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“Addition” and “Subtraction”: Selectivity Design for TypeII Maternal Embryonic Leucine Zipper Kinase Inhibitors Xin Chen, John Giraldes, Elizabeth R. Sprague, Subarna Shakya, Zhuoliang Chen, Yaping Wang, Carol Joud, Simon Mathieu, Christine Hiu-Tung Chen, Christopher Straub, Jose S. Duca, Kristen Hurov, Yanqiu Yuan, Wenlin Shao, and B. Barry Touré J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00033 • Publication Date (Web): 10 Feb 2017 Downloaded from http://pubs.acs.org on February 14, 2017

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Journal of Medicinal Chemistry

“Addition” and “Subtraction”: Selectivity Design for Type-II Maternal Embryonic Leucine Zipper Kinase Inhibitors Xin Chen,* John Giraldes, Elizabeth R. Sprague, Subarna Shakya, Zhuoliang Chen, Yaping Wang, Carol Joud, Simon Mathieu, Christine Hiu-Tung Chen, Christopher Straub, Jose Duca, Kristen Hurov, Yanqiu Yuan, Wenlin Shao, B. Barry Touré†* Novartis Institutes for BioMedical Research, Inc., Cambridge, Massachusetts 02139, United States ABSTRACT: While adding the structural features that are more favored by on-target activity is the more common strategy in selectivity optimization, the opposite strategy of subtracting the structural features that contribute more to off-target activity can also be very effective. Reported here is our successful effort of improving the kinase selectivity of type-II maternal embryonic leucine zipper kinase inhibitors by applying these two complementary approaches together, which clearly demonstrates the powerful synergy between them.

INTRODUCTION A sufficient selectivity between on-target and off-target activities is essential for a compound to potentially become a drug, given that the value of a therapeutic agent is largely determined by its desirable on-target effects (efficacy) versus undesirable off-target effects (toxicity). However, achieving such a demanded selectivity is often very challenging, especially among the closely related members of a target family, such as kinases. Although several rational approaches have been described in literature,1,2 selectivity design is still a much harder task to succeed compared to the design of many other properties such as potency, efficacy, solubility and permeability.

situ onto 5-bromo-2,4-dichloropyrimidine or 3-bromo-4chloropyridine to afford 1 or 2a. The former was condensed with aniline, 3-methoxy aniline or N-methylamine to yield 2b-d respectively. Then followed the coupling of 2a, 2b and 2d with ((4-methoxy-carbonyl)phenyl)boronic acid under the Suzuki-Miyaura cross-coupling conditions to afford 3a, 3b and 3d, which subsequently underwent hydrolysis to yield 4a, 4b and 4d. The conversion of 2c to 4c was carried out in one step by using 4-boronobenzoic acid as aryl donor. Coupling of 4a-d with the appropriate amine and Boc-removal led to the final products 5-14. Scheme 1. Synthesis of compounds 5-14.a

Maternal embryonic leucine zipper kinase (MELK) belongs to the AMPK subfamily of serine/threonine kinases.3 It is overexpressed in a broad spectrum of cancer cell lines, including breast, ovarian, lung and pancreas.4,5 Previous studies showed that MELK knockdown by shRNA led to selective G2/M cell cycle arrest and antiproliferation effect in vitro and in vivo.3 Collectively, the preclinical data suggested that MELK might be a promising antitumor target. Recently, a number of MELK inhibitors have been reported,6-10 including both type-I and type-II inhibitors which by definition bind to the DFG-in and DFG-out conformations of kinases, respectively.11 We lately discovered a series of phenylaminopyrimidine compounds as type-II MELK inhibitors, which unexpectedly showed a rather poor kinase selectivity profile. By introducing the structural features that enhance the MELK inhibition and removing those that drive the inhibition of other kinases, we successfully designed a series of pyridine compounds which display a very clean selectivity profile in our kinase screening panel. CHEMISTRY The synthesis began by treating N-Boc-piperidin-4-yl methanol with NaH; the ensuing alkoxide was added in

a Reagents and conditions: (a) N-Boc-4-hydroxymethyl piperidine, NaH, DMF, 0 oC; (b) amine, ethanol, AcOH, 100 oC, 16 hr; (c) boronic acid, Pd(amphos)Cl2, K3PO4, dioxane/water, 95 oC, 2 hr; or 4boronobenzoic acid, XPhos Pd G2, XPhos, Na2CO3, 120 0C, 25 min in microwave, dioxane/water; (d) LiOH, THF/water, 16-72 hr then HCl; (e) amine (or salt thereof), HATU, Et3N, DMF, r.t., 16 hr or amine EDCI, N-hydroxybenzotriazole, Et3N, r.t., 4-16 hr; (f) 4N HCl in dioxane, DCM, 0 oC to r.t. or TFA, DCM.

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RESULTS By X-ray crystallography, we found that MELK could adopt a DFG-out conformation under certain conditions. Consequently, compound 5 was designed to fit into this conformation and later confirmed by the X-ray as our first type-II MELK inhibitor. As shown in Figure 1(b), this compound uses an aminopyrimidine scaffold to form bidentate hydrogen-bonding with the backbone of Cys89 at the hinge region of MELK. Its 3-methoxyl aniline side chain is oriented towards the outside of ATP-binding pocket (“front pocket”), while its amide side chain extends into a “back pocket” created by the DFG-flipping.12 MELK is unique in that it has a bulky residue (Ile149) at the DFG-1 position, whereas most of the kinases that have shown DFG-out conformations in crystal structures have a much smaller residue at this position; typically alanine or glycine.13 Consequently, the side chain of Phe151 in the DFG motif of MELK is flipped into a position in-between what are usually observed in the DFG-in and DFG-out conformations of other kinases, and stabilized by a favorable cation-π interaction with the piperidine ring of 5.

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To rationalize this unexpected outcome, we speculated that compound 5 might bind to some other kinases as a type-I inhibitor while binding to MELK as a type-II, as illustrated in Figure 2. Such a switch between type-I and type-II binding modes is not unprecedented among kinase inhibitors. For example, as a prototypical type-II ABL inhibitor (PDB code: 1IEP),16 Gleevec/imatinib adopts a type-I binding mode in SYK (PDB code: 1XBB).17

Figure 2. Binding hypothesis of compound 5. X-ray crystallography reveals that 5 binds to MELK as a type-II inhibitor (at the left). When 5 approaches an off-target kinase that is incapable of adopting DFG-out conformation and therefore lacking of the “back pocket”, it may flip to adopt a type-I binding mode (at the right), burying its aniline side chain (in red) inside the binding pocket and leaving its amide side chain (in blue) into the solvent.

Figure 1. a) Structure of compound 5. b) Binding mode of 5 (colored in green) in MELK, based on the x-ray crystal structure. The surrounding residues are colored in pink. Hydrogen bond is represented by yellow dotted line and cation-π interaction by green dotted line. (For better visualization, the p-loop on top of the binding pocket is undisplayed.) c) Numbers of the off-targets that 5 inhibits in our screening panel of ~65 kinases, using IC50 < 10 µM, IC50 < 1 µM and IC50 < 0.1 µM as the cut-offs, respectively.

Despite binding to such a unique DFG-out conformation, compound 5 surprisingly showed a very poor kinase screening profile in our in-house panel of ~65 selected kinases (c.f., SI). As shown in Figure 1(c), it hits 40 kinases with IC50 < 10 µM, 13 kinases with IC50 < 1 µM, or 5 kinases with IC50 < 0.1 µM. This is contrary to the conventional belief that type-II kinase inhibitors are generally more selective,14 because the DFG-out conformation that these inhibitors target is less populated by kinases; in fact only ~10% of kinases in the kinome have demonstrated the capability of adopting DFG-out conformation in crystal structures.13,15 Moreover, poor kinase screening profiles were observed persistently for this phenylaminopyrimidine scaffold with more analogs being made, and we were unable to significantly reduce their off-target activities by optimization in the “back pocket”.

Based on this intuitive model, the aniline side chain of compound 5 should contribute more to the type-I binding mode than to type-II, since it is buried deeper inside the binding pocket in the type-I mode; in contrast, it is partially exposed to the solvent in the type-II mode. Likewise, the amide side chain should contribute more to the type-II binding mode than to type-I. This simple but instructive model significantly reduced the complexity of selectivity design in this case and provided us two practical approaches to improve the kinase selectivity of compound 5 and its analogs: 1) reducing the off-target binding affinity of the aniline side chain and 2) enhancing the ontarget binding affinity of the amide side chain. Due to the lack of crystal structures of off-target kinases complexed with compound 5 or its analog, the simplest way to reduce the off-target binding affinity of the aniline side chain is to truncate it. As shown in Figure 3, we first deleted the phenyl ring, thereby converting the aniline into methylamine (from 6 to 7). The number of kinase hits in our screening panel fell from 24 to 16 with IC50 < 10 µM, 8 to 4 with IC50 < 1 µM, or 4 to 0 with IC50 < 0.1 µM. Next, we removed the methylamine completely and also converted the pyrimidine into more basic pyridine for stronger hinge binding (from 7 to 8). The kinase screening profile became almost completely clean for compound 8, with only one hit showing IC50 < 1 µM (ROCK2: IC50 = 0.36 µM). Furthermore, similar SAR patterns were observed when the same structural transformations were applied to the other analogs, i.e., from 9 to 10 to 11, and from 12 to 13 to 14 (c.f., SI).


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However, ~10- to ~84-fold losses of MELK potency did occur during the truncation of aniline side chain. To enhance on-target binding affinity, we focused on the amide side chain. As shown in Figure 3, we first added a conformational constraint to the terminal amide by converting it into benzimidazole. This led to ~8-fold improvement in MELK potency, i.e., from 6 (IC50 = 8.8 nM) to 9 (IC50 = 1.1 nM). Then, we added a methoxyl group to the benzimidazole ring to fill a small allosteric site behind the “back pocket” as revealed by crystallography. This led to additional ~7-fold increase, i.e., from 9 (IC50 = 1.1 nM) to 12 (IC50 = 0.15 nM). Despite a total of ~59-fold improvement in the MELK potency, the kinase screening profiles of these compounds were largely unchanged. Once again, similar SAR patterns were observed when the same structural transformations were applied to the other analogs, i.e., from 7 to 10 to 13, and from 8 to 11 to 14 (c.f., SI).

Consequently, our lead series evolved from phenylaminopyrimidines to pyridines. With more analogs being made, a fair comparison between the two series becomes possible and is displayed in Figure 4 with 15 compounds of each series. Figure 4 clearly demonstrates that the selectivity difference as observed in Figure 3 is well preserved between these two series. Overall, the new pyridine series can achieve the same potency range of MELK inhibition with a much cleaner kinase screening profile, compared to the original phenylaminopyrimidine series.

Figure 4. Comparison of the kinase screening profiles of the phenylaminopyrimidine series (at the left) and the pyridine series (at the right). Each column represents a compound, and each row is a kinase. For each cell, colored in red is IC50 < 0.1 µM; in orange is 0.1 µM ≤ IC50 < 1 µM; in yellow is 1 µM ≤ IC50 < 10 µM, and in green is IC50 ≥ 10 µM. Blank cell is data missing. At the top, the MELK inhibition is colored from IC50 = 0.1 nM in bright red to IC50 = 1 µM in light red.

DISCUSSION

Figure 3. Numbers of the off-target hits in our screening panel of ~65 kinases, for the representative analogs made during the optimization process. Here, hits are defined by IC50 < 10 µM, IC50 < 1 µM and IC50 < 0.1 µM, respectively. MELK IC50 value is also listed below each structure.

Thus, by truncating the aniline side chain and decorating the amide side chain, we were able to achieve compound 14 (IC50 = 1.5 nM), which is not only slightly more potent than the original compound 6 (IC50 = 8.8 nM) but also much cleaner in our kinase screening panel. In fact, it hits only one kinase with IC50 < 10 µM (ROCK2: IC50 = 7.8 µM), while 6 hits 24 kinases in the same range of potency.

In principle, selectivity can be achieved by increasing on-target activity and/or decreasing off-target activity. Practically, the former is often accomplished by introducing structural features which lead to more favorable interactions with on-target (“addition”), while the latter is usually done by deleting structural features which contribute more to off-target interactions (“subtraction”). In structure-based drug design, the “addition” approach appears to be much more popular due to the availability of on-target crystal structures, which often reveal immediate opportunities to gain binding affinity, such as unfilled pockets, unmatched hydrogen-bonds, and so on. In contrast, off-targets usually come with no 3D structures available, making the rational “subtraction” design to reduce off-target binding much more difficult. However, as illustrated herein, the “addition” approach has its intrinsic limitations to overcome, especially when the binding modes (or pharmacophores) of on-target and off-target are significantly different. Furthermore, it inevitably increases molecular size, which may not only increase the chance of promiscuity18 but also lead to poor ADME/PK properties19. On the other hand, the “subtraction” ap-



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proach naturally reduces molecular size/complexity and therefore well complements the “addition” approach. It may not only bring better ADME/PK properties but also address the selectivity challenges that are intractable to the “addition” approach. Obviously, a combination of these two approaches should represent the best practice in selectivity optimization, as demonstrated in this work. Another interesting SAR in Figure 3 is that the two side chains appear to cooperate with each other in terms of MELK potency. First, weaker binding at the position of aniline side chain enhances the gain of binding affinity at the position of amide side chain. With the intact aniline group, the optimization of the amide chain leads to ~59fold improvement in potency from 6 to 12. With the smaller/weaker methylamine group, the same modification causes ~80-fold improvement from 7 to 13. When this side chain is completely removed, the potency gain further increases to ~493-fold from 8 to 14. Second, stronger binding at the position of amide side chain also reduces the loss of binding affinity at the position of aniline side chain. With the un-optimized amide side chain, the removal of aniline group leads to ~84-fold loss of potency from 6 to 8. With the optimized amide side chains, this loss is reduced to ~11-fold from 9 to 11, or ~10-fold from 12 to 14. This kind of cooperativity20 provides us additional benefit of applying the “addition” and “subtraction” approaches together in selectivity optimization. CONCLUSIONS Selectivity design is intrinsically challenging due to the usually limited scientific knowledge and research resource for off-targets. However, this report provides an example showing that selectivity can still be rationally optimized through creative modeling and careful designing. It also demonstrates the synergy between two complementary approaches: “addition” and “subtraction”, and the effectiveness of combining these two in selectivity design. The general strategy outlined herein should be applicable to other therapeutic targets in drug design. EXPERIMENTAL SECTION All final compounds were purified to >95% as determined by Waters AcQuity UPLC-UV system equipped with a Waters LCT premier mass spectrometer with UV detection at 254 nm. tert-butyl-4-(((5-bromo-2-chloropyrimidin-4-yl)oxy)methyl)piperidine-1-carboxylate (1). To a suspension of tert-butyl-4-(hydroxymethyl)piperidine-1-carboxylate (15.1 g, 70 mmol) in DMF (100 mL) was added NaH (4.0 g, 100 mmol). 5-bromo-2,4-dichloropyrimi-dine (15.2 g, 66.8 mmol) in DMF (30 mL) was then added. The reaction was quenched by dropwise addition of methanol followed by dilution with water (100 mL) and ethyl acetate (350 mL). After partition, the organic layer was washed with 4% NaCl brine, dried over Na2SO4, filtered and concentrated. The crude was purified and concentrated to give the title product as white solid (17.3 g, 62%). 1H NMR (400 MHz, DMSO-d6) δ 8.72 (s, 1H), 4.29 (d, J = 6.4 Hz, 2H), 4.04– 3.86 (m, 2H), 2.84–2.62 (m, 2H), 2.08–1.89 (m, 1H), 1.78– 1.65 (m, 2H), 1.40 (s, 9H), 1.19 (qd, J = 12.5, 4.3 Hz, 2H). MS calcd for C15H21BrClN3O3 [M]+ 405.1, found [M+H]+ 406.3.

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tert-butyl-4-(((3-bromopyridin-4-yl)oxy)methyl)piperidine-1-carboxylate (2a). To a solution of 3-bromo-4chloropyridine (3.0 g, 15.6 mmol) and tert-butyl-4-(hydroxymethyl)piperidine-1-carboxylate (3.7 g, 17.2 mmol) in DMF (50 mL) was added NaH (0.94 g, 23.4 mmol). The mixture was stirred at r. t. for 16 hr, quenched with water and diluted with ethyl acetate. After partition, the organic layer was washed with water, brine, dried over Na2SO4, filtered and concentrated. The resulting crude was purified to give the title product as white solid (5.58 g, 94%). 1 H NMR (400 MHz, DMSO-d6) δ 8.56 (s, 1H), 8.39 (d, J = 5.6 Hz, 1H), 7.17 (d, J = 5.6 Hz, 1H), 4.05 (d, J = 6.4 Hz, 2H), 4.02–3.93 (m, 2H), 2.75 (s, 2H), 1.99–1.93 (m, 1H), 1.82–1.67 (m, 2H), 1.40 (s, 9H), 1.23–1.15 (m, 2H). MS calcd for C16H23BrN2O3 [M]+ 370.1, found [M+H]+ 371.1. tert-butyl-4-(((5-bromo-2-(phenylamino)pyrimidin-4yl)oxy)methyl)piperidine-1-carboxylate (2b). To a suspension of 1 (1.0 g, 2.5 mmol) in 2-pentanol (5 mL) was added aniline (0.40 mL, 4.38 mmol) followed by acetic acid (0.25 mL, 4.37 mmol). The reaction was heated to 100 o C for 3 hr, cooled to r.t. and diluted with EtOAc. The solution was washed with 2.5% KHSO4, saturated NaHCO3, water, brine, dried over Na2SO4, filtered and concentrated. The resulting crude was purified to give the title product as white solid (0.95 g, 82%). 1H NMR (400 MHz, methanol-d4) δ 8.23 (s, 1H), 7.68–7.56 (m, 2H), 7.38– 7.22 (m, 2H), 7.08–6.96 (m, 1H), 4.33 (d, J = 6.3 Hz, 2H), 4.20–4.06 (m, 2H), 2.82 (s, 2H), 2.17–1.98 (m, 1H), 1.89–1.76 (m, 2H), 1.48 (s, 9H), 1.34–1.29 (m, 2H). MS calcd for C21H27BrN4O3 [M+H]+ 462.1, found [M+H]+ 463.1. tert-butyl-4-(((5-bromo-2-((3-methoxyphenyl)-amino )pyrimidin-4-yl)oxy)methyl)piperidine-1-carboxylate (2c). A mixture of 1 (1.5 g, 3.7 mmol) and 3-methoxyaniline hydrochloride salt (0.65 g, 4.1 mmol) in 2-pentanol was heated to 100 oC for 18 hr, then cooled to r.t., filtered and dried to afford 5-bromo-N-(3-methoxy-phenyl)-4-(piperidin-4-ylmethoxy)-pyrimidin-2-amine as a grey solid (1.47 g, 97%). To a solution of the above intermediate (0.8 g, 2.0 mmol) in DCM (50 ml) were added di-tert-butyl dicarbonate (0.45 g, 2.1 mmol) and triethylamine (1.0 g, 10.2 mmol), stirred at r.t. overnight and then filtered. The resulting solid was washed with diethyl ether and dried to afford the title product (0.50 g, 50 %) as white solid. 1H NMR (400 MHz, methanol-d4) δ 10.55 (s, 1H), 9.19 (s, 1H), 8.31 (t, J = 2.1 Hz, 1H), 8.17–7.91 (m, 2H), 7.36 (ddd, J = 7.6, 2.4, 1.4 Hz, 1H), 5.10 (d, J = 6.5 Hz, 2H), 4.55 (s, 3H), 4.16 (s, 2H), 3.71–3.41 (m, 2H), 2.81 (dd, J = 6.6, 3.1 Hz, 1H), 2.55 (d, J = 11.0 Hz, 2H), 2.21 (s, 9H), 2.10–1.93 (m, 2H). MS calcd for C22H29BrN4O4 [M]+ 492.1, found [M]+ 493.1. tert-butyl-4-(((5-bromo-2-(methylamino)pyrimidin-4 –yl)oxy)methyl)piperidine-1-carboxylate (2d). To a solution of 1 (1.0 g, 2.5 mmol) in THF (3 mL) was added methylamine (2.5 g, 12.3 mmol), heated to 50 oC for 2 hr, and then concentrated and purified to give the title product as white solid (0.6 g, 64%). 1H NMR (400 MHz, DMSO-d6) δ 8.14 (s, 1H), 7.22 (s, 1H), 4.17 (s, 1H), 4.01–3.93 (m, 2H), 3.32 (s, 1H), 2.82–2.69 (m, 5H), 1.99–1.87 (m, 1H), 1.74–1.65 (m, 2H), 1.39 (s, 9H), 1.23–1.09 (m, 2H). MS calcd for C16H25BrN4O3 [M]+ 400.1, found [M+H]+ 401.0.


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Procedure 1. Combined were the desired aryl halide (2, 1.0 equiv), boronic acid (1.2 equiv), bis(di-tert-butyl-(4dimethylaminophenyl)-phosphine)dichloropalladium(II) (0.1 equiv) and tripotassium phosphate (2.5 equiv) in a mixture of dioxane (10 mL) and water (1 mL), which was degassed under a stream of nitrogen for 5 min and then heated to 90 oC for 2 hr. The reaction was cooled to r.t. and diluted with ethyl acetate and water (10:1). After partition, the organic layer was washed with water and brine, dried over Na2SO4, filtered and concentrated. The crude was purified using silica gel chromatography with ethyl acetate/heptane to give the desired product. tert-butyl-4-(((3-(4-(methoxycarbonyl)phenyl)-pyridin-4-yl)oxy)methyl)piperidine-1-carboxylate (3a). Prepared from 2a (2.0 g, 5.4 mmol) and (4-(methoxycarbonyl)phenyl)boronic acid (1.1 g, 5.9 mmol) using Procedure 1. The title product was obtained as a white solid (1.9 g, 80%). 1H NMR (400 MHz, chloroform-d) δ 8.54–8.42 (m, 2H), 8.12 (d, J = 8.1 Hz, 2H), 7.60 (d, J = 8.2 Hz, 2H), 6.91 (d, J = 5.7 Hz, 1H), 4.27–4.06 (m, 2H), 3.96 (s, 3H), 3.95– 3.84 (m, 2H), 2.83–2.64 (m, 2H), 2.03–1.88 (m, 1H), 1.77– 1.67 (m, 2H), 1.47 (s, 9H), 1.29–1.20 (m, 2H). MS calcd for C24H30N2O5 [M]+ 426.2, found [M+H]+ 427.4. tert-butyl-4-(((5-(4-methoxycarbonylphenyl)-2-phenylaminopyrimidin-4-yl)oxy)methyl)piperidine-1-carboxylate (3b). Prepared from 2b (0.47 g, 1.0 mmol) and (4-(methoxycarbonyl)phenyl)boronic acid (0.22 g, 1.2 mmol) using Procedure 1. The title product was obtained as an orange solid (0.48 g, 90%). 1H NMR (400 MHz, DMSO-d6) δ 9.74 (s, 1H), 8.43 (s, 1H), 8.06–7.92 (m, 2H), 7.80–7.76 (m, 2H), 7.75–7.70 (m, 2H), 7.37–7.20 (m, 2H), 7.03–6.89 (m, 1H), 4.31 (d, J = 6.1 Hz, 2H), 4.07–3.94 (m, 2H), 3.87 (s, 3H), 2.91–2.63 (m, 2H), 2.09–1.90 (m, 1H), 1.78–1.63 (m, 2H), 1.39 (s, 9H), 1.27–1.09 (m, 2H). MS calcd for C29H34N4O5 [M]+ 518.3, found [M+H]+ 519.2. tert-butyl-4-(((5-(4-methoxycarbonylphenyl)-2-methylaminopyrimidin-4-yl)oxy)methyl)piperidine-1-carboxylate (3d). Prepared from 2d (0.50 g, 1.25 mmol) and (4-(methoxycarbonyl)phenyl)boronic acid (0.247 g, 1.37 mmol) using Procedure 1. The title product was obtained as white solid (0.450 g, 75%). 1H NMR (400 MHz, DMSOd6) δ 8.24 (s, 1H), 8.03–7.88 (m, 2H), 7.75–7.60 (m, 2H), 7.27 (s, 1H), 4.34–4.14 (m, 2H), 4.05–3.90 (m, 2H), 3.85 (s, 3H), 2.83 (d, J = 4.7 Hz, 3H), 2.77–2.62 (m, 2H), 2.03–1.85 (m, 1H), 1.74–1.62 (m, 2H), 1.38 (s, 9H), 1.19–1.06 (m, 2H). MS calcd for C24H32N4O5 [M]+ 456.2, found [M+H]+ 457.2. Procedure 2. To a solution of the ester (3, 1.0 equiv) in THF (20 mL) was added 4M aqueous LiOH (10 equiv), stirred for 72 hr at r.t. The reaction was diluted with water (25 mL), slowly acidified using 6N HCl until pH 2 was reached, and then cooled to 0 oC to facilitate full precipitation. The resulting white precipitate was collected by filtration giving the desired product. 4-(4-((1-(tert-butoxycarbonyl)piperidin-4-yl)methoxy )pyridin-3-yl)benzoic acid (4a). Prepared from 3a (1.8 g, 4.2 mmol) using Procedure 2. The title product was obtained as yellow solid (1.7 g, 97%). 1H NMR (400 MHz, DMSO-d6) δ 13.07 (s, 1H), 8.63 (d, J = 6.3 Hz, 1H), 8.59 (s, 1H), 8.07–7.98 (m, 2H), 7.73–7.65 (m, 2H), 7.43 (d, J = 6.3

Hz, 1H), 4.12 (d, J = 6.0 Hz, 2H), 4.03–3.88 (m, 2H), 2.82– 2.61 (m, 2H), 2.01–1.86 (m, 1H), 1.70–1.59 (m, 2H), 1.38 (s, 9H), 1.22–1.08 (m, 2H). MS calcd for C23H28N2O5 [M]+ 412.2, found [M+H]+ 413.1. 4-(4-((1-(tert-butoxycarbonyl)piperidin-4-yl)methoxy )-2-(phenylamino)pyrimidin-5-yl)benzoic acid (4b). Prepared from 3b (1.16 g, 2.2 mmol) using Procedure 2. The title product was obtained as a white solid (1.00 g, 88%). 1H NMR (400 MHz, DMSO-d6) δ 12.91 (s, 1H), 9.73 (s, 1H), 8.41 (s, 1H), 8.07–7.91 (m, 2H), 7.82–7.75 (m, 2H), 7.72–7.66 (m, 2H), 7.36–7.24 (m, 2H), 7.03–6.91 (m, 1H), 4.31 (d, J = 6.1 Hz, 2H), 4.14–3.85 (m, 2H), 2.85–2.60 (m, 2H), 2.10–1.87 (m, 1H), 1.78–1.56 (m, 2H), 1.38 (s, 9H), 1.18 (qd, J = 12.2, 3.8 Hz, 2H). MS calcd for C28H32N4O5 [M]+ 504.2, found [M+H]+ 505.2. 4-(4-((1-(tert-butoxycarbonyl)piperidin-4-yl)methoxy )-2-((3-methoxyphenyl)amino)pyrimidin-5-yl)benzoic acid (4c). In a mixture of dioxane (8 mL) and water (2 mL) was combined 2c (0.20 g, 0.41 mmol), 4-boronobenzoic acid (0.074 g, 0.45 mmol), chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl) [2-(2'amino-1,1'-biphen-yl)]-pall-adium(II), (0.02 g, 0.03 mmol), X-Phos (0.03 g, 0.06 mmol) and sodium carbonate (1.3 g, 4.1 mmol). The mixture was degassed under a stream of nitrogen for 5 min and heated to 120 oC for 25 min. The reaction was filtered and diluted with DCM and water (10:1). After partition, the organic layer was washed with water and brine, dried over Na2SO4, filtered and concentrated to give the title product as an orange solid. MS calcd for C29H34N4O6 [M]+ 534.3, found [M+1] 535.1. 4-(4-((1-(tert-butoxycarbonyl)piperidin-4-yl)methoxy )-2-(methylamino)pyrimidin-5-yl)benzoic acid (4d). Prepared from 3d (0.42 g, 0.92 mmol) using Procedure 2. The title product was obtained as an off-white solid (0.37 g, 89%). 1H NMR (400 MHz, DMSO-d6) δ 13.02 (s, 1H), 8.30 (s, 1H), 8.14 (s, 1H), 7.98 (d, J = 8.4 Hz, 2H), 7.63 (d, J = 8.4 Hz, 2H), 4.45–4.14 (m, 2H), 4.00–3.89 (m, 2H), 2.93 (s, 3H), 2.79–2.60 (m, 2H), 2.00–1.87 (m, 1H), 1.71–1.62 (m, 2H), 1.38 (s, 9H), 1.21–1.08 (m, 2H). MS calcd for C23H30N4O5 [M]+ 442.2, found [M+H]+ 443.2. Procedure 3. To a solution of the acid (4, 1.0 equiv), amine or salt thereof (1.0-2.2 equiv), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (1.5 equiv) and hydroxybenzotriazole (1.5 equiv) in DMF (3 mL) was added triethylamine (5.1 equiv) at r.t. and stirred for 4-16 hr. The reaction was diluted with ethyl acetate and washed with saturated NaHCO3, water, brine, dried over Na2SO4, filtered and concentrated. To a solution of the above product (1.0 equiv) in DCM (3 mL) was added dropwisely HCl (4M in dioxane, 10 equiv). The resulting suspension was slowly warmed up to r.t., stirred for 16 hr, concentrated and triturated from acetonitrile to afford the title product. 4-(2-((3-methoxyphenyl)amino)-4-(piperidin-4-ylmethoxy)-pyrimidin-5-yl)-N-(2-oxo-2-(phenylamino)ethyl)benzamide (5). To 4c (0.019 g, 0.036 mmol) in DMF (1.5 mL) was added HATU (0.021 g, 0.054 mmol). After stirred for 15 min, DIPEA (0.023 ml, 0.18 mmol) and 2-amino-N-phenylacetamide (0.010 g, 0.054 mmol) were added, followed by stirred overnight, diluted with water



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and extracted with ethyl acetate. The organic phase was washed with brine, dried over MgSO4 and concentrated. The residue was taken up in CH2Cl2 (0.3 mL) and TFA (0.3 mL) was added. The mixture was stirred for 2 hr, concentrated and purified to afford the title product as white solid (0.009 g, 36%). 1H NMR (400 MHz, methanol d4) δ 8.30 (d, J = 13.9 Hz, 2H), 8.00 (d, J = 8.5 Hz, 2H), 7.77-7.66 (m, 2H), 7.65-7.56 (m, 2H), 7.44 (dt, J = 10.5, 2.2 Hz, 1H), 7.38-7.04 (m, 4H), 6.67 (ddd, J = 37.8, 8.2, 2.1 Hz, 1H), 4.55-4.40 (m, 2H), 4.25 (s, 2H), 3.83 (s, 3H), 3.44 (d, J = 12.7 Hz, 2H), 3.15-2.94 (m, 2H), 2.39-2.19 (m, 1H), 2.08 (dd, J = 26.3, 14.1 Hz, 2H), 1.75-1.43 (m, 2H). HRMS calcd for C32H34N6O4 [M+H]+ 567.2719, found [M+1] 567.2696. N-(2-oxo-2-(phenylamino)ethyl)-4-(2-(phenylamino)4-(piperidin-4-ylmethoxy)pyrimidin-5-yl)benzamide (6). Prepared from ammonium salt of 4b (0.02 g, 0.04 mmol) and 2-amino-N-phenylacetamide (0.007 g, 0.04 mmol) using Procedure 3. The title product was obtained as a white solid (0.012 g, 50%). 1H NMR (400 MHz, Methanol-d4) δ 8.30 (s, 1H), 8.07–7.91 (m, 2H), 7.72–7.63 (m, 4H), 7.63–7.56 (m, 2H), 7.42–7.30 (m, 4H), 7.19–7.09 (m, 2H), 4.45 (d, J = 6.1 Hz, 2H), 4.25 (s, 2H), 3.48–3.39 (m, 2H), 3.10–2.94 (m, 2H), 2.31–2.14 (m, 1H), 2.09–1.98 (m, 2H), 1.65–1.48 (m, 2H). HRMS calcd for C31H32N6O3 [M+H]+ 537.2614, found [M+H]+ 537.2592. 4-(2-(methylamino)-4-(piperidin-4-ylmethoxy)pyrimidin-5-yl)-N-(2-oxo-2-(phenylamino)ethyl)benzamide (7). Prepared from 4d (0.08 g, 0.17 mmol), 2-amino-Nphenyl-acetamide (0.06 g, 0.37 mmol) using Procedure 3. The title product was obtained as a tan solid (0.063 g, 86%). 1H NMR (400 MHz, DMSO-d6/D2O) δ 8.37 – 8.22 (m, 1H), 7.99 (d, J = 8.4 Hz, 2H), 7.66 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 8.5 Hz, 2H), 7.33 (t, J = 7.9 Hz, 2H), 7.07 (t, J = 7.4 Hz, 1H), 4.48–4.23 (m, 2H), 4.09 (s, 2H), 3.36–3.22 (m, 2H), 3.01–2.82 (m, 5H), 2.17–2.08 (m, 1H), 1.92–1.78 (m, 2H), 1.55–1.39 (m, 2H). HRMS calcd for C26H30N6O3 [M+H]+ 475.2458, found [M+H]+ 475.2423. N-(2-oxo-2-(phenylamino)ethyl)-4-(4-(piperidin-4-ylmethoxy)pyridin-3-yl)benzamide (8). Prepared from 4a (0.040 g, 0.089 mmol) and 2-amino-N-phenyl-acetamide (0.020 g, 0.107 mmol) using Procedure 3. The title product was obtained (0.017 g, 39%). 1H NMR (400 MHz, methanol-d4) δ 8.67 (d, J = 6.9 Hz, 1H), 8.64 (d, J = 1.0 Hz, 1H), 7.98 (d, J = 8.3 Hz, 2H), 7.68 (d, J = 6.9 Hz, 1H), 7.64 (d, J = 8.4 Hz, 2H), 7.51–7.45 (m, 2H), 7.25–7.18 (m, 2H), 7.01 (t, J = 7.4 Hz, 1H), 4.27 (d, J = 6.1 Hz, 2H), 4.15 (s, 2H), 3.36–3.27 (m, 2H), 2.99–2.86 (m, 2H), 2.20–2.07 (m, 1H), 1.96–1.83 (m, 2H), 1.54–1.42 (m, 2H). HRMS calcd for C26H28N4O3 [M+H]+ 445.2240, found [M+H]+ 445.2245. N-((1H-benzo[d]imidazol-2-yl)methyl)-4-(2-(phenylamino)-4-(piperidin-4-ylmethoxy)pyrimidin-5-yl)benzamide (9). Prepared from 4b (0.07 g, 0.14 mmol) and (1H-benzo[d]imidazol-2-yl)methanamine dihydrochloride salt (0.04 g, 0.18 mmol) using Procedure 3. The product was obtained as a white solid (87%). 1H NMR (400 MHz, Methanol-d4) δ 8.25 (s, 1H), 8.13 (d, J = 8.5 Hz, 2H), 7.80 (dd, J = 6.2, 3.1 Hz, 2H), 7.76 (d, J = 8.5 Hz, 2H), 7.66–7.61 (m, 2H), 7.60–7.50 (m, 4H), 7.43–7.35 (m, 1H), 5.10 (d, J = 3.2 Hz, 2H), 4.50 (d, J = 6.2 Hz, 2H), 3.49–3.39 (m, 2H), 3.09–2.96 (m, 2H), 2.31–2.17 (m, 1H), 2.03–1.93

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(m, 2H), 1.68–1.49 (m, 2H). HRMS calcd for C31H31N7O2 [M+H]+ 534.2617, found [M+H]+ 534.2609. N-((1H-benzo[d]imidazol-2-yl)methyl)-4-(2-(methylamino)-4-(piperidin-4-ylmethoxy)pyrimidin-5-yl)benzamide (10). Prepared from 4d (0.070 g, 0.158 mmol) and (1H-benzo[d]imidazol-2-yl)methanamine dihydrochloride hydrate salt (0.045 g, 0.19 mmol) using Procedure 3. The title product was obtained as an off-white solid (0.07 g, 76%). 1H NMR (400 MHz, DMSO-d6) δ 9.80 (t, J = 5.1 Hz, 1H), 9.14 (d, J = 9.2 Hz, 1H), 9.04–8.89 (m, 1H), 8.63–8.36 (m, 1H), 8.09 (d, J = 8.4 Hz, 2H), 7.83–7.76 (m, 2H), 7.69 (d, J = 8.4 Hz, 2H), 7.59–7.51 (m, 2H), 4.99 (d, J = 5.2 Hz, 2H), 4.46–4.23 (m, 2H), 3.31–3.20 (m, 2H), 2.98 (s, 3H), 2.92–2.79 (m, 2H), 2.14–2.08 (m, 1H), 1.89– 1.76 (m, 2H), 1.58–1.45 (m, 2H). HRMS calcd for C26H29N7O2 [M+H]+ 472.2461, found [M+H]+ 472.2406. N-((1H-benzo[d]imidazol-2-yl)methyl)-4-(4-(piperidin-4-ylmethoxy)pyridin-3-yl)benzamide (11). Prepared from 4a (0.040 g, 0.097 mmol) and (1H-benzo[d]imidazol-2-yl)methanamine dihydrochloride salt (0.014 g, 0.097 mmol) using Procedure 3. The title product was obtained (0.007 g, 16%). 1H NMR (400 MHz, methanol-d4) δ 8.77– 8.62 (m, 2H), 8.07 (d, J = 8.1 Hz, 2H), 7.73–7.66 (m, 5H), 7.54–7.47 (m, 2H), 4.99 (s, 2H), 4.29 (d, J = 5.9 Hz, 2H), 3.39–3.28 (m, 2H), 3.01–2.87 (m, 2H), 2.21–2.06 (m, 1H), 1.96–1.83 (m, 2H), 1.60–1.44 (m, 2H). HRMS calcd for C26H27N5O2 [M+H]+ 442.2243, found [M+H]+ 442.2213. N-((5-methoxy-1H-benzo[d]imidazol-2-yl)methyl)-4(2-(phenylamino)-4-(piperidin-4-ylmethoxy)pyrimidin-5-yl)benzamide (12). Prepared from 4b (0.100 g, 0.198 mmol) and (5-methoxy-1H-benzo[d]imidazol-2-yl)methanamine dihydrochloride hydrate salt (0.064 g, 0.238 mmol) using Procedure 3. The title product was obtained as a white solid (0.102 g, 64%). 1H NMR (400 MHz, DMSO-d6) δ 9.75 (s, 1H), 9.42 (s, 1H), 8.51 (s, 1H), 8.23 (s, 1H), 8.01 (d, J = 8.5 Hz, 2H), 7.76 (dd, J = 15.8, 8.1 Hz, 4H), 7.64 (d, J = 8.9 Hz, 1H), 7.37–7.28 (m, 2H), 7.19 (d, J = 2.2 Hz, 1H), 7.11 (d, J = 9.0 Hz, 1H), 6.99 (t, J = 7.4 Hz, 1H), 4.89 (d, J = 5.1 Hz, 2H), 4.35 (d, J = 6.3 Hz, 2H), 3.85 (s, 3H), 3.31 (d, J = 13.0 Hz, 2H), 2.90 (d, J = 10.9 Hz, 2H), 2.46–2.43 (m, 1H), 2.13 (s, 1H), 1.89 (d, J = 13.6 Hz, 2H), 1.45 (d, J = 10.5 Hz, 2H). HRMS calcd for C32H33N7O3 [M+H]+ 564.2723, found [M+H]+ 564.2736. N-((5-methoxy-1H-benzo[d]imidazol-2-yl)methyl)-4(2-(methylamino)-4-(piperidin-4-ylmethoxy)pyrimidin-5-yl)benzamide (13). Prepared from 4d (0.100 g, 0.226 mmol) and (5-methoxy-1H-benzo[d]imidazol-2-yl)methanamine dihydrochloride salt (0.062 g, 0.249 mmol) using Procedure 3. The title product was obtained (0.067 g, 40%). 1H NMR (400 MHz, DMSO-d6/D2O) δ 8.25 (s, 1H), 8.01-7.94 (m, 2H), 7.69-7.65 (m, 3H), 7.23 (d, J = 2.3 Hz, 1H), 7.17 (dd, J = 9.0, 2.4 Hz, 1H), 4.91 (d, J = 3.2 Hz, 2H), 4.33 (s, 2H), 3.85 (s, 3H), 3.30 (d, J = 12.7 Hz, 2H), 2.96 2.84 (m, 5H), 2.08 (s, 1H), 1.87 (d, J = 13.8 Hz, 2H), 1.45 (t, J = 13.3 Hz, 2H). HRMS calcd for C27H31N7O3 [M+H]+ 502.2567, found [M+H]+ 502.2544. N-((6-methoxy-1H-benzo[d]imidazol-2-yl)methyl)-4(4-(piperidin-4-ylmethoxy)pyridin-3-yl)benzamide (14). Prepared from 4a (0.035 g, 0.085 mmol) and (6-


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methoxy-1H-benzo[d]imidazol-2-yl)-methanamine hydrochloride salt (0.023 g, 0.085 mmol) using Procedure 3. The title product was obtained (0.018 g, 35%). 1H NMR (400 MHz, methanol-d4) δ 8.75–8.61 (m, 2H), 8.12–8.01 (m, 2H), 7.73–7.63 (m, 3H), 7.60–7.52 (m, 1H), 7.18–7.04 (m, 2H), 4.94 (s, 2H), 4.28 (d, J = 6.2 Hz, 2H), 3.81 (s, 3H), 3.39–3.30 (m, 2H), 2.99–2.88 (m, 2H), 2.22–2.08 (m, 1H), 1.95–1.85 (m, 2H), 1.55–1.46 (m, 2H). HRMS calcd for C27H29N5O3 [M+H]+ 472.2349, found [M+H]+ 472.2344.

ASSOCIATED CONTENT Supporting Information. The supporting information is available free of charge at http://pubs.acs.org. Experimental and kinase screening data (Word) Molecular formula strings (CSV)

AUTHOR INFORMATION Corresponding Author * X.C.: e-mail, [email protected]; phone, 617-871-7631. * B.B.T.: e-mail, [email protected]; phone, 617-370-8818.

Present Addresses st

† Relay Therapeutics, 215 1 St., Cambridge, MA 02142, USA.

ABBREVIATIONS MELK, maternal embryonic leucine zipper kinase; AMPK, Adenosine Monophosphate-activated Protein Kinase. The atomic coordinates of MELK/compound 5 have been deposited in PDB as 5K00 and will be released upon publication.

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Selectivity Design for Type II Maternal Embryonic ... - ACS Publications

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