The Discovery of Orally Bioavailable Tyrosine Threonine Kinase (TTK

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The Discovery of Orally Bioavailable Tyrosine Threonine Kinase (TTK) Inhibitors: 3-(4-(heterocyclyl)phenyl)-1Hindazole-5-carboxamides as Anticancer Agents Yong Liu, Yunhui Lang, Narendra Kumar Patel, Grace Ng, Radoslaw Laufer, Sze Wan Li, Louise Edwards, Bryan Forrest, Peter Brent Sampson, Miklos Feher, Fuqiang Ban, Donald E Awrey, Irina Beletskaya, Guodong Mao, Richard Hodgson, Olga Plotnikova, Wei Qiu, Nickolay Y Chirgadze, Jacqueline M Mason, Xin Wei, Dan Chi-Chia Lin, Yi Che, Reza Kiarash, Brian Madeira, Graham C. Fletcher, Tak W. Mak, Mark R Bray, and Henry W Pauls J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501740a • Publication Date (Web): 12 Mar 2015 Downloaded from http://pubs.acs.org on March 13, 2015

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Journal of Medicinal Chemistry

Wei, Xin; University Health Network, Campbell Family Institute for Breast Cancer Research Lin, Dan; University Health Network, Campbell Family Institute for Breast Cancer Research Che, Yi; University Health Network, Campbell Family Institute for Breast Cancer Research Kiarash, Reza; University Health Network, Campbell Family Institute for Breast Cancer Research Madeira, Brian; University Health Network, Campbell Family Institute for Breast Cancer Research Fletcher, Graham; University Health Network, Campbell Family Institute for Breast Cancer Research Mak, Tak; University of Toronto, Princess Margaret Hospital; University Health Network, The Campbell Family Institute for Breast Cancer Research Bray, Mark; University Health Network, Campbell Family Institute for Breast Cancer Research Pauls, Henry; University Health Network, The Campbell Family Institute for Breast Cancer Research

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The Discovery of Orally Bioavailable Tyrosine Threonine Kinase (TTK) Inhibitors: 3-(4(heterocyclyl)phenyl)-1H-indazole-5-carboxamides as Anticancer Agents Yong Liu,*,† Yunhui Lang,† Narendra Kumar Patel,† Grace Ng,† Radoslaw Laufer,† Sze-Wan Li,† Louise Edwards,† Bryan Forrest,† Peter B. Sampson,† Miklos Feher,† Fuqiang Ban,† Donald E. Awrey,† Irina Beletskaya,† Guodong Mao,† Richard Hodgson,† Olga Plotnikova,† Wei Qiu,‡ Nickolay Y. Chirgadze,‡ Jacqueline M. Mason,† Xin Wei,† Dan Chi-Chia Lin,† Yi Che,† Reza Kiarash,† Brian Madeira,† Graham C. Fletcher,† Tak W. Mak,† Mark R. Bray,† and Henry W. Pauls*,† †

Campbell Family Institute for Breast Cancer Research, University Health Network, TMDT East Tower, MaRS Centre, 101 College Street, Toronto, Ontario, M5G 1L7, Canada ‡

Campbell Family Cancer Research Institute, University Health Network, Princess Margaret Cancer Center, 610 University Ave, Toronto, Ontario, M5G 2C4, Canada

RUNNING TITLE: The Discovery of Bioavailable TTK Inhibitors as Anticancer Agents. ABSTRACT:

The acetamido and carboxamido substituted 3-(1H-indazol-3-yl)benzenesulfonamides are potent TTK inhibitors. However, they display modest ability to attenuate cancer cell growth; their physicochemical properties, and attendant pharmacokinetic parameters, are not drug-like. By eliminating the polar 3sulfonamide group and grafting a heterocycle at the 4 position of the phenyl ring, potent inhibitors with ACS Paragon Plus Environment

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oral exposure were obtained. An X-ray co-crystal structure and a refined binding model allowed for a structure guided approach. Systematic optimization resulted in novel TTK inhibitors namely, 3-(4(heterocyclyl)phenyl)-1H-indazole-5-carboxamides.

Compounds

incorporating

the

3-hydroxy-8-

azabicyclo[3.2.1]octan-8-yl bicyclic system were potent (TTK IC50 < 10 nM, HCT116 GI50 < 0.1 µM), displayed low off-target activity (> 500X) and microsomal stability (T1/2 > 30 min). A subset was tested in rodent PK and mouse xenograft models of human cancer. Compound 75 (CFI-401870) recapitulated the phenotype of TTK RNAi, demonstrated in vivo tumor growth inhibition upon oral dosing and was selected for preclinical evaluation.

KEYWORDS: TTK inhibitors; Mps1 inhibitors; CFI-401870; 3-(4-(heterocyclyl)phenyl)-1H-indazole5-carboxamides; X-ray structure (PDB ID: 4O6L); antimitotic agents; HCT116 xenograft; Tyrosine Threonine Kinase; Monopolar Spindle 1 Kinase.

INTRODUCTION: A number of cell cycle and checkpoint kinases have been targeted as potential strategies for the treatment of cancer.1 The spindle assembly checkpoint (SAC) is an important and highly orchestrated control mechanism during mitosis; it assures the proper attachment of chromosomes to the mitotic spindle prior to anaphase.2 Most cancer cells are aneuploid,3 and thus take longer than normal diploid cells to align and attach their chromosomes to the mitotic spindle.4 It has been demonstrated that chromosomal instability (CIN) causes aneuploidy, a hallmark of cancer,5 therefore the selective suppression of aneuploid cells should be beneficial.6 The conserved dual-specificity kinase TTK (threonine tyrosine kinase, also known as Mps1) is essential for alignment of chromosomes to the metaphase plate7 and genomic integrity during cell division through establishment and maintenance of the SAC.8 TTK is overexpressed in a variety of solid cancers9-13 and elevated levels of TTK correlate with high histological grade in tumors11 and poor patient outcome.9 High levels of TTK in cancer cells may contribute to their tolerance of the aneuploidy state.11,14,15 Inhibition of TTK impairs the SAC and ACS Paragon Plus Environment

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allows cancer cells to progress through mitosis in the presence of misaligned chromosomes, resulting in an intolerably high degree of aneuploidy and eventual cell death.16 TTK has been identified as one of the top 25 genes overexpressed in tumors with chromosomal instability15 and aneuploidy.17,18 PTEN-deficient breast tumor cells are particularly dependent upon TTK for their survival such that RNAi-mediated knockdown leads to cell death. TTK inhibitors have been shown to target PTEN-deficient cells more acutely, presumably exploiting their underlying aneuploidy and genomic instability.17 Besides its role in mitosis, TTK has been implicated in the Hedgehog,19 EGFR20 and DNA damage signaling pathways.21-23 TTK controls DNA damage responses and genome stability by phosphorylating the Bloom syndrome helicase (BLM),21 CHK2 in the G2/M checkpoint22 and c-ABL after oxidative stress.23 TTK first came to our attention as a hit in a genome wide siRNA screen in which the growth of 7 out of 12 breast cancer cell lines tested was attenuated.24 Subsequently we were able to show that siRNA knockdown abrogated cancer cell growth and caused gross chromosome segregation errors resulting in aneuploidy and cell death. In vivo, interfering RNA mediated depletion suppressed the growth of tumor xenografts in mice.24 Taken together, these observations encouraged us to pursue TTK as a viable target for cancer therapy.25 TTK has received considerable attention from laboratories interested in the development of small molecule inhibitors of this potential cancer target. Representative structures are given in Chart 1. The structural feature most commonly incorporated in these inhibitors is the 2-(phenylamino)-pyrimidine motif found in AZ3146,26 Reversine,27 Mps1-IN-2 and its analog 1 (methoxy-Mps1-IN-2, Figure 1),28 2 (NMS-P715),29 and the Genentech inhibitor.30 The Shionogi-1 compound31 and CCT25145532 incorporate variations on this theme. These compounds have been used to further understanding of the cellular functions of TTK. A patent from Oncotherapy33 describes (alkylamino)imidazo[1,2b]pyridazines as a novel series of TTK inhibitors. The related (alkylamino)imidazo[1,2-a]pyrazines (Mps-BAY2a and Mps-BAY2b) are potent TTK inhibitors and the latter has demonstrated complete ACS Paragon Plus Environment

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inhibition of xenograft tumor growth in combination with paclitaxel.34 Recently a clinical study with a TTK inhibitor from Bayer, dosed in combination with paclitaxel, has been initiated. Our approach built upon hits obtained from a focused screening exercise which eventually lead to the identification of acetamido and carboxamido substituted 3-(1H-Indazol-3-yl)benzenesulfonamides.25 Independently, Shionogi has developed a series of 3-(1H-indazol-3-yl)benzenesulfonamides with very different substitution patterns on the indazole core (Shionogi-2, Chart 2).35 We found that the 5substituted acetamido and carboxamido 3-(1H-Indazol-3-yl)benzenesulfonamides such as 3 and 4 respectively (Chart 2), were potent inhibitors of TTK with modest ability to attenuate the growth of cancer cells. However, during the course of our in vivo evaluation of these compounds it became apparent that their physicochemical properties, and attendant pharmacokinetic parameters (Tables 1 and 2), were not sufficiently drug-like to move these compounds forward. We postulated that the polar nature of the sulfonamide group (e.g. compound 3, PSA = 130) was detrimental to the permeability of these molecules. We embarked upon a structure guided modification of phenyl ring substituents, while simultaneously exploring the 5 position of the indazole core. In moving the series forward, we were cognizant of the desirability of optimizing PK, while maintaining selectivity and potency, in order to further explore the unique role of TTK in tumor growth. This manuscript describes the successful realization of these objectives and the identification of selective, orally bioavailable inhibitors of TTK.

RESULTS and DISCUSSION:

Inhibitor Design. Figure 1 illustrates the design of our new inhibitors; the model shows an overlay of 3-sulfonamide 3 with the Dana Farber inhibitor 1.28 We sought to reduce the polar surface area of the molecules by morphing the sulfonamide (PSA = 130) to a sulfone (PSA = 104) moiety as found in compound 12 (Table 1). Subsequently, we explored the addition of heterocycles to the 4-position of the phenyl ring. Our modeling suggested that substitution at position 4 of the phenyl ring is tolerated and allows for the projection of polar moieties towards the enzyme/solvent interface. These heterocyclic ACS Paragon Plus Environment

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motifs were drawn from those found most frequently in known TTK inhibitors (Chart 1). We also contemplated the removal of the substituent at the 3-position of the phenyl ring since our computational analysis supported the hypothesis that 4-mono substituted compounds would be potent TTK inhibitors.

Chemical

Synthesis.

The

retrosynthesis

of

the

N-(3-(4-heterocyclyl

and/or

3-

(methylsulfonyl)phenyl)-1H-indazol-5-yl)acetamides of Tables 1 and 3 yields the requisite Bocprotected 3-iodo-1H-indazol-5-amine 9b or N-(3-iodo-1H-indazol-5-yl)acetamides 10 and boronates 7 (Scheme 1). When R1 was methyl (5c), direct borylation yielded the desired boronate 7c, however when R1 was a proton (5a,b), derivatization of the piperidine nitrogen as an amide preceded the borylation step. Aryl boronates 7 were generally prepared from aryl halides, such as 6a,b and 8d,e, using either pinacol borane36 or bis(pinocolato)diboran37, whereas, for the hydroxypiperidine R″, piperidinone 8f was borylated and reduced in situ. The acetamidoindazole iodide coupling partner 10a was assembled most readily by an amide bond formation from 3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-indol-5-amine 9a. The requisite carboxylic acids (R‴CO2H) were commercially available as racemic mixtures or prepared by Petasis reaction25 or α-alkylation of arylacetate (see Supporting Information). Compound 10b was prepared similarly, as previously described.25 Subsequent Suzuki-Miyaura coupling of boronates 7 (herein described) or commercially available boronic acids with 10b, yielded TTK inhibitors 12-18. A THP deprotection step of 10a, prior to Suzuki-Miyaura coupling, furnished compound 21. For compounds 19 and 20 the Suzuki-Miyaura coupling with 9b, followed by Boc deprotection gave 11; a final amide bond formation step gave the desired products. Scheme 2 describes the synthesis of racemic and achiral 3-(4-(heterocyclyl)phenyl)-1H-indazole-5carboxamide TTK inhibitors of Tables 2-5. Aromatic nucleophilic substitution of 1-fluoro-4iodobenzene with alcohols 22a-c provided ethers 23a-c.38 When R1 was Boc, 23a, TFA deprotection followed by amide coupling gave 24, which was converted to its corresponding boronate 25a. As for the tropine and pseudotropine derivatives 23b and 23c, direct borylation36 gave the desired boronates 25b and 25c, respectively. Compound 28, obtained by amide coupling of 26 and 27, was the precursor to the ACS Paragon Plus Environment

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achiral carboxamides of Tables 2, 3 and 5. When racemic amines 31 were not commercially available, they were prepared by reductive amination of ketones 29,39 or one-pot synthesis (Grignard addition and imine reduction)40 from 2-cyanopyridine 30. Suzuki-Miyaura cross-coupling of 28 or 32 with the requisite aryl boronates furnished the desired achiral compounds (33, 38 and 39) and racemates (34-37 and 40-48), respectively. Achiral compounds 49 and 50 (Table 5) were prepared by Suzuki-Miyaura cross-coupling of 28 with 3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-8-oxa-3-azabicyclo[3.2.1]octane

(see

Supporting Information) and boronate 60a, respectively (Scheme 3). Compound 51 (Table 2) was prepared using methods similar to those described in Scheme 3 from the enantiomerically enriched 1-(2chlorophenyl)propan-1-amine (see Supporting Information). Scheme

3

outlines

the

routes

used

to

prepare

the

chiral

N-(alkyl(aryl)methyl)-3-(4-

(heterocyclyl)phenyl)-1H-indazole-5-carboxamides of Table 5. The asymmetric center in these TTK inhibitors resides in the 5-substituent of the indazole and is derived from chiral amine precursors. For the preparation of chiral amines 55-S and 55-R, we employed both chiral resolution and enantioselective synthesis. For example, racemic 55 (Ar = pyridin-2-yl, R = isobutyl) was resolved with (D)- or (L)DBTA to provide both R and S enantiomers in good enantiomeric excess (ee) but poor yield. A more general and efficient synthesis made use of Ellman’s auxiliary following well established methods.41,42 Grignard reagent was added to sulfinamide 53-S in the presence of Me2Zn to give 54-S which was subsequently hydrolyzed to afford 55-S in high ee and moderate yield. Alternatively, for the preparation of (S)-cyclopentyl(pyridin-2-yl)methanamine, the (R)-2-methylpropane-2-sulfinamide 52-R was converted to the requisite sufinamide 56-R. Following a similar series of steps, using a pyridine-2-yl based Grignard reagent, the desired S enantiomer was obtained. The corresponding (R)alkyl(aryl)methanamines (R = cyclopropyl, cyclopentyl, isobutyl) were similarly prepared. Subsequent amide coupling of the chiral amines so prepared, with 3-iodo-1H-indazole-5-carboxylic acid 27, afforded the penultimate intermediates 32-S and 32-R. In some instances, racemic 32 was


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subjected to chiral HPLC separation to yield the desired enantiomeric iodoindazole intermediates 32-S and 32-R. For the bridged heterocycles, the precursor azabicycloalkanes 58a-d were arylated with 1,4diiodobenzene in the presence of CuI catalyst38 to give iodides 59a-d which were converted to pinacol boronic esters as described above. The homolog 60e and the tropine-morpholine hybrid 60f were prepared from ketones 62e and 62f respectively. Compounds 62e and 62f were obtained by a one pot Robinson synthesis43 from 61 using glutaraldehyde and 2-(2,2-diethoxyethoxy)-1,1-diethoxyethane, respectively. Reduction of the ketones with NaBH4 gave alcohols 63e and 63f which were subsequently borylated to provide 60e and 60f. Standard Suzuki-Miyaura coupling between 32-S or 32-R and boronates 60 completed the synthesis of the chiral 3-(4-(heterocyclyl)phenyl)-1H-indazole-5carboxamides 64-88. Lead Optimization. Table 1 summarizes our initial findings; the sulfonamide 3 to sulfone 12 modification was tolerated in that potent TTK inhibitory activity was maintained. As anticipated, the reduced polar surface area of sulfone 12 was reflected in a concomitant increase in exposure upon oral dosing as compared to the analogous sulfonamide 3. Unfortunately, this modification resulted in a 4fold decreased potency in the cell viability assay, a result also seen in sulfone 14. This tendency was observed for several additional sulfones (data not shown) with the lone exception being compound 13 wherein a basic dimethylaminopropyl group was grafted onto the 4-position of the phenyl ring. However, this modification served to abrogate oral exposure, therefore, we turned our attention to the 4monosubstituted analogs 15-18. The TTK IC50 values for compounds 14 and 15 supported the reasoning, presented in Figure 1 and predicted by molecular modeling, that substituents would be tolerated at position 4 of the phenyl ring. Furthermore, sulfonamides or sulfones, at position 3 of the phenyl ring, are not necessary for TTK inhibition. Subsequently, three additional compounds 16-18, lacking the 3-substituent, were made to further explore this hypothesis. All four compounds 15-18 potently inhibited TTK, had much reduced polar surface area (ranging from 68 to 84) and were effective in the cell viability assays (HCT116 GI50 ≤ ACS Paragon Plus Environment

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0.057 µM). In addition, these inhibitors achieved markedly higher exposures than the analogous sulfonamide 3 upon oral dosing in the mouse. Compounds 16 and 18 were of particular note with Cmax of 3.5 and 2.8 µg/mL and AUC of 7.9 and 6.8 µg.h/mL, respectively. Previously, we reported25 that the isomeric amide linker, i.e. carboxamido, was effective when incorporated into prototypical sulfonamides such as 4. Accordingly, we prepared representative carboxamides with the new heterocyclic moieties. The results, summarized in Table 2, indicate that carboxamides 33, 34 and 36-38, were also potent inhibitors of TTK with IC50 values ranging from single to double digit nanomolar. As was also seen for the acetamides, the (1-methylpiperidin-4-yl)oxy group yielded the most potent inhibitors, compounds 36 and 51. In contrast, and mirroring the acetamide result, the best exposure was achieved with compound 34, which incorporates the morpholino R″ group. Its regioisomer, compound 35, is twenty-fold less active indicating the preference for 4 versus 3 substitution on the phenyl ring. Plasma levels for a representative sulfonamide 3 were exceedingly low, over 2 orders of magnitude less than the morpholino analog 34. A priori, any one of the R″ heterocyclic moieties could be considered as viable starting points for subsequent lead optimization studies. Our initial effort focused on two: the (1-methylpiperidin-4-yl)oxy group for its potency and the morpholino group for its superior oral exposure. An additional consideration was the stereochemistry of the 5-indazole moiety. The acetamido and carboxamido substituted 3-(1H-Indazol-3-yl)benzenesulfonamides both incorporate an asymmetric center which is recapitulated with the new heterocyclic R″ groups. This raises the possibility of racemization, alpha to the acetamide carbonyl, especially if the substituent is attached by a nitrogen atom. One way to reduce this liability might be to substitute a carbon atom for nitrogen, as in the matched pair, compounds 17 and 20 (Table 1 and 3). However, the direct comparison illustrates the beneficial effect of the nitrogen on in vitro performance; the former is about 2.5-fold more potent against TTK, which is reflected in its superior activity in the cell viability assays. In the carboxamides, racemization of the chiral center, which is alpha to the nitrogen, is less of a concern. In addition, for the assembly of the asymmetric carboxamides, there are a number of routes available for the preparation of ACS Paragon Plus Environment

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the precursor chiral amines.41,42 This is in contrast to the paucity of suitable methods for the preparation of the chiral alpha-amino acids required for asymmetric acetamide synthesis. Finally, Table 3 summarizes the head to head comparison of three matched pairs of inhibitors: 19/39, 20/40 and 21/41. There is little difference in overall properties excepting a small but consistent advantage in potency for the carboxamides. Given these considerations, we decided to pursue the carboxamide linked inhibitors in our subsequent lead optimization efforts. Some published TTK inhibitors have a propensity to inhibit other mitotic kinases such as Aurora Kinase B (AURKB/INCENP) and strategies to mitigate this activity have been reported.44 Table 3 captures the off-target kinase activity of the indazole based compounds against Aurora Kinase A (AURKA), AURKB/INCENP and a receptor tyrosine kinase KDR (kinase insert domain receptor). Good selectivity against AURKA and KDR were observed, however, we continued to monitor the offtarget activity against AURKB/INCENP as the in vitro potency and eADME properties of the series were optimized. Our initial efforts on improving the properties of the 4-piperidin-4-yloxy class of TTK inhibitors are captured in Table 4. With the starting assumption that the basicity of the piperidine ring (calculated pKa 8.7) was contributing to low oral exposure, compounds were prepared that modulated this property. Formamides 42, 43 (calculated pKa ≤ 4.3) and amide 44 retained TTK potency and the latter two were of special interest given their effectiveness in the HCT116 cell viability assay. Unfortunately, compound 43 proved to be an inhibitor of AURKB/INCENP whereas the oral exposure of 44 was lower than its parent 36. During the course of our work we obtained a TTK co-complex X-ray structure with a 3-(4-((1piperidin-4-yl)oxy)phenyl)-1H-indazole-5-carboxamide, namely compound 51 (Figure 2). Inspection of this structure (vide infra) suggested that further modifications to the piperidine ring were possible. Accordingly, we prepared a pair of 8-azabicyclo[3.2.1]octane systems bridged in the endo 45 and exo 46 sense; modeling indicated that either of these modifications would be tolerated in the enzyme active site. We anticipated that these larger systems could make better contact with the enzyme surface thereby increasing TTK potency while decreasing off-target activity through steric clashes. The exo version 46, ACS Paragon Plus Environment

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was less active in vitro and, although TTK potency was retained for the endo compound 45, selectivity enhancements were not realized. The good activity for the endo system was carried over to the formylated versions 47 and 48. Compound 47 had poor oral exposure whereas compound 48 achieved respectable plasma levels but suffered from significant off-target activity (AURKB/INCENP and CYP 3A4). In fact, we were not able to achieve the desired potency and oral exposure, while minimizing offtarget effects, using this strategy. Consequently, we turned our attention to the morpholinophenyl containing compounds and, based on the reasoning above, prepared two bridged analogs, compounds 49 and 50 (Table 5). The former (i.e. the 8-oxa-3-azabicyclo[3.2.1]octan-3-yl system) was less active than its parent 39 (Table 3), however, the latter (i.e. the 3-oxa-8-azabicyclo[3.2.1]octan-8-yl system) was not only more potent but about an order of magnitude more selective against AURKB/INCENP. A number of additional variations incorporating the latter R'' group were examined, compounds 64-70. Potency against TTK was generally in the low nanomolar range; in addition to AURKB/INCENP selectivity, cell activity, microsomal stability and cytochrome P450 activity was routinely monitored and employed as filters to move compounds forward. In general, the S enantiomer was marginally more potent against TTK however, the differences were subtle and AURKB/INCENP selectivity sometimes favored the R enantiomer. TTK inhibitors with desirable potency (TTK IC50 < 10 nM), selectivity against AURKB/INCENP and CYP 3A4 (IC50 > 500 nM), rodent microsomal stability (T1/2 > 30 min) and cell activity (HCT116 GI50 < 0.1 µM) were selected for in vivo study. In common with other compounds incorporating the (1-morpholinocyclohexyl)methyl R‴ group, TTK inhibitor 50 had a low T1/2 in microsomal stability assays. This liability was generally mitigated by incorporating alkyl(aryl)methyl R‴ moieties as in compounds 64-66. However, due to their off-target activities, none of these compounds were further evaluated. In addition, concerns about their low calculated solubilities (ACD/Stucture Designer) prompted the investigation of compounds incorporating alkyl(pyridinyl)methyl R‴ groups. These mild bases (67-70) were effective inhibitors of TTK. Note that the meta and para pyridinyl analogs were not pursued since previous work with the phenyl sulfonamide ACS Paragon Plus Environment

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series25 had indicated a loss in activity for these congeners. The analog in which the R″ group had been expanded to the 3-oxa-9-azabicyclo[3.3.1]nonan-9-yl ring system, compound 71, was less potent and offered no other advantages over its closest analog 69. Compounds 68 and 69, deemed to incorporate the best features from this subclass, were moved into rodent PK studies. Concurrently, a similar approach to the hydroxyl-piperidine R″ yielded a series of bridged bicyclics, compounds 72-88 (Table 5). As a group, these compounds had good in vitro potency and were generally selective against AURKB/INCENP (>100 fold). The first two entries in the series, compounds 72 and 73, were moved quickly into mouse PK studies to gauge the impact of this new R″ group on oral exposure. The results (Table 6) were sufficiently encouraging to warrant further investigation of this chemotype. Given their solubility advantage (vide supra), subsequent analoging efforts concentrated on R‴ groups with nitrogen containing aromatics. Compounds 74 and 75, isolated as salts, demonstrated good in vitro potency, were stable to microsomes (Table 5) and otherwise met the criteria for in vivo study. A related set of compounds, the enantiomeric pair 76 and 77, incorporate the larger cyclopentyl ring, resulting in enhanced selectivity against AURKB/INCENP. The S-enantiomer 76 had the most favorable combination of properties. Further variations on this theme yielded pyrimidines 78 and 79, which showed comparable in vitro activity. They suffered somewhat from a lower selectivity; again, the more potent S-isomer was moved into a mouse PK study. Alternatively, the bulk of the alkyl side chain could be increased by incorporating a pendent isobutyl group alpha to the aromatic ring. Selectivity was maintained, however, enantiomers 80 and 81 demonstrated undesirable CYP 3A4 activity. This was mitigated by morphing the isobutyl group into a cyclopropylmethyl moiety e.g. compound 82 which was selected for in vivo study. Further variations to the R″ were also examined; ring expansion to a 9-azabicyclo[3.3.1]nonan-3-ol bicyclic system, compound 83, resulted in a loss in potency which could be recovered by replacing the additional carbon with oxygen as in 84. Nonetheless, the potent 3-oxa-9-azabicyclo[3.3.1]nonan-7-ols 84 (poor selectivity) and 85 (off-target CYP450 activity) did not meet the criteria for in vivo study. The stereochemistry of the substituent hydroxyl was probed by synthesis of the exo analogs 86-88. Potency ACS Paragon Plus Environment

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was maintained at least for the S-isomers and the compound with the best balance of in vitro properties, namely 86 was selected for rodent PK studies. Compound 88 was less stable upon incubation with rodent microsomes and inhibited CYP 2C9 with an IC50 of 120 nM. X-ray Structure. The co-complex x-ray structure of compound 51 with the kinase domain of TTK was obtained at a resolution of 2.38 Å (PDB: 4O6L, Figure 2). The present structure is the first reported for TTK in the C2 space group; it has two molecules in the asymmetric unit related by 2-fold noncrystallographic symmetry axis. Each protomer active site is located near the axis without blocking the other’s accessibility and in both molecules the activation loop is positioned away from any crystallographic contacts. In one of the protomer molecules (chain A) the activation loop is partially resolved (similar to our previous structure).25 The electron density is well-defined up to residue Ser677 and indicates three phosphorylated residues (Thr675, Thr676, and Ser677). The other protomer (chain B) is also partially resolved but is unphosphorylated and away from the ligand in a conformation reminiscent of inactive kinases. The ligand density is consistent with binding of the R enantiomer although the crystallization well contained a 68:32 mixture of R and S enantiomers (i.e. 36% ee, see the omit map in the supporting information, Figure S2). Key features of this structure (Figure 2) include Hbonds from the indazole nitrogens to the hinge region residues Glu603 and Gly605 and from the amide carbonyl to Lys553. For chain A, the chlorophenyl and ethyl groups occupy hydrophobic sub-pockets while the phenyl ring makes contact with the protein surface. The protonated piperdine ring extends toward the solvent interface and makes an interaction with the phosphorylated side chain of Thr675. Computational Chemistry. In the early phases of this work the protein structure, co-crystallized with a molecule from the sulfonamide series,25 was utilized for predictions (PDB code: 4JT3). However, with the availability of the 4O6L structure (Figure 2), a new predictive model was developed. We found that the raw GlideXP docking model using literature-standard docking protocols was unsatisfactory in that 10 of 86 molecules failed to dock. Achiral compounds such as 19 and 39 were particularly troublesome; in fact, inhibitors with the (1-morpholinocyclohexyl)methyl moiety at the 5 position of the indazole could not be scored. This result is not surprising when single copy docking is employed,45 however, the ACS Paragon Plus Environment

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model could be greatly improved by multiple copy docking.46-48 All inhibitor molecules dock when multiple copies of each input structure are generated via a conformational search,46 followed by a post docking minimization step and by selecting the top scoring solution for each ligand.47 The quality of predictions (R2:0.37, rmse: 0.86, n: 86, see Figure S3 in the supporting information) was further improved (Figure 3A, R2: 0.62, rmse: 0.68, n: 81) by removing the achiral outliers from the analysis. However, this outlier removal had little effect on ranking (Spearman's rho values of 0.79 and 0.71, respectively). Moving forward, this chemotype was not pursued thereby increasing our confidence in the working model which was used as an aid to prioritize target molecules for synthesis. This approach does not involve any training, hence it was found to be predictive across the entire range of TTK ligands in the project. In fact, the quality of predictions (Figure 3B, R2: 0.57, rmse: 0.69, n: 817) was sufficient for evaluating virtual libraries and to remove less promising target molecules from further consideration; the corresponding Spearman's rho value for rank correlation was 0.62. We also noted that the experimental pIC50’s are better described by non-linear models or by omitting points with very low Glide scores (e.g. > -7.5), however, in absence of an underlying explanation, only linear models with all available data were used. Rodent PK. The first two entries in Table 6 are from the 3-oxa-8-azabicyclo[3.2.1]octane sub-class of TTK inhibitors. Both demonstrated good oral exposure in the mouse and were selected for rat PK studies. The next 8 entries are from the 3-hydroxy-8-azabicyclo[3.2.1]octane sub-class, the first two of which showed significantly lower exposure than the rest. Compounds 72 and 73 were more hydrophobic than the latter entries which incorporated nitrogen in the R‴ aromatic moiety. Of these, two represent an enantiomeric pair 74 and 75; the latter isomer, with the best exposure, was subsequently tested in rat. The remaining compounds showed good mouse plasma levels and were also tested in rat. In the rat, compounds 68 and 82 had short half-lives whereas compounds 76, 78 and 82 had the lowest oral exposure. Thus a short list of three TTK inhibitors (69, 75 and 86) was identified for subsequent study in mouse HCT116 xenografts. A pharmacokinetic study of compound 75 was also performed in the dog with key PK parameters as follows: intravenous t1/2 = 1.2 h; oral BA 43%. ACS Paragon Plus Environment

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In vivo Efficacy. The colon HCT116 cell line was chosen for in vivo screening of the short listed TTK inhibitors, since the corresponding xenograft model was well characterized in our laboratory. The maximum tolerated dose (MTD) upon oral daily dosing (QD) was determined for each compound (69, 75 and 86) in athymic CD-1 nude mice. Compounds were dosed QD at their MTD and 2/3 MTD for 21 days. The results (Table 7) indicate that the best response, 77% and 55% tumor growth inhibition (TGI), was observed for compound 75 at 35 and 23 mg/kg, respectively (Figure 4). Mice were lost at the highest dose for all three compounds; one death was observed for compound 75 at its putative MTD. Based on this result compound 75 (designated CFI-401870) was tested in two additional xenograft models (breast cancer cell line MDA-MB-468 and colon cancer cell line SW48), however, the highest dose was reduced to 30 mg/kg. TGIs of 98% and 66%, respectively, were observed for these xenografts (Table 7), with minimal weight loss as compared to vehicle (see supporting information Table S5) and no signs of overt toxicity. This level of response is comparable to or better than those previously described for TTK inhibitors. For example, the Nerviano compound 2, given orally (90 mg/kg QD for 7 consecutive days), achieved a 53% tumor growth inhibition at the end of study (day 10) in an A2780 ovary carcinoma xenograft model.49 The Myriad/Myrexis compound (MPI-0479605 at 30 mg/kg QD for 21 days) had a TGI of 49% at day 22 in an HCT-116 xenograft model with one animal lost at day 15.50 It is possible that intermittent dosing strategies50 and/or combination studies34 will need to be explored to fully exploit the potential of TKK inhibitors in vivo. In vitro Characterization A kinetic analysis of TTK inhibitor 75 (Supporting Information) confirmed the potency (Ki = 0.7 ± 0.5 nM) and ATP competitive nature of the binding (i.e. KmApp increases with dose). However, the variation in Vmax over the concentration range indicates mixed kinetics for this inhibitor. Compound 75 was tested against an extensive panel of human kinases and the results are shown as a heat map in Figure 5. The compound was substantially more selective than sunitinib and comparable to imatinib, as measured by the number of off-target kinases hit. Half maximal inhibitory concentrations were determined for those kinases most potently inhibited (Table 8). Although mutant forms of cKit and Ret were inhibited the compound was ineffective against the wild-type ACS Paragon Plus Environment

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enzymes. Of the remaining, JNK3, MAPK2 and PLK4 were most significantly inhibited but there was a ≥1.3 log unit separation in activity and these kinases have been touted as anticancer targets in their own right. We were particularly interested in the activity of TTK inhibitor 75 against the mitotic kinases Aurora A and B. The fold separation was high and compound 75 did not inhibit the intracellular activity of these mitotic kinases up to 6 µM concentration. These results were reflected in the cell cycle progression of HCT116 colon cancer cells incubated with 75 (Figure 6); the profile recapitulated the phenotype of TTK silencing by RNAi. Compound 75 caused massive aneuploidy and cell death, as indicated by sub-G1 cells, at about 100-200 nM. Consistent with potent TTK inhibition and lack of intracellular Aurora B activity, 75 causes aneuploidy at low concentrations with no significant polyploidy at the highest concentration tested. Inhibition of cell growth by compound 75 was evaluated in an expanded panel of breast, colon, ovarian, prostate, melanoma and lung human cancer-derived cell lines (Table 9). Excepting human mammary epithelial cells (HMEC GI50 > 30 µM), the compound inhibited cell growth across the cell line panel; GI50 values ranged between 0.008 and 0.21 µM, with typical GI50 values in the double digit nanomolar range. The effect of compound 75 on chromosome segregation in colon cancer cells was examined by imaging HCT116 cells during mitosis (Figure 7). Cells treated with TTKi 75 for 2 hours prior to entry into mitosis exhibited severe defects in their ability to segregate chromosomes (>60% and >90% of cells at 100 and 500 nM of compound 75, respectively). Despite defects in chromosome segregation, the majority of TTKi 75 treated cells complete cytokinesis with a gain or loss in chromosomes (Figure 8). A decrease in cell viability was observed as early as 24 hours after treatment with compound 75, and at the same time, increases are induced in cleaved PARP (apoptosis marker), phospho-H2AX (DNA damage marker) and p53 (data not shown). Compound 75 was tested in several early ADME assays including CYP450 and hERG inhibition; separation in activity between these off-target effects and TTK inhibition approximated 3 orders of magnitude (Table S4, Supporting Information). In addition, compound 75 demonstrated good stability


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(T1/2 >60 min) in mouse, rat, dog and human microsomes. Plasma protein binding was observed to be 95.8, 93.1, 88.6 and 97.8% in mouse, rat, dog and human plasma respectively.

CONCLUSIONS:

The transformation of the 3-substituted sulfonamido phenyls to 4-substituted heterocyclic phenyl systems on the 1H-indazole 5-(carboxamide and acetamide) cores maintained TTK potency and greatly improved the PK properties of the molecules. This breakthrough set the stage for systematic lead optimization of this series; subsequent efforts focused on enantiomerically enriched carboxamides since they were more synthetically accessible and potent in vitro. A molecular model based on the TTK/compound 51 co-complex X-ray structure (pdb: 4O6L) was used to prioritize target compounds for synthesis. The SAR indicated that R″ moieties based on morpholino and hydroxypiperidinyl groups were of particular interest. Ten compounds with desirable potency (TTK IC50 < 10 nM), selectivity against AURBK/INCENP and CYP 3A4 (IC50 >500 nM), microsomal stability (T1/2 > 30 min) and cell activity (HCT116 GI50 < 0.1 µM) were identified for in vivo study. Compounds incorporating the 8-oxa3-azabicyclo[3.2.1]octan-3-yl and 3-oxa-9-azabicyclo[3.3.1]nonan-7-ol bicyclic systems demonstrated high exposure upon oral dosing in mice and were further evaluated in rat PK studies. Compounds 69, 75 and 86 are single digit nanomolar TTK inhibitors, with acceptable off-target activity and good oral exposure in rodents. The short listed compounds attenuate the growth of MDAMB-468 and HCT-116 cancer cells and are somewhat less effective against OVCAR-3. Compound 75 was the most efficacious of the shortlisted compounds in an HCT116 tumor xenograft model (77% TGI at 35 mg/kg PO). This compound inhibited off-target kinases (panel of 278 human kinases) with a frequency comparable to clinically used inhibitors, deemed to be selective (e.g. imatinib). Compound 75 inhibited the growth of a broad panel of human cancer cell lines including breast, ovarian, colon, prostate, lung and melanoma and recapitulated the phenotype of TTK interfering RNA in HCT116 cells. It demonstrated a reasonable separation of effects between TTK potency and human CYP and hERG ACS Paragon Plus Environment

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activity (≥1,000 fold). On the strength of the data presented herein, compound 75 was selected for preclinical development studies. EXPERIMENTAL SECTION: Computational methods. Ligands were prepared and docking results were analyzed in the MOE software.51 Appropriate protonation states were generated using the Wash process, all reasonable stereoisomers were produced using Corina.52 Low energy conformations were generated using the Conformer Import option (max. 30 conformers within a 3 kcal/mol window) and minimized using the MMFF94x force field within MOE. The protein structure was prepared using standard settings with the help of the Protein Preparation Wizard in the Schrodinger suite53 with all water molecules deleted. For each molecule, the pre-generated conformations were docked using the GlideXP program54 and the solutions with the highest docking score (gscore) were selected. In these docking runs hydrogen bonding to one of the two key hinge residues (Glu603 and Gly605) was used as a constraint and no state penalties for protomers / tautomers were added to the docking score, otherwise default settings were employed including post-docking minimization. General Synthetic Methods. Commercially available starting materials, reagents, and solvents were used as received. In general, anhydrous reactions were performed under nitrogen or argon. Microwave reactions were performed using a Biotage Initiator microwave reactor. Reaction progress was generally monitored by LCMS (Bruker Esquire 4000). Flash column chromatographic purification of intermediates or final products was performed on a Biotage Isolera One purification system using SNAP KP-sil cartridges or SNAP C-18 reverse-phase (RP) columns. Crude reactions were partially purified using Waters PoraPak™ Rxn CX cartridges. Preparative reverse-phase HPLC purification was performed on a Varian PrepStar model SD-1 HPLC system with a Varian Monochrom 10µ C-18 reverse-phase column. Elution was performed using a gradient of 10% MeOH/water to 90% MeOH/water (0.05% TFA) over a 46-min period at a flow rate of 40 mL/min. Fractions containing the


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desired material were concentrated and/or lyophilized to obtain the final products. All reported yields are un-optimized. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on a Bruker AV 400 MHz spectrometer. Chemical shifts were reported in parts per million (ppm) from tetramethylsilane. Proton coupling patterns were described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad (br). Low resolution mass spectra (MS ESI) were obtained using a Bruker Esquire 4000 spectrometer under electron spray ionization conditions. High resolution mass spectra (HRMS) were obtained by a Waters Acquity UPLC coupled with a Waters Synapt G2 time-of-flight mass spec, using an ESI source. Compounds in DMSO solutions (5 mg/mL) were diluted to 0.01 mg/mL; 5 µL was injected on to a Waters BEH C-18 1.7um 2.1x100 µm column equilibrated at 10% acetonitrile in water, spiked with 0.1 % formic acid at a flow rate of 0.5 mL/min. Bound analytes were eluted by a linear gradient of 10 to 95% acetonitrile in water, between 1 and 8 minutes. The mass spectrum was calibrated using formate clusters; leucine enkephalin was used as a "lock mass" for additional, real-time, accurate-mass correction. The purity of compounds described within was determined by HPLC (UV detection at λ = 214 and 254 nm) was performed on a Varian Prostar HPLC, using a 100 x 4.6 mm Phenomenex Luna 3µ C18 column with a gradient of 10-90% acetonitrile in water (with 0.1% TFA in each mobile phases). The purity of all final compounds was shown by HPLC to be ≥95%. Suzuki-Miyaura cross-coupling of iodoindazoles: General Procedure A. A mixture of 3iodo-1H-indazole (1.0 equiv), aryl boronic acid or boronate ester (1–2 equiv), base (2–3 equiv, 1 M aq Na2CO3) and palladium catalyst (0.05–0.2 equiv PdCl2dppf·CH2Cl2 or Pd(PPh3)4) in appropriate solvents (1:1 to 2:1 EtOH/PhMe or dioxane) was degassed with Ar, sealed and heated in a Biotage microwave reactor or oil bath at 100–130 oC for 2–48 h. The reaction was monitored by LCMS. After aqueous workup, the product was purified by Biotage silica gel column or by RP prep-HPLC to provide the desired compound. Ion-exchange PoraPak™ Rxn columns were used to assist the purification as ACS Paragon Plus Environment

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necessary. Salts (HCl) were prepared by dissolution of the free base in MeOH and treatment with 1–2 equiv. of 1 M HCl in Et2O. After evaporation of solvents, the process was repeated several times to obtain the desired mono or di-HCl salt. In some instances trifluoroacetic acid salts were prepared. Synthesis of indazole acetamides: General Procedure B. A solution of 3-iodo-1H-indazol-5amine 2,2,2-trifluoroacetate (1.0 equiv), DIPEA (3 equiv) and RCO2H (1.05 equiv) at 0 oC in DMF was treated with TBTU (1.05 equiv), added in one portion. The reaction was stirred and slowly warmed to rt. After several hours or overnight stirring the crude reaction was diluted with H2O. Generally, filtration of the precipitate followed by washing with water provided the desired material in high purity. Alternatively the material was purified directly by prep-HPLC and/or flash chromatography. N-(3-(3-(methylsulfonyl)phenyl)-1H-indazol-5-yl)-2-(pyrrolidin-1-yl)-2-(thiophen-3yl)acetamide (12). Prepared according to general procedure A using N-(3-iodo-1H-indazol-5-yl)-2(pyrrolidin-1-yl)-2-(thiophen-3-yl)acetamide

trifluoroacetate25

(290

mg,

0.51

mmol),

(3-

(methylsulfonyl)phenyl)boronic acid (120 mg, 0.6 mmol), PhCH3/EtOH (3 mL/9 mL), 1 M aq Na2CO3 (1.5 mL, 1.5 mmol) and Pd(PPh3)4 (11.6 mg, 0.01 mmol, 2 mol%), 1 h at 125 oC, 12 was obtained as a light brown solid (TFA salt, 176 mg, 58%). 1H NMR (400 MHz, CD3OD) δ 8.44 (s, 1H), 8.38 (s, 1H), 8.17 (d, J = 7.6 Hz, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.87 (s, 1H), 7.68 (t, J = 7.8 Hz, 1H), 7.62–7.57 (m, 1H), 7.51 (s, 2H), 7.39 (d, J = 5.2 Hz, 1H), 5.34 (s, 1H), 3.92–3.88 (m, 1H), 3.36–3.00 (m, 6H), 2.25– 1.90 (m, 4H). HRMS (ESI) m/z calcd for [C24H24N4O3S2 + H]+ 481.1368; found, 481.1360. N-(3-(4-(3-(dimethylamino)propyl)-3-(methylsulfonyl)phenyl)-1H-indazol-5-yl)-2-(pyrrolidin-1yl)-2-(thiophen-3-yl)acetamide (13). Prepared according to general procedure A using N-(3-iodo-1Hindazol-5-yl)-2-(pyrrolidin-1-yl)-2-(thiophen-3-yl)acetamide (60 mg, 0.13 mmol), N,N-dimethyl-3-(2(methylsulfonyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)propan-1-amine (54 mg, 0.15 mmol), PdCl2dppf·CH2Cl2 (5.4 mg, 0.007 mmol), Cs2CO3 (130 mg, 0.4 mmol) in DMF (2.5 mL) and H2O (0.6 mL), 2 h at 130 oC, 13 was obtained as an off white powder (11 mg, 15%). 1H NMR (400 MHz, CD3OD) δ 8.56 (d, J = 2.0 Hz, 1H), 8.36 (s, 1H), 8.20 (dd, J = 8.0, 2.0 Hz, 1H), 7.65 (d, J = 8.0 ACS Paragon Plus Environment

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Hz, 1H), 7.57–7.55 (m, 2H), 7.52 (dd, J = 3.0, 1.2 Hz, 1H), 7.43 (dd, J = 5.0, 3.0 Hz, 1H), 7.35 (dd, J = 5.1, 1.1 Hz, 1H), 4.14 (s, 1H), 3.25 (s, 3H), 3.14–3.07 (m, 2H), 2.74–2.65 (m, 2H), 2.62–2.56 (m, 2H), 2.56–2.48 (m, 2H), 2.37 (s, 6H), 2.03–1.93 (m, H), 1.86 (m, 4H). LCMS (ESI) m/z calcd for [C29H35N5O3S2 + H]+ 566.2; found, 566.1. N-(3-(4-(4-methylpiperazin-1-yl)-3-(methylsulfonyl)phenyl)-1H-indazol-5-yl)-2-(pyrrolidin-1yl)-2-(thiophen-3-yl)acetamide (14). Prepared according to general procedure A, using N-(3-iodo-1Hindazol-5-yl)-2-(pyrrolidin-1-yl)-2-(thiophene-3-yl)acetamide (75 mg, 0.165 mmol), 1-methyl-4-(2(methylsulfonyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) phenyl) piperazine (76 mg, 0.199 mmol), 1 M aq Na2CO3 (0.33 mL, 0.33 mmol), PhMe (2.2 mL), EtOH (1.1 mL) and Pd(PPh3)4 (10 mg, 0.008 mmol) and purified using a silica flash column (0–35% MeOH in DCM) followed by reverse phase C-18 flash chromatography (10–20% MeOH in 0.1% aqueous TFA) to give 14 as a white solid (TFA salt, 52 mg, 49%). 1H NMR (400 MHz, CD3OD) δ 8.63 (d, J = 2.0 Hz, 1H), 8.43 (d, J = 0.8 Hz, 1H), 8.32 (dd, J = 8.3, 2.3 Hz, 1H), 7.89–7.88 (m, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.66-7.64 (m, 1H), 7.60–7.53 (m, 2H), 7.39 (d, J =1.2, 1H), 5.27 (s, 1H), 3.91–3.88 (m, 1H), 3.68 (d, J = 12.0 Hz , 2H), 3.63 (d, J = 11.6 Hz , 2H), 3.55–3.41 (m, 5H), 3.35–3.31 (3H coincident with solvent peak), 3.28–3.13 (m, 2H), 3.01 (s, 3H), 2.18–2.13 (m, 3H), 2.01 (br s, 1H). HRMS (ESI) m/z calcd for [C29H34N6O3S2 + H]+ 579.2212; found 579.2214. N-(3-(4-(4-methylpiperazin-1-yl)phenyl)-1H-indazol-5-yl)-2-(pyrrolidin-1-yl)-2-(thiophen-3yl)acetamide (15). Prepared according to general procedure A, using N-(3-iodo-1H-indazol-5-yl)-2(pyrrolidin-1-yl)-2-(thiophen-3-yl)acetamide (50 mg, 0.11 mmol), 1-methyl-4-(4-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)phenyl)piperazine (40 mg, 0.13 mmol), Pd(PPh3)4 (6.35 mg, 0.005 mmol) and 1 M aq Na2CO3 (0.22 mL) in PhMe/EtOH (2.25 mL, 2:1 mixture), 15 was obtained as a pale yellow solid (TFA salt, 21 mg, 26%). 1H NMR (400 MHz, CD3OD) δ 8.35 (s, 1H), 7.85–7.83 (m, 3H), 7.64 (br s, 1H), 7.54–7.49 (m, 2H), 7.38 (dd, J = 4.4 Hz, 1H), 7.19 (d, J = 8.4 Hz, 2H), 5.26 (s, 1H), 3.98–3.86 (m, 3H), 3.67–3.60 (m, 2H), 3.41–3.25 (3H coincident with solvent peak), 3.17–3.11 (m, 4H), 3.00 (s, 3H) ,


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2.26–2.14 (m, 3H), 2.02–2.01 (br s, 1H). HRMS (ESI) m/z calcd for [C28H32N6OS + H]+ 501.2437; found 501.2426 N-(3-(4-morpholinophenyl)-1H-indazol-5-yl)-2-(pyrrolidin-1-yl)-2-(thiophen-3-yl)acetamide

(16).

Prepared according to general procedure A, using N-(3-iodo-1H-indazol-5-yl)-2-(pyrrolidin-1-yl)-2(thiophen-3-yl)acetamide (300 mg, 0.66 mmol) and (4-morpholinophenyl)boronic acid (191 mg, 0.93 mmol), 16 was obtained as a yellow powder (TFA salt, 265 mg, 56%). 1H NMR (400 MHz, CD3OD) δ 8.38 (d, J = 1.2 Hz, 1H), 7.87 (dd, J = 3.0, 1.25 Hz, 1H), 7.80 (d, J = 8.8 Hz, 2H), 7.67 (dd, J = 5.0, 3.0 Hz, 1H), 7.53 (d, J = 9.0 Hz, 1H), 7.48 (dd, J = 9.0, 1.7 Hz, 1H), 7.38 (dd, J = 5.0, 1.2 Hz, 1H), 7.20 (d, J = 8.8 Hz, 2H), 5.20 (s, 1H), 3.90–3.84 (m, 5H), 3.30–3.25 (m, 4H), 3.24–3.13 (m, 1H), 3.10 (br s, 1H), 2.27–2.13 (m, 3H), 1.99 (br s, 1H). HRMS (ESI) m/z calcd for [C27H29N5O2S + H]+ 488.2120; found, 488.2116. N-(3-(4-((1-methylpiperidin-4-yl)oxy)phenyl)-1H-indazol-5-yl)-2-(pyrrolidin-1-yl)-2-(thiophen3-yl)acetamide (17). Prepared according to general procedure A, using N-(3-iodo-1H-indazol-5-yl)-2(pyrrolidin-1-yl)-2-(thiophen-3-yl)acetamide (400 mg, 0.88 mmol) and 1-methyl-4-(4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)piperidine (368 mg, 1.05 mmol), 17 was obtained as an off-white solid (TFA salt, 545 mg, 28%). 1H NMR (400 MHz, CD3OD) δ 8.36 (s, 1H), 7.91–7.82 (m, 3H), 7.64 (dd, J = 5.0, 3.0 Hz, 1H), 7.57–7.49 (m, 2H), 7.38 (dd, J = 5.1, 1.1 Hz, 1H), 7.22–7.11 (m, 2H), 5.29 (s, 1H), 4.73–4.62 (m, 0.33H), 3.87 (br s, 1H), 3.60–3.69 (m, 0.67H), 3.49–3.34 (m, 3.33H), 3.27–3.02 (m, 3H), 2.93 (s, 3H), 2.43 (d, J = 14.3 Hz, 0.67H), 2.34–1.86 (m, 8H). HRMS (ESI) m/z calcd for [C29H33N5O2S + H]+ 516.2433; found, 516.2437. N-(3-(4-(4-hydroxypiperidin-1-yl)phenyl)-1H-indazol-5-yl)-2-(pyrrolidin-1-yl)-2-(thiophen-3yl)acetamide (18). Prepared according to general procedure A, using N-(3-iodo-1H-indazol-5-yl)-2(pyrrolidin-1-yl)-2-thiophen-3-yl) acetamide (250 mg, 0.55 mmol), 1-(4-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)phenyl)piperidin-4-ol (168 mg, 0.55 mmol), Pd(PPh3)4 (48 mg, 0.041 mmol), 1M aq Na2CO3 (1.11 mL) in toluene / EtOH (7.5 mL, 1:0.5 mixture), 3.5 h at 130 ºC, 18 was obtained as a light brown solid (TFA salt, 40 mg, 10%). 1H NMR (400 MHz, CD3OD) δ 8.42 (m, 1H), 8.13 (dd, J = 6.8, ACS Paragon Plus Environment

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2.0 Hz, 2H), 7.89 (dd, J = 2.8, 1.2 Hz, 1H), 7.79 (s, 1H), 7.77 (s, 1H), 7.64–7.62 (m, 1H), 7.59–7.53 (m, 2H), 7.39 (dd, J = 4.8, 1.2 Hz, 1H), 5.36 (s, 1H), 4.12–4.10 (m, 1H), 3.90–3.85 (m, 3H), 3.66–3.61(m, 2H), 3.30–3.05 (m, 2H), 2.29–2.01(m, 8H). HRMS (ESI) m/z calcd for [C28H31N5O2 S + H]+ 502.2277; found, 502.2270. 2-(1-morpholinocyclohexyl)-N-(3-(4-morpholinophenyl)-1H-indazol-5-yl)acetamide

(19).

Prepared according to general procedure B, using 2-(1-morpholinocyclohexyl)acetic acid hydrochloride (100 mg, 0.34 mmol), 3-(4-morpholinophenyl)-1H-indazol-5-amine (106 mg, 0.34 mmol), TBTU (109 mg, 0.34 mmol), DIPEA (0.3 mL, 1.70 mmol) and DMF (3 mL), 19 was obtained as a yellow solid (diTFA salt, 87 mg, 35%). 1H NMR (400 MHz, CD3OD) δ 10.62 (s, 1 H), 8.41 (s, 1H), 7.91 (d, J = 8.8 Hz, 2H), 7.58–7.52 (m, 2H), 7.30 (d, J = 8.8 Hz, 2H), 4.18 (d, J = 11.6 Hz, 2H), 3.93–3.81 (m, 6H), 3.64 (d, J = 12.0 Hz, 2H), 3.37–3.24 (m, 5H), 3.20 (s, 2H), 1.94–1.80 (m, 7H), 1.68–1.62 (m, 2H), 1.36–1.25 (m, 1H). LCMS (ESI) m/z calcd for [C29H37N5O3 + H]+ 504.3; found, 504.3. 2-cyclopentyl-N-(3-(4-((1-methylpiperidin-4-yl)oxy)phenyl)-1H-indazol-5-yl)-2-(thiophen-3yl)acetamide (20). Prepared according to general procedure B, using 2-cyclopentyl-2-(thiophen-3yl)acetic acid (19.0 mg, 0.09 mmol), 3-(4-((1-methylpiperidin-4-yl)oxy)phenyl)-1H-indazol-5-amine trifluoroacetate (50 mg, 0.09 mmol), TBTU (29.1 mg, 0.09 mmol), DIPEA (0.08 mL, 0.45 mmol) and DMF (1.5 mL), 20 was obtained as an off white solid (TFA salt, 31 mg, 54%). 1H NMR (400 MHz, CD3OD) δ 10.11–10.02 (m, 1H), 8.43 (d, J = 1.0 Hz, 1H), 7.91–7.82 (m, 2H), 7.51 (d, J = 9.0 Hz, 1H), 7.42–7.35 (m, 2H), 7.31 (dd, J = 2.9, 1.1 Hz, 1H), 7.24–7.11 (m, 3H), 4.88–4.83 (m, 2H), 4.71–4.61 (m, 1H), 3.65–3.54 (m, 2H), 3.44–3.24 (m, 2H), 2.94 (s, 3H), 2.45–2.28 (m, 2H), 2.15–2.07 (m, 3H), 1.75– 1.61 (m, 5H), 1.40–1.35 (m, 1H), 1.18–1.13 (m, 1H). LCMS (ESI) m/z calcd for [C30H34N4O2S+H]+ 515.2; found, 515.5. 2-cyclopentyl-N-(3-(4-((1-methylpiperidin-4-yl)oxy)phenyl)-1H-indazol-5-yl)-2-(pyridin-2yl)acetamide (21). Prepared according to general procedure A, using 2-cyclopentyl-N-(3-iodo-1Hindazol-5-yl)-2-(pyridin-2-yl)acetamide and 1-methyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)phenoxy)-piperidine, 21 was obtained as a white solid (18 mg, 32%). 1H NMR (400 MHz, CD3OD) δ ACS Paragon Plus Environment

22


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Page 24 of 76

8.53–8.47 (m, 1H), 8.37 (d, J = 1.0 Hz, 1H), 7.85–7.75 (m, 3H), 7.64 (d, J = 8.0 Hz, 1H), 7.52–7.43 (m, 2H), 7.31 (ddd, J = 7.2, 5.0, 1.0 Hz, 1H), 7.05 (d, J = 8.8 Hz, 2H), 4.46 (br s, 1H), 3.61 (d, J = 11.0 Hz, 1H), 2.87–2.64 (m, 3H), 2.39 (br s, 2H), 2.33–2.27 (m, 3H), 2.09–1.90 (m, 3H), 1.89–1.40 (m, 8H), 1.16–1.05 (m, 1H). LCMS (ESI) m/z calcd for [C31H35N5O2+ H]+ 510.3; found, 510.2. 3-(4-(4-methylpiperazin-1-yl)phenyl)-N-((1-morpholinocyclohexyl)methyl)-1H-indazole-5carboxamide

(33).

Prepared

according

to

general

procedure

A,

using

3-iodo-N-((1-

morpholinocyclohexyl)methyl)-1H-indazole-5-carboxamide trifluoroacetate (95 mg, 0.16 mmol) and 1methyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)piperazine (65 mg, 0.21 mmol), 33 was obtained as a white solid (TFA salt, 84 mg, 83%). 1H NMR (400 MHz, CD3OD) δ 8.70 (s, 1H), 8.01 (dd, J = 8.9, 1.6 Hz, 1H), 7.96 (d, J = 8.8 Hz, 2H), 7.66 (d, J = 8.8 Hz, 1H), 7.23 (d, J = 8.8 Hz, 2H), 4.21–4.12 (m, 2H), 4.04–3.96 (m, 2H), 3.94 (s, 2H), 3.94–3.83 (m, 2H), 3.71–3.64 (m, 4H), 3.283.38 (m, 4H), 3.22–3.08 (m, 2H), 3.01 (s, 3H), 2.05–1.55 (m, 9H), 1.40–1.22 (m, 1H). HRMS (ESI) m/z [C30H40N6O2+H]+ 517.3291; found, 517.3287. N-(cyclopropyl(phenyl)methyl)-3-(4-morpholinophenyl)-1H-indazole-5-carboxamide

(34).

Prepared according to general procedure A, using N-(cyclopropyl(phenyl)methyl)-3-iodo-1H-indazole5-carboxamide (50 mg, 0.12 mmol) and 4-morpholinophenylboronic acid pinacol ester (40 mg, 0.14 mmol), 34 was obtained as a beige solid (TFA salt, 11 mg, 16%). 1H NMR (400 MHz, CD3OD) δ 8.60 (s, 1H), 7.98–7.87 (m, 3H), 7.58 (d, J = 8.8 Hz, 1H), 7.47 (d, J = 7.0 Hz, 2H), 7.31 (t, J = 7.2 Hz, 2H), 7.22 (t, J = 6.8 Hz, 1H), 7.15 (d, J = 7.8 Hz, 2H), 4.48 (d, J = 9.5 Hz, 1H), 3.86 (br s, 4H), 3.25 (br s, 4H), 1.44–1.34 (m, 1H), 0.71–0.59 (m, 2H), 0.55–0.40 (m, 2H). HRMS (ESI) m/z calcd for [C28H28N4O2 + H]+ 453.2291; found, 453.2282. N-(cyclopropyl(phenyl)methyl)-3-(3-morpholinophenyl)-1H-indazole-5-carboxamide

(35).

Prepared according to general procedure A, using N-(cyclopropyl(phenyl)methyl)-3-iodo-1H-indazole5-carboxamide

(100

mg,

0.24

mmol)

and

4-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-

yl)phenyl)morpholine (69 mg, 0.24 mmol), 35 was obtained as a beige solid (39 mg, 36%). 1H NMR (400 MHz, CD3OD) δ 8.58 (s, 1H), 7.94 (dd, J = 8.8, 1.4 Hz, 1H), 7.62 (d, J = 8.8 Hz, 1H), 7.55–7.44 ACS Paragon Plus Environment

23

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(m, 5H), 7.34 (t, J = 7.6 Hz, 2H), 7.24 (t, J = 7.3 Hz, 1H), 7.10–7.05 (m, 1H), 4.48 (d, J = 8.5 Hz, 1H), 3.89–3.84 (m, 4H), 3.27–3.22 (m, 4H), 1.44–1.33 (m, 1H), 0.69–0.62 (m, 2H), 0.54–0.41 (m, 2H). LCMS (ESI) m/z calcd for [C28H28N4O2 + H]+ 453.2; found, 453.2. N-(cyclopropyl(phenyl)methyl)-3-(4-((1-methylpiperidin-4-yl)oxy)phenyl)-1H-indazole-5carboxamide (36). Prepared according to general procedure A, using N-(cyclopropyl(phenyl)methyl)-3iodo-1H-indazole-5-carboxamide

(200

mg,

0.48

mmol)

and

(4-((1-methylpiperidin-4-

yl)oxy)phenyl)boronic acid pinacol ester (152 mg, 0.48 mmol), 36 was obtained as a white solid (80 mg, 35%). 1H NMR (400 MHz, CD3OD) δ 8.60 (s, 1H), 7.95 (d, J = 9.0 Hz, 1H), 7.90 (d, J = 8.8 Hz, 2H), 7.59 (d, J = 8.8 Hz, 1H), 7.47 (d, J = 7.5 Hz, 2H), 7.31 (t, J = 7.5 Hz, 2H), 7.22 (t, J = 7.5 Hz, 1H), 7.08 (d, J = 8.8 Hz, 2H), 4.55 (br s, 1H), 4.47 (d, J = 9.3 Hz, 1H), 3.03–2.91 (m, 2H), 2.76–2.63 (m, 2H), 2.50 (s, 3H), 2.14–2.02 (m, 2H), 1.98–1.85 (m, 2H), 1.44–1.34 (m, 1H), 0.64 (d, J = 8.3 Hz, 2H), 0.45 (dd, J = 9.5, 4.5 Hz, 2H). LCMS (ESI) m/z calcd for [C30H32N4O2 + H]+ 481.3; found, 481.4. (R)-N-(1-(2-chlorophenyl)propyl)-3-(4-((1-methylpiperidin-4-yl)oxy)phenyl)-1H-indazole-5carboxamide (51). Prepared according to general procedure A, using (R)-N-(1-(2-chlorophenyl)propyl)3-iodo-1H-indazole-5-carboxamide (58 mg, 0.13 mmol 36 %ee) and 1-methyl-4-(4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy) piperidine (55 mg, 0.17 mmol), 51 was obtained as an orange solid (TFA salt, 39 mg, 59%). 1H NMR (400 MHz, CD3OD) δ 8.57 (s, 1H), 7.95–7.91 (m, 3H), 7.61 (d, J = 8.8 Hz, 1H), 7.55 (d, J = 7.0 Hz, 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.30–7.14 (m, 4H), 5.47– 5.43 (m, 1H), 4.87 (br s, 1H), 3.65–3.17 (m, 4H), 2.93–2.92 (m, 3H), 2.44–2.27 (m, 2H), 2.15–1.86 (m, 4H), 1.07 (t, J = 7.2 Hz, 3H). HRMS (ESI) m/z calcd for [C29H31ClN4O2 + H]+ 503.2214; found, 503.2214. N-(cyclopropyl(thiophen-3-yl)methyl)-3-(4-(4-hydroxypiperidin-1-yl)phenyl)-1H-indazole-5carboxamide (37).

Prepared according to general procedure A, using N-(cyclopropyl(thiophen-3-

yl)methyl)-3-iodo-1H-indazole-5-carboxamide (50 mg, 0.12 mmol) and 1-(4-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)phenyl)piperidin-4-ol (54 mg, 80% pure, 0.14 mmol), 37 was obtained as a pale pink solid (TFA salt, 27 mg, 39%). 1H NMR (400 MHz, CD3OD) δ 8.63 (s, 1H), 8.24 (d, J = 8.8 Hz, 2H), ACS Paragon Plus Environment

24


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Page 26 of 76

7.99 (dd, J = 8.9, 1.4 Hz, 1H), 7.78 (d, J = 8.8 Hz, 2H), 7.67 (d, J = 8.8 Hz, 1H), 7.40–7.36 (m, 2H), 7.21 (t, J = 3.3 Hz, 1H), 5.49 (s, 1H), 4.64 (d, J = 9.3 Hz, 1H), 4.11 (tt, J = 7.0, 3.3 Hz, 1H), 3.91 (ddd, J = 12.0, 8.4, 3.4 Hz, 2H), 3.65 (ddd, J = 11.9, 7.7, 3.5 Hz, 2H), 2.31–2.21 (m, 2H), 2.04 (dtd, J = 14.2, 7.2, 3.5 Hz, 2H), 1.51–1.41 (m, 1H), 0.74 (td, J = 8.3, 3.8 Hz, 1H), 0.65 (td, J = 8.3, 4.8 Hz, 1H), 0.56– 0.45 (m, 2H). HRMS (ESI) m/z calcd for [C27H28N4O2S + H]+ 473.2011; found, 473.2017. 3-(4-(4-hydroxypiperidin-1-yl)phenyl)-N-((1-morpholinocyclohexyl)methyl)-1H-indazole-5carboxamide

(38).

Prepared

according

to

general

procedure

A,

using

3-iodo-N-((1-

morpholinocyclohexyl)methyl)-1H-indazole-5-carboxamide trifluoroacetate (80 mg, 0.136 mmol), 1-(4(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)piperidin-4-ol (70 mg, 0.18 mmol) and Pd(PPh3)4 (19 mg, 0.016 mmol), 38 was obtained as a pale yellow solid (TFA salt, 31 mg, 30%). 1H NMR (400 MHz, CD3OD) δ 8.73 (s, 1H), 8.25 (d, J = 8.8 Hz, 2H), 8.04 (dd, J = 8.8, 1.6 Hz, 1H), 8.83 (d, J = 8.8 Hz, 2H), 7.71 (d, J = 8.8 Hz, 1H), 4.11–4.95 (m, 3H), 3.98–3.86 (m, 6H), 3.70–3.64 (m, 4H), 3.36–3.21 (1H coincident with solvent peak), 2.66 (s, 2H), 2.31–2.25 (m, 2H), 2.09–1.98 (m, 4H), 1.91– 1.81(m, 2H), 1.81–1.75 (m, 5H), 1.42–1.21 (m, 1H). LCMS (ESI) m/z calcd for [C30H39N5O3 + H]+ 518.3; found 518.2. N-((1-morpholinocyclohexyl)methyl)-3-(4-morpholinophenyl)-1H-indazole-5-carboxamide (39). Prepared according to general procedure A, using 3-iodo-N-((1-morpholinocyclohexyl)methyl)-1Hindazole-5-carboxamide trifluoroacetate (70 mg, 0.12 mmol) and (4-morpholinophenyl)boronic acid (35 mg, 0.17 mmol), 39 was obtained as pale yellow solid (TFA salt, 46 mg, 74%). 1H NMR (400 MHz, CD3OD) δ 8.54 (s, 1H), 7.89 (d, J = 9.0 Hz, 3H), 7.63 (d, J = 8.8 Hz, 1H), 7.13 (d, J = 9.0 Hz, 2H), 3.90–3.79 (m, 4H), 3.71–3.45 (m, 2H), 3.28–3.17 (m, 4H), 2.97–2.56 (m, 2H), 1.84–1.25 (m, 16H). LCMS (ESI) m/z calcd for [C29H37N5O3 + H]+ 504.3; found, 504.3. N-(cyclopentyl(thiophen-3-yl)methyl)-3-(4-((1-methylpiperidin-4-yl)oxy)phenyl)-1H-indazole-5carboxamide (40).

Prepared according to general procedure A, using N-(cyclopentyl(thiophen-3-

yl)methyl)-3-iodo-1H-indazole-5-carboxamide (150 mg, 0.332mmol) and 1-methyl-4-(4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)piperidine (106 mg, 0.334 mmol), 40 was obtained as an ACS Paragon Plus Environment

25

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off white solid (TFA salt, 60 mg, 28%). 1H NMR (400 MHz, CD3OD) δ 8.52 (s, 1H), 7.94–7.88 (m, 3H), 7.60 (d, J = 8.8 Hz, 1H), 7.36–7.32 (m, 2H), 7.20–7.13 (m, 3H), 5.08 (d, J = 6.0 Hz, 1H),4.85 (s, 1H), 3.66 (t, J = 13.6 Hz, 1H), 3.44–3.32 (m, 5H), 3.23 (br s, 1H), 2.59–2.53 (m, 1H), 2.45–2.27 (m, 2H), 2.16–1.87 (m, 3H), 1.74–1.44 (m, 6H), 1.28–1.25 (m, 1H). LCMS (ESI) m/z calcd for [C30H34N4O2S + H]+ 515.2; found 515.4. N-(cyclopentyl(pyridin-2-yl)methyl)-3-(4-((1-methylpiperidin-4-yl)oxy)phenyl)-1H-indazole-5carboxamide (41).

Prepared according to general procedure A, using N-(cyclopentyl(pyridin-2-

yl)methyl)-3-iodo-1H-indazole-5-carboxamide (100 mg, 0.22 mmol) and (4-((1-methylpiperidin-4yl)oxy)phenyl)boronic acid pinacol ester (70 mg, 0.22 mmol), Pd(PPh3)4 (13 mg, 0.011 mmol), 41 was obtained as a white solid (43 mg, 38%). 1H NMR (400 MHz, CD3OD) δ 8.57 (s, 1H), 8.51 (d, J = 4.3 Hz, 1H), 7.94–7.86 (m, 3H), 7.79 (t, J = 7.6 Hz, 1H), 7.59 (d, J = 8.8 Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.29 (dd, J = 7.0, 5.0 Hz, 1H), 7.10 (d, J = 8.5 Hz, 2H), 5.01 (d, J = 10.3 Hz, 1H), 4.52 (br s, 1H), 2.82 (br s, 2H), 2.58–2.46 (m, 3H), 2.39 (s, 3H), 2.12–1.81 (m, 5H), 1.76–1.45 (m, 5H), 1.41–1.20 (m, 2H). LCMS (ESI) m/z calcd for [C31H35N5O2 + H]+ 510.3; found, 510.2. N-(cyclopropyl(phenyl)methyl)-3-(4-((1-formylpiperidin-4-yl)oxy)phenyl)-1H-indazole-5carboxamide (42). Prepared according to general procedure A, using N-(cyclopropyl(phenyl)methyl)-3iodo-1H-indazole-5-carboxamide (43 mg, 0.10 mmol) and 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan2-yl)phenoxy)piperidine-1-carbaldehyde (41 mg, 0.12 mmol), 42 was obtained as a yellow solid (14 mg, 27%). 1H NMR (400 MHz, CD3OD) δ 8.61 (s, 1H), 8.03 (s, 1H), 7.98–7.89 (m, 3H), 7.59 (d, J = 8.8 Hz, 1H), 7.48 (d, J = 7.3 Hz, 2H), 7.32 (t, J = 7.5 Hz, 2H), 7.23 (m, J = 7.5 Hz, 1H), 7.12 (d, J = 8.8 Hz, 2H), 4.77–4.69 (m, 1H), 4.51–4.45 (m, 1H), 3.79–3.62 (m, 2H), 3.58–3.49 (m, 1H), 3.46–3.37 (m, 1H), 2.08–1.91 (m, 2H), 1.91–1.72 (m, 2H), 1.46–1.34 (m, 1H), 0.65 (d, J = 8.3 Hz, 2H), 0.53–0.41 (m, 2H). LCMS (ESI) m/z calcd for [C30H30N4O3 + H]+ 495.2; found 495.3. N-(cyclopropyl(pyridin-2-yl)methyl)-3-(4-((1-formylpiperidin-4-yl)oxy)phenyl)-1H-indazole-5carboxamide (43). Prepared according to general procedure A, using N-(cyclopropyl(pyridin-2yl)methyl)-3-iodo-1H-indazole-5-carboxamide (84 mg, 0.2 mmol) and 4-(4-(4,4,5,5-tetramethyl-1,3,2ACS Paragon Plus Environment

26


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Page 28 of 76

dioxaborolan-2-yl)phenoxy)piperidine-1-carbaldehyde (74 mg, 0.22 mmol), 43 was obtained as a white solid (36 mg, 29%). 1H NMR (400 MHz, CD3OD) δ 8.77 (dd, J = 5.7, 0.7 Hz, 1H), 8.70 (s, 1H), 8.56 (t, J = 7.1 Hz, 1H), 8.20 (d, J = 8.0 Hz, 1H), 8.05 (s, 1H), 8.01–7.90 (m, 4H), 7.63 (d, J = 8.8 Hz, 1H), 7.15 (d, J = 8.8 Hz, 2H), 4.80–4.72 (m, 1H), 4.49 (d, J = 10.0 Hz, 1H), 3.82–3.64 (m, 2H), 3.58–3.39 (m, 2H), 2.10–1.92 (m, 2H), 1.90–1.71 (m, 2H), 1.58–1.45 (m, 1H), 0.95–0.84 (m, 1H), 0.81–0.58 (m, 3H). HRMS (ESI) m/z calcd for [C29H29N5O3+ H]+ 496.2349; found 496.2355. N-(cyclopropyl(phenyl)methyl)-3-(4-((1-(2-(dimethylamino)acetyl)piperidin-4-yl)oxy)phenyl)1H-indazole-5-carboxamide

(44).

Prepared

according

to

cyclopropyl(phenyl)methyl)-3-iodo-1H-indazole-5-carboxamide

general (89

procedure

mg,

0.21

A, mmol)

using

N-

and

2-

(dimethylamino)-1-(4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)piperidin-1-yl)ethanone (91 mg, 0.24 mmol), 44 was obtained as a pale yellow solid (66 mg, 56%). 1H NMR (400 MHz, CD3OD) δ 8.60 (s, 1H), 7.95 (m, J = 8.5 Hz, 3H), 7.61 (d, J = 8.8 Hz, 1H), 7.50 (d, J = 7.0 Hz, 2H), 7.35 (t, J = 7.6 Hz, 2H), 7.25 (m, J = 7.3 Hz, 1H), 7.16 (d, J = 8.8 Hz, 2H), 4.80–4.73 (m, 1H), 4.49 (d, J = 9.5 Hz, 1H), 3.92–3.74 (m, 2H), 3.67 (s, 3H), 3.55–3.47 (m, 1H), 2.58 (s, 6H), 2.14–1.96 (m, 2H), 1.93–1.76 (m, 2H), 1.47–1.37 (m, 1H), 0.70–0.64 (m, 2H), 0.53–0.44 (m, 2H). LCMS (ESI) m/z calcd for [C33H37N5O3 + H]+ 552.3; found 552.5. N-(cyclopropyl(phenyl)methyl)-3-(4-((endo)-8-methyl-8-azabicyclo[3.2.1]octan-3yl)oxy)phenyl)-1H-indazole-5-carboxamide (45). Prepared according to general procedure A, using 2cyclopropyl-N-(3-iodo-1H-indazol-5-yl)-2-phenylacetamide (90 mg, 0.22 mmol) and (enso)-8-methyl3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)-8-azabicyclo[3.2.1]octane (0.14 g, 0.31 mmol), 45 was obtained as a white powder (7.5 mg, 7%). 1H NMR (400 MHz, CD3OD) δ 8.61 (s, 1 H), 8.00–7.89 (m, 3H), 7.61 (d, J = 8.8 Hz, 1H), 7.49 (d, J = 7.2 Hz, 2H), 7.33 (t, J = 7.5 Hz, 2H), 7.24 (t, J = 7.3 Hz, 1H), 7.04 (d, J = 8.8 Hz, 2H), 4.69 (t, J = 4.7 Hz, 1H), 4.48 (d, J = 9.2 Hz, 1H), 3.44 (br s, 2H), 2.50 (s, 3H), 2.37–2.02 (m, 8H), 1.48–1.37 (m, 1H), 0.70–0.62 (m, 2H), 0.52–0.42 (m, 2H). HRMS (ESI) m/z calcd for [C32H34N4O2 + H]+ 507.2760; found 507.2758.


27

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N-(cyclopropyl(phenyl)methyl)-3-(4-((exo)-8-methyl-8-azabicyclo[3.2.1]octan-3-yl)oxy)phenyl)1H-indazole-5-carboxamide (46). Prepared according to general procedure A,

using N-

(cyclopropyl(phenyl)methyl)-3-iodo-1H-indazole-5-carboxamide (208 mg, 0.5 mmol), (exo)-8-methyl3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)-8-azabicyclo [3.2.1]octane (137 mg, 0.4 mmol), 46 was obtained as a white solid (29 mg, 14%). 1H NMR (400 MHz, CD3OD) δ 8.62 (s, 1H), 7.96 (dd, J = 8.8, 1.6 Hz, 1H), 7.89 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 8.8 Hz, 1H), 7.47 (d, J = 7.2 Hz, 2H), 7.32 (t, J = 7.6 Hz, 2H), 7.22 (t, J = 7.2 Hz, 1H), 7.05 (d, J = 8.8 Hz, 2H), 4.70-4.60 (m, 1H), 4.48 (d, J = 9.6 Hz, 1H), 3.30-3.23 (m, 2H), 2.33 (s, 3H), 2.17–2.04 (m, 4H), 1.85–1.74 (m, 4H), 1.45–1.35 (m, 1H), 0.68–0.60 (m, 2H), 0.52–0.40 (m, 2H). HRMS (ESI) m/z calcd for [C32H34N4O2 + H]+ 507.2760; found 507.2752. N-(cyclobutyl(thiophen-3-yl)methyl)-3-(4-(((endo)-8-formyl-8-azabicyclo[3.2.1]octan-3yl)oxy)phenyl)-1H-indazole-5-carboxamide (47). Prepared according to general procedure A, using N(cyclobutyl(thiophen-3-yl)methyl)-3-iodo-1H-indazole-5-carboxamide (56 mg, 0.13 mmol) and (endo)-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)-8-azabicyclo[3.2.1]octane-8carbaldehyde (50 mg, 0.14 mmol), 47 was obtained as a white solid (17 mg, 25%). 1H NMR (400 MHz, CD3OD) δ 8.56 (s, 1H), 8.10 (s, 1H), 7.95–7.85 (m, 3H), 7.58 (d, J = 8.8 Hz, 1H), 7.32 (dd, J = 4.8, 3.0 Hz, 1H), 7.26 (s, 1H), 7.12 (d, J = 5.0 Hz, 1H), 6.98 (d, J = 8.8 Hz, 2H), 5.27 (d, J = 10.5 Hz, 1H), 4.74 (br s, 1H), 4.57–4.45 (m, 1H), 4.15 (br s, 1H), 2.93 (d, J = 9.8 Hz, 1H), 2.34–1.74 (m, 14H). HRMS (ESI) m/z calcd for [C31H32N4O3S + H]+ 541.2273; found 541.2275. 3-((4-((endo)-8-formyl-8-azabicyclo[3.2.1]octan-3-yl)oxy)phenyl)-N-(3-methyl-1-(pyridin-2yl)butyl)-1H-indazole-5-carboxamide (48). Prepared according to general procedure A, using 3-iodo-N(3-methyl-1-(pyridin-2-yl)butyl)-1H-indazole-5-carboxamide (55mg, 0.13 mmol) and (endo)-3-(4(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)-8-azabicyclo[3.2.1]octane-8-carbaldehyde

(50

mg, 0.14 mmol), 48 was obtained as a white solid (32 mg, 47%). 1H NMR (400 MHz, CD3OD) δ 8.65 (s, 1H), 8.49 (d, J = 5.0 Hz, 1H), 8.08 (s, 1H), 7.97 (dd, J = 8.8, 1.5 Hz, 1H), 7.90–7.86 (m, 2H), 7.78– 7.71 (m, 1H), 7.59 (d, J = 8.8 Hz, 1H), 7.47 (d, J = 7.8 Hz, 1H), 7.29–7.20 (m, 1H), 6.98–6.68 (m, 2H), ACS Paragon Plus Environment

28


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Page 30 of 76

5.36 (dd, J = 9.8, 5.3 Hz, 1H), 4.68 (br s, 1H), 4.50 (br s, 1H), 4.11 (br s, 1H), 2.29–2.15 (m, 2H), 2.15– 1.97 (m, 4H), 1.97–1.81 (m, 3H), 1.81–1.62 (m, 2H), 1.06–0.91 (m, 6H). LCMS (ESI) m/z calcd for [C32H35N5O3 + H]+ 538.3; found 538.4. 3-(4-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)phenyl)-N-((1-morpholinocyclohexyl)methyl)-1Hindazole-5-carboxamide (49). Prepared according to general procedure A, using 3-iodo-N-((1morpholinocyclohexyl)methyl)-1H-indazole-5-carboxamide (80 mg, 0.17 mmol), 3-(4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-8-oxa-3-azabicyclo[3.2.1]octane (54 mg, 0.17 mmol) and PdCl2dppf·CH2Cl2 (14 mg, 0.017 mmol), 49 was obtained as a yellow solid (di-TFA salt, 36 mg, 31%). 1

H NMR (400 MHz, CD3OD) δ 8.68 (s, 1 H), 8.00 (d, J = 9.3 Hz, 1H), 7.87 (d, J = 8.3 Hz, 2H), 7.64

(d, J = 8.8 Hz, 1H), 7.03 (d, J = 8.0 Hz, 2H), 4.51 (br s, 2H), 4.16 (d, J = 12.8 Hz, 2H), 3.98–3.82 (m, 4H), 3.66 (d, J = 12.3 Hz, 2H), 3.51 (d, J = 11.5 Hz, 2H), 2.98 (d, J = 11.5 Hz, 2H), 2.06–1.85 (m, 9H), 1.84–1.55 (m, 6H), 1.39–1.24 (m, 1H). HRMS (ESI) m/z calcd for [C31H39N5O3 + H]+ 530.3131; found 530.3125. 3-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)phenyl)-N-((1-morpholinocyclohexyl)methyl)-1Hindazole-5-carboxamide (50). Prepared according to general procedure A, using 3-iodo-N-((1morpholinocyclohexyl)methyl)-1H-indazole-5-carboxamide (100 mg, 0.21 mmol), 8-(4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3-oxa-8-azabicyclo[3.2.1]octane (67 mg, 0.21 mmol), 50 was obtained as a yellow solid (di-TFA salt, 70 mg, 44%). 1H NMR (400 MHz, CD3OD) δ 8.67 (s, 1H), 7.99 (dd, J = 8.9, 1.6 Hz, 1H), 7.86 (d, J = 8.8 Hz, 2 H), 7.63 (dd, J = 8.9, 0.6 Hz, 1H), 7.03 (d, J = 8.5 Hz, 2H), 4.21 (br s, 2H), 4.15 (d, J = 12.5 Hz, 2H), 3.95–3.82 (m, 6H), 3.65 (d, J = 11.8 Hz, 2H), 3.54 (d, J = 10.8 Hz, 2H), 3.33–3.30 (m, 2H), 2.13–2.00 (m, 4H), 1.99–1.93 (m, 2H), 1.92–1.84 (m, 2H), 1.83–1.70 (m, 3H), 1.69–1.55 (m, 2H), 1.36–1.23 (m, 1H). HRMS (ESI) m/z calcd for [C31H39N5O3 + H]+ 530.3131; found 530.3124. 3-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)phenyl)-N-((S)-cyclopropyl(2-fluorophenyl)methyl)1H-indazole-5-carboxamide (64). Prepared according to general procedure A, using (S)-N(cyclopropyl(2-fluorophenyl)methyl)-3-iodo-1H-indazole-5-carboxamide (75 mg, 0.17 mmol), 8-(4ACS Paragon Plus Environment

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(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3-oxa-8-azabicyclo[3.2.1]octane (55 mg, 0.17 mmol) and Pd(PPh3)4 (15 mg, 0.012 mmol), 64 was obtained as a yellow solid (HCl salt, 54 mg, 59%). 1

H NMR (400 MHz, CD3OD) δ 8.62 (s, 1H), 8.27 (d, J = 8.4 Hz, 2H), 7.99 (d, J = 8.8 Hz, 1H), 7.81 (d,

J = 8.4 Hz, 2H), 7.68 (d, J = 8.4 Hz, 1H), 7.58 (t, J = 6.4 Hz, 1H), 7.31–7.26 (m, 1H), 7.17 (t, J = 7.6 Hz, 1H), 7.11–7.06 (m, 1H), 4.77 (d, J = 9.6 Hz, 1H), 4.71 (br s, 2H), 4.25 (d, J = 12.4 Hz, 2H), 3.99 (d, J = 12.8 Hz, 2H), 2.37–2.35 (m, 2H), 2.22–2.19 (m, 2H), 1.48–1.46 (m, 1H), 0.73–0.70 ( m, 1H), 0.69– 0.59 (m, 1H), 0.54–0.45 (m, 2H). LCMS (ESI) m/z calcd for [C30H29FN4O2 + H]+ 497.2; found 497.4. 3-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)phenyl)-N-((R)-cyclopropyl(2-fluorophenyl)methyl)1H-indazole-5-carboxamide (65). Prepared according to general procedure A, using (R)-N(cyclopropyl(2-fluorophenyl)methyl)-3-iodo-1H-indazole-5-carboxamide (80 mg, 0.18 mmol), 8-(4(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3-oxa-8-azabicyclo[3.2.1]octane

(65

mg,

0.2

mmol) and Pd(PPh3)4 (10 mg, 0.009 mmol), 65 was obtained as a yellow solid (TFA salt, 13 mg, 12%). 1

H NMR (400 MHz, CD3OD) δ 8.61 (s, 1H), 7.97–7.84 (m, 3H), 7.63–7.50 (m, 2H), 7.31–7.22 (m, 1H),

7.16 (t, J = 7.2 Hz, 1H), 7.12–7.03 (m, 2H), 4.75 (d, J = 9.5 Hz, 1H), 4.24 (br s, 2H), 3.93 (d, J = 11.0 Hz, 2H), 3.57 (br s, 2H), 2.15–1.97 (m, 4H), 1.48–1.40 (m, 1H), 0.72–0.62 (m, 1H), 0.62–0.53 (m, 1H), 0.53–0.38 (m, 3H). LCMS (ESI) m/z calcd for [C30H29FN4O2 + H]+ 497.2; found 497.4. 3-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)phenyl)-N-((S)-1-(2-chlorophenyl)-2-methylpropyl)1H-indazole-5-carboxamide (66). Prepared according to general procedure A, using (S)-N-(1-(2chlorophenyl)-2-methylpropyl)-3-iodo-1H-indazole-5-carboxamide (100 mg, 0.22 mmol) and 8-(4(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3-oxa-8-azabicyclo[3.2.1]octane (69 mg, 0.22 mmol), 66 was obtained as a yellow solid (TFA salt, 51 mg, 37%). 1H NMR (400 MHz, CD3OD) δ 8.54 (s, 1H), 7.93 (br s, 2H), 7.90–7.86 (m, 1H), 7.58 (d, J = 8.5 Hz, 1H), 7.54 (dd, J = 7.7, 1.6 Hz, 1H), 7.38 (dd, J = 7.9, 1.4 Hz, 1H), 7.32–7.27 (m, 1H), 7.25–7.13 (m, 3H), 5.37 (d, J = 9.8 Hz, 1H), 4.32 (br s, 2H), 4.00–3.95 (m, 2H), 3.64 (br s, 2H), 2.33–2.22 (m, 1H), 2.18–2.02 (m, 4H), 1.16 (d, J = 6.8 Hz, 3H), 0.86 (d, J = 6.5 Hz, 3H). LCMS (ESI) m/z calcd for [C30H31ClN4O2 + H]+ 515.2; found 515.6.


30


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Page 32 of 76

3-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)phenyl)-N-((S)-cyclopropyl(pyridin-2-yl)methyl)-1Hindazole-5-carboxamide

(67).

Prepared

according

to

general

procedure

A,

using

(S)-N-

(cyclopropyl(pyridin-2-yl)methyl)-3-iodo-1H-indazole-5-carboxamide (100 mg, 0.24 mmol) and 8-(4(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3-oxa-8-azabicyclo[3.2.1]octane (76 mg, 0.24 mmol), 67 was obtained as a yellow solid (di-TFA salt, 64 mg, 38%). 1H NMR (400 MHz, CD3OD) δ 8.73–8.64 (m, 2H), 8.40 (td, J = 7.9, 1.8 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.96 (dd, J = 8.8, 1.5 Hz, 1H), 7.87 (d, J = 8.0 Hz, 2H), 7.83–7.77 (m, 1H), 7.60 (d, J = 8.8 Hz, 1H), 7.02 (br s, 2H), 4.48 (d, J = 10.0 Hz, 1H), 4.19 (br s, 2H), 3.90 (d, J = 10.8 Hz, 2H), 3.53 (br s, 2H), 2.12–1.96 (m, 4H), 1.54–1.42 (m, 1H), 0.89–0.80 (m, 1H), 0.75–0.56 (m, 3H). LCMS (ESI) m/z calcd for [C29H29N5O2 + H]+ 480.2; found 480.2. 3-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)phenyl)-N-((R)-cyclopropyl(pyridin-2-yl)methyl)-1Hindazole-5-carboxamide

(68).

Prepared

according

to

general

procedure

A,

using

(R)-N-

(cyclopropyl(pyridin-2-yl)methyl)-3-iodo-1H-indazole-5-carboxamide (100 mg, 0.24 mmol) and 8-(4(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3-oxa-8-azabicyclo[3.2.1]octane (76 mg, 0.24 mmol), 68 was obtained as a yellow solid (di-HCl salt, 68 mg, 51%). 1H NMR (400 MHz, CD3OD) δ 8.83–8.78 (m, 2H), 8.66 (t, J = 8.4 Hz, 1H), 8.32 (d, J = 8.0 Hz, 1 H), 8.28 (d, J = 8.5 Hz, 2H), 8.05– 8.00 (m, 2H), 7.75 (d, J = 8.5 Hz, 2H), 7.69 (d, J= 8.8 Hz, 1H), 4.68 (br s, 2H), 4.51 (d, J = 10.3 Hz, 1H), 4.21 (d, J = 12.5 Hz, 2H), 3.93 (d, J = 12.0 Hz, 2H), 2.38–2.30 (m, 2H), 2.24–2.16 (m, 2H), 1.65– 1.56 (m, 1H), 0.98–0.89 (m, 1H), 0.82–0.62 (m, 3H). HRMS (ESI) m/z calcd for [C29H29N5O2 + H]+ 480.2400; found 480.2399 3-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)phenyl)-N-((S)-cyclopentyl(pyridin-2-yl)methyl)-1Hindazole-5-carboxamide

(69).

Prepared

according

to

general

procedure

A,

using

(S)-N-

(cyclopentyl(pyridin-2-yl)methyl)-3-iodo-1H-indazole-5-carboxamide (100 mg, 0.22 mmol) and 8-(4(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3-oxa-8-azabicyclo[3.2.1]octane (69 mg, 0.22 mmol), 69 was obtained as a yellow solid (di-TFA salt, 32 mg, 20%, >99.9 %ee). 1H NMR (400 MHz, CD3OD) δ 8.78 (d, J = 6.0 Hz, 1H), 8.61 (s, 1H), 8.55 (t, J = 7.9 Hz, 1H), 8.09 (d, J = 8.0 Hz, 1H), ACS Paragon Plus Environment

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7.98–7.81 (m, 4H), 7.60 (d, J = 8.3 Hz, 1H), 7.08 (br s, 2H), 5.01 (d, J = 10.8 Hz, 1H), 4.25 (br s, 2H), 3.94 (d, J = 11.0 Hz, 2H), 3.58 (br s, 2H), 2.68–2.55 (m, 1H), 2.26–2.15 (m, 1H), 2.14–2.00 (m, 4H), 1.84–1.67 (m, 3H), 1.67–1.53 (m, 2H), 1.49–1.39 (m, 1H), 1.29–1.18 (m, 1H). HRMS (ESI) m/z calcd for [C31H33N5O2 + H]+ 508.2713; found 508.2709. Optical rotation (69-2HCl): [α]23D = +90.9° (c 0.47, MeOH). Chiral HPLC retention time (69-2HCl): 16.6 min; Daicel Chiralpak IA (150 mm × 4.6 mm); isocratic 40% iPrOH (0.5% Et2NH) in hexane; 1 mL/min, λ = 254 nm. 3-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)phenyl)-N-((R)-cyclopentyl(pyridin-2-yl)methyl)-1Hindazole-5-carboxamide

(70).

Prepared

according

to

general

procedure

A,

using

(R)-N-

(cyclopentyl(pyridin-2-yl)methyl)-3-iodo-1H-indazole-5-carboxamide (100 mg, 0.22 mmol) and 8-(4(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3-oxa-8-azabicyclo[3.2.1]octane (69 mg, 0.22 mmol), 70 was obtained as a yellow solid (di-TFA salt, 93 mg, 58%). 1H NMR (400 MHz, CD3OD) δ 8.76 (d, J = 6.0 Hz, 1H), 8.60 (s, 1H), 8.53–8.46 (m, 1H), 8.05 (d, J = 8.3 Hz, 1H), 7.93–7.83 (m, 4H), 7.60 (d, J = 8.8 Hz, 1H), 7.10–7.01 (m, 2H), 5.01 (d, J = 10.8 Hz, 1 H), 4.24 (br s, 2H), 3.93 (d, J = 11.3 Hz, 2H), 3.57 (d, J = 11.5 Hz, 2H), 2.66–2.55 (m, 1H), 2.23–2.13 (m, 1H), 2.10 (br s, 4H), 1.84–1.67 (m, 3H), 1.67–1.54 (m, 2H), 1.48–1.38 (m, 1H), 1.30–1.19 (m, 1H). LCMS (ESI) m/z calcd for [C31H33N5O2 + H]+ 508.3; found 508.3. 3-(4-(3-oxa-9-azabicyclo[3.3.1]nonan-9-yl)phenyl)-N-((S)-cyclopentyl(pyridin-2-yl)methyl)-1Hindazole-5-carboxamide

(71).

Prepared

according

to

general

procedure

A,

using

(S)-N-

(cyclopentyl(pyridin-2-yl)methyl)-3-iodo-1H-indazole-5-carboxamide (100 mg, 0.22 mmol) and 9-(4(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3-oxa-9-azabicyclo[3.3.1]nonane (74 mg, 0.22 mmol), 71 was obtained as a yellow solid (di-TFA salt, 49 mg, 30%). 1H NMR (400 MHz, CD3OD) δ 8.78 (d, J = 5.8 Hz, 1H), 8.61 (s, 1H), 8.56 (t, J = 7.3 Hz, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.95 (t, J = 6.8 Hz, 1H), 7.92–7.82 (m, 3H), 7.59 (d, J = 8.8 Hz, 1H), 7.04 (d, J = 8.5 Hz, 2H), 5.01 (d, J = 10.8 Hz, 1H), 4.05 (d, J = 11.0 Hz, 2H), 3.98 (d, J = 11.3 Hz, 2H), 3.92 (br s, 2H), 2.70–2.55 (m, 2H), 2.25–2.15 (m, 1H), 2.12–1.99 (m, 2H), 1.84–1.69 (m, 5H), 1.67–1.54 (m, 3H), 1.49–1.39 (m, 1H), 1.30–1.16 (m, 1H). LCMS (ESI) m/z calcd for [C32H35N5O2 + H]+ 522.3; found 522.4. ACS Paragon Plus Environment

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Page 34 of 76

N-((S)-1-(2-chlorophenyl)-2-methylpropyl)-3-(4-((endo)-3-hydroxy-8-azabicyclo[3.2.1]octan-8yl)phenyl)-1H-indazole-5-carboxamide (72). Prepared according to general procedure A, using (S)-N(1-(2-chlorophenyl)-2-methylpropyl)-3-iodo-1H-indazole-5-carboxamide (69 mg, 0.15 mmol) and (endo)-8-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol

(50

mg, 0.15 mmol), 72 was obtained as a yellow solid (15 mg, 19%). 1H NMR (400 MHz, CD3OD) δ 8.55 (dd, J = 1.6, 0.9 Hz, 1H), 7.87 (dd, J = 8.8, 1.5 Hz, 1H), 7.78 (d, J = 8.8 Hz, 2H), 7.58–7.50 (m, 2H), 7.36 (dd, J = 8.0, 1.3 Hz, 1H), 7.30–7.23 (m, 1H), 7.20 (dd, J = 8.0, 1.8 Hz, 1H), 6.89 (d, J = 8.8 Hz, 2H), 5.38 (d, J = 9.8 Hz, 1H), 4.21 (br s, 2H), 3.91 (s, 1H), 2.35 (d, J = 7.0 Hz, 2H), 2.31–2.23 (m, 1H), 2.19 (d, J = 14.8 Hz, 2H), 2.06–1.98 (m, 2H), 1.61 (d, J = 14.6 Hz, 2H), 1.14 (d, J = 6.5 Hz, 3H), 0.85 (d, J = 6.8 Hz, 3H). HRMS (ESI) m/z calcd for [C31H33ClN4O2 + H]+ 529.2370; found 529.2366. N-((S)-cyclopropyl(phenyl)methyl)-3-(4-((endo)-3-hydroxy-8-azabicyclo[3.2.1]octan-8yl)phenyl)-1H-indazole-5-carboxamide (73). Prepared according to general procedure A, using (S)-N(cyclopropyl(phenyl)methyl)-3-iodo-1H-indazole-5-carboxamide (115 mg, 0.28 mmol) and (endo)-8-(4(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol

(100 mg, 0.3

mmol), 73 was obtained as a yellow solid (40 mg, 29%). 1H NMR (400 MHz, CD3OD) δ 8.64 (d, J = 0.8 Hz, 1H), 7.95 (dd, J = 8.9, 1.4 Hz, 1H), 7.82 (d, J = 8.5 Hz, 2H), 7.56 (d, J = 8.8 Hz, 1H), 7.47 (d, J = 7.5 Hz, 2H), 7.30 (t, J = 7.5 Hz, 2H), 7.21 (m, J = 7.5 Hz, 1H), 6.90 (d, J = 8.8 Hz, 2H), 4.47 (d, J = 9.5 Hz, 1H), 4.20 (br s, 2 H), 3.92–3.87 (m, 1 H), 2.34 (m, J = 7.0 Hz, 2H), 2.23–2.13 (m, 2H), 2.06– 1.97 (m, 2H), 1.60 (d, J=14.3 Hz, 2H), 1.45–1.31 (m, 1H), 0.67–0.56 (m, 2H), 0.51–0.36 (m, 2H). HRMS (ESI) m/z calcd for [C31H32N4O2 + H]+ 493.2604; found 493.2604. N-((S)-cyclopropyl(pyridin-2-yl)methyl)-3-(4-((endo)-3-hydroxy-8-azabicyclo[3.2.1]octan-8yl)phenyl)-1H-indazole-5-carboxamide (74). Prepared according to general procedure A, using (S)-N(cyclopropyl(pyridin-2-yl)methyl)-3-iodo-1H-indazole-5-carboxamide (106 mg, 0.25 mmol) and (endo)-8-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol

(100

mg, 0.3 mmol), 74 was obtained as a yellow solid (13 mg, 10%, >99.9% ee). 1H NMR (400 MHz, CD3OD) δ 8.67 (d, J = 0.8 Hz, 1H), 8.51 (d, J = 4.0 Hz, 1H), 7.95 (dd, J = 8.8, 1.5 Hz, 1H), 7.88–7.76 ACS Paragon Plus Environment

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(m, 3H), 7.57 (d, J = 8.5 Hz, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.33–7.25 (m, 1H), 6.94 (d, J = 8.8 Hz, 2H), 4.51 (d, J = 9.5 Hz, 1H), 4.25 (br s, 2H), 3.93 (s, 1H), 2.36 (d, J = 7.3 Hz, 2H), 2.22 (d, J = 14.3 Hz, 2H), 2.09–2.00 (m, 2H), 1.63 (d, J = 14.6 Hz, 2H), 1.49–1.34 (m, 1H), 0.72–0.63 (m, 1H), 0.61–0.45 (m, 3H). HRMS (ESI) m/z calcd for [C30H31N5O2 + H]+ 494.2556; found 494.2565. [α]23D = +80.4° (c 0.746, MeOH). Chiral HPLC retention time (74-2HCl): 10.6 min; Daicel Chiralpak IA (150 mm × 4.6 mm); isocratic 40% iPrOH (0.5% Et2NH) in hexane; 1 mL/min, λ = 254 nm. N-((R)-cyclopropyl(pyridin-2-yl)methyl)-3-(4-((endo)-3-hydroxy-8-azabicyclo[3.2.1]octan-8yl)phenyl)-1H-indazole-5-carboxamide (75). A mixture of (R)-N-(cyclopropyl(pyridin-2-yl)methyl)-3iodo-1H-indazole-5-carboxamide (4.95 g, 11.8 mmol) and (endo)-8-(4-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol (4.29 g, 13.04 mmol) was treated with EtOH/PhMe (100 mL/50 mL), 1 M aq Na2CO3 (23.7 mL, 2.7 mmol, 2 equiv), followed by Pd(PPh3)4 (685 mg, 0.593 mmol, 5 mol%). The resulting mixture was refluxed (oil bath temperature at 110 oC) under Ar for 1.5 d; LCMS showed the reaction was incomplete. Additional portions of 1 M aq Na2CO3 (14.2 mL, 1.2 equiv) and Pd(PPh3)4 (685 mg, 0.59 mmol) were added and the resulting mixture was refluxed (oil bath temperature at 110 oC) for 1 d. Water (100 mL) and brine (50 mL) were added, followed by EtOAc (300 mL). Upon separation, the aqueous layer was extracted with EtOAc (2 x 60 mL) and the combined organic layers were dried over Na2SO4 and concentrated. The resulting dark brown oil was purified by flash chromatography using two Biotage 100g SiO2 columns (10–100% EtOAc in hexanes, followed by 0–10% MeOH in DCM). The impure fractions were combined and purified by flash chromatography for a second time. All clean fractions were combined and dried to give 75 (2.64 g, 45%, >99.9 %ee) as light brown foam. 1H NMR (400 MHz, CD3OD) 8.67 (s, 1H), 8.51 (d, J = 4.8 Hz, 1H), 7.95 (d, J = 8.4 Hz, 1H), 7.85 (d, J = 8.8 Hz, 2H), 7.80 (dt, J = 8.0, 1.6 Hz, 1H), 7.57 (d, J = 8.8 Hz, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.30 (dd, J = 5.2, 2.0 Hz, 1H), 6.94 (d, J = 8.8 Hz, 2H), 4.50 (d, J = 9.2 Hz, 1H), 4.25 (br s, 2H), 3.93 (br t, J = 4.8 Hz, 1H), 2.39–2.36 (m, 2H), 2.26–2.18 (m, 2H), 2.07–2.02 (m, 2H), 1.63 (d, J = 14.8 Hz, 2H), 1.46–1.36 (m, 1H), 0.71–0.66 (m, 1H), 0.61–0.50 (m, 3H). HRMS (ESI) m/z calcd for [C30H31N5O2 + H]+ 494.2556; found 494.2552. Optical rotation (75ACS Paragon Plus Environment

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2HCl): [α]23D = −81.2° (c 1.13, MeOH). Chiral HPLC retention time (75-2HCl): 7.9 min; Daicel Chiralpak IA (150 mm × 4.6 mm); isocratic 40% iPrOH (0.5% Et2NH) in hexane; 1 mL/min, λ = 254 nm. N-((S)-cyclopentyl(pyridin-2-yl)methyl)-3-(4-((endo)-3-hydroxy-8-azabicyclo[3.2.1]octan-8yl)phenyl)-1H-indazole-5-carboxamide (76). Prepared according to general procedure A, using (S)-N(cyclopentyl(pyridin-2-yl)methyl)-3-iodo-1H-indazole-5-carboxamide (86 mg, 0.19 mmol) and (endo)8-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol (70 mg, 0.21 mmol), 76 was obtained as a white solid (33 mg, 33%). 1H NMR (400 MHz, CD3OD) δ 8.61 (dd, J = 1.5, 0.8 Hz, 1H), 8.54–8.48 (m, 1H), 7.91 (dd, J = 8.8, 1.5 Hz, 1H), 7.84–7.73 (m, 3H), 7.56 (dd, J = 8.9, 0.6 Hz, 1H), 7.50–7.45 (m, 1H), 7.28 (ddd, J = 7.5, 5.0, 1.3 Hz, 1H), 6.90 (d, J = 8.8 Hz, 2H), 5.01 (d, J = 10.3 Hz, 1H), 4.22 (br s, 2H), 3.91 (t, J = 4.5 Hz, 1H), 2.58–2.46 (m, 1H), 2.40–2.29 (m, 2H), 2.19 (d, J = 11.0 Hz, 2H), 2.07–1.88 (m, 3H), 1.74–1.43 (m, 7H), 1.40–1.19 (m, 2H). HRMS (ESI) m/z calcd for [C32H35N5O2 + H]+ 522.2869; found 522.2844 N-((R)-cyclopentyl(pyridin-2-yl)methyl)-3-(4-((endo)-3-hydroxy-8-azabicyclo[3.2.1]octan-8yl)phenyl)-1H-indazole-5-carboxamide (77). Prepared according to general procedure A, using (R)-N(cyclopentyl(pyridin-2-yl)methyl)-3-iodo-1H-indazole-5-carboxamide (86 mg, 0.19 mmol) and (endo)8-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol (70 mg, 0.21 mmol), 77 was obtained as a yellow solid (34 mg, 24%). 1H NMR (400 MHz, CD3OD) δ 8.61 (dd, J = 1.5, 0.8 Hz, 1H), 8.54–8.48 (m, 1H), 7.91 (dd, J = 8.8, 1.5 Hz, 1H), 7.84–7.73 (m, 3H), 7.56 (dd, J = 8.9, 0.6 Hz, 1H), 7.50–7.45 (m, 1H), 7.28 (ddd, J = 7.5, 5.0, 1.3 Hz, 1H), 6.90 (d, J = 8.8 Hz, 2H), 5.01 (d, J = 10.3 Hz, 1H), 4.22 (br s, 2H), 3.91 (t, J = 4.5 Hz, 1H), 2.58-2.46 (m, 1H), 2.40–2.29 (m, 2H), 2.19 (d, J = 11.0 Hz, 2H), 2.07–1.88 (m, 3H), 1.74–1.43 (m, 7H), 1.40–1.19 (m, 2H). LCMS (ESI) m/z calcd for [C32H35N5O2 + H]+ 522.2869; found 522.2844. N-((S)-cyclopentyl(pyrimidin-2-yl)methyl)-3-(4-((endo)-3-hydroxy-8-azabicyclo[3.2.1]octan-8yl)phenyl)-1H-indazole-5-carboxamide (78). Prepared according to general procedure A, using (S)-N(cyclopentyl(pyrimidin-2-yl)methyl)-3-iodo-1H-indazole-5-carboxamide (70 mg, 0.16 mmol) and ACS Paragon Plus Environment

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(endo)-8-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol (52 mg, 0.16 mmol), 78 was obtained as a yellow solid (19 mg, 23%). 1H NMR (400 MHz, CD3OD) δ 8.76 (d, J = 5.0 Hz, 2H), 8.63 (d, J = 0.5 Hz, 1H), 7.92 (dd, J = 8.9, 1.6 Hz, 1H), 7.83 (d, J = 8.8 Hz, 2H), 7.57 (d, J = 8.8 Hz, 1H), 7.35 (t, J = 4.9 Hz, 1H), 6.93 (d, J = 8.8 Hz, 2H), 5.18 (d, J = 9.5 Hz, 1H), 4.24 (br s, 2H), 3.92 (t, J = 4.4 Hz, 1H), 2.57 (m, J = 9.0 Hz, 1H), 2.40–2.31 (m, 2H), 2.21 (d, J = 15.1 Hz, 2H), 2.08–2.00 (m, 2H), 1.92 (d, J = 7.3 Hz, 1H), 1.75–1.47 (m, 7H), 1.45–1.36 (m, 2H). HRMS (ESI) m/z calcd for [C31H34N6O2 + H]+ 523.2821; found 523.2801. N-((R)-cyclopentyl(pyrimidin-2-yl)methyl)-3-(4-((endo)-3-hydroxy-8-azabicyclo[3.2.1]octan-8yl)phenyl)-1H-indazole-5-carboxamide (79). Prepared according to general procedure A, using (R)-N(cyclopentyl(pyrimidin-2-yl)methyl)-3-iodo-1H-indazole-5-carboxamide (70 mg, 0.16 mmol) and (endo)-8-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol (52 mg, 0.16 mmol), 79 was obtained as a yellow solid (30 mg, 37%). 1H NMR (400 MHz, CD3OD) δ 8.80–8.72 (m, 2H), 8.65–8.60 (m, 1H), 7.92 (dd, J = 8.8, 1.5 Hz, 1H), 7.84 (d, J = 8.8 Hz, 2H), 7.57 (d, J = 8.8 Hz, 1H), 7.36 (t, J = 4.9 Hz, 1H), 6.94 (d, J = 8.8 Hz, 2H), 5.18 (d, J = 9.8 Hz, 1H), 4.25 (br s, 2H), 3.93 (s, 1H), 2.66–2.48 (m, 1H), 2.36 (d, J = 7.3 Hz, 2H), 2.21 (d, J = 14.6 Hz, 2H), 2.08–2.00 (m, 2H), 1.98– 1.88 (m, 1H), 1.75–1.47 (m, 7H), 1.46–1.33 (m, 2H). LCMS (ESI) m/z calcd for [C31H34N6O2 + H]+ 523.3; found 523.4. 3-(4-((endo)-3-hydroxy-8-azabicyclo[3.2.1]octan-8-yl)phenyl)-N-((S)-3-methyl-1-(pyridin-2yl)butyl)-1H-indazole-5-carboxamide (80). Prepared according to general procedure A, using (S)-3iodo-N-(3-methyl-1-(pyridin-2-yl)butyl)-1H-indazole-5-carboxamide (133 mg, 0.3 mmol) and (endo)-8(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol (120 mg, 0.36 mmol), 80 was obtained as a yellow solid (di-HCl salt, 44 mg, 25%). 1H NMR (free base, 400 MHz, CD3OD) δ 8.65 (d, J = 0.8 Hz, 1H), 8.52–8.49 (m, 1H), 7.95 (dd, J = 8.8, 1.6 Hz, 1H), 7.81 (d, J = 8.8 Hz, 2H), 7.77 (dt, J = 7.6, 1.7 Hz, 1H), 7.57 (d, J = 8.8 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.30–7.26 (m, 1H), 6.92 (d, J = 8.8 Hz, 2H), 5.34 (dd, J = 10.0, 5.2 Hz, 1H), 4.25–4.18 (m, 2H), 3.92 (t, J = 4.6 Hz, 1H), 2.38–2.31 (m, 2H), 2.23–2.16 (m, 2H), 2.06–1.98 (m, 2H), 1.96–1.85 (m, 1H), 1.80–1.66 (m, 2H), ACS Paragon Plus Environment

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1.62 (d, J = 14.4 Hz, 2H), 1.03–0.97 (m, 6H). LCMS (ESI) m/z calcd for [C31H35N5O2 + H]+ 510.3; found 510.4. 3-(4-((endo)-3-hydroxy-8-azabicyclo[3.2.1]octan-8-yl)phenyl)-N-((R)-3-methyl-1-(pyridin-2yl)butyl)-1H-indazole-5-carboxamide (81). Prepared according to general procedure A, using (R)-3iodo-N-(3-methyl-1-(pyridin-2-yl)butyl)-1H-indazole-5-carboxamide (130 mg, 0.3 mmol) and (endo)-8(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol (109 mg, 0.33 mmol), 81 was obtained as a yellow solid (di-HCl salt, 52 mg, 30%). 1H NMR (free base, 400 MHz, CD3OD) δ 8.66 (d, J = 0.8 Hz, 1H), 8.52–8.49 (m, 1H), 7.95 (dd, J = 8.8, 1.6 Hz, 1H), 7.82 (d, J = 8.8 Hz, 2H), 7.77 (dt, J = 7.6, 1.7 Hz, 1H), 7.57 (d, J = 8.8 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.29–7.25 (m, 1H), 6.91 (d, J = 8.8 Hz, 2H), 5.35 (dd, J = 10.0, 5.2 Hz, 1H), 4.25–4.18 (m, 2H), 3.91 (t, J = 4.6 Hz, 1H), 2.38–2.31 (m, 2H), 2.23–2.16 (m, 2H), 2.06–1.98 (m, 2H), 1.96–1.85 (m, 1H), 1.80–1.66 (m, 2H), 1.61 (d, J = 14.4 Hz, 2H), 1.02–0.97 (m, 6H). LCMS (ESI) m/z calcd for [C31H35N5O2 + H]+ 510.3; found 510.3. N-((R)-2-cyclopropyl-1-(pyridin-2-yl)ethyl)-3-(4-((endo)-3-hydroxy-8-azabicyclo[3.2.1]octan-8yl)phenyl)-1H-indazole-5-carboxamide (82). Prepared according to general procedure A, using (R)-N(2-cyclopropyl-1-(pyridin-2-yl)ethyl)-3-iodo-1H-indazole-5-carboxamide (166 mg, 0.4 mmol) and (endo)-8-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol

(132

mg, 0.4 mmol), 82 was obtained as a yellow solid (di-HCl salt, 67 mg, 29%). 1H NMR (free base, 400 MHz, CD3OD) δ 8.68–8.61 (m, 1H), 8.53 (d, J = 5.0 Hz, 1H), 7.94 (d, J = 8.5 Hz, 1H), 7.86 (d, J = 8.5 Hz, 2H), 7.82 (td, J = 7.7, 1.88 Hz, 1H), 7.59 (d, J = 9.0 Hz, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.34–7.28 (m, 1H), 6.99 (d, J = 8.8 Hz, 2H), 5.34 (dd, J = 8.7, 6.40 Hz, 1H), 4.32–4.26 (m, 2H), 3.99–3.93 (m, 1H), 2.42–2.35 (m, 2H), 2.30–2.22 (m, 2H), 2.10–2.03 (m, 2H), 1.99–1.90 (m, 1H), 1.88–1.78 (m, 1H), 1.70–1.63 (m, 2H), 0.85–0.76 (m, 1H), 0.56–0.39 (m, 2H), 0.25–0.17 (m, 1H), 0.11–0.03 (m, 1H); HRMS (ESI) m/z calcd for [C31H33N5O2 + H]+ 508.2713; found 508.2712. 3-(4-((endo)-3-hydroxy-9-azabicyclo[3.3.1]nonan-9-yl)phenyl)-N-((S)-3-methyl-1-(pyridin-2-yl)butyl)1H-indazole-5-carboxamide (83). Prepared according to general procedure A, using (S)-3-iodo-N-(3ACS Paragon Plus Environment

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methyl-1-(pyridin-2-yl)butyl)-1H-indazole-5-carboxamide (130 mg, 0.3 mmol) and (endo)-9-(4(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-9-azabicyclo[3.3.1]nonan-3-ol

(113

mg,

0.33

mmol), 83 was obtained as a yellow solid (di-HCl salt, 36 mg, 20%). 1H NMR (free base, 400 MHz, CD3OD) δ 8.66 (d, J = 0.4 Hz, 1H), 8.52–8.48 (m, 1H), 7.95 (dd, J = 9.0, 1.4 Hz, 1H), 7.80 (d, J = 8.4 Hz, 2H), 7.76 (dd, J = 7.8, 1.8 Hz, 1H), 7.58 (d, J = 8.8 Hz, 1H), 7.48 (d, J = 7.6 Hz, 1H), 7.29–7.25 (m, 1H), 7.98 (d, J = 8.8 Hz, 2H), 5.38–5.32 (m, 1H), 4.35–4.27 (m, 2H), 3.75–3.66 (m, 1H), 2.47–2.37 (m, 2H), 2.30–2.15 (m, 1H), 1.95–1.85 (m, 1H), 1.85–1.77 (m, 4H), 1.68–1.40 (m, 5H), 1.05–0.97 (m, 6H). LCMS (ESI) m/z calcd for [C32H37N5O2 + H]+ 524.3; found 524.3. 3-(4-((endo)-7-hydroxy-3-oxa-9-azabicyclo[3.3.1]nonan-9-yl)phenyl)-N-((S)-3-methyl-1(pyridin-2-yl)butyl)-1H-indazole-5-carboxamide (84). Prepared according to general procedure A, using (S)-3-iodo-N-(3-methyl-1-(pyridin-2-yl)butyl)-1H-indazole-5-carboxamide (130 mg, 0.3 mmol) and (endo)-9-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3-oxa-9-azabicyclo[3.3.1]nonan-7-ol (99 mg, 0.3 mmol), 84 was obtained as a yellow solid (di-HCl salt, 59 mg, 33%). 1H NMR (400 MHz, CD3OD) δ 8.88 (s, 1H), 8.32 (d, J = 4.8 Hz, 1H), 8.70–8.62 (m, 1H), 8.28 (d, J = 6.4 Hz, 1H), 8.20 (d, J = 8.8 Hz, 1H), 8.08–7.98 (m, 3H), 7.76 (d, J = 8.8 Hz, 1H), 7.22 (d, J = 6.4 Hz, 2H), 5.53–5.45 (m, 1H), 4.19 (br s, 2H), 4.07–3.95 (m, 4H), 3.92–3.87 (m, 1H), 2.40–2.31 (m, 3H), 1.91–1.78 (m, 4H), 1.08 (d, J = 5.2 Hz, 3H), 1.06 (d, J = 5.2 Hz, 3H). LCMS (ESI) m/z calcd for [C31H35N5O3 + H]+ 526.3; found 526.4. N-((R)-cyclopentyl(pyridin-2-yl)methyl)-3-(4-((endo)-7-hydroxy-3-oxa-9azabicyclo[3.3.1]nonan-9-yl)phenyl)-1H-indazole-5-carboxamide (85). Prepared according to general procedure A, using (R)-N-(cyclopentyl(pyridin-2-yl)methyl)-3-iodo-1H-indazole-5-carboxamide (100 mg,

0.22

mmol)

and

(endo)-9-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3-oxa-9-

azabicyclo[3.3.1]nonan-7-ol (76 mg, 0.22 mmol), 85 was obtained as a yellow solid (di-TFA salt, 68 mg, 40%). 1H NMR (400 MHz, CD3OD) δ 8.76 (d, J = 5.0 Hz, 1H), 8.60 (s, 1H), 8.48 (td, J = 7.9, 1.5 Hz, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.93–7.84 (m, 4H), 7.59 (d, J = 8.8 Hz, 1H), 7.05 (d, J = 8.5 Hz, 2H), 5.01 (d, J = 10.8 Hz, 1H), 4.09–4.03 (m, 2H), 4.02–3.91 (m, 4H), 3.88 (br s, 1H), 2.66–2.54 (m, 1H), ACS Paragon Plus Environment

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2.37–2.27 (m, 2H), 2.22–2.13 (m, 1H), 1.82–1.67 (m, 5H), 1.66–1.53 (m, 2H), 1.48–1.38 (m, 1H), 1.29– 1.18 (m, 1H). LCMS (ESI) m/z calcd for [C32H35N5O3 + H]+ 538.3; found 538.5. N-((S)-cyclopropyl(pyridin-2-yl)methyl)-3-(4-((exo)-3-hydroxy-8-azabicyclo[3.2.1]octan-8yl)phenyl)-1H-indazole-5-carboxamide (86). Prepared according to general procedure A, using (S)-N(cyclopropyl(pyridin-2-yl)methyl)-3-iodo-1H-indazole-5-carboxamide (125 mg, 0.3 mmol) and (exo)-8(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol

(99

mg,

0.3

mmol), 86 was obtained as a yellow solid (di-HCl salt, 65 mg, 38%, >99.9% ee). 1H NMR (free base, 400 MHz, CD3OD) δ 8.67 (s, 1H), 8.51 (d, J = 4.8 Hz, 1H), 7.95 (dd, J = 8.8, 1.2 Hz, 1H), 7.86 (d, J = 8.8 Hz, 2H), 7.79 (dt, J = 8.0, 1.7 Hz, 1H), 7.57 (d, J = 7.2 Hz, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.29 (dd, J = 7.4, 5.0 Hz, 1H), 6.96 (d, J = 8.8 Hz, 2H), 4.51 (d, J = 8.8 Hz, 1H), 4.33 (s, 2H), 4.20–4.08 (m, 1H), 2.14–1.97 (m, 2H), 1.90–1.68 (m, 6H), 1.45–1.35 (m, 1H), 0.72–0.64 (m, 1H), 0.60–0.48 (m, 3H). HRMS (ESI) m/z calcd for [C30H31N5O2 + H]+ 494.2556; found 494.2563. Optical rotation (86-2HCl): [α]23D = +82.0° (c 0.305, MeOH). Chiral HPLC retention time (86-2HCl): 13.7 min; Daicel Chiralpak IA (150 mm × 4.6 mm); isocratic 40% iPrOH (0.5% Et2NH) in hexane; 1 mL/min, λ = 254 nm. N-((R)-cyclopropyl(pyridin-2-yl)methyl)-3-(4-((exo)-3-hydroxy-8-azabicyclo[3.2.1]octan-8yl)phenyl)-1H-indazole-5-carboxamide (87). Prepared according to general procedure A, using (R)-N(cyclopropyl(pyridin-2-yl)methyl)-3-iodo-1H-indazole-5-carboxamide (92 mg, 0.22 mmol) and (exo)-8(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol

(80 mg, 0.24

mmol), 87 was obtained as a yellow solid (58 mg, 53%, >99.9% ee). 1H NMR (400 MHz, CD3OD) δ 8.68 (d, J = 0.8 Hz, 1H), 8.53–8.45 (m, 1H), 7.95 (dd, J = 8.9, 1.4 Hz, 1H), 7.84 (d, J = 8.5 Hz, 2H), 7.76 (td, J = 7.8, 1.8 Hz, 1H), 7.56 (d, J = 8.8 Hz, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.26 (ddd, J = 7.5, 5.0, 1.0 Hz, 1H), 6.92 (d, J = 8.8 Hz, 2H), 4.51 (d, J = 9.3 Hz, 1H), 4.29 (br s, 2H), 4.12 (tt, J = 11.0, 5.2 Hz, 1H), 2.07–1.97 (m, 2H), 1.84–1.65 (m, 6H), 1.48–1.28 (m, 1H), 0.70–0.62 (m, 1H), 0.59–0.46 (m, 3H). LCMS (ESI) m/z calcd for [C30H31N5O2 + H]+ 494.3; found 494.5. [α]23D = −85.8° (c 0.746, MeOH). Chiral HPLC retention time (87-2HCl): 9.15 min; Daicel Chiralpak IA (150 mm × 4.6 mm); isocratic 40% iPrOH (0.5% Et2NH) in hexane; 1 mL/min, λ = 254 nm. ACS Paragon Plus Environment

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N-((S)-cyclopentyl(pyridin-2-yl)methyl)-3-(4-((exo)-3-hydroxy-8-azabicyclo[3.2.1]octan-8-yl)phenyl)1H-indazole-5-carboxamide (88). Prepared according to general procedure A, using (S)-N(cyclopentyl(pyridin-2-yl)methyl)-3-iodo-1H-indazole-5-carboxamide (99 mg, 0.22 mmol) and (exo)-8(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-8-azabicyclo[3.2.1]octan-3-ol

(80 mg, 0.24

mmol), 88 was obtained as a yellow solid (66 mg, 57%). 1H NMR (400 MHz, CD3OD) δ 8.59 (s, 1 H), 8.53 (d, J = 4.0 Hz, 1H), 7.91 (d, J = 8.8 Hz, 1H), 7.86 (d, J = 8.8 Hz, 2H), 7.80 (m, J = 1.8 Hz, 1H), 7.58 (d, J = 8.8 Hz, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.35–7.27 (m, 1H), 7.01 (d, J = 8.8 Hz, 2H), 5.02 (d, J = 10.3 Hz, 1H), 4.38 (br s, 1H), 4.23–4.10 (m, 1H), 2.64–2.47 (m, 1H), 2.09 (d, J = 5.5 Hz, 2H), 2.04– 1.93 (m, 1H), 1.89–1.48 (m, 12H), 1.43–1.24 (m, 2H). LCMS (ESI) m/z calcd for [C32H35N5O2 + H]+ 522.3; found 522.5.

Purification of TTK-N protein substrate. An N-terminal fragment of human TTK (TTK-N; amino acids 1-275) was sub-cloned into the pET-SUMO vector (Life Technologies, Burlington, ON) for bacterial expression. The TTK- N protein was expressed in E. coli BL21 (DE3) CodonPlus-RIPL (Life Technologies) cells in TB Broth media overnight at 15 °C with 1.0 mM IPTG. The bacterial pellet was lysed in lysis buffer (30 mM Tris pH 7.5, 500 mM NaCl, 10% (v/v) Glycerol, 1 mM TCEP), supplemented with 1 mM PMSF, Complete Protease Inhibitor Tablets EDTA-free (Roche Diagnostics, Mississagua, ON) (1 tablet per 50 ml), and 0.01 µl/mL Benzonase nuclease (Sigma, Oakville, ON). The cell suspensions were stirred for 30 min on ice and kept at 4 °C for DNA cleavage and after that lysed by 3× passage using an EmulsiFlex C5 homogenizer (Avestin, Ottawa, ON). The lysed cells were centrifuged 40,000 ×g, 45 min, 4 °C. Cell lysate supernatants were treated with 1 g/50 mL Whatman DE52 resin (GE Life Sciences, Baie d’Urfe, QC), which had been pre-swollen and preequilibrated with lysis buffer. Lysate was allowed to contact the resin for 20 min on ice to bind and remove DNA. Lysate was vacuum-filtered to remove DE52 resin beads and loaded onto a 5 mL HisTrap column (GE Life Sciences) for FPLC purification of His-tagged proteins. The column was preequilibrated with lysis buffer after being charged with Ni2+ using 0.1 M NiSO4 as per manufacturer’s ACS Paragon Plus Environment

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instructions. The column was washed with 10 column volumes of lysis buffer, followed by 10 column volumes of lysis buffer supplemented with 10 mM imidazole. His6-SUMO-TTK-N protein was eluted with lysis buffer supplemented with 500 mM imidazole. Fractions were collected and analyzed by NuPAGE (Life Technologies). Appropriate fractions were pooled for size-exclusion chromatography on a Superdex 200 26/60 column (GE Life Sciences) equilibrated with 30 mM Tris pH 7.5, 200 mM NaCl, 0.5 mM TCEP. TTK inhibition assay: TTK activity was determined using the N-terminal portion (residues 1-275) as a polypeptide substrate.55 His6-SUMO-TTK-N (see above) was as effective as a substrate as the cleaved version and thus was used without cleavage of the His6-SUMO fusion tag. Active TTK was purchased as an amino terminal GST fusion of full length human TTK (Life Technologies, Burlington, ON). TTK activity was measured using an indirect ELISA detection system using 96-well microtitre plates, coated with 5 µg/well His6-SUMO-TTK-N, as previously described.25 Compound inhibition was determined at either a fixed concentration (10 µM) or at a variable inhibitor concentration (typically 50 µM to 0.1 µM in a 10 point dose response titration). Compounds were pre-incubated in the presence of enzyme for 15 minutes prior to addition of ATP and the activity remaining quantified using the above described activity assay. The % inhibition of a compound was determined using the following formula; % inhibition = 100 x (1 – (experimental value – background value)/(high activity control – background value)). The IC50 value was determined using a non-linear 4 point logistic curve fit (XLfit4, IDBS) with the formula; (A+(B/(1+((x/C)^D)))), where A = background value, B = range, C = inflection point, D = curve fit parameter. The apparent Ki for compound 75 was estimated to be 0.7 + 0.5 nM by varying the ATP concentration in a series of TTK inhibition experiments at 2.2, 6.6 and 11 nM of inhibitor and applying nonlinear regression analysis (see Supporting Material). Biochemical Assays. Active PLK4 was purified and used to measure PLK4 activity, using an indirect ELISA detection system, as previously described.56 KDR, AURKA and AUKB/INCENP compound


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inhibition were determined using FRET based homogenous assay kits from Invitrogen (Life Technologies, Burlington, Ontario). Recombinant Aurora A and KDR were purchased from Invitrogen; AURKB/INCENP was purchased from Millipore (Billerica, MA).

The assays were performed

according to the manufacturer’s specifications with ATP concentrations of 156, 20 and 128 µM, respectively for KDR, AURKA and AURKB/INCENP.25,56 Compound inhibition was determined at either a fixed concentration (10 µM) or at a variable inhibitor concentrations (as described above) to determine an IC50. The KDR, AURKA and AUKB/INCENP IC50 values given in the Tables 1-5 are the result of single determinations wherein standard fitting errors are given. The selectivity of inhibitor 75 against 278 protein kinases was assessed by Millipore (Dundee, Scotland) using a radiometric assay. Inhibition was determined at 0.1 µM inhibitor concentration with ATP at Km; % inhibition was determined (see Supporting Material). For compound 75 the inhibition constants (IC50s) were determined by Millipore, as per the suppliers specifications, against selected kinases: cKit (V560G), cKit, Ret (V804L), Ret (V804M),Ret, JNK3, MAPK1, MAPK2, MELK and MUSK. Fluorogenic Cytochrome P450 inhibition studies were conducted at 37 °C in 96-well plates, as previously described,25,56 for CYP 1A2, CYP 2D6, CYP 2C9, CYP 2C19 and CYP 3A4 (BFC and DBF substrates). Purification & Crystallization of TTK kinase domain. The kinase domain fragment of human TTK (TTK KD) (amino acids 513-795) was sub-cloned into the pET-SUMO vector (Life Technologies Inc., Burlington, ON) for bacterial expression. The protein was expressed in E.coli BL21 AI (Life Technologies) cells in Terrific Broth (TB) media overnight at 15 °C with 0.2% (w/v) L-arabinose and 1.0 mM IPTG. His6-SUMO-TTK-KD was purified as for His6-SUMO-TTK-N (see above) except the lysis buffer was 50 mM HEPES pH 7.5, 500 mM NaCl, 5% (v/v) glycerol, and 1 mM TCEP. Clarified lysates were loaded onto a 5 mL HisTrap equilibrated with lysis buffer containing 50 mM imidazole and the His6-SUMO-TTK-KD was eluted with 300 mM imidazole. Size exclusion chromatography was


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performed on a Superdex 200 26/60 column equilibrated with 50 mM HEPES pH 7.5, 250 mM NaCl, 1 mM TCEP. Elution was confirmed by SDS PAGE. SUMO protease (LifeSensors Inc., Malvern, PA) was added to fractions containing His6-SUMO-TTK KD to remove the His6-SUMO tag and the cleavage reaction was allowed to proceed overnight at 4 °C. The cleaved TTK-KD was separated from uncleaved protein, His6-SUMO protein, and His6-SUMO protease by loading onto a 5 mL HisTrap column. Uncleaved protein, His6-SUMO protein and His6SUMO protease were retained on the column whereas cleaved TTK KD was collected in the flowthrough. The TTK KD was further purified to homogeneity by ion-exchange chromatography (IEX). Cleaved TTK KD fractions were diluted 20x volume with IEX buffer (50 mM Tris pH 8.0, 1 mM TCEP) and loaded onto FPLC IEX Resource Q (GE Life Sciences), 6 ml column. Elution with a gradient 12.5-500 mM NaCl was monitored by NuPAGE and TTK KD peak, which eluted at approximately 250 mM NaCl, was pooled and concentrated. Purified TTK KD was crystallized in the presence of compound 51 using the hanging drop vapour diffusion method at 22 °C by mixing equal volumes of the protein solution and the reservoir solution. The TTK KD-compound 51 binary complex (protein:compound at a molar ratio of 1:5) was crystallized in 17% (v/v) PEG 10000, 0.2 M ammonium sulfate, and 0.1 M Bis-Tris pH 5.5. The crystals were soaked in the mother liquor supplemented with 15% (v/v) glycerol as cryoprotectant before freezing in liquid nitrogen. X-ray Structure Determination. The X-ray data set of the TTK-compound 51 complex was collected at a temperature of 100 oK using beam line 17-ID at the Advanced Photon Source, Argonne National Laboratory. The diffraction data was reduced and scaled with XDS software;57 the crystal of this TTK complex belongs to the C2 space group. The initial phases of the TTK-inhibitor complex was determined by molecular replacement using PDB:3CEK set of coordinates as a search model and Phaser from the Phenix program suite.58 Following the initial rigid body refinement, interactive cycles of model building and refinement were carried out using COOT,59 phenix.refine58 and buster-TNT.60 The coordinates and topology of the ligand from this study were generated using phenix.elbow58. The ligand ACS Paragon Plus Environment

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(compound 51) was introduced in the later stages of refinement after most of the protein model had been built. Water molecules as well as other solvent ligands were added based on the 2mFo-DFc map in COOT and refined with phenix.refine or buster-TNT. The final structure was refined to 2.38 Å resolution with an Rcryst of 20.97% and an Rfree of 24.99% and deposited in the PDB as 4O6L; additional crystallographic data and refinement statistics are presented in the Supporting Information. Cell Lines. All cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA), and maintained according to the supplier’s instructions. Short tandem repeat (STR) profiling was used to verify authenticity of the cell lines. Sixteen STR loci were simultaneously amplified in a multiplex PCR reaction (The Hospital for Sick Children, Toronto) and the ATCC database was used for comparison. Cell lines were routinely tested for mycoplasma and used at low passage numbers (60/>60

ND

68

0.002 ±0.0001

1.2 ±0.1

0.13

0.021

0.15

>60/>60

1.4

69

0.0012 ±5.8E-5

1.4 ±0.2

0.036 0.003

1.1

37/43

0.93

49

0.65


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70

0.0046 ±0.0006

1.4 ±0.1

0.11

0.017

0.16

>60/21

0.36

71

0.0083 ±0.0004

2.8 ±0.4

0.18

0.025

0.20

50/ND

ND

72

0.006 ±0.005

>6.3

0.021 0.009

0.007

34/>60

ND

73

0.0028 ±0.0001

1.1 ±0.1

0.070 0.022

0.12

47/35

ND

74

0.0012 ±0.0004

0.53 ±0.08

0.016 0.018

0.02

>60/>60

2.1

75

0.0031 ±0.0016

1.3 ±0.2

0.036 0.017

0.08

>60/>60

1.3

76

0.0043 ±0.003

>6.3

0.02

0.008

0.050

>60/>60

0.82

77

0.0041 ±0.0006

1.8 ±0.3

0.03

0.015

0.09

>60/22

0.23

78

0.0011 ±0.0005

0.62 ±0.09

0.014 0.001

0.02

>60/>60

0.94

79

0.0039 ±0.0005

0.85 ±0.26

0.15

0.02

0.16

>60/ND

ND

80

0.0017 ±0.0002

1.5 ±0.2

0.017 0.002

0.14

>60/48

0.32


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81

0.0027 ±0.0006

1.6 ±0.4

0.026 0.005

0.082

>60/25

0.29

82

0.0015 ±0.0001

1.2 ±0.1

0.027 0.016

ND

>60/>60

1.3

83

0.014 ±0.002

0.98 ±0.31

0.45

0.15

0.73

ND/ND

ND

84

0.004 ±0.001

0.23 ±0.06

0.05

0.005

0.14

37/ND

ND

85

0.0022 ±0.0002

0.74 ±0.19

0.14

0.015

0.16

60/33

0.33

86

0.0016 ±0.0007

0.79 ±0.06

0.090 0.018

0.17

>60/>60

1.7

87

0.0027 ±0.0002

1.7 ±0.2

0.21

.048

0.31

>60/>60

1.4

88

0.0018 ±0.0001

6.6 ±1.6

0.074 0.038

0.13

35/21

0.8

*Compounds

49 and 50 are achiral, the rest of the entries are chiral; **Compounds 69, 71, 72, 74, 75, 76, 78, 83, 84 and 86 are 2 or more replicates with standard deviation, the rest are single determinations with standard errors.


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Table 6. Rodent Pharmacokinetic Data for selected TTK Inhibitors

PK

Entry

68

69

72

73

74

75

76

78

82

86

Cmax (µg/mL) AUCINF

1.5

2.0

0.33

0.48

1.6

2.0

1.5

0.75

1.2

0.8

2.1

2.8

0.62

0.67

1.6

3.0

2.6

0.97

1.9

1.6

71

79

ND

ND

ND

66

91

125

57

46

0.4

0.72

ND

ND

ND

0.78

0.78

0.73

0.38

0.90

0.24

0.22

ND

ND

ND

0.25

0.19

0.13

0.29

0.36

0.89

0.43

ND

ND

ND

0.55

0.13

0.14

0.27

0.43

75

39

ND

ND

ND

44

13

21

19

24

Parameters Mouse PO (10 mg/kg)

.

(h µg/mL) Rat IV (1 mg/kg)

Rat PO (5 mg/kg)

Cl (mL min/kg) t1/2 (h) AUCINF /

(h.µg/mL) AUCINF

(h.µg/mL) Oral BA (%)

Table 7. Mouse Xenograft Results for TTK Inhibitors 69, 75 and 86 Percent Tumor Growth Inhibition Cell Line

HCT116

Entry

MDA-MB-468

SW48

69

17 @ 25 mg/kg

56 @ 35 mg/kg*

ND

ND

75

55 @ 23 mg/kg

77 @ 35 mg/kg**

98 @ 30 mg/kg

66 @ 30 mg/kg

86

5 @ 100 mg/kg

53 @ 150 mg/kg**

ND

ND

2/8* and 1/8** animals in this arm were lost during the study Table 8. Biochemical (IC50) and Cellular (EC50) Inhibition Constants for Compound 75 KINASE

IC50 (nM)

KINASE

IC50 (nM)

KINASE

IC50 (nM)

EC50 (nM)

cKit (V560G) cKit Ret (V804L) Ret (V804M) Ret

490 >30,000 250 280 2,800

JNK3 MAPK1 MAPK2 MELK MuSK

68 320 57 160 690

TTK PLK4 AURKA

3.1 130 1,300

14 ND >6,000

AURKB/ INCENP

1,300

>6,000


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Table 9: Growth Inhibition Data for TTK inhibitor 75 against HMECs and a Panel of Cancer Cell Lines Breast GI50 (µM)

HMEC GI50 (µM) >30

MDAMB-231

BT-474

SKBr-3

MCF-7

T-47D

A2780

0.015

0.008

0.015

0.020

0.020

0.070 Colon GI50 (µM)

COLO -205 0.060

Ovarian GI50 (µM) OV -90

-3

21G

0.030

0.017

0.016

Prostate GI50 (µM)

Melanoma GI50 (µM)

HT29

SW48

SW620

PC-3

DU145

SK-MEL-5

0.018

0.019

0.030

0.060

0.017

0.020

SKOV TOV-

SKMEL-28 0.21

Lung GI50 (µM) A549

H23

0.024

0.036

SUPPORTING INFORMATION Supporting Information Available: Synthesis of compound intermediates; Double reciprocal plot of TTK inhibition by compound 75; Inhibition of 278 Kinase Panel by 75; Summary of crystallographic data and refinement statistics for TTK-compound 51 co-complex (PDB accession code: 4O6L); Omit map for TTK-Compound 51 co-complex; Correlation of pIC50(TTK) vs GlideXP docking scores (n=86); Cell seeding data for cancer cell lines; Early ADME data for compound 75. Mouse weights upon oral QD dosing of compounds 69, 75 and 86. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION *Corresponding Authors: E-mail: [email protected], Phone: 416-581-7620; E-mail: [email protected], Phone: 416-581-8550 (8450) ACKNOWLEDGMENTS We thank Dr. Richard Brokx for editorial comment during manuscript preparation and Dr. Homer Pearce for helpful advice and discussion during the course of the work. The Canadian Institutes of ACS Paragon Plus Environment

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Health Research and the Princess Margaret Cancer Foundation are acknowledged for financial support. The use of the IMCA-CAT beam line 17-ID at the Advanced Photon Source was supported by the companies of the Industrial Macromolecular Crystallography Association through a contract with the Center for Advanced Radiation Sources at the University of Chicago. Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38. ABBREVIATIONS USED A%, area percent; ADME; absorption, distribution, metabolism and excretion; anh, anhydrous; aq, aqueous; Ar, argon; ATCC, American Type Culture Collection; ATP, Adenosine-5'-triphosphate; AURKA, Aurora

Kinase A; AURKB/INCENP Aurora kinase B/Inner centromere protein; b, broad; BA, bioavailability; BFC, 7-benzyloxy-4-trifluoromethylcoumarin; BLM, Bloom syndrome helicase; Boc, tert-butoxycarbonyl;

BSA, bovine serum albumin; calcd, calculated; CHK2, Checkpoint kinase 2; CIN, chromosomal instability; CYP, Cytochrome P450; d, doublet; DAPI, 4',6-diamidino-2-phenylindole; (D)- or (L)-DBTA, Dibenzoyl-D or L-tartaric acid; DBF, Dibenzylfluorescein; DBU, 1,8-diazabicycloundec-7-ene; DCM, dichloromethane; DI, deionized; DIPEA, diisopropylethylamine; DMAP, 4-dimethyl-aminopyridine; DME, dimethoxyethane; DMEM, Dulbecco's Modified Eagle's Medium; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; dppf, 1,1'- bis(diphenylphosphino) ferrocene; eADME, early ADME; ee, enantiomeric

excess; EGFR, epidermal growth factor receptor; EGTA, ethylene glycol tetraacetic acid; ELISA, enzyme-linked immunosorbent assay; FLT3, fms-related tyrosine kinase 3; GI50, half maximal cell growth inhibitory concentration; GST, Glutathione S-transferase; h, hour; hERG, human ether-a-go-go related gene; HEPES,

4-(2-hydroxyethyl)-1-piperazineethanesulfonic

acid;

HOBt,

Hydroxybenzotriazole;

HMBC,

Heteronuclear multiple-bond correlation spectroscopy; HPLC, high performance liquid chromatography; HRMS, high-resolution mass spectrometry; IEX, ion exchange; IP, intraperitoneal; IPTG, Isopropyl β-D--

thiogalactopyranoside; JNK3, c-Jun N-terminal kinase 3; KD, kinase domain; KDR, kinase insert domain receptor; LB, lysis buffer; LCMS, liquid chromatography coupled to mass spectrometry; MAPK2, mitogen-


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activated protein kinase 2; MBP, Myelin basic protein; min, minute; m, multiplet; Mps1, Monopolar Spindle 1; MS ESI, Electrospray Ionization mass spectrometry; MTD, maximum tolerated dose; NMP, Nmethylpyrrolidone; NMR, nuclear magnetic resonance; NBS, N-Bromosuccinimide; PAMPA, Parallel Artificial Membrane Permeability Assay; PARP, poly ADP ribose polymerase; PBS, Phosphate Buffered Saline; PCR polymerase chain reaction; PDB, protein data bank; PEG400, polyethylene glycol 400; pin, pinacol; PK,

pharmacokinetics; PLK4, Polo-like kinase 4; PMB, p-methoxybenzyl; PMSF, phenylmethanesulfonylfluoride; PPB, Plasma Protein Binding; prep, preparative; PTEN, Phosphatase and tensin homolog; PSA, polar surface area; QD, quaque die (once daily), QSAR, Quantitative structure–activity relationship; Ra-Ni, Raney nickel; RBF, round bottomed flask; (RIPA, radio-immunoprecipitation assay ; rmse, root-mean-square

error; rt, room temperature; RP, reverse phase; s, singlet; SAC, spindle assembly checkpoint; satd, saturated; SEM, 2-(Trimethylsilyl)ethoxy]methyl; siRNA, Small interfering RNA; SMs, starting materials; t, triplet; SRB,

sulforhodamine B; STR, short tandem repeat; SUMO, Small Ubiquitin-like Modifier; TB, Terrific Broth; TBAB, Tetra-n-butylammonium bromide; TBAF, Tetra-n-butylammonium fluoride; TBTU, O-(Benzotriazol-1-

yl)-N,N,N',N'-tetramethyluronium

tetrafluoroborate;

TCA,

trichloroacetic

acid;

TCEP,

tris(2-

carboxyethyl)phosphine; TEA, triethylamine; temp, temperature; TFA, trifluoroacetic acid; TGI, tumor growth inhibition; THF, tetrahydrofuran; THP, tetrahydropyran; TIE, tyrosine kinase with immunoglobulin-like and

EGF-like domains; TLC, thin layer chromatography;

TMB, 3,3’,5,5’-tetramethylbenzidine; TRK,

tropomyosin receptor kinase; TTK, threonine tyrosine kinase; TTK KD, TTK kinase domain; q, quartet. UPLC, ultra performance liquid chromatography; UHN IACUC, University Health Network Institutional Animal Care and Use Committee; Vmax maximum velocity; XDS, X-ray Detector Software, 1XPBS, phosphate

buffer saline containing 10 mM phosphate buffer, 2.7 mM potassium chloride, 137 mM sodium chloride, 1.76 mM potassium phosphate and adjusted to pH 7.4. REFERENCES 1.

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Abrieu, A.; Magnaghi-Jaulin, L.; Kahana, J. A.; Peter, M.; Castro, A.; Vigneron, S.; Lorca, T.; Cleveland, D. W.; Labbe, J. C. Mps1 is a kinetochore-associated kinase essential for the vertebrate mitotic checkpoint. Cell 2001, 106, 83–93.

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Liu, X.; Winey, M. The Mps1 family of protein kinases. Annu. Rev. Biochem. 2012, 81, 561– 585.

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Mills, G. B.; Schmandt, R.; McGill, M.; Amendola, A.; Hill, M.; Jacobs, K.; May, C.; Rodricks A. M.; Campbell, S.; Hogg, D. Expression of TTK, a novel human protein kinase, is associated with cell proliferation. J. Biol. Chem. 1992, 267, 16000–16006.

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Yuan, B.; Xu, Y.; Woo, J.-H.; Wang, Y.; Bae, Y. K.; Yoon, D.-S.; Wersto, R. P.; Tully, E.; Wilsbach, K.; Gabrielson, E. Increased expression of mitotic checkpoint genes in breast cancer cells with chromosomal instability. Clin. Cancer Res. 2006 12, 405–410.


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