Novel Small Molecule Inhibitors of Choline Kinase ... - ACS Publications

Dec 23, 2015 - ARIAD Pharmaceuticals, Inc., 26 Landsdowne Street, Cambridge, Massachusetts 02139, United States. •S Supporting Information. ABSTRACT...
0 downloads 0 Views 4MB Size
Article pubs.acs.org/jmc

Novel Small Molecule Inhibitors of Choline Kinase Identified by Fragment-Based Drug Discovery Stephan G. Zech,*,† Anna Kohlmann,† Tianjun Zhou,† Feng Li,† Rachel M. Squillace,‡ Lois E. Parillon,§ Matthew T. Greenfield, David P. Miller,∥ Jiwei Qi, R. Mathew Thomas,⊥ Yihan Wang,# Yongjin Xu, Juan J. Miret,∇ William C. Shakespeare, Xiaotian Zhu, and David C. Dalgarno ARIAD Pharmaceuticals, Inc., 26 Landsdowne Street, Cambridge, Massachusetts 02139, United States S Supporting Information *

ABSTRACT: Choline kinase α (ChoKα) is an enzyme involved in the synthesis of phospholipids and thereby plays key roles in regulation of cell proliferation, oncogenic transformation, and human carcinogenesis. Since several inhibitors of ChoKα display antiproliferative activity in both cellular and animal models, this novel oncogene has recently gained interest as a promising small molecule target for cancer therapy. Here we summarize our efforts to further validate ChoKα as an oncogenic target and explore the activity of novel small molecule inhibitors of ChoKα. Starting from weakly binding fragments, we describe a structure based lead discovery approach, which resulted in novel highly potent inhibitors of ChoKα. In cancer cell lines, our lead compounds exhibit a dose-dependent decrease of phosphocholine, inhibition of cell growth, and induction of apoptosis at low micromolar concentrations. The druglike lead series presented here is optimizable for improvements in cellular potency, drug target residence time, and pharmacokinetic parameters. These inhibitors may be utilized not only to further validate ChoKα as antioncogenic target but also as novel chemical matter that may lead to antitumor agents that specifically interfere with cancer cell metabolism.



INTRODUCTION Choline kinase (ChoK) is a cytosolic enzyme that catalyzes the MgATP-dependent phosphorylation of choline to phosphocholine (pCho) as the first step in the Kennedy pathway toward synthesis of the major membrane phospholipid, phosphatidylcholine (PtdCho). The mammalian choline kinase family is encoded by two distinct genes, ChoKα and ChoKβ, resulting in three different isoforms, namely, ChoKα1, ChoKα2, and ChoKβ. ChoKα1 (457 amino acids) and ChoKα2 (439 amino acids) are derived from a single gene (ChoKα) by alternative splicing. Apart from being a major structural component of mammalian cellular membranes, PtdCho serves as a precursor for the production of lipid second messengers that can activate growth and survival pathways.1,2 This implies that maintenance of PtdCho level is critical for cell growth because its oscillations would have implications not only in the structural homeostasis of the cell membranes but also in signaling processes involved in cell proliferation and apoptosis. Abnormal choline metabolism is characteristic of oncogenesis and cancer progression in an array of cancer types. Exogenous expression of ChoKα1, but not ChoKβ, is capable of driving tumor formation in nontransformed cells.3 It is known that increased phosphorylation of choline is a hallmark of certain malignant phenotypes. ChoKα overexpression, primarily ChoKα1, has been associated with certain human cancers, including breast, liver, lung, colorectal, ovarian, and prostate.4,5 © XXXX American Chemical Society

For example, ChoKα, pCho, and total choline were increased in breast carcinomas compared with normal breast tissue, and this was correlated with advanced tumor grades.6,7 This implies that elevated pCho or ChoKα itself may be a candidate biomarker for ChoKα inhibitor therapy across tumor types. Preclinical evidence suggests that inhibition of ChoKα expression in cell lines results in disruption of MAPK and AKT activity and decreased cell proliferation.8,9 It has also been found that cell lines expressing activated RAS exhibit sensitivity to ChoKα knockdown by siRNA or small molecule inhibition.10 More recently, ChoKα has been identified as a downstream mediator of EGFR/c-Src signaling in breast cancer cell lines.11 These studies indicate that tumors demonstrating aberrant MAPK, PI3K/AKT, RAS, or EGFR/c-Src signaling may also be targetable by ChoKα inhibitors. Finally, recent studies have implicated that ChoKα inhibition triggers apoptosis in cancer cells by inducing exacerbated endoplasmic reticulum stress.12 These observations have motivated efforts to develop anticancer agents targeting ChoKα.13,14 ChoKα inhibitors have been demonstrated to be potent antitumor agents both in vitro and in vivo. Hemicholinium-3 (1, HC-3),15 shown in Figure S1 (Supporting Information), is a known substrate site inhibitor that bears structural homology to Received: October 5, 2015

A

DOI: 10.1021/acs.jmedchem.5b01552 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

1152 diverse rule-of-three compliant fragments using STD NMR as the primary screening technique.27 Figure 1 shows an

choline, which contributes to its toxicity. A less toxic derivative of 1, MN58b (2),16 was reported to inhibit endogenous ChoKα activity and suppress breast cancer, colon cancer, and epidermoid carcinoma xenograft growth in vivo.16−19 Another ChoKα inhibitor, CK37 (3),9 identified through computational virtual screening, inhibited ChoKα activity both in enzymatic and in cellular assays. Compound 3 was also able to suppress pCho production and tumor growth in vivo.9 Intriguingly, both 2 and 3 showed inconsistencies between their relatively weak enzymatic activities and more potent cellular and in vivo tumor suppression activities, which may indicate these inhibitors possess activities on targets additional to ChoKα. On the other hand, V-11-0711 (4),20 also identified from a structure-directed approach, is a selective ChoKα inhibitor with an IC50 of 20 nM against ChoKα which exhibited excellent selectivity against a panel of 50 kinases. Although treatment of HeLa cells and other cancer cells with 4 led to a substantial reduction in the pCho level, this treatment caused a reversible growth arrest but not cell death.20 In addition, HeLa cells treated with 4 displayed only low levels of apoptotic markers. The precursor of this compound, VX-11-023907 (5),21 showed some unexpected properties with respect to its enzymatic mechanism of action. While the quinuclidine moiety binds to the choline site of ChoKα, the molecule has been found to be ATP competitive but not choline competitive.21 Recently RSM-932A (6, Figure S1),22,23 also known as TDC717, became the first compound to enter phase I clinical trials as a potential cancer therapeutic agent targeting choline metabolism.22 Compound 6 and its analogues23 are symmetric bis-quinolinium derivatives that were derived from 1 and that demonstrated ChoKα enzyme inhibition activities similar to those of 1 and 2. To overcome the toxicity problems associated with the 1 chemotype, pharmacophore-guided virtual screening has been used to identify novel chemical matter with binding affinities in the low μM range,24 starting from known choline site inhibitors. Alternatively, novel asymmetrical bispyridinium derivatives showed submicromolar enzyme activity, inhibited proliferation, and caused apoptosis in ChoK driven cancer cells.25 In our hands, an enzyme-based screening of a library of commercially available kinase inhibitors failed to identify potent (IC50 < 1 μM) ChoKα ATP site inhibitors. This is consistent with the low KM of ATP (∼300 μM),26 indicating that subtle structural differences between the ATP binding pockets of protein kinases and ChoKα attenuate binding of known tyrosine kinase templates to ChoKα. With this knowledge, we turned to alternative methods to identify ChoKα inhibitors. Here we describe a fragment-based approach to discovering novel and potent ChoKα inhibitors with the potential for a more favorable safety profile. We used NMR fragment screening as an unbiased hit-finding strategy followed by further biophysical validation and structure-driven chemical optimization. Novel symmetrical and nonsymmetrical ChoKα inhibitors were identified using an iterative structure-based approach with binding affinities against ChoKα of around 10 nM. Importantly, these inhibitors induced dose-dependent reduction of pCho and caused apoptotic cell death in breast cancer cell lines overexpressing ChoKα but not in control cell lines.

Figure 1. STD NMR fragment screening. (A) Reference NMR spectrum of 7 and STD NMR spectrum (red) in the presence of 30 μM ChoK indicating reversible binding of 7. (B) Reference spectrum and STD NMR spectrum of 8. The stronger STD signals observed for the diazepane moiety compared to the aromatic signals imply a close contact of the diazepane with the protein.

example of the NMR screen. Panel A shows the NMR reference spectrum of compound 7 together with the STD NMR spectrum in the lower trace, indicating reversible binding of 7 to ChoKα. In contrast DMSO and buffer signals are not observed in the difference spectrum. Panel B shows the reference and STD NMR spectra of 8. The resonances associated with the diazepane moiety are significantly more pronounced in the STD spectrum compared to the aromatic protons. The relative STD intensity can be used to deduce structural information,28 indicating that the diazepane is in close contact with the protein, thus facilitating more efficient magnetization transfer. The NMR screen returned 55 hits (4.8% hit rate) comprising a diversity of chemotypes. On the basis of structural features of those fragments (e.g., binding capability to the kinase hinge region), some hits were likely ATP site binders, while others, lacking those features, were thought to bind to alternative binding sites of the protein. Fluorine NMR detected competition experiments (FAXS) can offer a sensitive way to monitor compounds binding to a specific binding site that is occupied by the “spy” molecule,29 as an alternative to more resource demanding technologies such as SAR by NMR.30 In addition, FAXS competition experiments work well on larger proteins such as the homodimeric form of ChoKα1 (∼94 kDa) and require only weakly binding compounds as spy molecules (100−200 μM in this case). FAXS is therefore well suited for early stage discovery programs, where tight binding inhibitors may not be available. Figure S2 exemplifies the competition experiments with either 2-fluoroadenosine (9, Kd = 165 μM) or fluorocholine chloride (10, Kd = 113 μM) which were used to monitor binding to ATP and substrate sites, respectively. Table



RESULTS AND DISCUSSION Initial Hit Generation by Fragment Screening and Biophysical Validation. In the search for novel chemical matter targeting ChoKα1, we performed an unbiased screen of B

DOI: 10.1021/acs.jmedchem.5b01552 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 2. SPR validation of fragment hits. Sensograms for compounds 12, 11, and 13 binding to a surface with immobilized ChoKα (left) or the reference protein CA-II (right). The sensograms were obtained at 25 °C using a 2-fold dilution series with an upper concentration of 300 μM. Kd values were obtained by fitting the data to a 1:1 binding model (error margin of ∼2-fold, ∗ = injection artifact).

by the off-target affinity to carbonic anhydrase II (CA-II), a protein structurally and functionally unrelated to ChoK. An example is shown in Figure 2. Compound 12 showed significant selectivity for ChoK, with minor binding to CA-II. The other two compounds show affinity for both proteins. Nonspecific surface absorption of weakly binding fragments is often observed in early hit validation and gives rise to larger error margins for the Kd estimates and resulting derived ligand efficiencies. Ranking the hits based on SPR data alone would be difficult; however, taking data from NMR competition experiments into account enables a more confident prioritization for X-ray crystallography. In agreement with enzyme inhibition, compound 11 showed a strong affinity with Kd of 132 μM, despite some superstoichiometric binding at higher concentrations,31 and was among the compounds selected for cocrystallization. Structural Analysis of Fragment Hits. A selection of validated fragment hits was successfully soaked into ChoKα crystals, and crucial structural information was generated on both ATP and choline site binders. Surprisingly, during fragment optimization we found that the ATP site compounds were difficult to improve in potency, even though many of them were found to bind to the ATP site in typical ways (e.g., to the hinge region of the kinase ATP binding pocket). In contrast, we were able to progress the choline site binders rapidly using structure-guided approaches in hit-to-lead optimization.

S1 (Supporting Information) includes a selection of fragment hits and their respective binding sites. Out of 55 fragment hits investigated, 13 showed competition with choline site binder 10, while 21 compounds showed competition with ATP site binder 9. NMR monitored competition experiments allowed us not only to validate initial hits from either fragment or enzymebased screens (including high throughput and virtual screening campaigns) but also to locate screening hits to binding sites of interest. Compounds that did not compete with 9 or 10 were deprioritized because they may be very weak competitors, allosteric binders, or enzyme assay artifacts. Triaging primary screening hits using such competition assays results in both an increased confidence in the hits and improved success in downstream experiments, notably X-ray crystallography. Quantitative information on protein−ligand affinity was obtained by means of enzymatic assays and surface plasmon resonance (SPR). In the coupled choline kinase enzyme assay, most compounds showed only weak inhibition of the enzyme with IC50 values larger than 0.3 mM, as are typically found for initial fragment hits. One exception was fragment 11, which was confirmed as choline site binder by NMR competition experiments, with an IC50 of 87 μM. Protein−ligand interaction studied by SPR also revealed that the majority of the fragment hits exhibited mostly weak binding affinities with Kd between 0.5 and 2 mM. We also observed rapid association and dissociation for all fragments hits, consistent with this weak affinity. In addition, many fragments showed nonspecific binding to the protein surface as monitored C

DOI: 10.1021/acs.jmedchem.5b01552 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

carbon contact distances of 3.5−5.0 Å. The fourth side consists of mostly polar residues including D306, Q308, and E349, all of which are conserved among either choline kinase or eukaryotic protein kinase families and perform critical structural and functional roles.11,32 For example, D306 and Q308 were involved in the binding of the enzyme reaction product phosphocholine, and mutation D306A resulted in a total loss of activity.11,34 The diazepane methyl group is the most deeply bound portion of the inhibitor, filling a small hydrophobic pocket formed by Y333, W420, and W423 where it engages in strong aliphatic−aromatic edge-to-face contacts with the side chains of these residues (Figure 3B). The importance of the methyl group is supported by the SAR of the des-methyl analogs which are typically less potent. Of interest, the 1′ N of the diazepane to which the methyl is attached is within hydrogen bond distance to the side chain of D306 (3.1 Å). This implies that the N is protonated under the crystallization conditions (pH 7.0) and carries a positive charge. Indeed, the protonation status of the 1′ N atom was confirmed by NMR titration experiments (data not shown). This polar interaction is reminiscent of that formed by the ChoK inhibitor 5 between its quinuclidine-N and D306.21 The phenyl group of 11 binds to part of a hydrophobic groove located between a helix and a loop (connecting two β strands) in the C-lobe (Figure 3B), where it forms favorable π−π interactions with the aromatic side chains of Y437 and Y354 (edge-to-face) and F435 (face-to-face). Finally, the benzylamino group is partially disordered in the structure, likely due to the lack of favorable interactions in the hydrophobic groove. This observation prompted us to explore lipophilic substitutions on the phenyl ring to enhance the contacts with the hydrophobic residues in this groove. Evaluation of the SAR (e.g., Table S1) showed that compounds bearing a methylpiperazine headgroup (e.g., compound 22) have weaker affinity than their diazepane analogs (compound 11). This finding highlights the importance of the additional flexibility of diazepane which allows it to better fill the pocket and optimize the geometry of the hydrogen bonding interaction with D306. Structure Guided Hit Exploration. Examination of the previously published X-ray crystal structure of ChoKα (3G15)35 revealed a choline site with a deep enclosed base widening toward the solvent front. Similarly, SiteMap analysis of the X-ray structure of compound 11 demonstrates a hydrophobic patch spanning the length of the pocket: a deeply buried hydrophobic tip flanked on one side by buried hydrogen bond acceptors and donors, with only scarce polar interactions at the solvent front (Figure S4). This indicates that a potent inhibitor in the choline site must satisfy both the hydrophobic interactions and the precise arrangement of polar interactions in the pocket. To explore additional aspects of SAR around the initial fragment hits, commercially available analogs were selected based on docking studies into the binding site of ChoKα. Compounds exhibiting favorable interactions with the protein were purchased and tested for enzyme inhibition followed by SPR. Results for selected compounds are shown in Table S2. Compound 27, one of the purchased analogs, showed the highest potency (Kd = 7 μM, IC50 = 17 μM) and confirmed our observation that more lipophilic bulk in the vicinity of the phenyl ring would be favorable for binding. Compound 27 was chosen as a starting point for chemical optimization.

Figure 3 shows the cocrystal structure of 11 soaked into a crystal of N-terminally truncated ChoKα1 (see Experimental

Figure 3. Crystal structure of ChoKα bound by compound 11 at 2.4 Å (PDB entry 5EQE). (A) Overview of the ChoKα complex structure. Compound 11, binding in the choline site is shown as sticks and colored in green. The superimposed phosphochloline (PDB code 2CKQ) in cyan is included for comparison. The ATP site of the enzyme is also indicated. Structural figures were prepared using PyMol unless noted otherwise. (B) Binding mode of compound 11 in the substrate site of ChoKα. The protein is shown as cartoon with amino acid side chains as sticks. The hydrophobic residues lining the “hydrophobic groove” are labeled. The purple dashed line denotes hydrogen bonding interactions.

Section). The complex structure was determined at 2.4 Å by molecular replacement. As previously reported,32 ChoKα1 kinase adopts the typical eukaryotic protein kinase fold, including an N- and C-bilobal architecture with the ATP binding site located in a cleft between the two lobes and the choline substrate site in the lager C-lobe (Figure 3). There are two monomers in the crystallographic asymmetric unit, which assemble as a homodimer with the helix in the smaller N-lobe forming the major dimer interface. This is consistent with the findings that all the choline kinase isoforms exist either as dimers (homo- or hetero-) or as tetramers in solution and are not active in monomeric form.33 The electron density for 11 is well-defined in the structure despite its relatively low binding affinity (Figure S3). As predicted by competitive NMR studies, the inhibitor binds in the choline site located in the mostly helical C-lobe. The methyldiazepane moiety sits deep at the base of choline site which is predominantly hydrophobic in nature. W420 serves as the “floor” of the binding site, while W423, Y333, Y354, and Y440 form three of the four sides of the pocket, with carbon-toD

DOI: 10.1021/acs.jmedchem.5b01552 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 4. Key SAR elements of the fragment hit-to-lead progression. Ligand efficiency was calculated as LE = −1.4 log(Kd)/(no. of heavy atoms).

Figure 4 summarizes the key compounds in the hit-to-lead optimization process. Initially, the connectivity of the diazepane moiety was modified for ease of synthesis (compound 37, Table S3), with an insignificant change in potency. The cocrystal structure with this inhibitor showed that the second aromatic ring forms additional van der Waals contacts with residues lining the hydrophobic groove, specifically, a paralleldisplaced π−π stacking interaction with F435 (Figure S5). Subsequently, the quinoline moiety was modified to various fused bicyclic systems to probe the SAR in the solvent-frontdirected part of the molecule. No significant improvement in potency was achieved (Table S3). We then attempted to introduce flexibility into the solventdirected part of the molecule. A library of compounds was designed with flexible linkers at the para position of the phenyl ring, bearing various polar substituents (data not shown). Although a modest decrease in enzymatic potency was observed, the Kd values remained unchanged, and this subseries was not pursued further. Finally, we substituted a biphenyl in place of a fused bicycle (compound 43, Table 1) to retain hydrophobicity while introducing some flexibility into the aromatic system. Additionally, modifications at the para position of the second phenyl ring were introduced to probe polar interactions in the solvent front. An improvement in potency of 43 was observed when the second phenyl ring was substituted (Table 1). Addition of a heterocycle improved potency by 5- to 20-fold (compounds 44, 48, 49), while small, polar, or hydrophobic substituents brought no improvement (compounds 46, 47) Modifications were then introduced into the diazepane moiety (Table S4). Consistent with our earlier analysis of SAR in the initial hit series, most changes resulted in a drastic loss of potency (50- to >100-fold) compared to compound 38. This finding highlighted the methyldiazepane as a key pharmacophore feature for the binding of the inhibitor which is explained by the snug fit of the methyl group into the choline binding site observed in the cocrystal structure throughout fragment elaboration (Figure S6). Even conservative modifications at the 1′ position caused a drastic reduction in potency. Interestingly, adding a second methyl group at the 1′ position (compound 52 bearing a positively charged ammonium ion)

Table 1. Modifications of the Biphenyl Moiety at the Para Position (Error Margins: 2- to 3-Fold)

reduced the potency by 90-fold, indicating the importance of a hydrogen bonding donor at the 1′ position of the diazepane (see above). This suggests that methyldiazepane derivatives are better suited for the choline site binding site than quaternary ammonium ions present, e.g., in 1 and derivatives. Optimization of the Biphenyl System. We proceeded to optimize the biphenyl linker of 49 with substitutions at various E

DOI: 10.1021/acs.jmedchem.5b01552 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Table 2. SAR of Linker Optimization and Kinetic Evaluation of the Leadlike Seriesa

Association kinetics kon, protein−ligand residence time τ = 1/koff, and dissociation constant KD = 1/(konτ) were measured by surface plasmon resonance. IC50 values were obtained from ChoKα enzyme inhibition assays. Error margins for kon and τ are ∼2-fold, and error margins for Kd and IC50 are 2- to 3-fold. a

positions of one or both phenyl rings (Table 2) finally obtaining compound 65 with Kd = 10 nM. Additional or alternative substitutions to the biphenyl linker were also tested to engage in additional contacts to the neighboring protein residues. However, these compounds did not display improved potency over 65. As exemplified in Table 2, simple substitutions of the biphenyl linker were able to significantly improve potency and ligand efficiency of the lead series, (e.g., a gain in potency of about 40-fold was observed by adding a CN group to 49). The compound with the best potency, 65, also showed the longest target residence time (τ > 2 min), indicating that further elaboration of the biphenyl part could potentially improve both potency and dissociation rate. It is important to note that improving the affinity of compounds also led to better selectivity for ChoKα by reducing the nonspecific surface binding as monitored by SPR. In contrast to the early fragment hits (Figure 2 and Table S1), the lead series did not show significant binding affinity to the reference protein CA-II (Kd > 100 μM). As outlined in Figure 4 and Table 2, ligand efficiency of ChoKα binding was maintained or even slightly improved throughout the hit-tolead optimization process, indicating that all parts of the elaborated molecules contribute significantly to protein−ligand binding. Chemistry. Compounds in Tables S1 and S2 were purchased from vendors. Compounds 38, 50, 51, and 53−64

were synthesized in a parallel fashion (Table S5) as shown in Scheme 1 by reductive amination of 2-naphthaldehyde (71) and substituted 1H-1,4-diazepines (72). Scheme 1

Compound 52 was synthesized by treating amine 38 with iodomethane at room temperature and isolated as a salt form according to Scheme 2. Compounds 37, 39−43, 46, 47 were synthesized in a parallel fashion shown in Scheme 3, by reductive amination of NScheme 2

F

DOI: 10.1021/acs.jmedchem.5b01552 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

water molecules and polar protein residues (E309, D215) (Figure 5). There is a rotation of about 20° for the phenyl ring in 65 compared to those in 11 and 37 (Figure S6). This conformational change seems necessary not only to prevent the attached nitrile group from clashing to the protein molecule (Y354 in particular) but also to engage in molecular interactions through water molecules. While some of our compounds display a high degree of symmetry, resembling the dimeric structures of 1 and analogues, the SAR outlined in Tables 1 and 2 proves that this symmetry is not strictly required to improve affinity, as the most potent compounds display a break in symmetry compared to 49. The two diazepane moieties featured in our lead series serve a dual purpose: While one represents a novel pharmacophore with high affinity to the choline binding side, the other one is facing the solvent front. As shown in the cocrystal structure with compound 65 (Figure 5), both diazepane moieties make significant interactions with the protein, thus favorably contributing to protein−ligand binding which is reflected in the gain in potency along with maintained ligand efficiency during fragment elaboration. The architecture of the choline binding site can be viewed as a deep and wellformed hydrophobic groove with a rim composed of a number of negatively charged residues.32 These residues may function to guide the positively charged choline molecule into its binding site. The methyldiazepane of compound 65 that binds close to the solvent front is surrounded by E434 and E357 (Figures 5 and S6); thus, electrostatic interactions also play a role in its binding and further contribute to potency gain. We arrived at these quasi-symmetric structures driven by the SAR (Tables 1 and 2) rather than derivatization of existing chemical matter. In addition, the lack of symmetry within the compound binding pocket (Figure 5) supports that there is no need for symmetry in the druglike compounds. Further optimization for potency or pharmacologic parameters will determine whether this quasi-symmetry is necessary or just an artifact as suggested by the SAR presented here or for other choline site inhibitors.20,21,25 In addition to potency, the understanding of the binding kinetics of a drug to its target can be essential to define a drug’s efficacy and duration of target suppression, as well as its safety due to mechanism based toxicity.36−38 Biosensor techniques, such as SPR, have been proven powerful in evaluating both association (kon) and dissociation (koff) kinetics in vitro.39,40 Table 2 includes the kon rates and the residence times τ (= 1/ koff) for a number of compounds from the lead series. In comparison to other drugs with known drug−target kinetics36,41,42 our lead series exhibits relatively fast association

methyl-1H-1,4-diazepine (72a) and corresponding aromatic aldehydes (73). Scheme 3

Reductive amination of 72a and methyl 4′-formylbiphenyl-4carboxylate (74) gave compound 45, which was then treated with 72a under Weinreb conditions to afford 48. Amide 48 was reduced by LAH to give amine 49 (Scheme 4). Boronic ester 76 was synthesized by treating of 4(bromomethyl)benzeneboronic acid pinacol ester (75) with 72a. Suzuki coupling of 76 with 1-(4-bromophenyl)-4methylpiperazine (77) afforded 44 (Scheme 5). Compounds 66, 68−70 were synthesized in a parallel fashion. Suzuki coupling of 1-(4-bromobenzyl)-4-methylperhydro-1,4-diazepine (79) with subistitued 4-formylphenylboronic acids (78a−d) gave the corresponding intermediates (80a−d) followed by reductive amination with 72a (Scheme 6). 2-Bromo-5-(bromomethyl)benzonitrile (81) was treated with 72a to give 2-bromo-5-[(hexahydro-4-methyl-1H-1,4diazepin-1-yl)methyl]benzonitrile (82). Compound 65 was synthesized by Suzuki coupling of 82 with 79. Compound 67 was synthesized by Suzuki coupling of 82 and 2-fluoro-4formylbenzeneboronic acid pinacol ester (83) to give intermediate aldehyde 84 and followed by reductive amination with 72a as shown in Scheme 7. Structural and Kinetic Characterization of the Lead Series. To gain additional structural insights toward improved potency, we determined the crystal structure of ChoKα bound by 65 at 2.5 Å. The compound binds ChoKα vertically along the hydrophobic groove (Figure 5). While the bottom half of the inhibitor adopts a conformation similar to those of 11 (Figure 3) and 37 (Figure S5), including a hydrogen bond to D306, it engages in additional molecular interactions. The biphenyl linker adopts a roughly perpendicular dihedral angle and optimally fills the hydrophobic groove. The second methyldiazepane group of 65 binds close to the solvent front but still makes favorable contacts with F361 and I433 and the aliphatic side chain of E434, thus contributing to the improved potency. Finally the CN group attached to one of the phenyl rings points to the polar side of the choline binding site and participates in a hydrogen-bonding network involving several Scheme 4

G

DOI: 10.1021/acs.jmedchem.5b01552 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Scheme 5

Scheme 6

Scheme 7

Cellular Activity of Leadlike Compounds. Representative members of this lead series, compounds 49 and 67, were characterized in more detail and demonstrated potent enzyme inhibition against ChoKα as well as binding by SPR (Table 2). Figure 6 summarizes the cell growth inhibition data for compounds 67 (panel A) and 49 (panel B). Compound 67 inhibited the growth of ChoKα-expressing breast cancer lines, MDA-MB-468 and MDA-MB-415, with GI50 values of 7 and 2 μM, respectively. In contrast, compound 67 exhibited much lower activity against the nontransformed breast epithelial cell lines MCF-12A and MCF-10A, with a GI50 of >40 and 18 μM,

and dissociation kinetics for the given potency range. With target residence times between 0.5 and 2 min, these compounds could provide a balance between normal physiological function of ChoKα and inhibition of the target in ChoKα-overexpressed cancer cells, potentially reducing mechanism based toxicity. Further lead optimization for both potency and kinetics43 should aim at residence times that provide signal suppression until apoptosis is induced in ChoKα dependent tumor cells,12 while healthy cells are still able to recover their growth kinetics once the drug is removed (e.g., by metabolism or excretion). H

DOI: 10.1021/acs.jmedchem.5b01552 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

It is important to note that the breast cancer cell lines mentioned above were used solely because these or similar cell lines have been previously shown to be sensitive to ChoKα inhibition.3,12,22,44,45 This enables the comparison to other chemical matter, also on a cellular level. However, it does not imply that these cell lines are the most susceptible ones to ChoKα inhibition. Further evaluation and optimization of our lead series will encompass a broader screen of cancer cell lines22 to help identify even more sensitive cell lines with improved potency and selectivity for our compounds. This in turn will also indicate which cancers are the most vulnerable to small molecule ChoKα inhibition and thereby assist in patient selection. In summary, we demonstrated that small molecule inhibition of ChoKα resulted in a dose-dependent decrease of cellular pCho levels, inhibition of proliferation, and induction of apoptosis in ChoKα-expressing breast cancer cells at low micromolar concentrations. Taken together, these data further validate ChoKα as a potential therapeutic target in cancer and support the continued investigation into the utility of ChoKα inhibitors as antioncogenic agents.

Figure 5. Crystal structure of ChoKα bound by a lead compound 65 (2.5 Å, PDB entry 5EQY). ChoKα is colored in green, and compound 65 is in orange. Red spheres are water molecules that bridge the interactions between the protein and inhibitor molecules. The purple dashed line denotes hydrogen bonding interactions.

respectively. The activity of 49 against cancer cell lines was somewhat weaker, in agreement with weaker binding observed by SPR and in the ChoKα enzyme assay. However, in breast cancer cell lines 49 also resulted in cell death at low micromolar concentration, while nontransformed cells were not affected until concentrations of ∼30 μM were reached (Figure 6). Apoptosis signals were measured using the caspase-3/7 assay and are shown in Figure 6, panels D and E. The induction of apoptosis was consistent with the extent of proliferative inhibition. While 49 and 67 induced apoptosis at low μM concentrations in the ChoKα expressing cell line MDA-MB415, the epithelial cell line MCF-12A did not undergo apoptosis at concentrations up to 40 μM. Intracellular choline and pCho levels were measured by NMR after extraction44,45 of the nonmalignant cell line MCF12A as well as the cancer cell lines MDA-MB-468 and MDAMB-415. While the pCho levels of transformed cell lines were high and easily detectable by 1D proton NMR (Figure 7), the epithelial line MCF-12A showed very low levels of cellular choline and pCho (data not shown). After 24 h of treatment with 49, complete inhibition of pCho was observed in the MDA-MB-468 cell line, in a dose dependent manner with an IC50 of 2−3 μM. Similarly, 24 h treatment of MDA-MB-415 with 67 resulted in up to ∼80% inhibition of pCho with an IC50 of ∼0.75 μM (Figure 7). In both cases, the cellular choline levels were too small to allow quantification. The dosedependent inhibition of pCho in cells indicates that both compounds lead to inhibition of ChoKα in the cell lines MDAMB-415 and MDA-MB-468. Consistent with cell growth, only minor effects on choline levels were observed in MCF-12A cell lines as result of compound treatment with 67, while the pCho levels in this cell line were too small to be quantified by NMR (data not shown). Cellular activities in the low micromolar range have been previously reported for other ChoKα inhibitors.16,17,20,22 For our lead series, the potency for ChoKα driven cell lines and the selectivity over nontransformed cell lines represent a slight improvement over the current clinical compound 6, with reported IC50 values of 2.4 μM against MDA-MB-468 and 7.1 μM against the reference cell line MCF10-A.22 This improvement was achieved at a significantly reduced molecular weight of our compounds (49, 407 Da; 67, 450 Da) compared to 6 (878 Da) while also eliminating the permanent charges of the bis-quinolinium moieties.



CONCLUSIONS We successfully applied fragment based approaches targeting ChoKα to generate novel chemical matter as starting point for medicinal chemistry optimization. Following an unbiased NMR fragment screen, initial hits were further validated by biophysical methods that gathered information on fragment binding sites, potency, and specificity. We identified both ATP and choline site binders and gained structural insights by X-ray crystallography on a significant number of initial hits, leading to ideas on how to improve and elaborate fragments hits. Surprisingly, for ATP site hits, improvements in potency proved difficult to achieve. In contrast, the choline site inhibitors progressed rapidly from weak fragments to a series of leadlike compounds with binding affinities of 10−50 nM. Our lead series is optimizable for further improvements in drug−target residence time, cellular potency, and pharmacokinetic parameters. We demonstrated inhibition of cell growth in a dose-dependent manner and induction of apoptosis in ChoKα-expressing breast cancer cell lines. Inhibition of ChoKα in cancer cells was directly shown by monitoring intracellular phosphocholine production. Interestingly, many of the ChoKα inhibitors in late preclinical or clinical development are dimeric structures featuring quaternary ammonium ions that, similar to choline, exhibit strong affinity for the substrate site. Unfortunately, these double positively charged chemotypes are associated with significant toxicity due to off-target activities, in part caused by their structural homology to choline. In contrast, the lead series identified in this paper uses basic amines present in methyldiazepanes as major driver for binding to the choline site. The protonated 1′ nitrogen adds a key hydrogen bond, resulting in significantly improved potency compared to, for example, 1 (IC50 = 1.0 μM, Kd = 0.59 μM, LE = 0.29). It is important to note that the methyldiazepane moiety can be transiently protonated (i.e., charged) or be present as free base, depending on local conditions such as pH. Furthermore, diazepanes or piperazines are very commonly used in many approved drugs, mostly to improve solubility or ADME parameters. Usually those moieties do not give rise to major toxicity concerns. I

DOI: 10.1021/acs.jmedchem.5b01552 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 6. Cellular activity of elaborated ChoK inhibitors. Growth inhibition of breast cancer cell lines by ChoK inhibitors 67 (A) and 49 (B). Both compounds selectively induced cell death of breast cancer but not breast epithelial lines. (C) Summary of GI50 values and maximum growth inhibition. The markers identify the respective inhibition curves shown above. Numbers in red indicate cell death (>100% growth inhibition). Apoptosis signals measured for ChoK lead compounds 67 (D) and 49 (E) for cell lines MDA-MB-415 (top, solid red curves) and MCF-12A (bottom, solid blue curves).

cellular metabolism, allow further optimization and may be utilized to validate ChoKα as an antioncogenic target and beyond. In turn, these molecules may open a new avenue for the development of antitumor drugs or provide novel treatment for noncancer conditions such as rheumatoid arthritis.46,47

This paper highlights the power of fragment based drug design approaches to quickly identify diverse hot spots on the protein target and to generate novel chemical starting points. Our fragment screen identified methyldiazepane as a novel key pharmacophore for choline site binding. Using structure-driven chemical optimization, we rapidly progressed toward a series of leadlike molecules with cellular potency and a novel mode of action. The thorough biophysical and biochemical evaluation of our lead series led to a clear correlation between enzyme activity (compound 67: Kd ≈ IC50 = 0.09−0.1 μM), cellular pCho inhibition (∼0.75 μM), growth inhibition of ChoKdriven cancer cell lines (2−7 μM) resulting in cell death and induction of apoptosis (5−10 μM). In contrast, nontransformed epithelial cell lines were much less affected and did not undergo apoptosis. These molecules, which specifically inhibit



EXPERIMENTAL SECTION

Protein Expression, Purification, and Crystallography. An Nterminal truncated form of human choline kinase α1 (ChoKα1) that includes residues 75−457 (accession no. NM_001277.2) was cloned into the pET28a expression vector (Novagene) with an N-terminal 6xHis affinity tag followed by a thrombin site. The recombinant protein was purified via affinity, ion-exchange, and size-exclusion chromatography with >95% purity. For crystallography, the purified ChoKα1 protein was concentrated to ∼30 mg/mL and crystallized as J

DOI: 10.1021/acs.jmedchem.5b01552 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 7. NMR detected reduction of cellular pCho levels as a function of compound treatment. (A) Overlay of NMR spectra of cell extracts from cell line MDA-MB-468 treated with increasing amounts of 49. The level of pCho at ∼3.24 ppm shows a dose dependent inhibition. To account for slight variations in cell count and extraction efficiency, the spectral intensities have been calibrated to match the signals of lactate at ∼1.34 ppm. (B) NMR spectra of metabolites extracted from cell line MDA-MB-415 treated with 67. Metabolites at 3.26−3.29 ppm have been used as internal reference. The pCho levels show a dose dependent decrease. Spectral positions for glycerolphosphocholine (GPC) and choline (Ch) are indicated. Table: Approximate cellular IC50 of pCho inhibition evaluated from panels A and B. models. The structures were refined with CNX combined with model building in Quanta (Accelrys Inc.). The inhibitor molecules were built into the density after several cycles of refinement and model building. Refinement parameters are given in Table S6. Enzyme Assay. Enzyme inhibition measurements to obtain IC50 values were made using a coupled ATP regeneration assay26 with LDH/PK (5U/4U), choline chloride (200 μM), ATP (200 μM), PEP (500 μM), and NADH (250 μM) in MOPS buffer (100 mM MOPS, pH = 7.0, 150 mM NaCl, 10 mM MgCl2, 0.1% Triton X-100). The

apo form in a condition containing 0.1 M sodium formate, 8−12% PEG 3350 at 18 °C. The complex structures were obtained by soaking the inhibitors into the ChoKα1 crystals at a final concentration of 2−5 mM for 45−60 min before freezing in liquid nitrogen. Diffraction data were collected at 100 K on a Rigaku rotating anode and R-AXIS IV++ imaging system. The data were indexed and scaled using the HKL2000 package. The structures were determined by molecular replacement with CNX using published structures (e.g., PDB code 3G15) as search K

DOI: 10.1021/acs.jmedchem.5b01552 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

reaction was initiated by adding between 10 and 50 nM human ChoKα1 (monomeric concentration). Absorbance of NADH at 340 nm was measured over 10−30 min using a SpectraMax M5 (Molecular Devices). Initial reaction velocity (ΔA340/Δt) was calculated and plotted as a function of inhibitor concentration in a 2-fold dilution series. IC50 values were obtained by fitting the relative velocities to a dose−response model. NMR Fragment Screening and Competition Experiments. NMR fragment screening experiments were performed at a temperature of 300 K on a Bruker Avance DRX 600 MHz spectrometer (Bruker Biospin Corp.), equipped with a z-gradient 1.0 mm TXI microprobe and a Bruker SampleJet autosampler. STD NMR experiments were recorded with 2 s saturation time, applying 50 ms Gaussian shaped pulses with a nominal flip angle of 720°. The frequency of the on-resonance irradiation was set to 0.8 ppm, and the off-resonance pulse was at 30 ppm. The residual water signal was suppressed using excitation sculpting. For the primary screen, ChoKα1 (homodimeric form, 94 kDa) was dissolved at 30 μM monomer concentration in D2O based buffer containing 50 mM d-MOPS, 100 mM NaCl, pH 7.4, and 0.33 mM TSP as chemical shift reference. After addition of fragments from concentrated DMSO-d6 stock solution in pools of five compounds at a final concentration of ∼3 mM per compound, about 10 μL of solution was transferred to 1.0 mm NMR tubes using a Gilson liquid handler. Samples contained up to 7% DMSO-d6 and were kept at room temperature until measured. Data sets shown are the average of 320 scans. All data processing was performed using the Bruker TopSpin 2.0 software. 19 F-detected NMR competition experiments (FAXS29) were conducted on a 600 MHz Bruker Avance-III instrument using a 5 mm BBFO probe. The NMR samples (3 mm tubes, 170 μL) contained 10 μM ChoKα1 in D2O based buffer (50 mM d-MOPS, 100 mM NaCl, pH 7.4), with each 50 μM of reporter molecules 9 (SigmaAldrich) and 10 (SynQuest Laboratories). Compounds were added at 1 mM final concentration from DMSO-d6 stock. T2 relaxation weighted spectra were acquired using a CPMG pulse sequence with 20 ms echo delay and a total spin lock time of 160−200 ms to eliminate J-couplings and suppress 19F background signals. The transmitter frequency was placed close to the resonances of 9 (−52 ppm) or 10 (−193 ppm). Proton decoupling was applied only during acquisition. For each sample, 800 scans were acquired with 2 s relaxation delay. After magnitude Fourier transformation, the intensities of the 9 and 10 19F signals were analyzed by integration. In addition, a 1D proton reference spectrum with water suppression was acquired for every sample to ensure compound integrity, concentration, and proper shimming of the NMR magnet. Surface Plasmon Resonance Assay. Protein−ligand affinity (Kd) and kinetic rates were measured by surface plasmon resonance technology using either Biacore T100 (GE Healthcare) or SensiQ Pioneer (SensiQ Technologies) system. ChoKα1 protein was immobilized by standard amine coupling methods on a CM5 sensor chip (Biacore) or a COOH5 sensor chip installed in a SensiQ Pioneer system. ChoKα1 was diluted into 10 mM sodium acetate buffer (pH 4.0) with 10 mM MgCl2 and 1 mM ADP for the coupling step. As the reference protein, carbonic anhydrase II at a similar density was used. Surfaces were blocked with a 1:1 mixture of 1 M ethanolamine in the running buffer. Sensograms were measured using either the single step kinetics (Biacore) or FastStep screen method (SensiQ). Fragment hits were tested at 300 μM as the highest concentration in a 2-fold dilution series. The running buffer contained 100 mM Tris (pH 7.5), 100 mM NaCl, 10 mM MgCl2, 0.05% Tween-20, and 5% DMSO. All data were collected at 25 °C. Kinetics and affinity were obtained from fits to a 1:1 binding model. Molecular Modeling. The X-ray crystal structure of ChoKα reported here was used in modeling studies. To explore the binding cavity, SiteMap48,49 was applied to the crystal structure with default settings; cofactor and substrate were used to define binding site regions. An initial minimization and a molecular dynamics simulation were run for 9 ns on a solvated protein system in the absence of the ligand. The preparation of the protein for docking was performed with PrepWizard using the standard protocol, including the addition of

hydrogens, and the assignment of bond order and protonation states. The hydrogen bonding network of the protein was optimized. Docking experiments were performed using Glide XP.50 The fragments and ligands were treated with LigPrep51 to obtain 3D structures, and protonation states were assigned using Epik. MM-GB/ SA in Prime52 was performed on top scoring ligand poses, with default settings. Commercially available reagents were utilized to create virtual compounds according to a chemical synthesis scheme, and then compounds were docked and scored with Glide XP. Compounds with undesirable physicochemical properties were removed. Cellular Assays. Cell Lines. Cell lines MDA-MB-468, MDA-MB415, MCF12A, and MCF10A were obtained from American Type Culture Collection (Manassus, VA) and were used within 6 months without further cell line authentication. Cells were maintained and cultured using medium recommended by the supplier. Cell Growth Assay. Vehicle or compound treated cells were assessed for cell growth after 72 h of treatment using the CyQuant cell proliferation kit (Promega). To differentiate between a cytostatic and cytotoxic drug effect, the concentration that causes 50% growth inhibition (GI50) was determined by correcting for the cell count at time zero (time of treatment) and plotting data as percent growth relative to vehicle-treated cells. Data are shown as mean (±SD) from ≥2 separate experiments. Apoptosis Assay. Cells were treated with compounds for 24 h, then assessed for caspase activity using the Apo-ONE homogeneous caspase-3/7 assay (Promega). A representative experiment is shown with apoptosis plotted as a percentage relative to vehicle-treated cells. NMR Detected pCho Assay. 5 ×106 cells of each line were grown in the presence of increasing concentrations of the test compound or vehicle (DMSO) for 24 h, then were counted and assessed for viability using Trypan blue exclusion protocol. 5 ×106 viable cells were washed twice in ice cold d-PBS, then solubilized by vortexing for 30 s in 6 mL of ice-cold 100% methanol. 6 mL of chloroform and 6 mL of water were added, and the mixture was vortexed again. After centrifugation for 10 min at 5220 rpms, 10 mL of the aqueous phase was removed and lyophilized. The samples were redissolved in 50 μL of D2O, transferred into 1.7 mm NMR tubes, and analyzed by 1H NMR to determine phosphocholine levels. NMR measurements were performed at 600 MHz on a Bruker Avance III spectrometer equipped with a 1.7 mm PATXI probe using presaturation as water suppression and composite pulses for excitation (zgcppr). The relative spectral intensity was adjusted to match metabolites unrelated to choline metabolism, such as lactate. Chemistry. Commercially available chemicals and solvents were purchased and used without further purification. 1H NMR spectra were recorded on a Bruker Avance-III 400 spectrometer. Chemical shifts (δ) are reported relative to CDCl3 at 7.26 ppm or DMSO-d6 at 2.50 ppm as an internal standard. MS spectra were recorded on a Waters Micromass ZQ spectrometer. HPLC was performed on an Agilent 1100 HPLC system. All tested compounds were purified to >95% purity as determined by 1H NMR and HPLC (C-18 column, MeCN/H2O with 0.1% TFA as the mobile phase). General Procedure 1 for Reductive Amination. A mixture of aldehyde (1.0 mmol) and amine (1.0 mmol) in DCM (3.0 mL) was treated overnight with sodium triacetoxyborohydride (2.0 mmol) and molecular sieves (0.3 g) at room temperature. The mixture was diluted with aqueous NaHCO3 and extracted with DCM. Solvent was evaporated and the residue was purified by TLC plate (DCM/MeOH = 100/(5−10)) or preparative HPLC to give products. General Procedure 2 for Suzuki Coupling. A mixture of aryl bromide (0.50 mmol) and boronic acid or boronic ester (0.50 mmol), Pd(PPh3)4 (50 mg), K2CO3 (1.0 mmol) in dioxane (1.5 mL) and water (0.8 mL) was degassed and heated to reflux in sealed tube for 5 min. The mixture was extracted with DCM, and product was purified by TLC plate (DCM/MeOH = 100/(10−20)) or by preparative HPLC. Compounds 1−3, 7−36, 72−75, 77−79, 81, and 83 were obtained from commercial vendors and used without further purification. The following compounds were synthesized in a parallel fashion (Table S5) by utilizing general procedure 1. L

DOI: 10.1021/acs.jmedchem.5b01552 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

37: 7-((4-methyl-1,4-diazepan-1-yl)methyl)quinolone, C16H21N3, yellow oil, yield 63%. LCMS m/z 256 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 8.88 (dd, J = 1.76, 4.27 Hz, 1H), 8.13 (dd, J = 1.2, 8.4 Hz, 1H), 8.07 (d, J = 8.7 Hz, 1H), 7.78 (dd, J = 2.0, 8.8 Hz, 1H), 7.73 (s, 1H), 7.40 (dd, J = 4.27, 8.28 Hz, 1H), 3.82 (s, 2H), 2.74−2.84 (m, 4H), 2.70 (t, J = 5.6 Hz, 2H), 2.62−2.65 (m, 2H), 2.38 (s, 3H), 1.81−1.8 (m, 2H). 38: 1-methyl-4-(naphthalen-2-ylmethyl)-1,4-diazepane, C17H22N2, yellow oil, yield 82%. LCMS m/z 255 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 7.81−7.85 (m, 3H), 7.76 (s, 1H), 7.55 (dd, J = 1.6, 8.2 Hz, 1H), 7.44−7.51 (m, 2H), 3.82 (s, 2H), 2.77−2.82 (m, 4H), 2.73 (t, J = 5.6 Hz, 2H), 2.65−2.68 (m, 2H), 2.41 (s, 1H), 1.83−1.90 (m, 1H). 39: 6-((4-methyl-1,4-diazepan-1-yl)methyl)isoquinoline, C16H21N3, yellow oil, yield 49%. LCMS m/z 256 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 9.24 (s, 1H), 8.52 (d, J = 5.8 Hz, 1H), 7.94 (d, J = 8.5 Hz, 1H), 7.76 (s, 1H), 7.69 (dd, J = 1.2, 8.4 Hz, 1H), 7.63 (d, J = 5.7 Hz, 1H), 3.84 (s, 2H), 2.76−2.81 (m, 4H), 2.72 (t, J = 5.6 Hz, 2H), 2.63−2.67 (m, 2H), 2.40 (s, 3H), 1.82−1.90 (m, 2H). 40: 7-((4-methyl-1,4-diazepan-1-yl)methyl)isoquinoline, C16H21N3, yellow oil, yield 57%. LCMS m/z 256 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 9.24 (s, 1H), 8.52 (d, J = 5.7 Hz, 1H), 7.89 (s, 1H), 7.76−7.82 (m, 2H), 7.64 (td, J = 0.8, 5.7 Hz, 1H), 3.84 (s, 2H), 2.76− 2.81 (m, 4H), 2.73 (t, J = 5.6 Hz, 2H), 2.64−2.68 (m, 2H), 2.40 (s, 3H), 1.83−1.90 (m, 2H). 41: 1-methyl-6-((4-methyl-1,4-diazepan-1-yl)methyl)-1H-indazole, C15H22N4, yellow oil, yield 54%. LCMS m/z 259 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 7.95 (d, J = 0.8 Hz, 1H), 7.66 (dd, J = 0.8, 8.4 Hz, 1H), 7.39 (d, J = 0.8 Hz, 1H), 7.28 (s, 1H), 7.18 (dd, J = 1.2, 8.0 Hz, 1H), 4.09 (s, 3H), 3.80 (s, 2H), 2.75−2.81 (m, 4H), 2.71 (t, J = 5.6 Hz, 2H), 2.63−2.67 (m, 2H), 2.40 (s, 3H), 1.82−1.90 (m, 2H). 42: 6-((4-methyl-1,4-diazepan-1-yl)methyl)quinoxaline, C15H20N4, yellow oil, yield 69%. LCMS m/z 257 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 8.84 (d, J = 6.4 Hz, 1H), 8.82 (d, J = 6.4 Hz, 1H), 8.08 (d, J = 8.8 Hz, 1H), 8.03 (s, 1H), 7.88 (dd, J = 1.6, 8.8 Hz, 1H), 3.90 (s, 2H), 2.77−2.83 (m, 4H), 2.71 (t, J = 5.6 Hz, 2H), 2.64−2.67 (m, 2H), 2.40 (s, 3H), 1.83−1.90 (m, 2H). 43: 1-([1,1′-biphenyl]-4-ylmethyl)-4-methyl-1,4-diazepane, C19H24N2, yellow oil, yield 57%. LCMS m/z 281 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 7.60−7.65 (m, 2H), 7.54−7.59 (m, 2H), 7.42−7.48 (m, 4H), 7.35 (tt, J = 1.6, 7.2 Hz, 1H), 3.71 (s, 2H), 2.75−2.81 (m, 4H), 2.70−2.74 (m, 2H), 2.65−2.69 (m, 2H), 2.41 (s, 3H), 1.83−1.90 (m, 2H). 44: 1-methyl-4-((4′-(4-methylpiperazin-1-yl)-[1,1′-biphenyl]-4-yl)methyl)-1,4-diazepane, C24H34N4. Precursor hexahydro-1-methyl-4[[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]methyl]-1H1,4-diazepine, C19H31BN2O2 (76): To a solution of 72a (5.0 g, 43.8 mmol) in DCM (30 mL) 75 (6.0 g, 20.2 mmol) was added at room temperature. The mixture was stirred for 6 h and purified by chromatography on silica gel (DCM/MeOH = 100:12) to give white solid 76 (4.5 g, 67%). Compound 44 was synthesized by utilizing general procedure 2 from 76 and 77 as yellow solid (yield 20 mg, 11%). LCMS m/z 379 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 7.50−7.56 (m, 4H), 7.39 (d, J = 8.4 Hz, 1H), 6.99−7.04 (m, 2H), 3.68 (s, 2H), 3.27−3.32 (m, 4H), 2.74−2.80 (m, 4H), 2.69−2.73 (m, 2H), 2.65−2.68 (m, 2H), 2.60−2.64 (m, 4H), 2.40 (s, 3H), 2.39 (s, 3H), 1.86 (q, J = 6.0 Hz, 2H). 45: methyl 4′-((4-methyl-1,4-diazepan-1-yl)methyl)-[1,1′-biphenyl]-4-carboxylate, C21H26N2O2. A mixture of 72a (2.6 mL, 24.8 mmol) and 74 (5.0 g, 20.8 mmol) in DCM (150 mL) was added over molecular sieves (4 Å, 10 g). The mixture was stirred at room temperature for 1 h, and NaHB(OAc)3 (8.5 g, 40.0 mmol) was introduced. The mixture was stirred for an additional 5 h. After aqueous workup, the product was purified by flash column to give a yellow oil (yield 4.5 g, 64%). LCMS m/z 339 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 8.13 (td, J = 2.0, 12.8 Hz, 2H), 7.69 (td, J = 2.0, 8.4 Hz, 2H), 7.62 (br, s, 1H), 7.53 (td, J = 2.0, 7.6 Hz, 1H), 7.37−7.45 (m, 2H), 3.97 (s, 3H), 3.75 (s, 2H), 2.70−2.91 (m, 8H), 2.48 (s, 3H), 1.90−1.98 (m, 2H).

46: 1-((4′-methoxy-[1,1′-biphenyl]-4-yl)methyl)-4-methyl-1,4-diazepane, C20H26N2O, yellow oil, yield 27%. LCMS m/z 311 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 7.51−7.57 (m, 4H), 7.40 (d, J = 8.28 Hz, 2H), 6.97 (dd, J = 2.0, 8.8 Hz, 2H), 3.87 (s, 3H), 3.69 (s, 2H), 2.67−2.81 (m, 8H), 2.42 (s, 3H), 1.84−1.91 (m, 2H). 47: 1-methyl-4-((4′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)methyl)1,4-diazepane, C20H23F3N2, yellow oil, yield 32%. LCMS m/z 349 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 7.71 (s, 4H), 7.53−7.60 (m, 2H), 7.47 (d, J = 8.4 Hz, 2H), 3.72 (s, 2H), 2.75--2.80 (m, 4H), 2.73 (t, J = 6.0 Hz, 2H), 2.66−2.69 (m, 2H), 2.42 (s, 3H), 1.88 (quin, J = 6.0 Hz, 2H). 48: (4-methyl-1,4-diazepan-1-yl)(4′-((4-methyl-1,4-diazepan-1-yl)methyl)-[1,1′-biphenyl]-4-yl)methanone, C26H36N4O. To a mixture of 45 (30 mg, 0.086 mmol) and 72a (0.016 mL, 0.13 mmol) in 1,2dichloroethane (1.5 mL) Me3Al (0.1 mL, 2.0 M solution in toluene) was slowly added. The mixture was stirred at room temperature for 20 min and then heated to reflux for 5 min. The mixture was poured onto wet NaHCO3 and extracted with DCM. Solvent was removed and the residue was purified by preparative TLC plate (DCM/MeOH = 100:15) to give yellow oil (22 mg, 61%). LCMS m/z 421 [M + H+]. 1 H NMR (400 MHz, chloroform-d) δ 7.61−7.67 (m, 2H), 7.54−7.58 (m, 2H), 7.42−7.50 (m, 4H), 3.80−3.89 (m, 2H), 3.70 (s, 2H), 3.60 (m, 1H), 3.54 (t, J = 6.0 Hz, 1H), 2.57−2.81 (m, 12H), 2.37−2.45 (m, 6H), 2.04 (m, 1H), 1.82−1.92 (m, 3H). 49: 4,4′-bis((4-methyl-1,4-diazepan-1-yl)methyl)-1,1′-biphenyl, C26H38N4. To a solution of 48 (50 mg, 0.12 mmol) in dioxane (3.0 mL) LAH (20 mg, 0.53 mmol) was added at room temperature. The mixture was heated to reflux and then cooled to room temperature. The mixture was poured onto ice and extracted with DCM. Solvent was removed and the residue was purified by preparative TLC plate (DCM/MeOH = 100:15) to give a colorless oil (35 mg, 72%). LCMS m/z 407 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 7.56 (d, J = 8.2 Hz, 4H), 7.41 (d, J = 8.2 Hz, 4H), 3.65−3.76 (m, 4H), 2.74−2.80 (m, 8H), 2.71 (t, J = 6.0 Hz, 4H), 2.64−2.68 (m, 4H), 2.40 (s, 6H), 1.86 (quin, J = 6.0 Hz, 4H). 50: 1-allyl-4-(naphthalen-2-ylmethyl)-1,4-diazepane, C19H24N2, yellow oil, yield 39%. LCMS m/z 281 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 7.80−7.90 (m, 3H), 7.77 (s, 1H), 7.56 (dd, J = 1.6, 8.4 Hz, 1H), 7.43−7.52 (m, 2H), 5.92 (tdd, J = 6.4, 10.0, 16.8 Hz, 1H), 5.12−5.23 (m, 2H), 3.83 (s, 2H), 3.17 (td, J = 1.2, 6.4 Hz, 2H), 2.70−2.82 (m, 8H), 1.82−1.89 (m, 2H). 51: N,N-dimethyl-2-(4-(naphthalen-2-ylmethyl)-1,4-diazepan-1-yl)ethan-1-amine, C20H29N3, yellow oil, yield 28%. LCMS m/z 312 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 7.80−7.88 (m, 3H), 7.75 (s, 1H), 7.55 (dd, J = 1.6, 8.4 Hz, 1H), 7.43−7.49 (m, 2H), 3.81 (s, 2H), 2.83 (t, J = 6.0 Hz, 2H), 2.72−2.79 (m, 6H), 2.65−2.70 (m, 2H), 2.43 (dd, J = 6.8, 8.8 Hz, 2H), 2.27 (s, 6H), 1.79−1.83 (m, 2H). 52: 1,1-dimethyl-4-(naphthalen-2-ylmethyl)-1,4-diazepan-1-ium iodide, C18H25N2. A mixture of 38 (255 mg, 1.0 mmol) and iodomethane (1.0 mmol) in DCM (2.0 mL) was stirred at room temperature for 2 h. The product was purified by preparative TLC plate (DCM/MeOH = 100:15) to give a yellow solid (71 mg, yield 18%). LCMS m/z 269 [M+]. 1H NMR (400 MHz, chloroform-d) δ 7.80−7.87 (m, 3H), 7.73 (s, 1H), 7.45−7.52 (m, 2H), 3.87 (t, J = 5.2 Hz, 2H), 3.85 (s, 2H), 3.77 (t, J = 4.8 Hz, 2H), 3.51 (s, 6H), 3.01 (br, 3H), 2.88 (t, J = 5.6 Hz, 2H), 2.09 (br, 2H). 53: 4-(2-(dimethylamino)ethyl)-1-(naphthalen-2-ylmethyl)-1,4-diazepan-5-one, C20H27N3O, yellow oil, yield 31%. LCMS m/z 326 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 7.80−7.88 (m, 3H), 7.74 (s, 1H), 7.45−7.54 (m, 3H), 3.75 (s, 2H), 3.45−3.56 (m, 4H), 2.63− 2.75 (m, 6H), 2.45 (t, J = 7.0 Hz, 2H), 2.29 (s, 6H). 54: 4-(naphthalen-2-ylmethyl)-1,4-diazepane-1-carboxamide, C17H21N3O, white solid, yield 26%. LCMS m/z 284 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 7.80−7.88 (m, 3H), 7.76 (s, 1H), 7.44−7.56 (m, 3H), 4.41 (s, 2H), 3.83 (s, 2H), 3.43−3.59 (m, 4H), 2.67−2.80 (m, 4H), 1.93 (quin, J = 6.0 Hz, 2H). 55: 2-(4-(naphthalen-2-ylmethyl)-1,4-diazepan-1-yl)ethan-1-ol, C18H24N2O, yellow oil, yield 51%. LCMS m/z 285 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 7.81−7.88 (m, 3H), 7.75 (s, 1H), 7.55 (dd, J = 1.6, 8.4 Hz, 1H), 7.44−7.51 (m, 2H), 3.82 (s, 2H), 3.58 M

DOI: 10.1021/acs.jmedchem.5b01552 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(t, J = 5.6 Hz, 2H), 2.84 (t, J = 6.0 Hz, 2H), 2.70−2.80 (m, 9H), 1.81− 1.89 (m, 2H). 56: 2,4-dimethyl-1-(naphthalen-2-ylmethyl)-1,4-diazepane, C18H24N2, yellow oil, yield 23%. LCMS m/z 269 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 7.79−7.86 (m, 3H), 7.78 (s, 1H), 7.56 (dd, J = 1.6, 8.4 Hz, 1H), 7.43−7.50 (m, 2H), 3.96 (d, J = 13.9 Hz, 1H), 3.85 (d, J = 13.9 Hz, 1H), 3.08 (ddd, J = 3.8, 6.5, 7.8 Hz, 1H), 2.88 (ddd, J = 3.2, 8.0, 14.4 Hz, 1H), 2.71−2.78 (m, 2H), 2.68 (dd, J = 3.6, 13.6 Hz, 1H), 2.48−2.60 (m, 2H), 2.45 (s, 3H), 1.79− 1.89 (m, 1H), 1.61−1.71 (m, 1H), 1.17 (d, J = 6.53 Hz, 3H) 57: (1S,6R)-3-methyl-9-(naphthalen-2-ylmethyl)-3,9-diazabicyclo[4.2.1]nonane, C19H24N2, yellow oil, yield 35%. LCMS m/z 281 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 7.82−7.87 (m, 3H), 7.81 (s, 1H), 7.64 (dd, J = 1.2, 8.4 Hz, 1H), 7.43−7.51 (m, 2H), 3.93 (d, J = 14.0 Hz, 1H), 3.91 (d, J = 14.0 Hz, 1H), 3.37 (t, J = 8.0 Hz, 1H), 3.21−3.27 (m, 1H), 2.69 (ddd, J = 6.0, 8.0, 10.4 Hz, 1H), 2.55−2.63 (m, 1H), 2.47 (ddd, J = 1.6, 2.4, 11.6 Hz, 1H), 2.39 (dd, J = 2.4, 11.6 Hz, 1H), 2.36 (s, 3H), 2.20−2.30 (m, 1H), 2.06−2.16 (m, 1H), 1.89− 1.97 (m, 1H), 1.67−1.80 (m, 2H), 1.44−1.54 (m, 1H). 58: 1-(tert-butyl)-4-(naphthalen-2-ylmethyl)-1,4-diazepane, C20H28N2, yellow oil, yield 45%. LCMS m/z 297 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 7.80−7.88 (m, 3H), 7.77 (s, 1H), 7.55 (dd, J = 1.6, 8.4 Hz, 1H), 7.43−7.51 (m, 2H), 3.83 (s, 2H), 2.83 (t, J = 6.0 Hz, 2H), 2.74−2.78 (m, 4H), 2.69−2.72 (m, 2H), 1.81 (q, J = 6.0 Hz, 2H), 1.10 (s, 9H). 59: (S)-2-(naphthalen-2-ylmethyl)octahydro-1H-pyrrolo[1,2-a][1,4]diazepine, C19H24N2, yellow oil, yield 66%. LCMS m/z 281 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 7.80−7.88 (m, 3H), 7.77 (s, 1H), 7.55 (dd, J = 1.6, 8.4 Hz, 1H), 7.43−7.51 (m, 2H), 3.83 (s, 2H), 3.02−3.13 (m, 2H), 2.86−2.96 (m, 2H), 2.77−2.81 (m, 2H), 2.39−2.58 (m, 3H), 1.79−1.95 (m, 4H), 1.69−1.74 (m, 1H), 1.36− 1.42 (m, 1H). 60: cyclopropyl(4-(naphthalen-2-ylmethyl)-1,4-diazepan-1-yl)methanone, C20H24N2O, yellow oil, yield 53%. LCMS m/z 309 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 7.79−7.89 (m, 3H), 7.76 (br s, 1H), 7.55 (ddd, J = 1.2, 4.0, 8.0 Hz), 7.45−7.52 (m, 2H), 3.66− 3.84 (m, 6H), 2.82 (t, J = 5.2 Hz, 1H), 2.76 (t, J = 5.2 Hz, 1H), 2.72 (t, J = 5.6 Hz, 2H), 1.86−1.92 (m, 2H), 1.62−1.81 (m, 2H), 1.01−1.06 (m, 2H), 0.74−0.81 (m, 2H). 61: (3R,4S)-4-(4-(naphthalen-2-ylmethyl)-1,4-diazepan-1-yl)tetrahydrofuran-3-ol, C20H26N2O2, green oil, yield 61%. LCMS m/z 327 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 7.79−7.89 (m, 3H), 7.75 (s, 1H), 7.54 (dd, J = 1.6, 8.4 Hz, 1H), 7.44−7.52 (m, 2H), 4.36 (td, J = 4.05, 5.83 Hz, 1H), 4.06 (dd, J = 7.2, 9.2 Hz, 1H), 4.01 (dd, J = 6.0, 10.0 Hz, 1H), 3.84 (d, J = 13.2 Hz, 1H), 3.81 (d, J = 13.2 Hz, 1H), 3.71 (dd, J = 4.0, 9.6 Hz, 1H), 3.64 (dd, J = 6.8, 9.2 Hz, 1H), 3.20 (dt, J = 3.89, 7.03 Hz, 1H), 2.73−2.91 (m, 8H), 2.50 (br, 1H), 1.77−1.90 (m, 2H) 62: 1-(4-chlorobenzyl)-4-(naphthalen-2-ylmethyl)-1,4-diazepane, C23H25ClN2, yellow oil, yield 74%. LCMS m/z 365 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 7.79−7.88 (m, 3H), 7.76 (s, 1H), 7.56 (dd, J = 1.2, 8.4 Hz, 1H), 7.41−7.51 (m, 2H), 7.27−7.32 (m, 4H), 3.84 (s, 2H), 3.64 (s, 2H), 2.81 (t, J = 6.0 Hz, 2H), 2.73−2.78 (m, 4H), 2.60−2.71 (m, 2H), 1.84 (quin, J = 5.9 Hz, 2H). 63: N-methyl-2-(4-(naphthalen-2-ylmethyl)-1,4-diazepan-1-yl)acetamide, C19H25N3O, green oil, yield 72%. LCMS m/z 312 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 7.78−7.89 (m, 3H), 7.75 (s, 1H), 7.55 (dd, J = 1.2, 8.4 Hz, 1H), 7.44−7.52 (m, 2H), 7.29 (br, 1H), 3.83 (s, 2H), 3.18 (s, 2H), 2.87 (d, J = 4.8 Hz, 3H), 2.83 (t, J = 6.0 Hz, 2H), 2.71−2.80 (m, 6H), 1.83 (quin, J = 5.9 Hz, 2H). 64: 4-(naphthalen-2-ylmethyl)-1,4-diazepane-1-carbaldehyde, C17H20N2O, yellow oil, yield 41%. LCMS m/z 269 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 8.13 (d, J = 10.9 Hz, 1H), 7.80− 7.87 (m, 3H), 7.74 (br s, 1H), 7.53 (td, J = 2.0, 8.4 Hz, 1H), 7.45− 7.51 (m, 2H), 3.82 (d, J = 8.4 Hz, 2H), 3.57−3.62 (m, 2H), 3.46−3.53 (m, 2H), 2.78−2.82 (m, 1H), 2.67−2.76 (m, 3H), 1.87−1.98 (m, 2H). 65: 4,4′-bis((4-methyl-1,4-diazepan-1-yl)methyl)-[1,1′-biphenyl]-2carbonitrile, C27H37N5. To a solution of 72a (5.5 mL, 44.2 mmol) in DCM (50 mL) 81 (5.5 g, 20.0 mmol) was added portionwise. The mixture was stirred at room temperature for 15 min and then treated

with K2CO3 (15 g) and water (10 mL). The organic phase was evaporated and the residue was purified on a column (DCM/MeOH = 100:20) to give 82 (C14H18BrN3) as yellow oil (4.3 g, 70%). Suzuki coupling of 82 with 79 by utilizing general procedure 2 gave 65 as yellow oil, yield 59%. LCMS m/z 432 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 7.77 (d, J = 1.6 Hz, 1H), 7.63 (dd, J = 2.0, 8.0 Hz, 1H), 7.40−7.58 (m, 5H), 3.69−3.77 (m, 4H), 2.65−2.81 (m, 16H), 2.42 (s, 3H), 2.41 (s, 3H), 1.83−1.92 (m, 4H). 66, 68−70 were synthesized in a parallel fashion. Suzuki coupling of 79 with boronic acids 78a−d by utilizing general procedure 2 gave the corresponding intermediates 80a−d followed by reductive amination with 72a by utilizing general procedure 1. 66: 4,4′-((2,3-difluoro-[1,1′-biphenyl]-4,4′-diyl)bis(methylene))bis(1-methyl-1,4-diazepane), C26H36F2N4, yellow oil, yield 12% for two steps. LCMS m/z 443 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 7.49−7.57 (m, 2H), 7.43−7.46 (m, 2H), 7.23−7.27 (m, 1H), 7.15− 7.20 (m, 1H), 3.78 (s, 2H), 3.71 (s, 2H), 2.65−2.84 (m, 16H), 2.41 (s, 3H), 2.40 (s, 3H), 1.87(quin, J = 6.0 Hz, 4H). 67: 2′-fluoro-4,4′-bis((4-methyl-1,4-diazepan-1-yl)methyl)-[1,1′-biphenyl]-2-carbonitrile, C27H36FN5. Suzuki coupling of 82 with 83 utilizing general procedure 2 gave 2′-fluoro-4′-formyl-4-((4-methyl1,4-diazepan-1-yl)methyl)-[1,1′-biphenyl]-2-carbonitrile, C21H22FN3O (84) which was used in next step without fully characterization. Reductive amination of 84 with 72a (general procedure 1) gave 67 as brown oil. Yield 25 mg, 34% for two steps. LCMS m/z 450 [M + H+]. 1 H NMR (400 MHz, chloroform-d) δ 7.79 (d, J = 1.2 Hz, 1H), 7.64 (dd, J = 1.6, 8.0 Hz, 1H), 7.46 (dd, J = 0.8, 8.0 Hz, 1H), 7.37 (t, J = 7.6 Hz, 1H), 7.22−7.28 (m, 2H), 3.72 (s, 2H), 3.71 (s, 2H), 3.54 (s, 1H), 2.65−2.81 (m, 16H), 2.42 (s, 3H), 2.41 (s, 3H), 2.37−2.45 (m, 6H), 1.83−1.92 (m, 4H). 68: 4,4′-((3-chloro-[1,1′-biphenyl]-4,4′-diyl)bis(methylene))bis(1methyl-1,4-diazepane), C26H37ClN4, yellow oil, yield 12% for two steps. LCMS m/z 441 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 7.57−7.61 (m, 2H), 7.51−7.55 (m, 2H), 7.48 (dd, J = 2.0, 8.0 Hz, 1H), 7.41−7.45 (m, 2H), 3.80 (s, 2H), 3.70 (s, 2H), 2.63−2.86 (m, 16H), 2.42 (s, 3H), 2.40 (s, 3H), 1.82−1.94 (m, 4H). 69: 4,4′-((2-fluoro-[1,1′-biphenyl]-4,4′-diyl)bis(methylene))bis(1methyl-1,4-diazepane), C26H37FN4, yellow oil, yield 14% for two steps. LCMS m/z 425 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 7.50−7.54 (m, 2H), 7.36−7.45 (m, 3H), 7.16−7.22 (m, 2H), 3.70 (s, 2H), 3.68 (s, 2H), 2.64−2.80 (m, 16H), 2.41 (s, 3H), 2.40 (s, 3H), 1.86 (quin, J = 6.0 Hz, 4H). 70: 4,4′-((3,5-difluoro-[1,1′-biphenyl]-4,4′-diyl)bis(methylene))bis(1-methyl-1,4-diazepane), C26H36F2N4, yellow oil, yield 15% for two steps. LCMS m/z 443 [M + H+]. 1H NMR (400 MHz, chloroform-d) δ 7.49−7.53 (m, 2H), 7.44 (d, J = 8.4 Hz, 2H), 7.10−7.16 (m, 2H), 3.85 (s, 2H), 3.70 (s, 2H), 2.83−2.89 (m, 4H), 2.64−2.79 (m, 12H), 2.40 (s, 3H), 2.38 (s, 3H), 2.01 (s, 1H), 1.80−1.89 (m, 2H).



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b01552. Additional SAR tables, X-ray crystallography refinement data, structures of reference compounds, NMR spectra, and X-ray structures (PDF) Molecular formula strings (CSV) Accession Codes

The following X-ray crystal structures have been deposited in the RCSB Protein Data Bank: codes 5EQE, 5EQP, and 5EQY.



AUTHOR INFORMATION

Corresponding Author

*Phone: +1-617-621-2363. E-mail: [email protected]. N

DOI: 10.1021/acs.jmedchem.5b01552 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Present Addresses

magnetic resonance spectroscopy. Anticancer Res. 1999, 19, 1737− 1746. (8) Yalcin, A.; Clem, B.; Makoni, S.; Clem, A.; Nelson, K.; Thornburg, J.; Siow, D.; Lane, A. N.; Brock, S. E.; Goswami, U.; Eaton, J. W.; Telang, S.; Chesney, J. Selective inhibition of choline kinase simultaneously attenuates MAPK and PI3K/AKT signaling. Oncogene 2010, 29, 139−149. (9) Clem, B. F.; Clem, A. L.; Yalcin, A.; Goswami, U.; Arumugam, S.; Telang, S.; Trent, J. O.; Chesney, J. A novel small molecule antagonist of choline kinase-α that simultaneously suppresses MAPK and PI3K/ AKT signaling. Oncogene 2011, 30, 3370−3380. (10) Ramírez de Molina, A.; Rodríguez-González, A.; Penalva, V.; Lucas, L.; Lacal, J. C. Inhibition of ChoK Is an Efficient Antitumor Strategy for Harvey-, Kirsten-, and N-ras-Transformed Cells. Biochem. Biophys. Res. Commun. 2001, 285, 873−879. (11) Miyake, T.; Parsons, S. J. Functional interactions between Choline kinase α, epidermal growth factor receptor and c-Src in breast cancer cell proliferation. Oncogene 2012, 31, 1431−1441. (12) Sanchez-Lopez, E.; Zimmerman, T.; Gomez del Pulgar, T.; Moyer, M. P.; Lacal Sanjuan, J. C.; Cebrian, A. Choline kinase inhibition induces exacerbated endoplasmic reticulum stress and triggers apoptosis via CHOP in cancer cells. Cell Death Dis. 2013, 4, e933. (13) Janardhan, S.; Srivani, P.; Sastry, G. N. Choline kinase: an important target for cancer. Curr. Med. Chem. 2006, 13, 1169−1186. (14) Wu, G.; Vance, D. E. Choline kinase and its function. Biochem. Cell Biol. 2010, 88, 559−564. (15) Hamza, M.; Lloveras, J.; Ribbes, G.; Soula, G.; Douste-Blazy, L. An in vitro study of hemicholinium-3 on phospholipid metabolism of Krebs II ascites cells. Biochem. Pharmacol. 1983, 32, 1893−1897. (16) Hernández-Alcoceba, R.; Fernández, F.; Lacal, J. C. In vivo antitumor activity of choline kinase inhibitors a novel target for anticancer drug discovery. Cancer Res. 1999, 59, 3112−3118. (17) Hernández-Alcoceba, R.; Saniger, L.; Campos, J.; Núñez, M. C.; Khaless, F.; Gallo, M. A.; Espinosa, A.; Lacal, J. C. Choline kinase inhibitors as a novel approach for antiproliferative drug design. Oncogene 1997, 15, 2289−2301. (18) Rodríguez-Gonzalez, A.; Ramírez de Molina, A.; Fernández, F.; Ramos, M. A.; Nunez, M. d. C.; Campos, J.; Lacal, J. C. Inhibition of choline kinase as a specific cytotoxic strategy in oncogene-transformed cells. Oncogene 2003, 22, 8803−8812. (19) Ramirez de Molina, A.; Rodriguez-González, A.; Lacal, J. C. From Ras signalling to ChoK inhibitors: a further advance in anticancer drug design. Cancer Lett. 2004, 206, 137−148. (20) Falcon, S. C.; Hudson, C. S.; Huang, Y.; Mortimore, M.; Golec, J. M.; Charlton, P. A.; Weber, P.; Sundaram, H. A non-catalytic role of choline kinase alpha is important in promoting cancer cell survival. Oncogenesis 2013, 2, e38. (21) Hudson, C. S.; Knegtel, R. M.; Brown, K.; Charlton, P. A.; Pollard, J. R. Kinetic and mechanistic characterisation of Choline Kinase-α. Biochim. Biophys. Acta, Proteins Proteomics 2013, 1834, 1107−1116. (22) Lacal, J. C.; Campos, J. M. Preclinical characterization of RSM932A, a novel anticancer drug targeting the human choline kinase alpha, an enzyme involved in increased lipid metabolism of cancer cells. Mol. Cancer Ther. 2015, 14, 31−39. (23) Castro-Navas, F. F.; Schiaffino-Ortega, S.; Carrasco-Jimenez, M. P.; Ríos-Marco, P.; Marco, C.; Espinosa, A.; Gallo, M. A.; Mariotto, E.; Basso, G.; Viola, G.; Entrena-Guadix, A.; López-Cara, L. C. New more polar symmetrical bipyridinic compounds: new strategy for the inhibition of choline kinase α1. Future Med. Chem. 2015, 7, 417−436. (24) Serrán-Aguilera, L.; Nuti, R.; López-Cara, L. C.; Gallo Mezo, M. Á .; Macchiarulo, A.; Entrena, A.; Hurtado-Guerrero, R. Pharmacophore-based Virtual Screening to Discover New Active Compounds for Human Choline Kinase α1. Mol. Inf. 2015, 34, 458−466. (25) Rubio-Ruiz, B.; Figuerola-Conchas, A.; Ramos-Torrecillas, J.; Capitán-Cañadas, F.; Ríos-Marco, P.; Carrasco, M. P.; Gallo, M. Á .; Espinosa, A.; Marco, C.; Ruiz, C.; Entrena, A.; Hurtado-Guerrero, R.; Conejo-García, A. Discovery of a New Binding Site on Human



R.M.S.: Foundation Medicine, 150 Second Street, Cambridge, MA 02141, U.S. § L.E.P.: Siemens Healthcare Diagnostics, 333 Coney Street, Walpole, MA 02032, U.S. ∥ D.P.M.: Vaxess Technologies, 700 Main Street, Cambridge, MA 02139, U.S. ⊥ R.M.T.: Cerulean Pharma, Inc., 840 Memorial Drive, Cambridge, MA 02139, U.S. # Y.W.: Boston Ibio Inc., 32 Leonard Avenue, Newton, MA 02465, U.S. ∇ J.J.M.: Belfer Institute, Dana Farber Cancer Institute, 77 Louis Pasteur Avenue, Boston, MA 02115, U.S. Author Contributions †

S.G.Z., A.K., T.Z., and F.L. contributed equally to the work and manuscript preparation Notes

The authors declare the following competing financial interest(s): This work was funded by ARIAD Pharmaceuticals, Inc. All authors are employees and equity holders of ARIAD or were at the time that this study was conducted.



ACKNOWLEDGMENTS We thank D. Myszka (Biosensor Tools, LLC) and A. Vinitzky (Affinia Biotechnologies, Inc.) for SPR measurements, and S. Zhang and T. Padukkavidana for critical reading of the manuscript.

■ ■

DEDICATION The authors dedicate this work in friendship to Harvey Berger, MD, on the occasion of his 65th birthday. ABBREVIATIONS USED ChoK, choline kinase; STD NMR, saturation transfer difference nuclear magnetic resonance; FAXS, fluorine chemical shift anisotropy and exchange for screening; SPR, surface plasmon resonance; CA-II, carbonic anhydrase II; pCho, phosphocholine



REFERENCES

(1) Cuadrado, A.; Carnero, A.; Dolfi, F.; Jiménez, B.; Lacal, J. C. Phosphorylcholine: a novel second messenger essential for mitogenic activity of growth factors. Oncogene 1993, 8, 2959−2968. (2) Nishizuka, Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science 1992, 258, 607−614. (3) Gallego-Ortega, D.; Ramirez de Molina, A.; Ramos, M. A.; Valdes-Mora, F.; Barderas, M. G.; Sarmentero-Estrada, J.; Lacal, J. C. Differential role of human choline kinase alpha and beta enzymes in lipid metabolism: implications in cancer onset and treatment. PLoS One 2009, 4, e7819. (4) Glunde, K.; Bhujwalla, Z. M.; Ronen, S. M. Choline metabolism in malignant transformation. Nat. Rev. Cancer 2011, 11, 835−848. (5) Granata, A.; Nicoletti, R.; Perego, P.; Iorio, E.; Krishnamachary, B.; Benigni, F.; Ricci, A.; Podo, F.; Bhujwalla, Z. M.; Canevari, S.; Bagnoli, M.; Mezzanzanica, D. Global metabolic profile identifies choline kinase alpha as a key regulator of glutathione-dependent antioxidant cell defense in ovarian carcinoma. Oncotarget 2015, 6, 11216−11230. (6) Ramírez De Molina, A.; Gutiérrez, R.; Ramos, M. A.; Silva, J.; Silva, J. M.; Bonilla, F.; Sanchez, J.; Lacal, J. C. Increased choline kinase activity in human breast carcinomas: clinical evidence for a potential novel antitumor strategy. Oncogene 2002, 21, 4317−4322. (7) Gribbestad, I. S.; Sitter, B.; Lundgren, S.; Krane, J.; Axelson, D. Metabolite composition in breast tumors examined by proton nuclear O

DOI: 10.1021/acs.jmedchem.5b01552 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Choline Kinase α1: Design, Synthesis, Crystallographic Studies, and Biological Evaluation of Asymmetrical Bispyridinium Derivatives. J. Med. Chem. 2014, 57, 507−515. (26) Wittenberg, J.; Kornberg, A. Choline Phosphokinase. J. Biol. Chem. 1953, 202, 431−444. (27) Mayer, M.; Meyer, B. Characterization of Ligand Binding by Saturation Transfer Difference NMR Spectroscopy. Angew. Chem., Int. Ed. 1999, 38, 1784−1788. (28) Mayer, M.; Meyer, B. Group epitope mapping by saturation transfer difference NMR to identify segments of a ligand in direct contact with a protein receptor. J. Am. Chem. Soc. 2001, 123, 6108− 6117. (29) Dalvit, C.; Fagerness, P. E.; Hadden, D. T. A.; Sarver, R. W.; Stockman, B. J. Fluorine-NMR Experiments for High-Throughput Screening: Theoretical Aspects, Practical Considerations, and Range of Applicability. J. Am. Chem. Soc. 2003, 125, 7696−7703. (30) Shuker, S. B.; Hajduk, P. J.; Meadows, R. P.; Fesik, S. W. Discovering high-affinity ligands for proteins: SAR by NMR. Science 1996, 274, 1531−1534. (31) Giannetti, A. M.; Koch, B. D.; Browner, M. F. Surface plasmon resonance based assay for the detection and characterization of promiscuous inhibitors. J. Med. Chem. 2008, 51, 574−580. (32) Malito, E.; Sekulic, N.; Too, W. C. S.; Konrad, M.; Lavie, A. Elucidation of Human Choline Kinase Crystal Structures in Complex with the Products ADP or Phosphocholine. J. Mol. Biol. 2006, 364, 136−151. (33) Aoyama, C.; Liao, H.; Ishidate, K. Structure and function of choline kinase isoforms in mammalian cells. Prog. Lipid Res. 2004, 43, 266−281. (34) Liao, H.; Aoyama, C.; Ishidate, K.; Teraoka, H. Deletion and alanine mutation analyses for the formation of active homo-or heterodimer complexes of mouse choline kinase-α and-β. Biochim. Biophys. Acta, Mol. Cell Biol. Lipids 2006, 1761, 111−120. (35) Hong, B. S.; Allali-Hassani, A.; Tempel, W.; Finerty, P. J.; MacKenzie, F.; Dimov, S.; Vedadi, M.; Park, H.-W. Crystal Structures of Human Choline Kinase Isoforms in Complex with Hemicholinium3 Single Amino Acid near the Active Site Influences Inhibitor Sensitivity. J. Biol. Chem. 2010, 285, 16330−16340. (36) Copeland, R. A.; Pompliano, D. L.; Meek, T. D. Drug−target residence time and its implications for lead optimization. Nat. Rev. Drug Discovery 2006, 5, 730−739. (37) Swinney, D. C. The role of binding kinetics in therapeutically useful drug action. Curr. Opin. Drug Discovery Dev. 2009, 12, 31−39. (38) Zhang, R.; Monsma, F. The importance of drug-target residence time. Curr. Opin. Drug Discovery Dev. 2009, 12, 488−496. (39) Rich, R. L.; Myszka, D. G. Survey of the 2009 commercial optical biosensor literature. J. Mol. Recognit. 2011, 24, 892−914. (40) Wilson, W. D. Analyzing biomolecular interactions. Science 2002, 295, 2103. (41) Lu, H.; Tonge, P. J. Drug−target residence time: critical information for lead optimization. Curr. Opin. Chem. Biol. 2010, 14, 467−474. (42) Dahl, G.; Akerud, T. Pharmacokinetics and the drug−target residence time concept. Drug Discovery Today 2013, 18, 697−707. (43) Vilums, M.; Zweemer, A. J.; Yu, Z.; de Vries, H.; Hillger, J. M.; Wapenaar, H.; Bollen, I. A.; Barmare, F.; Gross, R.; Clemens, J. Structure−Kinetic Relationships: An Overlooked Parameter in Hit-toLead Optimization: A Case of Cyclopentylamines as Chemokine Receptor 2 Antagonists. J. Med. Chem. 2013, 56, 7706−7714. (44) Al-Saffar, N. M.; Troy, H.; Ramírez de Molina, A.; Jackson, L. E.; Madhu, B.; Griffiths, J. R.; Leach, M. O.; Workman, P.; Lacal, J. C.; Judson, I. R. Noninvasive magnetic resonance spectroscopic pharmacodynamic markers of the choline kinase inhibitor MN58b in human carcinoma models. Cancer Res. 2006, 66, 427−434. (45) Al-Saffar, N. M.; Jackson, L. E.; Raynaud, F. I.; Clarke, P. A.; Ramírez de Molina, A.; Lacal, J. C.; Workman, P.; Leach, M. O. The phosphoinositide 3-kinase inhibitor PI-103 downregulates choline kinase α leading to phosphocholine and total choline decrease

detected by magnetic resonance spectroscopy. Cancer Res. 2010, 70, 5507−5517. (46) Guma, M.; Sanchez-Lopez, E.; Lodi, A.; Garcia-Carbonell, R.; Tiziani, S.; Karin, M.; Lacal, J. C.; Firestein, G. S. Choline kinase inhibition in rheumatoid arthritis. Ann. Rheum. Dis. 2015, 74, 1399− 1407. (47) Bernard, N. J. Rheumatoid arthritis: Choline kinase: more than a cancer therapy target? Nat. Rev. Rheumatol. 2014, 10, 699−699. (48) Halgren, T. A. Identifying and characterizing binding sites and assessing druggability. J. Chem. Inf. Model. 2009, 49, 377−389. (49) SiteMap, version 2.4; Schrödinger, LLC: New York, NY, 2010. (50) Glide, version 5.5; Schrödinger, LLC: New York, NY, 2009. (51) LigPrep, version 2.3; Schrödinger, LLC: New York, NY, 2009. (52) Prime, version 2.1; Schrödinger, LLC: New York, NY, 2009.

P

DOI: 10.1021/acs.jmedchem.5b01552 J. Med. Chem. XXXX, XXX, XXX−XXX