pharmaceuticals Article

Search for Potent and Selective Aurora A Inhibitors Based on General Ser/Thr Kinase Pharmacophore Model Natalya I. Vasilevich *, Victor V. Tatarskiy Jr., Elena A. Aksenova, Denis N. Kazyulkin and Ilya I. Afanasyev ASINEX Company Group, Novie Nauchnie Tekhnologii Ltd., 20 Geroev Panfilovtsev Street, Moscow 125480, Russia; [email protected] (V.V.T.); [email protected] (E.A.A.); [email protected] (D.N.K.); [email protected] (I.I.A.) * Correspondence: [email protected]; Tel. +7-495-780-3410 Academic Editor: Jean Jacques Vanden Eynde Received: 21 December 2015; Accepted: 1 April 2016; Published: 13 April 2016

Abstract: Based on the data for compounds known from the literature to be active against various types of Ser/Thr kinases, a general pharmachophore model for these types of kinases was developed. The search for the molecules fitting to this pharmacophore among the ASINEX proprietary library revealed a number of compounds, which were tested and appeared to possess some activity against Ser/Thr kinases such as Aurora A, Aurora B and Haspin. Our work on the optimization of these molecules against Aurora A kinase allowed us to achieve several hits in a 3–5 nM range of activity with rather good selectivity and Absorption, Distribution, Metabolism, and Excretion (ADME) properties, and cytotoxicity against 16 cancer cell lines. Thus, we showed the possibility to fine-tune the general Ser/Thr pharmacophore to design active and selective compounds against desired types of kinases. Keywords: general pharmachophore model; Ser/Thr kinases; Aurora A

1. Introduction Serine/threonine protein kinases are enzymes that phosphorylate the OH group of serine or threonine. Among more than 500 human protein kinases, at least 125 appeared to be serine/threonine kinases (STK) [1]. Inhibitors of Ser/Thr kinases can possess potential therapeutic uses, from treating cancer to immune disorders. Since they were found in a number of mycobacterial organisms, they can be also used for treatment of bacterial infections such as tuberculosis. A number of attempts have been made to construct a pharmacophore model for various Ser/Thr kinase inhibitors, such as serine/threonine receptor kinase (STPK) inhibitors of tuberculosis, mTor kinase inhibitors, Aurora A and B inhibitors, B-Raf inhibitors, etc. [2–8]. Some of these models are based on the structure-based approach, i.e., docking [3]. Although docking is a promising tool for drug discovery, not all kinases (e.g., tuberculosis PknA) have an X-Ray–resolved structure, and, therefore, preliminary modeling of the binding site is necessary. This results in “double step prediction”: first the modeling of the binding site is done, followed by docking, which in turn decreases the obtained hit rate. Another group of these models has been developed based on the ligand-based approach [4–8]. In general, a more- or less-wide group of molecules is to be aligned and the common structure features are elucidated. Usually, common structure features are developed into more general pharmacophore models [4–7]. All cited articles are devoted to the search for specific pharmacophore models targeted to a specific group of kinases. We hypothesized that it is possible to find some general features of all serine-threonine inhibitors and to construct a general pharmacophore model. Then this general model might be adjusted to any specific kind of serine-threonine kinase. Pharmaceuticals 2016, 9, 19; doi:10.3390/ph9020019

www.mdpi.com/journal/pharmaceuticals

To construct the general pharmacophore model, we used known inhibitors of various serine-threonine kinases published in scientific literature and found in the ASINEX proprietary collection. Published STPK inhibitors included inhibitors of tuberculosis kinases, mTor kinase, Aurora A and B kinases, as well as B-Raf kinases [2–8]. ASINEX proprietary collections included Pharmaceuticals 2016, 9, 19 2 of 14 novel compounds tested and found to be active against pknA, pknB kinases. The conformers of all these compounds were generated in MOE (LowModeMD method (software produced by Chemical 2. Results and Discussion Computing Group ) with rejection limit of 100, iteration limit of 1000, RMS gradient of 0.005 and MM iteration limit of 500. Thegeneral RMSD limit was the default and the was set to The To construct the pharmacophore model, weconformation used knownlimit inhibitors of500). various pharmacophore elucidating module of MOE version 2010.10 was used for further pharmacophore serine-threonine kinases published in scientific literature and found in the ASINEX proprietary generation the conformer database obtained field set to “As-is”). collection. with Published STPK inhibitors included(conformations inhibitors of tuberculosis kinases,Other mTorsettings kinase, were theAdefaults. Aurora and B kinases, as well as B-Raf kinases [2–8]. ASINEX proprietary collections included resulting tested generaland pharmacophore has the shapepknA, of a rhomb with a side around 4–5 A, novelThe compounds found to be active against pknB kinases. The of conformers of all two occupying the opposite nodes, one aromatic groupproduced in the third and thesehydrophobic compounds groups were generated in MOE (LowModeMD method (software by node Chemical H-bond donor and/or acceptor projections located in the remaining corner rather close to each other Computing Group ) with rejection limit of 100, iteration limit of 1000, RMS gradient of 0.005 and MM (Figure The different of the thisdefault modeland is that it is more orlimit less was planar, the iteration1). limit of main 500. The RMSDfeature limit was the conformation set tounlike 500). The previously developed “volumetric ones” (see. version e.g., [1]).2010.10 was used for further pharmacophore pharmacophore elucidating module of MOE Application of this general pharmacophore to the ASINEX proprietary generation with the conformer database obtainedmodel (conformations field set to “As-is”).library Other allowed settings us to find a scaffold fitting to its requirements. This scaffold consisted of a thiazole group connected were the defaults. through amino general group topharmacophore a nitrogen-containing aromatic cycle, which in turn was The an resulting has the six-member shape of a rhomb with a side of around 4–5 A, bound to a pyrrolidine or piperidine ring directly or via a short linker. In this scaffold, thiazole two hydrophobic groups occupying the opposite nodes, one aromatic group in the third node and and saturated cycles served as a hydrophobic group, in while the middle-positioned six-member cycle H-bond donor and/or acceptor projections located the remaining corner rather close to each other presented a source for H-bond donor or acceptor projections (if one or more “A” moieties in the ring (Figure 1). The main different feature of this model is that it is more or less planar, unlike the previously means N). developed “volumetric ones” (see. e.g., [1]).

Figure 1. 1. General General pharmacophore pharmacophore of of serine-threonine serine-threonine kinase kinase inhibitors inhibitors (MOE (MOE 2010.10) 2010.10) and and results results of of Figure its application application to to ASINEX ASINEX library. library. its

To verify ourofhypothesis andpharmacophore the validity of the design, a series of compounds to this Application this general model to the ASINEX proprietarybelonging library allowed scaffold testedfitting against several Ser/Thr kinases available in-house: Aurora B and us to findwere a scaffold to its requirements. This scaffold consisted of a Aurora thiazoleA, group connected Haspin kinases. The results for the most active compounds are shown in Table 1. through an amino group to a nitrogen-containing six-member aromatic cycle, which in turn was bound to a pyrrolidine or piperidine ring directly or via a short linker. In this scaffold, thiazole and saturated cycles served as a hydrophobic group, while the middle-positioned six-member cycle presented a source for H-bond donor or acceptor projections (if one or more “A” moieties in the ring means N). To verify our hypothesis and the validity of the design, a series of compounds belonging to this scaffold were tested against several Ser/Thr kinases available in-house: Aurora A, Aurora B and Haspin kinases. The results for the most active compounds are shown in Table 1. To confirm our idea about the possibility of fine-tuning the general pharmacophore for selected types of kinases, we chose Aurora A kinase and tried to adjust the scaffold to its specific requirements. Compound 2, which showed the best activity against Aurora A (IC50 100 nM), was taken as a starting point and a series of iterations was performed. During the optimization process, three series of compound 2 analogues were synthesized. During the first step we kept the methyl substituent on the thiazole ring and the 2-fluoro-3-chloro-benzoyl substituent on the aliphatic nitrogen, varying both the six-member aromatic ring and an aliphatic N-containing cycle tethered to the aromatic one either directly or through a methylene linker. After choosing the best compound in this a series of compounds with methylene-4-piperidine linker, various N-acyl and N-alkyl substitutions were made. None of

Pharmaceuticals 2016, 9, 19 3 of 14 Pharmaceuticals 2016, 9, 19 3 of 14 Pharmaceuticals 2016, 9, 19 3 Pharmaceuticals 2016, 19 3of of14 14 Pharmaceuticals2016, 2016,9,9, 9,19 19 Pharmaceuticals 33ofof1414 Table 1. Activity of compounds fitting to general pharmacophore model against selected Ser/Thr kinases. Pharmaceuticals 2016, 9, 19 3 of 14

Table 1. Activity of compounds fitting to general pharmacophore model against selected Ser/Thr kinases.

Table Activity of compounds fitting general pharmacophore model against selected Ser/Thr kinases. Table 1.Activity Activity ofcompounds compounds fittingsuperiority togeneral generalpharmacophore pharmacophore model against selected Ser/Thrkinases. kinases. Pharmaceuticals 2016, 9,of 19 3 of During 14 Table 1.1. fitting toto model against Ser/Thr these substituents demonstrated compared 2-fluoro-3-chloro-benzoyl group. Aurora B,selected Haspin, Aurora to A, the No. Structure Pharmaceuticals 2016, 9, 19 3 of 14to be Table 1. Activity of compounds fitting to general pharmacophore model against selected Ser/Thr kinases. Aurora B, Haspin, Aurora A, the last step we varied the thiazole fragment and discovered pyrazole-containing derivatives 50 , μM IC 50 , μM IC 50 , μM IC Aurora B, Haspin, Aurora A, AuroraB, B, Haspin, AuroraA, A, Pharmaceuticals 2016, 9, 19 3 of 14 No. Structure Aurora Haspin, Aurora No. Structure No. Structure Table 1.No. Activity fitting general pharmacophore IC 50 IC IC Pharmaceuticals 2016, 9,of19compounds 3 of 14 Structure , μM μM model ICagainst 50,, μM μM selected IC50Ser/Thr 50,, μM μM kinases. IC5050,analogues. equally or even more active than to 4-methyl-thiazole μM IC5050, ,μM μM IC5050, ,μM μM IC5050, ,μM Aurora B, selected Haspin, Aurora A, model IC IC IC Table 1.No. ActivityH3of fitting to general pharmacophore against Ser/Thr kinases. N C compounds Structure Table 1. Activity of compounds fitting to general pharmacophore model against selected Ser/Thr Aurora B, Haspin, Aurora A, IC50, μM IC 50, μM kinases. IC50, μM HH3CCof compounds S NN No. Nof compounds N NHfitting Table 1. Activity fitting to general pharmacophore model against selected Ser/Thr NStructure 3 C Activity 2 Table 1. to general pharmacophore model against selected N 1 2.06 ± 0.15 1.45 ± 0.17 1.49 0.15 kinases. HH C 3 Aurora B, Haspin, Aurora A, H IC50, μM IC50, ±μM IC50, μM 3 N NH SS Structure No. NN NN Aurora B, Haspin, Aurora A, NH 2 CH SN NHN NN 1 2.06 ± 0.15 1.45 ± 0.17 1.49 ± 0.15 2 3 NH H C Ser/Thr kinases. S 1 2.06 ± 0.15 1.45 ± 0.17 1.49 ± 0.15 50 , μM IC 50 , μM IC 50 , μM IC NH 3 No. Structure H 2.06 ±0.15 0.15 1.45 ±0.17 0.17 1.49 ±0.15 0.15 2 23 Aurora B, Haspin, Aurora A, NN O CH 11 2.06 1.45 1.49 HH IC50,±μM IC50,±μM IC50,±μM CH No. H3C NN NH CH33 S N Structure N N CH 2 CH CH CH 333 3 50 , μM IC 50 , μM IC 50 , μM IC 1 2.06 ± 0.15 1.45 ± 0.17 1.49 ± 0.15 CH O CH H No. Aurora A, IC50 , µM Aurora B, IC50 , µM Haspin, IC50 , µM N OO ONH 3 3 H3C S N Structure N N CH 2 1 2.06 ± 0.15 1.45 ± 0.17 1.49 ± 0.15 O CH3 3 H3C NN H N N O NH S N N N N H3C 2 F CH 1 2.06 ± 0.15 1.45 ± 0.17 1.49 ± 0.15 3 O NH S SN N N NHN NNN OO CH 0.10±±0.15 0.005 1.45 3.46± ±0.17 0.36 105.2 ± 11.2 NO 2 3 NN O 12 2.06 1.49 ± 0.15 NN SH H NNN N FFNH CH N 3 N N 1 21 2.06±˘ 0.15 1.45 ˘ 0.17 105.2 1.49 ˘ 0.15 FCH SS NN N 2.06 ±0.005 0.15 1.45 0.17 1.49 ±±±0.15 Cl32 0.10 3.46 ±±±±0.36 11.2 N CH 2 0.10 ± 0.005 3.46 0.36 105.2 11.2 SN NHN NH ON OFCH N N 3 2 0.10 ± 0.005 3.46 0.36 105.2 11.2 S H 2 0.10 ± 0.005 3.46 ± 0.36 105.2 ±±11.2 2 2 2 22 3 32 333 3 3 33 3 34 444 4 4 44 4 4 45 555 5 55

5 5 5 56 666 66 6 6 6 6 67 7 77 77 7 7 7 7 78 8 888 8 8 8 8 8 89 9 99 99 9 9 9 9 1010 9 10 10 10 10 10

H3C HH3CC HH C3 C 3 3 H3C H3C H3C H3C H3C C H 3

HH3CC 3C HH C 3 3 H3C H3C H3C H3C H3C C H 3 HH3CC HH C3 C 3 3 H3C H3C H3C H3C H3C C H 3

HH3CC HH C3 C 3 3 H3C H3C H3C H3C H3C H3C

HH3CC 3C HH C 3 3 H3C H3C H3C H3C H3C C H 3 HH3CC 3C HH C 3 3 H3C H3C H3C H3C H3C

3CH N Cl O HH 3 Cl Cl 3 O F ClCH S N N N N O N H F O S N N NN N Cl N O N N H F S N NS NNN N N O F ClN N N N S N NH NN H N FCl S NN NN NN S H NS N N NN Cl SHSN NHH NN N H N H O Cl N N S N N N N O H N OO ON SN N N N H NO N S N NN N S NH N N N O NH S NN HN N S N N 2 N NN N H H SS NNO NH NN NN NH N NN NH222 SSN NHH O NH 2 NN HH O NO N S N N N NH2 O H N OO ONH SN N N 2 N H N O NH S N N N N 2 S NH N N ONH2 S NN HN N HN S N N NH2 N NN H H N SS N OHHNN NN NN S N NN H S N NHH NN NNOH HH O O NN H S N N N N O H N OO O SN N N HN N H N OHN S N NN N S NH N HN N O NH S NN HN N S N HN 2 N NN N H H NO NH SS N NN NN NH N NN NH222 SSN NHH O NH 2 NN HH O N O N S N N N NH2 O H N OO ONH SN N N 2 N H N O NH S NN N 2 S N NH N N N ONH2 S HN NN NH2 N N S NN H N N NH2 NN NN H NO NO NH SS NN NN NH 2 3 N NNN NH2CH SSN NHH 2 23 NN OONHCH HH N CH N N CH 33 S N N N NH CH 2 CH CH CH 333 3 CH H OO CH 3 3 N N O SN N O NHCH N 2 N N H CH3 3 N O NH S N N NN N N 2 CH 3 S CH NH N N N ONH 2 3 S NN HN N N NH S CH N NH NN N NN N 22 3 CH H H CH3 3 NO CH SS N NN NN NH NH CH 32 2 3 N NNN NH CH SSN NHH O NH CH 2 23 3 CH NN 3 HH O CH N O N CH 3 S N N N N NH CH CH 2 3 33 CH CH 33 CH H OO CH N OONH 3 3 SN N N N CH 2 N NN H CH3 3 N O NH S NN N N 2 CH N 3 H S N N N NH S NN CH N ONH 2 32 N HH N CH N NN NH N SSS NN N 2 3 NH CH N NH 2CH H 32 3 CH N NH SSN NHH 3 NN N O NH CH CH 2 23 NN 3CH HH NO O CH 3 CH N CH S N N N NH 3 33 CH 2 CH CH CH 3 O CH333 3 OO CH H 3 N O S N N O NHCH N 2 N H CH3 3 N O NH S N N NN 2 H CH 3 S CH NH N N ONH 2 3N NHN S N CH SNN H N NH N N 3 N 2H CH H H H H3NN CH SS 3 NNO CH N NN N O 3N N NN N CH SSN NHH O H3 3 CH N H N H O NO NN O O S N N N HO OO O H N N S N N OO O N H N H N O HN S NN N NOH H S N N N O N O S HN N N H NO O O O O

0.10 ± 0.005 0.10 ± 0.005 0.10 ± 0.005 1.95 ±0.005 0.22 0.10 0.10 ± ˘0.005 0.10 ±±±0.005 1.95 0.22 1.95 0.22 1.95±±0.22 0.22 1.95

3.46 ± 0.36 105.2 ± 11.2 3.46 ± 0.36 105.2 ± 11.2 3.46 ± 0.36 105.2 ± 11.2 1.76± ±0.36 0.16 7.06±±11.2 0.68 3.46 ˘ 0.36 3.46 105.2 3.46 ±± 0.16 0.36 105.2 ±0.68 11.2 1.76 ± 7.06 ± 1.76 0.16 7.06 ± 0.68 1.76±±0.16 0.16 7.06±±0.68 0.68 1.76 7.06

1.95 ± 0.22 1.95 ± 0.22 1.95±˘0.22 0.22 1.95 1.95 ± 0.22 2.08 ± 0.25 1.95 0.22 2.08 ± 0.25 2.08 0.25 2.08±±±0.25 0.25 2.08

1.76 ± 0.16 7.06 ± 0.68 1.76 ± 0.16 7.06 ± 0.68 1.76 ˘ 0.16 7.06 ± 0.68 1.76 ± 0.16 1.76 ± 0.16 7.06 ± 0.68 1.72 ± 0.16 0.19 0.08 1.76 7.06 ±± 0.008 0.68 1.72 ± 0.19 0.08 ±±±0.008 1.72 ± 0.19 0.08 0.008 1.72 ± 0.19 0.08 0.008 1.72 ± 0.19 0.08 ± 0.008

2.08 ± 0.25 2.08 ±˘0.25 0.25 2.08 2.08 ± 0.25 2.08 ± 0.25 1.44 ± 0.25 0.13 2.08 1.44 ± 0.13 1.44 ± 0.13 1.44±±0.13 0.13 1.44

1.72 ± 0.19 0.08 ± 0.008 1.72 ˘ 0.190.08 ± 0.008 1.72 ± 0.19 1.72 ± 0.19 0.08 ± 0.008 1.72 ± 0.19 0.08 ± 0.008 0.91 ± 0.19 0.08 0.43 ± 0.008 0.034 1.72 0.08 0.91 ± 0.08 0.43 ± 0.034 0.91 ± 0.08 0.43 ± 0.034 0.91±±0.08 0.08 0.43±±0.034 0.034 0.91 0.43

1.44±˘0.13 0.13 1.44 1.44 ± 0.13 1.44 ± 0.13 1.44 ± 0.13 1.67 ± 0.13 0.17 1.44 1.67 ± 0.17 1.67 ± 0.17 1.67 ±0.17 0.17 1.67±˘ 0.17 1.67

0.91 ˘ 0.080.43 ± 0.034 0.91 ± 0.08 0.91 ± 0.08 0.43 ± 0.034 0.91 ± 0.08 0.43 ± 0.034 0.91 ± 0.08 0.43 ± 0.034 1.75 ± 0.08 0.18 1.12±±0.034 0.11 0.91 0.43 1.75 ± 0.18 1.12 ± 0.11 1.75 ± 0.18 1.12 ± 0.11 1.75±±0.18 0.18 1.12±±0.11 0.11 1.75 ˘ 0.18 1.12 1.75

1.67 ± 0.17 1.67 ± 0.17 1.67 ± 0.17 1.67 ± 0.17 2.79 ± 0.17 0.30 1.67 2.79 0.30 2.79 ± ˘ 0.30 2.79 ± 0.30 2.79 ± 0.30 2.79 ± 0.30

1.75 ± 0.18 1.12 ± 0.11 1.75 ± 0.18 1.12 ± 0.11 1.75 ± 0.18 1.12 ± 0.11 1.75 ± 0.18 1.12 ± 0.11 2.25 ± 0.18 0.23 3.67 ± 0.11 0.37 1.75 1.12 2.25 ±±±0.23 ±±±0.37 2.25 ˘ 0.23 3.67 2.25 0.23 3.67 0.37 2.25 0.23 3.67 0.37 2.25 ± 0.23 3.67 ± 0.37

2.79 ± 0.30 2.79 ± 0.30 2.79 ± 0.30 2.79 ± 0.30 3.58 ± 0.30 0.43 2.79 3.58 ± 0.43 3.58 ± 0.43 3.58±˘ 0.43 3.58 ±0.43 0.43 3.58

2.25 ± 0.23 3.67 ± 0.37 2.25 ± 0.23 3.67 ± 0.37 2.25 ± 0.23 3.67 ± 0.37 2.25 ± 0.23 3.67 ± 0.37 2.05 ± 0.23 0.24 2.90 ± 0.37 0.43 2.25 3.67 2.05 ± 0.24 2.90 ± 0.43 2.05 ± 0.24 2.90 ± 0.43 2.05 ˘ 0.24 2.90 2.05±±0.24 0.24 2.90±±0.43 0.43 2.05

3.58 ± 0.43 3.58 ± 0.43 3.58 ± 0.43 3.58 ± 0.43 3.17 ± 0.43 0.33 3.58 3.17 ˘ 0.33 3.17 ± 0.33 3.17 0.33 3.17±±±0.33 0.33 3.17

2.05 ± 0.24 2.90 ± 0.43 2.05 ± 0.24 2.90 ± 0.43 2.05 ± 0.24 2.90 ± 0.43 2.05 ± 0.24 2.90 ± 0.43 8.02 ± 0.24 0.80 0.27 2.05 2.90 ±± 0.019 0.43 8.02 ˘ 0.80 8.02 ± 0.80 0.27 ±±±0.019 8.02 ± 0.80 0.27 0.019 8.02 ± 0.80 0.27 0.019 8.02 ± 0.80 0.27 ± 0.019

3.17 ± 0.33 3.17 ± 0.33 3.17 ± 0.33 3.17 ± 0.33 8.38 ˘ 8.38 0.84 3.17 ± 0.84 0.33 8.38 ± 0.84 8.38 ± 0.84 8.38±±0.84 0.84 8.38

8.02 ± 0.80 0.27 ± 0.019 8.02 ± 0.80 0.27 ± 0.019 8.02 ± 0.80 0.27 ± 0.019 8.02 ± 0.80 0.27 ± 0.019 0.84 ˘ 0.09 0.27 0.84 ± 0.80 0.09 7.80±±0.019 0.81 8.02 0.84 ± 0.09 7.80 ± 0.81 0.84 ± 0.09 7.80 ± 0.81 0.84±±0.09 0.09 7.80±±0.81 0.81 0.84 7.80

8.38 ± 0.84

0.84 ± 0.09

105.2 ˘ 11.2

7.06 ˘ 0.68

0.08 ˘ 0.008

0.43 ˘ 0.034

1.12 ˘ 0.11

3.67 ˘ 0.37

2.90 ˘ 0.43

0.27 ˘ 0.019

7.80 ˘ 0.81

7.80 ± 0.81

To confirm our idea about the possibility of fine-tuning pharmacophore 10 8.38 ± 0.84 the general 0.84 ± 0.09 7.80 ± 0.81 for selected To confirm our idea about the possibility of fine-tuning the general pharmacophore selected 10 8.38 ± 0.84 0.84 ± 0.09 7.80 ± 0.81 To confirm our idea about the possibility of fine-tuning the general pharmacophore for selected types of kinases, we chose Aurora A kinase and tried to adjust the scaffold to for itsselected specific Toconfirm confirm ouridea ideaabout aboutthe thepossibility possibilityofoffine-tuning fine-tuning thegeneral general pharmacophore for selected To our pharmacophore 10 8.38 ± 0.84 the 0.84 ± 0.09 7.80 ± 0.81 for types of kinases, we chose Aurora A kinase and tried to adjust the scaffold to its specific 10 8.38tried ± 0.84the 0.84 ± 0.09 7.80 ± to 0.81 types of kinases, we chose Aurora kinase and tried to adjust the scaffold to its specific types of kinases, we chose Aurora A kinase kinase and tried togeneral adjust the scaffold scaffold to for itsselected specific Toof confirm ourwe ideachose aboutAurora the possibility of fine-tuning pharmacophore types kinases, AA and to adjust the its specific To confirm our idea about the possibility of fine-tuning the general pharmacophore for selected types of kinases, we chose Aurora A kinase and tried to adjust the scaffold to its specific ourwe ideachose aboutAurora the possibility of fine-tuning pharmacophore typesToofconfirm kinases, A kinase and tried the to general adjust the scaffold to for its selected specific To confirm our idea about the possibility of fine-tuning the general pharmacophore for selected typesTo ofconfirm kinases,our we chose A kinase and tried the to adjust scaffold to its idea aboutAurora the possibility of fine-tuning generalthe pharmacophore for specific selected types of kinases, we chose Aurora A kinase and tried to adjust the scaffold to its specific

nitrogen, varying both the six-member aromatic ring and anan aliphatic N-containing cycle tethered toto nitrogen, varying both the six-member aromatic ring and an aliphatic N-containing cycle tethered to nitrogen, varying both the six-member aromatic ring and aliphatic N-containing cycle tethered nitrogen, varying both the six-member aromatic ring and an aliphatic N-containing cycle tethered to nitrogen, varying both the six-member aromatic ring and an aliphatic N-containing cycle tethered to nitrogen, varying both the six-member aromatic ring and an aliphatic N-containing cycle tethered to the aromatic one either directly or through a methylene linker. After choosing the best compound the aromatic one either directly or through a methylene linker. After choosing the best compound inin the aromatic one either directly or through a methylene linker. After choosing the best compound in the aromatic one either directly or through a methylene linker. After choosing the best compound the aromatic one either directly or through a methylene linker. After choosing the best compound in the aromatic one either directly through a methylene linker. After choosing the best compound the aromatic one either directly oror through a methylene linker. After choosing the best compound ininin this aseries series ofcompounds compounds with methylene-4-piperidine linker, various N-acyl andN-alkyl N-alkyl this a series of compounds with methylene-4-piperidine linker, various N-acyl and N-alkyl this a of with methylene-4-piperidine linker, various N-acyl and this aseries series compounds with methylene-4-piperidine linker, various N-acyl and N-alkyl this ofofof compounds with methylene-4-piperidine linker, various N-acyl and N-alkyl this series compounds with methylene-4-piperidine linker, various N-acyl and N-alkyl this aaaseries of compounds with methylene-4-piperidine linker, various N-acyl and N-alkyl substitutions were made.None None thesesubstituents substituentsdemonstrated demonstrated superiority compared the substitutions were made. None ofof these substituents demonstrated superiority compared toto the substitutions were made. of these superiority compared to the substitutions were made. None of these substituents demonstrated superiority compared to the substitutions were made. None of these substituents demonstrated superiority compared to the substitutionswere weremade. made.None Noneofofthese thesesubstituents substituentsdemonstrated demonstratedsuperiority superioritycompared comparedtotothe the substitutions 2-fluoro-3-chloro-benzoylgroup. group.During Duringthe thelast laststep stepwe wevaried variedthe thethiazole thiazolefragment fragmentand and 2-fluoro-3-chloro-benzoyl group. During the last step we varied the thiazole fragment and 2-fluoro-3-chloro-benzoyl 2-fluoro-3-chloro-benzoyl group. During the last step we varied the thiazole fragment and 2-fluoro-3-chloro-benzoyl group. During the last step we varied the thiazole fragment and 2-fluoro-3-chloro-benzoyl group.During Duringthe thelast laststep stepwewevaried variedthe thethiazole thiazolefragment fragment and 2-fluoro-3-chloro-benzoyl group. and Pharmaceuticals 2016, 9, 19 4 of 14 discovered pyrazole-containing derivatives be equally even more active than 4-methyl-thiazole discovered pyrazole-containing derivatives toto be equally oror even more active than 4-methyl-thiazole discovered pyrazole-containing derivatives to be equally or even more active than 4-methyl-thiazole discovered pyrazole-containing derivatives to be equally or even more active than 4-methyl-thiazole discovered pyrazole-containing derivatives to be equally or even more active than 4-methyl-thiazole discovered pyrazole-containing derivatives to be equally or even more active than 4-methyl-thiazole discovered pyrazole-containing derivatives to be equally or even more active than 4-methyl-thiazole analogues. analogues. analogues. analogues. analogues. analogues. analogues. As aresult result we obtained several compounds with excellent potency the biochemical assay As aresult we obtained several compounds with excellent potency inin the biochemical assay As we obtained several compounds with excellent potency in the biochemical assay result we obtained several compounds with excellent potency in the biochemical assay As aresult result we obtained several compounds with excellent potency in the biochemical assay As result we obtained several compounds with excellent potency in the biochemical assay As aresult we obtained several compounds with excellent potency inthe the biochemical assay As aaaa2). we obtained several compounds with excellent potency in biochemical assay (Table (Table 2). (Table 2). (Table 2). (Table (Table 2). (Table 2).2). (Table 2). Table 2.Compounds Compounds optimized for Aurora A kinase inhibition. Table optimized for Aurora kinase inhibition. Table 2. optimized for Aurora A inhibition. Table 2.2.Compounds Compounds optimized for Aurora AAkinase kinase inhibition. Table 2.Compounds Compounds optimized for Aurora A kinase inhibition. Table Compounds optimized for Aurora kinase inhibition. Table optimized for Aurora kinase inhibition. Table 2.2.2. Compounds optimized for Aurora AAA kinase inhibition. No. No. No. No.No. No. No. No.

2 22 22 2 2 2

1111 11 111111 11 11

1212 12 121212 12 12

1313 13 131313 13 13

1414 14 141414 14 14

1515 15 15 151515 15

1616 16 16 161616 16

Structure Structure Structure Structure Structure Structure Structure Structure

NNN NNN NNNN NNNN NN SSS NNN NNN SSSS NN NHN NN H H H H HH

NNN NNN NNNN NNNN NN SSS NNN NNN SSSS NN NHN NN H H H H HH

NNN NNN NNNN NNNN NN SSS NNNN N SSSS NN NH NNNN H H HHHH

NNN NNN NNNN NNNN Cl Cl Cl ClClCl SS NN NN Cl S NNHN NN NNN SSSS NN HH HHHH

HHH H NN HN HN H N N N N N NNNNN

NNN NNNN NNN NNNN N NH NH NNN NH HHHH

OOO O NNNOOO FF N F NNN FFF F ClCl Cl ClClCl Cl

F OOO FF OOOO FFF F ClCl Cl ClClCl NNN Cl N NNN

F OOO FF OOOO FFF F ClCl Cl ClClCl NNN Cl N N NN

F OOO FF OOOO FFF F ClCl Cl ClClCl NNN Cl N NNN

OOO FFF OOOO FFF F ClCl Cl ClClCl NNN Cl N N N N

HHH H HN HN H NNN N N NNNN NNN

OOO FFFFF OOOO FF ClCl Cl ClClCl NNN Cl N N NN

HHH H NN HN HN H N N N N N NNNNN

OOO FFFFF OOOO FF ClCl Cl ClClCl Cl NNN NNNN

NNN NNNN N NH NH NNN NH HHHH

NNN NNNN NNN NNNN N NH NH NNN NH HHHH

IC50,nM nM(Mean (Mean± ±SEM, SEM,four fourRepeats) Repeats) IC50, IC50, nM (Mean ± SEM, four Repeats) IC50, nM (Mean ±SEM, SEM, four Repeats) IC50, nM (Mean four Repeats) IC50, nM (Mean ±SEM, four Repeats) IC50, nM (Mean ˘ SEM, Repeats) IC50, nM (Mean ±±four SEM, four Repeats)

96.0± ±9.1 9.1 96.0 96.0 ± 9.1 96.0 ±9.1 9.1 96.0 96.0 ±9.1 96.0 ±±9.1 96.0 ˘ 9.1

12.0± ±1.1 1.1 12.0 12.0 ± 1.1 12.0 ±1.1 1.1 12.0 12.0 ˘ 1.1 12.0 ±1.1 12.0 ±±1.1

35.1± ±0.36 0.36 35.1 35.1 ± 0.36 35.1 ±0.36 0.36 35.1 35.1 ˘ 0.36 35.1 ±0.36 35.1 ±±0.36

4.1± ±0.35 0.35 4.1 4.1 ±± 0.35 4.1 ˘ 0.35 ±0.35 0.35 4.1 4.1 ±0.35 4.1 ±4.1 0.35

5.3± ±0.46 0.46 5.3 5.3 ˘ 0.46 5.3 ±± 0.46 ±0.46 0.46 5.3 5.3 ±0.46 5.3 ±5.3 0.46

17.5± ±2.1 2.1 17.5 ˘ 2.1 17.5 17.5 ± 2.1 17.5 ±2.1 2.1 17.5 17.5 ±2.1 17.5 ±±2.1

3.5± ±0.29 0.29 3.5 ˘ 0.293.5 3.5 ±± 0.29 ±0.29 0.29 3.5 3.5 ±0.29 3.5 ±3.5 0.29

Compounds 11–14were weresynthesized synthesizedinininaccordance accordancewith withScheme Scheme1.1.1.Amide Amide2′2′2′was wasprepared prepared Compounds 11–14 were synthesized accordance with Scheme Amide was prepared Compounds 11–14 Compounds 11–14 were synthesized in accordance with Scheme 1. Amide 2′ was prepared Compounds 11–14 were synthesized ininaccordance accordance with Scheme 1.1.Amide Amide 2′2′was was prepared Compounds 11–14 were synthesized accordance with Scheme Amide was prepared Compounds 11–14 were synthesized in with Scheme 1. 2′ prepared 1 from the corresponding Boc-protected amino acid. Compound 1′ was converted into amidine salt 3′ from the corresponding Boc-protected amino acid. Compound 1′ was converted into amidine salt Compounds 11–14 were synthesized in accordance with Scheme 1. Amide 2 was prepared from from the corresponding Boc-protected amino acid. Compound 1′ was converted into amidine salt from the corresponding Boc-protected amino acid. Compound 1′ was converted into amidine salt from the corresponding Boc-protected amino acid. Compound was converted into amidine salt 3′3′ from the corresponding Boc-protected amino acid. Compound was converted into amidine salt 3′3′ from the corresponding Boc-protected amino acid. Compound 1′1′1′ was converted into amidine salt 3′3′ 1 1 the corresponding Boc-protected amino acid. Compound 1 was converted into amidine salt 3 by reaction with triethyloxoniuntetrafluoroborate, followed by treatment with a solution of ammonia in methanol. Cyclization of the amidine obtained with acetoacetic ester in ethanol under reflux gave 6-methylpyrimidone 41 in 65% yield. This pyrimidones 41 were treated with three equivalents of phosphorus oxychloride and nine equivalents of dimethylaniline in toluene to give chloride 51 in 62% yield. A Buchwald reaction with aromatic amines (2-aminothiazoles or 3-aminopyrazoles) led to new derivatives 61 , and this reaction was performed in a toluene-water mixture with 2.5% mol Pd2 dba3 and 5% mol xantphos and potassium carbonate using a microwave reactor (65%–75% yield). For compound 13 with a 5-chloro-2-thiazole moiety, intermediate 61 (where R1 —unsubstituted 2-aminothiazole) was

by reaction with triethyloxoniuntetrafluoroborate, followed by treatment with a solution of ammonia in methanol. Cyclization of the amidine obtained with acetoacetic ester in ethanol under reflux gave 6-methylpyrimidone 4′ in 65% yield. This pyrimidones 4′ were treated with three equivalents of phosphorus oxychloride and nine equivalents of dimethylaniline in toluene to give chloride 5′ in 62% yield. A Buchwald reaction with aromatic amines (2-aminothiazoles or Pharmaceuticals 2016, 9, 19 5 of 14 3-aminopyrazoles) led to new derivatives 6′, and this reaction was performed in a toluene-water mixture with 2.5% mol Pd2dba3 and 5% mol xantphos and potassium carbonate using a microwave reactor (65%–75% yield). For compound 13 with a 5-chloro-2-thiazole moiety, intermediate 6′ (where treated with N-chlorosuccinimide in dichloroethane at room temperature (yield 70%). To synthesize R1—unsubstituted 2-aminothiazole) was treated with N-chlorosuccinimide in dichloroethane at compound 14 containing a 3-aminopyrazole moiety, the corresponding 3-amino-1H-pyrazole was room temperature (yield 70%). To synthesize compound 14 containing a 3-aminopyrazole moiety, protected by the tosyl group via reaction with tosyl chloride sodium hydrocarbonate in acetonitrile. the corresponding 3-amino-1H-pyrazole was protected by and the tosyl group via reaction with tosyl 1 by sodium hydroxide in methanol This chloride protective group was easily removed from intermediate 6 and sodium hydrocarbonate in acetonitrile. This protective group was easily removed from treatment. After 6′Boc-deprotection, amines 71 weretreatment. acylatedAfter by 3-chloro-2-fluorobenzoic intermediate by sodium hydroxide in methanol Boc-deprotection, amines 7′acid wereusing by 3-chloro-2-fluorobenzoic acid using TBTU as a coupling reagent (yields 85% and more). TBTUacylated as a coupling reagent (yields 85% and more). OH O

O OH

a

NH b

NH2

N boc

N boc

1' HN

NH2

N boc

2'

c

HN

N

e N

N boc

3'

N boc

4'

R1

5'

R1

N

N

f

N

d

N

HN

R1

N

Cl

N

g

N

N N

N boc

O

N H 7'a-d

6'a-d

F

11 - 14

Cl

R1=

S

* N

S

* N

11

12

S

*

Cl

*

H N

N

N

13

14

Scheme Synthesisofofcompounds compounds 11–14. 11–14. Conditions: 4)2CO 3, TBTU, Et3N, h; 8 h; Scheme 1. 1. Synthesis Conditions:(a) (a)(NH (NH EtCH CH3RT, CN,8 RT, 4 )2 CO 3 , TBTU, 3 N,3CN, (b) triethyloxoniumtetrafluoroborate,CH CHCl 2Cl2,, RT, RT, 22 h; NH 3/MeOH, RT,RT, 16 16 h; (c) CHCH 3COCH 2COOEt, (b) triethyloxoniumtetrafluoroborate, h; NH /MeOH, h; (c) COCH COOEt, 2 2 3 3 2 tBu, EtOH, RF, 10 h; (d) POCl3, N,N-dimethylaniline, toluene, RF, 4 h; (e) R1NH2, Na2CO3, NaOtNaO Bu, EtOH, RF, 10 h; (d) POCl3 , N,N-dimethylaniline, toluene, RF, 4 h; (e) R1 NH2 , Na2 CO3 , Pd2 dba3 , Pd2dba3, xantphos, toluene/H2O, MW, 140 °C, 2 h; (f) HCl/1,4-dioxane, RT, 4 h; (g) xantphos, toluene/H2 O, MW, 140 ˝ C, 2 h; (f) HCl/1,4-dioxane, RT, 4 h; (g) 3-chloro-2-fluoro-benzoic 3-chloro-2-fluoro-benzoic acid, TBTU, Et3N, CH3CN, RT, 8 h. acid, TBTU, Et3 N, CH3 CN, RT, 8 h.

Synthesis of compound 15 is shown in Scheme 2. Alkene 10′ was obtained from ketone 9′ via 1 was in reaction with iodide and sodium 10 hydride 75% yield. Subsequent Synthesis of methyltriphenylphosphonium compound 15 is shown in Scheme 2. Alkene obtained from ketone 91 via treatment 9-BBN in THF, and 2,6-dibromopyridine Suzuki reaction conditions gave bromide reaction with with methyltriphenylphosphonium iodide andinsodium hydride in 75% yield. Subsequent 11′ in 78% yield. A Buchwald reaction led to intermediate 12′ (70% yield). Compound 15 treatment with 9-BBN in THF, and 2,6-dibromopyridine in Suzuki reaction conditions gave was bromide by acylation reactions using TBTU as a coupling12 reagent. 1 (70% yield). Compound 15 was prepared 111 inprepared 78% yield. A Buchwald reaction led to intermediate Compound 16 was synthesized as shown in Scheme 3. A Buchwald reaction with by acylation reactions using TBTU as a coupling reagent. 2,4-dichloropyrimidine 15′ in conditions described above gave a mixture of regioisomers that were Compound 16 was synthesized as shown in Scheme 3. A Buchwald reaction with separated by column chromatography in 45% yield of target compound 16′. This compound was

2,4-dichloropyrimidine 151 in conditions described above gave a mixture of regioisomers that were separated by column chromatography in 45% yield of target compound 161 . This compound was involved in a Suzuki reaction with alkene 101 and 9-BBN, followed by Boc-deprotection, and this led to compound 181 which was further acylated to compound 16. Then three of the most active compounds were tested for selectivity over a number of other kinases and for some of their ADME properties, where they showed rather good results (Table 3).

Pharmaceuticals 2016, 9, 19

6 of 14

involved in a 2016, Suzuki Pharmaceuticals 9, 19reaction with alkene 10′ and 9-BBN, followed by Boc-deprotection, and this6 led of 14 to compound 18′ which was further acylated to compound 16. Then9,in three of thereaction most active were tested for selectivity over a number ofthis other involved a Suzuki with compounds alkene 10′ and 9-BBN, followed by Boc-deprotection, and led Pharmaceuticals 2016, 19 kinases and for18′ some of their they 16. showed rather good results (Table 3). to compound which was ADME further properties, acylated to where compound Then three of the most active compounds were tested for selectivity over a number of other kinases O and for some of their ADME properties, where they showed rather good results (Table 3). a NO boc

b N boc

a

Br

H N N

Br

N H

boc

N

c

O

N H

13'

N

N N Hts N N 12'

N

N HH N N

f

ts

N N

c

11' H N N

f

NH

boc

N

N 13'

N

11'

10' NH

N HH N N

N

b

10' N boc

9' N boc 9'

N

15

boc d, e boc d, e

N N H F 12' Cl

O

N

N

6 of 14

F Cl

N

N

+ I15 ´ , NaH, 2. Synthesis of compound Conditions: (a) 3P+I−P THF; (b)THF; 2,6-dibromo-pyridine, Scheme 2.Scheme Synthesis of compound 15.15. Conditions: (a)MePh MePh (b) 2,6-dibromo-pyridine, 3 , NaH, 9-BBN, Pd(Ph3P)4, K2CO3, THF, RF; (c) 5-methyl-2-(toluene-4-sulfonyl)-2H-pyrazol-3-ylamine, 9-BBN, Pd(Ph3 P)4 , K2 CO3 , THF, RF; (c) 5-methyl-2-(toluene-4-sulfonyl)-2H-pyrazol-3-ylamine, +I−, NaH, 2CO3, Pd dba3, xantphos, toluene/H O, MW, 140 2 h;3P(d) NaOH/MeOH; (e) HCl/1,4-dioxane, Na Scheme 2. 2Synthesis of compound 15. 2Conditions: (a)°C, MePh THF; (b) 2,6-dibromo-pyridine, ˝ C, Na2 CO3 , RT, Pd ,Rxantphos, toluene/H MW, 140 2 h; (d) NaOH/MeOH; (e) HCl/1,4-dioxane, 24dba 2 O, RT, h; (f)3Pd(Ph 2COOH, N, CHRF; 3CN, (c) 8 h. 9-BBN, 3P)4, TBTU, K2CO3,Et3THF, 5-methyl-2-(toluene-4-sulfonyl)-2H-pyrazol-3-ylamine, RT, 4 h; (f)Na R22CO COOH, TBTU, Et N, CH CN, RT, 8 h. 3 3 3, Pd2dba3, xantphos, toluene/H2O, MW, 140 °C, 2 h; (d) NaOH/MeOH; (e) HCl/1,4-dioxane, ts RT,N4 h; (f) R2COOH, TBTU, N Et N RT, 8 h. N3N, CH3CN, a

Cl

N

Cl N

Cl

b

15' N

Nts N NH

a

N N

16' N H

Cl

15'

Cl b

N

Cl

NH

e

16' H N N

N

HN N NH

N NH

N

e

N N

ts

N

Nts N N NH N 17' N N H 17' H N N

N

HN N NH

N18' N H

N H

boc

N N

c, d

F

O

Cl

N

N N

boc

O N

c, d

F Cl

N

16

Scheme 3. Synthesis of compound 16. Conditions: (a) 5-methyl-2-(toluene-4-sulfonyl)-2H-pyrazol18' 16 3-ylamine, Na2CO3, Pd2dba3, xantphos, toluene/H2O, MW, 140 °C, 2 h; (b) 9-BBN, Pd(Ph3P)4, K2CO3, THF, RF; (c) RT; (d) HCl/1,4-dioxane, 4 h; (e) R2COOH, TBTU, Et3N, CH3CN, RT, 3. NaOH/MeOH, Synthesis of compound 16. Conditions: RT, (a) 5-methyl-2-(toluene-4-sulfonyl)-2H-pyrazolScheme 3.Scheme Synthesis of compound 16. Conditions: (a) 5-methyl-2-(toluene-4-sulfonyl)-2H-pyrazol-3-ylamine, 83-ylamine, h. °C, 2 h; (b) 9-BBN, Pd(Ph3P)4, K2CO3, Na2CO3, Pd2dba3, xantphos, toluene/H2O, MW, ˝ C,140 Na2 CO3 , Pd , xantphos, toluene/H2 O, MW, 140RT, 2 hpp; (b) 9-BBN, Pd(Ph3 P) 2 dba 4 , K2 CO3 , THF, 3CN, RT, THF, RF;3(c) NaOH/MeOH, RT; (d) HCl/1,4-dioxane, 4 h; (e) R2COOH, TBTU, Et3N, CH Table 3. Selectivity and some ADME properties for compounds 13, 14 and 16. RF; (c) NaOH/MeOH, RT; (d) HCl/1,4-dioxane, RT, 4 h; (e) R2 COOH, TBTU, Et3 N, CH3 CN, RT, 8 h. 8 h. 13 14 16 Ser/Thr and and some ADME properties for compounds 13, 14 and 16. TableKinases 3. Selectivity IC50, nM IC50, nM IC50, nM 1 ADME Parameters 2) (Mean ±13 SEMproperties (Meanfor ±14SEM) (Mean13, ±16SEM) Table 3. Selectivity and some ADME compounds 14 and 16. Ser/Thr Kinases Aurora A and 4.1 ±500.35 5.3 ±500.46 3.5 ±500.29 IC , nM , nM IC , nM IC 1 Parameters ADME Aurora B 232.2 ±± 24.1 18,300.0 4182.5 ±± 40.1 13 (Mean 16 SEM 2) (Mean ±±14185.0 SEM) (Mean SEM) Ser/Thr Kinases and c-Kit 17,000.00 ± 165 21,000 ± 250 N/A 1 Aurora A 4.1 ± 0.35 5.3 ± 0.46 2 ADME Parameters IC50 , nM (Mean ˘ SEM) 3.5 ± 0.29IC50 , nM (Mean ˘ SEM) IC50 , nM (Mean ˘ SEM )3 EGFR B N/A± 24.1 N/A± 185.0 N/A± 40.1 Aurora 232.2 18,300.0 4182.5 Aurora A 4.1 ˘ 0.35 5.3 ˘ 0.46 3.5 ˘ 0.29 CSK 9500.0 ± 101.0 N/A± 250 N/A c-Kit 17,000.00 ± 165 21,000 N/A Aurora B 232.2 ˘ 24.1 18,300.0 ˘ 185.0 4182.5 ˘ 40.1 EPHA2 9000.0 ± 985.0 N/A N/A 3 EGFR N/A N/A N/A c-Kit 17,000.00 ˘ 165 21,000 ˘ 250 N/A GSK-3 N/A± 101.0 109.0 ± 12.1 292.8N/A ± 31.2 CSK 9500.0 N/A Haspin N/A± 985.0 N/A N/A EGFR N/A N/A N/A 3 9000.0 EPHA2 N/A N/A JNK3 85.18N/A ± 9.1 1634.0 ±± 167 572.5 GSK-3 9500.0 ˘ 101.0 109.0N/A 12.1 292.8±±56.1 31.2 CSK N/A LYN 3270.0 ± 324.0 N/A N/A Haspin N/A N/A EPHA2 9000.0 ˘ 985.0 N/A N/A N/A PIM1 N/A± 9.1 N/A± 167 N/A± 56.1 JNK3 85.18 1634.0 572.5 GSK-3 109.0 ˘ 12.1 292.8 ˘ 31.2 N/A LYN 3270.0 ± 324.0 N/A N/A Haspin N/A N/A N/A PIM1 N/A N/A N/A JNK3

85.18 ˘ 9.1

1634.0 ˘ 167

572.5 ˘ 56.1

LYN

3270.0 ˘ 324.0

N/A

N/A

PIM1

N/A

N/A

N/A

PLK1

N/A

N/A

N/A

RON

970.0 ˘ 101.0

N/A

N/A

CYP1A2 4

N/A

N/A

N/A

CYP2C19

N/A

9597.0 ˘ 960.0

965.7 ˘ 94.1

CYP 2C9

N/A

3900.0 ˘ 401

326.6 ˘ 33.0

CYP3A4

N/A

15070.0 ˘ 151

596.2 ˘ 60.1

HERG

N/A

Solubility (nephelometry)

100 µg/mL

>100 µg/mL

1

Absorption, distribution, metabolism and excretion; 2 Standard error of mean; 3 Not available; 4 cytochrome P450 isoforms.

Pharmaceuticals 2016, 9, 19

7 of 14

The five active compounds were screened against 16 cell lines of both tumor and non-tumor origin (Table 4). Overall, Aurora A inhibitors were active against most tumor cell lines with uM IC50, with colon carcinoma Colo320 and acute myeloid leukemia RS4;11 being the most sensitive with submicromolar activity and 10–30 fold selectivity against human fibroblast (HFB) and Mouse Embryonic Fibroblast (MEF) cell lines. Cell lines that were resistant to inhibitors, HL-60, H23 and MDA-MB-231, are also resistant to Aurora A siRNA knockdown [9,10]. Mouse Embryonic Fibroblasts (MEF) with bax´/´ bak´/´ double knockout were less sensitive than with wild-type MEF, indicating that cell death occurred via the mitochondrial apoptotic pathway. Table 4. Cytotoxicity of selected Aurora A inhibitors, µM. Cell Line

11

12

13

15

16

HL-60 H23 H1299 PC3 MDA-MB-453 RS4;11 A-549 HCT-116 HFB A431 Colo-320 MDA-MB231 BxPC3 MEFs Dko 48 h MEF WT 48 h

5.4 ˘ 0.3 3.9 ˘ 0.15 1.7 ˘ 0.2 3.2 ˘ 0.2 10.0 ˘ 1.2 0.5 ˘ 0.04 5.6 ˘ 0.5 3.0 ˘ 0.4 3.6 ˘ 0.4 4.6 ˘ 0.5 0.8 ˘ 0.09 7.3 ˘ 0.8 2.0 ˘ 0.021 3.1 ˘ 0.32 1.0 ˘ 0.2

39.8 ˘ 3.4 6.8 ˘ 0.5 5.0 ˘ 0.4 6.0 ˘ 0.7 16.3 ˘ 1.7 1.5 ˘ 0.2 10.0 ˘ 1.2 6.5 ˘ 0.7 3.8 ˘ 0.4 10.2 ˘ 1.2 2.0 ˘ 0.15 28.2 ˘ 0.3 6.0 ˘ 0.59 11.8 ˘ 1.2 5.8 ˘ 0.5

20.9 ˘ 2.1 1.2 ˘ 0.09 1.1 ˘ 0.1 3.9 ˘ 0.4 8.0 ˘ 0.79 1.1 ˘ 0.11 5.1 ˘ 0.4 4.4 ˘ 0.5 2.8 ˘ 0.3 6.1 ˘ 0.7 1.0 ˘ 0.11 10.5 ˘ 1.1 2.4 ˘ 0.14 3.6 ˘ 0.3 2.5 ˘ 0.3

38.8 ˘ 3.2 >50.0 3.7 ˘ 0.3 9.7 ˘ 1.0 5.7 ˘ 0.6 0.1 ˘ 0.02 5.7 ˘ 0.6 2.8 ˘ 0.3 3.0 ˘ 0.2 6.8 ˘ 0.7 1.0 ˘ 0.12 >50 7.2 ˘ 0.6 3.0 ˘ 0.2 0.9 ˘ 0.08

>50.0 >50.0 1.5 ˘ 0.12 8.3 ˘ 0.8 12.0 ˘ 1.2 0.3 ˘ 0.02 8.8 ˘ 0.9 4.2 ˘ 0.4 3.3 ˘ 0.3 9.0 ˘ 1.0 1.2 ˘ 0.03 >50 8.1 ˘ 0.8 4.5 ˘ 0.5 0.9 ˘ 0.1

3. Experimental Section 3.1. Synthesis of Compounds 3.1.1. General Procedure of Buchwald Reaction A mixture of aryl bromide or aryl chloride (1 mmol), aromatic amine (1 mmol), 9,9-dimethyl-4, 5-bis(diphenylphosphino)xantene (0.029 g, 0.05 mmol), tris(dibenzylideneacetone)dipalladium(0)chloroform complex (0.026 g, 0.025 mmol), Na2 CO3 (0.159 g, 1.5 mmol) in toluene (20 mL) and water (1 mL) was stirred at 140 ˝ C under argon atmosphere in microwave reactor for 2 h. The resulting mixture was cooled down, diluted with water and extracted with ethyl acetate. The organic extracts were evaporated to give a crude product, which was further purified by a silica gel column chromatography. 3.1.2. General Procedure of Boc-Deprotection A solution of Boc-protected compound in 18% HCl in 1,4-dioxane (20 mL) was stirred for 4 h at 25 ˝ C (TLC control). The solvent was removed in vacuo, the residue was triturated with ethyl acetate; a precipitate was filtered off, washed with ethyl acetate and dried. 3.1.3. General Procedure of Acylation First, 3-Chloro-2-fluoro-benzoic acid (63 mg, 0.36 mmol), TBTU (116 mg, 0.36 mmol), amine dihydrochloride (0.3 mmol) and triethylamine (0.168 mL, 1.2 mmol) were dissolved in dry acetonitrile (5 mL). The mixture was stirred for 8 h at room temperature (TLC control). After the reaction was completed the mixture was concentrated to dryness in vacuo. Residue was treated with 10% potassium carbonate (10 mL). The resulting mixture was extracted with dichloromethane (2 ˆ 20 mL), organic extract was concentrated and purified by column chromatography.

Pharmaceuticals 2016, 9, 19

8 of 14

Scheme 1 4-Carbamoylmethyl-piperidine-1-carboxylic acid tert-butyl Ester (21 ). The suspension of 4-carboxymethylpiperidine-1-carboxylic acid tert-butyl ester (11 ) (3.04 g, 12.5 mmol), TBTU (4.49 g, 14 mmol), ammonium carbonate (2.4 g, 25 mmol) and triethylamine (5.32 mL, 38 mmol) in dry acetonitrile (80 mL) was stirred for 8 h at room temperature (TLC control). After the reaction was completed the mixture was evaporated to dryness in vacuo. Residue was treated with 10% aqueous solution of potassium carbonate (50 mL). The resulting mixture was extracted with dichloromethane (3 ˆ 100 mL), organic extract was evaporated. Purification by column chromatography on silica gel (eluent: hexane/ethyl acetate—1/1) afforded (21 ) (2.42 g, 83%). 1 H-NMR δH (400 MHz, d6 -DMSO): 0.98 (m, 2H, CH2 ), 1.37 (s, 9H, BOC), 1.61 (m, 2H, CH2 ), 1.79 (m, 1H, CH), 1.96 (m, 2H, CH2 ), 2.70 (m, 2H, CH2 ), 3.87 (d, 2H, CH2 ), 6.61 (br.s., 1H), 7.16 (br.s., 1H). APCI-MS (m/z (intensity)): 243.2 ([M + H]+ , 100%). 4-(4-Hydroxy-6-methyl-pyrimidin-2-ylmethyl)-piperidine-1-carboxylic acid tert-butyl Ester (41 ). To a solution of amide (21 ) (2.42 g, 10 mmol) in dichloromethane (100 mL), triethyloxoniumtetrafluoroborate (1.78 g, 12 mmol) was added at constant stirring; the resulted mixture was stirred for 2 h at room temperature. Then the reaction mixture was concentrated at ~45 ˝ C. The residue was treated with solution of NH3 /CH3 OH (pH should be 10), and left to stay overnight. The reaction mixture was evaporated to dryness, and the obtained product (31 ) was used in the next step without purification. APCI-MS (m/z (intensity)): 242.1 ([M + H]+ , 100%). Crude compound (31 ) was added to a solution of NaOt Bu (1.15 g, 12 mmol) in anhydrous ethanol (100 mL). The mixture was stirred at room temperature for 10 min, and then methyl 3-oxobutanoate (1.95 g, 15 mmol) was added. The resulting mixture was refluxed for 10 h (TLC control). Then the reaction mixture was concentrated under reduced pressure, dissolved in water and acidified with 1 N HCl to pH = 5.0 and extracted with ethyl acetate (2 ˆ 100 mL). An organic layer was dried over Na2 SO4 and concentrated in vacuum. The residue was purified by flash-chromatography on silica gel (eluent: hexane/ethyl acetate—1/1). As a result, compound (41 ) (1.99 g, 65%) was obtained. APCI-MS (m/z (intensity)): 308.2 ([M + H]+ , 100%). 4-(4-Chloro-6-methyl-pyrimidin-2-ylmethyl)-piperidine-1-carboxylic acid tert-butyl Ester (51 ). The compound (41 ) (1.99 g, 6.5 mmol) and dimethylaniline (7.08 g, 58.5 mmol) were dissolved in toluene (100 mL). POCl3 (2.99 g, 19.5 mmol) was added dropwise. The reaction mixture was refluxed for 3 h, cooled down to room temperature and poured into water. Organic layer was separated, washed with 1 N HCl and water. Organic layer was concentrated under reduced pressure and purified with flash chromatography (eluent: hexane/ethylacetate 4/1). Yield 1.3 g (62%). 1 H-NMR δH (400 MHz, CDCl3 ): 1.20–1.29 (m, 2H, CH2 ), 1.44 (s, 9H, BOC), 1.61 (m, 2H, CH2 ), 2.11 (m, 1H, CH), 2.49 (s, 3H, CH3 ), 2.71 (t, 2H, CH2 ), 2.83 (d, 2H, CH2 ), 4.07 (d, 2H, CH2 ), 7.03 (s, 1H, ArH). APCI-MS (m/z (intensity)): 325.4 ([M + H]+ , 100%), 327.4 ([M + H]+ , 35%). 4-[4-Methyl-6-(thiazol-2-ylamino)-pyrimidin-2-ylmethyl]-piperidine-1-carboxylic acid tert-butyl Ester (61 a) was prepared using Buchwald reaction, yield 292 mg (75%). 1 H-NMR δH (400 MHz, d6 -DMSO): 1.10 (m, 2H, CH2 ), 1.32 (s, 9H, BOC), 1.60 (m, 2H, CH2 ), 2.21 (m, 1H, CH), 2.30 (s, 3H, CH3 ), 2.67 (m, 4H, CH2 ), 3.86 (d, 2H, CH2 ), 6.67 (s, 1H, ArH), 7.12 (m, 1H, ArH), 7.40 (m, 1H, ArH), 11.39 (br.s., 1H, NH). APCI-MS (m/z (intensity)): 390.12 ([M + H]+ , 100%). 4-[4-Methyl-6-(5-methyl-thiazol-2-ylamino)-pyrimidin-2-ylmethyl]-piperidine-1-carboxylic acid tert-butyl Ester (61 b) was prepared using Buchwald reaction, yield 290 mg (72%). 1 H-NMR δH (400 MHz, d6 -DMSO): 1.09 (m, 2H, CH2 ), 1.36 (s, 9H, BOC), 1.61 (m, 2H, CH2 ), 2.14 (m, 1H, CH), 2.28 (s, 3H, CH3 ), 2.31 (s, 3H, CH3 ), 2.69(m, 4H, CH2 ), 3.88 (d, 2H, CH2 ), 6.62 (s, 1H, ArH), 7.04 (s, 1H, ArH), 11.13 (s, 1H, NH). APCI-MS (m/z (intensity)): 404.2 ([M + H]+ , 100%). 4-[4-Methyl-6-(5-chloro-thiazol-2-ylamino)-pyrimidin-2-ylmethyl]-piperidine-1-carboxylic acid tert-butyl Ester (61 c) was prepared from (61 a) (194 mg, 0.5 mmol) by reaction with N-chlorosuccinimide (67 mg,

Pharmaceuticals 2016, 9, 19

9 of 14

0.5 mmol) in dichloromethane (20 mL) at room temperature for 4 h. Residue was treated with 10% aqueous solution of potassium carbonate (10 mL). The organic extract was evaporated and purified by column chromatography on silica gel (eluent: hexane/ethyl acetate—4/1) to afford (61 c) (148 mg, 70%). 1 H-NMR δ (400 MHz, d -DMSO): 1.12 (m, 2H, CH ), 1.36 (s, 9H, BOC), 1.61 (m, 2H, CH ), 2.13 (m, H 2 2 6 1H, CH), 2.32 (s, 3H, CH3 ), 2.69 (m, 4H, CH2 ), 3.87 (d, 2H, CH2 ), 6.62 (s, 1H, ArH), 7.41 (s, 1H, ArH), 11.66 (s, 1H, NH). APCI-MS (m/z (intensity)): 423.4 ([M + H]+ , 100%), 425.5 ([M + H]+ , 30%). 4-[4-Methyl-6-(2H-pyrazol-3-ylamino)-pyrimidin-2-ylmethyl]-piperidine-1-carboxylic acid tert-butyl Ester (61 d). 2-Tosyl-2H-pyrazol-3-ylamine was obtained by reaction between 2H-pyrazol-3-ylamine and tosyl chloride (1 mmol) in presence of sodium bicarbonate (1.2 mmol) in acetonitrile at room temperature for 2 h (yield 60% after recrystallization from ethanol). Buchwald reaction led to tosyl-derivative of (61 d), yield 45%. The protective tosyl group was removed by treatment with 0.12 M NaOH in wet methanol at room temperature for 6 h. After reaction completion (TLC control) the mixture was poured into water (20 mL) and extracted with dichloromethane (3 ˆ 20 mL), dried over Na2 SO4 and concentrated in vacuum to give the titled compound (61 d). Yield 92%. 1 H-NMR δH (400 MHz, d6 -DMSO): 1.04 (m, 2H, CH2 ), 1.35 (s, 9H, BOC), 1.56 (m, 2H, CH2 ), 1.99 (m, 1H, CH), 2.21 (s, 3H, CH3 ), 2.51-2.65 (m, 4H, CH2 ), 3.86 (m, 2H, CH2 ), 6.33 (m, 1H, ArH), 6.85 (m, 1H, ArH), 7.58 (s, 1H, ArH), 9.63 (s, 1H, NH), 12.21 (s, 1H, NH). APCI-MS (m/z (intensity)): 373.2 ([M + H]+ , 100%). (6-Methyl-2-piperidin-4-ylmethyl-pyrimidin-4-yl)-thiazol-2-yl-amine Dihydrochloride (71 a) was prepared by Boc-deprotection. Yield 287 mg (99%). 1 H-NMRδH (400 MHz, d6 -DMSO): 1.57 (m, 2H, CH2 ), 1.86 (m, 2H, CH2 ), 2.33 (m, 1H, CH), 2.56 (s, 3H, CH3 ), 2.84 (m, 2H, CH2 ), 3.07 (m, 2H, CH2 ), 3.19 (m, 2H, CH2 ), 7.03 (br.s, 1H, ArH), 7.41 (m, 1H, ArH), 7.56 (s, 1H, ArH), 8.96 (m, 1H), 9.52 (m, 1H). APCI-MS (m/z (intensity)): 290.2 ([M + H]+ , 100%). (6-Methyl-2-piperidin-4-ylmethyl-pyrimidin-4-yl)-(5-methyl-thiazol-2-yl)-amine Dihydrochloride (71 b) was prepared by Boc-deprotection. Yield 265 mg (98%). 1 H-NMR δH (400 MHz, d6 -DMSO): 1.56 (m, 2H, CH2 ), 1.86 (m, 2H, CH2 ), 2.33 (m, 1H, CH), 2.45 (s, 3H, CH3 ), 2.52 (s, 3H, CH3 ), 2.84 (m, 2H, CH2 ), 2.98 (m, 2H, CH2 ), 3.23 (m, 2H, CH2 ), 6.92 (br.s, 1H, ArH), 7.26 (s, 1H, ArH), 8.95 (m, 1H), 9.20 (m, 1H). APCI-MS (m/z (intensity)): 304.2 ([M + H]+ , 100%). (5-Chloro-thiazol-2-yl)-(6-methyl-2-piperidin-4-ylmethyl-pyrimidin-4-yl)-amine Dihydrochloride (71 c) was prepared by Boc-deprotection. Yield 136 mg (98%). 1 H-NMR δH (400 MHz, d6 -DMSO): 1.55 (m, 2H, CH2 ), 1.84 (m, 2H, CH2 ), 2.28 (m, 1H, CH), 2.51 (s, 3H, CH3 ), 2.83 (m, 2H, CH2 ), 2.98 (m, 2H, CH2 ), 3.23 (m, 2H, CH2 ), 6.92 (s, 1H, ArH), 7.54 (s, 1H, ArH), 8.77 (m, 1H), 9.02 (m, 1H). APCI-MS (m/z (intensity)): 323.5 ([M + H]+ , 100%), 325.5 ([M + H]+ , 32%). (6-Methyl-2-piperidin-4-ylmethyl-pyrimidin-4-yl)-(2H-pyrazol-3-yl)-amine Dihydrochloride (71 d) was prepared by Boc-deprotection. Yield 140 mg (99%). APCI-MS (m/z (intensity)): 273.1 ([M + H]+ , 100%). (3-Chloro-2-fluoro-phenyl)-{4-[4-methyl-6-(thiazol-2-ylamino)-pyrimidin-2-ylmethyl]-piperidin-1-yl}-methanone (12) was prepared by Boc-deprotection. Yield 113 mg (85%). M.p. 207–209 ˝ C. 1 H-NMR δH (400 MHz, d6 -DMSO): 1.09–1.26 (m, 2H), 1.64 (m, 1H), 1.80 (m, 1H), 2.23 (s, 3H), 2.35 (m, 1H), 2.65 (m, 2H), 2.84 (m, 1H), 3.04 (m, 1H), 3.33 (m, 1H), 4.43 (m, 1H), 6.45 (s, 1H), 6.89 (d, 1H), 7.28 (m, 3H), 7.62 (m, 2H). 13 C-NMR δ (400 MHz, d6 -DMSO): 23.45 (CH3 ), 31.30 (CH), 32.06 (CH2 ), 34.43 (CH2 ), 41.31 (CH2 ), 44.82 (CH2 ), 46.59 (CH2 ), 103.22 (CH), 112.25 (CH), 119.9 (d, J = 20 Hz, C), 125.80 (d, J = 4 Hz, CH), 125.91 (d, J = 20 Hz, C), 126.19 (CH), 127.09 (d, J = 4 Hz, CH), 131.08 (CH), 137.28 (CH), 152.77 (d, J = 247 Hz, C), 157.63 (C), 159.91 (C), 162.40 (C), 164.50 (C), 167.01 (C). APCI-MS (m/z (intensity)): 445.9 ([M + H]+ , 100%), 447.7([M + H]+ , 40%). HRMS (ESI): Calcd for C21 H21 ClFN5 OS ([M + H]+ ), 446.1212; found, 446.1219. (3-Chloro-2-fluoro-phenyl)-{4-[4-methyl-6-(5-methyl-thiazol-2-ylamino)-pyrimidin-2-ylmethyl]-piperidin-1-yl}methanone (11) was prepared by Boc-deprotection. Yield 120 mg (87%). M. p. 211–213 ˝ C. 1 H-NMR δH (400 MHz, d6 -DMSO): 1.11–1.27 (m, 2H), 1.62 (m, 1H), 1.78 (m, 1H), 2.27 (s, 3H), 2.34 (s, 3H), 2.72

Pharmaceuticals 2016, 9, 19

10 of 14

(m, 2H), 2.83 (m, 1H), 3.05 (m, 1H), 3.34 (m, 1H), 3.45 (m, 1H), 6.61 (s, 1H), 7.05 (s, 1H), 7.25–7.36 (m, 2H), 7.62 (m, 1H), 11.18 (br.s., 1H). 13 C-NMR δ (400 MHz, d6 -DMSO): 10.87 (CH3 ), 23.46 (CH3 ), 31.25 (CH), 31.98 (CH2 ), 34.43 (CH2 ), 41.29 (CH2 ), 44.71 (CH2 ), 46.56 (CH2 ), 102.41 (CH), 119.9 (d, J = 19 Hz, C), 125.48 (C), 125.76 (d, J = 4 Hz, CH), 126.09 (d, J = 19 Hz, C), 127.08 (d, J = 4 Hz, CH), 131.06 (CH), 134.43 (CH), 152.77 (d, J = 247 Hz, C), 156.81 (C), 156.91 (C), 162.38 (C), 162.03 (C), 167.16 (C). APCI-MS (m/z (intensity)): 459.8 ([M + H]+ , 100%), 461.7 ([M + H]+ , 37%). HRMS (ESI): Calcd for C22 H23 ClFN5 OS ([M + H]+ ), 460.1369; found, 460.1367. (3-Chloro-2-fluoro-phenyl)-{4-[4-(5-chloro-thiazol-2-ylamino)-6-methyl-pyrimidin-2-ylmethyl]-piperidin-1-yl}methanone (13) was prepared by Boc-deprotection. Yield 130 mg (90%). M. p. 221–223 ˝ C. 1 H-NMR δH (400 MHz, d6 -DMSO): 1.12–1.27 (m, 2H), 1.63 (m, 1H), 1.79 (m, 1H), 2.29 (s, 3H), 2.73 (m, 2H), 2.83 (m, 1H), 3.06 (m, 1H), 3.35 (m, 1H), 4.45 (m, 1H), 6.62 (s, 1H), 7.39 (s, 1H), 7.27–7.37 (m, 2H), 7.63 (m, 1H), 11.65 (s, 1H). APCI-MS (m/z (intensity)): 480.09 ([M + H]+ , 100%), 482.09 ([M + H]+ , 68%). HRMS (ESI): Calcd for C21 H20 Cl2 FN5 OS ([M + H]+ ), 480.0822; found, 480.0825. (3-Chloro-2-fluoro-phenyl)-{4-[4-methyl-6-(2H-pyrazol-3-ylamino)-pyrimidin-2-ylmethyl]-piperidin-1-yl}methanone (14) was prepared by Boc-deprotection. Yield 114 mg (89%). M. p. 125–128 ˝ C. 1 H-NMR δH (400 MHz, d6 -DMSO): 1.11–1.34 (m, 2H), 1.62 (m, 1H), 1.83 (m, 1H), 2.16 (m, 1H), 2.25 (s, 3H), 2.60 (m, 2H), 2.81 (m, 1H), 3.06 (m, 1H), 3.29 (m, 1H), 4.43 (m, 1H), 6.35 (s, 1H), 6.89 (s, 1H), 7.26–7.36 (m, 2H), 7.56-7.64 (m, 2H), 9.55 (s, 1H), 12.25 (s, 1H). APCI-MS (m/z (intensity)): 428.8 ([M + H]+ , 100%), 430.8 ([M + H]+ , 41%). HRMS (ESI): Calcd for C21 H21 ClFN6 O ([M + H]+ ), 429.1600; found, 429.1608. Scheme 2 4-Methylene-piperidine-1-carboxylic acid tert-butyl Ester (101 ). NaH in mineral oil (2.28 g, 57 mmol, 60%) was added portion-wise under argon atmosphere to DMSO (35 mL). The reaction mixture was stirred on a water bath up to 75 ˝ C for about 20 min until completion of hydrogen formation. The reaction mixture was cooled down to 20 ˝ C and treated with methyl triphenylsulfonium iodide (23.1 g, 57 mmol). After stirring for about 15 min, ketone (91 ) (9.96 g, 50 mmol) in THF (15 mL) was added. The mixture was stirred at 25 ˝ C for about 2 h and diluted with water, ethyl acetate and hexane. The organic layer was washed with water (2 ˆ 25 mL), diluted with dichloromethane and washed with brine. The combined aqueous layers were extracted with ethyl acetate (50 mL) and hexane (50 mL). The organic layer was filtered through SiO2 . The combined filtrate was concentrated and resulted in the title compound. Yield 7.4 g (75%). 4-(6-Bromo-pyridin-2-ylmethyl)-piperidine-1-carboxylic acid tert-butyl Ester (111 ). To a solution of alkene (101 ) (7.4 g, 37.5 mmol) in 50 mL of anhydrous THF 9-borabicyclo[3.3.1]nonane (90 mL, 0.5 M solution in THF, 45 mmol) was added under argon atmosphere. A resulted solution was stirred at room temperature for 24 h, then 2,6-dibromopyridine (8.9 g, 37.5 mmol), potassium carbonate (7.65 g, 56 mmol), water (15 mL), and catalyst Pd(PPh3 )4 (2.16 g, 1.87 mmol) were added therein. The reaction mixture was refluxed for 4 h, cooled to room temperature, poured into water (100 mL) and extracted with ethyl acetate (2 ˆ 100 mL). The combined organic extracts were evaporated to give a crude oil, which was purified by a silica gel column chromatography to give the titled compound. Yield 10.3 g (78%). 1 H-NMR δH (400 MHz, d6 -DMSO): 1.06 (m, 2H, CH2 ), 1.35 (s, 9H, BOC), 1.47 (m, 2H, CH2 ), 1.69–1.87 (m, 2H, CH2 ), 2.65 (m, 3H, CH, CH2 ), 3.84 (d, 2H, CH2 ), 7.26 (m, 1H, ArH), 7.46 (m, 1H, ArH), 7.64 (m, 1H, ArH). APCI-MS (m/z (intensity)): 355.1 ([M + H]+ , 100%), 357.0 ([M + H]+ , 80%). 4-{6-[5-Methyl-2-(toluene-4-sulfonyl)-2H-pyrazol-3-ylamino]-pyridin-2-ylmethyl}-piperidine-1-carboxylic acid tert-butyl Ester (121 ). Compound (121 ) was prepared by Buchwald reaction, yield 367 mg (70%). 5-Methyl-2-(toluene-4-sulfonyl)-2H-pyrazol-3-ylamine was obtained similar to compound (61 d) by reaction of 5-methyl-2H-pyrazol-3-ylamine and tosyl chloride (65%). APCI-MS (m/z (intensity)): 526.4 ([M + H]+ , 100%).

Pharmaceuticals 2016, 9, 19

11 of 14

(5-Methyl-2H-pyrazol-3-yl)-(6-piperidin-4-ylmethyl-pyridin-2-yl)-amine Dihydrochloride (131 ). Tosyl- and Boc-protective groups of the compound (121 ) were removed by subsequent treatment with NaOH in methanol and HCl in 1,4-dioxane). Yield 170 mg (90%). 1 H-NMR δH (400 MHz, d6 -DMSO): 1.11 (m, 2H, CH2 ), 1.38 (s, 9H, BOC), 1.61 (m, 2H, CH2 ), 1.93 (m, 1H, CH), 2.27 (s, 3H, CH3 ), 2.63–2.79 (m, 4H, CH2 ), 3.78 (d, 2H, CH2 ), 5.96 (s, 1H, ArH), 6.93 (d, 1H, ArH), 7.26 (d, 1H, ArH), 7.94 (m, 2H, ArH, NH), 11.4 (br.s., 1H, NH). APCI-MS (m/z (intensity)): 372.2 ([M + H]+ , 100%). (3-Chloro-2-fluoro-phenyl)-{4-[6-(5-methyl-2H-pyrazol-3-ylamino)-pyridin-2-ylmethyl]-piperidin-1-yl}-Methanone (15) was prepared by acylation general method. Yield 103 mg (80%). M. p. 120–122 ˝ C. 1 H-NMR δH (400 MHz, d6 -DMSO): 1.10–1.25 (m, 2H), 1.58 (m, 1H), 1.74 (m, 1H), 2.02 (m, 1H), 2.18 (s, 3H), 2.53 (m, 2H), 2.79 (m, 1H), 3.00 (m, 1H), 3.32 (m, 1H), 4.43 (m, 1H), 5.90 (s, 1H), 6.50 (d, 1H), 7.05 (s, 1H), 7.23–7.41 (m, 3H), 7.62 (m, 1H), 8.76 (s, 1H), 11.61 (s, 1H). APCI-MS (m/z (intensity)): 427.8 ([M + H]+ , 100%), 429.7 ([M + H]+ , 38%). HRMS (ESI): Calcd for C22 H23 ClFN5 O ([M + H]+ ), 428.1648; found, 428.1642. Scheme 3 (2-Chloro-pyrimidin-4-yl)-[5-methyl-2-(toluene-4-sulfonyl)-2H-pyrazol-3-yl]-amine (161 ). Compound (161 ) was prepared by Buchwald reaction with 5-methyl-2-(toluene-4-sulfonyl)-2H-pyrazol-3-ylamine. Yield 164 mg (45%). APCI-MS (m/z (intensity)): 363.6 ([M + H]+ , 100%), 365.4 ([M + H]+ , 30%). 4-{4-[5-Methyl-2-(toluene-4-sulfonyl)-2H-pyrazol-3-ylamino]-pyrimidin-2-ylmethyl}-piperidine-1-carboxylic acid tert-butyl Ester (171 ). Compound (171 ) was prepared by method described for compound (111 ) from alkene (101 ) (99 mg, 0.5 mmol) and chloride (161 ) (164 mg, 0.45 mmol), yield 149 mg (63%). APCI-MS (m/z (intensity)): 527.4 ([M + H]+ , 100%). (5-Methyl-2H-pyrazol-3-yl)-(2-piperidin-4-ylmethyl-pyrimidin-4-yl)-amine Dihydrochloride (181 ). Compound (181 ) was prepared by method described for compound (131 ), yield 91 mg (93%). APCI-MS (m/z (intensity)): 273.1([M + H]+ , 100%). (3-Chloro-2-fluoro-phenyl)-{4-[4-(5-methyl-2H-pyrazol-3-ylamino)-pyrimidin-2-ylmethyl]-piperidin-1-yl}-methanone (16) was prepared by acylation general method. Yield 96 mg (86%). M. p. 115–118 ˝ C. 1 H-NMR δH (400 MHz, d6 -DMSO): 1.11–1.25 (m, 2H), 1.60 (m, 1H), 1.76 (m, 1H), 2.17 (s, 3H), 2.62 (m, 2H), 2.81 (m, 1H), 3.04 (m, 1H), 3.33 (m, 1H), 4.45 (m, 1H), 6.08 (s, 1H), 7.01 (s, 1H), 7.25–7.36 (m, 2H), 7.61 (m, 1H), 8.14 (d, 1H), 9.50 (s, 1H), 11.85 (s, 1H). 13 C-NMR δ (400 MHz, d6 -DMSO): 10.55 (CH3 ), 31.27 (CH), 31.98 (CH2 ), 34.71 (CH2 ), 41.30 (CH2 ), 45.01 (CH2 ), 46.57 (CH2 ), 95.14 (CH), 103.17 (CH), 119.90 (d, J = 20 Hz, C), 125.77 (d, J = 4 Hz, CH), 126.09 (d, J = 20 Hz, C), 127.07 (d, J = 4 Hz, CH), 131.06 (CH), 138.28 (CH), 148.28 (C), 152.76 (d, J = 247 Hz, C), 155.36 (C), 159.22 (C), 162.36 (C), 167.90 (C). APCI-MS (m/z (intensity)): 428.8 ([M + H]+ , 100%), 430.7 ([M + H]+ , 40%). HRMS (ESI): Calcd for C21 H22 ClFN6 O ([M + H]+ ), 429.1600; found, 429.1602. 3.2. Analytical Data for Compounds 1–10 from the Table 1 2-Amino-2-methyl-1-(4-(6-(5-propylthiazole-2-ylamino)pyridin-2-yl)piperidin-1-yl)propan-1-one hydrochloride (1). 1 H-NMR δH (400 MHz, d6 -DMSO): 0.92 (t, 3H, CH3 ), 1.61 (m, 8H, CH3 , CH2 ), 1.74 (m, 2H, CH2 ), 1.97 (m, 2H, CH2 ), 2.63 (m, 2H, CH2 ), 3.03 (m, 3H, CH, CH2 ), 6.96 (d, 1H, ArH), 7.03 (d, 1H, ArH), 7.20 (s, 1H, ArH), 8.27 (br.s., 3H, NH, HCl). APCI-MS (m/z (intensity)): 388.4 ([M + H]+ , 100%). HRMS (ESI): Calcd for C20 H30 N5 OS+ ([M + H]+ ), 388.2166; found, 388.2171. (3-Chloro-2-flurophenyl)(3-((4-methylthiazol-2-ylamino)pyrimidin-2-yl)methyl)pirrolidine-1-yl)methanone (2). 1 H-NMR δ (400 MHz, d -DMSO): 1.67 (m, 1H, CH ), 2.02 (m, 1H, CH), 2.32 (s, 3H, CH ), 2.82 (m, H 2 3 6 2H, CH2 ), 2.90 (m, 1H, CH2 ), 3.22 (m, 1H, CH2 ), 3.35–3.47 (m, 2H, CH2 ), 3.56–3.72 (m, 1H, CH2 ), 6.78 (m, 1H, ArH), 7.07 (m, 1H, ArH), 7.17–7.30 (m, 1H, ArH), 7.37 (m, 1H, ArH), 7.56–7.66 (m, 1H, ArH),

Pharmaceuticals 2016, 9, 19

12 of 14

8.28 (m, 1H, ArH), 11.43 (s, 1H, NH). APCI-MS (m/z (intensity)): 432.7 ([M + H]+ , 100%). HRMS (ESI): Calcd for C20 H30 ClFN5 OS+ ([M + H]+ ), 432.1056; found, 432.1080. 2-(Dimethylamino)-1-(4-(6-(5-propylthiazol-2-ylamino)pyridin2-yl)piperidin-1-yl)ethanone (3). 1 H-NMR δH (400 MHz, d6 -DMSO): 0.91 (t, 3H, CH3 ), 1.65 (m, 2H, CH2 ), 1.72-1.85 (m, 4H, CH2 ), 2.26 (s, 6H, CH3 ), 2.66 (m, 3H, CH2 ), 2.91 (m, 1H, CH), 3.10–3.29 (m, 3H, CH2 ), 4.15 (d, 1H, CH2 ), 4.49 (d, 1H, CH2 ), 6.74 (d, 1H, ArH), 6.83 (d, 1H, ArH), 7.02 (s, 1H, ArH), 10.82 (br.s., 1H, NH). APCI-MS (m/z (intensity)): 388.4 ([M + H]+ , 100%). HRMS (ESI): Calcd for C20 H30 N5 OS+ ([M + H]+ ), 388.2166; found, 388.2159. 2-Amino-1-(4-(6-(5-propylthiazol-2-ylamino)pyridin-2-yl)piperidin-1-yl)ethanone (4). 1 H-NMR δH (400 MHz, d6 -DMSO): 1.91 (t, 3H, CH3 ), 1.45 (m, 2H, CH2 ), 1.65-1.91 (m, 4H, CH2 ), 2.65 (m, 2H, CH2 ), 2.90 (m, 1H, CH), 3.01–3.19 (m, 4H), 3.36 (m, 2H, CH2 ), 3.89 (m, 1H, CH2 ), 4.47 (m, 1H, CH2 ), 6.64 (d, 1H, ArH), 6.84 (d, 1H, ArH), 7.01 (s, 1H, ArH), 7.55 (m, 1H, ArH). APCI-MS (m/z (intensity)): 360.4 ([M + H]+ , 100%). HRMS (ESI): Calcd for C18 H26 N5 OS+ ([M + H]+ ), 360.1853; found, 360.1858. 2-(Methylamino)-1-(4-(6-(5-propylthiazol-2-ylamino)pyridin-2-yl)piperidin-1-yl)ethanone (5). 1 H-NMR δH (400 MHz, d6 -DMSO): 1.92 (t, 3H, CH3 ), 1.61 (m, 2H, CH2 ), 1.71–1.90 (m, 4H, CH2 ), 2.31 (m, 3H, CH3 ), 2.64 (m, 3H, CH2 ), 2.91 (m, 1H, CH), 3.02–3.33 (m, 4H, CH2 ), 3.95 (m, 1H, CH2 ), 4.52 (m, 1H, CH2 ), 6.74 (d, 1H, ArH), 6.30 (d, 1H, ArH), 7.01 (s, 1H, ArH), 7.55 (m, 1H, ArH). APCI-MS (m/z (intensity)): 374.3 ([M + H]+ , 100%). HRMS (ESI): Calcd for C19 H28 N5 OS+ ([M + H]+ ), 374.2009; found, 374.2015. (R)-2-Amino-1-(4-(6-(5-propylthiazol-2-ylamino)-pyridin-2-yl)piperidin-1-yl)propan-1-one (6). 1 H-NMR δH (400 MHz, d6 -DMSO): 0.91 (t, 3H, CH3 ), 1.11 (m, 3H), 1.57 (m, 3H), 1.67-1.96 (m, 6H), 2.63 (m, 3H), 2.91 (m, 1H, CH), 3.78 (m, 1H, CH2 ), 4.05 (m, 1H, CH2 ), 4.51 (m, 1H, CH2 ), 6.75 (d, 1H, ArH), 6.83 (d, 1H, ArH), 7.02 (s, 1H, ArH), 7.55 (m, 1H, ArH). APCI-MS (m/z (intensity)): 374.3 ([M + H]+ , 100%). HRMS (ESI): Calcd for C19 H28 N5 OS+ ([M + H]+ ), 374.2009; found, 374.2017. 2-Amino-1-(4-(4-isopropyl-6-(5-propylthiazol-2-ylamino)pyrimidin-2-yl)piperidin-1-yl)-2-methylpropan-1-one (7). 1 H-NMR δH (400 MHz, d6 -DMSO): 1.91 (t, 3H, CH3 ), 1.17 (d, 6H, CH3 ), 1.29 (s, 6H, CH3 ), 1.59 (q, 2H, CH2 ), 1.79–1.93 (m, 4H, CH2 ), 1.65 (m, 2H, CH2 ), 1.80 (m, 1H, CH), 2.92 (m, 3H, CH, CH2 ), 4.75 (m, 2H, CH2 ), 6.63 (s, 1H, ArH), 7.09 (s, 1H, ArH). APCI-MS (m/z (intensity)): 431.4 ([M + H]+ , 100%). HRMS (ESI): Calcd for C22 H35 N6 OS+ ([M + H]+ ), 431.2588; found, 431.2591. 2-Amino-1-(4-(4-ethyl-6-(5-propylthiazol-2-ylamino)pyrimidin-2-yl)piperidin-1-yl)-2-methylpropan-1-one (8). δH (400 MHz, d6 -DMSO): 0.93 (t, 3H, CH3 ), 1.14 (t, 3H, CH3 ), 1.31 (s, 6H, CH3 ), 1.57 (q, 2H, CH2 ), 1.81 (m, 2H, CH2 ), 1.90 (m, 2H, CH2 ), 2.57 (q, 2H, CH2 ), 1.64 (m, 2H, CH2 ), 2.93 (m, 3H, CH, CH2 ), 4.75 (m, 2H, CH2 ), 6.63 (s, 1H, ArH), 7.09 (s, 1H, ArH). APCI-MS (m/z (intensity)): 417.4 ([M + H]+ , 100%). HRMS (ESI): Calcd for C21 H33 N6 OS+ ([M + H]+ ), 417.2431; found, 417.2344.

1 H-NMR

2-Amino-2-methyl-1-(4-(6-(5-phenylthiazol-2-ylamino)pyridine-2-yl)propan-1-one hydrochloride (9). 1 H NMR δH (400 MHz, d6 -DMSO): 1.59 (s, 6H, CH3 ), 1.81 (m, 2H, CH2 ), 2.01 (m, 3H), 3.04 (m, 4H), 6.87 (d, 1H, ArH), 6.95 (d, 1H, ArH), 7.27 (m, 1H, ArH), 7.40 (t, 2H, ArH), 7.53 (m, 2H, ArH), 7.66 (t, 1H, ArH), 7.78 (s, 1H, ArH), 8.86 (m, 3H, NH, HCl). APCI-MS (m/z (intensity)): 422.4 ([M + H]+ , 100%). HRMS (ESI): Calcd for C23 H28 N5 OS+ ([M + H]+ ), 422.2009; found, 422.2014. Morpholin-2-yl(4-(6-(5-phenylthiazol-2-ylamino)pyridin-2-yl)piperidin-1-yl)methanone (10). 1 H-NMR δH (400 MHz, d6 -DMSO): 1.61–1.92 (m, 5H, CH2 ), 2.63–3.11 (m, 7H, CH, CH2 ), 3.51 (m, 1H, CH), 3.69 (m, 1H, CH2 ), 4.13 (m, 2H, CH2 ), 4.48 (m, 1H, NH), 6.82 (d, 1H, ArH), 6.89 (d, 1H, ArH), 7.25 (m, 1H, ArH), 7.38 (t, 2H, ArH), 7.52 (m, 2H, ArH), 7.61 (t, 1H, ArH), 7.74 (s, 1H, ArH), 11.20 (br.s., 1H, NH). APCI-MS (m/z (intensity)): 450.23 ([M + H]+ , 100%). HRMS (ESI): Calcd for C24 H28 N5 O2 S+ ([M + H]+ ), 450.1958; found, 450.1960.

Pharmaceuticals 2016, 9, 19

13 of 14

3.3. Cytotoxicity Assay 3.3.1. Cell Lines All cells lines were purchased in ATCC (Manassas, VA, USA), except for human primary fibroblasts (HFB), which were a generous gift from Dashinimaev E.B. (Koltsov Institute of Developmental Biology). H1299 (lung adenocarcinoma), H23 (lung adenocarcinoma), Colo320 (colon carcinoma), RS4;11 (acute lymphoblastic leukemia), PC3 (prostate adenocarcinoma) and HL-60 (acute promyelocytic leukemia) were cultivated in RPMI-1640 medium, with addition of 10% fetal calf serum (HyClone, South Logan, Utah, USA) and 2 mM L-Glutamine at 37 ˝ C, 5% CO2 . MDA-MB-453 (mammary adenocarcinoma), A549 (lung adenocarcinoma), HCT-116 (colon carcinoma), A431 (epidermoid carcinoma), MDA-MB-231 (mammary adenocarcinoma), BxPC3 (pancreatic adenocarcinoma), MEF (mouse embryonic fibroblasts, SV-40 immortalized), MEF Bax´/´ /Bak´/´ knockout cells (mouse embryonic fibroblasts, SV-40 immortalized, resistant to apoptosis) were cultivated in DMEM medium, with addition of 10% fetal calf serum (HyClone) and 2 mM L-Glutamine at 37 ˝ C, 5% CO2 . 3.3.2. Cytotoxicity Cells were seeded in 96-well plates (Corning, NY, USA): 5000 cells per well for adhesive cultures and cells, 25,000 cells per well for suspension cultures. Analyzed substances were added in 0.1–50 µM concentration range, with 2 ˆ dilutions, and incubated for 72 h at 37 ˝ C, 5% CO2 . After incubation 20 uL of 5 mg/mL of water solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, PANEKO, Moscow, Russia) was added to the cells and they were incubated for additional 4 h. After the incubation the medium was aspirated, cells were dissolved in 100 uL of DMSO and the optical density was measured at 570 nm with a MultiscanFC plate reader (ThermoScientific, Cambridge, MA, USA). The percentage of surviving cells at each dose was calculated by dividing the optical density of treated cells by the optical density of untreated control (which was taken as 100%). The IC50s were calculated in GraphPad Prism 5.0. 4. Conclusions Based on the literature and proprietary data on various Ser/Thr kinase inhibitors, a general pharmachophore model was developed. A search for the molecules fitting to this pharmacophore among the ASINEX library revealed a number of compounds, which were tested and appeared to possess some activity against Aurora A, Aurora B and HaspinSer/Thr kinases. During optimization of these molecules for Aurora A we found several hits with 3–5 nM activity, which were cytotoxic against 16 cancer cell lines with 10–30 fold selectivity against primary cells. Thus, we showed the possibility of fine-tuning the general Ser/Thr pharmacophore designed for desired types of kinase to obtain active and selective compounds. Acknowledgments: The authors gratefully acknowledge support from the Ministry of Education and Science of the Russian Federation for funding (agreement 14.576.21.0019 dated 27 July 2014). Author Contributions: Natalya I. Vasilevich and Ilya I. Afanasyev conceived and design the experiments, Natalya I. Vasilevich wrote the paper, Ilya I. Afanasyev performed the modeling; Victor V. Tatarskiy, Jr. performed biological experiments; Elena A. Aksenova and Denis N. Kazyulkin designed and performed synthesis and analyzed chemical compounds. Conflicts of Interest: The authors declare no conflict of interest.

References 1. 2.

Rask-Andersen, M.; Zhang, J.; Fabbro, D.; Schiöth, H.B. Advances in kinase targeting: Current clinical use and clinical trials. Trends Pharmacol. Sci. 2014, 11, 604–620. [CrossRef] [PubMed] Seal, A.; Yogeeswari, P.; Sriram, D.; Consortium, O.; Wild, D.J. Enhanced ranking of PknB Inhibitors using data fusion methods. J. Cheminform. 2013, 5. [CrossRef] [PubMed]

Pharmaceuticals 2016, 9, 19

3.

4. 5.

6.

7.

8.

9.

10.

14 of 14

Danilenko, V.N.; Osolodkin, D.I.; Lakatosh, S.A.; Preobrazhenskaya, M.; Shtil, A.A. Bacterial eukaryotic type serine-threonine protein kinases: From structural biology to targeted anti-infective drug design. Curr. Top. Med. Chem. 2011, 11, 1352–1369. [CrossRef] [PubMed] Tanneeru, K.; Guruprasad, L. Ligand-based 3-D pharmacophore generation and molecular docking of mTOR kinase inhibitors. J. Mol. Model. 2012, 18, 1611–1624. [CrossRef] [PubMed] Wang, H.Y.; Li, L.L.; Cao, Z.X.; Luo, S.D.; Wei, Y.Q.; Yang, S.Y. A specific pharmacophore model of Aurora B kinase inhibitors and virtual screening studies based on it. Chem. Biol. Drug Des. 2009, 73, 115–126. [CrossRef] [PubMed] Chavan, S.R.; Dash, R.C.; Alam, M.S.; Hirwani, R.R. Identification of new novel scaffold for Aurora A inhibition by pharmacophore modeling and virtual screening. Mol. Divers. 2014, 18, 853–863. [CrossRef] [PubMed] Deng, X.Q.; Wang, H.Y.; Zhao, Y.L.; Xiang, M.L.; Jiang, P.D.; Cao, Z.X.; Zheng, Y.Z.; Luo, S.D.; Yu, L.T.; Wei, Y.Q.; et al. Pharmacophore modelling and virtual screening for identification of new Aurora-A kinase inhibitors. Chem. Biol. Drug Des. 2008, 71, 533–539. [CrossRef] [PubMed] Xie, H.; Chen, L.; Zhang, J.; Xie, X.; Qiu, K.; Fu, J. A combined pharmacophore modeling, 3D QSAR and virtual screening studies on imidazopyridines as B-Raf inhibitors. Int. J. Mol. Sci. 2015, 16, 12307–12323. [CrossRef] [PubMed] Yang, H.; Lawrence, H.R.; Kazi, A.; Gevariya, H.; Patel, R.; Luo, Y.; Rix, U.; Schonbrunn, E.; Lawrence, N.J.; Sebti, S.M. Dual Aurora A and JAK2 kinase blockade effectively suppresses malignant transformation. Oncotarget 2014, 5, 2947–2961. [CrossRef] [PubMed] Liu, L.L.; Long, Z.J.; Wang, L.X.; Zheng, F.M.; Fang, Z.G.; Yan, M.; Xu, D.F.; Chen, J.J.; Wang, S.W.; Lin, D.J.; et al. Inhibition of mTOR pathway sensitizes acute myeloid leukemia cells to aurora inhibitors by suppression of glycolytic metabolism. Mol. Cancer Res. 2013, 11, 1326–1336. [CrossRef] [PubMed] © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons by Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).

Thr Kinase Pharmacophore Model.

Based on the data for compounds known from the literature to be active against various types of Ser/Thr kinases, a general pharmachophore model for th...
4MB Sizes 0 Downloads 8 Views