Bioorganic & Medicinal Chemistry Letters 22 (2012) 3223–3228

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

Isothiazole and isoxazole fused pyrimidones as PDE7 inhibitors: SARand pharmacokinetic evaluation

Abhisek Banerjee, Pravin S. Yadav, Malini Bajpai, Ramachandra Rao Sangana, Srinivas Gullapalli,Girish S. Gudi, Laxmikant A. Gharat ⇑Glenmark Pharmaceuticals Limited, Navi Mumbai, Maharashtra 400 709, India

a r t i c l e i n f o

Article history:Received 30 January 2012Revised 28 February 2012Accepted 7 March 2012Available online 23 March 2012

Keywords:Phosphodiesterase 7 inhibitorsIsothiazolopyrimidoneIsoxazolopyrimidoneParkinson’s diseaseCNS penetration

0960-894X/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.bmcl.2012.03.025

⇑ Corresponding author. Tel.: +91 022 6772 0000x3E-mail address: laxmikant_gharat@glenmarkpharm

a b s t r a c t

The synthesis and structure–activity relationship studies of isothiazole and isoxazole fused pyrimidonesas PDE7 inhibitors are discussed. The pharmacokinetic profile of 10 and 21 with adequate CNS penetra-tion, required for in vivo Parkinson’s disease models, are disclosed.

� 2012 Elsevier Ltd. All rights reserved.

NH

NNN

O

O

NH2

(1)

NH

NXN

OR1

(V)

R3 R2

NH

NNN

O

Ar

NH

NN

N

O

Ar

N NH

NN

O

ArN

NH

NN

O

Ar

(I) (II)

(III) (IV)

Ar: subsituted aryl or heteroaryl

Phosphodiesterase 7 (PDE7) an enzyme that selectively hydro-lyzes cAMP, has been extensively targeted for the treatment of ahost of immunological and autoimmune conditions.1 The publica-tion indicating the role of PDE7A in the activation and/or prolifer-ation of T-cells was inspirational in this context.2 However, thehypothesis was contradicted with the observation that PDE7Aknockout mice (PDE7A�/�) did not show deficiencies in T-cell func-tions.3 A number of structurally diverse, potent and selective PDE7inhibitors were identified to evaluate the potential of PDE7 aspharmacological target of immune response. However, to date,none has been advanced to the clinical trials.4

Two isoenzymes of PDE7, PDE7A and 7B are reported to be ex-pressed in rat brain. Within the brain, PDE7A mRNA is abundant inthe olfactory bulb, hippocampus, and several brain-stem nuclei.The highest concentrations of PDE7B transcripts in the brain arefound in the striatal complex, cerebellum, dentate gyrus of the hip-pocampus and in several thalamic nuclei.1 Since regulation ofcAMP-PKA-CREB pathway plays a key role in striatum controlledmotor functions, cognition, long term memory, neurogenesis,5

neuroprotection and neuroinflammatory responses,6 recent inter-ests have emerged with the PDE7 inhibitors in the context of Par-kinson’s disease (PD).7

Parkinson’s disease (PD) is a neurodegenerative disorder associ-ated with the loss of dopaminergic neurons in the substantia nigrapars compacta (SNc) leading to dysfunction of transmission of

ll rights reserved.

208.a.com (L.A. Gharat).

PDE7A IC50: 1.30 nM X: O, S; R1: cyclohexyl, aryl;

Figure 1. PDE7 inhibitors.

3224 A. Banerjee et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3223–3228

information between input (striatum), output (GPi/SNr) and intrin-sic nuclei (GPe, STN, SNc) in the basal ganglia. The primary symp-toms include bradykinesia, resting tremor, muscle rigidity andpostural instability.8 The fine motor function under a tonic dopa-minergic condition is controlled with a complex interplay of inte-gration of cortical and thalamic information in striatum,transmission to output nuclei to finally arrive at cerebral cortexvia a thalamic relay. The striatal information is projected to theoutput nuclei either through a dopamine D1 receptor dependent‘direct pathway’ to GPi/SNr or through D2 receptor dependent‘indirect pathway’ through GPe and STN.9 The current therapiesavailable provide symptomatic relief and there is no effectivedisease-modifying therapy. Levodopa (l-dopa), the metabolic pre-cursor of dopamine, has been used primarily to manage early mo-tor symptoms with limitation of development of dyskinesia upon

NH2

NH2

O(7)

SN

R1

R1 Cl

O

R1

HO

CN

CN

CN

NH2SN

R1

(2) (3)

(6)

a

f

N

N

O

SN

R1

(

R

R1: cyclohexyl, aryl

R3: OMe, OEt

R2: -NO2, -CN

CHOR3

R2(9a-c)

O

NH

OOHN

,

R2: -NH2(10, 36-43)

R2: -CONHMe (26)

X: -N(Ac)- (21-22), -N(SO2Me)

g

R2: -NH(CH2)2NH2 (11),

-NHCONHMe (32)

(29(30)

(27)

h

j

k

Scheme 1. Reagents and conditions: (a) malonitrile, NaH, THF, 0–5 �C, 2 h; (b) POCl3, refluconc. H2SO4, 80 �C, 0.5 h; (g) DMA, cat. I2, 130 �C, 1 h; (h) Fe, NH4Cl, MeOH, rt, 2 h; (j) ForCH2Cl2, rt, 1 h; For 32:(i) PhOCOCl, Et3N,THF, rt, 1 h; (ii) MeNH2–HCl, DMSO, 50 �C, 5 h;trimethylsulphonium iodide, NaH, DMSO, rt, 18 h; (ii) LiOH, THF–MeOH–H2O, rt, 2 h; (iii1 h; (v) MeNH2–HCl, PyBOP, DIPEA, DMF, rt, 12 h; (m) HCl in EtOAc, rt, 4 h; (n) LAH, TH

prolonged treatment. Moreover, it does not provide any benefitover the non-motor symptoms of PD such as dementia, depressionand others.10 Therefore, targeting novel pathways addressing bothmotor and non-motor symptoms of PD is in great demand.

Recently, in the lipopolysaccharide rat model of PD, it wasshown that PDE7 inhibition can protect dopaminergic neuronsagainst different insults, and thus provide support for the thera-peutic potential of PDE7 inhibitors in the treatment of neurodegen-erative disorders, particularly PD.11,12 Subsequently, dopaminereceptor intracellular signaling pathway has been proposed to bedown regulated by PDE7 and it has been shown that PDE7 inhibitoralone or in combination with l-dopa increased neuronal activationand restored paw stride length in MPTP treated mice model.13 Con-sidering this background, our initial goal was to identify potent,selective, CNS penetrant PDE7 inhibitors which could be evaluated

R1

H2N CN

SNH2

(4)

(5)

R1

H2N

CN

CNb, c

d

e

H

V)

3R2

, F

,

O

O NN THP

O

NBoc

m: 1-2; n: 1-3;

X: -N(Boc)-, -CH(NHBoc)-,

-CH(OH)- (16-18),

-N(Me)- (24), -CF2- (25),

-CH(CO2Et)-, -CONH- (31)

N Xm

n

m: 1-2; n: 1-3;

X: -NH- (12, 13), -CH(NH2)- (14-15, 34-35),

-CH(CONHMe)- (28), -CH(CH2OH)- (19)

N Xm

n

, ,

NNH

O

NH, ,

HN

- (23)

,

)(33) (20)

k

k for 28;m for 12-15, 34-35n for 19

o

l

x, 12–14 h; (c) aq. NH3, rt, 2 h; (d) H2S, Et3N, benzene; (e) H2O2, MeOH, 50 �C, 5 h; (f)11: (i) tert-butyl (2-oxoethyl)carbamate, NaBH(OAc)3, AcOH, DCE, rt, 12 h; (ii) TFA,

(k) (i) NaOH, MeOH, 50 �C, 2 h; (ii) MeNH2-HCl, PyBOP, DIPEA, DMF, rt, 12 h; (l) (i)) (R)-phenylglycinol, EDCl, HOBt, DIPEA, DMF, rt, 12 h; (iv) 6 N HCl, Dioxane, 100 �C,F, rt, 5 h; (o) AcCl (for 21–22) or MeSO2Cl (for 23), Et3N, CH2Cl2, rt, 1 h.

(8a)

CHOR3

F

R3: OMe, OEt, F

CHOR3

N

X n

n: 1-3; m: 1-2;

X: -N(Boc)-, -CH(OH)-, -CH(NHBoc)-,

-N(Me)-, -CF2-, -CH(CO2Et)-, -CONH-

(9a)

(8b)

OH

I

CHOO

R2

R2: -CN,

(9b)

a

O

ON

N THP

b and

(8c)

CHOOH

OH

e, f

(9c)

CHOOEt

O

NBoc

m

c or d

Scheme 2. Reagents and conditions: (a) amine, K2CO3, DMSO, 80–120 �C; (b) (i)(CH2O)n, MgCl2, Et3N, CH3CN, 80 �C; (ii) Mel, K2CO3, DMF; (c) CuCN, DMF, 90 �C; (d)methyl acrylate/boronate ester, Pd(PPh3)4, K2CO3, DMF, 120 �C; (e) (3R)-3-hydroxy-pyrrolidine-1-carboxylate, PPh3, DIAD, THF, rt; (f) Etl, K2CO3, DMF.

A. Banerjee et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3223–3228 3225

in animal models to increase the understanding of the target in thePD therapy.

Among the various heterocyclic structures that have been ex-plored for the discovery of novel and selective PDE7 inhibitors,1,4

we were particularly interested in the pyrimidone fused heterocy-clic scaffolds I–IV disclosed by Asubio Pharma (Fig. 1). The repre-sentative compound 1 was highly potent, more than 1000-foldselective against other PDE isoforms and had reasonable CNS pen-etration in mouse.14 Inspired by this novel structure, we decided toexplore isothiazole and isoxazole fused pyrimidones (V), to iden-tify various pyrimidone fused heterocyclic scaffolds as PDE7 inhib-itors similar to an approach described for PDE5 inhibitors.15 Also,we were keen on examining a possible replacement of the cyclo-alkyl group (R1) represented in I–IV with an aromatic group. Thiscommunication describes our preliminary efforts towards the goalmentioned above.

NH2

NH2

O

ON

a, b

(44)

CHOO

N

NBoc (9a)

+

Scheme 3. Reagents and conditions: (a) DMA, c

The synthetic strategy utilized in the preparation of isothiazol-opyrimidones (V: X = S; R1 = cyclohexyl, aryl) is described inScheme 1. The condensation of the appropriate acid chloride withmalononitrile in the presence of NaH provided 3 which was con-verted to 4 via sequential treatment with POCl3 and ammoniumhydroxide. The treatment of 4 with hydrogen sulfide gas provided5 which upon cyclization in presence of H2O2 provided 6.16 Thehydrolysis of 6 provided the intermediate 7 which was then con-densed with various aldehydes (9a–c) in presence of catalyticamount of iodine to provide V.

The synthesis of the aldehydes (9a–c) is briefly described inScheme 2. The nucleophilic aromatic substitution of 2-alkoxy-4-fluorobenzaldehyde (8a) with a variety of cyclic amines whichwere commercially available or prepared following the knownmethods, provided aldehyde 9a. The aldehyde 9b was preparedfrom 3-iodophenol (8b) following a three steps protocol: Casiraghiformylation,17 methylation and Heck or, Suzuki or copper cata-lyzed cyanation. The aldehyde 9c was synthesized from 2,4-dihy-droxybenzaldehyde (8c) following Mitsunobu and alkylationconditions subsequently.

As depicted in Scheme 1 the isothiazole analogs 16–18, 24, 25,and 32 were obtained directly by condensation of the intermediate7 with the appropriate aldehydes (9a). The synthesis of the com-pounds 10,18 36–43 were accomplished upon reduction of the nitrogroup in V using iron and NH4Cl in MeOH. The reductive aminationof 10 with tert-butyl (2-oxoethyl)carbamate followed by treatmentwith TFA provided 11. The synthesis of the compound 32 was per-formed following a two step protocol: treatment of 10 with phe-nylchloroformate and then with methylamine hydrochloride inDMSO at 50 �C. The synthesis of the compound 26 was performedby conversion of the nitrile in V to the corresponding acid and thenamidation with methylamine hydrochloride. Following the similartwo step protocol of hydrolysis and amidation of the esters in V,compounds 27 and 28 were obtained. The Corey–Chaykovskycyclopropanation of the acrylic ester in V and subsequent hydroly-sis provided the trans racemic cyclopropyl acids. The resolution ofthe racemic acids following the method described by Kozikowski19

and subsequent amide formation with methylamine hydrochlorideprovided the compounds 29 and 30. The removal of the protectinggroups (Boc or THP) in V (R2: heterocycle/O-linked heterocycle)with a saturated solution of HCl in ethyl acetate provided the com-pounds 1218–15, 34, and 35. The reduction of the ester in V (whereX: CH(CO2Et)) with lithium aluminium hydride provided the com-pound 19. The acylation of compounds 12 and 13 provided com-pounds 2118 and 22, respectively. Similarly, compound 23 wassynthesized from compound 12 under sulfonylation conditions,using methanesulfonyl chloride.

The synthesis of the isoxazolopyrimidone (45) was completedby condensation of the intermediate 4420 with aldehyde 9a fol-lowed by removal of the Boc group with HCl in ethyl acetate(Scheme 3).

The target compounds were tested for their inhibitory activityat the cloned human recombinant PDE7A, PDE7B and other PDE

NH

N

O

ON

O N

NH

(45)

PDE7A IC50: 31±8.05 nM

at, I2, 130 �C, 1 h; (b) HCl in EtOAc, rt, 1 h.

Table 1SAR of the R2 position of Isothiazolopyrimidone

NH

N

O

SN

R3 R2

Example R2 PDE7A IC50 (nM) ±SDc

10a –NH2 14 ± 4.5

11aHN NH2

46 ± 10.53

12a

NNH

9 ± 2.73

13a

N

NH4 ± 0.52

14a

NNH2 23 ± 4.12

15a

N

NH28 ± 1.79

16a

N

OH18 ± 3.97

17a

N

OH11 ± 4.51

18a

N

OH7 ± 1.12

19a

NOH

16 ± 5.76

20b NHO

35 ± 7.77

21a

NN

O

16 ± 4.59

22a

N

N

O

12 ± 2.10

23a

NN

SO

O66 ± 18.77

24b

NN

88 ± 11.96

25a

N

FF 106 ± 22.15

26aNH

O125 ± 11.99

27aNH

O47 ± 7.54

Table 1 (continued)

Example R2 PDE7A IC50 (nM) ±SDc

28a

NNH

O

18 ± 3.09

29a

HNO

35 ± 8.00

30a

HNO

38 ± 8.07

31a

NNH

O

15 ± 4.37

32a

HN

O

HN

17 ± 4.95

33aN

HN

25 ± 2.88

a R3 = OMe.b R3 = OEt.c IC50 values are means of three experiments.

Table 2SAR of the R3 position of Isothiazolopyrimidone

Example Structure PDE7A IC50 (nM)±SDa

15NH

N

O

SN

O N

NH2

8 ± 1.79

34NH

N

O

SN

O N

NH2

12 ± 4.76

35NH

N

O

SN

F N

NH2

101 ± 37.01

a IC50 values are means of three experiments.

3226 A. Banerjee et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3223–3228

Table 3SAR of the R1 position of Isothiazolopyrimidone

NH

N

O

SN

R1

O NH2

Example R1 PDE7A avg.%inhibition at 1 lMa

PDE7A IC50 (nM) ±SDb

36 70% ND

37F

95% 39 ± 6.36

38

F

75% ND

39

F

35% ND

40Cl

90% 80 ± 8.67

41F F

95% 30 ± 6.41

42

F

F

95% 46 ± 5.25

43F Cl

93% 14 ± 1.47

ND: not determined.a % Inhibition values are means of two experiments.b IC50 values are means of three experiments.

A. Banerjee et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3223–3228 3227

isozymes following a two step radiometric assay using 3H-cAMP asthe radioligand.21

A direct comparison between 1 and 10 indicated that thereplacement of the pyrazole moiety with isothaizole has an impacton the PDE7A activity as the later compound was nearly 10-fold

Table 4PDE Selectivity profile of 10, 13 and 21

Compound IC50 (nM)

PDE7A ±SDa PDE7Bb PDE 1A PDE 2A PDE 3A

10 14 ± 4.50 31 17 28 313 4 ± 0.52 17 38 16 421 16 ± 4.59 8 30 9 6

a IC50 values are means of three experiments.b IC50 values are means of two experiments.c % Inhibition values are means of two experiments.

less active. Thus, we examined the structure–activity relationshipat the R2 position of isothiazolopyrimidone (V, R1: cyclohexyl;R3: OMe, OEt) with the hope of improving the potency and the re-sults are summarized in Table 1. To determine the available spacefor expansion in the R2 region we decided to extend the H-bonddonor with a linker. The 1,2-diamino substitution (11) resulted athreefold loss of activity whereas the piperazine (12) and 3-amino-pyrrolidine substitutions (14) improved the PDE7A inhibitoryactivity. A further improvement in potency was observed withthe increase in corresponding ring sizes (13 and 15). Similar trendwas also observed with compounds 16, 17 and 18 where alcoholwas used as H-bond donor. The placement of the hydrogen bonddonor was also an important aspect as the potency was somewhatreduced in compounds 14, 19 and 20. Further investigation re-vealed that H-bond acceptor is also capable of retaining the po-tency (21 and 22). However, loss of activity was observed withmethylsulfonylpiperazine (23), methylpiperazine (24), and 4,4-difluorpiperidine (25) substitutions.

Next, we incorporated the amide group (26) which was ninefoldless active than the corresponding amine (10). However, as antici-pated the incorporation of a linker between the amide functionalgroup and the aromatic ring, improved the activity. The cinnamide(27) and the piperidine carboxamide derivatives (29) were threeand sevenfold more potent than 26, respectively. The appropriatelength of the linker and positioning of the functional group provedto be critical as the potency decreased in 29 and 30 compare to 28.Interestingly, the cyclic amide 31 which was designed to possessboth H-bond donor and acceptor further improved the activity.The urea derivative (32) and pyrazole derivative (33) also retainedthe activity.

Then the role of the R3 substituent in V was briefly investigatedand proved that the alkoxy substituent, capable of a strong hydro-gen bonding interaction with the pyridone NH, is essential sincethere was a significant loss in activity when R3 was substitutedwith fluoro (35) (Table 2). However, there was no significant differ-ence in potency between the methoxy (15) and the ethoxy substi-tuted compounds (34).

Then we turned our attention to develop a SAR at the R1 posi-tion by replacing the cyclohexyl moiety with the aryl groups. Thedata in Table 3 indicated that mono and di-orthosubstitution (37,40, 41, 43) increased potency against PDE7. In this SAR, the 2-chloro-6-fluoro substituted compound (43) was similar in potencyto compound (10) (Scheme 3).

Finally, we also compared the PDE7A inhibitory activity of iso-thiazole and isoxazole fused pyrimidones. The isoxazole compound(45) with the substitution pattern that provided the most potentisothiazole compound (13) was eightfold less potent (PDE7A IC50:31 nM).

The PDE selectivity profile of representative compounds 10, 13and 21 presented in Table 4 suggests that the compounds werenon-selective within PDE7 isoforms. However, they were selectiveagainst other PDEs.

% Inhibition at 10 lMc

PDE 4D PDE 5A PDE 8A1 PDE 9A2 PDE 10A PDE 11A

45 11 7 12 6 2867 3 16 8 3 3521 8 1 13 19 6

Table 5Pharmacokinetic profile of 10 and 21

Compound Metabolic stability(% remaining)a Rat PKb

1 mpk ivc,d 10 mpk orale

HLM RLM MLM Cbrain (ng/g) Cplasma (ng/mL) Ratio Cmax (ng/mL) AUC (ng h/mL) Tmax (h)

10 47 58 24 951 281 3.39 36 75 121 68 67 3 792 390 2.03 739 2877 2

a Percentage of test compound remaining after 60 min incubation with liver microsomes at 37 �C.b Male SD rats were used for the experiments.c iv formulation: 20% NMP, 20% EtOH, 10% Cremophor EL, 50% PEG 200/H2O (3:2, premix).d Brain and plasma samples were collected 5 min post iv dose.e Oral formulation: 2.5 lL/mL Tween 80 + 0.5% (w/v) methyl cellulose suspension (q.s.).

3228 A. Banerjee et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3223–3228

In the present set of active PDE7A inhibitors we further evalu-ated the pharmacokinetic profile of 10, and 21. The compoundswere metabolically stable in human and rat liver microsomes(Table 5) and more importantly, they had adequate CNS penetra-tion in male SD rats upon intravenous administration. Moreover,compound 21 exhibited an acceptable oral pharmacokinetic profilein rat (Table 5). Compounds 12, 13, 15 with basic nitrogen(pKa > 8), did not have adequate CNS penetration and were alsonon-selective in the panel of receptors (data not shown), thus nofurther evaluation was performed.

In conclusion, we have reported the synthesis and SAR evalua-tion of isothiazole and isoxazole fused pyrimidones as PDE7 inhib-itors. In the isothiazole series various groups with H-bond donorand/or acceptor at the R2 position of V provided potent PDE7 inhib-itors. An exploratory effort also demonstrated that the cyclohexylgroup at R1 position of V could be replaced with 2,6-disubstitutedaromatics. Furthermore, we identified two compounds (10 and 21)which had good CNS penetration and could be further profiled inanimal models to evaluate the potential of PDE7 as a target for PD.

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bmcl.2012.03.025.

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