Dacinostat | DNA RNA synthesis Signal

Optimization of the in Vitro Cardiac Safety of Hydroxamate-Based Histone Deacetylase Inhibitors
Michael D. Shultz,* Xueying Cao, Christine H. Chen, Young Shin Cho, Nicole R. Davis,†
Joe Eckman, Jianmei Fan, Alex Fekete, Brant Firestone, Julie Flynn,‡ Jack Green, Joseph D. Growney, Mats Holmqvist, Meier Hsu,§ Daniel Jansson, Lei Jiang, Paul Kwon, Gang Liu, Franco Lombardo, Qiang Lu,|| Dyuti Majumdar, Christopher Meta, Lawrence Perez, Minying Pu, Tim Ramsey,
Stacy Remiszewski, Suzanne Skolnik, Martin Traebert, Laszlo Urban, Vinita Uttamsingh,^ Ping Wang,
Steven Whitebread, Lewis Whitehead, Yan Yan-Neale, Yung-Mae Yao, Liping Zhou, and Peter Atadja
Novartis Institutes for Biomedical Research, Inc., 250 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States
bS Supporting Information

⦁ INTRODUCTION
The post-translational modiﬁcation of proteins via acetylation and deacetylation is a powerful mechanism of regulating protein function, stability, activity, and subcellular localization.1—12 In cancer and other diseases, enzymes that control posttranslational modiﬁcations have altered levels of activity due to genetic and epigenetic changes that accrue on a cellular level. One such mechanism of posttranslational modiﬁcation is the acetylation of the terminal amino group on lysine side chains, catalyzed by histone acetyl transferases and the deacetylation step that is catalyzed by a family of enzymes known as histone deacetylases (HDACs).13 Pharmacological inhibition of HDACs has been shown to be a successful strategy to treat certain forms of cancer, as evidenced by their approved use by regulatory agencies.14,15 In addition to their promise as antineoplastic agents, HDAC inhibi- tors are under extensive study to treat a wide range of disorders including Huntington’s disease, ﬁbrosis, cardiac hypertrophy, multiple sclerosis, spinal muscle atrophy, and as anti-infective agents.16—23 Several HDAC inhibitors have entered clinical devel-

hydroxamates such as vorinostat 1 (SAHA),24 belinostat 2 (PXD-101),25 dacinostat 3 (LAQ824),26 4 (SB-939),27 and ben- zamides such as mocetinostat 5 (MGCD-0103)28 and entinostat 6 (SNDX-275 formerly MS-275),29 while Romidepsin (FK228),24,30,31 a natural product not belonging to either class, has also received approval from regulatory agencies (Figure 1). There have been several excellent reviews detailing additional compounds under- going clinical investigation in this highly competitive ﬁeld.32—40 Although these agents hold great promise for a variety of indications, there has been concern about dose limiting toxicities observed in human trials including fatigue, thrombocytopenia, gastrointestinal toxicity, and QT prolongation.41—43 Episodes of QT prolongation have been reported for 2, 3, 4, romidepsin, givinostat, and JNJ-26481585, while atrial ﬁbrillation and peri- carditis were observed with CHR-3996 and 5, respectively.28 Because of the wide variety of chemical classes where QT prolongation, ventricular ﬁbrillation, and/or other cardiac toxicities have been reported, there is speculation that the

opment as anticancer treatments and studies with these and other

agents toward nononcology indications are gaining momentum. The two most prominent classes of HDAC inhibitors are
Received: April 1, 2011
Published: June 08, 2011

Figure 1. Select clinical and FDA approved HDAC inhibitors.

cardiovascular events are an on-target eﬀect, but this hypothesis had not yet been proven experimentally.44—46
Of the HDAC inhibitors to enter late stage clinical trials, 3 has the greatest in vitro potency against multiple HDAC isoforms as well as the hERG ion channel.47—52 Following a number of drug withdrawals from the market due to QT prolongation, torsade de pointes, and sudden death, the hERG channel has become linked with QT prolongation.53—57 As part of our eﬀort to develop a second-generation HDAC inhibitor, we initiated a program with the goal of enhancing the in vitro potency and in vivo eﬃcacy of a series of hydroxamate based HDAC inhibitors while simulta- neously eliminating the interaction with the cardiac ion channels that have been linked to QT prolongation.51 We hypothesized that a highly potent HDAC inhibitor devoid of activity against the hERG or other ion channels would either provide a superior clinical candidate or help determine if the observed cardiac eﬀects were a consequence of HDAC inhibition. None of the active clinical compounds demonstrated a substantial (>5000 fold) window between HDAC inhibition and hERG inhibition due to their limited HDAC potency and/or solubility. Com- pound 1, for which the most data is publicly available, reaches plasma exposures of only 0.96 μM following a 150 mg/kg oral dose in dog and 1.58 μM following an 800 mg oral dose in human, only marginally surpassing cellular IC50 values for cells sensitive to 1.58,59 The lack of cardiac ﬁndings in the preclinical and clinical telemetry studies of 1 therefore speaks more to the

low oral and cardiac exposure with this compound speciﬁcally rather than the behavior of HDAC inhibitors in general.59 Our eﬀorts were primarily focused at the hERG channel, but ad- vanced compounds were also proﬁled against sodium and calcium ion channels.60 To guide our eﬀorts, we employed homology models of both HDAC-1 and the hERG channel when previously reported approaches to resolve hERG binding were not successful. Herein we report the results of these eﬀorts which led to the identiﬁcation of several promising compounds including 11r and 25i, which demonstrated superior antitumor activity than both 1 and 3.

⦁ CHEMISTRY
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The N-hydroxy phenylacrylamide scaﬀold (2 4) has been employed extensively by many groups to generate potent HDAC inhibitors and has resulted in at least four clinical programs. This scaﬀold was further explored by a variety of N-alkyl derivatives based on the general structure 11. Structure-based drug design, physicochemical property modulation, and a matched molecular pair approaches were used in the design of these analogues. Scheme 1 outlines methods where R1-NH2, typically a trypta- mine or tryptamine analogue, was reacted with methyl 4-formyl- cinnamate (7) via reductive amination to generate substituted methyl 4-aminomethyl cinnamates (8). Subsequent alkylation of the secondary amines via reductive amination or reaction with

alkyl halides or epoxides yielded tertiary amines (9). Alterna- tively, when R1 precursors were more readily available as an aldehyde, they were reacted with methyl 4-aminomethylcinna- mate (8) under similar reductive amination conditions and could be further elaborated as described above. For some compounds,

Scheme 1. General Synthesis of Hydroxamate-Based HDAC Inhibitorsa

a Reagents: (a) NaBH(OAc)3, Et3N, THF (optional) (method A); (b) SiliaBond cyanoborohydride, (optional); (c) alkyl halide, base; (d) alkyl epoxide, base; (e) aldehyde, method A or SiliaBond cyanoborohydride
(f) NH2OH/H2O, NaOMe, MeOH (method B).

Scheme 2. Synthesis of 2-Substituted Tryptamine Intermediatesa
it was necessary to construct the scaﬀold using similar reactions but where sequence of the synthetic sequence was altered (e.g., 11r). Reductive amination with the 2-H indole moiety was not eﬃcient under all circumstances, therefore the 2-methyl indole moiety was optionally used due to higher yields under typical reaction conditions (e.g., the low yielding reductive amination reactions between ﬂuorinated alkyl amines and 1H-indole-3- acetaldehyde was dramatically improved by switching to 2-meth- yl-indole-3-acetaldehyde). The secondary (8) or tertiary amines
(9) were then converted to the corresponding hydroxamates (11) via aqueous hydroxylamine in methanolic sodium methoxide.
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The synthesis of 2-heteroaryl indoles was accomplished by N- bromosuccinimide mediated bromination of indole-3-acetoni- trile followed by Suzuki coupling to form 2-aryl and 2-heteroaryl indoles (12). Nitrile hydrogenation was accomplished with rhodium on alumina or borane THF to yield the modiﬁed tryptamine 13 that was used in the synthesis of target compounds (Scheme 2).
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3-(2-Amino-ethyl)-1H-indole-2-carboxylic acid ethyl ester 14 was synthesized using the Japp Klingemann reaction as pre- viously described (Scheme 3).61 Reductive amination with 4-bromo benzaldehyde proceeded smoothly using SiliaBond cyanoborohydride. To incorporate the 2-(1H-indol-2-yl)-pro- pan-2-ol motif, several approaches were tried, however under basic conditions, the tryptamine nitrogen formed a lactam with the indole 2-ethyl ester intermediate. The cyclization reaction was prevented by alkylation of the linker nitrogen.

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a Reagents: (a) NBS, DCM (67% yield); (b) Pd(PPh3)4, K2CO3; (c) H2 (50 psi), Rh Al2O3, EtOH NH4OH (optional) (method C); (d) BH3-THF (optional) (method D).

Scheme 3. Synthesis of (E)-N-Hydroxy-3-{4-[({2-[2-(1-hydroxy-1-methyl-ethyl)-1H-indol-3-yl]-ethyl}-isopropyl-amino)- methyl]-phenyl}-acrylamide 11ra

a Reagents: (a) (i) KOH, EtOH; (ii) benzenediazonium; (b) method A; (c) isopropyl iodide, Et3N, CH3CN; (d) methyllithium, THF; (E) methyl acrylate, Pd2dba3, 1 equiv H2O; (f) method B.

Scheme 4. General Synthesis of Azatryptaminesa

a Reagents: (a) (CH2O)n, DMA:butanol, dimethyl amine (method C); (b) (i) MeOSO3Me, THF; (ii) NaCN, H2O (method D); (c) B2H6, BF3, THF

Scheme 5. Synthesis of 2-tert-Butyl-1H-indole 17fa

a Reagents: (a) (CH ) COCl, K CO , toluene; (b) n-buLi, THF.
telemetry, measurement of myocardium concentrations, ex vivo cardiac preparations, isolated canine Purkinje ﬁber, etc.).62,63 The accepted deﬁnition of the cardiac safety index (CSI) is the ratio of hERG IC50 value (or another measure of adverse cardiac event) and the Cmax of the dose required to generate the desired in vivo eﬀect.57 To allow for decision making earlier in the lead optimization process, we introduced an in vitro cardiac safety

3 3 2 3

Following N-alkylation with isopropyl iodide, methyllithium addition resulted in 15. A Heck reaction with one equivalent of water yielded 16a, which was converted to the desired hydro- xamate 16b using aqueous hydroxylamine in methanolic sodium methoxide.
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The synthesis of several azaindole derivatives began with functionalization of the 3-position in a straightforward manner via a Mannich reaction (method E) between commercially available azaindoles (17a f) and dimethylamine in the presence of paraformaldehyde. Conversion of 18 to the quaternary ammo- nium salt followed by a retro-Michael addition reaction with sodium cyanide (method F) resulted in 19 in good yields. Nitrile reduction using rhodium on alumina led to the desired aza- tryptamine analogues 20 (Scheme 4). Conversion to the desired hydroxamates was accomplished as described in Scheme 1.
Several azaindoles that were not commercially available were synthesized as described below. The synthesis of 2-tert-butyl-1H- pyrrolo[2,3-b]pyridine 17f (Scheme 5) was accomplished using
index (iCSI) parameter deﬁned as the ratio of the hERG IC50, either radioligand binding or the cellular patch clamp, and the cellular IC50 used for compound proﬁling (antiproliferative activity in the HCT116 cell line). Other levels of inhibition, such as hERG IC20 or a cellular IC90, can be used and may be even more ideal. Similar to other oﬀ-target selectivity measurements, this approach helps identify discrete structural modiﬁcations that modulate the desired safety window and does not provide false comfort when compounds have weak in vitro activity.
¼
CSI hERG IC50
CmaxðunboundÞ
¼ ¼
iCSI hERG IC50 hERG IC50
cellular IC50 HCT116 IC50
It has been recommended that, at minimum, the hERG IC50 should be at least 30-fold higher than the free fraction Cmax of a given compound in vivo and a 100-fold margin is generally
preferred.57 Because it is likely that eﬃcacious concentrations at

modiﬁcations of previously reported methods. Subsequent ela-
boration to the azatryptamine 20g was performed as described
Cmax
will be several orders of magnitude higher than the cellular

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above. Reaction of 1-amino pyridinium iodide with a variety of substituted propiolic acid ethyl esters provided 2-substituted pyrazolo[1,5-a]pyrimidines 21a c (Scheme 6). Reduction of the ester with lithium aluminum hydride followed by manganese dioxide oxidation to the aldehyde proceeded in good to excellent yields. Conversion to the nitroethylene 23 was high yielding, however, diborane reduction to azatryptamines 24 were low yielding but scalable, so suﬃcient quantities of the azatryptamine could be prepared. Elaboration to 25h 25n proceeded as described in Scheme 1.
⦁ RESULTS AND DISCUSSION
The determination of cardiac safety risk with preclinical and clinical compounds has been an area of intense eﬀorts and debate.57 Unfortunately, the current standard for assessing the cardiac safety of a particular compound requires information available only after the initiation of clinical development (e.g., unbound concentrations at Cmax in human) or from an integrated risk assessment approach with extremely low throughput (in vivo
antiproliferative IC50s, an iCSI goal of 5000 was set to provide suﬃcient margin of safety prior to the initiation of in vivo studies. In part, this ratio was chosen to be approximately 10-fold higher than the iCSI calculated for 3. We proﬁled our compounds against the hERG channel in radioligand binding, manual patch clamp, and automated Q-Patch assays.64,65
hERG activity has been reported to be dependent on several calculable properties including lipophilicity (clogP), amine basi- city (pKa), and three-dimensional ﬁt into a pharmacophore model. A large number of HDAC inhibitors from our earlier eﬀorts were screened for hERG activity. For these compounds, the pKas were determined experimentally to determine how well hERG potency correlated with clogP and pKa. Whereas clogP and hERG activity appeared to be only weakly correlated (Supporting Information Figure 1), the basicity of the amine appeared to have some correlation with hERG patch clamp activity (data not shown). The interpretation of clogP SAR is complicated by potential errors in calculation as well as the inherent challenge of modulating this single parameter without aﬀecting other factors known to aﬀect aﬃnity for the hERG

Scheme 6. Synthesis of 2-Substituted Pyrazolo[1,5-a]pyridin-3-yl-ethylamine Analoguesa

a Reagents: (a) K2CO3, DMF; (b) LiAlH4, THF; (c) MnO2, THF; (d) NH4OAc, MeNO2; (e) B2H6, BF3, THF.

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channel such as distance between the amine and a hydrophobe, zwitterions, and the introduction of additional substituents such as β-carbonyl, β-hydroxyl, or other moieties that attenuate amine basicity. On the basis of the apparent pKa correlation and previously reported successes against hERG achieved by mod- ulating amine basicity, we decided to more closely investigate the role the linker amine basicity of 11 played in aﬀecting HDAC and hERG activity. The interpretation of pKa SAR may not be straightforward due to multiple molecular parameters that are concomitantly altered. To aid our investigation, we made a series of N-ethyl and N-propyl derivatives (Table 1, compounds 11b h) where the hydrogen atoms on the terminal methyl groups were replaced with ﬂuorine atoms in a stepwise manner. The inductive eﬀects of the ﬂuorine atoms decrease the basicity while having negligible eﬀects on the ligand size, ﬂexibility, clogP, or the overall pharmacophore. The amine basicity was thus modulated in relative isolation from other variables that may obfuscate the HDAC and hERG SAR interpretations.
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Within analogues of the general formula 11, an increase in hERG and HDAC binding potency is observed upon conversion to a tertiary amine (Table 1). The ethyl substituent of 11b increased both the HCT116 cellular and hERG potency nearly 5-fold relative to 11a. A single ﬂuorine atom on the terminal methyl group (11c) had no appreciable aﬀect on HDAC inhibi- tion or hERG aﬃnity. When a diﬂuoromethyl (11d) or triﬂu- oromethyl group (11e) was incorporated, hERG binding was completely abolished (<5% inhibition at 30 μM), and there was also a 100 200-fold decrease of HCT116 antiproliferative activity. With two or three ﬂuorine atoms incorporated, the amine basicity is more signiﬁcantly reduced and thus the equi- librium at physiological pH lies entirely on the side of the neutral amine species. In an attempt to attenuate the inductive eﬀects in a more subtle manner, an additional methylene group was intro- duced to insulate the amine from the di- and triﬂuoromethyl groups (11g and 11h). There was also a sharp reduction of both hERG and HDAC inhibition with both of these homologated analogues relative to 11f. Although it was predicted that the 3,3,3- triﬂuoropropyl group would adjust the pKa of 11h between that of 11c and 11d, the measured pKas of the ethyl series were higher
than predicted and the measured pKas of the propyl series were lower than predicted (Table 1). The modiﬁcations at R2 do not signiﬁcantly aﬀect the measured hydroxamate pKa (data not shown), therefore all changes in aﬃnity were attributed to the alterations in protonated amine equilibrium. The ammonium ion species distributions in this series was calculated (Figure 2), and only the ethyl and monoﬂuoro analogues (11b and 11c, respectively) were predicted to have any ammonium species present at physiological pH.
Although correlations between pKa and hERG binding have been well documented, we are unaware of a similar relationship with regard to HDAC inhibition. To better understand why HDAC inhibition was so highly sensitive to the basicity of the amine linker, an HDAC homology model was analyzed. The docked structure of 11a places Asp99 and the central amine of this scaﬀold within 3.4 Å, suggesting an ionic interaction (Figure 3). The dacinostat scaﬀold (11) has demonstrably greater potency than other hydroxamate and benzamide based HDAC inhibitors47 that lack a linker with a basic amine. The importance of this interaction is further illustrated when the measured pKa values were plotted against the HDAC-1 enzy- matic and HCT116 cellular IC50 values (Figure 4). The eﬀect of lipophilicity is negligible as a factor in HDAC potency between 11b and 11e (ΔclogP = 0.264), however a nearly 200-fold diﬀerence in cell growth inhibition is observed. It is interesting to note that when the amine basicity was reduced to eﬀectively eradicate the ammonium species at physiological pH, the anti- proliferative activity was indistinguishable from 1. The correla- tion between pKa, hERG, and HDAC inhibition indicates that modulation of amine basicity would be an unsuccessful approach for improving the iCSI within this series.
Another proven approach for reducing hERG activity is the formation of zwitterions by the incorporation of a carboxylic acid or similar motif (e.g., fexofenadine).66 The 2-position of the indole ring position was chosen for its ease of substitution and exposure to solvent in our docking models (Figure 3). The HDAC-1 enzymatic potency of 11j was slightly reduced com- pared to 11a, however in several cellular assays, no appreciable activity was observed, presumably due to decreased cellular and nuclear penetration (Table 2). It appeared that incorporation of

Table 1. Inhibitory Activity and iCSI of Compounds against HDAC-1, HCT116, and hERG in Relation to the pKa of the Linker Amine

a Radioligand binding assay. b Manual patch clamp assay.

zwitterions would not be a successful approach, thus alternate approaches were considered.
We next explored discrete structural modiﬁcations aimed at exploiting structural diﬀerences between the HDAC and hERG proteins and utilized a previously disclosed hERG homology model.67 11a was docked into this model (Figure 5), and several key interactions appeared to correlate with the hERG SAR in this series. In the hERG homology model, the amine sits in a hydrophilic pocket (gray), while the indole and phenyl rings bind within a hydrophobic belt (brown) in the hERG channel. The HDAC homology model was in agreement with data that the linker amine and the phenyl ring of 11 make key interactions with Asp99 and Phe205, respectively (Figure 3). The indole moiety lies on the surface of the HDAC enzyme but in a hydrophobic pocket when docked into the hERG homology model. We hypothesized that these diﬀerences could be used in

our favor with the introduction of amphiphilic modiﬁcations. The 2-phenyl indole analogue 11k improves HCT116 cell growth inhibition by an order of magnitude relative to 11a, however the iCSI is reduced due to a more dramatic increase in hERG inhibition. The 2-pyridyl analogue 11l has a lower iCSI than the phenyl analogue 11k, but the potential to form an internal hydrogen bond between the pyridyl nitrogen and indole NH may have rendered this analogue less polar than ﬁrst anticipated. The 3-pyridyl 5-methoxy moiety 11m was designed to force the pyridine ring out of planarity with the indole ring and thus prevent the formation of a similar internal hydrogen bond. The iCSI of this analogue is improved relative to 11k and 11l, and we therefore decided to further explore this tactic of utilizing amphiphilicity as a wedge between hERG and HDAC activity.
We next examined 5-membered heterocycles at the indole 2-position, and one of the most potent hERG blockers within this

series was 11n (IC50 = 480 nM). Low yielding Suzuki reactions hampered a more thorough exploration at this position, however 11o demonstrated that bulky, polar substituents at this position could signiﬁcantly decrease hERG activity. The loss of HDAC activity of 11o was surprising given the range of steric bulk that had previously been tolerated at this position (e.g., 11m). 11p has an improved iCSI relative to 11a. We wondered if replacing a methyl group of 11p with a hydroxyl group might provide the best balance of steric bulk while simultaneously introducing some lipophilicity to disfavor binding to the hERG channel. While the direct hydroxy analogue of 11p was not synthesized, 11r was tested in a manual patch clamp assay and the IC50 in this assay was determined to be >30 μM (36% inhibition at 30 μM) while being nearly 5-fold more potent in the HCT116 cell growth inhibition assay than 3, resulting in an iCSI greater than 7500. By way of a comparison of 11q and 11a, we conclude that the reduction in hERG binding was not due to the incorporation of the N- isopropyl moiety. Given this evidence that tertiary amines in- crease hERG channel blockade within this series, 11r validated the hypothesis of using targeted amphiphilicity to improve the iCSI. The PK parameters of 11r in HCT116 tumor bearing nude mice and na€ive Sprague—Dawley rats were determined. The

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Figure 2. Calculated species distribution plots of tertiary amines 11b e. The predicted amount of the protonated amine species is plotted as a function of pH.
in vivo PK is dominated by a high volume of distribution, rapid extra hepatic clearance, and a short half-life which limits the overall exposure when dosed orally or intravenously. The in vivo PK of 11r was similar to other compounds in this class that we have examined (data not shown), however the high in vivo clearance was greater than predicted by the stability observed in mouse liver microsomes. To investigate this apparent in vitro/ in vivo disconnect further, we examined the plasma stability of 11r. In mouse plasma, only 54% of the compound remained after three hours of incubation indicating that plasma instability was likely contributing to the rapid clearance. Compound 3, like other analogues based on scaﬀold 11, has low rodent plasma stability but is stable in human plasma, suggesting a species- dependent eﬀect that could overestimate the rate of human clearance. Internal studies demonstrated hydroxamates in this series are stable in thermally denatured mouse plasma. Taken together with recent publications point to enzymatic processing of esters and hydroxamates in rodent plasma, rat PK may be an extremely poor predictor of human PK with this class of compounds.68 For these reasons, rodent PK was a less decisive factor in compound progression with this class of compounds.
Because of the low oral exposure in mice, the in vivo eﬃcacy of 11r was evaluated in the HCT-116 human colon xenograft model using iv administration (Table 3). Following a determination of

Figure 4. Experimentally determined pKa versus in vitro activity. Triangles represent activity in the HDAC-1 biochemical assay. Squares represent activity in the HCT116 cellular proliferation assay.

Figure 3. HDAC-1 Homology model with the docked structure of 11a. Proposed interactions of 25i and HDAC-1 enzyme based on homology model.

Table 2. Inhibitory Activity and iCSI of Compounds against HDAC-1, HCT116, and hERG

a Radioligand binding assay IC50. b Manual patch clamp assay (% inhibition at 30 μM).

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the maximum tolerated dose (MTD: deﬁned as the dose that resulted in no deaths and less than 20% body weight loss following 5 days of dosing), animals were treated once daily with 11r at 10 mg/kg, iv, for a total of 7 days. Results are reported as %T/C, determined by the ratio of the change in tumor volume of treated animals to the change in tumor volume of the control animals, or % regression, determined by the ratio of the change in tumor volumes of the treated animals to the initial volume of the treated group. At this dose, the maximum eﬃcacy observed was 22% tumor regression and mean body weight change of 17.7% (Figure 6). Dosing was discontinued on day 7 due to body weight loss greater than 20% in a subset of animals. The positive control, 5-ﬂuorouracil, administered at 75 mg/kg, iv, resulted in T/C = 43%. Tumor regressions were also observed in the HH human cutaneous T-cell lymphoma (CTCL) xenograft model following once daily treatment with 11r at 5 mg/kg (data not shown).
Building on the success of amphiphilic modiﬁcations, we focused on improving the iCSI by replacing the indole moiety with a more polar heterocycle. A systematic exploration of azaindole analogues of 11a was initiated where single and double
nitrogen replacements were synthesized. While analogues with nitrogen replacements of C4 C7, either singly or in combina- tion (25a e), resulted in no appreciable hERG inhibition up to 30 μM (Table 4), the HCT116 antiproliferative activity in this series was also diminished to varying degrees. Of these analogues, the 7-azaindole analogue 25e retained the most HCT116 anti- proliferative activity and was explored further. Introduction of a tert-butyl group at the 2-position of 7-azaindole ring resulted in 25f with an iCSI at least 16-fold better than 3.
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For C8 and C9 azaindoles, the imidazo[1,2-a]pyridine mod- iﬁcation 25g was not well tolerated but more encouraging results were obtained with the pyrazolo[1,5-a]pyrine analogue 25h. This modiﬁcation was the only azaindole that increased the HDAC-1 enzymatic and cellular antiproliferative activity with a concomitant reduction of aﬃnity for the hERG ion channel relative to 3. The SAR was expanded at the 2-position of the pyrazolo[1,5-a]pyrine and on the amine nitrogen of the linker. Introduction of a 2-methyl group (25i) resulted in an additional 4-fold improvement in cellular activity without an apparent eﬀect on hERG activity, resulting in an iCSI of greater than 6667-fold.

Figure 5. hERG homology models of 11a with transparent binding surface (top) and binding preferences (bottom). Orange volumes represent areas in which hydrophobic groups are favored, while the light-blue volumes represent regions in which hydrophilic groups are favored.

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Table 3. PK Parameters of 11r in Sprague Dawley Rats and HCT116 Tumor Bearing Nude Mice

mean PK parameters rat mouse
route iv po iv po
dose (mg/kg) 5 10 5 20
AUC (μM 3 h) 1.307 0.336 0.463 0.0653
CL (mL/min/kg) 66 176
Vss (L/kg) 7 5.9
T1/2 (h) 1.3 1.0
Cmax (μM) 0.204 0.056
F % 5 4

Larger hydrophobic groups such as ethyl (25j) and phenyl (25k) had little eﬀect on cell growth, but in the case of 25k, apparently re-established the hERG pharmacophore (hERG IC50 = 10.2 μM). Introduction of the isopropyl group on the linker nitrogen resulted in increased aﬃnity for the hERG ion channel, conﬁrm- ing the diﬃculty of avoiding hERG activity with tertiary amines on this scaﬀold. It is noteworthy that in each and every case the azaindoles abrogated hERG activity and even facilitated the reintroduction of additional hydrophobic groups with a minimal

Figure 6. Tumor volumes and body weight changes in the HCT-116 human colon xenograft model in athymic female nude mice following treatment with vehicle, 11r, or 5-ﬂuorouracil. Treatments were initiated on day 13 post implantation when mean tumor volume reached approximately 180 mm3. 11r was administered in tartaric acid, iv, qd for 8 total doses. 5-Fluorouracil was administered in 1 phosphate buﬀered saline as a single dose. Data is expressed as mean ( standard error of the mean (SEM). p < 0.05 compared to vehicle controls using a one-way ANOVA.

×
above the HCT116 LD50 value (4 nM) for 24 h while plasma and tumor concentrations are well below the hERG IC50. Despite high in vivo clearance and low plasma stability, plasma levels of 25i remained above the cellular LD50 and well below the hERG IC50 (31% inhibition at 30 μM) for 8 h, suggesting the potential for achieving a suitable CSI following oral administration in higher species. Although the plasma exposure of 25f is slightly higher than 25i in mice when dosed orally, 25f could not be quantiﬁed in the tumor homogenate. On the basis of excellent solubility (>1 g/L at pH 6.8) and higher tumor exposure, 25i was selected for in vivo eﬃcacy studies.
The in vivo eﬃcacy of 11r and 25i were compared against previous studies of 1 and 3. Because of poor solubility, we were unable to formulate and dose 1 greater than 150 mg/kg iv. Although others have reported modest eﬃcacy at these doses, we did not observe signiﬁcant in vivo tumor growth inhibition in our eﬃcacy models at these doses (data not shown). Because 1 had demonstrated clinical eﬃcacy, we chose to compare 11r and 25i

increase in hERG blockade.
with the published HCT116
xenograft data from the FDA

Compounds 25f and 25i were selected for more extensive in vitro and in vivo characterization. Compound 25i has higher intravenous exposure, lower clearance, and a lower volume of distribution than 11r or 25f (Table 5). After a single 5 mg/kg intravenous dose, tumor concentrations of 25i are maintained
submission of 1, where 57% growth inhibition was observed (Figure 7). At 100 mg/kg, iv, 3 resulted in statistically signiﬁcant tumor growth inhibition with a T/C = 20%. Although 11r was not tolerated when dosed on a daily schedule for more than one week, signiﬁcant tumor regressions on a truncated dosing

Table 4. Inhibitory Activity and iCSI of Compounds against HDAC-1, HCT116, and hERGa of Azaindole Analogues

a Radioligand binding assay (% inhibition at 30 μM). b hERG patch clamp assay.

schedule were observed, suggesting that greater in vitro potency in the cellular antiproliferation assays may translate into greater in vivo eﬃcacy with a better tolerated compound.
On a daily intravenous dosing schedule, compound 25i dosed at 25 or 50 mg/kg resulted in antitumor activity and tumor
regressions, respectively, following 13 days of treatment in the HCT-116 human colon xenograft model (Figure 8). The anti- tumor activity of 25i corresponded to approximately 15% mean body weight loss at both dose levels. Common dose limiting toxicities of HDAC inhibitors include anorexia, fatigue, body

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Table 5. PK Parameters of 25i and 25f in Sprague Dawley Rats and HCT116 Tumor Bearing Nude Mice

route
dose (mg/kg) iv
2 po
10z iv
5 po
20 iv
5 po
20
AUC (μM 3 h) 2.56 5.10 0.362 2.44 0.474
CL (mL/min/kg) 36.5 46.4 95.0
Vss (L/kg) 1.99 1.46 4.5
T1/2 (h) 0.915 1.44 3.3
Cmax (μM) 0.061 0.202 0.849
F % nd 2 4

Figure 7. Plasma and tumor pharmacokinetics of 25i in HCT116 tumor bearing mice.

weight loss, thrombocytopenia, lethargy, and gastrointestinal eﬀects. In the current studies, the more pronounced eﬃcacy and body weight loss of 11r and 25i may be reﬂective of the greater HDAC potency of these compounds rather than any generalized toxicity of the compounds per se, however more deﬁnitive toxicology studies are warranted to determine if these speculations are accurate (Table 6).
The iCSI of several HDAC inhibitors that are undergoing clinical trials were determined and compared with several compounds in this series. Whereas 3 is the only clinical com- pound in this set that exhibits activity in the hERG patch clamp assay at concentrations less than 30 μM, the remaining com- pounds are signiﬁcantly less potent in the HCT116 proliferation assay, creating a challenge in the assessment of cardiac safety.

Figure 8. Tumor volumes and body weight changes in the HCT-116 human colon xenograft model in athymic female nude mice following treatment with vehicle or 25i at 25 or 50 mg/kg. Treatments were initiated on day 12 post implantation when mean tumor volume reached approximately 193 mm3. 25i was administered in tartaric acid, iv, qd for 13 total doses. Data is expressed as mean ( standard error of the mean (SEM). p < 0.05 compared to vehicle controls using a one-way ANOVA.

Table 6. Comparison of the in Vivo Eﬃcacy of 11r and 25i with 1 and 3 in the HCT116 Xenograft Model

1 150 ipb 21 43 NR NR
3 100 14 20 1.4 8/8
11r 10 8 22 >15 8/8
10 8 52 >15 8/8
25i 25 13 10 —14.2 8/8
50 13 9 —14.8 8/8

vehicle 100 —1.8 8/8

a Dosed iv on a QD schedule. b Dosed ip on a QD schedule (data from NDA no. 21991 study report PD005, FDA submission). NR: not reported.

Table 7. Comparison of Cellular Activity and iCSI of 11r, 25f, and 25i with Select Clinical HDAC Inhibitors

IC50 (nM)

compd
HCT116
H1299
hERGa hERG inhibition at 30 μM (%)
iCSId
1 810 8200 >30,000 18 >37
2 160 460 >30,000 18 >188
3 15 100 10,300b 57 542
5 310 1440 >30,000 30 >97
6 670 >4000 >30,000 38 >45
11r 4 26 >30,000b 5c >7500

Because cardiac safety is typically predicted based on ratios of 25f 3.4 21 >30,000 34 >9000
concentrations needed to aﬀect the hERG channel function and 25i 4.5 29 >30,000 31 >7000

those needed for target inhibition, low patch clamp activity does
not provide for a complete safety assessment and may lead to unexpected results in more advanced and costly animal models, especially with compounds of relatively low on-target activity or with the potential for high Cmax levels. Many organizations choose to use hERG IC20 instead of hERG IC50 for this very reason, and it is worth noting that in our assays 1 achieves 18% inhibition at 30 μM in the automated QPatch assay. For the clinical compounds in Table 7, the iCSI ranges from greater than
a Automated Q-patch assay. b Manual patch clamp. c Manual patch clamp % inhibition at 10 μM. d iCSI calculated from HCT116 cellular data.

37-fold for 1 to greater than 542-fold for 3. Compounds 11r, 25f, and 25i have at least another order of magnitude improvement in their CSI relative to 3. Combined with the enhanced potency of the compounds in this series, the iCSI of at least 4 orders of magnitude suggests that with a suitable ADME proﬁle, these compounds might be able to eﬀectively inhibit HDAC-1 without

signiﬁcant hERG blockade in vivo. By employing iCSI as a decision making tool during lead optimization, compounds were selected for in vivo studies that ultimately allowed toxicology studies to understand which cardiac observations may be oﬀ- target and which may be directly attributable to HDAC inhibi- tion in vivo.

⦁ CONCLUSIONS
In conclusion, utilizing previously published approaches to
Compounds 6, 11a, 11b, 11f, 11k, 11l, 11n, 11p, and 11q were prepared as previously described. The purity of all compounds was determined using high resolution HPLC/MS characterization and found to be >95% pure except as noted. High resolution LC/ESI-MS data were recorded using an Agilent 6220 mass spectrometer with electrospray ionization source and an Agilent 1200 liquid chromato- graph with a gradient from 5% to 95% acetonitrile in water on a C18 reverse phase column with a diode array detection. The resolution of the MS system was approximately 11000 (fwhm definition). HPLC separa- tion was performed using one of the methods (denoted in the header
text) listed at end of report. Purine and hexakis(1H,1H,3H-tetrafluor-

mitigate hERG aﬃnity in a series of hydroxamate based HDAC inhibitors, it was discovered that the pharmacophore for these two targets share a high degree of similarity and were not readily diﬀerentiated. A new approach toward reducing hERG activity was required and a more targeted approach utilizing homology models was employed. Compounds 11r and 25i were discovered through a novel approach of seeking divergence between the hERG and HDAC homology models based on the targeted introduction of amphiphilicity. This tactic may have broader application to other scaﬀolds that are recalcitrant to hERG optimizations, but the reliability of this approach needs to be evaluated more extensively before any general conclusions can be made. Analogues with an improved iCSI were generated and found to be eﬃcacious in mouse tumor xenograft models. When compared to several HDAC inhibitors in clinical trials, 11r and 25i are more potent, eﬃcacious, and provide a greater in vitro cardiac safety margin with which the QT eﬀects observed in clinical trials could been investigated in preclinical safety models with the aim of translating these ﬁndings into a clinical setting. Further work in this area is ongoing, and the results of these studies will be published in due course.

⦁ EXPERIMENTAL SECTION
Materials and Methods. Manual Patch Clamp Electrophysiol- ogy. Cell culture: HEK293 cells stably transfected with the R-subunits of the hERG (University of Wisconsin, USA) were continuously main- tained and passaged using standard cell culture media (Gibco-BRL, Switzerland) For experiments, the cells were plated onto sterile glass coverslips in 35 mm2 dishes at a density of 1.1—1.5 × 105 cells per dish. The dishes were stored in a humidified and gassed (5% CO2) incubator at 37 °C until use.
— —
Test system: The eﬀect on hERG currents was assessed by means of the patch clamp technique in the whole-cell conﬁguration at 35 ( 2 °C. The corresponding vehicle for all superfusion concentrations was 0.1% DMSO. The vehicle eﬀect was investigated in 5 cells. The eﬀect of the positive controls 100 nM E-4031 (Calbiochem, Switzerland) was investigated in 2 cells. Cells were exposed to the test item for approximately 10 min of hERG tail currents were elicited by voltage jumps from 75 mV to +10 mV (500 ms) and then to 40 mV (500 ms) at 0.1 Hz. The composition of the extracellular solutions was [mM]: NaCl 137; KCl 4; CaCl2 1.8; MgCl2 1.0; D-glucose 10; N-2-hydro-
xyethylpiperazine-N0-2-ethanesulfonic acid (HEPES) 10; pH 7.4 (adjusted with 5 M NaOH). The composition of the pipet solutions was (mM): KCl 130; MgCl2 1.0; ethylene glycol-bis(ss-aminoethyl ether)-N,N,N0,N0-tetraacetic acid (EGTA) 5; Mg-ATP 5; HEPES 10; pH 7.2 (adjusted with 1 M KOH). The compound eﬀect on the currents was corrected by the mean vehicle rundown which was observed (n = 5) in the two cell lines. Data capturing and analysis was performed by using Pulse (Heka Electronics, Germany) and Excel.
Enzymatic, cellular, and in vivo eﬃcacy studies were performed as previously described.52
opropoxy)phosphazine (protonated molecules m/z 121.05087 and 922.00979, respectively) were used as a reference. The mass accuracy of the system has been found to be <2 ppm.
Method A. Typical procedure for reductive amination using NaBH(OAc)3:
×
To a solution of the tryptamine analogue (1 mmol) and (E)-3-(4- formyl-phenyl)-acrylic acid methyl ester (0.96 mmol) in THF (4 mL) was added solid NaHCO3 (2.5 mmol), and the mixture was stirred for 48 h at ambient temperature. NaBH(OAc)3 (1.4 mmol) was then added and the mixture was stirred until judged complete by LCMS analysis, typically 5—16 h. It was quenched with aqueous NH4Cl and washed with excess EtOAc (2 ). The combined organic layers were dried (MgSO4), ﬁltered, concentrated, and puriﬁed by silica gel chromatography.
Method B. Typical procedure for the conversion of esters to hydroxamic acids:
—
To a stirred solution of a methyl ester (4 mmol) in methanol (5 mL) at 0 °C was added sodium methoxide (5 equiv, 20 mmol of a 25% solution in methanol) and 10 equiv of hydroxylamine (50% solution in water). The reaction is monitored by LCMS, and upon completion the reaction mixture was brought to pH 7 8 with 1 N hydrochloric acid whereupon a precipitate formed. The solid is ﬁltered, washed with water, and dried in vacuo to yield the desired hydroxamic acid.
Method C. Typical procedure for the formation of 3-dimethylami- nomethyl azaindoles:
A mixture of azaindole (22 mmol), dimethylamine hydrochloride (2.0 g, 25 mmol), and paraformaldehyde (0.75 g, 2.5 mmol) in 1-butanol (60 mL) were stirred at reﬂux for 3 h. The resulting solution was evaporated in vacuo and puriﬁed via ﬂash column chromatography.
Method D. Typical procedure for the formation of 3-azaindole acetonitriles from 3-dimethylaminomethyl indoles:
Sulfuric acid dimethyl ester (2.1 g, 16 mmol) was added to a solution of dimethyl 3-azindoylmethyl-amine (15 mmol) in THF (12 mL). The mixture was stirred at reﬂux for 30 min. The solution was then placed in an ice bath, and most of the THF is decanted out to give a gummy intermediate which was washed several times with ether. Water (6 mL) and sodium cyanide (950 mg, 19 mmol) were added, and the solution was then stirred at reﬂux for 1 h. Upon completion of the reaction, the solution was cooled and extracted with EtOH:DCM (1:4) several times. The combined organic layers were washed with brine and dried over Na2SO4. The organic solvent was removed to provide crude product, which was puriﬁed via ﬂash column chromatography (MeOH:DCM, 1:99 to 20:90).
Method E. (2-Methyl-1H-indol-3-yl)-acetaldehyde. Starting with (2-methyl-1H-indol-3-yl)-acetic acid ethyl ester (4.0 g, 18 mmol) the title compound (2.3 g, 74% yield) was prepared according to the previously described procedure.69 1H NMR (400 MHz, CDCl3) δ 9.69 (t, J = 2.9 Hz, 1 H), 7.99 (br, 1 H), 7.48 (d, J = 7.5 Hz, 1 H),
7.33 (d, J = 8.0 Hz, 1 H), 7.16 (ddt, J = 19.6, 10.4, 3.7 Hz, 2 H), 3.74 (d,
J = 3.0 Hz, 2 H), 2.42 (s, 3 H). MS m/z 174.1 (MH+).
(E)-3-{4-[(2-Fluoro-ethylamino)-methyl]-phenyl}-acrylic Acid Methyl Ester (8c). 2-Fluoro-ethylamine hydrochloride (110 mg, 1.1 mmol) was added to a solution of (E)-3-(4-formyl-phenyl)-acrylic acid methyl ester
(7) (200 mg, 1.1 mmol) and acetic acid (0.5 mL) in DMF (2 mL) at

room temperature. The mixture was stirred at room temperature for 10 min. SiliaBond cyanoborohydride (1.1 g, 1.1 mmol, 1 g/mmol) was added and stirred at room temperature for another 10 min. The mixture was then heated via microwave irradiation at 150 °C for 5 min, filtered, and concentrated under vacuum. The crude product was purified via flash column chromatography (EtOAc:hexanes, 10:90 to 100:0; MeOH: DCM, 1:99 to 10:90) to provide product (100 mg, 38% yield). 1H NMR (400 MHz, CDCl3) δ 7.70 (d, J = 15.9 Hz, 1 H), 7.50 (d, J = 8.1 Hz, 2 H),
7.42 (d, J = 8.2 Hz, 2 H), 6.44 (d, J = 16.0 Hz, 1 H), 4.55 (dt, J = 47.7,
5.0 Hz, 2 H), 3.83 (s, 3 H), 3.72 (s, 2 H), 2.82 (dt, J = 26.9, 5.1 Hz, 2 H). MS m/z 238.2 (MH+).
(E)-3-{4-[(2,2-Difluoro-ethylamino)-methyl]-phenyl}-acrylic Acid Methyl Ester (8d). Starting from 2,2-difluoro-ethylamine (250 mg,
3.1 mmol) the title compound was prepared following the procedure for 8c (550 mg, 70% yield). 1H NMR (400 MHz, CD3OD) δ 7.69 (d, J = 16.3 Hz, 1 H), 7.58 (d, J = 8.3 Hz, 2 H), 7.40 (d, J = 8.3 Hz, 2 H), 6.52 (d, J = 15.6 Hz, 1 H), 5.90 (tt, J = 56.3, 4.3 Hz, 1 H), 3.84 (s, 2 H), 3.78 (s, 3 H), 2.90 (td, J = 15.6, 4.6 Hz, 2 H). MS m/z 256.1 (MH+).
(E)-3-{4-[(3,3-Difluoro-propylamino)-methyl]-phenyl}-acrylic Acid Methyl Ester (8g). Starting from 3,3-difluoro-propylamine hydrochlor- ide (250 mg, 2.1 mmol) the title compound was prepared following the procedure for 8c (460 mg, 81% yield). MS m/z 270.1 (MH+).
(E)-3-[4-({(2-Fluoro-ethyl)-[2-(2-methyl-1H-indol-3-yl)-ethyl]-amino}- methyl)-phenyl]-acrylic Acid Methyl Ester (9c). Following method A, the title compound (100 mg, 60% yield) was prepared from 8c (100 mg,
—
0.42 mmol) and (2-methyl-1H-indol-3-yl)-acetaldehyde (80 mg, 0.46 mmol). 1H NMR (400 MHz, CDCl3) δ 7.80 (s, 1 H), 7.74 (d, J = 15.9 Hz, 1 H), 7.49 (d, J = 8.9 Hz, 2 H), 7.40 (d, J = 8.0 Hz, 2 H), 7.38 (d, J = 7.3 Hz, 1 H), 7.26 (d, J = 8.1 Hz, 1 H), 7.13 (t, J = 7.5 Hz, 1 H), 7.06 (t, J = 7.5 Hz, 1 H), 6.48 (d, J = 15.7 Hz, 1 H), 4.58 (dt, J = 47.6, 5.3 Hz, 2 H), 3.86 (s, 3 H), 3.83 (s, 2 H), 3.01 2.78 (m, 6 H), 2.32 (s, 3 H). MS m/z 395.2 (MH+).
—
(E)-3-[4-({(2,2-Difluoro-ethyl)-[2-(2-methyl-1H-indol-3-yl)-ethyl]- amino}-methyl)-phenyl]-acrylic Acid Methyl Ester (9d). Following method A, the title compound (400 mg, 81% yield) was prepared from 8d (550 mg, 2.1 mmol) and (2-methyl-1H-indol-3-yl)-acetaldehyde (200 mg, 1.2 mmol). 1H NMR (400 MHz, CDCl3) δ 7.75 (br, 1 H), 7.72 (d, J = 15.8 Hz, 1 H), 7.49 (d, J = 7.9 Hz, 2 H), 7.41 7.37 (m, 2 H), 7.34
(d, J = 7.8 Hz, 1 H), 7.27 (d, J = 8.2 Hz, 1 H), 7.11 (td, J = 7.5, 1.3 Hz,
1 H), 7.04 (td, J = 7.5, 0.9 Hz, 1 H), 6.46 (d, J = 15.6 Hz, 1 H), 5.81 (t, J =
56.2 Hz, 1 H), 3.85 (s, 2 H), 3.84 (s, 3 H), 3.00 (td, J = 14.4, 3.8 Hz, 2 H),
—
2.89 2.83 (m, 4 H), 2.32 (s, 3 H). MS m/z 413.2 (MH+).
(E)-3-(4-{[[2-(1H-Indol-3-yl)-ethyl]-(2,2,2-trifluoro-ethyl)-amino]- methyl}-phenyl)-acrylic Acid Methyl Ester (9e). 2,2,2-Trifluoro-ethane- 1,1-diol (100 mg, 0.90 mmol) and p-toluenesulfonic acid (6 mg,
0.03 mmol) were added to a solution of (E)-3-(4-{[2-(1H-indol-3-yl)- ethylamino]-methyl}-phenyl)-acrylic acid methyl ester (200 mg,
—
0.60 mmol) in toluene (2 mL). The mixture was stirred at reflux temperature under Dean Stark trap. Upon the completion of reaction, THF (2 mL) and sodium triacetoxyborohydride (150 mg, 0.72 mmol) were added. The resulting mixture was stirred at room temperature, and then sodium bicarbonate solution was added. The aqueous layer was extracted several times with EtOAc. The combined organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The resulting crude product was purified via flash column chromatography to afford a white powder (215 mg, 86% yield). 1H NMR (CDCl3) δ ppm 8.0 (br s, 1H), 7.72 (d, J = 16.0 Hz, 1 H), 7.61 (d, J = 8.6 Hz, 1 H), 7.54 (m, 4 H), 7.41 (d, J = 8.2 Hz, 1 H), 7.29 (m, 2 H), 7.20 (d, J = 7.7 Hz, 1 H), 6.48 (d, J = 16.0 Hz, 1 H), 4.29 (br s, 1 H), 4.14 (m, 5 H), 3.91 (s, 1 H), 3.84 (s, 4 H), 3.56 (m, 1 H), 3.35 (m, 1 H), 3.07 (br s, 1 H), 2.78 (m, 1 H), 2.07 (m, 2 H), 1.8 (m, 1 H).
(E)-3-[4-({(3,3-Difluoro-propyl)-[2-(2-methyl-1H-indol-3-yl)-ethyl]- amino}-methyl)-phenyl]-acrylic Acid Methyl Ester (9g). Following method A, the title compound (380 mg, 74% yield) was prepared from
8g (460 mg, 1.7 mmol) and (2-methyl-1H-indol-3-yl)-acetaldehyde (200 mg, 1.2 mmol). 1H NMR (400 MHz, CDCl3) δ 7.90 (br, 1 H), 7.74 (d, J = 16.0 Hz, 1 H), 7.51 (d, J = 8.4 Hz, 2 H), 7.40 (d, J = 8.0 Hz,
2 H), 7.32 (d, J = 7.8 Hz, 1 H), 7.24 (dt, J = 8.0, 0.9 Hz, 1 H), 7.10 (ddd, J =
8.1, 7.0, 1.2 Hz, 1 H), 7.03 (ddd, J = 7.8, 7.0, 1.0 Hz, 1 H), 6.48 (d, J = 15.9
—
Hz, 1 H), 3.87 (s, 2 H), 3.85 (s, 3 H), 2.96 (t, J = 13.2 Hz, 2 H), 2.89 2.79
(m, 4 H), 2.29 (s, 3 H), 1.65 (t, J = 18.8 Hz, 3 H). MS m/z 427.2 (MH+).
(E)-3-(4-{[[2-(1H-Indol-3-yl)-ethyl]-(3,3,3-trifluoro-propyl)-amino]- methyl}-phenyl)-acrylic Acid Methyl Ester (9h). Following method A, the title compound (160 mg, 41% yield) was prepared from 8h (300 mg,
0.90 mmol) and 3,3,3-trifluoro-propionaldehyde (105 mg, 0.94 mmol).
1H NMR (400 MHz, CDCl3) δ 7.97 (s, 1 H), 7.71 (d, J = 16.1 Hz, 1 H),
— —
7.49 7.46 (m, 3 H), 7.39 7.35 (m, 2 H), 7.20 (ddd, J = 8.1, 6.9, 1.3 Hz,
1 H), 7.09 (ddd, J = 8.0, 7.2, 1.0 Hz, 1 H), 7.00 (d, J = 2.9 Hz, 1 H), 6.46
—
—
(d, J = 16.3 Hz, 1 H), 3.84 (s, 3 H), 3.73 (s, 2 H), 2.99 2.93 (m, 2 H), 2.90 2.81 (m, 4 H), 2.36 (m, 2 H). MS m/z 431.2 (MH+).
(E)-3-[4-({(2-Fluoro-ethyl)-[2-(2-methyl-1H-indol-3-yl)-ethyl]-amino}- methyl)-phenyl]-N-hydroxy-acrylamide (11c). Following method B, the title compound (8.9 mg, 9.3% yield) was prepared from 9c (100 mg, 0.24 mmol). 1H NMR (400 MHz, CD3OD) δ 7.56 (d, J = 15.6 Hz, 1 H),
7.54 (d, J = 7.6 Hz, 1 H), 7.41 (d, J = 7.9 Hz, 2 H), 7.29 (d, J = 8.1 Hz, 2 H),
7.25 (d, J = 8.3 Hz, 1 H), 7.02 (ddd, J = 8.1, 7.0, 1.1 Hz, 1 H), 6.93 (ddd, J =
8.1, 7.0, 1.1 Hz, 1 H), 6.56 (d, J = 16.0 Hz, 1 H), 4.63 (dt, J = 47.4, 5.4 Hz,
—
2 H), 3.86 (s, 2 H), 3.03 (dt, J = 26.8, 5.2 Hz, 2 H), 2.95 2.78 (m, 4 H), 2.32 (s, 3 H). MS m/z 396.2 (MH+). HRMS calcd for C23H26N3O2F (MH+) 396.2087, found 396.2092.
(E)-3-[4-({(2,2-Difluoro-ethyl)-[2-(2-methyl-1H-indol-3-yl)-ethyl]- amino}-methyl)-phenyl]-N-hydroxy-acrylamide (11d). Following method B, the title compound (180 mg, 46% yield) was prepared from 9d (400 mg, 0.92 mmol). 1H NMR (400 MHz, CD3OD) δ 7.58 (d, J = 15.5 Hz, 1 H), 7.43 (d, J = 7.8 Hz, 2 H), 7.27 (d, J = 7.8 Hz,2 H),
7.22 (d, J = 5.3 Hz, 1 H), 7.20 (d, J = 4.7 Hz,1 H), 6.97 (t, J = 7.3 Hz,
1 H), 6.88 (t, J = 7.5 Hz, 1 H), 6.48 (d, J = 15.8 Hz, 1 H), 5.78 (tt, J =
56.2, 4.4 Hz, 1 H), 3.67 (s, 2 H), 2.88 (td, J = 14.9, 4.2 Hz, 2 H),
—
2.79 2.64 (m, 4 H), 2.23 (s, 3 H). MS m/z 414.2 (M + 1). 13C NMR (400 MHz, CD3OD) δ 166.50, 142.87, 141.54, 137.11, 135.14, 132.62, 130.52, 129.89, 128.79, 121.22, 119.3, 118.35, 117.98, 111.24, 109.32,
60.26, 57.37 (t), 56.29, 49.46, 23.23, 11.35. HRMS calcd for
C23H25N3O3F2 (M—) 412.1837, found 412.1854.
(E)-N-Hydroxy-3-(4-{[[2-(1H-indol-3-yl)-ethyl]-(2,2,2-trifluoro-ethyl)- amino]-methyl}-phenyl)-acrylamide (11e). Following method B, the title compound (26 mg, 19% yield) was prepared from 9e (140 mg, 0.39 mmol). 1H NMR (400 MHz, CD3OD) δ 7.58 (d, J = 15.8 Hz, 1 H), 7.54 (d, J = 8.2 Hz, 2 H), 7.50 (d, J = 8.2 Hz, 1 H), 7.45 (d, J = 7.6 Hz, 2 H), 7.34
(d, J = 8.2 Hz, 1 H), 7.13 (t, J = 8.2 H2, 1 H), 7.03 (t, J = 7.6 Hz, 1 H), 6.47
(d, J = 15.8 Hz, 1 H), 4.28 (q, J = 7.6 Hz, 1 H), 4.00 (d, J = 13.9 Hz, 1 H),
3.86 (d, J = 13.9 Hz, 1 H), 3.43 (m, 1 H), 3.15 (d, J = 13.2, 5.0 Hz, 1 H), 3.01
(m, 1 H), 2.61 (dd, J = 17.0, 3.2 Hz, 1 H). MS m/z (M + 1). 13C NMR (400 MHz, CD3OD) δ 142.09, 141.40, 138.40, 135.56, 130.56, 128.88, 127.78,
125.07, 123.23, 119.98, 119.19, 118.25, 112.20, 111.90, 79.04, 59.01, 46.65,
— —
17.72. HRMS calcd for C22H22N3O2F3 (MH)+ 418.1742, found 416.1611. (E)-3-[4-({(3,3-Difluoro-propyl)-[2-(2-methyl-1H-indol-3-yl)-ethyl]- amino}-methyl)-phenyl]-N-hydroxy-acrylamide (11g). Following method B, the title compound (200 mg, 53% yield) was prepared from 9g (380 mg, 0.85 mmol). 1H NMR (400 MHz, CD3OD) δ 7.59 (d, J = 15.4 Hz, 1 H), 7.51 (d, J = 7.9 Hz, 2 H), 7.38 (d, J = 8.2 Hz,2 H), 7.21 (d, J = 3.4 Hz, 1 H), 7.19 (d, J = 3.7 Hz, 1 H), 6.96 (td, J = 7.5, 1.2 Hz, 1 H), 6.86 (td, J = 7.5, 1.3 Hz, 1 H), 6.48 (d, J = 15.3 Hz, 1 H), 3.81 (s, 2 H), 2.93 (t, J = 13.2 Hz, 2 H), 2.86 2.82 (m, 2 H), 2.75 2.71 (m, 2 H), 2.26 (s, 3 H), 1.58 (t, J = 19.2 Hz, 3 H). MS m/z 428.3 (MH+). HRMS calcd for C24H27N3O2F2 (MH+) 428.2150, found 428.2148. (E)-N-Hydroxy-3-(4-{[[2-(1H-indol-3-yl)-ethyl]-(3,3,3-trifluoro-propyl)- amino]-methyl}-phenyl)-acrylamide (11h). Following method B, the title compound (33 mg, 21% yield) was prepared from 9h (160 mg,

—
0.37 mmol). 1H NMR (400 MHz, CD3OD) δ 7.53 7.46 (m, 3 H),
—
7.37 7.31 (m, 4 H), 7.06 (ddd, J = 8.1, 7.1, 1.0 Hz, 1 H), 7.01 (s, 1 H),
6.94 (ddd, J = 8.0, 7.0, 1.1 Hz, 1 H), 6.49 (d, J = 15.7 Hz, 1 H), 3.68 (s,
— — —
2 H), 2.94 2.90 (m, 2 H), 2.85 2.76 (m, 4 H), 2.39 2.30 (m, 2 H).
13C NMR (400 MHz, CD3OD) δ 166.5, 142.7, 141.52, 138.15, 135.12,
130.6, 128.79, 127.08, 123.22, 122.2, 119.44, 119.23, 118.0, 114.02,
112.2, 59.11, 55.59, 49.38 (q) 47.60 (q), 32.45 (q), 24.20. MS m/z 432.2 (MH+). HRMS calcd for C23H24N3O2F3 (MH+) 432.1899, found 432.1901.
3-(2-Amino-ethyl)-1H-indole-2-carboxylic Acid Ethyl Ester Hydro- chloride (14). Starting from 2-(3-chloro-propyl)-malonic acid diethyl ester (11.8 g, 49.9 mmol) the title compound (4.4 g, 33% yield) was prepared according to the procedure as previously described.70 1H NMR (400 MHz, DMSO-d6) δ 8.15 (br, 2 H), 7.79 (d, J = 8.3 Hz, 1 H), 7.45
(d, J = 8.7 Hz, 1 H), 7.29 (t, J = 7.6 Hz, 1 H), 7.11 (t, J = 7.5 Hz, 1 H),
—
4.38 (q, J = 7.1 Hz, 2 H), 3.38 (m, 2 H), 1.42 1.34 (m, 5 H). MS m/z
233 (MH+).
3-{2-[4-((E)-2-Methoxycarbonyl-vinyl)-bezylamino]-ethyl}-1H-in- dole-2-carboxylic Acid Ethyl Ester (9j). Following method A, the title compound was prepared from 14. 1H NMR (400 MHz, CDCl3) δ 8.67 (s, 1 H), 7.64 (dd, J = 8.1, 1.1 Hz, 1 H), 7.57 (d, J = 16.2 Hz, 1 H), 7.37 (d,
J = 8.0 Hz, 2 H), 7.29 (ddd, J = 9.2, 8.1, 1.2 Hz, 1 H), 7.24 (d, J = 8.0 Hz, 2
H), 7.07 (ddd, J = 8.1, 6.7, 1.2 Hz, 1 H), 6.31 (d, J = 16.0 Hz, 1 H), 4.32 (q,
J = 7.2 Hz, 2 H), 3.79 (s, 2 H), 3.73 (s, 3 H), 3.28 (t, J = 7.4 Hz, 2 H), 2.91
(t, J = 7.6 Hz, 2 H), 1.32 (t, J = 7.5 Hz, 3 H). MS m/z 313.2 (M + 1)
3-{2-[4-((E)-2-Hydroxycarbamoyl-vinyl)-benzylamino]-ethyl}-1H- indole-2-carboxylic Acid (11j). Powdered sodium hydroxide (100 mg,
2.5 mmol) was added to a solution of 9j (190 mg, 0.47 mmol) and hydroxylamine (0.16 mL, 5 mmol, 50% in water) in ethanol (3 mL). The mixture was stirred at room temperature overnight and purified via preparative HPLC to give both the title compound (13 mg, 7% yield) and (E)-N-hydroxy-3-[4-(1-oxo-1,3,4,9-tetrahydro-β-carbo- lin-2-ylmethyl)-phenyl]-acrylamide (9.5 mg, 5% yield). 1H NMR (400 MHz, CD3OD) δ 7.59 (dt, J = 8.0, 1.0 Hz, 1 H), 7.54 (d, J = 15.9 Hz, 1 H), 7.49 (d, J = 7.9 Hz, 2 H), 7.41 (dt, J = 8.2, 1.0 Hz, 1 H), 7.32 (d, J = 8.4 Hz, 2 H), 7.23 (ddd, J = 8.3, 7.0, 1.3 Hz, 1 H), 7.08 (ddd, J = 8.0, 6.9, 1.2 Hz, 1 H), 6.46 (d, J = 15.8 Hz, 1 H), 3.80 (s, 2 H), 3.24 (t, J = 6.5 Hz, 2 H), 3.07 (t, J = 6.1 Hz, 2 H). MS m/z 380.1 (M + 1)
—
(2-Bromo-1H-indol-3-yl)-acetonitrile. To a solution of (1H-indol-3- yl)-acetonitrile (5.0 g, 32.0 mmol) in DCM (300 mL) under N2 atmosphere was slowly added N-bromosuccinimide (5.98 g, 33.6 mmol), and the mixture was stirred for 30 min at ambient temperature. The reaction mixture was added with hexane, and the solvents were evaporated in vacuo (with no heat) and more hexane was added to make a slurry. The suspension was transferred to the top of a large SiO2 plug and filtered with 0 30% EtOAc in hexanes to give the title compound (5.03 g, 21.4 mmol, 67% yield) as a brown oil. 1H NMR (CDCl3) δ 8.23 (br s, 1H), 7.63 (d, J = 8.1 Hz, 1H), 7.32 (m, 1H), 7.22 (m, 2H), 3.79 (s, 2H); m/z 236.8 (MH+).
—
[2-(4-Methoxy-pyridin-3-yl)-1H-indol-3-yl]-acetonitrile (12m). In an oven-dried pressure tube under N2 atmosphere were taken (2- bromo-1H-indol-3-yl)-acetonitrile (100 mg, 0.43 mmol), 2-methoxy- 5-pyridine boronic acid (130 mg, 0.85 mmol), and K2CO3 (88 mg, 0.64 mmol) in toluene (4 mL), and N2 was bubbled through the suspension for 10 min. Then tetrakis(triphenylphosphine)palladium (49 mg, 0.04 mmol) was added to it, and the tube was sealed. The mixture was heated to 90 °C for 16 h, filtered through a Celite plug, and the plug was washed further with more DCM. All the filtrates were combined together and concentrated to a crude oil that was purified by flash column (0 30% EtOAc in heptane) to give the title compound (59 mg, 0.22 mmol, 53% yield) as a light-yellow oil. 1H NMR (CDCl3) δ 8.37 (d, J = 2.3 Hz, 1H), 8.26 (br s, 1H), 7.77 (dd, J = 2.5, 8.5 Hz, 1H), 7.73 (d, J = 7.7 Hz, 1H),
7.45 (d, J = 7.9 Hz, 1H), 7.29 (m, 2H), 6.93 (d, J = 8.5 Hz, 1H), 4.04 (s,
3H), 3.87 (s, 2H). m/z 263.9 (MH+).
[2-(3,5-Dimethyl-isoxazol-4-yl)-1H-indol-3-yl]-acetonitrile (12o). To a solution of (2-bromo-1H-indol-3-yl)-acetonitrile (2.0 g, 8.51 mmol) in toluene (15 mL) in a dry pressure tube under N2 were added 3,5-dimethyl-isoxazol-4-boronic acid (1.32 g, 9.36 mmol), trisdibenzy- lidinedipalladium (0.78 g, 0.85 mmol), tri-tert-butylphosphonium tetra- fluoroborate (0.49 g, 1.70 mmol), and potassium fluoride (1.48 g, 25.5 mmol), and the suspension was bubbled with N2. The tube was sealed and the mixture was heated to 50 °C for 16 h. The crude material was diluted with EtOAc and filtered through a Celite plug. The filtrate was concentrated and purified by column (0 40% EtOAc in heptane) to give the title compound (1.35 g, 5.37 mmol, 63% yield) as a yellow solid.
—
1H NMR (CDCl3) δ 8.21 (br s, 1H), 7.74 (d, J = 7.7 Hz, 1H), 7.44 (d, J =
8.0 Hz, 1H), 7.30 (m, 2H), 3.66 (s, 2H), 2.41 (s, 3H), 2.23 (s, 3H). m/z
251.9 (MH+).
—
2-[2-(4-Methoxy-pyridin-3-yl)-1H-indol-3-yl]-ethylamine (13m). In a dry Parr bottle was dissolved 12m (592 mg, 2.25 mmol) in EtOH (15 mL). Then aqueous NH4OH (7.5 mL) followed by rhodium on alumina (400 mg) were added to the mixture and stirred at room temperature for 40 h under 45 psi of H2 pressure when LCMS showed complete product formation. The reaction mixture was filtered through a Celite pad, and the pad was washed with excess MeOH. The combined filtrates were concentrated to give the title compound (590 mg, 2.21 mmol, 98% yield) as a crude yellow brown solid that was used in the next step directly. 1H NMR (CD3OD) δ 8.40 (m, 1H), 7.94 (dd, J = 2.3, 8.5 Hz, 1H), 7.63 (d, J = 7.9 Hz, 1H), 7.42 (d, J = 7.9 Hz, 1H), 7.19 (m,
1H), 7.11 (m, 1H), 6.98 (d, J = 8.7 Hz, 1H), 4.01 (s, 3H), 3.23 (m, 4H).
m/z 267.9 (MH+).
2-[2-(3,5-Dimethyl-isoxazol-4-yl)-1H-indol-3-yl]-ethylamine (13o).
To a solution of 12o (830 mg, 3.3 mmol) in THF (20 mL) was added BH3 3 THF (9.9 mL, 1M, 9.9 mmol), and the mixture was stirred for 5 h at ambient temperature. It was then cooled in ice bath and quenched
with MeOH. The crude material was concentrated to an oil, combined with 5 mL of aqueous 6N NaOH solution and stirred for 30 min. Then EtOAc was added and the layers were separated. The aqueous layer was washed twice with EtOAc, and the combined organic layers were dried with MgSO4, concentrated, and purified by flash column (100% EtOAc, then 10% MeOH in DCM with a few drops of NH4OH) to give the title compound (388 mg, 1.52 mmol, 48% yield, 90% pure) as a crude brown solid. 1H NMR (CD2Cl2) δ 8.31 (br s, 1H), 7.67 (d, J = 8.6 Hz, 1H), 7.41 (d, J = 7.9 Hz, 1H), 7.24 (m, 1H), 7.15 (m, 1H), 3.44 (s, 1H), 2.89 (m,
2H), 2.77 (m, 2H), 2.36 (s, 3H), 2.20 (s, 3H). m/z 255.9 (MH+).
(E)-3-[4-({2-[2-(4-Methoxy-pyridin-3-yl)-1H-indol-3-yl]-ethylamino}- methyl)-phenyl]-acrylic Acid Methyl Ester (8m). Following method A, 20m (200 mg, 0.75 mmol) was converted to the title compound (120 mg, 0.27 mmol, 36% yield) as a yellow oil. 1H NMR (CDCl3) δ 8.41 (m, 1H), 8.08 (br s, 1H), 7.83 (m, 1H), 7.63 (m, 2H), 7.40 (m, 3H),
7.30 (m, 2H), 7.24 (m, 1H), 7.16 (m, 1H), 6.86 (d, J = 8.5 Hz, 1H), 6.37
(d, J = 15.8 Hz, 1H), 4.02 (s, 3H), 3.83 (m, 5H), 3.05 (m, 4H). m/z
441.9 (MH+).
(E)-3-[4-({2-[2-(3,5-Dimethyl-isoxazol-4-yl)-1H-indol-3-yl]-ethylamino}- methyl)-phenyl]-acrylic Acid Methyl Ester (8o). Following method A, 20o (279 mg, 1.09 mmol) was converted to the title compound (142 mg,
0.33 mmol, 36% yield) as a yellow oil. 1H NMR (CDCl3) δ 7.65 (m, 2H), 7.42 (d, J = 7.7 Hz, 2H), 7.37 (d, J = 7.7 Hz, 1H), 7.23 (m, 3H), 7.14 (m, 1H), 6.40 (d, J = 16.5 Hz, 1H), 3.79 (s, 3H), 3.73 (s, 2H), 2.84 (br s, 2H), 2.25 (s, 3H), 2.10 (s, 3H). m/z 430.1 (MH+).
(E)-N-Hydroxy-3-[4-({2-[2-(4-methoxy-pyridin-3-yl)-1H-indol-3-yl]- ethylamino}-methyl)-phenyl]-acrylamide (11m). Following method B, 8m (120 mg, 0.27 mmol) was converted to the title compound (46 mg,
0.10 mmol, 38% yield) as a yellow solid after HPLC purification. 1H NMR (CD3OD) δ 8.35 (s, 1H), 7.86 (d, J = 8.9 Hz, 1H), 7.54 (d, J = 7.6 Hz, 1H), 7.46 (m, 3H), 7.37 (d, J = 8.0 Hz, 1H), 7.23 (d, J = 7.8 Hz,1H), 7.13 (m, 1H), 7.02 (m, 1H), 6.88 (d, J = 8.9 Hz,1H), 6.48 (d, J = 15.6 Hz,1H), 3.97 (s, 3H), 3.73 (s, 2H), 3.09 (m, 2H), 2.85 (m, 2H).

13C NMR (CD3OD) δ 166.13, 164.86, 146.95, 141.95, 140.06, 139.11,
137.96, 135.68, 132.97, 130.17, 129.94, 128.65, 124.37, 122.97, 120.15,
119.63, 119.53, 112.08, 111.72, 110.75, 54.27, 53.69, 50.18, 25.41. m/z
443.1 (MH+). HRMS (MH+) calcd for C26H26N4O3, 443.2083, found 443.2074.
(E)-3-[4-({2-[2-(3,5-Dimethyl-isoxazol-4-yl)-1H-indol-3-yl]-ethylamino}- methyl)-phenyl]-N-hydroxy-acrylamide (11o). Following method B, 8o (180 mg, 0.42 mmol) was converted to the title compound (145 mg,
0.337 mmol, 80% yield) TFA salt as a yellow solid after RP-HPLC purification. 1H NMR (CD3OD) δ 7.61 (m, 4H), 7.46 (d, J = 8.1 Hz, 2H), 7.38 (d, J = 7.7 Hz, 1H), 7.19 (m, 1H), 7.10 (m, 1H), 6.53 (d, J = 15.3 Hz, 1H), 4.23 (s, 2H), 3.18 (m, 2H), 3.04 (m, 2H), 2.34 (s, 3H), 2.17 (s, 3H). 13C NMR (CD3OD) δ 169.4, 165.9, 161.2, 140.4, 138.4, 137.5, 134.1, 131.5, 131.4, 129.7, 129.5, 128.6, 125.3, 123.6, 120.6, 120.0, 119.2, 112.5, 110.3, 109.9, 51.7, 23.0, 11.6, 10.5. m/z 430.9 (MH+). HRMS (MH+) calcd for C25H26N4O3, 431.2083, found 431.2085.
—
2-(3-{2-[(4-Bromo-benzyl)-isopropyl-amino]-ethyl}-1H-indol-2-yl)- propan-2-ol (15). 3-[2-(4-Bromo-benzylamino)-ethyl]-1H-indole-2- carboxylic acid ethyl ester: 3-(2-Amino-ethyl)-1H-indole-2-carboxylic acid ethyl ester hydrochloride (8.5 g, 27 mmol) was added to a solution of 4-bromo-benzaldehyde (4.7 g, 26 mmol) and acetic acid (2.3 mL, 41 mmol) in THF (120 mL). The mixture was stirred at room temperature for 1 h. To this mixture, sodium triacetoxyborohydride (8.4 g, 38 mol) was added, and the reaction mixture was stirred at room temperature overnight. Satd NaHCO3 solution was added, and the aqueous layer was extracted with EtOAc several times. The organic layer was combined, dried over Na2SO4, filtered, and evaporated off. The crude product was purified via flash column chromatography (EtOAc:hexanes, 10:90 to 60:40) to provide product (9 g, 83% yield). 1H NMR (400 MHz, CDCl3) δ 8.77 (br, 1 H), 7.72 (d, J = 8.2 Hz, 1 H), 7.54 7.09 (m, 7 H), 4.39 (q,
J = 7.0 Hz, 2 H), 3.88 (s, 2 H), 3.42 (t, J = 7.2 Hz, 2 H), 3.06 (t, J = 7.0 Hz, 2 H), 1.40 (t, J = 7.2 Hz, 3 H). MS m/z 402 (MH+).
—
3-{2-[(4-Bromo-benzyl)-isopropyl-amino]-ethyl}-1H-indole-2-car- boxylic acid ethyl ester: 3-[2-(4-Bromo-benzylamino)-ethyl]-1H-in- dole-2-carboxylic acid ethyl ester (9.0 g, 222 mmol) was added to a solution of 2-iodo-propane (76 g, 448 mmol) and triethylamine (62 mL, 448 mmol) in CH3CN (200 mL). The mixture was reﬂuxed overnight. The solvent was then removed under vacuum. EtOAc and water were added. The aqueous layer was extracted several times with EtOAc. The combined organic layer was washed with brine, dried over Na2SO4, ﬁltered, and concentrated in vacuo. The crude product was puriﬁed via ﬂash column chromatography (EtOAc:hexanes, 10:90 to 90:10) to provide slightly yellow solid (6.5 g, 65% yield). 1H NMR (400 MHz, CDCl3) δ 8.64 (br, 1 H), 7.45 (d, J = 8.3 Hz, 1 H), 6.87 7.27 (m, 4 H),
7.15 (d, J = 8.1 Hz, 2 H), 7.09 (t, J = 7.5 Hz, 1 H), 4.35 (q, J = 7.1 Hz, 2
—
H), 3.59 (s, 2 H), 3.18 (t, J = 7.9 Hz, 2 H), 2.09 2.00 (m, 1 H), 2.69 (t,
J = 7.5 Hz, 2 H), 1.37 (t, J = 7.3 Hz, 3 H), 1.02 (d, J = 6.9 Hz, 6 H). MS m/z 444 (MH+).
—
—
Methyl lithium (18 mL, 44 mmol, 2.5 M in diethoxymethane) was added to a solution of 3-{2-[(4-bromo-benzyl)-isopropyl-amino]- ethyl}-1H-indole-2-carboxylic acid ethyl ester (6.5 g, 15 mmol) in THF (65 mL) at 65 °C. The mixture was stirred at same temperature for 2 h. Ice water was added slowly. The aqueous layer was then extracted several times with EtOAc. The combined organic layer was washed with brine, dried over Na2SO4, ﬁltered, and evaporated oﬀ. The crude product was puriﬁed via ﬂash column chromatography (EtOAc:hexanes, 10:90 to 100:0) to provide yellow oil (5.5 g, 87% yield). 1H NMR (400 MHz, CDCl3) δ 8.05 (s, 1 H), 7.32 (d, J = 7.9 Hz, 1 H), 6.78 6.73 (m, 3
—
H), 6.64 6.60 (m, 3 H), 6.54 (t, J = 7.3 Hz, 1 H), 3.44 (s, 2 H),
—
3.01 2.94 (m, 1 H), 2.89 (t, J = 7.0 Hz, 2 H), 2.68 (t, J = 6.9 Hz, 2 H),
1.58 (s, 6 H), 1.01 (d, J = 6.7 Hz, 6 H). MS m/z 430 (MH+).
(E)-3-{4-[({2-[2-(1-Hydroxy-1-methyl-ethyl)-1H-indol-3-yl]-ethyl}- isopropyl-amino)-methyl]-phenyl}-acrylic Acid Methyl Ester (16).
2-(3-{2-[(4-Bromo-benzyl)-isopropyl-amino]-ethyl}-1H-indol-2-yl)- propan-2-ol (5.0 g, 12 mmol) was added to a suspension of tris- (dibenzylideneacetone)dipalladium(0) (100 mg, 0.12 mmol) and tribu- tylphosphine tetrafluoroborate (140 mg, 0.47 mmol) in dioxane (10 mL). The suspension was degassed and filled with nitrogen several times, and then dicyclohexyl-methylamine (3 mL, 14 mmol) was added under nitrogen. The mixture was stirred at room temperature for 10 min and then acrylic acid methyl ester (2.1 mL, 23 mmol) and water (0.21 mL, 12 mmol) was added. It was then heated via microwave at 100 °C for 12 min. The crude product mixture was purified via flash column chromatography directly (EtOAc:hexanes, 10:90 to 100:0) to provide yellow syrup product (4.2 g, 83% yield). 1H NMR (400 MHz, CD3OD) δ 7.47 (d, J = 15.7 Hz, 1 H), 7.32 (d, J = 7.8 Hz, 2 H), 7.26 7.18 (m, 4 H),
—
7.03 (t, J = 7.3 Hz, 1 H), 6.93 (t, J = 7.3 Hz, 1 H), 6.45 (d, J = 15.7 Hz,
—
1 H), 4.02 (s, 2 H), 3.37 (s, 3 H), 3.34 3.32 (m, 2 H), 3.18 (t, J = 6.3 Hz,
2 H), 1.66 (s, 6 H), 1.40 (d, J = 6.3 Hz, 6 H). MS m/z 436.2 (MH+).
—
(E)-N-Hydroxy-3-{4-[({2-[2-(1-hydroxy-1-methyl-ethyl)-1H-indol- 3-yl]-ethyl}-isopropyl-amino)-methyl]-phenyl}-acrylamide (11r). Following method A, the title compound (45 mg, 32% yield) was prepared from 16 (140 mg, 0.32 mmol). 1H NMR (400 MHz, DMSO- d6) δ10.74 (s, 1 H), 10.36 (s, 1 H), 9.03 (br, 1 H), 7.54 7.42 (m, 5 H),
7.26 (d, J = 8.1 Hz, 1 H), 7.10 (d, J = 7.8 Hz, 1 H), 6.93 (t, J = 7.4 Hz,
—
1 H), 6.81 (t, J = 7.3 Hz, 1 H), 5.24 (s, 1 H), 3.65 (s, 2 H), 3.34 3.31
— —
(m, 2 H), 3.04 3.00 (m, 1 H), 2.87 2.83 (m, 2 H), 1.45 (s, 6 H), 1.04 (d, J = 6.0 Hz, 6 H). 13C NMR (400 MHz, CD3OD) δ166.44, 142.7, 142.36, 141.25, 136.21, 135.05, 130.91, 130.42, 128.66, 121.95,
119.56, 118.78, 118.24, 112.06, 108.12, 71.18, 58.41, 55.53, 52.41,
52.12, 31.44, 25.78, 18.49, 18.43. MS m/z 436 (MH+). HRMS calcd
for C26H34N3O3 (MH+) 436.2600, found 436.2607.
—
2-tert-Butyl-1H-pyrrolo[2,3-b]pyridine (17f). To an ice-cold solution of 3-methyl-pyridin-2-ylamine (25 g, 0.23 mol) in toluene (500 mL) under N2 atmosphere was added potassium carbonate (32.0 g, 0.23 mol). The mixture was stirred at room temperature for 30 min, followed by dropwise addition of trimethylacetyl chloride (31.3 mL, 0.25 mol). The mixture was stirred at 25 °C for 16 h and filtered. The precipitate was washed EtOAc. The combined filtrates were washed with aqueous NH4Cl and brine. The organic layer was dried (MgSO4) and concen- trated to give 2,2-dimethyl-N-(3-methyl-pyridin-2-yl)-propionamide (21.3 g, 0.11 mol, 48% yield) as an off-white solid. To a cold suspension ( 20 °C) of this solid (21.3 g, 0.11 mol) in THF (300 mL) under N2 was slowly added butyllithium (10 M in hexane, 33 mL, 0.33 mol), and the flask was allowed to warm up to room temperature and stand for 16
h. The mixture was then cooled below 0 °C and quenched with 25 mL of
aqueous 1N HCl, diluted with ether, and the layers were separated. The organic layer was washed with aqueous NaHCO3. The aqueous wash- ings were extracted with additional ether, and the combined organic layers were washed with brine, dried (MgSO4), and concentrated to give the title compound (22.7 g) as a crude white solid that was used directly in the next step without additional purification. 1H NMR (CDCl3) δ 8.25 (d, J = 4.4 Hz, 1H), 7.87 (br s, 1H), 7.56 (d, J = 6.9 Hz, 1H), 7.11
(dd, J = 4.8, 7.2 Hz, 1H), 2.23 (s, 3H), 1.35 (s, 9H). m/z 193.06 (MH+).
Dimethyl-(1H-pyrrolo[3,2-b]pyridin-3-ylmethyl)-amine (18a). The title compound (1.1 g, >99% yield) was prepared according to method C from 1H-pyrrolo[3,2-b]pyridine (750 mg, 6.4 mmol). 1H NMR (400
MHz, CD3OD) δ 8.45 (d, J = 4.4 Hz, 1 H), 7.93 (dd, J = 8.4, 1.6 Hz, 1 H), 7.85 (s, 1 H), 7.29 (dd, J = 8.2, 4.8 Hz, 1 H), 4.46 (s, 2 H), 2.81 (s,
6 H). MS m/z 176.1 (MH+).
Dimethyl-(1H-pyrrolo[3,2-c]pyridin-3-ylmethyl)-amine (18d). The title compound (2.6 g, 68% yield) was prepared according to method C from 1H-pyrrolo[3,2-c]pyridine (2.6 g, 22 mmol). 1H NMR (400 MHz, CD3OD) δ 9.13 (s, 1 H), 8.23 (d, J = 6.3 Hz, 1 H), 7.77 (s, 1 H), 7.56 (d,
J = 6.4 Hz, 1 H), 4.50 (s, 2 H), 2.82 (s, 6 H). MS m/z 176.2 (MH+).
(2-tert-Butyl-1H-pyrrolo[2,3-b]pyridin-3-ylmethyl)-dimethyl-amine (18f). In a dry vial was taken paraformaldehyde (517 mg, 17.2 mmol) in

dimethylacetamide:n-butanol (29:1 v/v, 3 mL) and subjected to micro- wave irradiation (100 °C for 13 min). Dimethylamine hydrochloride (1.4 g, 17.2 mmol) was added, and the mixture was heated and sonicated. To the resulting clear solution was added 17f (2.0 g, 11.5 mmol), and the mixture was subjected to microwave irradiation (100 °C for 20 min). The mixture was diluted with MeOH and concentrated in vacuo. The residue was dissolved in DCM and washed with a small volume of aqueous Na2CO3. The aqueous washings were rewashed (2×) with DCM, and the combined organic layers were dried (MgSO4), concen- trated, and dried to give the title compound (2.31 g, 9.99 mmol, 87% yield) as a yellow solid. 1H NMR (CDCl3) δ 9.4 (br s, 1H), 8.21 (dd, J = 1.4, 4.8 Hz, 1H), 7.99 (d, J = 7.9 Hz, 1H), 7.02 (dd, J = 4.8, 7.9 Hz, 1H),
3.63 (s, 2H), 2.23 (s, 6H), 1.53 (s, 9H). m/z 232.0 (MH+).
Imidazo[1,2-a]pyridin-3-ylmethyl-dimethyl-amine (18g). The title compound (900 mg, 68% yield) was prepared according to method C from imidazo[1,2-a]pyridine (880 mg, 7.5 mmol). 1H NMR (400 MHz, CDCl3) δ 8.19 (d, J = 6.8 Hz, 1 H), 7.60 (d, J = 9.4 Hz, 1 H), 7.53 (s, 1
H), 7.22 (ddd, J = 9.2, 6.7, 1.3 Hz, 1 H), 6.84 (t, J = 6.7 Hz, 1 H), 4.78 (s, 2 H), 3.60 (s, 6 H). MS m/z 176.1 (MH+).
(1H-Pyrrolo[3,2-b]pyridin-3-yl)-acetonitrile (19a). The title com- pound (340 mg, 34% yield) was prepared according to method D from 18a (1.1 g, 6.3 mmol). MS m/z 158.1 (MH+).
1H-Pyrrolo[3,2-c]pyridin-3-yl)-acetonitrile (19d). The title com- pound (600 mg, 26% yield) was prepared according to method D from 18d (2.6 g, 15 mmol).
—
(2-tert-Butyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-acetonitrile (19f). To a solution of 18f (2.0 g, 8.66 mmol) in THF (40 mL) was slowly added dimethyl sulfate (900 μL, 9.51 mmol), and the mixture was refluxed for 30 min. The solvent was removed in vacuo, and the residue was suspended in 30 mL water to which NaCN (0.57 g, 11.2 mmol) was added. After refluxing for 1 h, the aqueous suspension was washed with excess EtOAc (3×), and the combined organic layers were dried (MgSO4) and concentrated to give a residue that was purified by flash column (0 40% EtOAc in Hex) to give the title compound (0.33 g,1.55 mmol, 18% yield) as a white solid. 1H NMR (CDCl3) δ 9.87 (br s, 1H), 8.31 (dd, J = 1.4, 4.9 Hz, 1H), 7.92 (d, J = 7.4 Hz, 1H), 7.12 (dd, J = 4.9,
8.1 Hz, 1H), 3.95 (s, 2H), 1.56 (s, 9H). m/z 214.1 (MH+).
Imidazo[1,2-a]pyridin-3-yl-acetonitrile (19g). The title compound (110 mg, 5.1% yield) was prepared according to method D from 18g (2.6 g, 15 mmol). MS m/z 158.0 (MH+).
2-(1H-Pyrrolo[3,2-b]pyridin-3-yl)-ethylamine (20a). The title com- pound (340 mg, 98% yield) was prepared according to same procedure as 13m from 19a (340 mg, 2.2 mmol). MS m/z 162.1 (MH+).
—
2-(6-Methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-ethylamine (20b). The title compound (140 mg, 72% yield) was prepared according to same procedure as 13m from (6-methyl-7H-pyrrolo[2,3-d]pyrimidin-5- yl)-acetonitrile (200 mg, 1.1 mmol). 1H NMR (400 MHz, DMSO-d6) δ 8.81 (s, 1 H), 8.60 (s, 1 H), 2.77 2.75 (m, 4 H), 2.30 (s, 3 H). MS m/z
177.1 (MH+).
2-(6-Methyl-5H-pyrrolo[2,3-b]pyrazin-7-yl)-ethylamine (20c). The title compound (200 mg, 98% yield) was prepared according to same procedure as 13m from (6-methyl-5H-pyrrolo[2,3-b]pyrazin-7-yl)-acet- onitrile (200 mg, 1.1 mmol). MS m/z 177.1 (MH+).
2-(1H-Pyrrolo[3,2-c]pyridin-3-yl)-ethylamine (20d). Rhodium on alumina (200 mg, 0.1 mmol) was added to a solution of 19d (200 mg,
1.3 mmol) in ammonium hydroxide (6 mL) and ethanol (12 mL). The mixture was stirred under hydrogen atmosphere (50 psi) in a Parr shaker overnight. Upon completion of the reaction, the solution was filtered and evaporated off. The resulting product (200 mg, 98% yield) was used directly in the next step.
2-(2-tert-Butyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-ethylamine (20f). The title compound (524 mg, 2.03 mmol, 86% yield, 84% purity) was prepared according to same procedure as 13m from 19f (500 mg,
2.34 mmol) and was directly used in the next step. m/z 218.2 (MH+).
2-Imidazo[1,2-a]pyridin-3-yl-ethylamine (20g). The title com- pound (140 mg, 79% yield) was prepared according to same procedure as 20d from 20g (180 mg, 1.1 mmol). 1H NMR (400 MHz, CD3OD) δ 8.32 (d, J = 6.9 Hz, 1 H), 7.56 (d, J = 8.9 Hz, 1 H), 7.44 (s, 1 H), 7.32
(ddd, J = 9.1, 6.8, 1.2 Hz, 1 H), 6.99 (t, J = 6.8 Hz, 1 H), 3.12 (t, J = 6.8 Hz, 2 H), 3.05 (t, J = 6.8 Hz, 2 H). MS m/z 162.1 (MH+).
Typical Procedure for the Formation of 2-Ylpyrazolo[1,5- a]pyridine-3-carboxylic Acid Ethyl Esters. To a solution of amino- pyridinium iodide (22 g, 99 mmol) and anhydrous potassium carbonate (17.8 g, 128.7 mmol) in dimethylformamide (225 mL) was added an alkyl- or aryl-2-pentynoate (198 mmol). The solution was stirred at room temperature for 24 h. The reaction mixture was poured into 1.2 L of ice water. The resulting light-brown precipitate was collected by vacuum filtration and air-dried overnight to yield 11.9 g (55% yield) of crude product, which was used without further purification.
— — —
2-Methyl-pyrazolo[1,5-a]pyridine-3-carboxylic Acid Ethyl Ester (21i). The title compound (6.2 g, 52% yield) was prepared according to the above procedure from amino-pyridinium iodide (13.0 g, 58.5 mmol) and methyl-2-butynoate (13.6 mL, 117 mmol). 1H NMR (400 MHz, CDCl3) δ 8.42 8.39 (m, 1 H), 8.09 8.06 (m, 1 H), 7.37 7.33
—
(m, 1 H), 6.89 6.86 (m, 1 H), 4.39 (q, J = 7.9 Hz, 2 H), 2.68 (s, 3 H), 1.43 (t, J = 9.0 Hz, 3 H). MS m/z 205 (MH+).
2-Ethyl-pyrazolo[1,5-a]pyridine-3-carboxylic Acid Ethyl Ester (21j). The title compound (11 g, 55% yield) was prepared according to the above procedure from amino-pyridinium iodide (22 g, 99 mmol) and ethyl-2-butynoate (24.98 mL, 198 mmol). 1H NMR (400 MHz, CDCl3) δ 8.43 (ddd, J = 6.9, 1.1, 1.1 Hz, 1 H), 8.10 (ddd, J = 8.8, 1.2, 1.3 Hz, 1 H),
7.35 (ddd, J = 9.0, 6.8, 1.2 Hz, 1 H), 6.88 (ddd, J = 7.7, 6.1, 0.7 Hz, 1 H),
4.39 (q, J = 7.1 Hz, 2 H), 3.13 (q, J = 7.5 Hz, 2 H), 1.43 (t, J = 7.0 Hz, 3 H), 1.36 (t, J = 7.8 Hz, 3 H). MS m/z 219 (MH+).
— —
2-Phenyl-pyrazolo[1,5-a]pyridine-3-carboxylic Acid Ethyl Ester (21k). The title compound (20 g, 74% yield) was prepared according to the above procedure from amino-pyridinium iodide (13.0 g, 58.5 mmol) and phenyl-propynoic acid ethyl ester (19.3 mL, 117 mmol). 1H NMR (400 MHz, CDCl3) δ 8.52 (ddd, J = 7.0, 1.2, 1.2 Hz, 1 H), 8.21 (ddd, J = 8.9, 1.2, 1.2 Hz, 1 H), 7.80 7.77 (m, 2 H), 7.48 7.38 (m, 3 H),
6.95 (ddd, J = 7.7, 6.2, 0.8 Hz, 1 H), 4.32 (q, J = 7.2 Hz, 2 H), 1.31 (t, J =
7.1 Hz, 3 H). MS m/z 267 (MH+).
—
Typical Procedure for the Formation of 2-Ylpyrazolo[1,5-a]- pyridine-3-carbaldehydes. A solution of pyrazolo[1,5-a]pyridine-3- carboxylic acid ethyl ester (68.7 mmol) in THF (300 mL) was cooled in an ice bath, and lithium aluminum hydride (2.6 g, 68.7 mmol) was added. The reaction mixture was stirred at room temperature overnight. Water was added until bubbling stopped. Silica gel was added, and the solvent was evaporated in vacuo. The resulting residue was purified via silica gel chromatography eluted with 20% EtOAc:hexanes to 100% to yield 9.67 g (80% yield) of product as a yellow brown oil.
To a solution of (pyrazolo[1,5-a]pyridin-3-yl)-methanol (54.9 mmol) in THF (500 mL) was added MnO2 (23.88 g, 274.5 mmol, Aldrich cat. no. 217646, 85%, dried overnight in oven at 120 °C) and stirred at reﬂux for 1.5 h. The reaction mixture was ﬁltered through Celite and concentrated under reduced pressure to give 9.4 g (98% yield) of product, which was used without further puriﬁcation.
2-Methyl-pyrazolo[1,5-a]pyridine-3-carbaldehyde (22i). (2-Meth- yl-pyrazolo[1,5-a]pyridin-3-yl)-methanol (3.9 g, 80% yield) was pre- pared according to same procedure as above from 21i (6.1 g, 30 mmol). 1H NMR (400 MHz, CDCl3) δ 8.28 (dt, J = 7.0, 1.0 Hz, 1 H),
7.47 (dt, J = 9.0, 1.2 Hz, 1 H), 7.06 (ddd, J = 8.9, 6.8, 1.1 Hz, 1 H),
6.65 (td, J = 6.9, 1.6 Hz, 1 H), 4.78 (s, 2 H), 2.43 (s, 3 H). MS m/z 163.1 (MH+).
The title compound (3.8 g, 97% yield) was prepared according to same procedure as above from (2-methyl-pyrazolo[1,5-a]pyridin-3-yl)- methanol (3.9 g, 24 mmol). 1H NMR (400 MHz, CDCl3) δ 10.02 (s, 1 H), 8.40 (dt, J = 6.8, 1.0 Hz, 1 H), 8.14 (dt, J = 8.7, 1.3 Hz, 1 H), 7.42

(ddd, J = 8.7, 7.0, 1.1 Hz, 1 H), 6.93 (ddd, J = 7.7, 6.2, 0.8 Hz, 1 H), 2.62 (s, 3 H). MS m/z 161.1 (MH+).
2-Ethyl-pyrazolo[1,5-a]pyridine-3-carbaldehyde (22j). (2-Ethyl- pyrazolo[1,5-a]pyridin-3-yl)-methanol (9.67 g 80% yield) was pre- pared according to same procedure as above from 22i (15 g, 68.7 mmol) 1H NMR (400 MHz, CDCl3) δ 8.35 (dt, J = 8.3, 1.3 Hz, 1 H), 7.51 (dt, J = 8.9, 1.3 Hz, 1 H), 7.09 (ddd, J = 9.1, 6.5, 1.2 Hz,
1 H), 6.68 (ddd, J = 7.5, 6.2, 0.6 Hz, 1 H), 4.83 (s, 2 H), 2.79 (q, J = 7.5
Hz, 2 H), 1.28 (t, J = 7.6 Hz, 3 H). MS m/z 177.1 (MH+).
The title compound (9.4 g 98% yield) was prepared according to same procedure as above from (2-ethyl-pyrazolo[1,5-a]pyridin-3-yl)- methanol (9.67 g, 54.9 mmol). 1H NMR (400 MHz, CDCl3) δ 10.04 (s, 1 H), 8.41 (dt, J = 6.8, 1.3 Hz, 1 H), 8.15 (dt, J = 8.9, 1.5 Hz, 1 H), 7.41
(ddd, J = 8.8, 6.8, 1.1 Hz, 1 H), 6.93 (ddd, J = 7.8, 6.1, 0.8 Hz, 1 H), 3.03
(q, J = 7.6 Hz, 2 H), 1.35 (t, J = 7.5 Hz, 3 H). MS m/z 175.1 (MH+).
2-Phenyl-pyrazolo[1,5-a]pyridine-3-carbaldehyde (22k). (2-Phen- yl-pyrazolo[1,5-a]pyridin-3-yl)-methanol (3.6 g, 43% yield) was pre- pared according to same procedure as above from 21k (10 g, 38 mmol).
1H NMR (400 MHz, CDCl3) δ 8.49 (dt, J = 7.0, 1.1 Hz, 1 H), 7.91 (dt,
—
J = 6.7, 1.6 Hz, 2 H), 7.65 (dt, J = 8.9, 1.1 Hz, 1 H), 7.54 7.49 (m, 2 H),
7.45 (dt, J = 7.2, 1.9 Hz, 1 H), 7.19 (ddd, J = 8.9, 6.7, 1.1 Hz, 1 H), 6.81
(ddd, J = 7.5, 6.2, 0.8 Hz, 1 H), 4.96 (s, 2 H). MS m/z 225.0 (MH+).
The title compound (870 mg, 98% yield) was prepared according to same procedure as above from (2-phenyl-pyrazolo[1,5-a]pyridin-3-yl)- methanol (900 mg, 4.0 mmol). 1H NMR (400 MHz, CDCl3) δ 10.04 (s, 1 H), 8.53 (dt, J = 6.9, 1.0 Hz, 1 H), 8.36 (dt, J = 8.8, 1.2 Hz, 1 H),
— —
7.73 7.70 (m, 2 H), 7.50 7.44 (m, 4 H), 7.03 (ddd, J = 7.6, 6.3, 0.6 Hz,
1 H). MS m/z 223.0 (MH+).
—
Typical Procedure for the Formation of 2-Yl-3-((E)-2-nitro-vinyl)- pyrazolo[1,5-a]pyridines. To a solution of 2-yl-pyrazolo[1,5-a]pyridine- 3-carbaldehyde (48 mmol) in nitromethane (29 g, 480 mmol) was added ammonium acetate (18 g, 240 mmol) and heated at 100 °C for 1 h. The reaction mixture was concentrated under vacuum, and the resulting residue was chromatographed (silica gel, 10% EtOAc/hexane to 100% EtOAc/hexane) to obtain the title compound (62 83% yields).
2-Methyl-3-((E)-2-nitro-vinyl)-pyrazolo[ 1,5-a]pyridine (23i). The ti- tle compound (1 g, 83% yield) was prepared according to same procedure as above from 22i (950 mg, 5.9 mmol). 1H NMR (400 MHz, CDCl3) δ 8.50 (dt, J = 6.9, 1.1 Hz, 1 H), 8.31 (d, J = 13.6 Hz, 1 H),
7.70 (dt, J = 8.8, 1.0 Hz, 1 H), 7.58 (d, J = 13.7 Hz, 1 H), 7.49 (ddd, J =
8.8, 6.9, 1.2 Hz, 1 H), 7.00 (ddd, J = 7.6, 6.1, 0.6 Hz, 1 H), 2.62 (s, 3 H). MS m/z 204.0 (MH+).
2-Ethyl-3-((E)-2-nitro-vinyl)-pyrazolo[1,5-a]pyridine (23j). The title compound (6.3 g, 62% yield) was prepared according to same procedure as above from 22j (8.3 g, 48 mmol). 1H NMR (400 MHz, CDCl3) δ 8.52 (dt, J = 6.9, 1.0 Hz, 1 H), 8.31 (d, J = 13.6 Hz, 1 H), 7.70 (dt, J = 8.9, 1.2
Hz, 1 H), 7.58 (d, J = 13.5 Hz, 1 H), 7.49 (ddd, J = 8.7, 6.9, 1.1 Hz, 1 H),
7.01 (ddd, J = 7.6, 6.2, 0.7 Hz, 1 H), 2.99 (q, J = 7.4 Hz, 2 H), 1.42 (t, J =
7.6 Hz, 3 H). MS m/z 218.0 (MH+).
3-((E)-2-Nitro-vinyl)-2-phenyl-pyrazolo[ 1,5-a]pyridine (23k). The title compound (1 g, 99% yield) was prepared according to same procedure as above from 22k (850 mg, 3.8 mmol) 1H NMR (400 MHz, CDCl3) δ 8.55 (dt, J = 6.9, 1.1 Hz, 1 H), 8.26 (d, J = 13.5 Hz, 1 H),
—
7.72 (dt, J = 8.8, 1.2 Hz, 1 H), 7.61 7.58 (m, 2 H), 7.53 (d, J = 13.7 Hz,
—
1 H), 7.50 7.44 (m, 4 H), 7.01 (ddd, J = 7.6, 6.3, 0.7 Hz, 1 H). MS m/z
265.9 (MH+).
Typical Procedure for the Formation of 2-Yl-pyrazolo[1,5-a]pyridin- 3yl Ethylamines. To a solution of 2-yl-3-((E)-2-nitro-vinyl)-pyrazolo- [1,5-a]pyridine (31 mmol) in THF (300 mL) was added BF3 (93 mL, 1 M in THF) and BH3 (188 mL, 1 M in THF) and stirred at 65 °C overnight. The reaction was quenched with MeOH until bubbling ceased. The solution was concentrated in vacuo, and aqueous NaOH (1N) was added followed by extraction with CH2Cl2:EtOH (4:1) several times until no product was detected by TLC in the aqueous
solution. The organic layer was washed with brine. The brine layer was further extracted with CH2Cl2:EtOH (4:1) several times until no product was detected by TLC in brine layer. The organic layers were combined, concentrated under vacuum, and purified on a short silica gel column, eluting with CH2Cl2 to 25% MeOH/CH2Cl2 with 1% NH4OH, (82.5 95% yield).
—
2-(2-Methyl-pyrazolo[1,5-a]pyridin-3-yl)-ethylamine (24i). The title compound (820 mg, 95% yield) was prepared according to same procedure as above from 23i (1.0 g, 4.9 mmol). 1H NMR (400 MHz, CD3OD) δ 8.69 (dt, J = 7.0, 0.9 Hz, 1 H), 8.00 (dt, J = 9.1, 1.2 Hz, 1 H), 7.76 (ddd, J = 9.0,
—
7.1, 1.0 Hz, 1 H), 7.40 (ddd, J = 7.7, 6.3, 0.6 Hz, 1 H), 3.36 3.31 (m, 2 H),
—
3.23 3.18 (m, 2 H), 2.61 (s, 3 H). MS m/z 176.1 (MH+).
2-(2-Ethyl-pyrazolo[1,5-a]pyridin-3-yl)-ethylamine (24j). The title compound (5.75 g, 85% pure, 82.5% yield) was prepared according to same procedure as above from 23i (6.8 g, 31 mmol) and used in the next step without further purification. 1H NMR (400 MHz, CD3OD) δ 8.43 (dt, J = 7.1, 1.1 Hz, 1 H), 7.60 (dt, J = 8.8, 1.3 Hz, 1 H), 7.24 (ddd, J = 8.9,
—
6.8, 1.0 Hz, 1 H), 6.86 (ddd, J = 7.6, 6.0, 0.7 Hz, 1 H), 3.35 3.31 (m, 2
—
H), 3.14 3.09 (m, 2 H), 2.86 (q, J = 7.4 Hz, 2 H), 1.36 (t, J = 7.8 Hz, 3 H). MS m/z 190.1 (MH+).
—
2-(2-Phenyl-pyrazolo[1,5-a]pyridin-3-yl)-ethylamine (24k). The ti- tle compound (700 mg, 78% yield) was prepared according to same procedure as above from 23k (1.0 g, 3.8 mmol). 1H NMR (400 MHz, CD3OD) δ 8.55 (dt, J = 7.1, 1.1 Hz, 1 H), 7.76 7.70 (m, 3 H),
—
7.57 7.53 (m, 2 H), 7.49 (tt, J = 7.3, 1.8 Hz, 1 H), 7.33 (ddd, J = 9.1, 6.8,
1.1 Hz, 1 H), 6.97 (ddd, J = 7.5, 6.2, 0.7 Hz, 1 H), 3.30 (t, J = 8.7 Hz, 2 H),
3.09 (t, J = 8.0 Hz, 2 H). MS m/z 238.0 (MH+).
(E)-N-Hydroxy-3-(4-{[2-(1H-pyrrolo[3,2-b]pyridin-3-yl)-ethylamino]- methyl}-phenyl)-acrylamide (25a). Following method A, (E)-3-(4-
—
{[2-(1H-pyrrolo[3,2-b]pyridin-3-yl)-ethylamino]-methyl}-phenyl)-acrylic acid methyl ester (200 mg, 48% yield) was prepared from 20a (200 mg, 1.2 mmol). 1H NMR (400 MHz, CD3OD) δ 8.27 (dd, J = 4.7, 1.4 Hz, 1 H), 7.86 (dd, J = 8.2, 1.4 Hz, 1 H), 7.75 (s, 1 H), 7.72 7.69 (m, 2 H), 7.56
(d, J = 8.3 Hz, 2 H), 7.51 (s, 1 H), 7.22 (dd, J = 8.1, 4.7 Hz, 1 H), 6.61 (d,
J = 15.9 Hz, 1 H), 4.24 (s, 2 H), 3.81 (s, 3 H), 3.33 (t, J = 6.1 Hz, 2 H), 3.22 (t, J = 6.5 Hz, 2 H). MS m/z 336.2 (MH+).
The title compound (10 mg, 5.5% yield) was prepared according to method B from (E)-3-(4-{[2-(1H-pyrrolo[3,2-b]pyridin-3-yl)- ethylamino]-methyl}-phenyl)-acrylic acid methyl ester (190 mg, 0.54 mmol). 1H NMR (400 MHz, CD3OD) δ 8.28 (dd, J = 4.7, 1.4 Hz, 1 H), 7.80 (dd, J = 8.3, 1.4 Hz, 1 H), 7.55 (d, J = 16.0 Hz, 1 H), 7.51 (d, J = 8.3
Hz, 2 H), 7.43 (s, 1 H), 7.35 (d, J = 8.6 Hz, 2 H), 7.17 (dd, J = 8.3, 4.9 Hz,
1 H), 6.46 (d, J = 16.3 Hz, 1 H), 3.87 (s, 2 H), 3.10 (t, J = 6.8 Hz, 2 H),
3.01 (t, J = 6.7 Hz, 2 H). MS m/z 336.9 (MH+). HRMS calcd for C19H20N4O2 (M—) 335.1508, found 335.1518.
(E)-N-Hydroxy-3-(4-{[2-(6-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)- ethylamino]-methyl}-phenyl)-acrylamide (25b). Following method A, (E)-3-(4-{[2-(6-methyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-ethylamino]- methyl}-phenyl)-acrylic acid methyl ester (60 mg, 21% yield) was prepared from 20b (140 mg, 0.80 mmol). 1H NMR (400 MHz, CD3OD) δ 8.76 (s, 1 H), 8.61 (s, 1 H), 7.68 (d, J = 16.2 Hz, 1 H), 7.55 (d, J = 8.3 Hz,
2 H), 7.35 (d, J = 8.4 Hz, 2 H), 6.52 (d, J = 15.8 Hz, 1 H), 3.82 (s, 2 H),
3.80 (s, 3 H), 2.96 (t, J = 7.6 Hz, 2 H), 2.85 (t, J = 7.5 Hz, 2 H), 2.43 (s, 3 H). MS m/z 351.1 (MH+).
The title compound (4 mg, 7% yield) was prepared according to method B from (E)-3-(4-{[2-(6-methyl-7H-pyrrolo[2,3-d]pyrimidin-5- yl)-ethylamino]-methyl}-phenyl)-acrylic acid methyl ester (60 mg, 0.17 mmol). 1H NMR (400 MHz, DMSO-d6) δ 8.81 (s, 1 H), 8.60 (s, 1 H),
7.48 (d, J = 8.2 Hz, 2 H), 7.42 (d, J = 15.9 Hz, 1 H), 7.33 (d, J = 7.8 Hz, 2
H), 6.43 (d, J = 16.1 Hz, 1 H), 3.75 (s, 2 H), 2.83 (t, J = 6.6 Hz, 2 H), 2.73 (t, J = 7.0 Hz, 2 H), 2.34 (s, 3 H). MS m/z 351.8 (MH+). HRMS calcd for C19H21N5O2 (MH+) 352.1774, found 352.1784.
(E)-N-Hydroxy-3-(4-{[2-(6-methyl-5H-pyrrolo[2,3-b]pyrazin-7-yl)- ethylamino]-methyl}-phenyl)-acrylamide (25c). Following method A,

(E)-3-(4-{[2-(6-methyl-5H-pyrrolo[2,3-b]pyrazin-7-yl)-ethylamino]- methyl}-phenyl)-acrylic acid methyl ester (130 mg, 41% yield) was prepared from 20c (160 mg, 0.91 mmol). 1H NMR (400 MHz,
CD3OD) δ 8.76 (s, 1 H), 8.61 (s, 1 H), 7.68 (d, J = 16.2 Hz, 1 H),
7.54 (d, J = 7.9 Hz, 2 H), 7.34 (d, J = 8.1 Hz, 2 H), 6.51 (d, J = 16.0 Hz,
1 H), 3.82 (s, 2 H), 3.80 (s, 3 H), 2.96 (t, J = 7.3 Hz, 2 H), 2.85 (t, J = 7.1
Hz, 2 H), 2.42 (s, 3 H). MS m/z 350.8 (MH+).
The title compound (33 mg, 25% yield) was prepared according to method B from (E)-3-(4-{[2-(6-methyl-5H-pyrrolo[2,3-b]pyrazin-7- yl)-ethylamino]-methyl}-phenyl)-acrylic acid methyl ester (130 mg, 0.37 mmol). 1H NMR (400 MHz, DMSO-d6) δ 8.80 (s, 1 H), 8.60 (s, 1 H), 7.47 (d, J = 8.1 Hz, 2 H), 7.42 (d, J = 15.2 Hz, 1 H), 7.33 (d, J =
8.7 Hz, 2 H), 6.42 (d, J = 15.5 Hz, 1 H), 3.74 (s, 2 H), 2.82 (t, J = 6.7 Hz,
2 H), 2.71 (t, J = 7.2 Hz, 2 H), 2.33 (s, 3 H). 13C NMR (400 MHz,
CD3OD) δ 166.35, 152.32, 150.57, 146.53, 141.23, 136.83, 135.35,
130.18, 128.96, 128.28, 120.93, 118.36, 108.64, 53.75, 50.03, 24.51,
11.30. MS m/z 351.7 (MH+). HRMS calcd for C19H21N5O2 (MH+) 352.1774, found 352.1775.
(E)-N-Hydroxy-3-(4-{[2-(1H-pyrrolo[3,2-c]pyridin-3-yl)-ethylamino]- methyl}-phenyl)-acrylamide (25d). Following method A, (E)-3-(4-
{[2-(1H-pyrrolo[3,2-c]pyridin-3-yl)-ethylamino]-methyl}-phenyl)-acrylic acid methyl ester (270 mg, 65% yield) was prepared from 20d (200 mg, 1.2 mmol). 1H NMR (400 MHz, CD3OD) δ 8.80 (s, 1 H), 8.14 (d, J =
6.2 Hz, 1 H), 7.68 (d, J = 16.1 Hz, 1 H), 7.58 (d, J = 7.9 Hz, 2 H),
—
7.43 7.39 (m, 3 H), 7.27 (s, 1 H), 6.53 (d, J = 16.2 Hz, 1 H), 3.96 (s, 2 H),
— —
3.80 (s, 3 H), 3.33 3.32 (m, 2 H), 2.13 2.06 (m, 2 H). MS m/z 336.1 (MH+).
The title compound (20 mg, 7.8% yield) was prepared according to method B from (E)-3-(4-{[2-(1H-pyrrolo[3,2-c]pyridin-3-yl)- ethylamino]-methyl}-phenyl)-acrylic acid methyl ester (270 mg, 0.77 mmol). 1H NMR (400 MHz, DMSO-d6) δ 8.79 (s, 1 H), 8.12 (d, J = 5.8 Hz, 1 H), 7.48 (d, J = 7.8 Hz, 2 H), 7.43 (d, J = 16.0 Hz, 1 H), 7.36 (d, J =
8.2 Hz, 2 H), 7.30 (dd, J = 5.6, 1.2 Hz, 1 H), 7.21 (s, 1 H), 6.43 (d, J =
17.2 Hz, 1 H), 3.77 (s, 2 H), 2.91 (t, J = 7.3 Hz, 2 H), 2.82 (t, J = 7.0 Hz, 2 H). 13C NMR (400 MHz, CD3OD) δ 166.33, 142.06, 141.99, 141.91,
141.19, 140.12, 135.33, 130.18, 128.95, 125.76, 125.63, 118.34, 114.21,
108.25, 53.72, 50.19, 25.68. MS m/z 337 (MH+). HRMS calcd for
C19H20N4O2 (MH+) 337.1665, found 337.1657.
(E)-3-(4-{[2-(2-tert-Butyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-ethylamino]- methyl}-phenyl)-N-hydroxy-acrylamide (25f). Following method A, 20f (524 mg, 2.03 mmol, approximately 84% pure) was converted into (E)-3-(4-{[2-(2-tert-butyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-ethylamino]- methyl}-phenyl)-acrylic acid methyl ester (392 mg, 1.0 mmol, 49% yield). 1H NMR (DMSO-d6) δ 10.99 (br s, 1H), 8.09 (dd, J = 1.5, 5.0 Hz,
1H), 7.77 (d, J = 7.8 Hz, 1H) 7.66 (m, 3H), 7.40 (d, J = 7.9 Hz, 2H), 6.95
(dd, J = 4.8, 7.8 Hz, 1H), 6.61 (d, J = 15.6 Hz, 1H), 3.83 (s, 2H), 3.73 (s,
3H), 2.99 (m, 2H), 2.73 (m, 2H), 1.40 (s, 9H). m/z 392.0 (MH+).
Following method B, (E)-3-(4-{[2-(2-tert-butyl-1H-pyrrolo[2,3- b]pyridin-3-yl)-ethylamino]-methyl}-phenyl)-acrylic acid methyl ester (400 mg, 1.02 mmol) was converted to 25f (52 mg, 0.13 mmol, 13% yield) after HPLC puriﬁcation. 1H NMR (CD3OD) δ 8.07 (d, J = 4.7 Hz, 1H) 7.82 (d, J = 8.0 Hz, 1H), 7.51 (m, 3H), 7.35 (d, J = 8.8 Hz, 2H),
6.99 (dd, J = 5.0, 8.0 Hz, 1H), 6.47 (d, J = 15.9 Hz, 1H), 3.82 (s, 2H),
3.09 (m, 2H), 2.80 (m, 2H), 1.46 (s, 9H). 13C NMR (CD3OD) δ 166.0,
148.3, 146.1, 142.7, 140.7, 136.9, 131.1, 129.4, 127.2, 123.5, 119.5, 116.4,
104.6, 52.5, 34.7, 31.1, 24.1. m/z 393.2 (MH+). HRMS (MH+) calcd for C23H28N4O2, 393.2291, found 393.2291.
(E)-N-Hydroxy-3-{4-[(2-imidazo[1,2-a]pyridin-3-yl-ethylamino)-
methyl]-phenyl}-acrylamide (25g). (E)-3-{4-[(2-Imidazo[1,2-a]- pyridin-3-yl-ethylamino)-methyl]-phenyl}-acrylic acid methyl ester (50 mg, 17% yield) was prepared according to general method A from 20g (140 mg, 0.87 mmol). MS m/z 335.7 (MH+).
The title compound (13 mg, 26% yield) was prepared according to general procedure method B from (E)-3-{4-[(2-imidazo[1,2-a]pyridin-
3-yl-ethylamino)-methyl]-phenyl}-acrylic acid methyl ester (50 mg,
—
—
0.15 mmol). 1H NMR (400 MHz, CD3OD) δ 8.24 (d, J = 7.7 Hz, 1 H), 7.57 7.49 (m, 4 H), 7.39 (d, J = 14.4 Hz, 1 H), 7.36 (d, J = 8.0 Hz, 2 H), 7.30 (t, J = 7.8 Hz, 1 H), 6.96 6.92 (m, 1 H), 6.46 (d, J = 15.5 Hz, 1 H), 3.87 (s, 2 H), 3.17 (t, J = 7.7 Hz, 2 H), 3.01 (t, J = 7.4 Hz, 2 H). 13C NMR (400 MHz, CD3OD) δ 166.30, 146.67, 141.44, 141.19, 135.53, 131.21, 130.31, 129.03, 126.09, 125.17, 123.60, 118.47, 117.70, 113.85, 53.61, 46.96, 24.27. MS m/z 336.7 (MH+). HRMS calcd for C19H20N4O2 (MH+) 337.1665, found 337.1652.
(E)-N-Hydroxy-3-(4-{[2-(2-methyl-pyrazolo[1,5-a]pyridin-3-yl)-
ethylamino]-methyl}-phenyl)-acrylamide (25i). (E)-3-(4-{[2-(2- Methyl-pyrazolo[1,5-a]pyridin-3-yl)-ethylamino]-methyl}-phenyl)- acrylic acid methyl ester (510 mg, 31% yield) was prepared according to general procedure method A from 24i (820 mg, 4.7 mmol). 1H NMR (400 MHz, CDCl3) δ 8.28 (d, J = 6.7 Hz, 1 H), 7.53 (d, J = 16.6 Hz, 1 H),
—
7.44 (d, J = 7.2 Hz, 2 H), 7.40 7.37 (m, 3 H), 7.02 (dd, J = 8.9, 6.8 Hz,
1 H), 6.63 (t, J = 7.0 Hz, 1 H), 6.32 (d, J = 16.3 Hz, 1 H), 3.81 (s, 3 H),
—
3.70 (s, 2 H), 3.00 2.98 (m, 4 H), 2.39 (s, 3 H). MS m/z 349.9 (MH+).
The title compound (50 mg, 25% yield) was prepared according to method B from (E)-3-(4-{[2-(2-methyl-pyrazolo[1,5-a]pyridin-3-yl)- ethylamino]-methyl}-phenyl)-acrylic acid methyl ester (200 mg, 0.57 mmol). 1H NMR (400 MHz, CD3OD) δ 8.38 (d, J = 7.1, 1 H), 7.63 (d, J = 7.6 Hz, 2 H), 7.61 (d, J = 9.6, 1 H), 7.54 (dd, J = 13.1, 3.5 Hz, 1 H),
7.50 (d, J = 7.6, 2 H), 7.20 (dd, J = 7.6, 7.1 Hz, 1 H), 6.81 (t, J = 7.1 Hz,
1 H), 6.52 (d, J = 15.6 Hz, 1 H), 4.18 (s, 2 H), 3.12 (m, 4 H), 2.42 (s,
3 H). 13C NMR (400 MHz, CD3OD) δ 166.2, 150.89, 141.00, 140.75,
140.05, 135.87, 130.52, 129.09, 128.83, 124.65, 118.72, 117.11, 112.69,
106.03, 53.31, 49.71, 22.90, 11.81. MS m/z 351.0 (MH+). HRMS calcd for C12H25N4O2 (MH+) 351.1821, found 351.1831.
(E)-3-(4-{[2-(2-Ethyl-pyrazolo[1,5-a]pyridin-3-yl)-ethylamino]-
methyl}-phenyl)-N-hydroxy-acrylamide (25j). (E)-3-(4-{[2-(2-Ethyl- pyrazolo[1,5-a]pyridin-3-yl)-ethylamino]-methyl}-phenyl)-acrylic acid methyl ester (1.0 g, 99% yield) was prepared according to general procedure method A from 24j (530 mg, 2.8 mmol). 1H NMR (400 MHz, CDCl3) δ 8.33 (dt, J = 7.0, 1.2 Hz, 1 H), 7.51 (d, J = 15.9 Hz, 1 H),
— — —
7.48 7.39 (m, 5 H), 7.07 7.03 (m, 1 H), 6.69 6.64 (m, 1 H), 6.31 (d,
J = 15.9 Hz, 1 H), 3.82 (s, 3 H), 3.71 (s, 2 H), 3.45 (t, J = 6.4 Hz, 2 H),
2.92 (t, J = 7.0 Hz, 2 H), 2.75 (q, J = 7.7 Hz, 2 H), 1.28 (t, J = 7.7 Hz, 3 H). MS m/z 363.9 (MH+).
—
The title compound (47 mg, 47% yield) was prepared according to general procedure method B from (E)-3-(4-{[2-(2-ethyl-pyrazolo[1,5- a]pyridin-3-yl)-ethylamino]-methyl}-phenyl)-acrylic acid methyl ester (100 mg, 0.27 mmol). 1H NMR (400 MHz, CD3OD) δ 8.43 (d, J = 7.0, 1 H), 7.68 (d, J = 8.1 Hz, 2 H), 7.63 7.57 (m, 4 H), 7.23 (dd, J = 8.7, 6.8
Hz, 1 H), 6.85 (td, J = 6.8, 1.0 Hz, 1 H), 6.56 (d, J = 16.0 Hz, 1 H), 4.31
—
(s, 2 H), 3.25 3.16 (m, 4 H), 2.85 (q, J = 7.7 Hz, 2 H), 1.34 (t, J = 7.6 Hz, 3 H). 13C NMR (400 MHz, CD3OD) δ 165.94, 156.47, 140.82, 140.48,
134.16, 131.7, 129.5, 129.12, 125.11, 119.94, 117.07, 113.08, 103.05,
51.97, 49.75, 20.88, 20.63, 14.57. HRMS C12H25N4O2 (MH+). calcd
365.1978, found 365.1985.
(E)-N-Hydroxy-3-(4-{[2-(2-phenyl-pyrazolo[1,5-a]pyridin-3-yl)-
—
—
ethylamino]-methyl}-phenyl)-acrylamide (25k). (E)-3-(4-{[2-(2- Phenyl-pyrazolo[1,5-a]pyridin-3-yl)-ethylamino]-methyl}-phenyl)- acrylic acid methyl ester (640 mg, 53% yield) was prepared according to general procedure method A from 24k (700 mg, 3.0 mmol). 1H NMR (400 MHz, CDCl3) δ 8.44 (d, J = 6.9 Hz, 1 H), 7.74 7.70 (m, 3 H), 7.52 7.41 (m, 4 H), 7.38 (d, J = 7.6 Hz, 2 H), 7.29 (d, J = 7.9
Hz, 2 H), 7.11 (dd, J = 8.8, 6.8 Hz, 1 H), 6.76 (ddd, J = 7.5, 6.2, 0.7 Hz,
1 H), 6.28 (d, J = 16.1 Hz, 1 H), 3.86 (s, 3 H), 3.83 (s, 2 H), 3.09 (t, J =
7.9 Hz, 2 H), 2.94 (t, J = 7.5 Hz, 2 H). MS m/z 412.1 (MH+).
The title compound (35 mg, 35% yield) was prepared according to general procedure method B from (E)-3-(4-{[2-(2-phenyl-pyrazolo- [1,5-a]pyridin-3-yl)-ethylamino]-methyl}-phenyl)-acrylic acid methyl ester (100 mg, 0.24 mmol). 1H NMR (400 MHz, CD3OD) δ 8.48

—
(dt, J = 7.0, 1.1 Hz, 1 H), 7.70 7.67 (m, 2 H), 7.62 (dt, J = 9.2, 1.2 Hz,
—
1 H), 7.54 7.43 (m, 6 H), 7.27 (d, J = 8.0 Hz, 2 H), 7.21 (ddd, J = 9.0,
6.6, 1.2 Hz, 1 H), 6.89 (dt, J = 9.7, 3.5 Hz, 1 H), 6.47 (d, J = 15.8 Hz, 1 H),
3.74 (s, 2 H), 3.13 (t, J = 7.8 Hz, 2 H), 2.78 (t, J = 7.8 Hz, 2 H). 13C NMR (400 MHz, CD3OD) δ 166.35, 153.31, 142.23, 141.45, 141.14, 135.23, 134.58, 131.05, 130.07, 129.78, 129.47, 129.07, 128.92, 124.66, 118.35,
118.03, 113.71, 106.78, 53.54, 49.86, 24.03. MS m/z 412.9 (MH+). HRMS calcd for C25H24N4O2 (M—) 411.1821, found 411.1830.
(E)-N-Hydroxy-3-[4-({isopropyl-[2-(2-methyl-pyrazolo[1,5-a]pyridin-
3-yl)-ethyl]-amino}-methyl)-phenyl]-acrylamide (25l). (E)-3-[4- ({Isopropyl-[2-(2-methyl-pyrazolo[1,5-a]pyridin-3-yl)-ethyl]-amino}- methyl)-phenyl]-acrylic acid methyl ester (400 mg, 70% yield) was prepared according to same procedure as the intermediate for 25m below from (E)-3-(4-{[2-(2-methyl-pyrazolo[1,5-a]pyridin-3- yl)-ethylamino]-methyl}-phenyl)-acrylic acid methyl ester (900 mg, 2.48 mmol). 1H NMR (400 MHz, CDCl3) δ 8.27 (dt, J = 7.0, 1.2 Hz, 1 H),
7.68 (d, J = 16.7 Hz, 1 H), 7.40 (d, J = 7.5 Hz, 2 H), 7.30 (d, J = 8.0 Hz, 2
H), 7.09 (dt, J = 8.8, 1.2 Hz, 1 H), 6.89 (ddd, J = 8.8, 6.7, 1.1 Hz, 1 H), 6.56
(td, J = 6.9, 1.4 Hz, 1 H), 6.41 (d, J = 16.0 Hz, 1 H), 3.81 (s, 3 H), 3.62 (s,
2 H), 3.05 (sept, J = 6.5 Hz, 1 H), 2.69 (t, J = 7.6 Hz, 2 H), 2.56 (t, J = 7.9
Hz, 2 H), 2.32 (s, 3 H), 1.05 (d, J = 6.3 Hz, 6 H). MS m/z 391.9 (MH+).
The title compound (280 mg, 72% yield) was prepared according to method B from (E)-3-[4-({isopropyl-[2-(2-methyl-pyrazolo[1,5- a]pyridin-3-yl)-ethyl]-amino}-methyl)-phenyl]-acrylic acid methyl es- ter (390 mg, 0.99 mmol). 1H NMR (400 MHz, CD3OD) δ 8.29 (dt, J = 7.0, 1.1 Hz, 1 H), 7.48 (d, J = 16.1 Hz, 1 H), 7.35 (d, J = 8.3 Hz, 2 H),
—
7.21 7.18 (m, 3 H), 7.00 (ddd, J = 8.9, 6.7, 1.0 Hz, 1 H), 6.70 (dt, J = 9.5,
3.5 Hz, 1 H), 6.44 (d, J = 16.0 Hz, 1 H), 3.60 (s, 2 H), 3.13 (sept, J = 6.6
Hz, 1 H), 2.72 (t, J = 7.1 Hz, 2 H), 2.61 (t, J = 7.2 Hz, 2 H), 2.27 (s, 3 H),
1.11 (d, J = 6.5 Hz, 6 H). MS m/z 393.0 (MH+). HRMS calcd for C23H28N4O2 (MH+) 393.2291, found 39.2289.
(E)-3-[4-({[2-(2-Ethyl-pyrazolo[1,5-a]pyridin-3-yl)-ethyl]-isopropyl- amino}-methyl)-phenyl]-N-hydroxy-acrylamide (25m). (E)-3-(4-
{[2-(2-Ethyl-pyrazolo[1,5-a]pyridin-3-yl)-ethylamino]-methyl}-phenyl)- acrylic acid methyl ester (900 mg, 2.48 mmol) was added to a solution of 2-iodo-propane (4.21 g, 24.8 mmol) and triethylamine (3.44 mL,
—
24.8 mmol) in CH3CN (20 mL). The mixture was refluxed overnight. The solvent was then removed under vacuum. EtOAc and water were added. The aqueous layer was extracted several times with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and evaporated off. The crude product was purified via flash column chroma- tography (EtOAc:hexanes, 10:90 to 100:0) to provide (E)-3-[4-({[2-(2- ethyl-pyrazolo[1,5-a]pyridin-3-yl)-ethyl]-isopropyl-amino}-methyl)- phenyl]-acrylic acid methyl ester as a slightly yellow oil (300 mg, 30% yield). 1H NMR (400 MHz, CDCl3) δ 8.30 (dt, J = 7.0, 1.2 Hz, 1 H), 7.69 (d, J = 16.1 Hz, 1 H), 7.42 (d, J = 7.9 Hz, 2 H), 7.34 (d, J = 8.0 Hz, 2 H), 7.11 (dt, J = 8.8, 1.3 Hz, 1 H), 6.90 (ddd, J = 8.9, 6.6, 1.2 Hz,1 H), 6.57 (ddd, J = 7.8, 6.0, 0.7 Hz, 1 H), 6.42 (d, J = 15.9 Hz, 1 H), 3.81 (s, 3 H), 3.64 (s, 2 H), 3.06 (sept, J = 6.6 Hz, 1 H), 2.72 2.66 (m, 4 H), 2.56 (t, J = 8.1 Hz, 2 H), 1.24 (t, J = 7.6 Hz, 3 H), 1.07 (d, J = 6.8 Hz, 6 H). MS m/z 406.6 (MH+).
The title compound (160 mg, 54% yield) was prepared according to the general procedure method B from (E)-3-[4-({[2-(2-ethyl-pyrazolo- [1,5-a]pyridin-3-yl)-ethyl]-isopropyl-amino}-methyl)-phenyl]-acrylic acid methyl ester (300 mg, 0.73 mmol). 1H NMR (400 MHz, CD3OD) δ 8.30 (d, J = 6.9 Hz, 1 H), 7.54 (d, J = 15.8 Hz, 1 H), 7.40 (d, J = 8.3 Hz,
2 H), 7.26 (d, J = 7.8 Hz,2 H), 7.21 (d, J = 8.7 Hz, 1 H), 7.01 (ddd, J =
8.9, 6.7, 1.0 Hz, 1 H), 6.70 (dt, J = 9.5, 3.5 Hz, 1 H), 6.43 (d, J = 15.8 Hz,
1 H), 3.66 (s, 2 H), 3.18 (sept, J = 6.4 Hz, 1 H), 2.72 (t, J = 7.2 Hz, 2 H),
—
2.66 2.60 (m, 4 H), 1.19 (t, J = 7.6 Hz, 3H), 1.14 (d, J = 6.4 Hz, 6H).
13C NMR (400 MHz, CD3OD) δ 166.48, 155.99, 141.52, 140.59,
134.96, 130.65, 128.71, 128.63, 123.98, 117.96, 117.47, 112.45, 107.15,
54.77, 52.10, 51.68, 23.36, 20.64, 18.36, 14.63. MS m/z 407.0 (MH+). HRMS C24H30N4O2 (M—) calcd 405.2291, found 405.2323
(E)-N-Hydroxy-3-[4-({isopropyl-[2-(2-phenyl-pyrazolo[1,5-a]pyridin-
⦁ yl)-ethyl]-amino}-methyl)-phenyl]-acrylamide (25n). (E)-3-[4- ({Isopropyl-[2-(2-phenyl-pyrazolo[1,5-a]pyridin-3-yl)-ethyl]-amino}- methyl)-phenyl]-acrylic acid methyl ester (300 mg, 52% yield) was prepared according to same procedure as above from (E)-3-(4-{[2-(2- phenyl-pyrazolo[1,5-a]pyridin-3-yl)-ethylamino]-methyl}-phenyl)-ac- rylic acid methyl ester (520 mg, 1.26 mmol). 1H NMR (400 MHz, CDCl3) δ 8.39 (dt, J = 7.1, 1.1 Hz, 1 H), 7.69 (d, J = 14.5 Hz, 1 H),
— —
7.66 7.64 (m, 2 H), 7.42 7.37 (m, 5 H), 7.27 (d, J = 8.6 Hz, 2 H), 7.20
(dt, J = 9.0, 1.4 Hz, 1 H), 6.96 (ddd, J = 8.9, 6.6, 1.1 Hz, 1 H), 6.67 (ddd,
J = 7.5, 6.3, 0.7 Hz, 1 H), 6.42 (d, J = 15.8 Hz, 1 H), 3.82 (s, 3 H), 3.57 (s,
2 H), 2.99 (sept, J = 5.2 Hz, 1 H), 2.92 (t, J = 7.6 Hz, 2 H), 2.62 (t, J = 7.8
Hz, 2 H), 1.00 (d, J = 6.4 Hz, 6 H). MS m/z 453.9 (MH+).
The title compound (270 mg, 92% yield) was prepared according to the general procedure method B from (E)-3-[4-({isopropyl-[2-(2- phenyl-pyrazolo[1,5-a]pyridin-3-yl)-ethyl]-amino}-methyl)-phenyl]- acrylic acid methyl ester (300 mg, 0.66 mmol). 1H NMR (400 MHz, CD3OD) δ 8.41 (dt, J = 7.0, 1.1 Hz,1 H), 7.62 (dt, J = 6.0, 2.0 Hz, 2 H),
—
7.47 7.40 (m, 0 H), 7.34 (d, J = 8.0 Hz, 2 H), 7.30 (dt, J = 8.9, 1.3 Hz,
1 H), 7.15 (d, J = 8.1 Hz, 2 H), 7.08 (ddd, J = 9.0, 6.7, 1.0 Hz,1 H), 6.82
(dt, J = 9.7, 3.5 Hz, 1 H), 6.47 (d, J = 15.8 Hz, 1 H), 3.53 (s, 2 H), 3.02
(sept, J = 6.6 Hz, 1 H), 2.93 (t, J = 7.2 Hz, 2 H), 2.60 (t, J = 7.7 Hz, 2 H), 1.00 (d, J = 6.5 Hz, 6 H). 13C NMR (400 MHz, CD3OD) δ 166.56,
153.26, 144.31, 141.67, 141.3, 134.79, 130.36, 129.79, 129.67, 129.62,
129.35, 129.30,128.94, 128.9, 128.51, 128.41, 126.34, 124.18, 118.13,
117.73, 113.46, 108.14, 54.69, 54.43, 51.47, 51.34, 23.83, 18.28. HRMS calcd for C28H30N4O2 (MH+) 455.2447, found 455.2442.

⦁ ASSOCIATED CONTENT
bS Supporting Information. Scatter plot of clogP and hERG radioligand displacement IC50. This material is available free of charge via the Internet at http://pubs.acs.org.
⦁ AUTHOR INFORMATION
Corresponding Author
*Phone: 617-871-7551. Fax: 617-871-4081. E-mail: michael. [email protected].
Present Addresses
†Department of Chemistry, Massachusetts Institute of Technol-
ogy, Cambridge, Massachusetts, United States.
‡Alkermes, 852 Winter Street, Waltham, Massachusetts 02451, United States.
§Memorial Sloan Kettering Cancer Center, New York, New York,
)
United States.
WuXi AppTec, Shanghai, China.
^Concert Pharmaceuticals Inc., 99 Hayden Avenue, Lexington Massachusetts, 02421, United States.

⦁ ABBREVIATIONS USED
CSI, cardiac safety index; fwhm, full width at half-maximum; H1299, human nonsmall cell lung cancer cell line; HCT-116, human colorectal carcinoma cell line; HDAC, histone deacety- lase; hERG, human ether-a-go-go related gene product; iCSI, in vitro cardiac safety index; PK, pharmacokinetic; SAHA, suberoy- lanilide hydroxamic acid; SAR, structure—activity relationships
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