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European Journal of Medicinal Chemistry 56 (2012) 210e216

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European Journal of Medicinal Chemistry

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

Original article

Synthesis and biological evaluation of selective and potent cyclin-dependentkinase inhibitors

Joannah N’gompaza-Diarra a,b, Karima Bettayeb c, Nohad Gresh b, Laurent Meijer c,**, Nassima Oumata a,*

aManRos Therapeutics, Centre de Perharidy, Hôtel de recherche, 29680 Roscoff, Franceb Laboratoire de Chimie Organique 2, UMR CNRS 8601, Université Paris Descartes, Sorbonne Paris Cité, Faculté de pharmacie, Paris, FrancecC.N.R.S., ‘Protein Phosphorylation & Human Disease’ Group, Station Biologique, B.P. 74, 29682 Roscoff, France

a r t i c l e i n f o

Article history:Received 9 June 2012Received in revised form21 August 2012Accepted 22 August 2012Available online 1 September 2012

Keywords:KinaseDYRK1ACancerProdrugPurineRoscovitine

* Corresponding author. Tel.: þ33 (0)2 98 72 94 94** Corresponding author. Tel.: þ33 (0)6 08 60 58 34

E-mail addresses: meijer@sb-roscoff.fr (L.therapeutics.com (N. Oumata).

0223-5234/$ e see front matter � 2012 Elsevier Mashttp://dx.doi.org/10.1016/j.ejmech.2012.08.033

a b s t r a c t

A new series of 2,6,9-trisubstituted purines, structurally related to the cyclin-dependent kinase (CDK)inhibitor Roscovitine, has been synthesized. These compounds mainly differ by the substituent on theC-2 position which encompasses a diol group. These compounds were screened for kinase inhibitoryactivities and antiproliferative effects. They were shown to be potent inhibitors of cyclin-dependentkinases but also, for some of them of casein kinase 1 (CK1) and dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A). The inhibition of kinases was accompanied by anantiproliferative effect against several tumor cell-lines. The most potent derivatives inhibited SH-SY5Y(neuroblastoma) tumor cell line with an IC50 < 0.5 mM which means approximately a 30 fold increasecompared to Roscovitine. A valine ester was also prepared from the most potent inhibitor to serve asa prodrug.

� 2012 Elsevier Masson SAS. All rights reserved.

1. Introduction

Cyclin-dependent kinases (CDKs) play an important role in cellcycle regulation [1] but are also implicated in the regulation ofapoptosis [2], transcription [3] and neural functions [4]. CDKs arederegulated in numerous proliferative diseases such as cancer [5],viral infections [6] and kidney disease [7] but also in non prolifer-ative disease, such as Alzheimer disease in the case of CDK5 [8].

These implications have stimulated the search for pharmaco-logical inhibitors [9]. Roscovitine [10] and Flavopiridol [11] (Fig. 1)were the first two to enter clinical trials in the late nineties but up tonow, no product has reached the market. However the interest forRoscovitine and Flavopiridol remains high as these two productshave now reached phase II/III clinical tests [12]. On the other handmany products have been terminated due to several toxicities [13].Most of the ongoing clinical tests are targeting leukemia [14].

Inspired by the continuous interest for Roscovitine we haveconducted several optimization studies whichweremostly devotedto the selection of the substituent at the position 6 of the purine

.

.Meijer), oumata@manros-

son SAS. All rights reserved.

scaffold [15]. This led us to the identification of CR8 a potent andselective kinase inhibitor [16].

We now focus on the optimization of groups on the C-2 positionof the purine scaffold. In particular, with the aim to develop lesshydrophobic compounds which could be more soluble and there-fore display an improved bioavailability.

2. Results and discussion

2.1. Chemistry

Two different approaches were adopted to prepare those of theaminodiols, which were not commercially available (Scheme 1). Inthe first route reaction of cyclohexenylbromide in THF at 60 �C inthe presence of K2CO3 led to the formation of the tertiary amine.The alkene 1 was then subjected to asymmetric dihydroxylationwith AD-mix b� as described by Sharpless [17] to obtain the diol 2.In a third step, benzyl groups were removed using catalytichydrogenation conditions. This approach was exemplified in thesynthesis of 3-aminocyclohexanediol which was isolated as itscorresponding hydrochloride 3. The second route starts from thecommercial amino acids which were first converted to their ethylesters using thionyl chloride in EtOH (4aeb) and the ester groupwas reduced into alcohol using AlLiH4 and afforded compounds

FlavopiridolAlvocidib

(R)-Roscovitine Seliciclib

Fig. 1. Representation of (R)-Roscovitine and Flavopiridol the first two CDK inhibitorswhich reached clinical tests.

J. N’gompaza-Diarra et al. / European Journal of Medicinal Chemistry 56 (2012) 210e216 211

(5aeb). Both methods proved to be reproducible in various scales,providing the desired compounds in good yields.

The substituted purines were then obtained through a classicalthree-step synthetic route outlined in Scheme 2. Starting fromcommercially available 2,6-dichloropurine, alkylation of N-9 with2-bromopropane provided 2,6-dichloro-9-iso-propylpurine 6.Displacement of the 6-chloride by nucleophilic substitution with4-(2-pyridyl)benzylamine led to compound 7 according to a previ-ously described procedure [18]. In the final step, reaction of thechloropurine with diols led to compounds 8aed in moderate yield:12e53% (Table 1).

Fig. 2. Molecular docking of compound 10 with CDK9. The construction of the compl

The selective esterification of one of the alcohols of compound8a has been investigated with Bocevaline. A mono ester 9 wasformed under classical acylation conditions (DCC, HOBt). Finally,the Boc protecting group was eliminated using an ethereal hydro-chloric acid solution and provided compound 10 (Scheme 3).

2.2. Biological evaluation

The prepared compounds were first evaluated against a subsetof CDKs but also against GSK3-a/b (Glycogen synthase kinase 3),DYRK1A (dual-specificity tyrosine-(Y)-phosphorylation regulatedkinase 1A) and CK1 (casein kinase 1). Compounds were tested fortheir antiproliferative effects. Human neuroblastoma SH-SY5Y cellswere exposed for 48 h to various concentrations of each compound.Viability was then estimated by the MTS reduction assay.

The results of the kinase inhibition assays and cell anti-proliferative effects are gathered in Table 1. The products can beconsidered as rather selective CDK inhibitors. Indeed, the productsdo not inhibit GSK3 a/b which is frequently inhibited by most ofCDK inhibitors such as AT5147, a CDK inhibitor which was intro-duced in clinical phase 1 and showed major toxic effects [19].Inhibition of GSK3 is generally considered potentially carcinogenicin relation with the inhibition of the Wnt pathway [20].

The products exhibited roughly the same selectivity as Rosco-vitine. In particular, some of the prepared compounds appeared tobe also inhibitors of DYRK1A. The role of DYRK1A in cancer is far to

ex was based on the crystal structure determination of the CR8eCDK9 complex.

d

Cl

a

_

b

a

5a-b4a-b

b

e

Route A

c

21 3

Scheme 1. Preparation of amino-diols. Reagents and reaction conditions route A. (a)Dibenzylamine, K2CO3, THF, 60 �C, 6 h; (b) Ad-mix b, MeSONH2, NaHCO3, H2O, tert-BuOH, r.t, 72 h; (c) 1-HCl/Et2O, 2-H2/Pd/C. Reagents and conditions route B: (d) SOCl2,EtOH, 60 �C. (e) LiAlH4, THF, H2O, 0 �C- 20 �C.

Table 1Kinase inhibition by compounds 8 and 10 against CDKs (Cyclin-dependent kinases):CDK1/cyclin B, CDK2/cyclin A, CDK7/cyclin H, CDK9/cyclin T, CDK5/p25, CK1, CLK1,DYRK1A and against the tumor cell line SH-SY5Y. The IC50 (mM) are themean of threeexperiments. (R)-Roscovitine (Ros) was used as a positive control.

Cpd CDK1 CDK2 CDK5 CDK9 GSK3 CK1 CLK1 DYRK1A SHSY-5Y

Ros 0.33 0.21 0.28 0.23 60 4 4.3 3 178a 0.60 0.48 0.19 0.15 7.1 0.18 4.0 0.71 0.528b 0.87 0.61 0.27 0.37 >10 0.60 e 0.89 0.918c 0.45 0.65 0.61 0.45 >10 0.39 e 1.6 0.488d 0.37 0.98 0.82 1.01 >10 0.66 e 0.79 1.410 1.4 0.87 0.18 0.18 >10 0.15 12 1.1 1.1

J. N’gompaza-Diarra et al. / European Journal of Medicinal Chemistry 56 (2012) 210e216212

be well understood however some leukemia have been associatedwith overexpression of DYRK1A [21]. In contrast to Roscovitine,they inhibited CK1, a property that probably efficiently contributeto their high antiproliferative activity.

Several valyl esters have been previously described to improvebioavailability of the parent compound. The most known is Vala-cyclovir (Valtrex�), the L-valyl ester prodrug of acyclovir (ACV),which is widely used to treat viral infections mainly caused byherpes simplex virus [22].

In the particular case of ester 10, it was observed that 10 dis-played antiproliferative activity against cancer cells. This is notreally surprising as the ester may undergo a slow hydrolysis in theculture media. The ester was also found to inhibit kinases. Thisresult was not anticipated as, up to now, rather small groups havebeen introduced in position 2 of the purine.

2.3. Docking analysis

A docking analysis was performed with the ester 10 for a betterunderstanding of the unexpected activity of 10 against kinases.

8a 8b

R1

R2

6

a b

Scheme 2. Synthesis of trisubstituted purines. Reagents and conditions: (a) 2-bromopropR1R2NH, neat or R1R2NH, NEt3,160e170 �C.

The complexes were constructed using the previously reportedcrystal structure of CR8eCDK9 complex [16]. The binding of ligand10 is stabilized by (Fig. 1):

- two hydrogen bonds with the Cys106 backbone. As is the casewith CR8, the unsubstituted N of the pentacyclic ring acceptsa proton from the Cys NH group, while the extracyclic Ndonates its proton to the Cys carbonyl group;

- a hydrogen bond between the pyridine nitrogen and the Lys35side-chain;

- an ionic bond between the protonated ammonium group of theligand and the side-chain of Asp 109. There are in addition vander Waals interactions between:

- the bicyclic ring and Phe 105, Ile 25, and Leu 156, while itssubstituting isopropyl group interacts with Phe 103;

- the phenyl group and both Phe 105 and Ile 25.

The substituent in position 2 of the kinase scaffold is partlyoutside the ATP pocket. Such a result could open the way to thedesign of new inhibitors with rather diverse substitutions in position2. Finally to further evaluate the potential interest of 8a, which is themost potent compound of the series, several preliminary ADMEparameters were determined. Inhibition against a series of CYP 450enzymes was found higher than 5 mM and plasma binding proteinwas also favorable with a moderate binding to serum proteins(Table 2). Evaluation of the ester 10 requires in vivo experiment fora better understanding of the dual effect of this compound (Fig. 2).

3. Conclusion

The attractive inhibitory profile against kinase of derivative 8awith in particular a strong inhibition of CK1 led us to select 8a and

8d 8c

R1

R2

7 8

c

ane, K2CO3, DMSO, 10e15 �C, 55%; (b) ArCH2NH3þ. TFA�, NEt3, BuOH, 100 �C, 2 h; (c)

H3N

Cl

a

_

b

8a 9 10

Scheme 3. Synthesis of valine mono ester from 8a. Reagents and conditions: (a) BocevaleOH, DCC/HOBt, THF/AcOEt, rt, 72 h, (b) HCl/Et2O.

J. N’gompaza-Diarra et al. / European Journal of Medicinal Chemistry 56 (2012) 210e216 213

its ester 10 for further evaluations. This choice is also supported thehigh potency of this product compared to Roscovitine, approxi-mately 30 fold at inhibiting cell proliferation. Moreover thepreliminary ADME assays in particular high solubility andmoderateserum protein binding were found very favorable.

4. Experimental

4.1. General methods

Melting points were determined on a Kofler hot-stage (Reichert)and are uncorrected. NMR spectra were recorded on Bruker Avance400 MHz. Chemical shifts are given in ppm downfield of tetrame-thylsilane (TMS) used as an internal standard. Reactions weremonitored by TLC using SDS silica gel 60F-254, 60 A-15 mm thinlayer plates. Column chromatography was carried out on SDSChromagel 60 ACC, 40e63 mm. Compounds 8e10 gave satisfactorymicroanalyses �0.4 calculated values.

4.2. Preparation and dihydroxylation of dibenzylaminocyclohex-2-ene

4.2.1. N,N-Dibenzylaminocyclohex-2-ene (1)3-Bromocyclohexenyl (10 mL, 86 mmol) in 20 mL THF was

added to a mixture of dibenzylamine (40 mL, 210 mmol) and thenK2CO3 (14.26 g, 103 mmol) in 120 mL THF. The mixture was stirredand heated at reflux. The reaction was followed by TLC. THF wasconcentrated in vacuo. The residue was extracted by 20mL H2O andAcOEt (3 � 20 mL). The organic layer was washed with H2O, driedover anhydrous Na2SO4, filtered and concentrated in vacuo to affordviscous yellow oil which was purified by silica gel column chro-matography, Cyclohexane/AcOEt (95:5), Rf ¼ 0.7,m¼ 21.86 g, yield:92%. 1H NMR (400 MHz, CDCl3) d : 1.4e1.6 (m, 3H, CH2eCH2), 1.75(m, 1H, CHeCHeN), 1.8e2 (m, 2H, CH2eCH]CH), 3.3 (m, 1H, CH2e

CHeN), 3.5 (d, J ¼ 14.05 Hz, 2H, CH2eAr), 3.7 (d, J ¼ 14.05 Hz, 2H,CH2eAr), 5.6e5.8 (m, 2H, CH]CH), 7.1e7.4 (m,10H, HeAr). 13C NMRd : 21.87 (CH2), 23.19 (CH2), 25.38 (CH2), 53.84 (CH2), 54.51 (CH),126.61 (CH), 127.88 (C), 128.12 (CH), 128.23 (CH), 128.34 (C), 128.49(CH), 128.75 (C), 130.06 (CH), 130.87 (CH), 140.89 (CH).

4.2.2. (1S-2R-3R)-3-(Dibenzylamino)cyclohexane-1,2-diol (2)AD-mix b� (73.6 g, 52 mmol) was added to a solution of NaHCO3

(10.16 g, 120 mmol), Methanesulfonamide (3.8 g, 95.11 mmol) in50 mL H2O: tert-butanol (1:1). The mediumwas stirred at 20 �C for

Table 2Preliminary ADME study. CYP 450 inhibitions. IC50 are expressed in mM. Serumprotein binding (rat and human) in %.

CYP450 inhibition Serum protein binding

1A2 2C19 2C9 2D6 3A4 Rat Human

>5 >5 >5 >5 >5 97.4 94.6

15 min and then cooled to 0 �C. The alkene 1, (10 g, 36 mmol) wasadded and the reaction mixture was stirred for 3 days at 20 �C. Thereaction was quenched with Na2SO3 and the suspension wasallowed to stir for 1e2 h. The suspension was diluted H2O andAcOEt The organic layer was extracted, washed with water andbrine three times, dried over anhydrous Na2SO4, filtered andevaporated in vacuum to afford crude product which was purifiedby silica gel column chromatography. Cyclohexane/AcOEt (6/4),Rf ¼ 0.33,m ¼ 7 g, yield: 62%. 1H NMR (400 MHz, CDCl3) d : 1.15 (m,1H, CH2), 1.29 (m, 1H, CH2), 1.8 (m, 2H, CH2), 2.10 (d, J ¼ 1.85 Hz, 1H,CHeNeBn2), 2.79 (m, 1H, CH2eCHOH), 3.32 (d, J ¼ 13.24 Hz, 2H,CH2eAr), 3.44 (dd, J ¼ 3.12 Hz, J ¼ 10.64 Hz, 1H, CH2eCHOH), 3.6 (s,1H, CHOH), 3.75 (d, J ¼ 13.28 Hz, 2H, CH2eAr), 4.07 (s, 1H, CHOH),7.1e7.3 (m, 10H, HeAr). 13C NMR d : 19.48 (CH2), 22.03 (CH2), 29.51(CH2), 53.64 (CH2), 57.45 (CH), 68.28 (CH), 70.85 (CH), 127.26 (CH),127.46 (C), 128.52 (C), 128.98 (CH), 139.29 (C).

4.2.3. (1S-2R-3R)-3-Aminocyclohexane-1,2-diol hydrochloride (3)The dibenzylamine derivative (7 g, 21.8 mmol) was mixed with

Et2O and a solution of HCl in Et2O was added to the medium,a white solid precipitated. The solid was filtered, dried in vacuo,then diluted in methanol and submitted to hydrogenation (H2/Pd/C). The reaction mixture was stirred for 48 h. The solution wasfiltered over celite, washed with MeOH and evaporated to givea viscous oil. Yield: 93%. 1H NMR (400 MHz, CDCl3) d : 1.1e1.8 (m,6H, CH2CH2CH2), 2.9 (m, 1H, CHOH), 3 (m, 1H, CHeNH2), 3.3 (m, 1H,CHOH), 5.5 (s, 2H, NH2).

4.3. Preparation of aminodiols from amino-acids

4.3.1. Esterification of amino-acidsThionyl chloride (7.71 mL, 76.2 mmol) was added droplet in cold

(�10 �C) ethanol (100mL), the solutionwas left at�10 �C for 15min.The acid (38.1mmol) was added to themedium and themixturewasrefluxed at 60 �C for 4 h. The reactionwas followedwith TLC and theplates revealed with ninhydrin. The medium was concentrated andthe white crystals were dissolved in DCM, washed with NH4OH aq.The aqueous layer was extracted with DCM. The organic layer wasdried over anhydrous Na2SO4, filtered and evaporated.

4.3.1.1. (2S-4R)-Ethyl-4-hydroxypyrrolidine-2-carboxylate (4a).This ester was obtained as viscous yellow oil, 5.27 g, yield: 87%following the above procedure starting with HeHypeOH. 1H NMR(400 MHz, CDCl3) d : 1.1 (t, 3H, CH2eCH3), 1.8 (m, 1H, CH2eCHeCO2Et), 2 (m, 1H, CH2eCHeCO2Et), 2.2 (m, 1H, CH2eNH), 2.9 (m,1H, CH2eNH), 3 (m, 1H, CH2eNH), 3.4 (m, 1H, CHeCOH), 4.6 (q, 2H,CH2eCH3).

4.3.1.2. (S)-Ethyl 4-amino-hydroxy-butanoate (4b). Starting with(S)-4-amino-2-hydroxy-butanoic acid (10 g, 0.083 mol) 4b, wasobtained as a colorless oil, m ¼ 6.98 g, 57%. 1H NMR (400 MHz,

J. N’gompaza-Diarra et al. / European Journal of Medicinal Chemistry 56 (2012) 210e216214

CDCl3) d : 1.25 (t, 3H, CH2eCH3), 2e2.3 (m, 2H, CH2eCHOH), 3.3 (m,2H, CH2eNH2), 4.2 (q, 2H, CH2eCH3), 4.5 (t, 1H, CHOH), 5.3 (s, 1H,NH2). 13C NMR (400 MHz, DMSO) d : 14.08 (CH3), 31.28(CH2),35.73(CH2), 60.30 (CH2), 67.34(CH), 173.21(C).

4.3.2. Reduction of estersAnhydrous THF (35 mL) was added dropwise to LiAlH4 (5.51 g,

145 mmol) at 0 �C. A solution of ester (33 mmol) in THF was addeddropwiseunder stirring at 0 �C. The reactionmixturewaswarmed tor.t thenheated to60 �C for 3h. Themediumwas cooled to�15 �C andH2Owas addeddropwise into themixtureuntil it becamewhite. Thereaction mixture was then stirred overnight at r.t. The solid wasfiltered and aluminawas washedwith CH2Cl2 and THF. The solutionwas concentrated to provide the diol derivative as viscous oil.

4.3.2.1. (3R,5S)-5-(Hydroxymethyl)-pyrrolidin-3-ol (5a). The reduc-tion of (2S-4R)-Ethyl-4-hydroxypyrrolidine-2-carboxylate (5.27 g,0.033mol) afforded 5a as yellow oil (1.3 g, 34%) following the aboveprocedure. 1H NMR (400 MHz, CDCl3) d : 1.6 (m, 1H, CH2e

CHCH2OH), 1.85 (m, 1H, CH2eCHCH2OH), 2 (s, 1H, NH), 2.55 (m,1H, CH2eNH), 2.85 (m, 1H, CH2eNH), 2.9 (m, 1H, CHeCH2OH), 3.3(m, 1H, CHeOH), 3.4e3.55 (m, 2H, CH2OH), 4.4 (s, 1H, OH).

4.3.2.2. (S)-4-Aminobutane-1,2-diol (5b). The reduction of (S)-Ethyl-4-amino-hydroxy-butanoate (6.9 g, 0.046mol) afforded 5b asviscous yellow oil (3.07 g, 63%) following the above procedure. 1HNMR (400MHz, DMSO) d : 1.3 (m, 1H, CH2eCH2eCHOH), 1.5 (m,1H,CH2eCH2eCHOH), 2.6 (m, 2H, CH2eNH2), 3.2 (m, 1H, CH2eCHOH),3.3 (m, 1H, CH2OH), 3.4 (m, 1H, CH2OH).

4.4. 2,6-Dichloro-9-iso-propylpurine (6)

This compound was prepared as described previously [15]. Amixtureof2,6-dichloropurine (10g, 53mmol) inDMSO(80mL)K2CO3(39.25 g, 280 mmol) was cooled to 15 �C. 2-Bromopropane (35 mL,60.05 mmol) was introduced droplet. The reactionwas monitored byTLC until completion. The solutionwas diluted with 100 mL H2O andextractedwithAcOEt (3� 25mL). The organic layerwaswashedwithH2O (3 � 10 mL) and dried over anhydrous Na2SO4, filtered andevaporated under vacuum to provide a yellow solid which was puri-fied by chromatography on silica gel column (toluene/AcOEt) (9/1) toyield awhite solid (6.8 g, 55%). 1HNMR (400MHz, CDCl3) d 1.67 (s, 6H,2(CH3)), 4.93 (s, 1H, CH(CH3)2), 8.17 (s, 1H, Hearyl).

4.5. Preparation of 2-chloro-9-iso-propyl-N-[[4-(2-pyridyl)phenyl]methyl]purin-6-amine (7)

To a solution of 6 (2.31 g, 10 mmol) in n-BuOH was added theprimary amine (12 mmol) and NEt3 (2.20 mL, 16 mmol). Afterheating at 100 �C for 2 h, n-BuOH was evaporated in vacuo. Afterdilution with H2O (10 mL), the mixture was extracted with AcOEt(3 � 20 mL). The combined organic extracts were dried (Na2SO4)and the solvent was removed in vacuo. The residue was chroma-tographied on a silica gel column using CH2Cl2/AcOEt, 2:1 to 1:1 aseluent. Yield 56%, mp: 176e179 �C. 1H NMR (CDCl3) d :1.58 (d, 6H,J ¼ 6.8 Hz, CH(CH3)2), 4.79 (hept, 1H, CH(CH3)2), 4.85 (bs, 2H,NHCH2), 6.59 (bs, 1H, NHCH2), 7.20e7.23 (m, 1H, Hpyridyl), 7.49 (d,2H, J ¼ 8 Hz, Hphenyl), 7.73e7.71 (m, 2H, Hpyridyl), 7.79 (s, 1H, H-8),7.98 (d, 2H, Hphenyl), 8.71 (d, 1H, J ¼ 4.8 Hz, Hpyridyl).

4.6. Nucleophilic substitution at C-2 position

A mixture of compound 7 (0.756 g, 2 mmol) and aminodiol(16 mmol) was heated neat or in presence of NEt3 at 170 �C untilcompletion of the reaction as indicated by TLC. The mixture was

cooled to r.t; water and CH2Cl2 were added into the medium. Theorganic layer was extracted, dried over anhydrous Na2SO4, filteredand evaporated to provide brown solid. Purification was achievedby flash chromatography or trituration with diethyl ether toprovide the desired product.

4.6.1. (2R)-3-[[9-iso-propyl-6-[[4-(2-pyridyl)phenyl]methylamino]purin-2-yl]amino]propane-1,2-diol (8a)

Yield 53%, 1H NMR, (CDCl3) d : 1.5 (d, J¼ 6.72 Hz, 6H, 2CH3), 3.4e3.6 (m, 4H, CH2NHCHCH2OH), 4.1 (m, 1H, CHOH), 4.6 (s, 1H, NCHN),4.8 (s, 2H, ArCH2NH), 5.2 (s, 1H, CHeiPr), 6.2 (s, 1H, NH), 7.2 (m, 1H,Hpyridyl), 7.4 (d, J¼ 8.28 Hz, 2H, Hphenyl), 7.5 (s, 1H, H-8), 7.75 (m, 2H,Hpyridyl), 7.95 (d, J ¼ 8.28 Hz, 2H, Hphenyl), 8.5 (d, J ¼ 4.12 Hz, 1H,Hpyridyl). NMR 13C (CDCl3) d : 22.64 (CH3), 44.78 (CH), 46.46 (CH),63.61 (CH2), 72.55 (CH), 114.75 (C), 120.54 (CH), 122.13 (CH), 127.18(CH), 127.98 (CH), 128.23 (C), 129.04 (CH), 134.64 (CH), 136.80 (CH),138.55 (C), 139.48 (C), 149.66 (CH), 154.88 (C), 157.12 (C).

4.6.2. (1S,2R,3R)-3-[[9-iso-propyl-6-[[4-(2-pyridyl)phenyl]methylamino]purin-2yl]amino]cyclohexane-1,2-diol (8b)

Compound3wasfirst converted into the free base upon extractionwith CH2Cl2 of amixture of 3 in a saturated solution of Na2CO3 in H2O.Yield 13%,mp:100e101 �C,1HNMR(DMSO) d : 1.5 (d, J¼ 6.722Hz, 6H,2CH3), 1.8 (m, 4H, CH2CH2CH2), 2 (m, 2H, CH2CH2CH2), 2.8 (m, 1H,CHNH), 3.45 (dd, J ¼ 3 Hz, J ¼ 9.32 Hz, 1H, CHOH), 3.7 (m, 1H,CH2NHAr), 4 (d, J¼ 2.74 Hz,1H, CHOH), 4.6 (m,1H, NHN), 4.75 (m,1H,CHeiPr), 5.7 (s, 1H, NH), 6 (s, 1H, NH), 7.1 (m, 1H, Hpyridyl), 7.4 (d,J¼ 8.36Hz, 2H, Hphenyl), 7.6 (s,1H, Hphenyl), 7.7 (m, 2H, Hpyridyl, Hphenyl),8 (d, J¼ 8.32Hz, 2H,Hpyridyl, H-8), 8.5 (d, J¼ 5Hz,1H,Hpyridyl).13CNMR(CDCl3) d : 18.57 (CH2), 19.05 (CH2), 21.29 (CH), 22.52 (CH3), 22.64(CH3), 23.55 (CH), 29.12(CH2), 31.48 (CH2), 46.67 (CH), 52.75 (CH),69.33 (CH), 72.71 (C), 79.4 (CH), 120.53 (CH), 122.13 (CH), 127.22 (CH),127.98 (CH), 134.90 (C), 136.78 (CH), 138.62 (C), 149.68 (CH).

4.6.3. (3R,5S)-5-(Hydroxymethyl)-1-[9-iso-propyl-6-[[4-(2-pyridyl)phenyl]methylamino]- purin-2-yl]pyrrolidin-3-ol (8c)

Yield 43%, mp: 210e212 �C, 1H NMR (DMSO) d : 1.1e1.2 (t,J ¼ 7 Hz, 4H, CH2CHCH2OH), 1.55 (d, J ¼ 6.76 Hz, 6H, 2CH3), 4.15 (m,1H, CHeCH2OH), 4.4 (m, 1H, CHOH), 4.45 (d, J ¼ 5.08 Hz, 2H, CH2e

Ar), 4.6 (m,1H, CH2OH), 4.7 (m,1H, CH2eOH), 4.8 (d, J¼ 4.08 Hz,1H,CHeiPr), 7.35 (m, 1H, Hpyridyl), 7.55 (d, J ¼ 8.24 Hz, 2H, Hphenyl), 7.8e8 (m, 3H, Hpyridyl, H-8), 8.1 (d, J ¼ 5.6 Hz, 2H, Hphenyl), 8.7 (m, 1H,Hpyridyl). 13C NMR (DMSO, CDCl3) d : 21.93(CH3), 37.21 (CH2), 40.10(CH), 45.86 (CH), 58.29(CH), 68.00 (CH), 119.99 (CH), 122.34 (CH),126.26 (CH), 127.82 (CH), 135.38 (C), 136.96 (C), 137.12 (CH), 149.42(CH), 155.93 (C), 157.54 (C).

4.6.4. (2S)-4-[[9-iso-propyl-6-[[4-(2-pyridyl)phenyl]methylamino]purin-2-yl]amino]butane-1,2-diol (8d)

Yield 12%, mp: 186e187 �C, 1H NMR (DMSO) d : 1.1e1.3 (m, 2H,CH2eCH2), 1.5 (d, J ¼ 6.92 Hz, 6H, 2CH3), 1.9 (m, 1H, CH2eNH), 2 (m,1H, CH2NH), 4.4 (s, 1H, CH2eCHeOH), 4.6 (spt, 2H, CH2eOH), 4.8 (s,2H, CH2eAr), 4.9 (m, CHeIsop), 7.45 (m, 1H, Hpyridyl), 7.55 (d,J ¼ 8.2 Hz, 1H, Hphenyl), 7.85 (s, 1H, Hepyr), 7.9 (m, 1H, H-8), 8 (m,2H, Hphenyl), 8.1 (d, J ¼ 8.28 Hz, 2H, Hphenyl), 8.7 (d, J ¼ 4.72 Hz, 1H,Hpyridyl).13C NMR (CDCl3) d : 21.93 (CH3), 33.53 (CH2), 40.10 (CH),44.44 (CH2), 45.66, 54.95 (CH2), 69.2, 120.00 (CH), 122.34 (CH),126.23 (CH), 127.9 (CH), 135.09 (CH), 136.92 (C), 137.14 (CH), 141.97(C), 149.42 (CH), 155.93 (C), 157.17 (C).

4.7. Preparation of the valyl ester from 8a

4.7.1. Esterification of 8a with Boc-L-valineA solution of BocevaleOH (0.434 g, 2 mmol), HOBt (0.229 g,

1.7 mmol) in 30 mL THFeAcOEt (2:1), the medium was cooled to

J. N’gompaza-Diarra et al. / European Journal of Medicinal Chemistry 56 (2012) 210e216 215

0 �C and DCC (0.364 g, 1.7 mmol) was added. The mixture was thenstirred to r.t for 2.5 h. The solid (dicyclohexylurea) was filtered offand the solution introduced into a round-necked flask containingwith the diol (0.257 g, 0.59 mmol) and triethylamine (0.2 mL) inTHF 20 mL. The solution was allowed to stir 24 h at r.t. THF wasevaporated, the residue dissolved in ethyl acetate, washed with20 mL citric acid 1 M, a saturated solution of Na2CO3 and finallywith 20 mL H2O. The organic layer was dried over anhydrousNa2SO4, filtered and evaporated under vacuum to provide yellowlight crystals which were purified by chromatography on silica gelcolumn, eluent: AcOEt/Toluene/EtOH (5/2/0.3e0.5) and affordedwhite crystals (190 mg, 51%).1H NMR (CDCl3) d : 0.8e1 (m, 6H,2CH3), 1.4 (s, 9H, 3CH3),1. 5 (s, 6H, 2CH3), 2.1 (m, 1H, NH2eCHeiPr),3.5e3.8 (m, 2H, CH2eNH), 4.05 (m, 1H, CHOH), 4.2 (m, 2H,CH2OCO�), 4.65 (m, 1H, CONCHCO�), 4.8 (s, 2H, CH2ephe), 5.05 (s,1H, CHeiPr), 7 (m, 1H, Hpyridyl), 7.5 (d, 2H, Hphenyl), 7.65 (s, 1H, H-8),7.75 (m, 2H, Hpyridyl), 7.95 (d, 2H, Hphenyl), 8.7 (m, 1H, Hpyridyl). 13CNMR (CDCl3) d : 17.65(CH3), 19.07 (CH3), 22.55 (CH3), 22.57 (CH3),22.75(CH3), 28.33, 31.22, 45.88 (CH2), 46.33 (CH), 46.58 (CH), 58.71(CH), 66.34 (CH2), 70.78 (CH), 79.85(CH), 120.47 (CH), 122.10 (CH),125.3 (C), 127.15 (C), 128.01 (C), 128.06 (C), 128.23 (CH), 129.04 (CH),134.82 (C), 136.75 (CH), 138.55 (CH), 139.49 (CH), 149.68 (CH),154.88 (C), 155.76 (C), 157.10 (C), 160.21(C).

4.7.2. [(2R)-2-hydroxy-3-[[9-iso-propyl-6-[[4-(2-pyridyl)phenyl]methylamino]purin-2yl]amino]propyl] (2S)-valyl esterhydrochloride (10)

1H NMR (CDCl3) d : (CH2CHCH2), 4.25 (m, 1H, CHOH), 4.75 (m,1H, NCHN), 4.8 (m, 2H, CH2eVal), 4.9 (m, 1H, CHeiPr), 7.45 (m, 1 H,H-8), 7.6 (d, J ¼ 4.68 Hz, 2H, Hpyridyl), 7.9 (m, 2H, H-8), 8.1 (d,J ¼ 3.6 Hz, 2H, Hphenyl), 8.25e8.4 (m, 2H, Hpyridyl, Hphenyl), 8.7 (d,J ¼ 4.8 Hz, 1H, Hpyridyl). 13C NMR (DMSO) d : 17.44(CH3), 18.03(CH3),21.65(CH3), 29.24 (CH), 57.42 (CH), 60.15 (CH2), 66.87 (CH), 67.63(CH2), 71.96 (CH2), 120.86 (CH), 122.93 (CH), 126.73(CH), 127.87(CH), 138.42 (CH), 148.60, 155.15 (C), 168.60 (C).

4.8. Docking analysis

Energy-minimization resorted to the 3 Å resolution X-raycrystal structure of CDK9 complexed with a sub-micromolarinhibitor (PDB access code RSCB 057590) [16]. It was done usingthe DISCOVER (Accelrys) software with the Cff91 force-field [23]and the BFGS minimizer with a dielectric constant of 1. As inour previous work using this software [24], the protein backbonewas frozen, while the side-chains of the residues of the recogni-tion site and the entirety of the ligand were relaxed. The structuralfeatures resulting from these computations are, as such, prelimi-nary and only aim to show a structural compatibility of this ligandwith the CDK9 binding site. Detailed energy balances bearing ona series of ligands related to it, also involving structural watermolecules, are planned in the context of the polarizable molecularmechanics procedure SIBFA [25]. This procedure has previouslybeen used to investigate the complexation of several relatedligands to protein targets such as Znemetalloeproteins [26] andthe FAK kinase [27].

4.9. Kinase inhibition assays

CDK1/cyclin B (native affinity purified from starfish oocytes),CDK2/cyclin A, CDK7/cyclin H, CDK9/cyclin T (human recombinant,expressed in baculovirus-infected insect cells), and CDK5/p25(human recombinant, expressed in E. coli) were assayed in thepresence of 15 mM ATP as previously described [16]. IC50 valueswere determined from dose-response curves and are expressed inmM. DYRK1A and CLK Kinase activities were assayed in duplicate

using Buffer D (10 mM MgCl2, 1 mM EGTA (ethyleneglycoltetra-acetic acid), 1 mM DTT (dithiothreitol), 25 mM Tris/HCl, 50 mg/mLheparin) at 30 �C, at a final ATP concentration of 15 mM. Blankvalues were subtracted and activities expressed as % of themaximalactivity in the absence of inhibitors. Controls were performed withappropriate dilutions of DMSO. IC50 values were calculated fromdose-response curves. DYRKs (human) and CLKs (mouse)(recombinant, expressed in E. coli) were extracted from bacteriaand purified by affinity chromatography on glutathione agarose.Kinase activity was assayed with either 1 mg/mL of RS peptide(GRSRSRSRSRSR, Proteogenix, Oberhausbergen, France), in thepresence of 15 mM [g-33P] ATP (3,000 Ci/mmol; 10 mCi/mL) ina final volume of 30 mL. After 30 min incubation at 30 �C, thereaction was stopped by harvesting onto P81 phosphocellulosepapers (Whatman) using a FilterMate harvester (Packard) andpapers were washed in 1% phosphoric acid. Scintillation fluid wasadded and the radioactivity measured in a Packard counter.

4.10. Cell culture

SH-SY5Y human neuroblastoma cell line was grown in DMEMsupplemented with 2 mM L-glutamine (Invitrogen, Cergy Pontoise,France) plus antibiotics and a 10% volume of FCS (Invitrogen). Cellviability was determined by means of the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium) method as previously described [28].

4.11. Cytochrome P450 inhibition (CYP 450)

The CYP inhibition was determined by a specialized companyfollowing a classical procedure. Briefly microsomal enzyme inhi-bition was determined by incubation of microsomes the corre-sponding substrate and the compound under study (5 mM).

4.12. Plasma binding protein

The product was added to a chamber containing mouse orhuman plasma separated from a buffer chamber by a dialysismembrane. The quantification of the concentration in buffer wasused to calculate the fraction bound to plasma proteins.

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