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Design, Synthesis and Biological Activity of Substrate Competitive SMYD2 Inhibitors Scott D. Cowen, Daniel Russell, Leslie A. Dakin, Huawei Ray Chen, Nicholas A. Larsen, Robert Godin, Scott Throner, Xiaolan Zheng, Audrey Molina, Jiaquan Wu, Tony Cheung, Tina Howard, Renee GarciaArenas, Nicholas Keen, Christopher S. Pendleton, Jennifer A. Pietenpol, and Andrew D. Ferguson J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01303 • Publication Date (Web): 14 Nov 2016 Downloaded from http://pubs.acs.org on November 15, 2016

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Journal of Medicinal Chemistry

Design, Synthesis and Biological Activity of Substrate Competitive SMYD2 Inhibitors Scott D. Cowen,†,* Daniel Russell,† Leslie A. Dakin,† Huawei Chen,‡ Nicholas A. Larsen,§ Robert Godin,‡ Scott Throner,† Xiaolan Zheng,† Audrey Molina,† Jiaquan Wu,‡ Tony Cheung,‡ Tina Howard,ǁ Renee Garcia-Arenas,‡ Nicholas Keen,‡ Christopher S. Pendleton,⊥ Jennifer A. Pietenpol,⊥ and Andrew D. Ferguson§,* †

Department of Chemistry, ‡ Department of Bioscience, Oncology Innovative Medicines Unit, §

Structure and Biophysics, Discovery Sciences, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, United States ǁ

Structure and Biophysics, Discovery Sciences, AstraZeneca, Mereside, Alderley Park,

Cheshire, SK10 4TG United Kingdom ⊥

Department of Biochemistry, Vanderbilt-Ingram Cancer Center, Vanderbilt University,

Nashville, TN 37232 United States Correspondence: [email protected] Supporting Information included with this manuscript

ACS Paragon Plus Environment


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ABSTRACT: Protein lysine methyltransferases (KMTs) have emerged as important regulators of epigenetic signaling. These enzymes catalyze the transfer of donor methyl groups from the cofactor S-adenosylmethionine to specific acceptor lysine residues on histones, leading to changes in chromatin structure and transcriptional regulation. These enzymes also methylate an array of non-histone proteins, suggesting additional mechanisms by which they influence cellular physiology.

SMYD2 is reported to be an oncogenic methyltransferase that represses the

functional activity of the tumor suppressor proteins p53 and RB.

HTS screening led to

identification of five distinct substrate-competitive chemical series. Determination of liganded crystal structures of SMYD2 contributed significantly to ‘hit-to-lead’ design efforts, culminating in the creation of potent and selective inhibitors that were used to understand the functional consequences of SMYD2 inhibition. Taken together these results have broad implications for inhibitor design against KMTs, and clearly demonstrate the potential for developing novel therapies against these enzymes.


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INTRODUCTION Protein lysine methyltransferases (KMTs) are important regulators of epigenetic signaling. This enzyme class catalyzes the transfer of donor methyl groups from the cofactor Sadenosylmethionine to specific acceptor lysine residues on histones, resulting in discrete patterns of mono, di- or tri-methylation on target substrates. Methylation marks act as recognition elements for additional effector proteins that induce changes in chromatin structure and transcriptional regulation. KMTs are also known to methylate numerous non-histone protein substrates, revealing additional mechanisms to regulate cellular physiology, and this area has been the subject of several reviews1. The KMT protein, SMYD2, has been implicated in a variety of cancers. Komatsu et al2 have shown that overexpression of SMYD2 is observed in KYSE150 cells with amplification at 1q3241.1 and in other esophageal squamous cell carcinoma (ESCC) cell lines. Overexpression of SMYD2 messenger RNA and protein has also been detected in cell lines and primary tumor tissues2. This study also showed that patients with SMYD2-overexpressing ESCC tumors had a lower survival rate when compared to those with non-SMYD2 expressing ESCC tumors. Knockdown of SMYD2 gene expression inhibits ESCC cell proliferation in a TP53 mutationindependent manner3. p53 is a transcription factor that functions as a tumor suppressor that regulates cell cycle arrest through both transcription-dependent and independent signaling pathways.

Inactivating

mutations in the p53 gene are observed in roughly 50% of all human cancers.

SMYD2

preferentially methylates residue K370 of p53 when compared to histone substrates. Endogenous levels of monomethylated p53 are significantly elevated when SMYD2 is



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overexpressed in vivo3, which decreases the ability of p53 to transcriptionally activate target genes involved in cell cycle arrest. These findings suggest that SMYD2 inhibition may provide a potential therapeutic option for cancers that utilize SMYD2 to inactivate p53 signaling. Recent studies have shown that SMYD2 acts upon additional non-histone targets, including estrogen receptor α (ERα)4, heat shock protein 90 (HSP90)5, poly (ADP-ribose) polymerase 1 (PARP1)6 and the retinoblastoma tumor suppressor (RB)7. These target proteins are involved in a variety of cellular processes, including epigenetic gene regulation, cellular differentiation, apoptosis, the DNA damage response and cell cycle progression.

In each of these cases,

evidence has been presented that SMYD2-mediated methylation represses the functional activities of these proteins. A recent SILAC-based proteomics study8 indicates that the number of target proteins that are methylated by SMYD2 is even larger than previously appreciated, thus the ability to discern which SMYD2 substrates may contribute to a given cellular phenotype has become increasingly complex. Here we report the discovery and ‘hit-to-lead’ efforts leading to the development of potent and selective SMYD2 inhibitors (Figure 1) that will serve as valuable tools to further elucidate the physiological role of SMYD2.


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Figure 1. Chemical structures of SMYD2 inhibitors9,10

RESULTS AND DISCUSSION Screening. We undertook an internal hit identification campaign to identify chemical equity against a panel of histone methyltransferases using a combination of high throughput screening and high concentration fragment screening. We previously reported the details of that screening campaign9,11. Those screening efforts identified a variety of chemotypes with activity against SMYD2 with IC50 values ranging from 0.3 to 20 µM. Two generalized modes of inhibition are possible with KMTs, substrate-competitive as observed with G9a and GLP12, or cofactorcompetitive as seen with DOT1L (overlaps with the cofactor) and EZH2 (partially overlaps the cofactor)13.

However, allosteric binding modes are also possible.

Isothermal titration

calorimetry (ITC) was used to validate the biophysical binding properties of exemplar compounds from the most chemically attractive clusters. Those experiments demonstrated clear binding for five distinct substrate-competitive chemotypes (Figure 2). Intriguingly, these five series do not share any obvious structural or chemical similarities that would define a pharmacophore.



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5

6

x

7

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8

Figure 2. ITC validated hits identified from the screening campaign.

Hit Evaluation. With these validated hits, we initiated a ‘hit-to-lead’ chemistry program with the goal of improving biochemical and cellular potency, and to assess the associated pharmacokinetic profiles.

We initially expanded the scope of the SAR of these series by

evaluating near neighbors from the AstraZeneca corporate collection, and through the synthesis of focused SAR expansion sets. Compound 5 (IC50 = 5.6 µM) was particularly attractive as a starting point. It had modest enzymatic activity and a favorable physicochemical profile with excellent solubility, but had a higher than desirable rat microsomal clearance (Figure 3). The other hits were also pursued. The series related to compound 6 was pursued and eventually furnished potent SMYD2 inhibitors, which will be discussed in another publication. Analogs of compound 7 were found to be unstable as the piperidine ring was prone to aromatization, so that series was discontinued. Compound 8 and its analogs were also deprioritized as they are potent kinase inhibitors.


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Figure 3. ITC titration of compound 5. 34 µM SMYD2 protein solution and 400 µM compound 5 were used. Kd of 1.73 µM, ∆H° = -6.90 kCal/mol, T∆S° = 3.14 kCal/mol and N = 0.92.

At the onset of the chemistry campaign, no crystallographic data was available for SMYD2. However, by analogy to the published structures of G9a and EHMT112 inhibitors, we hypothesized that the propyl pyrrolidine moiety of 5 functioned as a methyl lysine mimetic that would be positioned in the lysine binding channel of SMYD2. The synthetic route to generating 5 and analogs thereof was conveniently carried out in a two-step, one-pot reaction starting from pyruvic acid, and the appropriate aldehydes and aminopyrazoles (see chemistry section). Although the cyclocondensation step is low yielding, the simplicity of the reaction sequence allowed for rapid preparation of multiple intermediates. The resulting intermediates were then functionalized with a variety of amines (Table 1). After preparing more than 50 analogs within this chemical series, we were unable to improve the biochemical potency of the initial hit. The



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IC50 values for this series of analogs demonstrated steep SAR at this position, and the superiority of the propyl pyrrolidine substituent relative to other analogs. In parallel, we also examined the SAR of the C6 aryl functionality (Table 2), and likewise, observed that the orthomethyl phenyl seen in 5 was the preferred substituent at this position. Finally, we performed a simple scan of heteroatom and substituents on the benzamide ring (A-Ring in Table 3).

Although many

combinations were evaluated, only the most potent compounds are included here in Table 3. We observed that incorporation of an electron withdrawing group such as cyano at R1 or a nitrogen at para to the carboxamide improved activity. These results were used to inform the design of subsequent analogs featured in Tables 4 and 5. Table 1. Amide SAR

Cmpd

IC50 (µ µM)

LE

>61

NA

10

>83

NA

11

41.3

0.21

13.5

0.24

12.1

0.24

3.6

0.22

16.9

0.22

9

12

R1 N H

N

N H

N

13

14

N

N H

Ph

15


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19.6

16

0.23

Table 2. Aryl SAR

Cmpd

R1

R2

IC50 (µ µM)

LE

17

Br

H

7.3

0.26

18

Cl

H

8.0

0.26

19

F

H

16.8

0.24

20

Et

H

8.4

0.25

21

OMe

H

14.5

0.24

22

Me

Me

8.9

0.25

23

Me

cPro

13.3

0.22

Table 3. A-Ring SAR

Cmpd

X

R1

IC50 (µ µM)

LE

24

C

H

17.4

0.27

25

C

CN

7.3

0.27

26

C

OMe

22.1

0.25

27

N

H

8.8

0.29

28

N

OMe

9.9

0.26

29

N

NHMe

17.7

0.25



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Structure-Guided Design. The determination of the X-ray crystal structures of the five ITC validated substrate competitive chemical series in complex with SMYD2 illustrated how these inhibitors achieved enzymatic inhibition, which proved to be invaluable in informing the chemistry plan. In the following section, we discuss the structure-guided design, synthesis and biological activity of our hit-to lead efforts based on compounds 1 and 5. The crystal structure of SMYD2 in complex with 5 (Figure 4) confirmed our initial hypothesis that the propyl pyrrolidine moiety would act as a methyl lysine mimetic (Table S1). Structural overlay of 5 and the p53 substrate peptide show that the pyrrolidine moiety occupies the same region as the p53 peptide K370 terminus within the lysine binding channel.

Figure 4. Overlay of the X-ray crystal structures of SMYD2 in complex with 5 (green) and a peptide derived from p53 (magenta). The bound inhibitor molecule and peptide are shown as stick models, and the protein backbone as presented as a white ribbon (PDB accession code: 5KJL).


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We previously described the binding mode of 19 (Figure 5a). Compound 1 is composed of three distinct moieties: benzooxazinone, cyclohexyl and dichlorophenethyl substituents. The benzooxazinone group is positioned deep within the lysine binding channel of SMYD2, and forms an intricate series of electrostatic and hydrophobic packing interactions with the donor methyl group of the cofactor SAM, and main chain and side chain atoms of SMYD2. The ketone oxygen of the benzooxazinone moiety is placed 2.8 Å from the donor methyl of SAM. The hydroxyl substituent and the ring amine nitrogen jointly coordinate a water molecule, which mediates electrostatic interactions with the backbone carbonyl oxygens of residues V202 and M205. On the opposing side of the benzooxazinone ring, the ether oxygen is coordinated by residue Y258. A highly coordinated water molecule bridges additional interactions between the ether oxygen and the amine linker of 1, and the backbone carbonyls of residues N180, G183 and Y258. The cyclohexyl group of 1 is positioned in a primary hydrophobic pocket. The amine linker connecting the cyclohexyl and dichlorophenethyl elements of 1 forms a hydrogen bond with the side chain of residue N180. Perhaps the most interesting feature of the compound 1 binding mode is that the dichlorophenethyl moiety of the inhibitor extends across the peptide binding groove of SMYD2, and is inserted into a secondary hydrophobic pocket adjacent to the cofactor binding site. The secondary hydrophobic pocket is adjacent to the SAM binding site, and residues that form electrostatic interactions with the adenosyl group of SAM also make contacts with the dichlorophenethyl moiety of 1. This observation suggests the possibility of cooperativity, as the presence of SAM may contribute to inhibitor binding. The overlay of compounds 1 and 5 (Figure 5a) revealed opportunities for scaffold hybridization. The methyl group of 5 presents a convenient functional handle and is ideally



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situated to append a spacer to enable the placement of a hydrophobic moiety into the secondary hydrophobic pocket that is occupied by the dichlorophenyl moiety of compound 1. To evaluate possible analogs, we enumerated a virtual library of compounds combining features of both 1 and 5. Docking studies using GLIDE helped to select compounds that were likely to span the binding site. Compound 30 illustrates a representative hybrid (Figure 5b). The basic nitrogen of the piperazine moiety in the docked binding pose is positioned to form a hydrogen bond with residue N180, and would also present the phenethyl group in the appropriate direction to occupy the secondary hydrophobic binding pocket.

Figure 5. Hybridization strategy. (a) Overlay of the X-ray crystal structures of SMYD2 in complex with 1 (light blue) and 5 (green). The bound inhibitor molecules are shown as stick models. SMYD2 is colored white. (b) Chemistry schematic describing the structure-based hybridization approach.

We synthesized a variety of analogs including 30, and were pleased to find that the new compounds had improved enzymatic and cellular activity relative to the progenitor compounds.


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Indeed, 30 showed an IC50 value of 65 nM with cellular activity that was superior to either compound 1 or 5.

In addition, determination of the crystal structure of SMYD2 with 30

confirmed the expected binding mode (Figure 6). However, this compound has a poor in vitro DMPK profile with a LogD value >3.5, huMic Clint >300 and high plasma protein binding. To address these issues, we designed subsequent analogs with the intention of reducing polarity and metabolic clearance (Table 4).

Figure 6. X-ray crystal structure of SMYD2 in complex with 30. The bound inhibitor is colored magenta, and cofactor SAM is colored orange. All residues within 3.2 Å of the inhibitor are shown as sticks, and hydrogen bonds are shown as black dashed lines (PDB accession code 5KJM).

The SAR from compounds 24-29 (Table 3) taken together with the crystal structure of SMYD2 in complex with 30, suggest that the fused pyrazole ring in this series does not



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contribute significantly to binding affinity. We therefore, focused analog design on a tricyclic ring system exemplified in Tables 4 and 5. Table 4. SAR and In vitro DMPK values for Bis-Aryl inhibitors.

Clint Hu Micsb

Sol (µ µM)c

Hu PPB (% free)d

R1

R2

IC50 (µ µM)

Cell EC50 (µ µM)a

31

H

3-Me-phenethyl

0.211

4.14

35

722

5.3

32

H

3-MeO-phenethyl

0.273

3.50

36

815

10

33

H

4-Cl-phenethyl

0.114

3.12

30

332

1.2

34

H

Phenethyl

0.339

4.91

30

851

8.0

35

H

3-(2-ethyl)-indole

0.041

3.46

16

246

3.3

36

H

3,4-di-Cl-phenethyl

0.165

3.23

68

34

1000

34

43

N

C

4-Cl-phenethyl

0.305

1.62

>1000

13

44

N

C

3,4-di-Cl-phenethyl

0.120

5.57

790

1.4

45

N

C

3-(2-ethyl)-indole

0.062

5.61

923

34

46

C

N

3,4-di-Cl-phenethyl

0.038

0.450

393

1.1

47

C

N

3-(2-ethyl)-indole

0.017

1.02

858

17

a

Immunofluorescence assay measuring inhibition of SMYD2 mediated methylation of monomethyl p53 peptide in U2OS cells. b24 h solubility of solid sample determined in 0.1M phosphate buffer (pH 7.4) at 25 oC cFraction unbound (fu%) in human plasma, determined from DMSO stock solution of compound by equilibrium dialysis.

In addition to the SAR findings summarized in Tables 4 and 5, we also examined how replacement of the piperazine linker might affect SAR. We prepared analogs with various alternatives to the piperazine and observed that some of these modifications were tolerated, although in general, they did not offer any advantage with respect to cellular activity or DMPK profile. The crystal structures of SMYD2 in complex with two of the more potent examples from this effort, compounds 47 and 48, were determined (Figure 7 and Table S1).

The

superposition of SMYD2 in complex with the indole compound 47 with 48 (Table 5), showed that the binding mode is shared with the piperazine analogs.



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Figure 7. Structural superposition. (a) Overlay of the X-ray crystal structures of SMYD2 in complex with 48 (white) and 47 (green) illustrating alternate linkers. The bound inhibitors are shown as stick models, and the binding groove of SMYD2 is shown as a surface and has been colored by electrostatic potential (PDB accession codes 5KJN and 5KJK). (b) Chemical structures of compounds 48 and 49.

In Vivo Pharmacokinetics. Two compounds, 39 and 40, with good cellular activity and reasonable in vitro DMPK profiles and were selected for in vivo pharmacokinetic studies. The results from this study are presented in Table 6. Although they are di-basic, both compounds have good oral bioavailability (F = 18% and 81% respectively). Compound 39 has a relatively higher clearance and a correspondingly shorter half-life. The half-life of 40 is estimated to be 28 hours based on extrapolation from the final time point at 24 hours. Table 6. Rat Pharmacokinetics of Compounds 39 and 40a

Cmpd

F (%)

Cl (mL/min/kg)

Vdss (L/kg)

t1/2 (h)

39

18

74

68

5

40

81

5.8

8.5

>24


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a

Rat pharmacokinetics in Han Wistar rats followed a dose of 14 µMols/kg (po) and 4 µMols/kg (iv).

Selectivity. The selectivity of a set of compounds was evaluated against a focused panel of KMTs with previously defined selective tool compounds: EZH2, NSD1, G9a SMYD2 and DOT1L. The data shows that these hybrid compounds are selective for SMYD2 but do possess limited activity against DOT1L at high inhibitor concentrations (Table S2).

Cell Data. To verify that compounds inhibited SMYD2 activity in cells, a polyclonal antibody against a p53 peptide containing the mono-methylated K370 epitope3 was used in an immunofluorescence assay as described previously9. U2OS cells transfected with a SMYD2 expression vector were treated with compound for 24 hours and a reduction in the SMYD2mediated methylation signal was observed (Table 7). Staining with the anti-FLAG tag antibody was used to control for loss of cell number during compound treatment. Quantification of fluorescent intensity per cell was determined, and the IC50 values for these compounds are shown in Table 7. There was at least a >2-fold shift in the IC50 values relative to the direct effect on SMYD2 methylation.

These results demonstrate that these compounds do inhibit SMYD2

methyltransferase activity in cells, leading to a decrease in the SMYD2-mediated methylation signal.

Table 7. Inhibition of SMYD2-Mediated Methylation. U2OS cells were transfected with a SMYD2 expression vector and treated with compound for 24 hours. The calculated IC50 values for each compound are based on fluorescent intensity per cell. DAPI staining was used to calculate the cell count IC50 value. Cmpd

p53-me Cell Assay IC50 (µM)

Cell Count IC50 (µM)

1

9.98

23.59



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40 47

0.67 2.43

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5.0 13.26

Figure 8. Effect of the indicated compounds on cell proliferation is not p53-dependent. HCT116 +/+ and RKO-neo represent p53 wild type cells; HCT116 -/- and RKO-E6a represent p53deficient cells. Cells were treated with the indicated compounds (1, 40 and 47; listed in Table 7) between 0.03 to 30 µM for 72 hours, and cell viability was determined using Alamar Blue. No significant differences in IC50 values were observed between p53 wild-type and p53-deficient cell lines. The assay was performed in triplicate in two separate experiments. Values shown are mean ±SEM.

To evaluate if the SMYD2 inhibitors affect tumor cell viability in a p53-dependent manner, IC50 values were determined using a panel of matched paired p53 wild-type and p53-null colorectal cancer cell lines (Figure 8). The parental HCT116 cell line (+/+) contains a wild-type p53 gene, whereas the TP53 gene has been removed through homologous recombination in the isogenic derivative HCT 116 (-/-) cell line14. The colorectal cancer cell line RKO-E6a expresses a human papillomavirus E6 gene that binds p53 and targets it for degradation14. The RKO-neo


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cell line is the empty vector control pair to the RKO-E6a line. There was not an appreciable difference in the sensitivity of the p53-wild type or p53-deficient cell lines to the SMYD2 inhibitors evaluated (Figure 8). There was a strong correlation between the IC50 values obtained from the 24-hour p53-methylation and cell viability assays across all cell lines (either p53 wildtype or p53-deficient cells), suggesting that the effects on viability are not due to SMYD2mediated modulation of p53 activity by the inhibitors.

These results do not support the

hypothesis that SMYD2 inhibitors work in a p53-dependent manner to affect cell proliferation under the conditions evaluated. However, a recent study has shown that SMYD2 suppresses p53-dependent cardiomyocyte apoptosis, indicating that SMYD2 functions as a cardioprotective protein by methylating p53 in cardiomyocytes15.

Chemistry. The synthesis of the pyrazolopyridine 5 and related analogs is outlined in Scheme 1. The cyclocondensation reaction of an appropriately substituted benzaldehyde with pyruvic acid and 5-aminopyrazoles readily afforded the 6-aryl-pyrazolopyridine-4-carboxylic acid16 intermediates 50a-h. Intermediate 50a was coupled with a variety of amines to give compounds 9-16 in found in Table 1. Compounds 17-23 in Table 2 were similarly prepared from the acids 50b-h

by

coupling

with

3-(pyrrolidin-1-yl)propan-1-amine.

Although

the

initial

cyclocondensation step is low yielding, the simplicity of the reaction sequence allowed for rapid preparation of analogs to investigate the SAR of this series.

Scheme 1. Synthesis of Pyrazolopyridine Analogsa



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R3

H N

O

N O H R1

HO

R2 H 2N

N H

N

O R2

a

N H

N

5, 9-16

b

R1

N N

N H

H N

N

O R2

50a: R1 = Me, R2 = H 50b: R1 = Br, R2 = H 50c: R1 = Cl, R2 = H 50d: R1 = F, R2 = H 50e: R1 = Et, R2 = H 50f: R1 = OMe, R2 = H 50g: R1 = Me, R2 = Me 50h: R1 = Me, R2 = cPr

R1

N N

N H

17-23

a

Reagents and conditions: (a) pyruvic acid, AcOH, reflux, 8-24%; (b) Amine, HATU or EDC, DIPEA, DMF, 50 °C, 24-90%. See Table 1 for amines for 9-16.

Entry into the truncated analogs lacking the fused pyrazole ring is described in Scheme 2. Amide formation starting with commercially available 3-bromobenzoic acids 51a-c or isonicotinic acids 51d-f proceeded in good yield to give carboxamides 52a-f.

Palladium

mediated cross-coupling with o-tolylboronic acid gave rise to final compounds 24-29 in the range of yields indicated.

Scheme 2. Synthesis of Truncated Biaryl Amidesa


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b

a

51a: X = Br, Y = C, R = H 51b: X = Br, Y = C, R = CN 51c: X = Br, Y = C, R = OMe 51d: X = Br, Y = N, R = H 51e: X = Cl, Y = N, R = OMe 51f: X = Cl, Y = N, R = Cl

52a: X = Br, Y = C, R = H 52b: X = Br, Y = C, R = CN 52c: X = Br, Y = C, R = OMe 52d: X = Br, Y = N, R = H 52e: X = Cl, Y = N, R = OMe 52f: X = o-MePh, Y = N, R = Cl

24: Y 25: Y 26: Y 27: Y 28: Y 29: Y

= = = = = =

C, C, C, N, N, N,

R= R= R= R= R= R=

H CN OMe H OMe NHMe

a

Reagents and conditions: (a) 3-(pyrrolidin-1-yl)propan-1-amine, EDC, HOBt, TEA, CH2Cl2, rt, 80-84%; (b) o-tolylboronic acid, Pd(PPh3)4, K2CO3, DME/H2O, 80 °C, 14-84%.

The preparation of the extended pyrazolopyridine 30 is outlined in Scheme 3. The requisite benzaldehyde 54 was prepared in two-steps starting from commercially available 53a. Bocdeprotection with HCl in dioxane afforded the piperazine 53b, which was subsequently alkylated with 3,4-dichlorophenethyl methanesulfonate17. With 54 in hand, the stage was set to perform the cyclocondensation reaction to afford the elaborated pyrazolopyridine-4-carboxylic acid 55 in moderate yield. The propyl pyrrolidine moiety was introduced via amide coupling to afford the desired compound 30. Although the amide coupling occurred with good conversion to the carboxamide, material was lost under purification resulting in the poor overall recovery.

Scheme 3. Synthesis of Extended Pyrazolopyridinea



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b

53a R=Boc 53b R=H

54 a

d

c

55

30

a

Reagents and conditions: (a) HCl in dioxane, rt, quant; (b) K2CO3, ACN, 70 °C, 65%; (c) 1Hpyrazol-3-amine, pyruvic acid, AcOH, reflux, 24%; (d) 3-(pyrrolidin-1-yl)propan-1-amine, EDC, HOBt, DMF, 60 °C, 5%.

Biphenylpiperazine analogs 31-36 were synthesized according to the route outlined in Scheme 4. The aryl piperazine 56 was Boc-protected to generate 57, which was then subject to a Suzuki coupling reaction with the 3-(ethoxycarbonyl)phenylboronic acid, giving rise to 58a in high yield over the two steps. The acid 58b was obtained by ester hydrolysis under basic conditions and the product was subjected to amide coupling to produce 59. Removal of the Boc group provided piperazine 60 which could be alkylated under a variety of conditions (f or g in Scheme 4) to afford the final compounds 31-36.

Scheme 4. Synthesis of Biphenylpiperazinesa


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56

57

58a R=Et 58b R=OH

c

f or g

e

59

d

b

a

60

31-36

a

Reagents and conditions: (a) Boc2O, TEA, Et2O/THF, rt, 98%; (b) 3-(ethoxycarbonyl)phenylboronic acid, Pd(PPh3)4, K2CO3, dioxane/H2O, 90 °C, 98%; (c) LiOH, MeOH/THF/H2O, rt, 92%; (d) 3-(pyrrolidin-1-yl)propan-1-amine, EDC, HOBt, DIPEA, CH2Cl2, rt, 91%; (e) HCl in dioxane, MeOH, rt, 90%; (f) R-Br, TEA, ACN, reflux, 31-50%; (g) 3,4-dichlorophenethyl methanesulfonate, K2CO3, ACN, 72%.

The 3-cyano-biphenylpiperazine derivatives were synthesized as shown in Scheme 5. Palladium mediate cross-coupling of aryl bromide 52b (previously described in Scheme 2) with 2-[4-(N-Boc)piperazin-1-yl]phenylboronic acid pinacol ester generated 62a in high yield. Subsequent Boc-deprotection with TFA in DCM gave piperazine intermediate 62b. This was then treated with the appropriate alkyl bromides in the presence of potassium carbonate in acetonitrile at high temperatures under microwave heating or reflux conditions to afford the final compounds 37-39. Likewise, alkylation with 3,4-dichlorophenethyl methanesulfonate in the presence of sodium iodide gave rise to 40. The 2-aryl isonicotinic acid derivatives 41-45 were prepared in the same fashion starting from the pyridyl bromide 52d.



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Scheme 5. Synthesis of 3-Cyanophenyl and Pyridyl Analogsa

c or d or e

a 52b 62a R = Boc 62b R = H

37-40

b

a 61

52d

c or d or e

63a R = Boc 63b R = H

b

31-45

a

Reagents and conditions: (a) 52b or 52d, Pd(PPh3)4, K2CO3, DME/H2O, 70 °C, 86%; (b) TFA, CH2Cl2, rt, quant; (c) R-Br, K2CO3, ACN, µwave 100-150 °C, 36-42%; (d) R-Br 3-(2bromoethyl)-1H-indole, K2CO3, ACN, reflux, 18-36%; (e) 3,4-dichlorophenethyl methanesulfonate, NaI, K2CO3, ACN, 70 °C, 69%.

The 3,5-disubstituted pyridyl analogs 46 and 47 were synthesized as described in Scheme 7. Palladium mediated cross-coupling between 3-(ethoxycarbonyl)pyridine-5-boronic acid pinacol ester 64 and piperazinyl aryl bromide 57 (Scheme 4) gave the pyridyl ester 65. Direct amidation of the ester facilitated by catalytic TBD18 in hot toluene gave the amide 66a in high yield, followed by Boc-deprotection to furnish 66b. Alternatively, 66a could be obtained starting from 5-bromonicotininc acid 67 via amide coupling to afford 68, and subsequent Suzuki coupling with


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boronate ester 61 (Scheme 5). Alkylation of the piperazine in a manner previously described was then performed to obtain 46 and 47.

Scheme 6. Synthesis of 3,5-Disubstituted-Pyridyl A-Ring Biarylpiperazinesa Method A

a 57

b

64

65 f or g

Method B d 66a

c

67

66b

e

46: R1 = 3,4-di-Cl-phenethyl 47: R1 = 3-(2-ethyl)-indole

68

a

Reagents and conditions: (a) 57, Pd(PPh3)4, Cs2CO3, dioxane/H2O, 75 °C, 69%; (b) 3(pyrrolidin-1-yl)propan-1-amine, TBD, Toluene, 90 oC, 94%; (c) 3-(pyrrolidin-1-yl)propan-1amine, EDC, HOBt, TEA, CH2Cl2, rt, 85%; (d) 61, Pd(PPh3)4, K2CO3, DME/H2O, 75 °C, 99%; (e) TFA, CH2Cl2, rt, quant; (f) mesylate, K2CO3, ACN, 70 °C, 46%; (g) 3-(2-bromoethyl)-1Hindole, TEA, ACN, 75 °C, 20%.

CONCLUSIONS Our lead generation efforts have led to the discovery of five novel chemical series of SMYD2 inhibitors. Our original disclosure of compounds 19 and 3919, representing the first potent and selective SMYD2 inhibitors, paved the way for further studies10 leading to the development of 2 (LLY-507)10 (an analog of 39), 3 (A-893) (an analog of 1)10 and 4 (BAY-598)10. In this study, we used structure-guided design and medicinal chemistry to understand the SAR in the lysine binding channel and the selectivity pockets of the substrate binding groove of



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SMYD2. Importantly, we utilized scaffold hybridization techniques to combine the attractive features from two distinct chemical scaffolds to improve biochemical and cellular potency, as well as the pharmocokinetic profile of the resulting piperazine-based hybrid molecules. X-ray crystallography was used to demonstrate that these compounds form optimal interactions in the lysine binding channel and form a favorable series of hydrophobic interactions in both selectivity pockets of SMYD2. Despite having highly potent biochemical inhibitors with attractive physicochemical properties and cellular activity, our data does not support the hypothesis that SMYD2 inhibitors work in a p53-dependent manner to affect cell proliferation. These findings agree with recent in vivo xenograft and DMPK data on 410, which failed also to show a direct effect on p53-dependent cell proliferation10. The utility and value of SMYD2 inhibitors to understand the complex cell biology of SMYD2 is best exemplified by the recent findings of Reynoird et al20. In this study, it was found that levels of SMYD2 are elevated in pancreatic ductal adenocarcinoma (PDAC), indicating that SMYD2 promotes pancreatic cancer. Pharmalogical inhibition of SMYD2 was shown to restrict PDAC proliferation. Moreover, inhibition of SMYD2 enhances the efficacy when used in combination with standard chemotherapy to treat PDAC.

EXPERIMENTAL SECTION General Experimental. All reagents and solvents used were purchased from commercial sources and used without further purification (the 3-(pyrrolidin-1-yl)propan-1-amine was purchased from Acros; 3-(ethoxycarbonyl)pyridine-5-boronic acid pinacol ester was purchased from Frontier Scientific; 5-bromo-2-fluoronicotinic acid was purchased from Ark Pharma; 5bromo-2-fluorobenzoic acid was purchased from Combi-Blocks; 2-chloro-6-methoxyisonicotinic


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acid was purchased from Maybridge. All other solvents and chemicals were purchased from Sigma-Aldrich). All reactions were performed under a nitrogen or argon atmosphere unless otherwise noted.

1

H NMR spectra were obtained using a Bruker 300 MHz or 400 MHz

spectrometer at 27 °C unless otherwise noted; chemical shifts are expressed in parts per million (ppm, δ units) and are referenced to the residual protons in the deuterated solvent used. Coupling constants are given in units of hertz (Hz).

Splitting patterns describe apparent

multiplicities and are designated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and br s (broad singlet). Mass spectrometry analyses were performed with a Waters ZQ mass spectrometer connected to an Agilent 1100 equipped with Waters (Atlantis T3, 2.1 x 50 mm, 3 µm; or Atlantis C18, 2.1 x 50 mm, 5 µm) or Varian (Polaris C18A, 2mm x 50mm, 3µm) columns eluted with a gradient mixture of water and acetonitrile with either formic acid or ammonium acetate added as a modifier - overall run length correspond to: LCMS procedure A = 5-95% of acetonitrile over 4.5 min, LCMS Procedure B = 5-95% of acetonitrile over 2.5 min. High resolution mass spectra (HRMS) were recorded on a Bruker Daltonics microTOF-Q II hybrid quadrupole time-of-flight mass spectrometer using positive electrospray ionization. Reversephase chromatography was performed on a Gilson system using an Atlantis Prep T3 OBD, Xbridge C18 or Phenyl OBD column - reverse-phase HPLC column (19 mm x 100 mm) in water/acetonitrile with 0.1% trifluoroacetic acid as mobile phase, unless otherwise noted. The reported molecular ion corresponds to the [M+H]+; for molecules with multiple isotopic patterns (Br, Cl, etc.) the reported value is the one obtained for the lowest isotope mass unless otherwise specified. Thin layer chromatography (TLC) was performed using EMD silica gel 60 F254 plates, which were visualized using either UV light, reversibly stained with iodine (I2 absorbed on silica) or a stain prepared by dissolving 2 g KMnO4 and 12 g Na2CO3 in 200 mL H2O. Column



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chromatography was performed using SiliCycle SiliaSep or RediSep Rf preloaded silica gel cartridges on Teledyne ISCO CombiFlash Companion automated purification systems. Unless otherwise indicated, all final compounds were purified to ≥95% purity as assessed by analytical HPLC using an Agilent 1100 equipped with Waters columns (Atlantis T3, 2.1x50 mm, 3 µm; or Atlantis C18, 2.1 x 50 mm, 5 µm) eluted for >10 minutes with a gradient mixture of water and acetonitrile with either formic acid or ammonium acetate added as a modifier, monitored at wavelengths of 220, 254, and 280 nm. Compounds are listed in the order in which they appear in the Tables or Chemistry schemes. N-(3-(Pyrrolidin-1-yl)propyl)-6-(o-tolyl)-1H-pyrazolo[3,4-b]pyridine-4-carboxamide

(5).

A stirred mixture of the 6-o-tolyl-1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid 50a (0.143 g, 0.56 mmol), DIPEA (0.394 mL, 2.26 mmol), 3-(pyrrolidin-1-yl)propan-1-amine (0.145 g, 1.13 mmol), and HATU (0.537 g, 1.41 mmol) in DMF (2 mL) was warmed to 50 °C for 48 h. The reaction was cooled to rt, diluted with water (100 mL), and extracted with ethyl acetate (2 x 100 mL). The combined organic extracts were washed with brine, dried with MgSO4, filtered, and concentrated in vacuo. The residue was purified by reverse-phase HPLC (gradient elution: 1270% ACN in water containing 0.1% trifluoroacetic acid) to afford title compound after lyophilization as a white solid (0.155 g, 58%). 1H NMR (400 MHz, DMSO-d6) δ 1.64-2.14 (m, 6H), 2.21-2.44 (m, 3H), 2.82-3.09 (m, 2H), 3.09-3.28 (m, 2H), 3.43 (q, J = 6.4 Hz, 2H), 3.57 (dd, J = 10.6, 5.3 Hz, 2H), 7.13-7.43 (m, 3H), 7.52 (d, J = 7.1 Hz, 1H), 7.62-7.87 (m, 1H), 8.43 (s, 1H), 8.99 (t, J = 5.6 Hz, 1H), 9.27-9.84 (m, 1H), 13.88 (br, s, 1H). HRMS (ES+, TOF) m/z calcd for C21H26N5O (M + H)+, 364.2132; found, 364.2143.


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The following compounds were prepared in an analogous manner to compound 5 using the appropriate amine. N-(3-(Dimethylamino)propyl)-6-o-tolyl-1H-pyrazolo[3,4-b]pyridine-4-carboxamide 1

(9).

H NMR (400 MHz, DMSO-d6) δ 1.87-1.98 (m, 2H), 2.37 (s, 3H), 2.78 (d, J = 4.8 Hz, 6H), 3.07-

3.17 (m, 2H), 3.40 (q, J = 6.4 Hz, 2H), 7.31-7.41 (m, 3H), 7.50 (d, J = 7.1 Hz, 1H), 7.70 (s, 1H), 8.41 (s, 1H), 8.98 (t, J = 5.81 Hz, 1H), 9.30-9.44 (m, 1H). LCMS using procedure A: MS m/z (ES+)+ (M+H)+ = 338.2. tR = 1.53 min. HRMS (ES+, TOF) m/z calcd for C19H24N5O (M + H)+, 338.1975; found, 338.1989. N-(3-(Methylamino)propyl)-6-o-tolyl-1H-pyrazolo[3,4-b]pyridine-4-carboxamide hydrochloride (10). A vial was charged with tert-butyl methyl(3-(6-o-tolyl-1H-pyrazolo[3,4b]pyridine-4-carboxamido)propyl)carbamate (85 mg, 0.20 mmol) and 0.5 M HCl in MeOH (5 mL). The reaction was stirred at room temperature for 2 h then concentrated in vacuo to afford the hydrochloride salt of the title compound as white solid (69 mg, 96%). 1H NMR (400 MHz, DMSO-d6) δ 1.92 (quin, J = 7.1 Hz, 2H), 2.38 (s, 3H), 2.50-2.55 (m, 3H), 2.87-3.01 (m, 2H), 3.42 (q, J = 6.5 Hz, 2H), 7.30-7.41 (m, 3H), 7.53 (d, J = 7.1 Hz, 1H), 7.77 (s, 1H), 8.42 (s, 1H), 8.97 (br s, 2H), 9.13 (t, J = 5.7 Hz, 1H). LCMS using procedure A: MS m/z (ES+)+ (M+H)+ = 324.2. tR = 1.48 min. HRMS (ES+, TOF) m/z calcd for C18H22N5O (M + H)+, 324.1819; found, 324.1831. N-(3-Morpholinopropyl)-6-o-tolyl-1H-pyrazolo[3,4-b]pyridine-4-carboxamide

(11).

1

H

NMR (400 MHz, DMSO-d6) δ 1.85-1.96 (m, 2H), 2.31 (s, 3H), 2.93-3.06 (m, 2H), 3.12 (dd, J = 10.5, 5.9 Hz, 2H), 3.32-3.42 (m, 4H), 3.57 (t, J = 11.9 Hz, 2H), 3.91 (d, J = 11.6 Hz, 2H), 7.257.37 (m, 3H), 7.44 (d, J= 7.1 Hz, 1H), 7.64 (s, 1H), 8.35 (s, 1H), 8.93 (t, J = 5.8 Hz, 1H), 9.57



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(br s, 1H). LCMS using procedure A: MS m/z (ES+)+ (M+H)+ = 380.1. tR = 1.54 min. HRMS (ES+, TOF) m/z calcd for C21H26N5O2 (M + H)+, 380.2081; found, 380.2089. N-(3-(Piperidin-1-yl)propyl)-6-o-tolyl-1H-pyrazolo[3,4-b]pyridine-4-carboxamide (12). 1H NMR (300 MHz, DMSO-d6) δ 1.52-1.72 (m, 4H), 1.75-1.87 (m, 2H), 1.89-2.01 (m, 2H), 2.37 (s, 3H), 2.79-2.95 (m, 2H), 3.04-3.17 (m, 2H), 3.35-3.51 (m, 4H), 7.33-7.39 (m, 3H), 7.47-7.53 (m, 1H), 7.69 (s, 1H), 8.41 (s, 1H), 8.92-9.05 (m, 2H). LCMS using procedure A: MS m/z (ES+)+ (M+H)+ = 378.2. tR = 1.64 min. HRMS (ES+, TOF) m/z calcd for C22H28N5O (M + H)+, 378.2288; found, 378.2285. N-(2-Hydroxy-3-(pyrrolidin-1-yl)propyl)-6-o-tolyl-1H-pyrazolo[3,4-b]pyridine-4carboxamide (13). 1H NMR (400 MHz, DMSO-d6) δ 1.79-1.90 (m, 2H), 1.93-2.03 (m, 2H), 2.37 (s, 3 H), 2.96-3.09 (m, 2H), 3.11-3.20 (m, 1H), 3.23-3.32 (m, 1H), 3.40 (q, J = 5.8 Hz, 2H), 3.47-3.60 (m, 2H), 4.00-4.11 (m, 1H), 5.95 (br s, 1H), 7.33- 7.42 (m, 3H), 7.51 (d, J = 7.1 Hz, 1H), 7.75 (s, 1H), 8.41 (s, 1H), 9.00 (t, J = 5.8 Hz, 1H), 9.46 (br s, 1H). LCMS using procedure A: MS m/z (ES+)+ (M+H)+ = 380.2. tR = 1.54 min. HRMS (ES+, TOF) m/z calcd for C21H26N5O2 (M + H)+, 380.2081; found, 380.2094. N-(2-Benzyl-3-(pyrrolidin-1-yl)propyl)-6-o-tolyl-1H-pyrazolo[3,4-b]pyridine-4carboxamide (14). 1H NMR (300 MHz, DMSO-d6) δ 1.85-2.02 (m, 4H), 2.38 (s, 3H), 2.39-2.44 (m, 1H), 2.64-2.84 (m, 2H), 2.93 (br d, J = 7.0 Hz, 1H), 2.97-3.13 (m, 2H), 3.15-3.30 (m, 1H), 3.36-3.47 (m, 2H), 3.68 (d, J = 4.5 Hz, 2H), 7.20 (app dq, J = 8.5, 4.3 Hz, 1H), 7.30 (d, J= 4.3 Hz, 4H), 7.33-7.40 (m, 3H), 7.47-7.55 (m, 1H), 7.70 (s, 1H), 8.40 (s, 1H), 9.00 (t, J = 5.7 Hz, 1H), 9.44-9.62 (m, 1H). LCMS using procedure A: MS m/z (ES+)+ (M+H)+ = 454.1. tR = 2.11 min. HRMS (ES+, TOF) m/z calcd for C28H32N5O (M + H)+, 454.2601; found, 454.2611.


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1,4'-Bipiperidin-1'-yl(6-o-tolyl-1H-pyrazolo[3,4-b]pyridin-4-yl)methanone (15). 1H NMR (300 MHz, DMSO-d6) δ 1.22-1.43 (m, 1H), 1.44-1.69 (m, 5H), 1.70-1.89 (m, 3H), 2.00-2.14 (m, 1H), 2.32 (s, 3H), 2.75-2.96 (m, 3H), 3.00-3.18 (m, 1H), 3.23-3.64 (m, 4H), 4.58-4.74 (m, 1H), 7.21-7.35 (m, 4H), 7.42 (d, J = 7.0 Hz, 1H), 8.11 (s, 1H), 9.06 (br s, 1H). LCMS using procedure A: MS m/z (ES+)+ (M+H)+ = 404.3. tR = 1.62 min. HRMS (ES+, TOF) m/z calcd for C24H30N5O (M + H)+, 404.2445; found, 404.2462. N-Methyl-N-(3-(pyrrolidin-1-yl)propyl)-6-o-tolyl-1H-pyrazolo[3,4-b]pyridine-4carboxamide (16). 1H NMR (400 MHz CD3OD) δ 2.06-2.28 (m, 6H), 2.41 (s, 3H), 3.08 (s, 3H), 3.13 (m, 2H), 3.32-3.41 (m, 2H), 3.70-3.82 (m, 4H), 7.33-7.49 (m, 4H), 7.61 (s, 1H), 8.52 (s, 1H) - NH not observed. LCMS using procedure A: MS m/z (ES+)+ (M+H)+ = 378.2. tR = 1.54 min. HRMS (ES+, TOF) m/z calcd for C22H28N5O (M + H)+, 378.2288; found, 378.2292. The following compounds were prepared in an analogous manner to compound 5 using the appropriate benzaldehyde. 6-(2-Bromophenyl)-N-(3-(pyrrolidin-1-yl)propyl)-1H-pyrazolo[3,4-b]pyridine-4carboxamide (17). 1H NMR (400 MHz, DMSO-d6) δ 1.69-2.09 (m, 6H), 2.87-3.07 (m, 2H), 3.15-3.30 (m, 2H), 3.42 (q, J = 6.3 Hz, 2H), 3.50-3.68 (m, 2H), 7.06-7.49 (m, 1H), 7.54-7.66 (m, 2H), 7.74-7.92 (m, 2H), 8.22-8.60 (m, 1H), 8.97 (t, J = 5.6 Hz, 1H), 9.65 (br s, 1H), 13.99 (br s, 1H). HRMS (ES+, TOF) m/z calcd for C20H23BrN5O (M + H)+, 428.1080; found, 428.1091. 6-(2-Chlorophenyl)-N-(3-(pyrrolidin-1-yl)propyl)-1H-pyrazolo[3,4-b]pyridine-4carboxamide (18). 1H NMR (400 MHz, DMSO-d6) δ 1.55-2.08 (m, 6H), 2.81-3.01 (m, 2H), 3.06-3.19 (m, 2H), 3.36 (q, J = 6.3 Hz, 2H), 3.41-3.57 (m, 2H), 7.26-7.51 (m, 2H), 7.51-7.69 (m, 2H), 7.72 (s, 1H), 7.77 (s, 1H), 7.68-7.89 (m, 1H), 8.38 (s, 1H), 8.91(t, J = 5.9 Hz, 1H), 9.49 (br



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s, 1H), 13.99 (br s, 1H). HRMS (ES+, TOF) m/z calcd for C20H23ClN5O (M + H)+, 384.1586; found, 364.1593. 6-(2-Fluorophenyl)-N-(3-(pyrrolidin-1-yl)propyl)-1H-pyrazolo[3,4-b]pyridine-4carboxamide (19). 1H NMR (300 MHz, DMSO-d6) δ 1.67-2.23 (m, 6H), 2.82-3.11 (m, 2H), 3.11-3.31 (m, 2H), 3.44 (q, J = 6.3 Hz, 2H), 3.50-3.67 (m, 2H), 7.22-7.49 (m, 2H), 7.49-7.67 (m, 2H), 7.82-8.06 (m, 2H), 8.43 (s, 1H), 8.85-9.11 (m, 1H). HRMS (ES+, TOF) m/z calcd for C20H23FN5O (M + H)+, 368.1881; found, 368.1889. 6-(2-Ethylphenyl)-N-(3-(pyrrolidin-1-yl)propyl)-1H-pyrazolo[3,4-b]pyridine-4carboxamide (20). 1H NMR (400 MHz, DMSO-d6) δ 1.05 (t, J = 7.4 Hz, 3H), 1.72-2.22 (m, 6H), 2.67-2.83 (m, 2H), 2.90-3.05 (m, 2H), 3.18-3.25 (m, 2H), 3.42 (q, J = 7.4 Hz, 2H), 3.56 (dd, J = 10.4, 5.3 Hz, 2H), 7.04-7.54 (m, 4H), 7.64-7.82 (m, 1H), 8.22-8.58 (m, 1H), 9.00 (t, J = 5.7 Hz, 1H), 9.74 (br s, 1H). HRMS (ES+, TOF) m/z calcd for C22H28N5O (M + H)+, 378.2288; found, 378.2287. 6-(2-Methoxyphenyl)-N-(3-(pyrrolidin-1-yl)propyl)-1H-pyrazolo[3,4-b]pyridine-4carboxamide (21). 1H NMR (300 MHz, DMSO-d6) δ 1.75 (m, 4H), 1.80-1.90 (m, 2H), 2.762.86 (m, 6H), 3.39 (q, J = 6.7 Hz, 2H), 3.82 (s, 3H), 7.10 (td, J = 7.5, 1.0 Hz, 1H), 7.20 (d, J = 8.3 Hz, 1H), 7.47 (ddd, J = 8.3, 7.4, 1.8 Hz, 1H), 7.65 (dd, J = 7.6, 1.8 Hz, 1H), 7.87 (s, 1H), 8.34 (s, 1H), 8.82 - 8.90 (m, 1H), 13.82 (br s, 1H). HRMS (ES+, TOF) m/z calcd for C21H26N5O2 (M + H)+, 380.2081; found, 380.2085. The following compounds were prepared in an analogous manner to compound 5 using the appropriate pyrazoles. 3-Methyl-N-(3-(pyrrolidin-1-yl)propyl)-6-(o-tolyl)-1H-pyrazolo[3,4-b]pyridine-4carboxamide (22). 1H NMR (400 MHz, DMSO-d6) δ 1.70-2.00 (m, 6H), 2.31 (s, 3H), 2.42 (s,


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3H), 2.81-3.01 (m, 2H), 3.03-3.21 (m, 2H), 3.33 (q, J = 6.6 Hz, 2H), 3.50 (dd, J = 10.4, 5.1 Hz, 2H), 7.06-7.35 (m, 4H), 7.42 (d, J = 7.1 Hz, 1H), 8.80 (t, J = 5.7 Hz, 1H), 9.68 (br s, 1 H). HRMS (ES+, TOF) m/z calcd for C22H28N5O (M + H)+, 378.2288; found, 378.2299. 3-Cyclopropyl-N-(3-(pyrrolidin-1-yl)propyl)-6-(o-tolyl)-1H-pyrazolo[3,4-b]pyridine-4carboxamide (23). 1H NMR (400 MHz, DMSO-d6) δ 0.71-0.95 (m, 4H), 1.71-2.01 (m, 6H), 2.16-2.37 (m, 4H), 2.79-3.02 (m, 2H), 3.03-3.23 (m, 2H), 3.34 (q, J = 6.6 Hz, 2H), 3.49 (dd, J = 10.4, 5.3 Hz, 2H), 7.07-7.35 (m, 4H), 7.42 (d, J = 7.3 Hz, 1H), 8.82 (t, J = 5.7 Hz, 1H), 9.81 (br s, 1H). HRMS (ES+, TOF) m/z calcd for C24H30N5O (M + H)+, 404.2445; found, 404.2454. N-(3-(Pyrrolidin-1-yl)propyl)-2-(o-tolyl)isonicotinamide (24). A 50 mL vial was charged with the 2-bromo-N-(3-(pyrrolidin-1-yl)propyl)isonicotinamide 52a (280 mg, 0.90 mmol), otolylboronic acid (183 mg, 1.35 mmol), K2CO3 (372 mg, 2.69 mmol), and Pd(PPh3)4 (155 mg, 0.13 mmol) and a mixture of dioxane/water (10:1, 3 mL). The vial was purged and placed under argon then the stirred mixture was heated to 90 °C for 24 h. The reaction was diluted with water (50 mL) and extracted with EtOAc (2 x 50 mL). The combined organic extract was dried with MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by reverse-phase HPLC (gradient elution: 0-65% ACN in water containing 0.1% trifluoroacetic acid) to provide the trifluoroacetate of the title compound as a white solid (55 mg, 14%).

1

H

NMR (300 MHz, DMSO-d6) δ 1.79-1.91 (m, 4H), 1.92-2.05 (m, 2H), 2.18 (s, 3H), 2.88-3.00 (m, 2H), 3.07-3.17 (m, 2H), 3.32 (t, J = 6.6 Hz, 2H), 3.44-3.56 (m, 2H), 3.83 (m, 1H), 7.13-7.22 (m, 1H), 7.22-7.31 (m, 3H), 7.44-7.49 (m, 1H), 7.50-7.58 (m, 1H), 7.73 (s, 1H), 7.76-7.84 (m, 1H). LCMS using procedure A: MS m/z (ES+)+ (M+H)+ = 323.3. tR = 2.07 min. HRMS (ES+, TOF) m/z calcd for C21H27N2O (M + H)+, 323.2118; found, 323.2134.



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5-Cyano-2'-methyl-N-(3-(pyrrolidin-1-yl)propyl)biphenyl-3-carboxamide (25). A solution of 3-bromo-5-cyano-N-(3-(pyrrolidin-1-yl)propyl)benzamide 52b (477 mg, 1.42 mmol), otolylboronic acid (251 mg, 1.85 mmol), Pd(PPh3)4 (164 mg, 0.14 mmol) and K2CO3 (589 mg, 4.26 mmol) in DME/water (10:1, 11 mL) was at 80 °C for 3 h. The reaction mixture was concentrated in vacuo and the residue purified directly by silica gel chromatography (gradient elution: 0-10% of 3% NH4OH/MeOH in EtOAc). The desired fractions were collected and concentrated in vacuo. The residue obtained was then treated with 0.5 M HCl in MeOH and concentrated under reduced pressure.

Drying on high vacuum overnight afforded the

hydrochloride of the title compound as a white solid (389 mg, 71%).

1

H NMR (400 MHz

CD3OD) δ 1.99-2.21 (m, 6H), 2.26 (s, 3H), 3.00-3.17 (m, 2H), 3.27 (d, J = 8.1 Hz, 2H), 3.53 (t, J = 6.6 Hz, 2H), 3.60-3.78 (m, 2H), 7.17-7.41 (m, 4H), 7.88 (d, J = 1.5 Hz, 1H), 8.09 (t, J = 1.4 Hz, 1H), 8.21 (t, J = 1.3 Hz, 1H) - exchangeable NH not observed. LCMS using procedure A: MS m/z (ES+) (M+H)+ = 348.1. tR = 2.08 min. HRMS (ES+, TOF) m/z calcd for C22H26N3O (M + H)+, 348.2070; found, 348.2082. 5-Methoxy-2'-methyl-N-(3-(pyrrolidin-1-yl)propyl)biphenyl-3-carboxamide

(26).

A

solution of 3-bromo-5-methoxy-N-(3-(pyrrolidin-1-yl)propyl)benzamide 52c (200 mg, 0.59 mmol), o-tolylboronic acid (104 mg, 0.76 mmol), K2CO3 (243 mg, 1.76 mmol), and Pd(PPh3)4 (67.7 mg, 0.06 mmol) in a mixture of DME/Water (10:1, 6 mL) was allowed to stir for 3 h under an argon atmosphere at 80 °C. The reaction mixture was cooled then concentrated under reduced pressure. The crude product was purified by silica gel chromatography (gradient elution: 0-10% of 3% NH4OH/MeOH in EtOAc). The desired fractions were collected and concentrated under reduced pressure and the product residue was then treated with 0.5 M HCl in MeOH and concentrated to dryness. The residue was treated with water and subject to lyophilization to


Page 35 of 69

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afford the hydrochloride salt of the title compound as a tacky solid (50 mg, 22%). 1H NMR (400 MHz CD3OD) δ 2.06 (m, 6H), 2.24 (s, 3H), 3.23-3.27 (m, 2H), 3.28-3.30 (m, 4H), 3.50 (t, J = 6.4 Hz, 2H), 3.87 (s, 3H), 7.03 (dd, J = 2.4, 1.4 Hz, 1H), 7.17-7.25 (m, 2H), 7.25-7.29 (m, 2H), 7.35 (t, J = 1.4 Hz, 1H), 7.39-7.42 (m, 1H) – exchangeable NH not observed. LCMS using procedure A: MS m/z (ES+)+ (M+H)+ = 353.3. tR = 2.13 min. HRMS (ES+, TOF) m/z calcd for C22H29N2O2 (M + H)+, 353.2224; found, 353.2237. N-(3-(Pyrrolidin-1-yl)propyl)-2-(o-tolyl)isonicotinamide (27). A 50 mL vial was charged with the 2-bromo-N-(3-(pyrrolidin-1-yl)propyl)isonicotinamide 52d (280 mg, 0.90 mmol), otolylboronic acid (183 mg, 1.35 mmol), K2CO3 (372 mg, 2.69 mmol), and Pd(PPh3)4 (155 mg, 0.13 mmol) and a mixture of dioxane/water (10:1, 3 mL). The vial was purged and placed under an argon atmosphere and the stirred mixture was heated to 90 °C for 24 h. The reaction was diluted with water (50 mL) and extracted with ethyl acetate (2 x 50 mL). The combined organic extract was dried with MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by reverse-phase HPLC (gradient elution: 0-65% ACN in water containing 0.1% trifluoroacetic acid) to provide the trifluoroacetate of the title compound as a white solid (55 mg, 14%). 1H NMR (400 MHz, DMSO-d6) δ 1.97 (br m, 6H), 2.34 (br s, 3H), 2.95 (m, 2H), 3.16 (m, 2H), 3.39 (m, 2H), 3.50 (m, 2H), 7.19-7.59 (m, 4H), 7.90 (br s, 1H), 8.03 (br s, 1H), 8.86 (m, 1H), 9.18 (s., 1H), 10.75 (m, 1H). HRMS (ES+, TOF) m/z calcd for C20H26N3O (M + H)+, 324.2070; found, 324.2083 2-Methoxy-N-(3-(pyrrolidin-1-yl)propyl)-6-o-tolylisonicotinamide (28). A solution of 2chloro-6-methoxy-N-(3-(pyrrolidin-1-yl)propyl)isonicotinamide 52e (353 mg, 1.19 mmol), otolylboronic acid (210 mg, 1.54 mmol), K2CO3 (492 mg, 3.56 mmol), and Pd(PPh3)4 (137 mg, 0.22 mmol) in a mixture of DME/water (10:1, 5.5 mL) was stirred at 80 °C for 3 h. The reaction



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mixture was concentrated in vacuo and purified directly by silica gel chromatography (gradient elution: 0-10% of 3% NH4OH/MeOH in EtOAc). The desired fractions were collected and concentrated in vacuo. The residue obtained was then treated with 0.5 M HCl in MeOH and concentrated under reduced pressure.

Drying on high vacuum overnight afforded the

hydrochloride of the title compound as a white solid (388 mg, 84%).

1

H NMR (400 MHz

CD3OD) δ 1.98-2.27 (m, 6H), 2.40 (s, 3H), 2.97-3.18 (m, 2H), 3.26 (d, J = 7.7 Hz, 2H), 3.52 (t, J = 6.6 Hz, 2H), 3.60-3.79 (m, 2H), 3.89-4.08 (m, 3H), 7.17 (d, J = 1.3 Hz, 1H), 7.24-7.38 (m, 3H), 7.39-7.52 (m, 2 H) - exchangeable NH not observed. LCMS using procedure A: MS m/z (ES+) (M+H)+ = 354.3. tR = 2.07 min. HRMS (ES+, TOF) m/z calcd for C21H28N3O2 (M + H)+, 345.2176; found, 345.2177. 2-(Methylamino)-N-(3-(pyrrolidin-1-yl)propyl)-6-o-tolylisonicotinamide (29). A mixture of 2-chloro-N-(3-(pyrrolidin-1-yl)propyl)-6-o-tolylisonicotinamide (180 mg, 0.50 mmol) and methylamine (102 mg, 1.51 mmol) in NMP (1.2 mL) was subject to microwave heating at 200 °C for 1 h. The crude mixture was directly subject to reverse-phase HPLC purification (gradient elution: 10-30% ACN in water containing 0.1% trifluoroacetic acid) to afford the bistrifluoroacetate salt of the title compound as pale yellow solid (132 mg, 46%) following lyophilization. 1H NMR (400 MHz CD3OD) δ 2.01-2.18 (m, 6H), 2.36 (s, 3H), 3.06-3.11 (m, 2H), 3.12 (s, 3H), 3.27-3.30 (m, 2H), 3.53 (t, J = 6.2 Hz, 2H), 3.64-3.71 (m, 2H), 7.14 (s, 1H), 7.33-7.41 (m, 2H), 7.42 (s, 1H), 7.43-7.52 (m, 3H) – exchangeable NH not observed. LCMS using procedure A: MS m/z (ES+)+ (M+H)+ = 353.2. tR = 1.06 min. HRMS (ES+, TOF) m/z calcd for C21H29N4O (M + H)+, 353.2336; found, 353.2338. 6-(2-(4-(3,4-Dichlorophenethyl)piperazin-1-yl)phenyl)-N-(3-(pyrrolidin-1-yl)propyl)-1Hpyrazolo[3,4-b]pyridine-4-carboxamide (30). A mixture of EDC (145 mg, 0.81 mmol), HOBt


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(223 mg, 0.81 mmol), 6-(2-(4-(3,4-dichlorophenethyl)piperazin-1-yl)phenyl)-1H-pyrazolo[3,4b]pyridine-4-carboxylic acid 55 (200 mg, 0.40 mmol) and 3-(pyrrolidin-1-yl)propan-1-amine (207 mg, 1.61 mmol) in DMF (2 mL) was stirred at 60 °C for 2 h. The mixture was directly purified by reverse-phase HPLC (gradient elution: 5-85% ACN in water containing 0.1% ammonium acetate) to afford the title compound as a dry film (8 mg, 5%). 1H NMR (400 MHz, DMSO-d6) δ 1.53 (br s, 4H), 1.60-1.74 (m, 2H), 2.18-2.32 (m, 5H), 2.32-2.40 (m, 5H), 2.61 (t, J = 7.4 Hz, 3H), 2.72 (br s, 4H), 3.27-3.38 (m, 3H), 6.98-7.16 (m, 3H), 7.21-7.38 (m, 1H), 7.387.48 (m, 2H), 7.53 (dd, J = 7.9, 1.6 Hz, 1H), 8.22-8.30 (m, 2H), 8.82 (t, J = 5.4 Hz, 1H), 13.74 (s, 1H). LCMS using procedure A: MS m/z (ES+) (M+H)+ = 606.2. tR = 1.68 min. 2'-(4-(3-Methylphenethyl)piperazin-1-yl)-N-(3-(pyrrolidin-1-yl)propyl)biphenyl-3carboxamide

(31).

To

a

stirred

solution

of

2'-(piperazin-1-yl)-N-(3-(pyrrolidin-1-

yl)propyl)biphenyl-3-carboxamide dihydrochloride 60 (220 mg, 0.26 mmol) and triethylamine (0.180 mL, 1.29 mmol) in ACN (1.5 mL) was added the 1-(2-bromoethyl)-3-methylbenzene (0.043 mL, 0.28 mmol). The solution was then brought to reflux for 16 h. Upon cooling the mixture was diluted with MeOH, filtered and purified by reverse-phase HPLC (gradient elution: 15-40% ACN in water containing 0.1% trifluoroacetic acid). Lyophilization gave rise to bistrifluoroacetate of the title compound as a white solid (97 mg, 50%).

1

H NMR (400 MHz

CD3OD) δ 1.95-2.08 (m, 4H), 2.10-2.19 (m, 2H), 2.32 (s, 3H), 2.94-3.09 (m, 8H), 3.23-3.28 (m, 4H), 3.32-3.38 (m, 2H), 3.51 (app t, J = 6.6 Hz, 4 H), 3.65 (m, 2 H), 7.00-7.08 (m, 2H), 7.09 (s, 1H), 7.17-7.25 (m, 3H), 7.33 (dd, J = 7.6, 1.5 Hz, 1H), 7.35-7.44 (m, 1H), 7.56 (t, J = 7.7 Hz, 1H), 7.78-7.82 (m, 1H), 7.82-7.86 (m, 1H), 8.11 (t, J = 1.6 Hz, 1H) - exchangeable NH not observed. LCMS using procedure A: MS m/z (ES+) (M+H)+ = 511.3. tR = 1.92 min. HRMS (ES+, TOF) m/z calcd for C33H43N4O (M + H)+, 511.3431; found, 511.3432.



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2'-(4-(3-Methoxyphenethyl)piperazin-1-yl)-N-(3-(pyrrolidin-1-yl)propyl)biphenyl-3carboxamide (32). The compound was prepared in an analogous manner to compound 31 using 3-methoxyphenethyl bromide to afford the bis-trifluoroacetate salt of the title compound as a colorless semi-solid (44 mg, 41%). 1H NMR (400 MHz CD3OD) δ 1.87-2.15 (m, 6H), 2.82-3.06 (m, 8H), 3.09-3.21 (m, 4H), 3.25-3.34 (m, 2H), 3.36- 3.49 (m, 4H), 3.57 (d, J = 5.1 Hz, 2H), 3.70 (s, 3H), 6.71-6.81 (m, 3H), 7.07-7.20 (m, 3H), 7.25 (dd, J = 7.5, 1.6 Hz, 1H), 7.28-7.35 (m, 1H), 7.49 (t, J = 7.7 Hz, 1 H), 7.68-7.79 (m, 2H), 8.03 (t, J = 1.6 Hz, 1H), 8.71 (t, J = 5.7 Hz, 1H). LCMS using procedure A: MS m/z (ES+) (M+H)+ = 527.3. tR = 1.73 min. HRMS (ES+, TOF) m/z calcd for C33H43N4O2 (M + H)+, 527.3381; found, 527.3379. 2'-(4-(4-Chlorophenethyl)piperazin-1-yl)-N-(3-(pyrrolidin-1-yl)propyl)biphenyl-3carboxamide (33). The material was prepared in an analogous manner to compound 31 using 1(2-bromoethyl)-4-chlorobenzene to afford the title compound as the bis-trifluoroacetate salt (97 mg, 50%). 1H NMR (400 MHz CD3OD) δ 1.97-2.23 (m, 6H), 2.95-3.15 (m, 8H), 3.19-3.31 (m, 4H), 3.35-3.46 (m, 2H), 3.54 (t, J = 6.6 Hz, 4H), 3.63-3.74 (m, 2H), 7.18-7.25 (m, 2H), 7.26-7.31 (m, 2H), 7.32-7.44 (m, 4H), 7.58 (t, J = 7.8 Hz, 1H), 7.81-7.88 (m, 2H), 8.14 (t, J = 1.6 Hz, 1H) exchangeable NH not observed. LCMS using procedure A: MS m/z (ES+) (M + H)+ = 531.2. tR = 1.72 min. HRMS (ES+, TOF) m/z calcd for C32H40ClN4O (M + H)+, 531.2885; found, 531.2888. 2'-(4-Phenethylpiperazin-1-yl)-N-(3-(pyrrolidin-1-yl)propyl)biphenyl-3-carboxamide (34). The material was prepared in an analogous manner to compound 31 using (2bromoethyl)benzene to afford the bis-trifluoroacetate salt of the title compound as a gum (65 mg, 35%). 1H NMR (400 MHz CD3OD) δ 1.94-2.28 (m, 6H), 2.96-3.14 (m, 8H), 3.18-3.31 (m, 4H), 3.35-3.43 (m, 2H), 3.54 (app t, J = 6.6 Hz, 4H), 3.62-3.77 (m, 2H), 7.17-7.25 (m, 2H), 7.25-7.31 (m, 3H), 7.32-7.43 (m, 4H), 7.58 (t, J = 7.7 Hz, 1H), 7.84 (app tt, J = 7.8, 1.2 Hz, 2H), 8.14 (t, J


Page 39 of 69

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= 1.6 Hz, 1H) - exchangeable NH not observed. LCMS using procedure A: MS m/z (ES+) (M+H)+ = 497.3. tR = 1.67 min. HRMS (ES+, TOF) m/z calcd for C32H41N4O (M + H)+, 497.3275; found, 497.3273. 2'-(4-(2-(1H-Indol-3-yl)ethyl)piperazin-1-yl)-N-(3-(pyrrolidin-1-yl)propyl)biphenyl-3carboxamide (35). The title compound was prepared in an analogous manner to compound 31 using 3-(2-bromoethyl)-1H-indole.

Following reverse-phase HPLC purification (gradient

elution: 5-55% ACN in water containing 0.1% trifluoroacetic acid) the fractions containing the desired product were combined and concentrated under reduced pressure to remove the organics. The aqueous remains were treated with NH4OH (aq) dropwise and extracted with CH2Cl2 (3x). The combined organics were dried over Na2SO4, filtered and concentrated under reduced pressure, to give rise to the title compound (34 mg, 31%) as an off-white solid. 1H NMR (400 MHz CD2Cl2) δ 1.18 (s, 2H), 1.61 (app ddd, J = 6.3, 3.5, 3.3 Hz, 4H), 1.76 (quin, J = 6.0 Hz, 2H), 2.35 (br s, 3H), 2.49-2.62 (m, 5H), 2.65-2.72 (m, 2H), 2.73-2.83 (m, 6H), 3.40-3.51 (m, 2H), 6.91-7.01 (m, 4H), 7.05 (td, J = 7.6, 1.3 Hz, 1H), 7.14-7.28 (m, 3H), 7.37 (t, J = 7.7 Hz, 1H), 7.46 (d, J = 7.8 Hz, 1H), 7.66 (dt, J = 7.8, 1.4 Hz, 1H), 7.73 (ddd, J = 7.7, 1.4, 1.3 Hz, 1H), 7.92 (t, J = 1.5 Hz, 1H), 8.27 (br s, 1H), 8.47 (br s, 1H). LCMS using procedure A: MS m/z (ES+) (M+H)+ = 536.3. tR = 1.86 min. HRMS (ES+, TOF) m/z calcd for C34H42N5O (M + H)+, 536.3384; found, 536.3380. 2'-(4-(3,4-Dichlorophenethyl)piperazin-1-yl)-N-(3-(pyrrolidin-1-yl)propyl)biphenyl-3carboxamide (36). The 3,4-dichlorophenethyl methanesulfonate (211 mg, 0.78 mmol) was added to a stirred mixture of 2'-(piperazin-1-yl)-N-(3-(pyrrolidin-1-yl)propyl)biphenyl-3carboxamide dihydrochloride 60 (243 mg, 0.39 mmol) and K2CO3 (433 mg, 3.13 mmol) in ACN (3 mL). The mixture was heated to 70 °C for 20 h. The mixture was cooled, filtered and the



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Page 40 of 69

filtrate concentrated under reduced pressure. The crude product was subject to silica gel chromatography (gradient elution: 0-10% of 3% NH4OH/MeOH in EtOAc).

The resultant

impure solid was again subject to silica gel chromatography (isocratic: 5% of 3% NH4OH/MeOH in EtOAc) and the resultant product was treated with 4 M HCl in dioxane. Concentration and vacuum drying yielded the dihydrochloride of the title compound as a pale tan solid (159 mg, 72%).

1

H NMR (400 MHz CD3OD) δ 2.01-2.19 (m, 6H), 3.00-3.17 (m, 8H),

3.25-3.30 (m, 2H), 3.34-3.42 (m, 2H), 3.49-3.56 (m, 4H), 3.63-3.71 (m, 4H), 7.16-7.22 (m, 2H), 7.27 (d, J = 8.1 Hz, 1H), 7.31-7.40 (m, 2H), 7.48 (d, J = 8.3 Hz, 1H), 7.52-7.61 (m, 2H), 7.84 (t, J = 8.0 Hz, 2H), 8.14 (s, 1H) - exchangeable NH not observed. LCMS using procedure A: MS m/z (ES+) (M + H)+ = 565.2. tR = 1.50 min. 5-Cyano-2'-(4-(3-methylphenethyl)piperazin-1-yl)-N-(3-(pyrrolidin-1-yl)propyl)biphenyl3-carboxamide

(37)

A

mixture

of

5-cyano-2'-(piperazin-1-yl)-N-(3-(pyrrolidin-1-

yl)propyl)biphenyl-3-carboxamide bis-trifluoroacetate 62b (75 mg, 0.18 mmol), 1-(2bromoethyl)-3-methylbenzene (34 mg, 0.2 mmol) and K2CO3 (99 mg, 0.072 mmol) in ACN (1 mL) was subject to microwave heating at 100 °C for 20 min then 150 °C for 30 min. The mixture was filtered then concentrated under reduced pressure. The crude product was subject to silica gel chromatography (gradient elution: 0-10% of 1% NH4OH/MeOH in CH2Cl2) to afford the impure desired compound. The residue was purified via reverse-phase HPLC (gradient elution: 10-70% ACN in water containing 0.1% trifluoroacetic acid). The appropriate fractions were collected and set to lyophilize to afford the bis-trifluoroacetate of title compound as white solid (57 mg, 42%). 1H NMR (400 MHz, CD3OD) δ 1.96-2.20 (m, 6H), 2.32 (s, 3H), 2.94-3.15 (m, 8H), 3.16-3.30 (m, 4H), 3.33-3.41 (m, 2H), 3.48-3.59 (m, 4H), 3.61-3.71 (m, 2H), 7.00-7.14 (m, 3H), 7.34-7.40 (m, 1H), 7.16-7.31 (m, 3H), 7.41-7.51 (m, 1H), 8.15 (dt, J = 10.3, 1.6 Hz,


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2H), 8.46 (t, J = 1.8 Hz, 1H) - exchangeable NH not observed. LCMS using procedure A: MS m/z (ES+) (M+H)+ = 536.2. tR = 1.86 min. HRMS (ES+, TOF) m/z calcd for C34H42N5O (M + H)+, 536.3384; found, 536.3378. 5-Cyano-2'-(4-(3-methoxyphenethyl)piperazin-1-yl)-N-(3-(pyrrolidin-1yl)propyl)biphenyl-3-carboxamide (38). A vial was charged with 5-cyano-2'-(piperazin-1-yl)N-(3-(pyrrolidin-1-yl)propyl)biphenyl-3-carboxamide bis-trifluoroacetate 62b (74 mg, 0.18 mmol), 3-methoxyphenethyl bromide (0.033 mL, 0.21 mmol), K2CO3 (98 mg, 0.71 mmol) and ACN (2 mL). The stirred mixture was subject to microwave heating at 150 °C for 1 h. The mixture was filtered and purified by reverse-phase HPLC (gradient elution: 10-70% ACN in water containing 0.1% trifluoroacetic acid) to afford the desired product as the bistrifluoroacetate following lyophilization. The resulting gum was treated with excess 4 M HCl in dioxane to afford the dihydrochloride salt of the title compound (40 mg, 36%). 1H NMR (400 MHz CD3OD) δ 1.96-2.22 (m, 6H), 2.96-3.17 (m, 8H), 3.18-3.29 (m, 4H), 3.35-3.43 (m, 2H), 3.47-3.59 (m, 4H), 3.61-3.70 (m, 2H), 3.79 (s, 3H), 6.71-6.92 (m, 3H), 7.22-7.31 (m, 3H), 7.357.51 (m, 2H), 8.15 (dt, J = 19.7, 1.5 Hz, 2H), 8.47 (t, J = 1.6 Hz, 1H) - exchangeable NH not observed. LCMS using procedure A: MS m/z (ES+) (M+H)+ = 552.3. tR = 2.1 min. HRMS (ES+, TOF) m/z calcd for C34H42N5O2 (M + H)+, 552.3333; found, 552.3326. 2'-(4-(2-(1H-Indol-3-yl)ethyl)piperazin-1-yl)-5-cyano-N-(3-(pyrrolidin-1-yl)propyl)-[1,1'biphenyl]-3-carboxamide (39). Potassium carbonate (596 mg, 4.31 mmol) was added to a stirred solution of 5-cyano-2'-(piperazin-1-yl)-N-(3-(pyrrolidin-1-yl)propyl)-[1,1'-biphenyl]-3carboxamide bis-trifluoroacetate 62b (655 mg, 0.86 mmol) in ACN (7 mL). The mixture was stirred for 3 min then 3-(2-bromoethyl)-1H-indole (199 mg, 0.86 mmol) was added and the mixture was heated at 80 °C for 14 h. The mixture was cooled to ambient temperature, diluted



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with EtOAc, filtered and concentrated under reduced pressure. The resultant gum was subject to silica gel chromatography (40 g; gradient elution: 0.5-10% 2 M NH3/MeOH in CH2Cl2) to afford the impure product as a white solid. The resulting solid was dissolved in DMSO and purified by reverse-phase HPLC (gradient elution: 25-50% ACN in water containing 10 mM NH4OAc). The appropriate fractions were dried and subject lyophilization to afford the title compound (111 mg, 18%) as the white solid of the acetate salt (~2.5 eq). 1H NMR (400 MHz, DMSO-d6) δ 1.61 (td, J = 6.8, 3.1 Hz, 4H), 1.66-1.77 (m, 2H), 1.90 (s, 2.5H, acetate salt), 2.34-2.40 (m, 7H), 2.79 (d, J = 5.0 Hz, 6H), 3.32-3.39 (m, 7H), 6.89-6.98 (m, 1H), 7.00-7.08 (m, 1H), 7.22 (d, J = 2.3 Hz, 1H), 7.14-7.23 (m, 2H), 7.32 (d, J = 8.0 Hz, 1H), 7.34-7.43 (m, 2H), 7.47 (d, J = 7.8 Hz, 1H), 8.17 (t, J = 1.5 Hz, 1H), 8.23 (t, J = 1.5 Hz, 1H), 8.48 (t, J = 1.6 Hz, 1H), 8.77 (t, J = 5.3 Hz, 1H), 10.75 (s, 1H). LCMS using procedure B: MS m/z (ES+) (M+H)+ = 562. tR = 0.60 min. HRMS (ES+, TOF) m/z calcd for C35H41N6O (M + H)+, 561.3336; found, 561.3323. 5-Cyano-2'-(4-(3,4-dichlorophenethyl)piperazin-1-yl)-N-(3-(pyrrolidin-1-yl)propyl) biphenyl-3-carboxamide (40). A mixture of 5-cyano-2'-(piperazin-1-yl)-N-(3-(pyrrolidin-1yl)propyl)biphenyl-3-carboxamide bis-trifluoroacetate 62b (183 mg, 0.44 mmol), 3,4dichlorophenethyl methanesulfonate (236 mg, 0.88 mmol), and K2CO3 (484 mg, 3.50 mmol) in ACN (3 mL) was allowed to stir at 70 °C for 20 h. The reaction was cooled to ambient temperature, sodium iodide (66 mg, 0.44 mmol) was added and heating resumed. After 24 h the reaction mixture was directly subject to silica gel chromatography (gradient elution: 0-10% of 3% NH4OH/MeOH in EtOAc) and the desired fractions were collected and concentrated in vacuo.

The resulting gum was treated with 4 M HCl in dioxane and the mixture was

concentrated under reduced pressure. Drying in vacuo gave rise to the dihydrochloride salt of title compound as a tan solid (200 mg, 69%). 1H NMR (400 MHz CD3OD) δ 1.98-2.25 (m, 6H),


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2.94-3.27 (m, 10 H), 3.35-3.46 (m, 2H), 3.46-3.85 (m, 8H), 7.28 (dd, J = 6.2, 3.8 Hz, 3H), 7.347.61 (m, 4H), 8.07-8.25 (m, 2H), 8.48 (br s, 1H) - exchangeable NH not observed. LCMS using procedure A: MS m/z (ES+) (M+H)+ = 590.3. tR = 1.98 min. HRMS (ES+, TOF) m/z calcd for C33H38Cl2N5O (M + H)+, 590.2448; found, 590.2448. 2-(2-(4-(3-Methoxyphenethyl)piperazin-1-yl)phenyl)-N-(3-(pyrrolidin-1yl)propyl)isonicotinamide (41). A solution of 2-(2-(piperazin-1-yl)phenyl)-N-(3-(pyrrolidin-1yl)propyl)isonicotinamide dihydrochloride 63b (100 mg, 0.21 mmol), 1-(2-bromoethyl)-3methoxybenezene (0.050 mL, 0.32 mmol), and DIPEA (0.150 mL, 0.86 mmol) in DMF (1 mL) was stirred at 90 °C overnight.

The crude reaction was directly subject to silica gel

chromatography (gradient elution: 0-10% of 1% NH4OH/MeOH in CH2Cl2). The resultant was dissolved in MeOH and treat with 1 M HCl in Et2O. The solution was concentrated in vacuo and the residue was dissolved in water. Lyophilization afforded the dihydrochloride salt of the title compound (23mg, 16%) as a white solid.

1

H NMR (400 MHz CD3OD) δ 1.98-2.21 (m, 6H),

3.03-3.18 (m, 4H), 3.20-3.29 (m, 6H), 3.34-3.40 (m, 2H), 3.42-3.50 (m, 2H), 3.54-3.63 (m, 4H), 3.65-3.72 (m, 2H), 3.79 (s, 3H), 6.79-6.93 (m, 3H), 7.25 (t, J = 7.8 Hz, 1H), 7.45 (t, J = 7.4 Hz, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.66-7.74 (m, 1H), 7.77 (d, J = 7.6 Hz, 1H), 8.41 (d, J = 5.3 Hz, 1H), 8.77 (s, 1H), 9.11 (d, J = 5.8 Hz, 1H) - exchangeable NH not observed. LCMS using procedure A: MS m/z (ES+) (M+H)+ = 528.2. tR = 1.46 min. HRMS (ES+, TOF) m/z calcd for C32H42N5O2 (M + H)+, 528.3333; found, 528.3326. 2-(2-(4-(4-Methoxyphenethyl)piperazin-1-yl)phenyl)-N-(3-(pyrrolidin-1-yl)propyl) isonicotinamide (42). The title compound was prepared in an analogous manner to compound 41 using 1-(2-bromoethyl)-4-methoxybenezene to yield the dihydrochloride as a white solid (40 mg, 30%). 1H NMR (400 MHz CD3OD) δ 1.92-2.24 (m, 6H), 2.99-3.06 (m, 2H), 3.07-3.16 (m,



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2H), 3.16-3.28 (m, 4H), 3.33-3.46 (m, 2H), 3.53-3.64 (m, 4H), 3.53-3.64 (m, 4H), 3.68 (m, 2H), 3.77 (s, 3H), 6.90 (d, J = 8.6 Hz, 2H), 7.23 (d, J = 8.6 Hz, 2H), 7.40-7.57 (m, 2H), 7.67-7.82 (m, 2H), 8.34-8.47 (m, 1H), 8.77 (s, 1H), 9.09 (d, J = 6.1 Hz, 1H). LCMS using procedure A: MS m/z (ES+) (M+H)+ = 528.3. tR = 1.47 min. HRMS (ES+, TOF) m/z calcd for C32H42N5O2 (M + H)+, 528.3333; found, 528.3321. 2-(2-(4-(4-Chlorophenethyl)piperazin-1-yl)phenyl)-N-(3-(pyrrolidin-1yl)propyl)isonicotin-amide (43). A solution of 2-(2-(piperazin-1-yl)phenyl)-N-(3-(pyrrolidin-1yl)propyl)iso-nicotinamide dihydrochloride 63b (150 mg, 0.32 mmol), 1-(2-bromoethyl)-4chlorobenzene (0.049 mL, 0.34 mmol), and DIPEA (0.225 mL, 1.29 mmol) in DMF (2 mL) was heated to 70 °C overnight. Additional 1-(2-bromoethyl)-4-chlorobenzene (50 µL) and DIPEA (200 µL) were added and the reaction was heated 95 °C for 3 h. The solution was allowed to cool and directly subject to silica gel chromatography (gradient elution: 0-10% of 1% NH4OH/MeOH in CH2Cl2). The resultant residue was treated with 4 M HCl in dioxane and concentrated under reduced pressure. The solid was triturated with Et2O/hexanes to afford the dihydrochloride of the title compound as a white solid (80 mg, 41%).

1

H NMR (400 MHz

CD3OD) δ 2.17 (m, 6H), 3.11 (d, J = 4.6 Hz, 4H), 3.20-3.29 (m, 6H), 3.35 (s, 2H), 3.44 (br s, 2H), 3.52-3.64 (m, 4H), 3.68 (d, J = 4.0 Hz, 2H), 7.34 (d, J = 4.8 Hz, 4H), 7.40-7.55 (m, 2H), 7.71 (d, J = 10.6 Hz, 2H), 8.28-8.40 (m, 1H), 8.72 (s, 1H), 8.98-9.13 (m, 1H) - exchangeable NH not observed. LCMS using procedure A: MS m/z (ES+) (M+H)+ = 532.2. tR = 1.58 min. HRMS (ES+, TOF) m/z calcd for C31H39ClN5O (M + H)+, 532.2838; found, 532.2832. 2-(2-(4-(3,4-Dichlorophenethyl)piperazin-1-yl)phenyl)-N-(3-(pyrrolidin-1yl)propyl)isonicotinamide (44). A suspension of 2-(2-(piperazin-1-yl)phenyl)-N-(3-(pyrrolidin1-yl)propyl)isonicotinamide dihydrochloride 63b (100 mg, 0.21 mmol), 3,4-dichlorophenethyl


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methanesulfonate (115 mg, 0.43 mmol), and K2CO3 (237 mg, 1.72 mmol) in ACN (15 ml) was stirred at 70 °C for 24 h then at reflux for 16 h. The reaction mixture was cooled, filtered, and the filter cake washed with MeOH. The filtrate was concentrated and the purified by reversephase HPLC (gradient elution of ACN in water containing 0.1% trifluoroacetic acid) to afford the title compound as a pale yellow solid (41 mg, 24%). 1H NMR (400 MHz, DMSO-d6) δ 1.721.97 (m, 6H), 2.75-3.02 (m, 8H), 3.10 (dt, J = 10.4, 5.4 Hz, 4H), 3.19-3.35 (m, 4H), 3.35-3.53 (m, 4H), 7.01-7.29 (m, 3H), 7.29-7.44 (m, 1H), 7.44-7.62 (m, 3H), 7.68 (dd, J = 5.1, 1.5 Hz, 1H), 8.32 (s, 1H), 8.77 (d, J = 5.31 Hz, 1H), 8.89 (t, J = 5.7 Hz, 1H), 9.78 (br s, 1H), 10.06 (br s, 1H). HRMS (ES+, TOF) m/z calcd for C31H38Cl2N5O (M + H)+, 566.2448; found, 566.2445. 2-(2-(4-(2-(1H-Indol-3-yl)ethyl)piperazin-1-yl)phenyl)-N-(3-(pyrrolidin-1yl)propyl)isonicotinamide (45). A mixture of 2-(2-(piperazin-1-yl)phenyl)-N-(3-(pyrrolidin-1yl)propyl)isonicotinamide dihydrochloride 63b (100 mg, 0.21 mmol), 3-(2-bromoethyl)-1Hindole (29 mg, 0.13 mmol), DIPEA (0.75 mL, 0.43 mmol) and K2CO3 (59 mg, 0.43 mmol) in DMF (1 mL) was subject to microwave heating at 100 oC for 30 min then at 150 °C for 30 min. The mixture was allowd to cool, filtered and the crude product solution purified by silica gel chromatography (gradient elution: 0-10% of 1% NH4OH/MeOH in CH2Cl2). The resultant product was dissolved in MeOH and treated with 1 M HCl in Et2O.

The solution was

concentrated in vacuo and the residue was dissolved in water. Lyophilization afforded the dihydrochloride salt of the title compound as a white solid (27 mg, 40%). 1H NMR (400 MHz CD3OD) δ 1.20-1.43 (m, 2H), 1.97-2.03 (m, 2H), 2.11 (br s, 4H), 3.03 (br s, 2H), 3.26 (br s, 8H), 3.43-3.53 (m, 2H), 3.55-3.72 (m, 6H), 6.99-7.08 (m, 1H), 7.12 (t, J = 7.6 Hz, 1H), 7.22 (s, 1H), 7.37 (d, J = 8.1 Hz, 1H), 7.40-7.54 (m, 2H), 7.55-7.82 (m, 3H), 8.35 (br s, 1H), 8.77 (br s, 1H), 9.04 (br s, 1H), 9.38 (br s, 1H) - exchangeable NH not observed. LCMS using procedure A: MS



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Page 46 of 69

m/z (ES+) (M+H)+ = 537.2. tR = 1.50 min. HRMS (ES+, TOF) m/z calcd for C33H41N6O (M + H)+, 537.3336; found, 537.3324. 5-(2-(4-(3,4-Dichlorophenethyl)piperazin-1-yl)phenyl)-N-(3-(pyrrolidin-1yl)propyl)nicotinamide (46). A mixture of 5-(2-(piperazin-1-yl)phenyl)-N-(3-(pyrrolidin-1yl)propyl)nicotinamide bis-trifluoroacetate 66b (727 mg, 1.17 mmol) in ACN (4 mL) was treated with a solution of 3,4-dichlorophenethyl methanesulfonate (630 mg, 2.34 mmol) in ACN (1 mL), followed by K2CO3 (1.29 g, 9.36 mmol). The mixture was heated to 70 °C for 4 h. The mixture was cooled, filtered and concentrated. The residue was purified by silica gel chromatography (gradient elution: 0-10% of 3% NH4OH/MeOH in EtOAc) to afford the desired product. The residue was then treated with 4 M HCl in dioxane, filtered and dried in vacuo to afford the dihydrochloride salt of title compound (344 mg, 46%) as a white solid.

1

H NMR (400 MHz,

CD3OD) δ 1.97-2.06 (m, 2H), 2.08-2.17 (m, 4H), 3.05-3.15 (m, 4H), 3.15-3.27 (m, 6H), 3.313.37 (m, 2H), 3.38-3.48 (m, 2H), 3.52-3.62 (m, 4H), 3.65-3.73 (m, 2H), 7.28 (dd, J = 8.3, 1.9 Hz, 1H), 7.37-7.45 (m, 2H), 7.49 (d, J = 8.3 Hz, 1H), 7.53-7.61 (m, 3H), 9.25 (d, J = 1.5 Hz, 1H), 9.27-9.32 (m, 2H) - exchangeable NH not observed. LCMS using procedure A: MS m/z (ES+) (M+H)+ = 566.3. tR = 1.66 min. HRMS (ES+, TOF) m/z calcd for C31H38Cl2N5O (M + H)+, 566.2448; found, 566.2453. 5-(2-(4-(2-(1H-Indol-3-yl)ethyl)piperazin-1-yl)phenyl)-N-(3-(pyrrolidin-1yl)propyl)nicotin-amide (47). The title compound was prepared in a similar manner as described21.

Briefly, to a stirred mixture of 5-(2-(piperazin-1-yl)phenyl)-N-(3-(pyrrolidin-1-

yl)propyl)nicotinamide, bis-trifluoroacetate 66b (605 mg, 0.97 mmol), TEA (0.723 mL, 5.19 mmol) and 4 Å molecular sieves (powdered) in anhydrous ACN (4 mL) was added the 3-(2bromoethyl)-1H-indole (320 mg, 1.43 mmol) and sodium iodide (16 mg, 0.11 mmol). The


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reaction was warmed to 75 °C for 40 h then was cooled, diluted with MeOH, filtered and concentrated under reduced pressure.

The resultant residue was subject to silica gel

chromatography (gradient elution: 1-10% 2 M NH3/MeOH in CH2Cl2) to afford the title compound (115 mg) in 94% purity (by HPLC). The solid was dissolved in MeOH and purified by reverse-phase HPLC (gradient elution: 10-45% ACN in water containing 0.1% trifluoroacetic acid).

After lyophilization, the resultant trifluoroacetate salt was dissolved in CH2Cl2 and

washed successively with saturated K2CO3 (aq), water, brine, dried over Na2SO4, filtered and concentrated under reduced pressure to afford the title compound. The resultant free-base was then dissolved in MeOH and added to a methanolic solution of citric acid (1 eq, 37 mg). The solution was concentrated via a N2 stream, then dried in vacuo overnight giving rise to a white solid of the title compound as the citrate salt (141 mg, 20%).

1

H NMR (400 MHz, CD3OD) δ

1.95-2.22 (m, 6H), 2.66-2.82 (m, 4H – citric acid), 2.83-2.89 (br s, 2H) 2.97 (m, 2H), 2.98-3.10 (m, 6H), 3.14-3.19 (m, 2H), 3.24-3.31 (m, 6H), 3.55 (t, J = 6.5 Hz, 2H), 6.98-7.06 (m, 1H), 7.087.16 (m, 2H), 7.21-7.31 (m, 2H), 7.36 (d, J = 8.1 Hz, 1H), 7.40 (dd, J = 7.6, 1.5 Hz, 1H), 7.427.49 (m, 1H), 7.56 (d, J = 7.6 Hz, 1H), 8.61 (t, J = 2.2 Hz, 1H), 8.97 (dd, J = 7.2, 2.2 Hz, 2H) two exchangeable NHs and citric acid OHs not observed. LCMS using procedure A: MS m/z (ES+) (M+H)+ = 537.2. tR = 1.59 min. HRMS (ES+, TOF) m/z calcd for C33H41N6O (M + H)+, 537.3336; found, 537.3340. 6-(o-Tolyl)-1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid (50a). The title compound was prepared as described15. Briefly, a round-bottom flask was charged with a magnetic stir bar, 1Hpyrazol-5-amine (1.000 g, 12.03 mmol), 2-oxopropanoic acid (0.841 mL, 12.03 mmol), acetic acid (17 mL), and 2-methylbenzaldehyde (1.446 g, 12.03 mmol). The reaction was heated to reflux for 12 h then allowed to cool to ambient temperature. Water (400 mL) was added and the



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resulting tan precipitate was collected via vacuum filtration. The solid was washed with ethanol, collected and dried in vacuo to afford the title compound as an off-white solid (0.655 g, 21.5%). This material was used directly without further purification. 1H NMR (400 MHz, DMSO-d6) δ 2.36 (s, 3H), 7.31-7.40 (m, 3H), 7.49 (d, 1H), 7.72 (s, 1H), 8.39 (s, 1H), 13.98 (s, 1H). LCMS using procedure A: MS m/z (ES+) (M+H)+ = 254.2. tR = 2.23 min. 2,6-Dichloro-N-(3-(pyrrolidin-1-yl)propyl)isonicotinamide (51f). A solution of 2,6dichloroisonicotinic acid (3.0 g, 15.622 mmol) in CH2Cl2 (50 mL) was treated in succession with 3-(pyrrolidin-1-yl)propan-1-amine (2.5 mL, 17.19 mmol), EDC (3.59 g, 1.8.75 mmol), TEA (6.53 mL, 46.88 mmol) and HOBt (2.87 g, 18.75 mmol). The reaction mixture was stirred at room temperature for 12 h. The mixture was concentrated and the crude product purified by silica gel chromatography (gradient elution: 0-10% of 3% NH4OH/MeOH in EtOAc) to afford the title compound as an orange solid (3.367 g, 71%). LCMS using procedure A: MS m/z (ES+) (M+H)+ = 303.9. tR = 1.28 min. 2-Bromo-N-(3-pyrrolidin-1-ylpropyl)pyridine-4-carboxamide (52a). A mixture of 3(pyrrolidin-1-yl)propan-1-amine (3.9 mL, 27.2 mmol), 2-bromoisonicotinic acid (5 mL, 24.8 mmol), EDC (5.22 g, 27.2 mmol), HOBt (4.17 mg, 27.2 mmol), and TEA (10.35 mL, 74.3 mmol) in CH2Cl2 (70 mL) was stirred overnight. The solution was further diluted with CH2Cl2 and washed with saturated aq NaHCO3. The phases were separated and the organics were dried over dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (gradient elution: 0-10% of 3% NH4OH/MeOH in EtOAc) to afford the title compound as a white solid upon standing (6.34 g, 82%). LCMS using procedure A: MS m/z (ES+) (M+H)+ = 311.9. tR = 1.02 min.


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3-Bromo-5-cyano-N-(3-(pyrrolidin-1-yl)propyl)benzamide (52b). The triethylamine (1.9 mL, 13.63 mmol) was added to a stirred solution of 3-bromo-5-cyanobenzoic acid 51b (1.026 g, 4.54 mmol), 3-(pyrrolidin-1-yl)propan-1-amine (0.621 mL, 4.77 mmol) and EDC (1.044 g, 5.45 mmol) in anhydrous CH2Cl2 (15 mL). After 17 h the reaction was concentrated to ~1/3 volume and directly subject to silica gel chromatography (gradient elution: 2-7.5% of 2 M NH3/MeOH in CH2Cl2) to afford the titled compound as a clear gum (0.75 g, 49%). This material used without further purification. 1H NMR (400 MHz, DMSO-d6) δ 1.65-1.75 (m, 6H), 2.37-2.47 (m, 4H), 3.27-3.36 (m, 4H), 8.25 (t, J = 1.4 Hz, 1H), 8.30 (t, J = 1.8 Hz, 1H), 8.32-8.37 (m, 1H), 8.81 (t, J = 5.1 Hz, 1H). LCMS using procedure B: MS m/z (ES+) (M+H)+ = 336.0. tR = 0.58 min. 3-Bromo-5-methoxy-N-(3-(pyrrolidin-1-yl)propyl)benzamide

(52c).

The

triethylamine

(0.593 mL, 4.25 mmol) was added to a stirred solution of 3-bromo-5-methoxybenzoic acid 51c (328 mg, 1.42 mmol), 3-(pyrrolidin-1-yl)propan-1-amine (0.22 mL, 1.56 mmol) and EDC (326 mg, 1.70 mmol) in CH2Cl2 (15 mL) under a nitrogen atmosphere. After 12 h the reaction was concentrated to ~1/3 volume and directly subject to silica gel chromatography (gradient elution: 0-10% of 3% NH4OH/MeOH in EtOAc) to afford the titled compound as a clear gum (485 mg, quant). This material used as-is without further purification. LCMS using procedure B: MS m/z (ES+) (M+H)+ = 342.9. tR = 1.56 min. 2-Bromo-N-(3-pyrrolidin-1-ylpropyl)pyridine-4-carboxamide (52d). A mixture of 3(pyrrolidin-1-yl)propan-1-amine (3.9 mL, 27.2 mmol), 2-bromoisonicotinic acid 51d (5 mL, 24.8 mmol), EDC (5.22 g, 27.2 mmol), HOBt (4.17 mg, 27.2 mmol), and TEA (10.35 mL, 74.3 mmol) in CH2Cl2 (70 mL) was stirred overnight. The solution was further diluted with CH2Cl2 and washed with saturated aq NaHCO3. The phases were separated and the organics were dried over dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica



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gel chromatography (gradient elution: 0-10% of 3% NH4OH/MeOH in EtOAc) to afford the title compound as a white solid upon standing (6.34 g, 82%). LCMS using procedure A: MS m/z (ES+) (M+H)+ = 311.9. tR = 1.02 min. 2-Chloro-6-methoxy-N-(3-(pyrrolidin-1-yl)propyl)isonicotinamide (52e). A solution of 2chloro-6-methoxyisonicotinic acid 51e (266 mg, 1.42 mmol) in CH2Cl2 (5 mL) was treated in succession with 3-(pyrrolidin-1-yl)propan-1-amine (0.222 mL, 1.56 mmol), EDC (326 mg, 1.70 mmol), TEA (0.593 mL, 4.25 mmol) and HOBt (261 mg, 1.70 mmol). The reaction mixture was stirred at room temperature for 16 h. The mixture was subject to silica gel chromatography (gradient elution: 0-10% of 3% NH4OH/MeOH in EtOAc) to afford the title compound as a white solid (353 mg, 84%). LCMS using procedure A: MS m/z (ES+) (M+H)+ = 298.0. tR = 2.08 min. 2-Chloro-N-(3-(pyrrolidin-1-yl)propyl)-6-o-tolylisonicotinamide (52f). A solution of 2,6dichloro-N-(3-(pyrrolidin-1-yl)propyl)isonicotinamide 51f (1.89 g, 6.24 mmol), o-tolylboronic acid (0.81 g, 5.93 mmol), K2CO3 (2.59 g, 18.71 mmol), and Pd(PPh3)4 (0.72 g, 0.62 mmol) in a mixture of DME/water (10:1, 5.5 mL) was stirred at 80 °C for 12 h. The reaction mixture was concentrated in vacuo and the crude product purified silica gel chromatography (gradient elution: 0-10% of 3% NH4OH/MeOH in EtOAc) to afford the title compound as a solid (371 mg, 17%). LCMS using procedure A: MS m/z (ES+) (M+H)+ = 358.1. tR = 1.92 min. 2-(Piperazin-1-yl)benzaldehyde (53b). A solution of 4 M HCl in dioxane (10 mL, 288 mmol) was added to the solid tert-butyl 4-(2-formylphenyl)piperazine-1-carboxylate 53a (1 g, 3.44 mmol). The resulting suspension was stirred at ambient temperature for 16 h. The reaction mixture was diluted with hexane (20 mL), the solid from the reaction mixture was collected by filtration and dried in vacuo to yield the hydrochloride salt of the title compound as a yellow


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solid (0.780 g, 100%). LCMS using procedure B: MS m/z (ES+) (M+H)+ = 191.2. tR = 0.36 min. 2-(4-(3,4-Dichlorophenethyl)piperazin-1-yl)benzaldehyde (54). To a mixture of 3,4dichlorophenethyl methanesulfonate (722 mg, 2.65 mmol), prepared as described16 and 2(piperazin-1-yl)benzaldehyde 53b (600 mg, 2.65 mmol) in ACN (30 mL) was added K2CO3 (1829 mg, 13.23 mmol) in one portion. The suspension was stirred at 70 °C for overnight. Water was added to the mixture and the aqueous phase was extracted with CH2Cl2. The organics were dried over MgSO4, filtered and concentrated. The residue was purified by silica gel chromatography (gradient elution: 15-50% EtOAc in hexanes) to yield the title compound as a light yellow oil (620 mg, 65%). 1H NMR (400 MHz, DMSO-d6) δ 2.58-2.71 (m, 6H), 2.72-2.88 (m, 2H), 2.91-3.18 (m, 4H), 7.15 (t, J = 7.4 Hz, 1H), 7.22 (d, J = 7.8 Hz, 1H), 7.28 (dd, J = 8.1, 2.0 Hz, 1H), 7.45-7.64 (m, 3H), 7.70 (dd, J = 7.7, 1.6 Hz, 1H), 10.21 (s, 1H). LCMS using procedure A: MS m/z (ES+)+ = 364.9. tR = 2.16 min. 6-(2-(4-(3,4-Dichlorophenethyl)piperazin-1-yl)phenyl)-1H-pyrazolo[3,4-b]pyridine-4carboxylic acid (55). A mixture of 2-(4-(3,4-dichlorophenethyl)piperazin-1-yl)benzaldehyde 54 (611 mg, 1.68 mmol), 1H-pyrazol-3-amine (140 mg, 1.68 mmol), 2-oxopropanoic acid (148 mg, 1.68 mmol) in acetic acid (5 mL) was stirred at reflux for 16 h. The reaction was concentrated under reduced pressure. The residue was treated with Et2O and sonicated. The resultant solid was then collected by filtration. The solid was dissolved in DMSO and purified by reverse-phase HPLC (gradient elution: 10-75% ACN in water containing 0.1% trifluoroacetic acid) to afford the title compound (200 mg, 24%). LCMS using procedure A: MS m/z (ES+)+ (M+H)+ = 496.1. tR = 2.17 min.



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Tert-butyl 4-(2-bromophenyl)piperazine-1-carboxylate (57). To a stirred solution of 1-(2bromophenyl)piperazine 56 (650 mg, 2.70 mmol) and TEA (0.451 mL, 3.23 mmol) in a mixture of Et2O/THF (1:1, 10 mL) was added a solution of Boc2O (706 mg, 3.23 mmol) in Et2O (2 mL) dropwise. After 30 min the mixture was diluted with Et2O (10 mL), washed with water (10 mL) then brine (10 mL). The organic phase was dried over MgSO4, filtered through a plug of silica and concentrated to dryness to give rise to the desire product as a white semi-solid (898 mg, 98%). This material was used directly without further purification. 1H NMR (400 MHz, CDCl3)

δ 1.44-1.53 (m, 10H), 2.90-3.05 (m, 4H), 3.55-3.67 (m, 4H), 6.90-6.98 (m, 1H), 7.00-7.08 (m, 1H), 7.23-7.32 (m, 1H), 7.58 (dd, J = 8.1, 1.5 Hz, 1H). LCMS using procedure A: MS m/z (ES+) (M+H)+ = 340.9. tR = 3.40 min. Tert-butyl 4-(3'-(ethoxycarbonyl)biphenyl-2-yl)piperazine-1-carboxylate

(58a). To a

stirred mixture of tert-butyl 4-(2-bromophenyl)piperazine-1-carboxylate 57 (5.00 g, 14.65 mmol), 3-(ethoxycarbonyl)phenylboronic acid (3.55 g, 18.32 mmol) and K2CO3 (4.05 g, 29.30 mmol) in dioxane/water (10:1, 80 mL) was added Pd(PPh3)4 (1.693 g, 1.47 mmol). The reaction vessel was capped with a septa, purged with argon, and heated to 90 °C for 22 h. The reaction was diluted with water (100 mL) and extracted with ethyl acetate (2 x 100 mL). The combined organic extract was dried with MgSO4, filtered, and concentrated in vacuo. The crude product purified via silica gel chromatography (80 g; 20% EtOAc/hexanes as the eluent) to provide the title compound (5.90 g, 98%). LCMS using procedure A: MS m/z (ES+) (M+H)+ = 411.0. tR = 3.62 min. 2'-(4-(Tert-butoxycarbonyl)piperazin-1-yl)biphenyl-3-carboxylic acid (58b). To a stirred solution of tert-butyl 4-(3'-(ethoxycarbonyl)-biphenyl-2-yl)piperazine-1-carboxylate 58a (4.90 g, 11.94 mmol) in a mixture of THF/MeOH/water (2:2:1, 50 mL) was added the LiOH (0.858 g,


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35.81 mmol). The reaction was stirred at room temperature overnight. The volatiles were removed in vacuo and water (50 mL) was added. The flask was cooled in an ice bath and acidified with 1 M HCl (35 mL). A precipitate formed which was collected via vacuum filtration and the solid dried in vacuo to afford the desired compound as an off-white solid (4.18 g, 92%). 1

H NMR (400 MHz, DMSO-d6) δ 1.18-1.30 (m, 11H), 2.62 (br s, 5H), 3.03-3.15 (m, 5H), 6.96-

7.10 (m, 3H), 7.20 (dd, J = 7.5, 1.6 Hz, 1H), 7.22-7.36 (m, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.627.87 (m, 3H), 8.19 (t, J = 1.6 Hz, 1 H), 22.89 (br s, 1 H). LCMS using procedure A: MS m/z (ES+) (M+H)+ = 383.0. tR = 3.42 min. Tert-butyl

4-(3'-(3-(pyrrolidin-1-yl)propylcarbamoyl)biphenyl-2-yl)piperazine-1-

carboxylate (59). The HOBt (2.51 g, 16.39 mmol) and EDC (3.14 g, 16.39 mmol) were added to a stirred solution of 2'-(4-(tert-butoxycarbonyl)piperazin-1-yl)biphenyl-3-carboxylic acid 58b (4.18 g, 10.93 mmol), DIPEA (5.7 mL, 32.8 mmol) and 3-(pyrrolidin-1-yl)propan-1-amine (4.20 g, 32.8 mmol) in CH2Cl2 (38 mL). The reaction was stirred at room temperature overnight. The mixture was poured into water (250 mL) and extracted with CHCl3/IPA (10:1, 2 x 250 mL). The combined organic extracts were dried over MgSO4, filtered, and concentrated in vacuo. The crude product was purified via silica gel chromatography (10% MeOH/CH2Cl2 as eluent) to provide the title compound as a white solid (4.90 g, 91%).

1

H NMR (400 MHz, DMSO-d6) δ

1.37 (s, 9H), 1.58-1.79 (m, 6H), 2.51-2.59 (m, 5H), 2.70 (br s, 4H), 3.25-3.37 (m, 4H), 6.64 (s, 1H), 7.04-7.17 (m, 2 H), 7.25-7.38 (m, 2H), 7.44-7.60 (m, 2H), 7.74-7.82 (m, 2H), 8.09-8.20 (m, 2H), 8.48-8.67 (m, 1H). LCMS using procedure A: MS m/z (ES+) (M+H)+ = 493.2. tR = 2.47 min. 2'-(Piperazin-1-yl)-N-(3-(pyrrolidin-1-yl)propyl)biphenyl-3-carboxamide (60). The 4 M HCl in dioxane (10 mL, 40 mmol) was added to a stirred solution of tert-butyl 4-(3'-(3-



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(pyrrolidin-1-yl)propylcarbamoyl)biphenyl-2-yl)piperazine-1-carboxylate 59 (2.0 g, 4.06 mmol) in MeOH (6 mL). The mixture was stirred overnight at ambient temperature before being concentrated in vacuo. This material was triturated with Et2O, sonicated, and filtered to afford the dihydrochloride salt of the title compound as a white solid upon drying (1.710 g, 90%). 1H NMR (400 MHz, DMSO-d6) δ 1.79-2.06 (m, 4H), 2.83-3.05 (m, 7H), 3.08-3.22 (m, 2H), 3.303.42 (m, 2H), 3.45-3.57 (m, 2H), 4.80 (br s, 2H), 7.22-7.22 (m, 1H), 7.31-7.40 (m, 1H), 7.507.60 (m, 1H), 7.85 (dd, J = 19.7, 7.8 Hz, 1H), 8.20 (s, 1H), 8.64-8.92 (m, 1H), 8.79 (t, J = 5.8 Hz, 1H), 9.10-9.37 (m, 2H), 9.24 (br s, 2H), 10.73 (br s, 1H), 10.65-10.86 (m, 1H). LCMS using procedure A: MS m/z (ES+) (M+H)+ = 393.1. tR = 1.50 min. Tert-butyl

4-(3'-cyano-5'-((3-(pyrrolidin-1-yl)propyl)carbamoyl)-[1,1'-biphenyl]-2-

yl)piperazine-1-carboxylate (62a). A round bottom flask was charged with the 3-bromo-5cyano-N-(3-(pyrrolidin-1-yl)propyl)benzamide

52b

(745

mg,

2.22

mmol),

2-[4-(N-

Boc)piperazin-1-yl]phenylboronic acid pinacol ester 61 (946 mg, 2.44 mmol), Pd(PPh3)4 (77 mg, 0.07 mmol), K2CO3 (919 mg, 6.65 mmol) and a stir bar. The flask was evacuated and back-filled with N2 three-times. A mixture of DME/water (5:1, 22 mL - previously degassed via N2 sparge) was added and the mixture was placed in a pre-heated oil bath at 70 °C for 16 h. The reaction was cooled to ambient temperature, diluted with ethyl acetate and the aqueous phase was separated. The organics were washed with brine and concentrated under reduced pressure. The resultant gum was subject to silica gel chromatography (gradient elution:

2-10% of 2 M

NH3/MeOH in CH2Cl2) to afford the title compound as a tacky solid (986 mg, 86%). 1H NMR (400 MHz, DMSO-d6) δ 1.37 (s, 9H), 1.59-1.79 (m, 6H), 2.43 (br s, 6H), 2.70 (br s, 4H), 3.19 (br s, 4H), 3.41 (br s, 2H), 7.07-7.23 (m, 2H), 7.33-7.47 (m, 2H), 8.19 (d, J = 9.3 Hz, 2H), 8.49 (s, 1H), 8.76 (br s, 1H). LCMS using procedure B: MS m/z (ES+) (M+H)+ = 518.2. tR = 0.94 min.


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5-Cyano-2'-(piperazin-1-yl)-N-(3-(pyrrolidin-1-yl)propyl)-[1,1'-biphenyl]-3-carboxamide (62b). Trifluoroacetic acid (3 mL, 38.94 mmol) was added to a stirred solution of tert-butyl 4(3'-cyano-5'-((3-(pyrrolidin-1-yl)propyl)carbamoyl)-[1,1'-biphenyl]-2-yl)piperazine-1carboxylate 62a (960 mg, 1.85 mmol) in CH2Cl2 (5 mL) at ambient temperature. After 45 min the reaction was concentrated under reduced pressure and the gum reconstituted in CH2Cl2 and concentrated again. Drying in vacuo afforded the title compound as the bis-trifluoroacetate salt (1.725 g). This material was used directly without further purification. LCMS using procedure B: MS m/z (ES+) (M+H)+ = 418.3. tR = 0.52 min. Tert-butyl 4-(2-(4-(3-(pyrrolidin-1-yl)propylcarbamoyl)pyridin-2-yl)phenyl) piperazine-1carboxylate (63a). A mixture of 2-bromo-N-(3-(pyrrolidin-1-yl)propyl)iso-nicotinamide 52d (2.475 g, 7.93 mmol), 2-[4-(N-Boc)piperazin-1-yl]phenylboronic acid pinacol ester (3.14 g, 8.09 mmol), and K2CO3 (3.29 g, 23.78 mmol) in DME/water (10:1, 40 mL) was charged with Pd(PPh3)4 (0.733 g, 0.63 mmol) under a N2 sparge. The stirred mixture was heated at 75 °C for 8 h, at which time TLC (10% MeOH + 3% NH4OH/EtOAc) revealed some starting bromide was still present. Additional 2-[4-(N-Boc)piperazin-1-yl]phenylboronic acid pinacol ester 61 (0.5 equiv), K2CO3 (1.5 equiv), and Pd(PPh3)4 (0.04 equiv) were added and the mixture was set to heat 80 °C for an additional 22 h. The reaction was cooled to room temperature, filtered and the crude product purified by silica gel chromatography (gradient elution: 1-10% of 1% NH4OH/MeOH in CH2Cl2) to afford the title compound as a white waxy solid (2.02 g, 52%). This material was used directly without further characterization. 2-(2-Piperazin-1-ylphenyl)-N-(3-pyrrolidin-1-ylpropyl)pyridine-4-carboxamide (63b). To a

solution

of

tert-butyl

4-(2-(4-(3-(pyrrolidin-1-yl)propylcarbamoyl)pyridin-2-

yl)phenyl)piperazine-1-carboxylate 63a (2.02 g, 4.09 mmol) in CH2Cl2 (25 mL) was added TFA



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(10 mL). The solution was stirred at room temperature until the complete consumption of the starting material as judged by TLC analysis (15% MeOH/CH2Cl2).

The solution was

concentrated under reduced pressure, reconstituted in CH2Cl2 and concentrated again. The residue was treated with 4 M HCl in dioxane to afford the dihydrochloride salt of the title compound as a tacky gum (2.385 g, quant). This material was taken forward without further purification. LCMS using procedure A: MS m/z (ES+) (M+H)+ = 394.2. tR = 1.02 min. Tert-butyl4-(2-(5-(ethoxycarbonyl)pyridin-3-yl)phenyl)piperazine-1- carboxylate (65). A mixture of 3-(ethoxycarbonyl)pyridine-5-boronic acid pinacol ester 64 (526 mg, 1.90 mmol), tert-butyl 4-(2-bromophenyl)piperazine-1-carboxylate 57 (648 mg, 1.90 mmol), and Cs2CO3 (928 mg, 2.85 mmol) was stirred in a mixture of dioxane/water (4:1, 15 mL). The stirred solution was degassed by nitrogen (g) sparge at room temperature for 5 min prior to the addition of Pd(PPh3)4 (43.9 mg, 0.04 mmol). The mixture was then heated to 75 °C overnight, cooled to room temperature, and concentrated under reduced pressure.

The residue was partitioned

between water (10 mL) and CH2Cl2 (30 mL). The organic phase was separated and the aqueous phase was extracted with CH2Cl2 (10 mL). The combined organics were washed with water (10 mL), brine (10 mL), dried over MgSO4, filtered and concentrated to dryness. The crude product mixture was purified by silica gel chromatography (gradient elution of 1-5% MeOH/CH2Cl2) to afford the title compound as an off-white powder (540 mg, 69%). 1H NMR (400 MHz, CDCl3) δ 1.40-1.47 (m, 12H), 2.78 (br s, 4H), 3.32 (br s, 4H), 4.45 (q, J = 7.2 Hz, 2H), 7.09-7.14 (m, 1H), 7.15-7.23 (m, 1H), 7.31 (dd, J = 7.6, 1.5 Hz, 1H), 7.39 (td, J = 7.7, 1.8 Hz, 1 H), 8.65 (t, J = 2.2 Hz, 1H), 9.03 (d, J = 2.0 Hz, 1H), 9.16 (d, J = 2.0 Hz, 1H). LCMS using procedure A: MS m/z (ES+) (M+H)+ = 422.1. tR = 3.19 min.


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Tert-butyl 4-(2-(5-(3-(pyrrolidin-1-yl)propylcarbamoyl)pyridin-3-yl)phenyl) piperazine 1-carboxylate

A

(66a).

solution

of

tert-butyl

4-(2-(5-(ethoxycarbonyl)pyridin-3-

yl)phenyl)piperazine-1-carboxylate 65 (400 mg, 0.97 mmol) and 3-(pyrrolidin-1-yl)propan-1amine (374 mg, 2.92 mmol) were stirred in toluene (10 mL). A catalytic amount of TBD (~15 mg) was added and the reaction was warmed to 90 oC. After 15 h, the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (5% MeOH in CH2Cl2 as the eluent) to give rise to the title compound as a light tan solid (450 mg, 94%). 1H NMR (400 MHz, C6D6) δ 1.34 (dt, J = 11.5, 5.9 Hz, 3H), 1.41 (s, 9H), 1.46-1.55 (m, 4H), 2.15 (br s, 4H), 2.17-2.24 (m, 2H), 2.38-2.41 (m, 4H), 3.13-3.45 (m, 4H), 3.52-3.59 (m, 2H), 6.626.67 (m, 1H), 6.83-6.91 (m, 1H), 7.02-7.08 (m, 2H), 8.82 (t, J = 2.0 Hz, 1H), 8.89 (br s, 1H) 9.08 (d, J = 2.3 Hz, 1H), 9.18 (d, J = 2.0 Hz, 1H). LCMS using procedure A: MS m/z (ES+) (M+H)+ = 493. tR = 2.03 min. 5-(2-(Piperazin-1-yl)phenyl)-N-(3-(pyrrolidin-1-yl)propyl)nicotinamide (66b). Method

A.

To

a

stirred

solution

of

tert-butyl

4-(2-(5-(3-(pyrrolidin-1-

yl)propylcarbamoyl)pyridin-3-yl)phenyl)piperazine-1-carboxylate 66a (400 mg, 0.81 mmol) in CH2Cl2 (3 mL) was added a mixture of TFA/CH2Cl2 (1:1, 6 mL) dropwise at ambient temperature. After 15 h the solvent was removed in vacuo and the resulting gum was purified by reverse-phase HPLC (C18, gradient elution, 10-75% ACN in water with 0.1% trifluoroacetic acid).

The fractions were lyophylized to yield the the bis-trifluoroacetate salt of the title

compound as a glassy solid (520 mg, quant). This material carried forward without further characterization. Method B. A mixture of 5-bromo-N-(3-(pyrrolidin-1-yl)propyl)nicotinamide 68 (365 mg, 1.17 mmol), 2-[4-(N-Boc)piperazin-1-yl]phenylboronic acid pinacol ester 61 (500 mg, 1.29 mmol),



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Page 58 of 69

and K2CO3 (485 mg, 3.51 mmol), in DME/water (10:1, 3.3 mL) was treated with Pd(PPh3)4 (135 mg, 0.22 mmol) and set to heat at 70 °C for 16 h. The reaction was cooled to room temperature and partitioned between water (10 mL) and CH2Cl2 (30 mL). The organic phase was separated and the aqueous phase was extracted with CH2Cl2 (3 x 10 mL). The combined organics were dried over MgSO4, filtered and concentrated to dryness. The crude product mixture was purified by silica gel chromatography (gradient elution: 1-10% of 1% NH4OH/MeOH in CH2Cl2) to give rise to the intermediate tert-butyl 4-(2-(5-(3-(pyrrolidin-1-yl)propyl-carbamoyl)pyridin-3yl)phenyl) piperazine-1-carboxylate 66a. The residue was reconstituted in CH2Cl2 (10 mL) and treated with TFA (10 mL) at ambient temperature. Concentration under reduced pressure and drying in vacuo afforded the bis-trifluoroacetate salt title compound as a semi-solid (727 mg, 99%). LCMS using procedure A: MS m/z (ES+) (M+H)+ = 494.4. tR = 2.14 min. 5-Bromo-N-(3-pyrrolidin-1-ylpropyl)pyridine-3-carboxamide (68). A mixture of 3(pyrrolidin-1-yl)propan-1-amine (0.222 mL, 1.56 mmol), 5-bromopyridine-3-carboxylic acid 67 (286 mg, 1.42 mmol), EDC (326 mg, 1.70 mmol), HOBt (260 mg, 1.70 mmol), and TEA (0.592 mL, 4.25 mmol) in CH2Cl2 (3 mL) stirred overnight at room temperature. The reaction was further diluted with CH2Cl2 and washed with saturated aq. NaHCO3.

The organics were

separated and dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (gradient elution: 1-10% of 3% NH4OH/MeOH in EtOAc) to afford the title compound (1.31 g, 85%). LCMS using procedure A: MS m/z (ES+) (M+H)+ = 311.9. tR = 1.10 min. ITC. ITC experiments were conducted using a Microcal ITC200 at 23 ºC in 25 mM Tris (pH 8.5), 50 mM NaCl and 2% DMSO. Approximately 300 µL of a 15 µM SMYD2 protein solution was loaded into the sample cell, and a 200 µM compound solubilized in DMSO was loaded into


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the injection syringe. Eight 4 µL injections of compound were sequentially added to the sample cell, producing a final compound-to-protein molar ratio of approximately 1.4:1. Thermodynamic parameters N (stoichiometry), Ka (association constant) and ∆H° (enthalpic change) were obtained by nonlinear least-square fitting using Origin software. Crystallography. SMYD2 protein expressed and purified as described9. All compounds were co-crystallized with SMYD2 using previously established crystallization conditions9. Crystals typically appeared within three days and achieved their full size in one week. All crystals were vitrified in liquid nitrogen using a cryoprotectant solution consisting of a 4:1 ratio of reservoir solution and L-(+)-2,3-butanediol.

All X-ray diffraction data were collected at cryogenic

temperature using synchrotron radiation at beam line ID 23.1 at the European Radiation Synchrotron Facility (Grenoble, France) (Table S1). Diffraction data was processed with XDS and scaled using SCALA22 as implemented in the autoPROC routines from Global Phasing. These crystals belong to the tetragonal space group I4 and contain one molecule per asymmetric unit. All liganded structures were solved by molecular replacement. The resulting difference density maps unambiguously showed connected difference density with the expected molecular features for these inhibitors. Sequential rounds of model building using Coot and refinement using autoBUSTER22 produced models that contain all residues except for the first four Nterminal residues. The final refinement statistics are shown in Table S1. All figures were prepared using PyMOL (The PyMOL Molecular Graphics System, Schrödinger, LLC.). Cell Assay. U2OS cells were plated at a density of 8,000 cells/well in 96-well plates and transfected using the Fugene system with constructs expressing wild-type SMYD2. Compounds were solubilized in 100% DMSO and diluted with cell culture media (final DMSO concentration of 0.2%) before being added to cells. Cells were incubated for 24 hours following transfection,



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washed and fixed in 4% PFA, then stained with anti-FLAG Tag antibody at a dilution of 1:800, and a custom polyclonal antibody (21st Century, Marlboro, MA) generated against the monomethylated p53 epitope at a dilution of 1:1000. Cells were stained with DAPI and images were captured on an Image Express instrument. Fluorescent intensity and cell number was quantified using Accuity and MetaExpress software. Proliferation Assay. HCT116 p53+/+, HCT116 p53-/-, RKO-neo and RKO-E6a cells were seeded in triplicate in 96-well plates, and allowed to attach overnight. Media was then replaced with either DMSO control media or media containing compounds 1, 40, or 47 at concentrations ranging from 0.03 to 30 µM. Cells were incubated in the presence of compound for 72 hours. Viability was determined by measuring fluorescent intensity following incubation with AlamarBlue. IC50 values were determined as previously described14. Graphical representations were created using Prism.

ASSOCIATED CONTENT Supporting Information. Tables S1 and S2. PDB Accession Codes. The coordinates and structure factor amplitudes of the SMYD2 inhibitor complexes have been deposited in the Protein Data Bank with the following accession codes: 5KJM (30), 5KJK (48), 5KJN (47) and 5KJL (5), and will be released upon publication. AUTHOR INFORMATION Corresponding Authors


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*E-mail: [email protected] (Tel: 774-278-8014) and [email protected] (Tel: 908-5287078) Author Contributions The manuscript was written through contributions of all authors.

All authors have given

approval to the final version of the manuscript. Notes The authors declare no competing ﬁnancial interest. ACKNOWLEDGEMENTS The authors wish to thank J. Debreczeni and J. Tucker for their assistance with data collection; and E. Code, H. Pollard and X. Zhu for protein preparation and A. Nielsen for additional chemical synthesis support. We thank the staff of the European Radiation Synchrotron Facility for their assistance. Use of the IMCA-CAT beamline 17-ID at the Advanced Photon Source was supported by the companies of the Industrial Macromolecular Crystallography Association through a contract with Hauptman-Woodward Medical Research Institute. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. ABBREVIATIONS ADME, adsorption, distribution, metabolism and excretion; PK, pharmacokinetics; IC50, halfmaximal inhibitory concentration; HTS, high-throughput screening; ITC, isothermal titration calorimetry; ACN, acetonitrile; DIPEA, diisopropylethylamine; DME, dimethoxyethane; DMF,



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N,N-dimethylformamide; DMA, N,N-dimethylacetamide; DMSO, dimethyl sulfoxide; EDC, 1ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; EtOAc, ethyl acetate; GMF, glass membrane filter; HATU, (dimethylamino)-N,N-dimethyl(3H-[1,2,3]triazolo[4,5-b]pyridin-3loxy)methaniminium hexafluoro-phosphate; HOBt, 1-hydroxybenzotriazole; IPA, isopropyl alcohol; NMP, N-methyl-2-pyrrolidone; TBD, 1,5,7-triazabicyclo[4.4.0]dec-5-ene; TEA, triethylamine; TFA, trifluoroacetic acid; THF, tetrahydrofuran

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C.; Keller, P.; Paciorek, W.; Roversi, P.; Smart, O.S.; Vonrhein, C.; Womack, T.O. BUSTER version 2.8.0. Cambridge, United Kingdom: Global Phasing Ltd. 2009.


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