127 g, 30%); Rf 0.56 (petroleum ether/AcOEt 8:2); mp 63e65 C; 1H NMR (CDCl3) 8.07e8.02 (m, 1H), 7.74e7.70 (m, 1H), 7.54e7.50 (m, 2H), 7.05 (s, 1H), 6.82 (s, 1H), 4.98 (s, 2H), 3.63 (s, 3H), 2.55 (s, 3H); 13C NMR (CDCl3) 150.7, 139.7, 134.9, 128.9, 127.7, 127.0, 126.9, 123.5, 123.4, 108.0, 97.4, 69.2, 59.3, 14.2; API-ES m/z: [MþH]þ calcd for C14H15N2O: 227.1, found: 226.9. 4.1.4. General procedure III. Synthesis of hydroxymethyl-based tricyclic pyrazoles (23e26) To a cooled solution (0 C) of the appropriate methyl ether (1 eq, 1.76 mmol) in dry DCM (24 mL), under nitrogen atmosphere, BBr3 (1 M in DCM, 3 eq, 5.28 mmol) was added and the mixture warmed to room temperature and stirred for 1.5 h. The whole was cooled to 0 C and treated with saturated aqueous NaHCO3 solution (7 mL); the aqueous phase was extracted with DCM. The combined organic layer was washed with water, brine, dried over Na2SO4, and evaporated. The crude product was purified by FC (petroleum ether/ AcOEt 3:7 / AcOEt). 4.1.4.1. (2-Methylpyrazolo[5,1-f][1,6]naphthyridin-5-yl)methanol (23). Synthesized from the methyl ether 18. White solid (0.303 g, 81%); mp 188e189 C; 1H NMR (CDCl3) 8.85 (d, J ¼ 4.4 Hz, 1H), 8.33 (d, J ¼ 8.0 Hz, 1H), 7.45 (dd, J ¼ 4.4 Hz, 8.0 Hz, 1H), 7.19 (s, 1H), 6.86 (s, 1H), 5.08 (s, 2H), 2.55 (s, 3H); API-ES m/z: [MþH]þ calcd for C12H12N3O: 214.1, found: 213.9. 4.1.4.2. Ethyl 5-(hydroxymethyl)pyrazolo[5,1-f][1,6]naphthyridine-2carboxylate (24). Synthesized from the methyl ether 19. White solid (0.300 g, 63%); mp 183 C dec.; 1H NMR (CDCl3) 9.0e8.85 (m, 1H), 8.42 (d, J ¼ 7.6 Hz, 1H), 7.62 (s, 1H), 7.60e7.48 (m, 1H), 7.46 (s, 1H), 5.19 (s, 2H), 4.50 (q, J ¼ 7.2 Hz, 2H), 1.47 (t, J ¼ 7.2 Hz, 3H). APIES m/z: [MþH]þ calcd for C14H14N3O3: 272.1, found: 272.0. 4.1.4.3. (9-Methylpyrazolo[5,1-a][2,7]naphthyridin-6-yl)methanol (25). Synthesized from the methyl ether 21. White solid (0.168 g, 45%); mp 189e191 C dec.; 1H NMR (CDCl3) 9.39 (s, 1H), 8.65 (d, J ¼ 5.2 Hz, 1H), 7.53 (d, J ¼ 5.6 Hz, 1H), 6.94 (s, 1H); 6.88 (s, 1H), 5.06 (s, 2H), 2.56 (s, 3H); API-ES m/z: [MþH]þ calcd for C12H12N3O: 214.1, found: 214.0. 4.1.4.4. (2-Methylpyrazolo[5,1-a]isoquinolin-5-yl)methanol (26). Synthesized from the methyl ether 22. White solid (0.238 g, 64%); mp 157e158 C; 1H NMR (CDCl3) 8.04 (m, 1H), 7.62 (m, 1H), 7.55 (m, 2H), 7.09 (s, 1H), 6.85 (s, 1H), 4.97 (s, 2H), 2.58 (s, 3H); API-ES m/z: [MþH]þ calcd for C13H13N2O: 213.1, found: 213.0. 4.1.5. General procedure IV. Synthesis of chloromethyl-based tricyclic pyrazoles (27e30) To a cooled (0 C) solution of the appropriate alcohol (1 eq, 0.80 mmol) in dry DCM (5 ml), SOCl2 (10 eq, 8.0 mmol) was added and the whole stirred at room temperature for 12 h. The solution was evaporated to afford a crude product which was used in the following step with no further purification.

189

4.1.6. General procedure V. Synthesis of triphenylphosphonium chlorides (31e34) A mixture of the appropriate chloride (1 eq, 0.80 mmol), PPh3 (1.5 eq, 1.2 mmol), in dry MeCN (9 mL) was refluxed for 12 h. The solvent was evaporated and the residue was triturated with Et2O, the solid filtered to give the desired triphenylphosphonium chloride in almost quantitative yields as white solid, which was used in the following step with no further purification. 4.1.7. Synthesis of ((1-methyl-4-phenyl-1H-imidazol-2-yl)methyl) triphenylphosphonium chloride Step 1. A mixture of 1-methyl-4-phenyl-1H-imidazole-2carbaldehyde [25] (1 eq, 2.70 mmol), NaBH4 (3 eq, 8.10 mmol) in dry MeOH (34 mL) was stirred at room temperature for 4 h. The solvent was evaporated and the residue was solubilized in AcOEt. The organic solution was washed with water, brine, dried over Na2SO4, and evaporated to give (1-methyl-4-phenyl-1H-imidazol2-yl)methanol as white solid (0.510 g, 100%). 1H NMR (CDCl3) 7.62e7.75 (m, 2H), 7.18e7.39 (m, 3H), 6.95 (s, 1H), 4.71 (s, 2H), 3.52 (s, 3H). Step 2. (1-methyl-4-phenyl-1H-imidazol-2-yl)methanol (1 eq, 2.71 mmol) was converted into the titled phosphonium salt following the above described General procedures IV and V. 4.1.8. General procedure VI. Synthesis of carbaldehyde-based tricyclic pyrazoles (35, 36) To a solution of the appropriate alcohol (1 eq, 0.90 mmol) in dry DCE (22 mL), under nitrogen atmosphere, DMP (1.1 eq, 0.99 mmol) was added and the whole stirred at room temperature for 12 h. Saturated aqueous NaHCO3 solution (18 mL) was added and the aqueous phase extracted with Et2O. The combined organic layer was washed with water, brine, dried over Na2SO4, and evaporated. The crude product was used in the following step with no further purification. 4.1.8.1. 2-Methylpyrazolo[5,1-f][1,6]naphthyridin-5-carbaldehyde (35). Synthesized from the alcohol 23. Pale yellow solid (0.157 g, 83%); mp 182e183 C; 1H NMR (CDCl3) 10.90 (s, 1H), 8.93 (dd, Jm ¼ 1.6 Hz, Jo ¼ 4.6 Hz, 1H), 8.35 (d, J ¼ 8.2 Hz, 1H), 7.83 (s, 1H), 7.56 (dd, J ¼ 4.4 Hz, 8.2 Hz, 1H), 6.92 (s, 1H), 2.60 (s, 3H); API-ES m/z: [MþH]þ calcd for C12H10N3O: 212.0, found: 211.9. 4.1.8.2. 2-Methylpyrazolo[5,1-a]isoquinoline-5-carbaldehyde (36). Synthesized from the alcohol 26. Pale yellow solid (0.185 g, 98%); mp 163e164 C; 1H NMR (CDCl3) 10.86 (s, 1H), 8.06 (d, J ¼ 7.8 Hz, 1H), 7.85 (d, J ¼ 7.8 Hz, 1H), 7.60 (m, 3H), 6.86 (s, 1H), 2.58 (s, 3H); API-ES m/z: [MþH]þ calcd for C13H11N2O: 211.1, found: 211.0. 4.1.9. General procedure VII. Synthesis of alkenes (37e40, 45e48) To a suspension of 60% NaH in mineral oil (2 eq, 1.42 mmol) in dry DMF (4e6 mL) cooled to 5 C, the appropriate phosphonium salt (1 eq, 0.71 mmol) was portionwise added, and the resulting solution stirred for 30 min at the same temperature. The appropriate aldehyde (1 eq, 0.71 mmol) was portionwise added, and the resulting mixture stirred at room temperature for 5 h. The whole was quenched with H2O, and the crude product extracted with AcOEt. The combined organic layer was washed with water, brine, dried over Na2SO4, and evaporated to afford a crude residue purified by FC (petroleum ether/AcOEt 3:7). 4.1.9.1. Ethyl 5-(2-(1-methyl-4-phenyl-1H-imidazol-2-yl)vinyl)pyrazolo[5,1-f][1,6]naphthyridine-2-carboxylate (37). Synthesized from the phosphonium salt 32 and 1-methyl-4-phenyl-1H-imidazole-2carbaldehyde. Yellow solid (0.189 g, 63%); API-ES m/z: [MþH]þ calcd for C25H22N5O2: 424.2, found: 424.0.

190


4.1.9.2. 9-Methyl-6-(2-(1-methyl-4-phenyl-1H-imidazol-2-yl)vinyl) pyrazolo[5,1-a][2,7]naphthyridine (38). Synthesized from the phosphonium salt 33 and 1-methyl-4-phenyl-1H-imidazole-2carbaldehyde. Yellow solid (0.259 g, 100%); API-ES m/z: [MþH]þ calcd for C23H20N5: 366.2, found: 366.0. 4.1.9.3. 2-Methyl-5-(2-(1-methyl-4-phenyl-1H-imidazol-2-yl)vinyl) pyrazolo[5,1-f][1,6]naphthyridine (39). Synthesized from ((1methyl-4-phenyl-1H-imidazol-2-yl)methyl)triphenylphosphonium chloride and the aldehyde 35. Yellow solid (0.168 g, 65%); APIES m/z: [MþH]þ calcd for C23H20N5: 366.2, found: 366.0. 4.1.9.4. 2-Methyl-5-(2-(1-methyl-4-phenyl-1H-imidazol-2-yl)vinyl) pyrazolo[5,1-a]isoquinoline (40). Synthesized from ((1-methyl-4phenyl-1H-imidazol-2-yl)methyl)triphenylphosphonium chloride and the aldehyde 36. Purified by FC (petroleum ether/AcOEt 7:3). Yellow solid (0.144 g, 56%); API-ES m/z: [MþH]þ calcd for C24H21N4: 365.2, found: 365.0. 4.1.9.5. 2-Methyl-5-(2-(1-methyl-1H-benzo[d]imidazol-2-yl)vinyl) pyrazolo[5,1-f][1,6]naphthyridine (45). Synthesized from the phosphonium salt 31 and 1-methyl-1H-benzo[d]imidazole-2carbaldehyde. Yellow solid (0.238 g, 99%); API-ES m/z: [MþH]þ calcd for C21H18N5: 340.1, found: 340.0. 4.1.9.6. Ethyl 5-(2-(1-methyl-1H-benzo[d]imidazol-2-yl)vinyl)pyrazolo[5,1-f][1,6]naphthyridine-2-carboxylate (46). Synthesized from the phosphonium salt 32 and 1-methyl-1H-benzo[d]imidazole-2carbaldehyde. Yellow solid (0.211 g, 75%); API-ES m/z: [MþH]þ calcd for C23H20N5O2: 398.1, found: 398.0. 4.1.9.7. 9-Methyl-6-(2-(1-methyl-1H-benzo[d]imidazol-2-yl)vinyl) pyrazolo[5,1-a][2,7]naphthyridine (47). Synthesized from the phosphonium salt 33 and 1-methyl-1H-benzo[d]imidazole-2carbaldehyde. Yellow solid (0.240 g, 100%); API-ES m/z: [MþH]þ calcd for C21H18N5: 340.1, found: 340.0. 4.1.9.8. 2-Methyl-5-(2-(1-methyl-1H-benzo[d]imidazol-2-yl)vinyl) pyrazolo[5,1-a]isoquinoline (48). Synthesized from the phosphonium salt 34 and 1-methyl-1H-benzo[d]imidazole-2-carbaldehyde. Purified by FC (petroleum ether/AcOEt 7:3). Yellow solid (0.230 g, 96%); API-ES m/z: [MþH]þ calcd for C22H19N4: 339.1, found: 339.0. 4.1.10. General procedure VIII. Synthesis of final compounds (41e44, 49e52) A mixture of the appropriate alkene (1 eq, 0.35 mmol), PTSH (3 eq, 1.05 mmol), in dry DMF (6 mL) was heated at 120 C for 4 h under nitrogen atmosphere. PTSH (1.5 eq, 0.53 mmol) was further added and the whole heated for 2 h. The mixture was cooled to room temperature, quenched with H2O and basified with saturated aqueous NaHCO3 solution. The crude product was extracted with AcOEt; the combined organic layer was washed with water, brine, dried over Na2SO4, and evaporated to afford a crude residue which was purified by FC (petroleum ether/AcOEt 3:7 / AcOEt). 4.1.10.1. Ethyl 5-(2-(1-methyl-4-phenyl-1H-imidazol-2-yl)ethyl)pyrazolo[5,1-f][1,6]naphthyridine-2-carboxylate (41). Synthesized from the alkene 37. Purified by FC (CHCl3/MeOH 9.5:0.5); white solid (0.077 g, 52%); Rf 0.57 (CHCl3/CH3OH 95:5); mp 172e174 C; 1H NMR (CDCl3) 8.91e8.89 (m, 1H), 8.40 (d, J ¼ 8.0 Hz, 1H), 7.75 (d, J ¼ 6.8 Hz, 2H), 7.64 (s, 1H), 7.51 (dd, J ¼ 4.6 Hz, 8.0 Hz, 1H), 7.41e7.36 (m, 3H), 7.29e7.19 (m, 1H), 7.13 (s, 1H), 4.49 (q, J ¼ 7.0 Hz, 2H), 3.83 (s, 3H), 3.75e3.55 (m, 2H), 3.50e3.25 (m, 2H), 1.47 (t, J ¼ 7.0 Hz, 3H); 13C NMR (CDCl3) 162.6, 151.2, 147.4, 146.1, 144.9, 141.8, 140.3, 138.9, 134.4, 131.3, 128.5, 126.5, 124.8, 122.1, 119.6, 116.7,

114.7, 101.9, 61.4, 33.1, 30.7, 24.7, 14.4; API-ES m/z: [MþH]þ calcd for C25H24N5O2: 426.2, found: 426.0. Anal. (C25H23N5O2) C, H, N. 4.1.10.2. 9-Methyl-6-(2-(1-methyl-4-phenyl-1H-imidazol-2-yl)ethyl) pyrazolo[5,1-a][2,7]naphthyridine (42). Synthesized from the alkene 38. White solid (0.073 g, 57%); Rf 0.6 (CHCl3/CH3OH 95:5); mp 163e165 C; 1H NMR (CDCl3) 9.37 (s, 1H), 8.60 (d, J ¼ 5.0 Hz, 1H), 7.75 (d, J ¼ 7.0 Hz, 2H), 7.46 (d, J ¼ 5.0 Hz, 1H), 7.37 (t, J ¼ 7.4 Hz, 2H), 7.22 (t, J ¼ 7.2 Hz, 1H), 7.06 (s, 1H), 6.96 (s, 1H), 6.79 (s, 1H), 3.63e3.59 (m, 2H), 3.57 (s, 3H), 3.39e3.32 (m, 2H), 2.58 (s, 3H); 13C NMR (CDCl3) 151.6, 147.6, 146.6, 142.2, 140.3, 137.8, 134.3, 133.9, 128.6, 126.5, 124.7, 119.6, 118.9, 116.5, 107.8, 97.8, 32.7, 31.0, 24.6, 14.2; API-ES m/z: [MþH]þ calcd for C23H22N5: 368.2, found: 368.0. Anal. (C23H21N5) C, H, N. 4.1.10.3. 2-Methyl-5-(2-(1-methyl-4-phenyl-1H-imidazol-2-yl)ethyl) pyrazolo[5,1-f][1,6]naphthyridine (43). Synthesized from the alkene 39. White solid (0.079 g, 62%); Rf 0.29 (petroleum ether/AcOEt 2:8); mp 148e150 C; 1H NMR (CDCl3) 8.79 (dd, Jm ¼ 1.8 Hz, Jo ¼ 4.8 Hz, 1H), 8.27 (dd, Jm ¼ 1.8 Hz, Jo ¼ 8.1 Hz, 1H), 7.74 (d, J ¼ 7.2 Hz, 2H), 7.40e7.33 (m, 3H), 7.23 (t, J ¼ 7.2 Hz, 1H), 7.08 (s, 1H), 7.06 (s, 1H), 6.85 (s, 1H), 3.62 (s, 3H), 3.60e3.55 (m, 2H), 3.36e3.30 (m, 2H), 2.55 (s, 3H); 13C NMR (CDCl3) 151.3, 150.4, 147.6, 146.3, 141.6, 140.3, 138.7, 134.4, 131.1, 128.5, 126.5, 124.8, 121.3, 118.9, 116.5, 110.9, 98.8, 32.7, 30.7, 24.6, 14.2; API-ES m/z: [MþH]þ calcd for C23H22N5: 368.2, found: 368.1. Anal. (C23H21N5) C, H, N. 4.1.10.4. 2-Methyl-5-(2-(1-methyl-4-phenyl-1H-imidazol-2-yl)ethyl) pyrazolo[5,1-a]isoquinoline (44). Synthesized from the alkene 40. Purified by FC (petroleum ether/AcOEt 6:4); white solid (0.103 g, 81%); Rf 0.57 (petroleum ether/AcOEt 2:8); mp 123e125 C; 1H NMR (CDCl3) 8.06e7.98 (m, 1H), 7.76 (dd, Jm ¼ 1.2 Hz, Jo ¼ 7.4 Hz, 2H), 7.67e7.58 (m, 1H), 7.55e7.44 (m, 2H), 7.41e7.33 (m, 2H), 7.29e7.18 (m, 1H), 7.05 (s, 1H), 6.83 (s, 1H), 6.81 (s, 1H), 3.62e3.47 (m, 2H), 3.55 (s, 3H), 3.42e3.30 (m, 2H), 2.56 (s, 3H); 13C NMR (CDCl3) 150.4, 148.1, 140.0, 139.6, 137.2, 134.3, 129.2, 128.6, 127.7, 126.7, 126.5, 124.8, 123.4, 123.4, 116.4, 109.9, 97.4, 32.7, 31.0, 24.7, 14.2; API-ES m/z: [MþH]þ calcd for C24H23N4: 367.2, found: 367.0. Anal. (C24H22N4) C, H, N. 4.1.10.5. 2-Methyl-5-(2-(1-methyl-1H-benzo[d]imidazol-2-yl)ethyl) pyrazolo[5,1-f][1,6]naphthyridine (49). Synthesized from the alkene 45. White solid (0.085 g, 71%); Rf 0.27 (petroleum ether/AcOEt 3:7); mp 188e190 C; 1H NMR (CDCl3) 8.81 (dd, Jm ¼ 1.6 Hz, Jo ¼ 4.6 Hz, 1H), 8.31 (dd, Jm ¼ 1.8 Hz, Jo ¼ 8.2 Hz, 1H), 7.77e7.73 (m, 1H), 7.41 (dd, J ¼ 4.6 Hz, 8.2 Hz, 1H), 7.31e7.23 (m, 3H), 7.12 (s, 1H), 6.88 (s, 1H), 3.81 (s, 3H), 3.76e3.67 (m, 2H), 3.59e3.49 (m, 2H), 2.55 (s, 3H); 13 C NMR (CDCl3) 154.0, 151.3, 150.4, 146.3, 142.7, 141.4, 138.7, 135.9, 131.1, 122.2, 122.0, 121.4, 119.3, 119.0, 111.0, 109.0, 98.8, 30.2, 29.7, 25.4, 14.2; API-ES m/z: [MþH]þ calcd for C21H20N5: 342.2, found: 342.1. Anal. (C21H19N5) C, H, N. 4.1.10.6. Ethyl 5-(2-(1-methyl-1H-benzo[d]imidazol-2-yl)ethyl)pyrazolo[5,1-f][1,6]naphthyridine-2-carboxylate (50). Synthesized from the alkene 46. Purified by FC (CHCl3/MeOH 9.5:0.5); white solid (0.092 g, 66%); Rf 0.31 (petroleum ether/AcOEt 3:7); mp 135e137 C; 1H NMR (CDCl3): 8.91e8.89 (m, 1H), 8.41 (d, J ¼ 7.8 Hz, 1H), 7.73e7.71 (m, 1H), 7.64 (s, 1H), 7.56e7.47 (m, 1H), 7.38 (s, 1H), 7.35e7.26 (m, 3H), 4.50 (q, J ¼ 7.2 Hz, 2H), 3.97 (s, 3H), 3.80e3.76 (m, 2H), 3.67e3.65 (m, 2H), 1.48 (t, J ¼ 7.2 Hz, 3H); 13C NMR (CDCl3) 162.5, 153.6, 151.2, 146.0, 144.9, 142.0, 141.4, 138.8, 135.7, 131.4, 122.5, 122.2, 122.1, 119.6, 119.0, 114.8, 109.3, 101.9, 61.4, 30.3, 30.0, 25.2, 14.4; API-ES m/z: [MþH]þ calcd for C23H22N5O2: 400.2, found: 400.0. Anal. (C23H21N5O2) C, H, N.


191

4.1.10.7. 9-Methyl-6-(2-(1-methyl-1H-benzo[d]imidazol-2-yl)ethyl) pyrazolo[5,1-a][2,7]naphthyridine (51). Synthesized from the alkene 47. White solid (0.046 g, 39%); Rf 0.54 (CHCl3/CH3OH 95:5); mp 170e171 C; 1H NMR (CDCl3) 9.36 (s, 1H), 8.59 (d, J ¼ 4.8 Hz, 1H), 7.74e7.65 (m, 1H), 7.45 (d, J ¼ 4.8 Hz, 1H), 7.35e7.20 (m, 3H), 6.95 (s, 1H), 6.79 (s, 1H), 3.74 (s, 3H), 3.74e3.67 (m, 2H), 3.56e3.48 (m, 2H), 2.56 (s, 3H); 13C NMR (CDCl3) 153.9, 151.7, 146.7, 146.6, 142.6, 141.9, 137.8, 135.9, 133.8, 122.2, 122.0, 119.6, 119.2, 118.9, 109.1, 107.8, 97.8, 30.5, 29.7, 25.4, 14.2; API-ES m/z: [MþH]þ calcd for C21H20N5: 342.2, found: 342.0. Anal. (C21H19N5) C, H, N.

following the general synthetic procedure I, heating the reaction mixture using microwave irradiation at 50 C for 20 min. Purified by FC (petroleum ether/AcOEt 4:6 / AcOEt); yellow solid (0.243 g, 60%); Rf 0.13 (petroleum ether/AcOEt 4:6); mp 98e100 C; 1H NMR (CDCl3): 10.45 (s, 1H), 8.74 (dd, Jm ¼ 1.2 Hz, Jo ¼ 4.4 Hz, 1H), 8.14 (dd, Jm ¼ 1.2 Hz, Jo ¼ 8.0 Hz, 1H), 7.74 (d, J ¼ 7.2 Hz, 2H), 7.50e7.32 (m, 3H), 7.27e7.21 (m, 1H), 7.10 (s, 1H), 3.68 (s, 3H), 3.10 (s, 4H); 13C NMR (CDCl3) 191.1, 154.3, 146.5, 146.2, 140.3, 134.7, 134.2, 131.9, 128.5, 126.6, 124.8, 124.7, 123.1, 116.7, 96.7, 32.9, 25.7, 18.8; API-ES m/z: [MþH]þ calcd for C20H18N3O: 316.1, found: 316.0.

4.1.10.8. 2-Methyl-5-(2-(1-methyl-1H-benzo[d]imidazol-2-yl)ethyl) pyrazolo[5,1-a]isoquinoline (52). Synthesized from the alkene 48. Purified by FC (petroleum ether/AcOEt 6:4); white solid (0.092 g, 77%); Rf 0.42 (petroleum ether/AcOEt 4:6); mp 120e122 C; 1H NMR (CDCl3) 8.06e7.96 (m, 1H), 7.80e7.74 (m, 1H), 7.66e7.57 (m, 1H), 7.55e7.43 (m, 2H), 7.33e7.19 (m, 3H), 6.82 (s, 1H), 6.81 (s, 1H), 3.73 (s, 3H), 3.68e3.59 (m, 2H), 3.58e3.46 (m, 2H), 2.55 (s, 3H); 13C NMR (CDCl3) 154.4, 150.5, 142.7, 139.7, 137.1, 135.9, 129.1, 127.7, 126.7, 123.4, 122.1, 121.9, 119.2, 109.9, 109.1, 97.4, 30.5, 29.7, 25.6, 14.2; API-ES m/z: [MþH]þ calcd for C22H21N4: 341.2, found: 341.0. Anal. (C22H20N4) C, H, N.

4.1.13. Synthesis of final compounds (41 and 57) 4.1.13.1. Ethyl 5-(2-(1-methyl-4-phenyl-1H-imidazol-2-yl)ethyl)pyrazolo[5,1-f][1,6]naphthyridine-2-carboxylate (41). Synthesized from 56 (1 eq, 0.97 mmol) and ethyl pyruvate following the general procedure II (0.292 g, 71%).

4.1.11. Synthesis of 2-(but-3-yn-1-yl)-1-methyl-4-phenyl-1Himidazole (55) Scheme 3, Step a. Cs2CO3 (0.5 eq, 5.10 mmol) was added to a solution of 4-pentynoic acid (53) (1 eq, 10.20 mmol) in EtOH/H2O 1:1 (20 mL), and the whole stirred at room temperature for 1 h. The solvent was evaporated and the crude residue solubilized in dry DMF (12.5 mL); a solution of 2-bromoacetophenone (1 eq, 10.20 mmol) in dry DMF (6.5 mL) was dropwise added and the whole stirred at room temperature for 15 min. The solvent was evaporated, the residue taken up with AcOEt and the inorganic salt was filtered off. The filtrate was dried over Na2SO4, and the solvent evaporated to afford a yellow oily residue. Step b. The oily residue was solubilized in xylene (50 mL). NH4OAc (15 eq, 153.00 mmol) was added and the resulting solution refluxed for 12 h using a DeaneStark trap. The solution was cooled to room temperature, washed with H2O and the organic phase evaporated to give a residue which was taken up with AcOEt. The organic phase was washed with saturated aqueous NaHCO3 solution, brine, dried with Na2SO4 and evaporated to give 2-(but-3-yn-1-yl)-4-phenyl-1Himidazole (54) as crude residue purified by FC (petroleum ether/ AcOEt 6:4). Orange solid (1.960 g, 98%); Rf 0.10 (petroleum ether/ AcOEt 6:4); mp 53e55 C; 1H NMR (CDCl3) 7.67 (d, J ¼ 7.4 Hz, 2H), 7.40e7.23 (m, 4H), 3.02 (t, J ¼ 6.8 Hz, 2H), 2.63 (dt, J ¼ 2.4, 6.8 Hz, 2H), 2.12 (t, J ¼ 2.4 Hz, 1H). Step c. 60% NaH in mineral oil (1.5 eq, 15.00 mmol) was added to a cooled solution (0 C) of 54 (1 eq, 9.98 mmol) in dry THF (50 mL), and the whole stirred at the same temperature for 30 min. MeI (1.1 eq, 11.00 mmol) was dropwise added and the resulting solution warmed to room temperature and stirred for 12 h. The solution was washed with H2O, brine, dried with Na2SO4 and evaporated to give 55 as crude residue purified by FC (petroleum ether/AcOEt 1:1). Orange oil (1.572 g, 75%); Rf 0.59 (petroleum ether/AcOEt 4:6); 1H NMR (CDCl3): 7.72 (d, J ¼ 7.4 Hz, 2H), 7.39e7.20 (m, 3H), 7.07 (s, 1H), 3.65 (s, 3H), 2.97 (t, J ¼ 7.6 Hz, 2H), 2.70 (dt, J ¼ 2.4, 7.6 Hz, 2H), 2.00 (t, J ¼ 2.4 Hz, 1H). 13C NMR (CDCl3) 147.1, 140.2, 134.3, 128.5, 126.5, 124.7, 116.5, 83.3, 69.2, 32.9, 26.1, 17.9; API-ES m/z: [MþH]þ calcd for C14H15N2: 211.1, found: 211.0. 4.1.12. Synthesis of 2-(4-(1-methyl-4-phenyl-1H-imidazol-2-yl) but-1-yn-1-yl)nicotinaldehyde (56) Synthesized from 2-bromonicotinaldehyde (6) (1 eq, 1.29 mmol) and 2-(but-3-yn-1-yl)-1-methyl-4-phenyl-1H-imidazole (55),

4.1.13.2. 5-(2-(1-Methyl-4-phenyl-1H-imidazol-2-yl)ethyl)-2-(trifluoromethyl)pyrazolo[5,1-f][1,6]naphthyridine (57). Synthesized from 56 (1 eq, 0.97 mmol) and 1,1,1-trifluoroacetone, following the general procedure II. Purified by FC (AcOEt / AcOEt/ MeOH 9:1); white solid (0.301 g, 75%); Rf 0.38 (petroleum ether/ AcOEt 2:8); mp 159e161 C; 1H NMR (CDCl3) 8.93 (dd, Jm ¼ 1.6 Hz, Jo ¼ 4.6 Hz, 1H), 8.39 (dd, Jm ¼ 1.6 Hz and 8.0 Hz, 1H), 7.74 (d, J ¼ 7.2 Hz, 2H), 7.52 (dd, J ¼ 4.6 Hz, 8.0 Hz, 1H), 7.36 (t, J ¼ 7.4 Hz, 2H), 7.35 (s, 1H), 7.27e7.21 (m, 2H), 7.11 (s, 1H), 3.76 (s, 3H), 3.71e3.62 (m, 2H), 3.39e3.31 (m, 2H). 13C NMR (CDCl3) 151.5, 147.2, 146.1, 144.5, 144.1, 143.7, 143.4, 141.6, 140.2, 138.9, 134.2, 131.3, 128.5, 126.5, 124.7, 122.2, 119.1, 116.7, 114.7, 97.5, 32.8, 30.5, 24.6; API-ES m/ z: : [MþH]þ calcd for C23H19F3N5: 422.1, found: 422.0. Anal. (C23H18F3N5) C, H, N. 4.1.14. Synthesis of final compound 2-(methoxymethyl)-5-(2-(1methyl-4-phenyl-1H-imidazol-2-yl)ethyl)pyrazolo[5,1-f][1,6] naphthyridine (59) Scheme 3, Step f. NaBH4 (2.2 eq, 0.88 mmol) was added to a solution of 41 (1 eq, 0.40 mmol) in dry EtOH (4 mL), and the whole refluxed for 12 h. The solvent was evaporated to give (5-(2-(1methyl-4-phenyl-1H-imidazol-2-yl)ethyl)pyrazolo[5,1-f][1,6]naphthyridin-2-yl)methanol (58) as crude residue purified by FC (CHCl3/ MeOH 9.5:0.5). White solid (0.093 g, 61%); Rf 0,28 (CHCl3/CH3OH 95:5); 1H NMR (CDCl3) 8.77 (dd, Jm ¼ 1.6 Hz, Jo ¼ 4.6 Hz, 1H), 8.24 (d, J ¼ 8.4 Hz, 1H), 7.73 (d, J ¼ 7.0 Hz, 2H), 7.39e7.17 (m, 5H), 7.05 (s, 1H), 4.96 (s, 2H), 3.59 (s, 3H), 3.53e3.49 (m, 2H), 3.31e3.23 (m, 2H), 2.87 (br s, 1H). Step c. Compound 58 (1 eq, 0.20 mmol) was converted into 59 following the procedure used for the synthesis of 55 (Scheme 3, Step c). Purified by FC (CHCl3/MeOH 9.5:0.5). Yellow solid (0.046 g, 58%); Rf 0.53 (CHCl3/CH3OH 9.5:0.5); mp 133e134 C; 1H NMR (CDCl3) 8.84 (d, J ¼ 4.4 Hz, 1H), 8.35 (d, J ¼ 7.8 Hz, 1H), 7.75 (d, J ¼ 8.0 Hz, 2H), 7.48e7.40 (m, 1H), 7.36 (t, J ¼ 8.0 Hz, 2H), 7.30e7.20 (m, 1H), 7.17 (s, 1H), 7.13 (s, 1H), 7.10 (s, 1H), 4.74 (s, 2H), 3.70e3.55 (m, 2H), 3.66 (s, 3H), 3.50 (s, 3H), 3.40e3.25 (m, 2H); 13C NMR (CDCl3) 152.0, 150.6, 147.5, 146.2, 141.7, 140.1, 138.9, 134.1, 131.2, 128.6, 126.6, 124.8, 121.6, 119.2, 116.6, 112.0, 98.3, 68.7, 58.6, 32.8, 30.5, 24.6; API-ES m/z: [MþH]þ calcd for C24H24N5O 398.2, found: 398.0. Anal. (C24H23N5O) C, H, N. 4.1.15. Synthesis of 3-(4-(1-methyl-4-phenyl-1H-imidazol-2-yl) but-1-yn-1-yl)isonicotinaldehyde (60) Synthesized from 3-bromoisonicotinaldehyde (7) (1 eq, 1.39 mmol) and the alkyne 55, following the general synthetic procedure I, heating the reaction mixture using microwave irradiation at 70 C for 2 h. Purified by FC (petroleum ether/AcOEt 4/6). Yellow solid (0.346 g, 79%); Rf 0.22 (petroleum ether/AcOEt 4:6); mp 118e119 C; 1H NMR (CDCl3) 10.42 (s, 1H), 8.81 (s, 1H), 8.67 (d,

192


J ¼ 5 Hz, 1H), 7.74 (d, J ¼ 7.8 Hz, 2H), 7.64 (d, J ¼ 5 Hz, 1H), 7.36 (t, J ¼ 7.4 Hz, 2H), 7.25e7.21 (m, 1H), 7.11 (s, 1H), 3.68 (s, 3H), 3.08 (s, 4H); 13C NMR (CDCl3) 191.0, 154.8, 149.0, 146.5, 140.8, 140.4, 134.2, 128.6, 126.6, 124.7, 121.7, 119.0, 116.6, 99.5, 74.4, 32.9, 25.8, 18.9; APIES m/z: [MþH]þ calcd for C20H18N3O: 316.1, found: 316.0. 4.1.16. Synthesis of 3-(4-(1-methyl-4-phenyl-1H-imidazol-2-yl) but-1-yn-1-yl)isonicotinaldehyde oxime (61) A mixture of 60 (1 eq, 4.12 mmol), NH2OH$HCl (1.5 eq, 6.18 mmol) and AcONa$H2O (1.5 eq, 6.18 mmol) in EtOH (24 mL) was stirred at room temperature for 4 h. The solvent was evaporated and the residue was extracted with Et2O. The organic phase was dried with Na2SO4 and evaporated to give a crude residue used without purification. Yellow solid (0.816 g, 60%); Rf 0.45 (petroleum ether/AcOEt 3:7); mp 145e147 C; 1H NMR (CDCl3) 8.58 (s, 1H), 8.47 (s, 1H), 8.39 (d, J ¼ 5.2 Hz, 1H), 7.76e7.68 (m, 2H), 7.65 (d, J ¼ 5.2 Hz, 1H), 7.39e7.22 (m, 3H), 7.11 (s, 1H); 3.70 (s, 3H), 3.15e3.11 (m, 2H), 3.00e2.95 (m, 2H); API-ES m/z: [MþH]þ calcd for C20H19N4O: 331.1, found: 331.0. 4.1.17. Synthesis of 3-(2-(1-methyl-4-phenyl-1H-imidazol-2-yl) ethyl)-2,6-naphthyridine 2-oxide (62) A mixture of 61 (1 eq, 1.82 mmol) and dry K2CO3 (1.5 eq, 2.73 mmol) in dry EtOH (36 mL) was stirred at room temperature for 6 h. The solvent was evaporated and the residue extracted with DCM. The organic phase was dried with Na2SO4 and evaporated to give a crude residue used in the following step with no further purification. Yellow solid (0.558 g, 93%); Rf 0.0 (petroleum ether/ AcOEt 4:6); mp 194e196 C; 1H NMR (CDCl3) 9.16 (s, 1H), 8.80 (s, 1H), 8.60 (d, J ¼ 5.8 Hz, 1H), 7.96 (s, 1H), 7.73 (d, J ¼ 7.2 Hz, 2H), 7.47 (d, J ¼ 5.8 Hz, 1H), 7.40e7.22 (m, 3H), 7.07 (s, 1H), 3.65 (s, 3H), 3.53 (t, J ¼ 7 Hz, 2H), 3.26 (t, J ¼ 7 Hz, 2H); API-ES m/z: [MþH]þ calcd for C20H19N4O: 331.1, found: 331.0. 4.1.18. Synthesis of 1-chloro-3-(2-(1-methyl-4-phenyl-1Himidazol-2-yl)ethyl)-2,6-naphthyridine (63) POCl3 (5 eq, 15 mmol) was added to a solution of 62 (1 eq, 3.0 mmol) in dry MeCN (20 mL) and the whole refluxed for 2 h. The solution was evaporated, the residue taken up with ice, the resulting solution basified with conc. NH4OH and extracted with DCM. The organic phase was dried with Na2SO4 and evaporated to give 63 as crude residue purified by FC (petroleum ether/AcOEt 3:7). White solid (0.396 g, 40%); Rf 0.24 (petroleum ether/AcOEt 3:7); mp 171e172 C; 1H NMR (CDCl3) 9.25 (s, 1H), 8.74 (d, J ¼ 5.8 Hz, 1H), 8.02 (d, J ¼ 5.8 Hz, 1H), 7.73 (d, J ¼ 7 Hz, 2H), 7.66 (s, 1H), 7.36 (t, J ¼ 7 Hz, 2H), 7.25e7.21 (m, 1H), 7.03 (s, 1H), 3.58 (s, 3H), 3.49 (t, J ¼ 7.6 Hz, 2H), 3.25 (t, J ¼ 7.6 Hz, 2H); 13C NMR (CDCl3) 155.5, 151.7, 150.3, 147.5, 145.4, 140.1, 134.4, 132.6, 128.5, 128.0, 126.5, 124.7, 117.8, 117.7, 116.3, 35.8, 32.8, 26.2; API-ES m/z: [MþH]þ calcd for C20H18ClN4: 349.1, found: 349.0. 4.1.19. Synthesis of 1-(3-(2-(1-methyl-4-phenyl-1H-imidazol-2-yl) ethyl)-2,6-naphthyridin-1-yl)prop-1-en-2-ol (64) A solution of 63 (1 eq, 1.15 mmol) in dry toluene (5 mL) was deoxygenated with bubbling N2. Isopropenyl acetate (1.5 eq, 1.73 mmol) was added to the reaction mixture, followed by the addition of DavePhos (0.1 eq, 0.11 mmol) and then tributyl(methoxy)stannane (1.5 eq, 1.72 mmol) and tris(dibenzylideneacetone)dipalladium (0) (0.01 eq, 0.01 mmol). The solution was heated to 100 C and stirred for 12 h. The solution was evaporated and the residue purified by FC (CHCl3/MeOH 9.5:0.5). Orange solid (0.384 g, 96%); Rf 0.46 (CHCl3/CH3OH 95:5); mp 73e75 C; 1H NMR (CDCl3) 15.45 (br s, 1H), 8.86 (s, 1H), 8.56 (d, J ¼ 6.0 Hz, 1H), 7.74e7.73 (m, 3H), 7.39e7.21 (m, 3H), 7.07 (s, 1H), 6.62 (s, 1H), 6.06

(s, 1H), 3.61 (s, 3H), 3.30e3.12 (m, 4H), 2.26 (s, 3H). API-ES m/z: [MþH]þ calcd for C23H23N4O 371.2, found 371.0. 4.1.20. Synthesis of 1-(3-(2-(1-methyl-4-phenyl-1H-imidazol-2-yl) ethyl)-2,6-naphthyridin-1-yl)propan-2-one oxime (65) Synthesized from 64 (1 eq, 0.97 mmol) following the procedure used for the synthesis of 61. Purified by FC (CHCl3/MeOH 9.5:0.5). Yellow solid (0.321 g, 86%); Rf 0.33 (petroleum ether/AcOEt 1:9); mp 84e86 C; 1H NMR (CDCl3) 9.17 (s, 1H), 8.56 (d, J ¼ 5.8 Hz, 1H), 7.90 (d, J ¼ 6 Hz, 1H), 7.72 (d, J ¼ 7.4 Hz, 2H), 7.51 (s, 1H), 7.35 (t, J ¼ 8 Hz, 2H), 7.24e7.02 (m, 1H), 7.00 (s, 1H), 4.18 (s, 2H), 3.51 (s, 3H), 3.45e3.38 (m, 2H), 3.30e3.22 (m, 2H), 1.95 (s, 3H). API-ES m/z: [MþH]þ calcd for C23H24N5O 386.2, found: 386.0. 4.1.21. Synthesis of 2-methyl-5-(2-(1-methyl-4-phenyl-1Himidazol-2-yl)ethyl)pyrazolo[5,1-a][2,6]naphthyridine (66) To a cooled solution (0 C) of 65 (1.0 eq, 0.8 mmol) in dry DME (5 mL) was added TFAA (1 eq, 0.80 mmol) and the mixture stirred for 10 min. Et3N (3.1 eq, 2.4 mmol) was added dropwise over 15 min, and then the whole was warmed to room temperature and stirred for 0.5 h. FeCl2 (0.05 eq, 0.039 mmol) was added and the mixture heated to 75 C for 12 h. The mixture was poured into water, basified with a saturated aqueous K2CO3 solution and extracted with AcOEt. The organic phase was dried under Na2SO4 and evaporated to afford a crude residue purified by FC (petroleum ether/AcOEt 4:6). White solid (0.023 g, 8%). Rf 0.30 (petroleum ether/AcOEt 2:8); mp 99e100 C; 1H NMR (CDCl3) 9.00 (s, 1H), 8.63 (d, J ¼ 5.4 Hz, 1H), 7.81 (d, J ¼ 5.4, 1H), 7.74 (d, J ¼ 7.6 Hz, 2H), 7.37 (t, J ¼ 7.6 Hz, 2H), 7.27e7.22 (m, 1H), 7.06 (s, 1H), 6.96 (s, 1H), 6.91 (s, 1H), 3.62e3.51 (m, 5H), 3.38e3.31 (m, 2H), 2.58 (s, 3H). 13C NMR (CDCl3) 151.0, 149.7, 147.6, 145.6, 140.3, 139.7, 137.0, 134.4, 128.6, 128.0, 126.5, 124.7, 123.8, 116.5, 116.2, 107.8, 99.6, 32.7, 30.8, 24.6, 14.2; API-ES m/z: [MþH]þ calcd for C23H22N5: 368.2, found: 368.0. Anal. (C23H21N5) C, H, N. 4.2. Biology experimental 4.2.1. PDE10A enzyme PDE10A can be prepared in different cell types, for example, insect cells or Escherichia coli. Catalytically active PDE10A was obtained as follows: human PDE10A (amino acids 14e779 from the sequence with accession number NP_006652) was amplified from total human brain total RNA by standard RT-PCR and cloned into the BamH1 and Not1 sites of the pFastBac-HTb (Invitrogen). Expression in Sf9 cells using the Bac-to-Bac® Baculovirus Expression System (Gibco). Sf9 cells were grown at 27 C in Sf-900 II serum free medium containing 50 units/ml penicillin and 50 mg/ml streptomycin and were infected with 1 ml of virus for 25 ml of media. At 72 h, cells were harvested and disrupted in lysis buffer (50 mM Tris8.0 þ 1 mM MgCl2 þ PI þ 0.5%triton) for 15 min on ice and then centrifuged at 20,000g for 20 min PDE10A was partially purified on Q sepharose and the most active fractions were pooled. 4.2.2. PDE10A inhibition assay A PDE10A assay was performed as follows: the assay was performed in 60 mL samples containing a fixed amount of the relevant PDE enzyme (sufficient to convert 20e25% of the cyclic nucleotide substrate), a buffer (50 mM HEPES 7.6; 10 mM MgCl2; 0.02% Tween20), 0.1 mg/ml BSA, 225 pCi of 3H-labelled cyclic nucleotide substrate, tritium labelled cAMP to a final concentration of 5 nM and varying amounts of inhibitors. Reactions were initiated by addition of the cyclic nucleotide substrate, and reactions were allowed to proceed for 1 h at room temperature before being terminated through mixing with 15 mL 8 mg/ml yttrium silicate SPA beads (Amersham). The beads were allowed to settle for 1 h in the


dark before the plates were counted in a Wallac 1450 Microbeta counter. The measured signals were converted to activity relative to an uninhibited control (100%) and IC50 values were calculated using the Xlfit extension to EXCEL. [10]

Acknowledgements Financial support from the Regione Autonoma della Sardegna (CRP-26417) is acknowledged.

[11]

Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2014.07.020.

[12]

References [1] M. Conti, J. Beavo, Biochemistry and physiology of cyclic nucleotide phosphodiesterases: essential components in cyclic nucleotide signalling, Annu. Rev. Biochem 76 (2007) 481e511. [2] J.A. Beavo, S.H. Francis, M.D. Houslay, Cyclic Nucleotide Phosphodiesterases in Health and Disease, Crc Press, 2006. [3] a) S.H. Soderling, S.J. Bayuga, J.A. Beavo, Isolation and characterization of a dual-substrate phosphodiesterase gene family: PDE10A, Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 7071e7076; b) K. Fujishige, J. Kotera, H. Michibata, K. Yuasa, S. Takebayashi, K. Okumura, K. Omori, Cloning and characterization of a novel human phosphodiesterase that hydrolyzes both cAMP and cGMP (PDE10A), J. Biol. Chem. 274 (1999) 18438e18445; c) K. Loughney, P.B. Snyder, L. Huher, G.J. Rosman, K. Ferguson, V.A. Florio, Isolation and characterization of PDE10A, a novel human 3', 5'-cyclic nucleotide phosphodiesterase, Gene 234 (1999) 109e117. [4] V. Lakics, E.H. Karran, F.G. Boess, Quantitative comparison of phosphodiesterase mRNA distribution in human brain and peripheral tissues, Neuropharmacology 59 (2010) 367e374. [5] a) T.F. Seeger, B. Bartlett, T.M. Coskran, J.S. Culp, L.C. James, D.L. Krull, J. Lanfear, A.M. Ryan, C.J. Schmidt, C.A. Strick, A.H. Varghese, R.D. Williams, P.G. Wylie, F.S. Menniti, Immunohistochemical localization of PDE10A in the rat brain, Brain Res. 985 (2003) 113e126; b) T.M. Coskran, D. Morton, F.S. Menniti, W.O. Adamowicz, R.J. Kleiman, A.M. Ryan, C.A. Strick, C.J. Schmidt, D.T. Stephenson, Immunohistochemical localization of phosphodiesterase 10A in multiple mammalian species, J. Histochem. Cytochem. 54 (2006) 1205e1213; c) Z. Xie, W.O. Adamowicz, W.D. Eldred, A.B. Jakowski, R.J. Kleiman, D.G. Morton, D.T. Stephenson, C.A. Strick, R.D. Williams, F.S. Menniti, Cellular and subcellular localization of PDE10A, a striatum-enriched phosphodiesterase, Neuroscience 139 (2006) 597e607. [6] F.S. Menniti, W.S. Faraci, C.J. Schmidt, Phosphodiesterases in the CNS: targets for drug development, Nat. Rev. Drug. Discov. 5 (2006) 660e670. n, D.R. Greve, The potential therapeutic use of phospho[7] a) J. Kehler, A. Ritze diesterase 10 inhibitors, Expert Opin. Ther. Patents 17 (2007) 147e158; b) F.S. Menniti, T.A. Chappie, J.M. Humphrey, C.J. Schmidt, Phosphodiesterases 10A inhibitors: a novel approach to the treatment of the symptoms of schizophrenia, Curr. Opin. Invest. Drugs 8 (2007) 54e59; c) T. Chappie, J. Humphrey, F. Menniti, C. Schmidt, PDE10A inhibitors: an assessment of the current CNS drug discovery landscape, Curr. Opin. Drug. Discov. Devel. 12 (2009) 458e467; d) J. Kehler, J.P. Kilburn, Patented PDE10A inhibitors: novel compounds since 2007, Expert Opin. Ther. Patents 19 (2009) 1715e1725; e) J. Kehler, J. Nielsen, PDE10A inhibitors: novel therapeutic drugs for schizophrenia, Curr. Pharm. Des. 17 (2011) 137e150; f) T.A. Chappie, C.J. Helal, X. Hou, Current landscape of phosphodiesterase 10A (PDE10A) inhibition, J. Med. Chem. 55 (2012) 7299e7331; g) N. Hoefgen, C. Grunwald, B. Langen, Targeting PDE10A in schizophrenia, Drugs Future 37 (2012) 577e589; h) J. Kehler, Phosphodiesterase 10A inhibitors: a 2009-2012 patent update, Expert Opin. Ther. Patents 23 (2013) 31e45; i) B. Asproni, A. Dore, G. Murineddu, G.A. Pinna, Chemistry of tricyclic-based heterocycles as useful scaffolds for phosphodiesterase 10A ligands, MiniRev. Org. Chem. 10 (2013) 123e140. [8] J.A. Siuciak, D.S. Chapin, J.F. Harms, L.A. Lebel, S.A. McCarthy, L. Chambers, A. Shrikhande, S. Wong, F.S. Menniti, C.J. Schmidt, Inhibition of the striatumenriched phosphodiesterase PDE10A: a novel approach to the treatment of psychosis, Neuropharmacology 51 (2006) 386e396. [9] P.R. Verhoest, D.S. Chapin, M. Corman, K. Fonseca, J.F. Harms, X. Hou, E.S. Marr, F.S. Menniti, F. Nelson, R. O'Connor, J. Pandit, C. Proulx-LaFrance,

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20] [21]

[22]

[23] [24]

[25]

[26]

193

A.W. Schmidt, C.J. Schmidt, J.A. Suiciak, S. Liras, Discovery of a novel class of phosphodiesterase 10A inhibitors and identification of clinical candidate 2-[4(1-methyl-4-pyridin-4-yl-1H-pyrazol-3-yl)-phenoxymethyl]-quinoline (PF2545920) for the treatment of schizophrenia, J. Med. Chem. 52 (2009) 5188e5196. S.M. Grauer, V.L. Pulito, R.L. Navarra, M.P. Kelly, C. Kelley, R. Graf, B. Langen, S. Logue, J. Brennan, L. Jiang, E. Charych, U. Egerland, F. Liu, K.L. Marquis, M. Malamas, T. Hage, T.A. Comery, N.J. Brandon, Phosphodiesterase 10A inhibitor activity in preclinical models of the positive, cognitive, and negative symptoms of schizophrenia, J. Pharmacol. Exp. Ther. 331 (2009) 574e590. C.J. Schmidt, D.S. Chapin, J. Cianfrogna, M.L. Corman, M. Hajos, J.F. Harms, W.E. Hoffman, L.A. Lebel, S.A. McCarthy, F.R. Nelson, C. Proulx-LaFrance, M.J. Majchrzak, A.D. Ramirez, K. Schmidt, P.A. Seymour, J.A. Siuciak, F.D. Tingley III, R.D. Williams, P.R. Verhoest, F.S. Menniti, Preclinical characterization of selective phosphodiesterase 10A inhibitors: a new therapeutic approach to the treatment of schizophrenia, J. Pharmacol. Exp. Ther. 325 (2008) 681e690. N. DeMartinis, A. Banerjee, V. Kumar, S. Boyer, C. Schmidt, S. Arroyo, Results of a phase 2A proof-of-concept trial with a PDE10A inhibitor in the treatment of acute exacerbation of schizophrenia, in: Abstracts of the 3rd Biennial Schizophrenia International Research Conference, Schizophrenia Research, 136, 2012. Suppl. 1 S262 poster 212. For implications in Huntington's disease see: a) C. Giamp a, S. Patassini, A. Borreca, D. Laurenti, F. Marullo, G. Bernardi, F.S. Menniti, F.R. Fusco, Phosphodiesterase 10 inhibition reduces striatal excitotoxicity in the quinolinic acid model of Huntington's disease, Neurobiol. Dis. 34 (2009) 450e456; , D. Laurenti, S. Anzilotti, G. Bernardi, F.S. Menniti, F.R. Fusco, b) C. Giampa Inhibition of the striatal specific phosphodiesterase PDE10A ameliorates striatal and cortical pathology in R6/2 mouse model of Huntington's disease, PLoS ONE 5 (2010) e13417; c) R.J. Kleiman, L.H. Kimmel, S.E. Bove, T.A. Lanz, J.F. Harms, A. Romegialli, K.S. Miller, A. Willis, S. des Etages, M. Kuhn, C.J. Schmidt, Chronic suppression of phosphodiesterase 10A alters striatal expression of genes responsible for neurotransmitter synthesis, neurotransmission, and signaling pathways implicated in Huntington's disease, J. Pharmacol. Exper. Ther. 336 (2011) 64e76. B. Asproni, G. Murineddu, A. Pau, G.A. Pinna, M. Langgård, C.T. Christoffersen, J. Nielsen, J. Kehler, Synthesis and SAR study of new phenylimidazole-pyrazolo[1,5-c]quinazolines as potent phosphodiesterase 10A inhibitors, Bioorg. Med. Chem. 19 (2011) 642e649. n, M. Langgård, S.L. Petersen, M.M. Farah, C. Bundgaard, J. Kehler, A. Ritze C.T. Christoffersen, J. Nielsen, J.P. Kilburn, Triazoloquinazolines as a novel class of phosphodiesterase 10A (PDE10A) inhibitors, Bioorg. Med. Chem. Lett. 21 (2011) 3738e3742. Q. Ding, J. Wu, Lewis acid- and organocatalyst-cocatalyzed multicomponent reactions of 2-alkynylbenzaldehydes, amines, and ketones, Org. Lett. 9 (2007) 4959e4962. Z. Chen, X. Yang, J. Wu, AgOTf-catalyzed tandem reaction of N'-(2alkynylbenzylidene)hydrazide with alkyne, Chem. Commun. 23 (2009) 3469e3471. X. Yu, Z. Chen, X. Yang, J. Wu, Tandem reactions of N'-(2-alkynylbenzylidene) hydrazides with silyl enolates: a facile route to H-pyrazolo[5,1-a]isoquinolines, J. Comb. Chem. 12 (2010) 374e378. A.C. Bissember, M.G. Banwell, Microwave-assisted trans-halogenation reactions of various chloro-, bromo-, trifluoromethanesulfonyloxy- and nonafluorobutanesulfonyloxy-substituted quinolines, isoquinolines, and pyridines leading to the corresponding iodinated heterocycles, J. Org. Chem. 74 (2009) 4893e4895. Glaxosmithkline, Fitzgerald RN, Jung DK, Eaddy JF, Process for the preparation of chemical compounds, US20030212275A1 Novemb., 13 (2003). A. Numata, Y. Kondo, T. Sakamoto, General synthetic method for naphthyridines and their N-oxides containing isoquinolinic nitrogen, Synthesis 2 (1999) 306e311. P. Liu, T.J. Lanza Jr., J.P. Jewell, C.P. Jones, W.K. Hagmann, L.S. Lin, A highly active catalyst system for the heteroarylation of acetone, Tetrahedron Lett. 44 (2003) 8869e8871. € dinger, LLC, New York, NY, 2011. Glide, Version 5.7, Schro R. Risgaard, A. Ettrup, T. Balle, A. Dyssegaard, H.D. Hansen, S. Lehel, J. Madsen, H. Pedersen, A. Püschl, L. Badolo, B.B. -Andersen, G.M. Knudsen, J.L. Kristensen, Radiolabelling and PET brain imaging of the a₁-adrenoceptor antagonist Lu AE43936, Nucl. Med. Biol. 40 (2013) 135e140. S.F. Poon, D.J. St Jean Jr., P.E. Harrington, C. Henley III, J. Davis, S. Morony, F.D. Lott, J.D. Reagan, J.Y.-L. Lu, Y. Yang, C. Fotsch, Discovery and optimization of substituted 1-(1-phenyl-1H-pyrazol-3-yl)methanamines as potent and efficacious type II calcimimetics, J. Med. Chem. 52 (2009) 6535e6538. T. Godet, C. Vaxelaire, C. Michel, A. Milet, P. Belmont, Silver versus gold catalysis in tandem reactions of carbonyl functions onto alkynes: a versatile access to furoquinoline and pyranoquinoline cores, Chem. Eur. J. 13 (2007) 5632e5641.

Discovery of Novel Imidazo[4,5-b]pyridines as Potent and Selective Inhibitors of Phosphodiesterase 10A (PDE10A).

Synthesis and SAR study of potent and selective PI3Kδ inhibitors.

Novel benzimidazole derivatives as phosphodiesterase 10A (PDE10A) inhibitors with improved metabolic stability.

Design, synthesis and SAR studies of NAD analogues as potent inhibitors towards CD38 NADase.

Pyrazole derivatives as potent inhibitors of c-Jun N-terminal kinase: synthesis and SAR studies.

Discovery of benzylidene derivatives as potent Syk inhibitors: synthesis, SAR analysis, and biological evaluation.

Discovery and SAR of a novel series of 2,4,5,6-tetrahydrocyclopenta[c]pyrazoles as N-type calcium channel inhibitors.

Discovery and SAR of novel tetrahydropyrrolo[3,4-c]pyrazoles as inhibitors of the N-type calcium channel.

Synthesis of novel indenoquinoxaline derivatives as potent α-glucosidase inhibitors.

Synthesis of novel compounds as new potent tyrosinase inhibitors.

Isoquinoline derivatives as potent CRTH2 antagonists: design, synthesis and SAR.

CC isosterism in borazaronaphthalenes towards phosphodiesterase 10A (PDE10A) inhibitors.

Discovery of novel pyrazolopyrimidinone analogs as potent inhibitors of phosphodiesterase type-5.

Synthesis and SAR of Thiazolylmethylphenyl Glucoside as Novel C-Aryl Glucoside SGLT2 Inhibitors.

Synthesis and SAR assessment of novel Tubathian analogs in the pursuit of potent and selective HDAC6 inhibitors.

Design and synthesis of tricyclic JAK3 inhibitors with picomolar affinities as novel molecular probes.

Tricyclic pyrazoles part 7. Discovery of potent and selective dihydrothienocyclopentapyrazole derived CB2 ligands.

Synthesis and biological evaluation of novel pyridine derivatives as potential anticancer agents and phosphodiesterase-3 inhibitors.

Design and synthesis of highly potent and isoform selective JNK3 inhibitors: SAR studies on aminopyrazole derivatives.

SAR studies on curcumin's pro-inflammatory targets: discovery of prenylated pyrazolocurcuminoids as potent and selective novel inhibitors of 5-lipoxygenase.

Discovery of Novel Tricyclic Heterocycles as Potent and Selective DPP-4 Inhibitors for the Treatment of Type 2 Diabetes.

Discovery and SAR study of piperidine-based derivatives as novel influenza virus inhibitors.

Synthesis of N-benzenesulfonamide-1H-pyrazoles bearing arylsulfonyl moiety: novel celecoxib analogs as potent anti-inflammatory agents.

Discovery of a novel class of potent HCV NS4B inhibitors: SAR studies on piperazinone derivatives.