Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx

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Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl

Pim kinase inhibitory and antiproliferative activity of a novel series of meridianin C derivatives Kunal N. More a, Hyo Weon Jang b, Victor S. Hong a,⇑, Jinho Lee a,⇑ a b

Department of Chemistry, Keimyung University, Daegu 704-701, Republic of Korea Department of Chemistry, Sunchon National University, Sunchon, Chonnam 540-742, Republic of Korea

a r t i c l e

i n f o

Article history: Received 5 February 2014 Revised 27 March 2014 Accepted 9 April 2014 Available online xxxx Keywords: Pim kinase Meridianin C 3,5-Disubstituted indole Antiproliferative activity Inhibitor

a b s t r a c t A novel series of meridianin C derivatives substituted at C-5 position were prepared. These derivatives were tested for their kinase inhibitory potencies against all three family members of the pim kinases (Pim-1, Pim-2 and Pim-3). In addition, their antiproliferative activity towards three human leukemia cell lines as MV4-11, Jurkat clone E6-1 and K562 has been evaluated. Structure activity relationships at C-3 and C-5 positions of indole were performed to better understand the mechanism behind the enhanced potency. Compound 7f, the most active compound of the series showed a single-digit nanomolar IC50 with selectivity towards Pim-1 kinase. Ó 2014 Elsevier Ltd. All rights reserved.

Pim kinases are a family of kinases comprising Pim-1, Pim-2 and Pim-3. Pim-1 was first identified as proviral integration Moloney Murine Leukemia Virus associated with T-cell lymphomogenesis.1 It is reported that they possess serine/threonine kinase activity, involved synergistically with c-Myc in STAT3-mediated cell cycle progression and antiapoptosis.2,3 They have high affinity towards substrates which have a binding mode very similar to members of the calmodulin-dependent protein kinase family.4 They play a pivotal role in cell cycle progression and signal transduction by phosphorylating c-Myc-driven transcription and capdependent translation processes.2,4 Pim kinases act as downstream target for the JAK/STAT signaling pathway. Pim kinases are regulated by cytokines and hormones carrying signal through structurally related receptors such as IL-3, GM-CSF, G-CSF or IL-6, causing phosphorylation of JAK (janus kinase) and activating STAT (signal transducer and activator of transcription) proteins in series.5,6 Activated STATs then translocate to the nucleus and serve as transcription factors for the Pim genes. Pim-kinases are emerging as important targets for cancer treatment because their overexpression or deregulation of Pim-1 and Pim-2 kinases leads to hematologic cancer such as lymphocyte leukemia and solid tumors and Pim-3 leads to adenocarcinomas.7,8 According to the crystal structures of Pim-1 and pairwise sequence alignment, the pim kinase family has a unique proline (Pro123 in Pim-1) residue in the hinge ⇑ Corresponding authors. Tel.: +82 53 580 5184 (V.S.H.), +82 53 580 5183 (J.L.). E-mail addresses: [email protected] (V.S. Hong), [email protected] (J. Lee).

region and lacks a H-donor which makes one hydrogen bond interaction with ATP. Therefore pim kinases can make only one hydrogen bond to the adenine of ATP, which is a distinct character of pim kinases compared to others.9,10 Most of the reported pim kinase inhibitors utilize interaction with lysine that is Lys67 as an alternative driving force for binding. In addition, Pim kinase has a number of hydrophobic residues such as Leu44, Phe49, Val52, Ala65, Ile104, Leu120, Val126, Leu174 and Ile185 in the ATP binding site. The interactions of inhibitors in the reported crystal structures suggested the crucial role of the hydrophobic interactions with these residues in determining the binding affinity of inhibitors.11 Therefore, it would be possible to design highly selective inhibitors for this drug target. Pim kinases also show high homology within the family resulting in functional redundancy which can help to develop inhibitors that would be potent for all three members of family.12 To date, a total of 16 compounds are known to have entered in preclinical development and clinical trials, although the one of them, SGI-1776 (Astex) failed in phase I clinical trial due to its cardiotoxicity.13,14 Indole is a naturally occurring scaffold found in many marine derived anticancer agents.15–18 It was reported that aminopyrimidylindoles exhibit potent antiproliferative activity.19,20 Meridianin alkaloids isolated from marine source such as south Atlantic tunicate Aplidium meridianum are also known to be potent inhibitors of protein kinases.21,22 Meridianins (A–G) contain a 2-aminopyrimidyl ring at 3-position with variation at 4-, 5-, 6- and 7-positions of indole ring. In one recent report 3,5-disubstituted indole was

http://dx.doi.org/10.1016/j.bmcl.2014.04.035 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: More, K. N.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.04.035

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K. N. More et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx

H2N N

N

R

N R2

study. This preliminary result encouraged us to pursue modification of meridianin C to find more potent inhibitors against the pim kinases. Previous reports and modeling study suggested that pyrazine-2-yl group at 5-position of indole in meridianin C is suitable to make interaction with Lys67 in pim kinase pocket.12 Substitution of 6-ethoxypyrazine at C-5 of indole improved inhibitory activity against Pim-1 kinase more than an order of magnitude (Table 1). To further improve the potency and hydrophilicity of 7a, 6-ethoxy group on pyrazine was replaced with aminoalkyl groups using either amine or ether linkage. Addition of 2-aminoethyl group by amine linkage provided the compounds showing significantly improved potency against Pim-1 kinase. The activity gain may be attributed by two factors. First, the difference in basicity of pyrazine depending on the type of atom at 2-position of pyrazine may affect the interaction with Lys67. Alkoxy group at 2-position of pyrazine is less basic than amino group by 2 pKb units.25 Secondly, the interaction of aminoalkyl substituent with carbonyl and/or carboxyl groups of enzyme is influenced by the position of amino group. Two methylene units between amino group of aminoalkyl substituent and nitrogen linker were found better than three methylene units. The potency of compound against Pim-3 kinase was similar to Pim-1 and showed very close substituent dependency. However, the inhibitory activity of compound against Pim-2 kinase was lower by more than an order of magnitude compared to other isozymes. Replacement of 2-aminopyrimidine ring at C-3 position of indole with substituted ketone group considerably decreased the Pim-1 potency by more than 50 fold (Tables 1 and 2. 7b vs 7k, 7f vs 7l). The effect of 2-aminopyrimidine on the potency could be due to both hydrophobic interaction26 and hydrogen bond interaction with enzyme,16 as demonstrated by docking study (Fig. 2). A molecular modeling based on the reported X-ray structure (PDB code: 3BGQ, Human Pim-1 kinase in complex with an triazolopyridazine inhibitor VX2) showed that compound 7f was bound in the ATP binding pocket of Pim-1 (Fig. 2).27 As expected, one nitrogen atom of pyrazine forms a hydrogen bonding interaction (3.13 Å) with the e-amino group in the side chain of Lys67. 2-Aminopyrimidine rotates about 4 degree relative to the indole ring, which makes its amino group to form a hydrogen bond (3.28 Å) with the backbone carbonyl group of Pro123. In addition, there

NH2

H2N

N

N N F

N

Br

R N R1

N H

(A)

N H

(B)

(C)

Figure 1. Structures of meridianin C and synthesized indole analogues. (A) synthesized meridianin analogues, (B) meridianin C, (C) 3,5-disubstituted indole as an inhibitor of pan-pim kinases.

presented as very potent and selective inhibitor of pan-pim kinases (Fig. 1).12 Since meridianin C (3,5-disubstituted indole) exhibited a single digit micromolar potency for Pim-1, we sought to optimize its scaffold for in vitro potency against Pim-1 as well as other family members such as Pim-2 and 3. Further modification and structure activity relationship towards meridianin C core gave us potent compounds with single digit nanomolar potency. The general synthetic route for synthesis of 3-(2-aminopyrimidine)-5-substituted indole series is depicted in Scheme 1. The protection of 5-bromoindole with tosyl chloride under basic conditions followed by acetylation at C-3 position by general Friedel–Crafts acylation provided compound 3. Reaction of compound 3 with N,N-dimethylformamide diethyl acetal in N,N-dimethylformamide provided enaminone 4, which was used in the last step for the synthesis of 2-aminopyrimdine ring at C-3 position of the indole.23 Pinacol ester was introduced at C-5 position by Miyaura borylation.24 Compound 6 was obtained by palladium catalyzed Suzuki type coupling of compound 5 with 6-substituted-2-chloropyrazine (Het-Cl). Cyclization to 2-aminopyrimidine with simultaneous cleavage of tosyl group using guanidine and K2CO3 facilitated the preparation of final compound. Synthesis of compound 7k and 7l was done by removal of tosyl group from compound 6f and 6b, respectively. Meridianin C bearing 2-aminopyrimidine at C-3 and bromine at C-5 position was chosen as starting point in our study. Meridianin C was found to exhibit IC50 of 1.04 lM in Pim-1 kinase inhibition

O NH

(a)

N Ts

NH 2 N

(b) N Ts

N

Br

Br

NH

Br

2

1

3

R

O N

O

N N Ts

(d)

(e)

O B O

7a-j

N Ts

N Ts Br

N N

(f )

N (c)

X

O

R

X

O

N

N

(g) N

4 5 Cl

N N

Cl

(h)

NH

6a-j R

X

N N

Cl

R

X

N N

X : O, NH or NCH3

7k, 7l

Het-Cl Scheme 1. Synthesis of compounds 7a–l. Reagents and experimental conditions: (a) NaH, TsCl, DMF, 0 °C to RT; (b) AcCl, AlCl3, CS2, RT under N2; (c) DMF-DEA, DMF, microwave; (d) bis(pinacolato)diboron, PdCl2(dppf), KOAc, 1,4-dioxane, microwave; (e) Het-Cl, PdCl2(PPh3)2, 2 M K2CO3, 1,4-dioxane/EtOH (2:3), microwave; (f) guanidine carbonate, K2CO3, 2-methoxyethanol, microwave; (g) K2CO3, 2-methoxyethanol, microwave; (h) NaH, ROH, THF, microwave or RNH2 (or RNHCH3), K2CO3, DMF, RT.

Please cite this article in press as: More, K. N.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.04.035

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K. N. More et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx Table 1 Pim-kinase inhibitory activity of compounds 7a–7j

Meridianin C 7a 7b 7c 7d 7e 7f 7g 7h 7i 7j

R

X

— Et Me2N(CH2)2 Et2N(CH2)2 Me2N(CH2)3 Et2N(CH2)3 Me2N(CH2)2 Et2N(CH2)2 Me2N(CH2)3 Et2N(CH2)3 Me2N(CH2)2

— O O O O O NH NH NH NH NCH3

IC50 (nM)a Pim-1

Pim-2

Pim-3

1040 45.1 ± 0.8* 20.4 ± 0.2 25.8 ± 0.4 18.7 ± 2.7 34.5 ± 4.2* 3.4 ± 0.5** 2.8 ± 0.2** 18.3 ± 2.9 17.2 ± 5.8 8.0 ± 0.1**

NDb 740 380 590 540 660 110 310 630 920 180

ND 50.9 ± 10.7* 21.7 ± 5.8 30.0 ± 2.3 20.5 ± 0.9 55.7 ± 14.6* 6.5 ± 1.4 9.9 ± 0.1 31.3 ± 3.4 30.2 ± 4.9 7.8 ± 2.2

a

Values are reported as means ± SD. Not determined. p