Discovery of Potent KIFC1 Inhibitors Using a Method of Integrated

Nov 19, 2014 - ... Li Sha, Mark Zambrowski, Michael H. Block, James E. Dowling, Nancy Su, ... Nicholas J. Weise , Sabine L. Flitsch , and Nicholas J. ...
0 downloads 0 Views 2MB Size
Article pubs.acs.org/jmc

Discovery of Potent KIFC1 Inhibitors Using a Method of Integrated High-Throughput Synthesis and Screening Bin Yang,* Michelle L. Lamb, Tao Zhang, Edward J. Hennessy, Gurmit Grewal, Li Sha, Mark Zambrowski, Michael H. Block, James E. Dowling, Nancy Su, Jiaquan Wu,† Tracy Deegan, Keith Mikule, Wenxian Wang, Rüdiger Kaspera, Claudio Chuaqui, and Huawei Chen Oncology Innovative Medicine Unit, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States S Supporting Information *

ABSTRACT: KIFC1 (HSET), a member of the kinesin-14 family of motor proteins, plays an essential role in centrosomal bundling in cancer cells, but its function is not required for normal diploid cell division. To explore the potential of KIFC1 as a therapeutic target for human cancers, a series of potent KIFC1 inhibitors featuring a phenylalanine scaffold was developed from hits identified through high-throughput screening (HTS). Optimization of the initial hits combined both design−synthesis−test cycles and an integrated high-throughput synthesis and biochemical screening method. An important aspect of this integrated method was the utilization of DMSO stock solutions of compounds registered in the corporate compound collection as synthetic reactants. Using this method, over 1500 compounds selected for structural diversity were quickly assembled in assay-ready 384well plates and were directly tested after the necessary dilutions. Our efforts led to the discovery of a potent KIFC1 inhibitor, AZ82, which demonstrated the desired centrosome declustering mode of action in cell studies.



INTRODUCTION Kinesins play critical roles in the cytoskeletal rearrangements required for formation of the mitotic spindle and for the transport of organelles including mitochondria, lysosomes, and synaptic vesicles.1 Kinesin inhibitors have long been sought after as valuable anticancer agents, as they interfere with spindle formation to cause mitotic arrest. Kinesin spindle protein (known as KSP, HsEg5, or Eg5) from the kinesin-5 family was the first target of this type to be investigated in cancer drug discovery,2 and inhibition of KSP was found to arrest cell mitosis by preventing normal bipolar spindle formation,3 inducing apoptosis or cell death following the well-documented monoastral phenotype.4 A number of KSP inhibitors have been reported, including monastrol,4a the quinazolinone analogue ispinesib,4b MK-0731,5a filanesib (ARRY-520),5b AZD4877,6 and EMD-534185 (Figure 1).7 An inhibitor (GSK-923295) for another mitotic kinesin, centromere-associated protein-E (CENP-E; kinesin-7), has also been reported (Figure 2).8 KIFC1 is a member of kinesin-14 family of motor proteins.9 Similar to other kinesins, it contains a microtubule binding domain, a stalk domain, and a motor domain.10 Contrary to kinesin-5 proteins, KIFC1 and other kinesin-14 proteins have their motor domains located at the C-terminus rather than the N-terminus. Functionally, the KIFC1 motor protein slides and © 2014 American Chemical Society

cross-links microtubules from the minus-end of spindles during mitosis,11 thus opposing the function of plus-end directed motor proteins, such as KSP. It is possible that in normal cells KIFC1 does not play an essential role in mitosis because ablation of KIFC1 by a KIFC1-specific antibody in cultured cells did not change the microtubule architecture.12 In contrast, in cancer cells with supernumerary centrosomes, KIFC1 functions as the main force to cluster the amplified centrosomes, allowing cells to pass mitosis. KIFC1 knockdown by siRNA in MDA-MB-231 and BT549 cells (with centrosome amplification) was observed to cause multipolar spindles and eventually cell death but not in HeLa and MCF-7 cells (without centrosome amplification).13 These data suggest that small molecule inhibitors of KIFC1 may be effective in selectively targeting centrosome-amplified tumor cells. To identify KIFC1 inhibitors, we conducted a highthroughput screen of over a million compounds from our corporate compound collection using a Malachite Green (MG) based ATPase assay.14 The initial hits were triaged using a ratio test15 and were confirmed by an orthogonal KIFC1 assay (a pyruvate kinase/lactate dehydrogenase coupled assay, PK/ Received: August 1, 2014 Published: November 19, 2014 9958

dx.doi.org/10.1021/jm501179r | J. Med. Chem. 2014, 57, 9958−9970

Journal of Medicinal Chemistry

Article

compounds to extensively explore the SAR of the Phe scaffold. Particularly, we developed an integrated high speed synthesis and screening method, and using this method we synthesized and tested over 1500 compounds in less than a week. Herein we report a full account of the hit optimization effort on the Phe series that ultimately led to the discovery of potent KIFC1 inhibitors.



CHEMISTRY The initial chemistry strategy for the Phe series was to retain the phenylalanine core and to modify the three peripheral groups (R1, R2, and R3 in 7, Scheme 1). As illustrated in Scheme 1, synthetic routes were developed from the racemic version of 4-bromophenylalanine 3 to allow diversification of each of the R- groups at the last step. To explore R1, coupling of 3 with aryl or heteroaryl boronic acids afforded the biaryl intermediates 4. A coupling reaction of 4 with amines at the carboxyl group of the phenylalanine provided biaryl amides 5. Subsequent Boc deprotection generated the amine intermediates 6, setting the stage for the installation of a variety of R1 groups through amide coupling reactions with carboxylic acids to produce the Phe series analogues 7. To focus instead on R3, the Boc deprotection step of 4 and the subsequent reaction with pentafluorophenyl activated esters (R1COOC6F5)18 in the presence of organic base installed the R1 groups to produce the carboxylic acid intermediates 8. Amide coupling of 8 with amines produced compounds 7, allowing the installation of a variety of R3-amines at the C-terminus of the Phe core. Diversification at R2 was achieved by coupling R3-amines directly to the starting intermediate 3 gave to give compounds 9. Boc deprotection of 9 and the subsequent amide coupling reaction on the amine product with a R1-carboxylic acid produced the bisamide intermediate 10. With the presence of the 4-bromo group in 10, Suzuki coupling reactions were carried out with various R2-boronic acid reagents to afford analogues 7. Alternatively, compounds 10 could be also synthesized from 3 by sequential Boc deprotection reactions to generate amino acids, and their reaction with the activated esters R1COOC6F5 to install the R1 group afforded carboxylic acid intermediates. Coupling the acid intermediates with amines also generated compounds 10. However, we found this reaction sequence would result in partial racemization if an enantiomerically pure starting material 3 was used. The same tendency toward racemization was also observed in the conversion of compounds 8 to compounds 7. Pure enantiomers of compounds 7 were synthesized when enantiopure phenylalanine 3 was used as the starting material. To secure the chiral

Figure 1. Examples of known KSP inhibitors.

LDH). A number of compounds containing a phenylalanine scaffold, represented by compounds 1 and 2 (Figure 2), showed moderate KIFC1 inhibitory activities in both MG and PK/LDH assays. The kinesin motor domain is well conserved among various kinesins.16 While there are a number of inhibitor and ADPbound KSP X-ray structures available in public domain, the published crystal structures of KIFC1 and CENP-E are bound to ADP only.17 We previously reported that the binding of our series of inhibitors to KIFC1 protein requires the formation of KIFC1/microtubule complex, and an inhibitor-bound KIFC1 X-ray crystal structure proved to be difficult to obtain.14 We observed that 1 remarkably resembles the known CENP-E inhibitor (Figure 2).8 Recognizing the similarity in 3D structure between KIFC1 and CENP-E, we initially planned to leverage the structure−activity relationships (SAR) reported in the discovery of GSK-923295 to facilitate the design and synthesis of more potent KIFC1 inhibitors in this series. However, it was also possible that the SAR of CENP-E inhibitors might not be transferrable to KIFC1, as it was reported that the reported CENP-E inhibitors demonstrated minimal inhibition activity against a panel of human mitotic kinesins, including KIFC1.8 Moreover, three KSP inhibitors (monastrol, ispinesib, and AZD4877) were all inactive (IC50 >100 μM) in the KIFC1 MG assay. While utilizing the SAR of CENP-E inhibitors was a rational approach in drug design, in parallel we also undertook a serendipity-based chemistry strategy to synthesize libraries of

Figure 2. Structures of CENP-E inhibitor GSK-923295 and KIFC1 hits identified from HTS. 9959

dx.doi.org/10.1021/jm501179r | J. Med. Chem. 2014, 57, 9958−9970

Journal of Medicinal Chemistry

Article

Scheme 1a

a Reagents and conditions: (a) ArB(OH)2, Pd(PPh3)4, Cs2CO3, 1,4-dioxane/water, reflux; (b) amine, HATU, DIPEA, DMF, rt; (c) 4 N HCl in 1,4dioxane, MeOH, rt; (d) carboxylic acids, HATU, DIPEA, DMF, rt; (e) R1COO(C6F5), DIPEA, DMF, 50 °C.

Scheme 3a

integrity of the product, the preferred synthetic route was through compounds 5 in which the amide coupling reaction with different amines was carried out prior to the removal of the Boc protecting group. The high-throughput exploration of R1 groups utilized the reaction shown in Scheme 2. The coupling reaction condition was modified using DMSO as the solvent and triethylamine as the base. Scheme 2

a

Reagents and conditions: (a) Zn, I2, 5-Br-2-R2-pyridine, DMF, PdCl2(PPh3)2, 50 °C; (b) LiOH, MeOH/water; (c) R3NH2, HATU, DIPEA, DMF, rt; (d) HCl (4 N in 1,4-dioxane), MeOH, rt; (e) R1 carboxylic acid, HATU, DIPEA, DMF, rt.

formed by an induced-fit pocket between helix α3 and the L5 insertion loop as determined by X-ray crystallography.19 The binding site of CENP-E inhibitors was mapped to a similar region in CENP-E17b through cross-linking and mutagenesis studies, between helices α2 and α3, adjacent to loop L5.20 These results suggest that this same site in KIFC1 could be susceptible to small molecule inhibitors. Comparison of KSP− ligand−ADP and KSP−ADP structures suggested that ligand binding results in a more open site between two helices (α2 and α3) than is seen in the structure with ADP alone. Illustrated in Figure 3 is a previously described homology model of KIFC114 that was built by incorporating insight from the structure of KSP−EMD-534085−ADP (PDB: 3L9H, an

To replace the Phe phenyl ring with a pyridyl group, a very effective Negishi coupling reaction was employed (Scheme 3). Utilizing commercially available iodoalanine (13), reaction with substituted bromopyridines gave good yields and retained enantiomeric integrity of the chiral center. Subsequent ester hydrolysis afforded the carboxylic acid 15. Amide coupling and Boc deprotection provided the amine intermediate 16, which coupled with R1 carboxylic acids to generate the pyridyl analogues 17.



RESULTS AND DISCUSSION Initial Screen of R1 Groups. Many known KSP inhibitors, including monastrol, share a common allosteric binding site 9960

dx.doi.org/10.1021/jm501179r | J. Med. Chem. 2014, 57, 9958−9970

Journal of Medicinal Chemistry

Article

potency of CENP-E inhibitors,8 they were detrimental to the potency of KIFC1 inhibitors. Similarly, the incorporation of the N,N-dimethyl glycinamide side chain of GSK923295 to the Phe scaffold (compound 22, synthesis information in Scheme S1, Supporting Information) also caused the complete loss of potency. Encouragingly, the installation of the 3-chloro-4isopropyl-benzamide group as R1 into the Phe KIFC1 scaffold gave compound 21 that largely retained the potency of the original hits (1 and 2). An intriguing aspect is that the 3-chloro-4-isopropylbenzamide group had been derived from fragment-based screening, and it appeared to be the characteristic structural moiety in the disclosed CENP-E inhibitors.8 We hypothesized that a similar group could induce a pocket in KIFC1, as illustrated in the proposed binding model in Figure 3, where the substituted benzamide group R1 is immersed in a deep hydrophobic pocket. To further evaluate the impact of various R1 groups on the potency of the Phe scaffold, we undertook a synthetic campaign to investigate the SAR at this position. The selection of carboxylic acids to replace the benzamide group in compound 12 was first diversity-based, with a variety of alkyl, aryl, heteroaryl, and heterocyclic carboxylic acids being installed through amide coupling reactions (Scheme 1, synthesis of compounds 7 from compounds 6). The diversity of R1 groups is characterized in Figure 5. Out of the total of 190 R1 groups installed, a clear trend illustrated in Figure 5A is that more polar R1 groups (low ClogP) diminished the potency of the Phe compounds (IC50 >100 μM). Further analysis of these groups indicated that the presence of hydrogen bond donors reduced the activity of the Phe compounds. The number of rings in the R1 groups was plotted against the KIFC1 IC50 in Figure 5B. Evidently, none of the acyclic R 1 groups demonstrated KIFC1 inhibitory activity. A variety of bicyclic and tricyclic R1 groups were also incorporated in this synthetic campaign, without meaningfully improving the potency. Among the carboxylic acids with just one aromatic ring, substituted benzoic acids were systematically explored, with the

Figure 3. Homology model of KIFC1 bound to 21 (gray sticks), illustrating its location relative to ADP (gold sticks) and the L5 loop (purple) and the proposed position of the substituted benzamide group in a pocket induced between helices α2 and α3 (yellow ribbon). The model is constructed based on the EMD-534085-bound KSP (PDB: 3L9H) and ADP-bound KIFC1 (PDB: 2REP) crystal structures.

inhibitor which contained an aryl-CF3 group) and the inhibited CENP-E model proposed by Wood et al.20 Without an experimental inhibitor-bound KIFC1 structure or a known KIFC1 inhibitor to aid rational design,21 but using our model and the CENP-E SAR8 as a guide, we quickly assembled a number of compounds 7 in which about a dozen heteroaryl groups were incorporated at R2. The meta-CF3-Ph group in 1, which is associated with poor physical properties, such as high lipophilicity, high plasma protein binding, and low solubility, was first replaced by an imidazolpyridine group and a variety of imidazole and pyrazole groups (shown in Figure 4 are three exemplary compounds 18, 19, and 20). However, while the imidazole groups had been reported to significantly increase the

Figure 4. Biochemical activities of initial hybrid analogues molecules. 9961

dx.doi.org/10.1021/jm501179r | J. Med. Chem. 2014, 57, 9958−9970

Journal of Medicinal Chemistry

Article

Figure 5. (A) ClogP of R1 groups and potency of compounds 12. (B) Number of rings in R1 and the potency of compounds 12. Each dot in (A) is colored by the number of hydrogen bond acceptors (HBA) and in (B) by the number of hydrogen bond donors (HBD).

purification difficulty of final compounds from solution-phase based methods. However, Burgess et al. reported an interesting solution-phase combinatorial method for the synthesis of labeled bivalent β-turn mimics.25 The library of compounds was formed by pipetting aliquots of each reaction component into each well, without coupling agents or protecting group chemistry, resulting in covalently assembly of highly pure products, thus further purification of the compounds was deemed unnecessary. More recently, an efficient synthetic method was developed for the synthesis of histone deacetylase (HDAC) inhibitors in 96-well plates. In particular, the compounds synthesized were used directly in high-throughput screening without further purification.26 However, both of these solution-phase methods avoided the use of additional reagents to prevent possible interference in the subsequent biological assays, which in turn limited the choice of suitable reactions, as well as the size of their respective libraries. Recognizing the efficient amide coupling reactions for the installation of carboxylic acids to the phenylalanine core shown in Scheme 2, we anticipated that a large number of carboxylic acids could be directly used for the synthesis of compounds 12. An accessible source of these carboxylic acids is our compound collection, in which thousands of carboxylic acids are available as 10 mM solutions in DMSO. Moreover, the fixed concentration of the acids was expected to determine the concentration of the products formed, which would be directly screened in the KIFC1 MG assay. A concern was the possible interference in the assay of the coupling reagents or the side products resulting from their degradation. To establish this method, we initially tested a small set of benzoic acids that had been previous installed to the Phe scaffold with known potency. Plate-Synthesized Samples Demonstrated Validated KIFC1 IC50. The IC50s of the plate-synthesized compounds 12 were obtained in the KIFC1 MG assay, and they were plotted against the IC50s of the pure compounds that were obtained earlier. The activity of the pure compounds spread from 2 μM to greater than 100 μM. As illustrated in Figure 6, the measured potency of the plate-synthesized samples is very well correlated to the potency of those corresponding pure samples. As negative controls, a few benzoic acids whose corresponding pure products showed IC50s >100 μM (omitted from Figure 6) were included, and none of their corresponding platesynthesized products showed biochemical activity. To rule

KIFC1 IC50 of this subset of compounds varying from 2 to >100 μM. The initial hit identified from the R1 group screening that demonstrated a 10-fold improvement in potency was compound 23, which featured a 4-methoxy and 3-methyoxymethyl substitution on the phenyl ring of the benzamide group (Table 1). Replacing the MeOCH2− moiety on C-3 of Table 1. Optimization on R1 Improved Potency

23 24 25 26

R1

KIFC1 IC50 (μM)

4-MeO-3-MeOCH2-Ph 3-EtO-4-MeO-Ph 3-EtO-5-EtO-Ph 3-EtO-Ph

1.9 1.3 2.1 3.2

the benzamide with an ethoxy substituent (24) retained potency. The equipotent diethoxy-substituted (25) and mono-EtO-substituted analogues (26) indicated that the meta substitution of EtO− or MeOCH2− is critical to the potency of these compounds. Indeed, other close analogues (not shown) that contain MeO, iPrO−, or n-BuO− as the meta substituent showed significantly reduced potency (IC50 >15 μM). Integrated High-Throughput Synthesis and Screening. Encouraged by the emerging SAR when different carboxylic acids were installed as R1 in structure 12, we searched for a feasible method to extensively explore substituted aryl and heteroaryl acids to further improve the potency of this series. The field of parallel synthesis and combinatorial chemistry initially received wide application since the publication of a series of seminal papers,22 but today its limitations are more recognized and its use less prevalent. One key challenge for these methods has been the purification of compounds,23 resulting in false positive and false negative results in biological assays.24 Solid supported high-speed synthesis (e.g., covalent attachment, ionic attachment, and scavenger resins) has been utilized more because of the 9962

dx.doi.org/10.1021/jm501179r | J. Med. Chem. 2014, 57, 9958−9970

Journal of Medicinal Chemistry

Article

Figure 6. IC50s of the plate-synthesized compounds correlate well with the IC50s of pure compounds synthesized separately. The blue line represents equipotency. Detailed structural information and KIFC1 IC50 data of these compounds is in Table S1, Supporting Information.

Figure 7. A diverse set of R1 groups were selected to explore properties such as molecular weight, ClogP, and the number of hydrogen bond acceptors (HBA). Calculated polar surface area (PSA), number of hydrogen bond donors (HBD), and number of heavy atoms of the R1 groups are shown in Figure S1, Supporting Information.

out any false positive signals that could stem from the multiple reaction components (Scheme 2), the protocol was carried out first in the absence of compound 11 and then without any of the carboxylic acids, and no inhibition of KIFC1 activity was observed. Thus, the activity observed in the MG assay accurately reflected the potency of the products formed in the reactions on the plate, and the method could be confidently applied to a large number of carboxylic acids. Selection of Carboxylic Acids and Screening Results. Given the substantial number and structural diversity of carboxylic acids in the corporate compound collection, acids were selected using the following criteria. First, existing SAR indicated particular substitutions of a phenyl ring of the benzamide motif (R1 group, structure 12) demonstrated strong impact on potency, while alkyl R1 groups were devoid of activity. Hence only benzoic acids, heteroaryl carboxylic acids, and a very limited number of nonaromatic, cyclic carboxylic acids were selected. Second, taking into consideration the high capacity of this method and the need for rapid SAR exploration, we decided to select over 1500 acids. Lastly, recognizing the possibility for serendipity, the acids were selected to cover a diverse range of structural features and physical properties such as polar surface area (PSA), lipophilicity, number of hydrogen bond donors and acceptors, and number of rings (Figure 7). The high-throughput synthesis was conducted in multiple plates. The reactants were added to each well of the plates in three solutions, each with fixed concentration and volume. The selected carboxylic acids solutions were first dispensed, followed by the solution mixture of amine 11 and TEA in DMSO. Finally, freshly prepared HATU solution (10 mM in DMSO) was dispensed to the plates. After incubation, an LC/ MS purity analysis was conducted for the solutions in all of the plates. Desired products were detected in more than 94% of the reactions, and there were tentative products in another 1% of the reactions, thus leaving about 5% of the reactions that afforded no observed products (Figure S2, Supporting Information). The presumed concentration of the products generated in this method is 3.3 mM, and the solutions were diluted to 16.6 and 1.66 μM for the biochemical assay. Out of the total of 1549 reactions that were tested, 130 showed equal or greater than 50% inhibition at 16.6 μM (hit rate 8.3%). At 1.66 μM concentration, five of the reactions demonstrated greater than 50% inhibition. The activities obtained in these two concentrations correlated well, and compounds shown to

have greater than 50% inhibition at 1.6 μM all demonstrated greater than 80% inhibition at 16.6 μM. The top 32 hits were followed-up with 10-point IC50 measurements to confirm their potency (Figure 8). Satisfac-

Figure 8. IC50 of the selected hits and their corresponding percent inhibition at 1.6 μM. Potent inhibitors 27 and 28 are indicated.

torily, the top two hits, 27 and 28, were shown to have submicromolar IC50s. Upon independent resynthesis, their potency against KIFC1 was indeed confirmed to be 0.18 and 0.32 μM, respectively (Table 2). These two hits contain distinct substitutions pattern on their respective 5-membered heteroaryl rings (thiophene and furan). Two additional analogues (29, 30) were also synthesized, in which the thiophene ring was replaced by a phenyl ring, and the optimal methyl and n-propyl substitutions were retained, as was their KIFC1 enzyme activity (Table 2). Investigation of the R2 Groups through Suzuki Coupling. Having rapidly explored R1, we turned out attention to the R2 position. In addition to imidazole and pyrazole groups at R2, we also investigated another 95 groups through Suzuki coupling reactions. Plotted in Figure 9 is a representation of these R2 groups based on their ClogP and molecular weight. The number of hydrogen bond acceptors varied from 0 to 4. To mimic the hydrophobic feature of the m-CF3-Ph group in the initial hit 2, most R2 possess no H-bond donors. The vast majority of the R2 groups were substituted phenyl groups, with a small set featuring bicyclic or tricyclic structures. 9963

dx.doi.org/10.1021/jm501179r | J. Med. Chem. 2014, 57, 9958−9970

Journal of Medicinal Chemistry

Article

compounds. Aiming to adjust the dihedral angle of the biphenyl motif, we also synthesized analogues with ortho substituents (e.g., F−, Me−) on the phenyl group, but the potency of these analogues was not improved (data not shown). We speculate that the trifluoromethyl moiety interacts with KIFC1 through a hydrophobic pocket at the base of the L5 loop that also fits it in size, and this would be consistent with the sensitivity to any change in size or position of this group, as illustrated in Figure 3. It is well recognized that this loop is not highly conserved among the family of kinesins, and indeed the 3D overlay of X-ray structures of KSP, KIFC1, and CENP-E showed distinct difference in length among the L5 loops of these three kinesins (KSP > KIFC1 > CENP-E).27 Additionally, the amino acid residues surrounding the possible induced-fit pocket among these three kinesins differ substantially despite their similar fold. Consistent with the selectivity observed for KSP inhibitors over other kinesins, we later tested GSK-923295 and confirmed its inactivity in the KIFC1 MG assay (IC50 > 100 μM). Potency of Enantiomers and the Basic Amine Side Chain SAR. To further understand the SAR of compound 23, both enantiomers (23a and 23b) were synthesized using the enantiomerically pure version of compound 3 but surprisingly did not demonstrate meaningful potency differences with respect to KIFC1 inhibition (Table 4). We suspected that the

Table 2. Phenylalanine Compounds from High-Throughput Synthesis and Screening

27 28 29 30

R1

KIFC1 IC50 (μM)

a b c d

0.18 0.32 0.14 0.25

Table 4. Enantiomeric Preference of Phe Compounds on Potency

Figure 9. Diversity-based selection of R2 groups. As indicated in the graph, the dots are colored by the number of hydrogen bond acceptors in the R2 groups, and the shape represents the number of rings in R2.

Surprisingly, structural changes to the terminal m-CF3-Ph moiety were not well tolerated, with only the m-CF3O-Ph retaining low micromolar potency (31, Table 3). A 3-Et-Ph group (32) remained weakly active against KIFC1, while a naked phenyl group (35) was inactive. Replacing the phenyl of m-CF3-Ph in compound 12 with pyridyls also gave inactive

23a 23b 27a 27b 36a 36b 37a 37b 38a 38b

Table 3. R2 Groups and Their KIFC1 Inhibition Activity

a

23 31 32 33 34 35

R2

KIFC1 IC50 (μM)

3-CF3-Ph 3-CF3O-Ph 3-Et-Ph 3-iPr-Ph 3-t-Bu-Ph Ph

1.9 0.96 6.0 41 50 >100

chiral center

R1

R3

KIFC1 IC50 (μM)

R S R S R S R S R S

A a b b b b c c c c

b b b b a a c c a a

2.6 2.0 0.15 4.5 1.7 18.0 0.19 0.52 4.8 10.2

ClogP

solubility (μM)

5.15

20

6.99

6

7.0

>684a

6.34

1

6.90

12

The solubility is the average of two enantiomers 36a and 36b.

activity differences of enantiomers would be significant with more potent compounds. Indeed, with the installation of the substituted thiophene-2-carboxamide as R1, two enantiomers (27a and 27b) demonstrated 30-fold difference in potency. The potency difference between their acid analogues (36a and 36b) was less significant, but the R configuration consistently gave better potency against KIFC1. Compared to the thiophene amide enantiomeric pairs (27 and 36), the benzamide version of the pairs of enantiomers (37a and 37b, 38a and 38b) were 9964

dx.doi.org/10.1021/jm501179r | J. Med. Chem. 2014, 57, 9958−9970

Journal of Medicinal Chemistry

Article

more similar in potency, although again R enantiomers were more potent. A possible explanation for the lack of enantiomeric preference for the less potent Phe analogues as KIFC1 inhibitors is that the basic amine side chain (R3) could be exposed to solvent when the ligand binds and thus lack a specific interaction with the kinesin or ADP. Earlier in our SAR exploration, R3 in compound 7 had been modified and replaced by simple N-methyl, N,N-dimethyl, or primary amides. To our surprise, these analogues all lost potency (data not shown), even though the carboxylic acid group in compound 2 identified from HTS was active. We set out to explore a set of primary and secondary amines (R3 groups in Table 4) to investigate the impact on potency (Scheme 1, synthesis from 8 to 7 through amide coupling reactions). Surrogates of the basic amine group, such as nitrogen-containing heteroaryls, were not tolerated (data not shown). However, we found a wide range of alkyl substituted ethylene diamines as the R3 group retained the potency of compound 12; in particular, the alkyl substituents on the terminal nitrogen could be successfully fused into a pyrrolidine ring (R3, c in Table 4). Replacement of the Phenyl Group of the Phenylalanine Core. A number of analogues replacing the central phenyl group of the Phe scaffold with heteroaryl groups were synthesized utilizing the synthetic route illustrated in Scheme 3, aiming to potentially enhance potency and lower log P to improve physical properties (Table 5). Compounds 39−42 maintained similar potency compared to their phenyl analogues, and possibly as a result of decreased lipophilicity, their solubility is also marginally improved. In a human plasma protein binding assay, however, the compounds were shown to be less than 1% free.

Compound 39 (AZ82) demonstrated better cellular potency in our initial cell activity assessments and was more extensively tested in a series of enzymatic and cellular studies.14 We confirmed 39 is a specific inhibitor of KIFC1 (Ki = 0.043 μM) by biochemical and biophysical experiments. In HeLa cell studies, compound 39 (0.4 μM) effectively engaged with the minus-end directed KIFC1 motor inside cells to reverse the monopolar spindle phenotype induced by the inhibition of the plus end-directed kinesin KSP. When we treated BT-549 cells with 39 (0.4 μM), robust induction of multipolar spindle formation was observed, whereas minimal effects were seen in HeLa cells. Consistent with the hypothesis that aneuploid cells with unbundled centrosomes will eventually become apoptotic, we observed evidence of increased metaphase to anaphase time and mitotic catastrophe only in BT-549 cells when mitotic catastrophe was monitored both in BT-549 and MCF7 cells for 44 h following 39 treatments (0.4 μM). Our studies supported the hypothesis that inhibiting KIFC1 could specifically impact the survival of cancer cells with amplified centrosomes.14 To further evaluate the potential of 39 as an in vivo probe, we first tested the pharmacokinetics of 39 in rats using both intravenous (dosed at 1 mg/kg) and oral (dosed at 2.5 mg/kg) routes. The compound was found to have low clearance (0.6 L· h−1·kg−1), a moderate volume of distribution (1.3 L·kg−1), and a half-life of 8.4 h. The bioavailability of 39 was negligible, likely due to its low solubility and an active efflux as measured in an MDCK-MDR1 and Caco-2 cell assays (efflux ratio >120). We further tested its exposure in mice (C57bl/6) after oral and intraperitoneal injection doses. As shown in Table 6, very Table 6. Mouse Exposure of Compound 39 after PO and IP Dosea

Table 5. Pyridyl Analogues Demonstrate Similar Potency and Improved Physical Properties

a

route

dose (mg/kg(

AUCinf (ng·h/mL)

Cmax (ng/mL)

t1/2 (h)

PO PO IP

30 100 30

211 5.6 × 103 1.2 × 105

67 1.0 × 103 3.8 × 104

3.3 7.5b

30% cremophor/captisol at pH 4.0 for formulation. bEstimated.

limited exposure was obtained after oral administration of 39 at 30 and 100 mg/kg, consistent with the low bioavailability observed in rats. However, following a single IP dose at 30 mg/ kg, substantial exposure of 39 was observed in mouse plasma, and the compound had a reasonable half-life of 7.5 h.



39 40 41 42

KIFC1 IC50 (μM)

ClogP

solubility (μM)

hPPB (% free)

0.31 0.50 0.70 2.0

5.39 5.95 5.74 4.78

5.5 64 12 7

98%. For enantiomer 27b; ee >98%. 2-Cyclopropyl-N-(1-(2-(dimethylamino)ethylamino)-1-oxo-3-(3′(trifluoromethyl)biphenyl-4-yl)propan-2-yl)-5-methylfuran-3-carboxamide (28). 1H NMR (300 MHz, DMSO-d6) d ppm 9.32 (br s, 1 H), 8.31 (s, 1 H), 7.88−8.08 (m, 3 H), 7.65−7.75 (m, 4 H), 7.43 (d, J = 8.10 Hz, 2 H), 6.47−6.58 (m, 1 H), 4.57−4.71 (m, 1 H), 3.37−3.51 (m, 2 H), 2.97−3.17 (m, 4 H), 2.81 (d, J = 4.52 Hz, 6 H), 2.60−2.75 (m, 1 H), 2.16 (s, 3 H), 0.72−0.92 (m, 4 H). LCMS (M + H) = 528. (E)-N-(1-(2-(Dimethylamino)ethylamino)-1-oxo-3-(3 ′(trifluoromethyl)biphenyl-4-yl)propan-2-yl)-4-methyl-3-(prop-1enyl)benzamide (29). 1H NMR (300 MHz, DMSO-d6) δ ppm 9.25− 9.42 (m, 1 H), 8.67 (d, J = 8.10 Hz, 1 H), 8.34 (s, 1 H), 7.85−8.00 (m, 3 H), 7.68 (d, J = 9.23 Hz, 4 H), 7.58 (d,J = 7.91 Hz, 1 H), 7.45 (d, J = 8.10 Hz, 2 H), 7.21 (d, J = 7.91 Hz, 1 H), 6.60 (s, 1 H), 6.14−6.34 (m, 1 H), 4.61−4.77 (m, 1 H), 3.45 (m, J = 6.00 Hz, 2 H), 3.03−3.24 (m,

4 H), 2.81 (br s, 6 H), 2.33 (s, 3 H), 1.90 (d, J = 6.59 Hz, 3 H). LCMS (M + H) = 538. N-(1-(2-(Dimethylamino)ethylamino)-1-oxo-3-(3′(trifluoromethyl)biphenyl-4-yl)propan-2-yl)-4-methyl-3-propylbenzamide (30). 1H NMR (300 MHz, DMSO-d6) δ ppm 9.27−9.45 (m, 1 H), 8.57 (d, J = 8.10 Hz, 1 H), 8.34 (t, J = 5.46 Hz, 1 H), 7.87−8.00 (m, 2 H), 7.62−7.74 (m, 4 H), 7.51−7.61 (m, 2 H), 7.45 (d, J = 8.29 Hz, 2 H), 7.20 (d, J = 7.72 Hz, 1 H), 4.69 (d, J = 3.39 Hz, 1 H), 3.39− 3.49 (m, 2 H), 3.02−3.24 (m, 4 H), 2.81 (d, J = 4.14 Hz, 6 H), 2.53− 2.63 (m, 2 H), 2.29 (s, 3 H), 1.44−1.67 (m, 2 H), 0.85−1.01 (m, 3 H). LCMS (M + H) = 540. (S)-2-(5-Methyl-4-propylthiophene-2-carboxamido)-3-(3′(trifluoromethyl)biphenyl-4-yl)propanoic Acid (36). Both enantiomers were synthesized utilizing the route summarized in Scheme 1, through the intermediates 5 and 6. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.49−8.59 (m, 1 H), 7.93 (s, 2 H), 7.65−7.72 (m, 4 H), 7.58 (s, 1 H), 7.42 (d, J = 8.28 Hz, 2 H), 4.53−4.64 (m, 1 H), 3.17−3.28 (m, 1 H), 3.05−3.13 (m, 1 H), 2.41−2.47 (m, 2 H), 2.32 (s, 3 H), 1.55 (m, J = 7.30 Hz, 2 H), 0.91 (t, J = 7.28 Hz, 3 H). LCMS (M + H) = 476. Enantiomer 36a ee >96%. Enantiomer 36b ee >95%. 4-Methyl-N-((R)-1-oxo-1-((R)-pyrrolidin-3-ylamino)-3-(3′(trifluoromethyl)biphenyl-4-yl)propan-2-yl)-3-((E)-prop-1-enyl)benzamide (37a). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.57 (d, J = 8.28 Hz, 1 H), 8.18−8.25 (m, 1 H), 7.91−8.01 (m, 2 H), 7.88 (d, J = 1.51 Hz, 1 H), 7.63−7.73 (m, 4 H), 7.56 (d, J = 1.76 Hz, 1 H), 7.46 (d, J = 8.28 Hz, 2 H), 7.21 (d, J = 8.03 Hz, 1 H), 6.57−6.66 (m, 1 H), 6.21−6.33 (m, 1 H), 4.66−4.76 (m, 1 H), 4.09−4.21 (m, 1 H), 3.04− 3.15 (m, 2 H), 2.97 (m, J = 6.80 Hz, 2 H), 2.80−2.89 (m, 1 H), 2.54− 2.60 (m, 1 H), 2.31 (s, 3 H), 1.93−2.03 (m, 1 H), 1.90 (dd, J = 6.53, 1.51 Hz, 3 H), 1.56−1.67 (m, 1 H). LCMS (M + H) = 536; ee >98%. 4-Methyl-N-((S)-1-oxo-1-((R)-pyrrolidin-3-ylamino)-3-(3′(trifluoromethyl)biphenyl-4-yl)propan-2-yl)-3-((E)-prop-1-enyl)benzamide (37b). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.54 (d, J = 8.53 Hz, 1 H), 8.15 (d, J = 7.03 Hz, 1 H), 7.83−7.99 (m, 3 H), 7.64−7.73 (m, 4 H), 7.57 (m,1 H), 7.46 (d, 2 H), 7.20 (d, J = 7.78 Hz, 1 H), 6.62 (dd, J = 15.56, 1.76 Hz, 1 H), 6.29 (d, J = 6.53 Hz, 1 H), 4.65−4.78 (m, 1 H), 4.06−4.18 (m, 1 H), 3.02−3.14 (m, 2 H), 2.91− 2.99 (m, 1 H), 2.71−2.86 (m, 2 H), 2.57 (d, J = 11.29 Hz, 1 H), 2.31 (s, 3 H), 1.90 (m, J = 6.50, 1.50 Hz, 4 H), 1.38−1.53 (m, 1 H). LCMS (M + H) = 536; ee >98%. (R,E)-2-(4-Methyl-3-(prop-1-enyl)benzamido)-3-(3′(trifluoromethyl)biphenyl-4-yl)propanoic Acid (38). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.70 (d, J = 8.28 Hz, 1 H), 7.90−8.00 (m, 2 H), 7.87 (d, J = 1.76 Hz, 1 H), 7.63−7.74 (m, 4 H), 7.57 (dd, J = 8.03, 1.76 Hz, 1 H), 7.45 (d, J = 8.28 Hz, 2 H), 7.22 (d, J = 8.03 Hz, 1 H), 6.62 (dd, J = 15.81, 1.76 Hz, 1 H), 6.25 (dd, J = 15.69, 6.65 Hz, 1 H), 4.60−4.72 (m, 1 H), 3.21−3.31 (m, 1 H), 3.08−3.20 (m, 1 H), 2.31 (s, 3 H), 1.90 (dd, J = 6.53, 1.51 Hz, 3 H). LCMS (M + H) = 468. Enantiomeric excess was not determined for either enantiomers. Synthetic Procedure of Compound 39, AZ82. (R)-Methyl 3-(5Bromopyridin-2-yl)-2-(tert-butoxycarbonylamino)propanoate (Scheme 3, Step a). A two-neck 100 mL round-bottomed flask was charged with zinc (0.61 g, 9.24 mmol) and iodine (0.084 g, 0.33 mmol). The flask was evacuated under vacuum and heated with a heat gun for 10 min, flushed three times with nitrogen, and allowed to cool to rt. After addition of DMF (5 mL), a solution of (S)-methyl 2-(tertbutoxycarbonylamino)-3-iodopropanoate (2.390 g, 7.26 mmol) (7.26 mmol) in DMF (10 mL) was added dropwise to the well-stirred suspension of zinc at 0 °C in an ice/water bath. The mixture was stirred at 0 °C under nitrogen for 30 min. After the ice bath was removed, 5-bromo-2-(3-(trifluoromethoxy)phenyl)pyridine (2.1 g, 6.60 mmol) and dichlorobis(triphenylphosphine)-palladium(II) (0.232 g, 0.33 mmol) were added. The reaction mixture was heated at 50 °C for 5 h under nitrogen protection. The mixture was cooled to rt, and the excess amount of zinc was filtered off. Concentration in vacuo removed the solvent. To the residue was added water (400 mL) and EtOAc (300 mL). After partition and extraction with EtOAc (2 × 100 mL), the combined organic layers were dried (Na2SO4) and concentrated in vacuo. The crude product was added onto a silica gel column (330 g) and was eluted with (0−50% EtOAc/hexane) to give the product (1.6 g, 51% yield). LCMS (M + H) = 441. 9967

dx.doi.org/10.1021/jm501179r | J. Med. Chem. 2014, 57, 9958−9970

Journal of Medicinal Chemistry

Article

(R)-2-(tert-Butoxycarbonylamino)-3-(6-(3-(trifluoromethoxy)phenyl)pyridin-3-yl)propanoic Acid (Scheme 3, Step b). A solution of (R)-methyl 2-(tert-butoxycarbonylamino)-3-(6-(3(trifluoromethoxy)phenyl)pyridin-3-yl)propanoate (0.42 g, 0.95 mmol) in MeOH (5 mL) was placed in a 20 mL round-bottomed flask. Water (5.0 mL) and lithium hydroxide (0.027 g, 1.14 mmol) were added. The mixture was stirred at 60 °C for 12 h. Concentration removed the solvent to give the product, and it was carried forward without further purification (0.405 g, quantitative yield). LCMS (M − H) = 425. (R)-Benzyl 3-((R)-2-(tert-Butoxycarbonylamino)-3-(6-(3(trifluoromethoxy)phenyl)pyridin-3-yl)propanamido)pyrrolidine-1carboxylate (Scheme 3, step c). A solution of (R)-2-(tertbutoxycarbonylamino)-3-(6-(3-(trifluoromethoxy)phenyl)pyridin-3yl)propanoic acid (0.405 g, 0.95 mmol) and (R)-benzyl 3-aminopyrrolidine-1-carboxylate (0.209 g, 0.95 mmol) in DMF (3 mL) was placed in a 5 mL vial. HATU (0.397 g, 1.05 mmol) and Hunig’s base (0.498 mL, 2.85 mmol) were added. The reaction was stirred at rt for 2 h. Concentration in vacuo removed the solvent. To the residue was added EtOAc (80 mL) and water (200 mL). After partition and extraction with EtOAc (1 × 50 mL), the combined organic layers were dried (Na2SO4) and concentrated. The crude product was added to a silica gel column (120 g) and was eluted with MeOH (0.7 N NH3)/ DCM (0−8%). The collected fractions were concentrated to give the product as a brown solid (0.45 g, 75% yield). LCMS (M + H) = 629. (R)-Benzyl 3-((R)-2-Amino-3-(6-(3-(trifluoromethoxy)phenyl)pyridin-3-yl)propanamido)pyrrolidine-1-carboxylate (Scheme 3, Step d). (R)-Benzyl 3-((R)-2-(tert-butoxycarbonylamino)-3-(6-(3(trifluoromethoxy)phenyl)pyridin-3-yl)propanamido)pyrrolidine-1carboxylate (0.6 g, 0.95 mmol) was placed in a round-bottomed flask equipped with magnetic stirring bar. DCM (60 mL) was added at rt. The solution was stirred at rt for 2 h after the addition of 4 N HCl solution in dioxane (3 mL). Concentration in vacuo removed the solvent to give the product as its HCl salt. The product was carried forward without further purification (0.54 g, quantitative yield). LCMS (M + H) = 529. (R)-Benzyl 3-((R)-2-(5-Methyl-4-propylthiophene-2-carboxamido)-3-(6-(3-(trifluoromethoxy)phenyl)pyridin-3-yl)propanamido)pyrrolidine-1-carboxylate (Scheme 3, Step e). 5-Methyl-4-propylthiophene-2-carboxylic acid (0.045 g, 0.25 mmol) and (R)-benzyl 3((R)-2-amino-3-(6-(3-(trifluoromethoxy)phenyl)pyridin-3-yl)propanamido)pyrrolidine-1-carboxylate (0.13g, 0.25 mmol) were added in a 5 mL vial in DMF (1 mL) to give a brown solution. Hunig’s base (0.129 mL, 0.74 mmol) and HATU (0.094 g, 0.25 mmol) were added. The reaction mixture was stirred at rt for 0.5 h. The crude reaction solution was added to a prep HPLC column (Atlantis T3, 19 mm × 100 mm, 5 um) and was eluted with acetonitrile/water (0.1% TFA, 14 min run). The collected fractions were concentrated to give the product (0.031 g, 20% yield). LCMS (M + H) = 695. 5-Methyl-N-((R)-1-oxo-1-((R)-pyrrolidin-3-ylamino)-3-(6-(3(trifluoromethoxy)phenyl)pyridin-3-yl)propan-2-yl)-4-propylthiophene-2-carboxamide (39, AZ82). (R)-Benzyl 3-((R)-2-(5-methyl-4propylthiophene-2-carboxamido)-3-(6-(3-(trifluoromethoxy)phenyl)pyridin-3-yl)propanamido)pyrrolidine-1-carboxylate (0.020 g, 0.03 mmol) in MeOH (3 mL) was added in a round-bottomed flask equipped with magnetic stirring bar. Pd/C (10% powder, 0.02 g) was added. A hydrogen balloon was equipped to the flask, and the reaction was stirred at rt overnight. The solid residue was filtered off, and the crude reaction solution was added to a prep HPLC column (Atlantis T3, 19 mm × 100 mm, 5 μm) and was eluted with acetonitrile/water (0.1% TFA, 14 min run). The collected fractions were concentrated to give the product as its TFA salt (0.011 g, 52% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.55−8.63 (m, 2 H), 8.47 (d, 1 H), 8.09 (d, 1 H), 7.96−8.05 (m, 2 H), 7.83 (m,1 H), 7.58−7.66 (m, 2 H), 7.38− 7.45 (m, 1 H), 4.57−4.71 (m, 1 H), 4.32 (m, 1 H), 3.25−3.41 (m, 2 H), 3.17−3.26 (m, 1 H), 3.12 (m, 1 H), 3.05 (m, 1 H), 2.86−2.95 (m, 1 H), 2.40−2.48 (m, 3 H), 2.31 (s, 3 H), 2.15 (s, 1 H), 1.78−1.90 (m, 1 H), 1.48−1.63 (m, 2 H), 0.91 (t, 3 H). LCMS (M + H) = 561. Compounds 40, 41, and 42 were prepared using a similar procedure to compound 39.

(R)-N-(1-(2-(Dimethylamino)-1-oxo-3-(6-(3-(trifluoromethoxy)phenyl)pyridin-3-yl)propan-2-yl)-5-methyl-4-propylthiophene-2carboxamide (40). 1H NMR (400 MHz, DMSO-d6) δ ppm 9.44 (br s, 2 H), 8.55−8.66 (m, 6 H), 8.37 (t, J = 5.77 Hz, 2 H), 8.08 (d, J = 8.03 Hz, 3 H), 7.95−8.04 (m, 6 H), 7.83 (dd, J = 8.28, 2.26 Hz, 3 H), 7.59−7.66 (m, 5 H), 7.38−7.45 (m, 3 H), 4.58−4.70 (m, 3 H), 3.44 (d, J = 6.02 Hz, 4 H), 3.44 (d, J = 18.57 Hz, 2 H), 3.02−3.24 (m, 15 H), 2.81 (d, J = 3.76 Hz, 16 H), 2.40−2.48 (m, 7 H), 2.26−2.34 (m, 9 H), 1.50−1.62 (m, 6 H), 0.83−0.96 (m, 9 H). LCMS (M + H) = 563. (R)-N-(1-(2-(Dimethylamino)ethylamino)-1-oxo-3-(6-(3(trifluoromethyl)phenyl)pyridin-3-yl)propan-2-yl)-5-methyl-4-propylthiophene-2-carboxamide (41). 1H NMR (400 MHz, DMSO-d6) δ ppm 9.44 (br s, 3 H), 8.62 (m, 6 H), 8.30−8.44 (m, 9 H), 8.04 (d, J = 8.03 Hz, 3 H), 7.84 (dd, J = 8.03, 2.26 Hz, 3 H), 7.78 (d, J = 7.78 Hz, 3 H), 7.71 (t, J = 7.78 Hz, 3 H), 7.62 (s, 3 H), 4.64 (m, 3 H), 3.44 (d, J = 6.02 Hz, 4 H), 3.44 (d, J = 18.57 Hz, 2 H), 3.01−3.26 (m, 12 H), 2.81 (d, J = 3.76 Hz, 18 H), 2.44 (t, J = 7.53 Hz, 6 H), 2.30 (s, 9 H), 1.55 (dd, J = 7.53, 1.25 Hz, 4 H), 1.48−1.62 (m, 2 H), 0.86−0.95 (m, 9 H). LCMS (M + H) = 547. 3-Ethoxy-4-methyl-N-((R)-1-oxo-1-((R)-pyrrolidin-3-ylamino)-3(6-(3-(trifluoromethoxy)phenyl)pyridin-3-yl)propan-2-yl)benzamide (42). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.62−8.67 (m, 2 H), 8.48 (d, J = 6.27 Hz, 1 H), 8.09 (d, J = 8.03 Hz, 1 H), 7.97−8.05 (m, 2 H), 7.85 (dd, J = 8.28, 2.26 Hz, 1 H), 7.61 (t, J = 8.03 Hz, 1 H), 7.38− 7.45 (m, 1 H), 7.34 (dd, J = 7.65, 1.38 Hz, 1 H), 7.28−7.32 (m, 1 H), 7.20 (d, J = 8.03 Hz, 1H), 4.72 (m, J = 3.30 Hz, 1 H), 4.33 (m, J = 6.30 Hz, 1 H), 4.01−4.13 (m, 2 H), 3.27−3.43 (m, 2 H), 3.13−3.27 (m, 2 H), 3.06−3.13 (m, 1 H), 2.93 (d, J = 5.27 Hz, 1 H), 2.11−2.23 (m, 4 H), 1.86 (m, J = 7.00 Hz, 1 H), 1.35 (t, J = 6.90 Hz, 3 H). LCMS (M + H) = 557. High-Throughput Synthesis and Screening Method. Carboxylic acids in their solution form (10 mM in DMSO) were ordered from the corporate compound collection (20 μL each). The acid solutions were delivered in plastic 384-well plates (Thermo Scientific). The DMSO solution of the mixture of the amine starting material 11 (HCl salt) and TEA was prepared, and the concentrations of the amine and TEA in the solution were at 10 and 25 mM, respectively. Separately, a solution of HATU in DMSO (10 mM) was freshly prepared before its use. Sequentially, the amine and TEA solution (20 μL) and HATU solution (20 μL) were added into the wells in the plates that contained 20 μL of the acid solutions in DMSO. The reactions in the plate were incubated at room temperature overnight. The conversion of the reactions was presumed to be 100%, and the concentration of the products in each of the wells was calculated to be at 3.3 mM. The product solutions were diluted to two concentrations (33.3 and 1.6 μM) for the KIFC1 biochemical assay and screened as described previously.14



ASSOCIATED CONTENT

S Supporting Information *

Annotation of the table of contents graphic; synthetic scheme of compound 22; graphic representation of the diversity-based screening of the R1 groups; LCMS detection of products from the high throughput synthesis and screening; IC50s measured for both the isolated pure compounds and their corresponding crude samples synthesized in the high throughout synthesis method; experimental data for compound 22 and the pure compounds used in the method validation. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

*Phone: 781-839-4135. Fax: 781-839-4230. E-mail: bin.yang@ astrazeneca.com. Present Address †

For J.W.: E. J. Corey Institute of Biomedical Research, Jiangyin, Jiangsu 214437, P. R. China 9968

dx.doi.org/10.1021/jm501179r | J. Med. Chem. 2014, 57, 9958−9970

Journal of Medicinal Chemistry

Article

Notes

(HHPQs) as potent and selective inhibitors of the mitotic kinesin-5. Bioorg. Med. Chem. Lett. 2010, 20, 1491−1495. (8) Qian, X.; McDonald, A.; Zhou, H.; Adams, N. D.; Parrish, C. A.; Duffy, K. J.; Fitch, D. M.; Tedesco, R.; Ashcraft, L. W.; Yao, B.; Jiang, H.; Huang, J. K.; Marin, M. V.; Aroyan, C. E.; Wang, J.; Ahmed, S.; Burgess, J. L.; Chaudhari, A. M.; Donatelli, C. A.; Darcy, M. G.; Ridgers, L. H.; Newlander, K. A.; Schmidt, S. J.; Chai, C.; Colon, M.; Zimmerman, M. N.; Lad, L.; Sakowicz, R.; Schauer, S.; Belmont, L.; Baliga, R.; Pierce, D. W.; Finer, J. T.; Wang, Z.; Morgan, B. P.; Morgans, D. J.; Auger, K. R.; Sung, C.; Carson, J. D.; Luo, L.; Hugger, E. D.; Copeland, R. A.; Sutton, D.; Elliott, J. D.; Jackson, J. R.; Wood, K. W.; Dhanak, D.; Bergnes, G.; Knight, S. D. Discovery of the first potent and selective inhibitor of centromere-associated protein E: GSK923295. ACS Med. Chem. Lett. 2010, 1, 30−34. (9) Kuriyama, R.; Kofron, M.; Essner, R.; Kato, T.; Dragas-Granoic, S.; Omoto, C. K.; Khodjakov, A. J. Characterization of a minus enddirected kinesin-like motor protein from cultured mammalian cells. J. Cell Biol. 1995, 129, 1049−1059. (10) Lockhart, A.; Cross, R. A. Origins of reversed directionality in the NCD molecular motor. EMBO J. 1994, 13, 751−757. (11) (a) Walczak, C. E.; Verma, S.; Mitchison, T. J. XCTK2: a kinesin-related protein that promotes mitotic spindle assembly in Xenopus laevis egg extracts. J. Cell Biol. 1997, 136, 859−870. (b) Fink, G.; Hajdo, L.; Skowronek, K. J.; Reuther, C.; Kasprzak, A. A.; Diez, S. The mitotic kinesin-14 NCD drives directional microtubule-microtubule sliding. Nature Cell Biol. 2009, 11, 717−723. (12) Mountain, V.; Simerly, C.; Howard, L.; Andod, A.; Schatten, G.; Compton, D. A. The kinesin-related protein, HSET, opposes the activity of Eg5 and cross-links microtubules in the mammalian mitotic spindle. J. Cell Biol. 1999, 147, 351−66. (13) Kwon, M.; Godinho, S. A.; Chandhok, N. S.; Ganem, N. J.; Azioune, A.; Thery, M.; Pellman, D. Mechanisms to suppress multipolar divisions in cancer cells with extra centrosomes. Genes Dev. 2008, 22, 2189−2203. (14) Wu, J.; Mikule, K.; Wang, W.; Su, N.; Petteruti, P.; Gharahdaghi, F.; Code, E.; Zhu, X.; Jacques, K.; Lai, Z.; Yang, B.; Lamb, M. L.; Chuaqui, C.; Keen, N.; Chen, H. Discovery and mechanistic study of a small molecule inhibitor for motor protein KIFC1. ACS Chem. Biol. 2013, 8, 2201−2208. (15) Ratio test was designed to remove nonspecific inhibitors. HTS screening hits were tested under two parallel KIFC1 MG assays, with one being the regular MG assay and the other one tweaked to have 10 times enzyme concentration while the enzymatic reaction was onetenth compared to the regular MG assay. The ratio of the IC50 values from these two parallel KIFC1 MG assays was computed, and screening hits with a ratio greater than 3 were removed as promiscuous compounds. For reference, see: Feng, B. Y.; Shelat, A.; Doman, T. N.; Guy, R. K.; Shoichet, B. K. High-throughput assays for promiscuous inhibitors. Nature Chem. Biol. 2005, 1, 146−148. (16) Bergnes, G.; Brejc, K.; Belmont, L. Mitotic kinesins: prospects for antimitotic drug discovery. Curr. Top. Med. Chem. 2005, 5, 127− 145. (17) (a) Zhu, H.; Tempel, W.; He, H.; Shen, Y.; Wang, J.; Brothers, G.; Landry, R.; Arrowsmith, C. H.; Edwards, A. M.; Sundstrom, M.; Weigelt, J.; Bochkarev, A.; Park, H.; Crystal Structure of the Motor Domain of Human Kinesin Family Member C1; Protein Data Bank: Piscataway, NJ, 2007; DOI: 10.2210/pdb2rep/pdb. (b) Garcia-Saez, I.; Yen, T.; Wade, R. H.; Kozielski, F. Crystal structure of the motor domain of the human kinetochore protein CENP-E. J. Mol. Biol. 2004, 340, 1107−1116. (18) The activated esters was prepared from the corresponding acids and perfluorophenyl 2,2,2-trifluoroacetate in the presence of triethylamine in DCM at room temperature. (19) (a) Cox, C. D.; Breslin, M. J.; Mariano, B. J.; Coleman, P. J.; Buser, C. A.; Walsh, E. S.; Hamilton, K.; Huber, H. E.; Kohl, N. E.; Torrent, M.; Yan, Y.; Kuo, L. C.; Hartman, G. D. Kinesin spindle protein (KSP) inhibitors. Part 1: The discovery of 3,5-diaryl-4,5dihydropyrazoles as potent and selective inhibitors of the mitotic kinesin KSP. Bioorg. Med. Chem. Lett. 2005, 15, 2041−2045. (b) Yan,

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We thank Christie Grande for her contribution in biochemical assay development and compound screening, Alan Rosen and Olga Seltser for their automation assistance, and Kelly Goodwin for her bioanalysis of DMPK studies.



ABBREVIATIONS USED KSP, also names as hsEg5, kinesin spindle protein; KIFC1, a member of kinesin-14 family, also named as HSET; CENP-E, centromere-associated protein E; MG, malachite green ATPase; PK/LDH, pyruvate kinase/lactate dehydrogenase; HTS, highthroughput screening; PDB, Protein Data Bank; Phe, phenylalanine; SAR, structure−activity relationship



REFERENCES

(1) Hirokawa, N.; Noda, Y. Intracellular transport and kinesin superfamily proteins, KIFs: structure, function, and dynamics. Physiol. Rev. 2008, 88, 1089−1118. (2) (a) Kapitein, L. C.; Peterman, E. J.; Kwok, B. H.; Kim, J. H.; Kapoor, T. M.; Schmidt, C. F. The bipolar mitotic kinesin Eg5 moves on both microtubules that it crosslinks. Nature 2005, 435, 114−118. (b) Kwok, B. H.; Yang, J. G.; Kapoor, T. M. The rate of bipolar spindle assembly depends on the microtubule-gliding velocity of the mitotic kinesin Eg5. Curr. Biol. 2004, 14, 1783−1788. (3) Heald, R. Motor function in the mitotic spindle. Cell 2000, 102, 399−402. (4) (a) Mayer, T. U.; Kapoor, T. M.; Haggarty, S. J.; King, R. W.; Schreiber, S. L.; Mithison, T. J. Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. Science 1999, 286, 971−974. (b) Lad, L.; Luo, L.; Carson, J. D.; Wood, K. W.; Hartman, J. J.; Copeland, R. A.; Sakowicz, R. Mechanism of inhibition of human KSP by ispinesib. Biochemistry 2008, 47, 3576−3585. (c) Sakowicz, R.; Finer, J. T.; Beraud, C.; Crompton, A.; Lewis, E.; Fritsch, A.; Lee, Y.; Mak, J.; Moody, R.; Turincio, R.; Chabala, J. C.; Gonzales, P.; Roth, S.; Weitman, S.; Wood, K. W. Antitumor activity of a kinesin inhibitor. Cancer Res. 2004, 64, 3276. (5) (a) Cox, C. D.; Coleman, P. J.; Breslin, M. J.; Whitman, D. B.; Garbaccio, R. M.; Fraley, M. E.; Buser, C. A.; Walsh, E. S.; Hamilton, K.; Schaber, M. D.; Lobell, R. B.; Tao, W.; Davide, J. P.; Diehl, R. E.; Abrams, M. T.; South, V. J.; Huber, H. E.; Torrent, M.; Prueksaritanont, T.; Li, C.; Slaughter, D. E.; Mahan, E.; FernandezMetzler, C.; Yan, Y.; Kuo, L. C.; Kohl, N. E.; Hartman, G. D. Kinesin Spindle Protein (KSP) Inhibitors. 9. Discovery of (2S)-4-(2,5Difluorophenyl)-N-[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]-2-(hydroxymethyl)-N-methyl-2-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide (MK-0731) for the Treatment of Taxane-Refractory Cancer. J. Med. Chem. 2008, 51, 4239−4252. (b) Woessner, R.; Tunquist, B.; Lemieux, C.; Chlipala, E.; Jackinsky, S.; Dewolf, W., Jr.; Voegtli, W.; Cox, A.; Rana, S.; Lee, P.; Walker, D. ARRY-520, a novel KSP inhibitor with potent activity in hematological and taxane-resistant tumor models. Anticancer Res. 2009, 29, 4373−4380. (6) Theoclitou, M.; Aquila, B.; Block, M. H.; Brassil, P. J.; Castriotta, L.; Code, E.; Collins, M. P.; Davies, A. M.; Deegan, T.; Ezhuthachan, J.; Filla, S.; Freed, E.; Hu, H.; Huszar, D.; Jayaraman, M.; Lawson, D.; Lewis, P. M.; Nadella, M. V. P.; Oza, V.; Padmanilayam, M.; Pontz, T.; Ronco, L.; Russell, D.; Whitston, D.; Zheng, X. J. Discovery of (+)-N(3-aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-[1,2]thiazolo[5,4-d]pyrimidin-6-yl)-2-methylpropyl]-4-methylbenzamide (AZD4877), a kinesin spindle protein inhibitor and potential anticancer agent. J. Med. Chem. 2011, 54, 6734−6750. (7) Schiemann, K.; Finsinger, D.; Zenke, F.; Amendt, C.; Knochel, T.; Bruge, D.; Buchstaller, H. P.; Emde, U.; Stahle, W.; Anzali, S. The discovery and optimization of hexahydro-2H-pyrano[3,2-c]quinolines 9969

dx.doi.org/10.1021/jm501179r | J. Med. Chem. 2014, 57, 9958−9970

Journal of Medicinal Chemistry

Article

Y.; Sardana, V.; Xu, B.; Homnick, C.; Halczenko, W.; Buser, C. A.; Schaber, M.; Hartman, G. D.; Huber, H. E.; Kuo, L. C. Inhibition of a mitotic motor protein: where, how, and conformational consequences. J. Mol. Biol. 2004, 335, 547−554. (20) Wood, K. W.; Lad, L.; Luo, L.; Qian, X.; Knight, S. D.; Nevins, N.; Brejc, K.; Sutton, D.; Gilmartin, A. G.; Chua, P. R. Antitumor activity of an allosteric inhibitor of centromere-associated protein-E. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 5839−5844. (21) Following the publication of ref 14, another group has since disclosed structurally different KIFC1 inhibitors. Watts, C. A.; Richards, F. M.; Bender, A.; Bond, P. J.; Korb, O.; Kern, O.; Riddick, M.; Owen, P.; Myers, R. M.; Raff, J.; Gergely, F.; Jodrell, D. I.; Ley, S. V. Design, synthesis, and biological evaluation of an allosteric inhibitor of HSET that targets cancer cells with supernumerary centrosomes. Chem. Biol. 2013, 20, 1399−1410. (22) (a) Houghten, R. A.; Pinilla, C.; Blondelle, S. E.; Appel, J. R.; Dooley, C. T.; Cuervo, J. H. Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery. Nature 1991, 354, 84−86. (b) Lam, K. S.; Salmon, S. E.; Hersh, E. M.; Hruby, V. J.; Kazmierski, W. M.; Knapp, R. J. A new type of synthetic peptide library for identifying ligand-binding activity. Nature 1991, 354, 82− 84. (23) Edward, P. J. Purification strategies for combinatorial and parallel chemistry. Comb. Chem. High Throughput Screening 2003, 6, 11−27. (24) (a) Kiplinger, J. P.; Cole, R. O.; Robinson, S.; Roskamp, E. J.; Ware, R. S.; O’Connell, H. J.; Brailsford, A.; Batt, J. Structure controlled automated purification of parallel synthesis products in drug discovery. Rapid Commun. Mass. Spectrom. 1998, 12, 658−664. (b) Ripka, W. C.; Barker, G.; Krakover, J. High-throughput purification of compound libraries. Drug Discovery Today 2001, 6, 471−477. (25) Angell, Y.; Chen, D.; Brahimi, F.; Saragovi, H. U.; Burgess, K. A combinatorial method for solution-phase synthesis of labeled bivalent beta-turn mimics. J. Am. Chem. Soc. 2008, 130, 556−565. (26) Tang, W.; Luo, T.; Greenberg, E. F.; Bradner, J. E.; Schreiber, S. L. Discovery of histone deacetylase 8 selective inhibitors. Bioorg. Med. Chem. Lett. 2011, 21, 2601−2605. (27) Zhang, Y.; Sperry, A. O. Comparative analysis of two C-terminal kinesin motor proteins: KIFC1 and KIFC5A. Cell Motil. Cytoskeleton 2004, 58, 213−230.

9970

dx.doi.org/10.1021/jm501179r | J. Med. Chem. 2014, 57, 9958−9970