Minimizing CYP2C9 Inhibition of Exposed-Pyridine NAMPT

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Minimizing CYP2C9 Inhibition of Exposed-Pyridine NAMPT (Nicotinamide Phosphoribosyltransferase) Inhibitors Mark Zak, Po-Wai Yuen, Xiongcai Liu, Snahel Patel, Deepak Sampath, Jason Oeh, Bianca M. Liederer, Weiru Wang, Thomas O'Brien, Yang Xiao, Nicholas Skelton, Rongbao Hua, Jasleen Sodhi, Yunli Wang, Lei Zhang, Guiling Zhao, Xiaozhang Zheng, Yen-Ching Ho, Kenneth W. Bair, and Peter S. Dragovich J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00697 • Publication Date (Web): 19 Aug 2016 Downloaded from http://pubs.acs.org on August 20, 2016

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Journal of Medicinal Chemistry

Minimizing CYP2C9 Inhibition of Exposed-Pyridine NAMPT (Nicotinamide Phosphoribosyltransferase) Inhibitors Mark Zaka,*, Po-wai Yuenc, Xiongcai Liuc, Snahel Patela, Deepak Sampatha, Jason Oeha, Bianca M. Liederera, Weiru Wanga, Thomas O’Briena, Yang Xiaoa, Nicholas Skeltona, Rongbao Huac, Jasleen Sodhia, Yunli Wangc, Lei Zhangc, Guiling Zhaoa, Xiaozhang Zhengb, Yen-Ching Hob, Kenneth W. Bairb, Peter S. Dragovicha

a

Genentech Inc., 1 DNA Way, South San Francisco, California 94080, USA

b

FORMA Therapeutics Inc., 500 Arsenal Street, Watertown, Massachusetts 02472, USA

c

Pharmaron Beijing Co. Ltd., 6 Taihe Road, BDA, Beijing 100176, PR China

*

Corresponding Author. E-mail address: [email protected]. Phone: 650-467-4533

1

ACS Paragon Plus Environment


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ABSTRACT: NAMPT inhibitors may show potential as therapeutics for oncology. Throughout our NAMPT inhibitor program, we found that exposed pyridines or related heterocyclic systems in the left-hand portion of the inhibitors are necessary pharmacophores for potent cellular NAMPT inhibition. However, when combined with a benzyl group in the center of the inhibitors, such pyridine-like moieties also led to consistent and potent inhibition of CYP2C9. In an attempt to reduce CYP2C9 inhibition a parallel synthesis approach was used to identify central benzyl group replacements with increased Fsp3. A spirocyclic central motif was thus discovered that was combined with left-hand pyridines (or pyridine-like systems) to provide cellularly potent NAMPT inhibitors with minimal CYP2C9 inhibition. Further optimization of potency and ADME properties led to the discovery of compound 68, a highly potent NAMPT inhibitor with outstanding efficacy in a mouse tumor xenograft model, and lacking measurable CYP2C9 inhibition at the concentrations tested. INTRODUCTION: Nicotinamide adenine dinucleotide (NAD) is a ubiquitously expressed biomolecule implicated in multiple biological processes including cell metabolism, calcium homeostasis, cell survival, and aging.1 NAD acts as both an electron-carrying cofactor for oxidoreductases, and as an ADP-ribose donator in multiple enzymatic reactions.1 In two examples of the latter mode of action, NAD participates in reactions catalyzed by PARPs (poly-ADP ribosylation)2 and Sirtuins (deacylation and ADP ribosylation).3 Such reactions consume NAD, while liberating nicotinamide (NAM) as a byproduct (see Figure 1a).

The wide ranging biological functions of NAD, coupled with its

consumption by enzymes such as the PARPs and Sirtuins requires cells to replenish NAD in order to survive.4 Multiple pathways for NAD biosynthesis have been elucidated, including a mechanism that recycles NAM back to NAD (see Figure 1a).5

This pathway relies on nicotinamide 2


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phosphoribosyltransferase (NAMPT) to catalyze its rate-determining step, a condensation reaction between NAM and phosphoribosyl pyrophosphate (PRPP) to generate nicotinamide mononucleotide (NMN).6

NMN, the phosphoribosylated adduct of NAM, is then converted to NAD in a reaction

catalyzed by an additional enzyme, nicotinate/nicotinamide mononucleotide adenyltransferase (NMNAT).

Rapidly proliferating cells such as cancers possess a high metabolic load and a

corresponding high demand for NAD. Reduction of intracellular NAD concentrations via blockade of the NAM-NAD recycling may, thus, be an appropriate mechanism to target such cells.7 As such, there is interest in identifying NAMPT inhibitors as potential therapies for oncology indications.4,8

3



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Figure 1a. The NAM-NAD recycling pathway. Figure 1b. NMN-like phosphoribose adducts generated by co-incubating NAMPT inhibitors 1, 2, 3, or 7 with NAMPT, ATP, and PRPP.9a,9c,20,21

As shown in Figure 2, multiple NAMPT inhibitors have been previously reported by both our own group (compounds 1-69), as well as by other organizations (compounds 710 and 811).

These

compounds share similar molecular features, and as exemplified by compound 1, can be divided into four general regions. The left-hand side is comprised of a pyridine or pyridine-like heterocycle containing an exposed and nucleophilic nitrogen atom. The linker group is an amide, urea, or cyanoguanidine. The central region is a lipophilic group comprised of a benzyl group, a phenyl, or an aliphatic chain. The right-hand side may encompass a wide array of groups, including aromatic and non-aromatic cycles, with or without polar heteroatoms. One recurring issue with all these molecules is persistent inhibition of CYP2C9. Indeed, as exemplified in Table 1, the majority of the previously reported NAMPT inhibitors exhibited potent CYP2C9 inhibition, with IC50 values typically less than 1 µM ((S)-warfarin probe). Also notable is the CYP isoform selectivity exhibited by certain classes of NAMPT inhibitors.

Compounds containing bicyclic left-hand moieties,

exemplified by 1, 3, 5, and 6, were typically potent inhibitors of only CYP2C9, and exhibited little or no inhibition of the other CYP isoforms tested. Another notable finding was the importance of the probe used to assess CYP2C9 inhibition. Indeed, as exemplified by compounds 1 and 3, and consistent with what has previously been reported by others,12 much different inhibition profiles were observed depending on whether (S)-warfarin or diclofenac was used. As (S)-warfarin was the more sensitive of the two probes for this class of inhibitors, it was used to assess CYP2C9 inhibition for the remainder of this work.

4


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Figure 2. Structures of previously reported NAMPT inhibitors.9,10,11

Table 1. NAMPT and CYP2C9 Inhibition Data for Previously Reported NAMPT inhibitors.9,10,11

Cmpd.

NAMPT A2780 IC50a IC50b (µM) (µM)

CYP Inhibition IC50c (µM) 2C9

2C9

3A4

3A4

((S)-Warfarin) (Diclofenac) (Testosterone) (Midazolam)

1

0.005

0.002

0.20

2

0.007

0.032

0.42

3

0.006

0.004

0.07

4

0.004

0.006

5

0.011

6

>10

2D6 (Dextromethorphan)

1A2

2C19

(Tacrine) (Mephenytoin)

>10

>10

>10

>10

>10

>10

>10

>10

>10

>10

7.9

9.0

>10

>10

6.5

0.72

0.09

0.68

>10

>10

>10

0.011

0.08

>10

>10

>10

>10

>10

0.039

0.011

0.16

>10

>10

>10

>10

>10

7

0.002

0.001

0.12

0.22

0.32

0.19

>10

0.63

8

0.003

0.001

3.6

1.5

0.54

>10

4.8

>10

5



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All assay results are reported as the arithmetic mean of at least two separate runs (n ≥ 2). See reference 9d for experimental details. a

NAMPT biochemical inhibition. The average coefficient of variation (CV) for all compounds reported in this work is

0.48. Individual CV values are reported in the Supporting Information. b

Antiproliferation activity determined in cell culture experiments using A2780 cell line. This inhibition can be reversed

by addition of 0.33 µM of NMN, strongly implicating NAMPT inhibition as the causative MOA. The average coefficient of variation (CV) for all compounds reported in this work is 0.23. Individual CV values are reported in the Supporting Information. c

Reversible Cytochrome P450 2C9 inhibition. The probe used to assess inhibition is indicated in parentheses.

CYP2C9 inhibition is undesirable for a number of reasons.13 CYP2C9 comprises a substantial proportion of human liver microsomal P450 content, second only to CYP3A4 in abundance. Additionally CYP2C9 is implicated in the metabolism of a significant percentage of drugs undergoing phase 1 metabolism, with values of 15 – 20% reported in the literature.14 Included amongst these are several non-steroidal anti-inflammatory drugs, oral antidiabetics, and angiotensin II receptor blockers. CYP2C9 is also the primary enzyme responsible for metabolism of the oral anticoagulant (S)-warfarin, a drug with a low therapeutic index. Drugs metabolized by CYP2C9, especially those with low therapeutic indexes, will be susceptible to drug-drug interactions and potential toxicity when co-administered with potent CYP2C9 inhibitors. Finally the CYP2C9 gene is highly polymorphic with common variants exhibiting poor metabolism phenotypes. Patients carrying such genetic variants and expressing defective or partially defective forms of CYP2C9 are especially susceptible to CYP2C9 inhibitors. As such, we wished to discover NAMPT inhibitors with reduced CYP2C9 inhibition profiles.

6


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Developing strategies to reduce CYP inhibition remains an emerging area of drug discovery research. Although several computational approaches have been proposed,15 it remains challenging to develop a general model to predict CYP2C9 inhibition due to several factors.16 The active sites of CYPs are generally large and flexible, consistent with their role of accommodating and metabolizing a wide array of xenobiotics and endogenous substrates. Multiple binding modes and a spectrum of binding sites are possible, spanning direct interaction with the heme, to water mediated heme binding, to binding in close proximity to the heme, or binding to allosteric sites distant from the heme. Additionally, multiple ligands may bind simultaneously, and covalent attachment is also possible.

Despite not yet being routine, examples of structure based drug design influencing

CYP2C9 inhibition are also beginning to emerge in the literature. Indeed, a recent disclosure by scientists at AstraZeneca and the University of Gothenburg has demonstrated the use of X-ray crystal structures of modified forms of CYP2C9 and CYP3A4 in complex with inhibitors.16 While two successful examples were presented, the authors also highlight the technical challenges associated with routine collection of CYP-inhibitor complexed crystal structures and the fact that iterative CYP structural support is not realistic at the present time. Physical chemical properties and shape also play a role in CYP inhibition. Indeed, reducing the number of aromatic rings and increasing the fraction of sp3 centers (Fsp3)17 may improve a number of drug developability parameters, including reduced CYP inhibition.18 Additionally, a recent review summarizes common strategies used to circumvent inhibition of CYP2C9 and other CYP isoforms from a medicinal chemist’s perspective.19 Although no general solutions are identified, successful examples are presented of modulating properties such as lipophilicity and molecular weight, and blocking or replacing common CYP inhibitory pharmacophores including exposed pyridines and related aromatic heterocycles. 7



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Conspicuously, all of the compounds depicted in Figure 2 contain an archetypal CYP pharmacophore in the left-hand portion of the inhibitors, an exposed pyridine or related heterocycle with an exposed and nucleophilic nitrogen atom. Such heterocycles are typically thought to cause inhibition by binding to the heme center of the CYP enzymes.15f Unfortunately, these exposed and nucleophilic left-hand groups are also a necessary pharmacophore for potent NAMPT cell based inhibition. We9a,9c,20 and others,21 have shown that such compounds mimic NAMPT’s endogenous substrate, nicotinamide, by undergoing enzyme-catalyzed condensation with PRPP as in step 1 of Figure 1a. Indeed, as shown in Figure 1b, the NMN-like phosphoribose adducts 1′, 2′, 3′, and 7′ have been generated and characterized by incubating inhibitors 1, 2, 3, and 7 with NAMPT, ATP, and PRPP.9a,9c,20,21 Additionally, it has been demonstrated that the phosphoribose adduct 7′ generated in HeLa cells was retained within the cell, whereas the parent compound 7 was readily washed out.21 The formation and subsequent retention of highly impermeable phosphoribose adducts within the cellular environment appears to be a critical factor associated with the cell based potency of NAMPT inhibitors. Thus, the pharmacophores for CYP2C9 inhibition and potent cellular NAMPT inhibition are closely linked.

A previous publication by our group has described a partial solution to the CYP2C9 inhibition issue in the urea series of NAMPT inhibitors related to compound 2.9a,22 Indeed, as shown in Table 2, compound 9, containing an exposed left-hand pyridine moiety was both a relatively potent cellular NAMPT inhibitor and an extremely potent inhibitor of CYP2C9. Consistent with the hypothesis that the pyridine motif was the CYP2C9 pharmacophore, blocking the pyridine N with an ortho methyl group eliminated measurable CYP inhibition at the concentrations measured (compound 10). 8


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However, cellular NAMPT inhibition was also reduced dramatically. Multiple other groups were screened in an attempt to reduce CYP inhibition while maintaining potent cellular NAMPT activity. The only such modification to produce the desired result was an ortho amino group (compound 11). The increased steric bulk adjacent to the pyridine center eliminated CYP2C9 inhibition, while the strongly donating amino group presumably allowed the less accessible pyridine to maintain the required nucleophilicity for phosphoribose adduct formation and cell activity. Unfortunately the increased electron richness present in 11 also led to potent time dependent inhibition (TDI) of CYP3A4, limiting the utility of this group. Interestingly, in the course of this work it was also found that modification of the right-hand side of the inhibitor, a site distal to the nucleophilic pyridine, could dramatically reduce CYP2C9 inhibition. Regrettably, such modifications were also found to introduce a new liability into the inhibitors. As exemplified by compound 12, the highly polar and often basic groups that were effective in reducing CYP2C9 inhibition also led to extremely low MDCK permeability and hampered oral bioavailability.

One final improvement to CYP2C9

inhibition was previously noted in the optimization of bicyclic azabenzofuran NAMPT inhibitors, again at a site distal to the nucleophilic pyridine center.9f In this case moving from sulfone 13 to the more polar sulfoxide 14 led to a minor decrease in CYP2C9 inhibition, but once again, this CYP2C9 improvement was accompanied by an additional negative outcome in the form of reduced metabolic stability and increased in vivo clearance. To summarize our previous findings, certain modifications in the immediate proximity of, as well as distal to, the left-hand nucleophilic aromatic center of NAMPT inhibitors were found to improve CYP2C9 inhibition.

This observation implied a

cooperative effect between the left-hand pyridine and the central or right-hand portion of the molecule in producing the CYP2C9 inhibition. However, all modifications to both the pyridines and the center / right-hand sides of previously reported molecules led to additional undesired liabilities. 9



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Thus, a solution to the CYP2C9 inhibition problem that did not introduce additional liabilities into the inhibitors was still required.

Table 2. NAMPT and CYP2C9 Inhibition Data for Previously Reported NAMPT Inhibitors.9a,9f,22

Y

NAMPT IC50a (µM)

A2780 CYP2C9 IC50a LogD7.4b Inhibition (µM) IC50a,c (µM)

Cmpd.

Scaffold

X

9

A

H

0.003

0.144

2.7i

0.07

10

A

CH3

0.059

>2

2.4 i

>10

11

A

NH2

0.003

0.070

2.7 i

>10

12

A

H

0.006

0.076

10

CYP3A4 TDI (%AUC shift)d te 10

28

>2

>2

1.7

0.33

>10

0.002e

0.013

2.0

0.43

0.08

Cmpd.

R

N

26 O

29

NAf

14


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Unless otherwise indicated, all assay results are reported as the arithmetic mean of at least two separate runs (n ≥ 2). a

See footnotes from Table 1 for assay descriptions.

b

See footnotes from Table 2 for assay description.

c

Fraction of sp3 centers. (number of sp3 hybridized carbons ÷ total carbon count)

d

(S)-Warfarin probe used.

e

Single determination (n=1).

f

Not applicable.

To aid further optimization, X-ray crystal structures of compounds 1 – 6 (or closely related analogs) and compound 8 in complex with NAMPT were examined. These structures showed that the central portions of the various molecules were not perfectly coincident, and indeed swept out a diverse volume of space (see Figure 3). This observation suggested the NAMPT protein could be tolerant of inhibitors with different shapes in this region. Additionally the interactions between the central region of the inhibitors and the NAMPT protein were primarily hydrophobic in nature (see Figure 4a for a discussion of the residues in this region). The lack of specific polar interactions with strict requirements for directionality, coupled with the observation that a variety of shapes could be tolerated in the central portion of the NAMPT inhibitors led us to focus on a parallel synthesis approach as opposed to rigorous structure based design. We believed parallel synthesis could allow us to rapidly explore an array of shapes extending into the central portion of the NAMPT protein. The primary goal was to find high Fsp3 central moieties to improve potency relative to 27 and 28 while maintaining the favorable lack of CYP2C9 inhibition. Thus, we designed a 119-membered

15



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library (see Table 5 for general structure and selected examples) heavily focused on maintaining a high degree of saturation in the central and right-hand portions of the inhibitors.23

Figure 3. Overlay of indicated ligands from previously reported X-ray structures in complex with NAMPT. The proteins are omitted for clarity. Accession codes are as follows: 1: 4KFO,9b 2a: 4JR5,9a 3: 4O13,24 4: 4LVG,9d 5a: 4KFP,9e 6a: 4WQ6,9f 8: 2GVJ.25 1X-ray structures of compounds 2, 5, and 6 in complex with NAMPT were not available at the time of this work. Thus, structures of related analogs 2a, 5a, and 6a were used as surrogates.

After synthesis, all of the library compounds were initially tested at a single concentration of 1 µM to assess for NAMPT inhibition. Unfortunately, the majority were found to be virtually inactive, with less than 50% inhibition at 1 µM observed for 117 of the 119 analogs prepared. The two noteworthy exceptions were spirocycles (±)-30 and (±)-31. These compounds showed significant NAMPT inhibition at the 1 µM screening concentration, which further translated to IC50 values of 830 nM ((±)-30) and 74 nM ((±)-31). Notably, the spirocyclic system was found to be crucial to NAMPT inhibition, as related open chain analogs such as 32 and (±)-33 were found to be far less potent NAMPT inhibitors. The final significant finding was the greatly reduced CYP2C9 inhibition of the 16


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compounds in Table 5. Importantly, the compound with the best NAMPT potency (compound (±)31) also exhibited negligible CYP2C9 inhibition at the concentrations tested. These results validated the hypothesis that a high degree of saturation in the central portion of the scaffold was a viable approach to separate the overlapping NAMPT and CYP2C9 pharmacologies observed with earlier inhibitors.

Table 5. NAMPT and CYP2C9 Inhibition Data of Selected Compounds From a 119-Membered Parallel Synthesis Library.

% NAMPT Inhibition at 1 µMa

NAMPT IC50 (µM)b

Fsp3c

CYP2C9 Inhibition IC50b,d (µM)

(±)-30

64

0.830

0.61

>10

(±)-31

96

0.074

0.57

>10

32

26

>1.7

0.55

>10

(±)-33

17

>2

0.55

3.4

Cmpd.

R

All assay results are reported as the arithmetic mean of at least two separate runs (n ≥ 2). All compounds are racemic. a

Biochemical NAMPT inhibition at a single inhibitor concentration of 1 µM.

17



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b


c

See footnotes from Table 4 for description.

d


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As shown in Table 6, the most promising spirocyclic moiety identified from the parallel synthesis approach was then appended to several additional left-hand groups mimicking nicotinamide.26 The 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine group present in compound (±)-40 was found to combine best with the spirocyclic moiety. Indeed (±)-40 was not only 10-fold more potent than (±)-31 in the biochemical assay, but was also found to be the most cell potent NAMPT inhibitor, with an IC50 value of 2 nM in the A2780 anti proliferation assay. The outstanding cell potency of (±)-40 did not appear to be related to an increase in membrane permeability relative to the other spirocyclic inhibitors tested. Indeed, all of the compounds in Table 6 tested in the MDCK assay were found to be highly permeable, including compound (±)-34 which lacked measurable cell based NAMPT inhibition. The difference in cell activity is likely related to the positioning of the nucleophilic pyridine or related heterocycle in the left-hand portion of the inhibitor. We believe the pyridine present in (±)-40 is most ideally situated to form the phosphoribose adducts similar to NMN (see Figure 1) previously shown to be a necessary prerequisite to NAMPT cell based potency.20 This hypothesis is supported by the X-ray crystal structure of the enantiomerically pure form of compound (±)-40 in complex with NAMPT (see Figure 4 and accompanying discussion).

Table 6. NAMPT and CYP2C9 Inhibition Data for Spirocyclic Analogs.

18


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Cmpd.

R

NAMPT IC50a (µM)

A2780 IC50a (µM)

CYP2C9 Inhibition IC50a.b (µM)

MDCKc A:B, B:A (x10-6 cm/s)

0.074

0.101

>10

15, 14

0.059

>2

>10

17, 16

O

(±)-31 NH

N

(±)-34 O N

(±)-35

>2

HN N

(±)-36

0.024

0.070

>10

(±)-37

0.018

0.119

>10

0.009

0.203

7.2

0.010

0.036

>10

16, 15

0.007

0.002

>10

15, 20

17, 18

O S

(±)-38 N

O O

(±)-39 N

(±)-40

19



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All assay results are reported as the arithmetic mean of at least two separate runs (n ≥ 2). All compounds are racemic. a


b


c


As shown in Table 7, the individual enantiomers of (±)-40 were found to have differing levels of NAMPT potency, with the (S) isomer (41) being more potent than the (R) (42). Both enantiomers were found to maintain the favorable lack of CYP2C9 inhibition observed in the racemate, and both had similar levels of stability in human liver microsomes.

Table 7. Characterization of Individual Enantiomers of (±)-40.

O N N

NAMPT IC50a (µM)

A2780 IC50a (µM)

CYP2C9 Inhibition IC50a,b (µM)

HLMc Clpred (mL/min/kg)

41

0.013

0.001

>10

17

42

0.035

0.034

>10

16

Cmpd.

R

All assay results are reported as the arithmetic mean of at least two separate runs (n ≥ 2).

20


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a


b


c


Figure 4a shows the X-ray crystal structure of compound 41 in complex with NAMPT, and a summary of the notable interactions is as follows. The bicyclic left-hand side of the inhibitor participates in face to face pi-stacking interactions with Tyr18’ and Phe193. The pyridine N of 41 forms a favorable polar interaction with Arg196 and also engages in an H-bond interaction with a crystallographic water molecule. The pyrrolidine portion of the bicyclic ring system participates in a favorable Van der Waals interaction with Arg311, while the pyridine ring makes a similar interaction with the sidechain methylene of Asp219. The urea carbonyl of the linker forms an Hbond interaction with the sidechain OH of Ser275, while the NH portion of the urea linker forms a similar interaction with a tightly bound crystallographic water stabilized by H-bonds with Asp219, Val242, and Ser241. These linker interactions are a key pharmacophore for NAMPT inhibitors, and often involve a bridging water molecule between the ligand and Ser275 or Asp219. In the central region of 41 additional Van der Waals contacts are formed between the methylene group appended to the cyclopropane and the sidechain CH2 of Ser275, while the cyclopropane itself makes further Van der Waals contacts with sidechains of His191, Val242, and Ile351. The piperidine portion of spirocyclic system makes favorable Van der Waals interactions with sidechains of Ile351 and Ala379. Finally, the carbamate carbonyl in the right-hand side of the inhibitor forms an H-bond with a crystallographic water, while the tert-butyl group protrudes into a relatively open and solventexposed region of the protein.

21



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A potential explanation for the potent NAMPT inhibition, yet lack of significant CYP2C9 inhibition exhibited by compound 41 is as follows. Compounds 1, 8, and 41 were three of the most cellularly potent NAMPT inhibitors profiled in our assays, each possessing A2780 IC50 values of 1–2 nM. The ligands from each compound’s X-ray structure in complex with NAMPT are overlaid in Figure 4b. It is notable that the nucleophilic N in the left-hand moiety of each inhibitor is placed in virtually the same location within the active site of NAMPT (circled in Figure 4b). Thus, the inhibitors are ideally poised to form the corresponding phosphoribose adducts, leading to the outstanding cell based NAMPT potency observed in each case. Despite possessing similarly potent NAMPT cellular activity, compounds 1, 8, and 41 displayed a range of CYP2C9 IC50’s spanning potent (1, 0.2 µM) to moderate (8, 3.6 µM) to negligible (41, >10 µM) inhibition. As shown in Figure 4b, notable differences were observed in the topology of the central and right-hand regions, which may contribute to each compound’s ability to bind to and inhibit CYP2C9. Additionally, the spirocyclic moiety in 41 (negligible CYP2C9 inhibition: IC50 >10 µM) is much more rigid than the saturated yet conformationally unrestrained central regions of compounds such as 7 and 8 (significant CYP2C9 inhibition: IC50’s 0.12 µM and 3.6 µM, respectively). Thus, the unique shape and rigidity of the spirocyclic central motif within 41 allow it to bind productively to NAMPT, but may preclude it from binding effectively to CYP2C9.

4a

22


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4b

23



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Figure 4a. X-ray crystal structure of 41 in complex with NAMPT. The ligand 41 is shown in orange. Protein sidechains forming interactions with the ligand are shown in teal. Hydrogen bonding interactions are shown as dashed lines. Crystallographic waters forming H-bonds with the ligand are shown as red spheres. PDB code: 5KIT, resolution = 1.60 Å. Figure 4b. Overlay of the small molecule ligand (41, orange) from PDB code 5KIT (37) with the ligands from previously reported structures 1 (green): 4KFO,9b and 8 (white): 2GVJ.25 The location of the nucleophilic N in the lefthand side of each ligand is circled.

As noted in Table 7, one issue associated with compound 41 is poor liver microsome stability. As shown in Figure 4a, the tert-butyl carbamate group protrudes into a relatively open region of the protein and was expected to be tolerant of substitution. A strategy of reducing cLogD7.4 as a potential means of improving metabolic stability was undertaken by modifying this portion of the inhibitor. Additionally, since both enantiomers of (±)-40 possessed similar metabolic stability, the optimization work was done with the racemic spirocyclic system, with only key analogs being resolved into their constituent enantiomers.

As exemplified by the entries in Table 8, a number of tert-butyl carbamate replacements were prepared using either a subsequent round of parallel synthesis, or by targeted singleton synthesis for more synthetically challenging analogs.

Compounds (±)-43 – (±)-59 summarize the efforts at

replacing the tert-butyl carbamate with amides. A wide array of aliphatic and aromatic amides were prepared. Although reasonable to excellent NAMPT potency was maintained, it was very difficult to substantively improve metabolic stability while maintaining high membrane permeability. Multiple analogs such as (±)-47 – (±)-49, and (±)-56 – (±)-58 were found to be metabolically stable, yet 24


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possessed poor MDCK permeability. By contrast, amides maintaining promising levels of MDCK permeability were found to suffer from poor metabolic stability (analogs (±)-43, (±)-44, and (±)-51). Compound (±)-60, the urea isostere of (±)-40, was found to moderately improve metabolic stability, although cell-based potency was reduced by over 10-fold. Non carbonyl linked groups such as alkylated analog (±)-61 suffered from a marked decrease in NAMPT inhibition. Having failed to achieve the required balance of potency, metabolic stability, and membrane permeability with carbamate replacements, we then investigated whether substituted carbamates ((±)-62 – (±)-66) could provide the desired profile.27 These analogs maintained high levels of MDCK permeability, and gratifyingly, the most polar example ((±)-66) also exhibited significantly improved metabolic stability relative to the starting point (±)-40. Finally, an in vitro metabolite identification study in human liver microsomes (data not shown) indicated the left-hand side bicycle to be a site of metabolism. Thus, the deuterated molecule (±)-67 was prepared in an attempt to further improve metabolic stability, but was not found to be meaningfully different than the proto parent (±)-66. Compound (±)-66 was, therefore, selected as the most promising compound for further characterization and progression.

One final point to emphasize about the compounds in Table 8 is the consistent lack of CYP2C9 inhibition. The saturated and rigid spirocyclic central moiety offered a general solution to the CYP2C9 inhibition issue.

Table 8. SAR of Carbamate Replacements and Substitutions.

25



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Page 26 of 92

cLogD7.4a

NAMPT IC50b (µM)

A2780 IC50b (µM)

CYP2C9 Inhibition IC50b,c (µM)

HLMd Clpred (mL/min/kg)

MDCKd A:B, B:A (x10-6 cm/s)

(±)-40

2.2

0.0072

0.0016

>10

17

15, 20

(±)-43

2.1

0.0212

0.0016

>10

14

9.2, 13

(±)-44

1.8

0.0128

0.0044

>10

13

7.3, 14

(±)-45

1.4

0.0060

0.0004

>10

13

1, 9.1

(±)-46

1.6

0.0161

0.0058

>10e

11

1.6, 11

(±)-47

1.1

0.0219

0.0069

>10

6

1.4, 7

(±)-48

1

0.0589

0.0133

>10

5

0.2, 1

(±)-49

0.5

0.0478

0.0112

>10

6

1.4e, 4e

(±)-50

1.7

0.0265

0.0248

>10e

9

(±)-51

1.6

0.0210

0.0035

>10

10

7.9e, 19e

(±)-52

0.6

0.0061

0.0053

>10e

7

0.2, 1

Cmpd.

R

26


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O

N

-0.7

0.0410e

0.0312e

>10e

8

1.4

0.0142

0.0038

>10e

9

1.4, 11

0.9

0.0274

0.0065

>10

7

0.3, 2.7

0.5

0.0204

0.0040

>10

6

0.3, 2.6

0.6

0.0086

0.0062

>10e

4

0.2, 1.9

0.2

0.0412

0.0075

>10

5

0.2, 1.1

(±)-59

0.9

0.0044

0.0016

>10

9

1.4, 9

(±)-60

1.6

0.0481

0.0207

>10e

11

(±)-61

1.2

0.5554

0.7535

>10e

15

(±)-62

1.8

0.0151

0.0043

>10

14

(±)-63

1.5

0.0387

0.0294

>10e

12

(±)-64

2.3

0.0222e

0.0048

>10e

18

(±)-65

1.6

0.0067

0.0016

>10e

18

(±)-66

0.9

0.0171

0.0022

>10

7

(±)-53

N O S

(±)-54

N

(±)-55 O

(±)-56

N N O

(±)-57

N N O

(±)-58

N N

13, 14

15e, 12e

11, 16

27



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(±)-67

Page 28 of 92

NAf

0.9

0.0119e

0.0010

>10

7

Unless otherwised indicated, all assay results are reported as the arithmetic mean of at least two separate runs (n ≥ 2). All compounds are racemic. a

Calculated LogD at pH 7.4.28

b


c


d


e


f

Not applicable

The racemic compound with the best balance of potency, metabolic stability, and membrane permeability was found to be (±)-66. The enantiopure versions (68 and 69) were, thus, isolated and profiled. As shown in Table 9, the in vitro metabolic stability and membrane permeability was virtually identical among each enantiomer (68 and 69) and the racemate ((±)-66). However, one enantiomer (68) was notably more potent against NAMPT than the other, with a sub-nM IC50 observed in the A2780 cell based antiproliferation assay. As a precursor to a tumor xenograft study, the cell potency of the active enantiomer 68 was further evaluated in a relevant cell line (HT1080), where a potent antiproliferative IC50 value of 1.5 nM was observed.

Table 9. Characterization of Single Enantiomers of (±)-66.

28


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O N N

Cmpd.

R

Fsp3

a

NAMPT A2780 IC50b IC50b (µM) (µM)

68

0.67

0.0055

69

0.67

0.075

0.0006

HT1080 HLMe MDCKe IC50c Clpred A:B, B:A (µM) (mL/min/kg) (x10-6 cm/s) 0.0015d

8

8.8, 12

7

11f, 15f

All assay results are reported as the arithmetic mean of at least two separate runs (n ≥ 2). a


b


c

Antiproliferation activity determined in cell culture experiments using HT1080 cell line. This inhibition can be reversed

by addition of 0.33 µM of NMN, strongly implicating NAMPT inhibition as the causative MOA. See reference 9d for experimental details. d

The coefficient of variation for this value is 0.03 (n=3).

e


f


The in vitro ADME properties of compound 68 are shown in Table 10. Compound 68 did not significantly inhibit any CYP isoform tested (including CYP2C9) at the concentrations evaluated. Similarly, no time dependent CYP inhibition was observed against any isoform tested. 68 was found to have moderate in vitro liver microsome stability in all species except for mouse, where poor stability (high predicted clearance) was observed. Compound 68 was generally more metabolically 29



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Page 30 of 92

stable in in vitro hepatocyte assays, with low clearance predicted in human, rat, and dog, and moderate clearance predicted in mouse and cyno. Plasma protein binding was moderate, with values in the 50 – 60% range observed across species. MDCK permeability was on the border of moderate and high, with no evidence of significant efflux. Finally pH 7.4 aqueous solubility was high in both kinetic and thermodynamic assay formats.

Table 10. ADME Properties of Compound 68.

CYP CYP TDI Inhibition (%AUC IC50a shift)b (µM) >10

c

LM, HRMDC Hep, HRMDCd

% PPB, HRMDCe

8 19

79

17

27

2 17

51

6

19

MDCKf pH 7.4 pH 7.4 A:B, B:A Kinetic Thermo g LogD7.4 (x10-6 Solubility Solubility cm/s) (µM)h (µM)i

56 56 53 66 54

100

>6000

All assay results are reported as the arithmetic mean of at least two separate runs (n ≥ 2). See reference 9d for experimental details. a

Reversible Cytochrome P450 inhibition. Isoforms tested: 3A4 (testosterone probe), 3A4 (midazolam probe), 2D6, 1A2,

2C19, 2C9 ((S)-Warfarin probe). b

Time-dependent inhibition of cytochrome P450’s. Isoforms tested: 3A4 (testosterone probe), 3A4 (midazolam probe),

2D6, 1A2, 2C19, 2C9. >15% AUC shift considered possible TDI risk. c

Hepatic clearance predicted from human, rat, mouse, dog, and cyno liver microsomes, respectively. Numerical values

represent predicted hepatic clearance (mL/min/kg). Colors represent stability categories: green = stable (Clpred 70% of liver blood flow). d

Hepatic clearance predicted from hepatocytes. Species, numerical values, and colors as above.

e

% Plasma protein binding in human, rat, mouse, dog, and cyno, respectively.

f

Apparent permeability in MDCK transwell culture. Permeability categories are as follows (units of 10-6 cm/s): low < 1,

moderate = 1-10, high >10. A:B, apical-to-basolateral. B:A, basolateral-to-apical.

30


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g

Measured LogD at pH 7.4.

h

10 mM DMSO stock solution diluted to 200 µM with pH 7.4 aqueous buffer. Final DMSO concentration: 2% (v/v).

Solubility measured after shaking for 24 h. i

Thermodynamic solubility of crystalline powder after shaking for 24 h in aqueous pH 7.4 buffer.

With 68 exhibiting a promising in vitro ADME profile, in vivo PK experiments were carried out. These results are summarized in Table 11, and Figures 5 and 6. Despite moderate to low predicted clearance from in vitro liver microsome and hepatocyte stability experiments (Table 10), high in vivo clearance and relatively low oral bioavailability was observed in the rat. Similarly, compound 68 was rapidly eliminated from the plasma of mice. However, due to its outstanding cellular potency and relatively low plasma protein binding, a 25 mg/kg BID oral dose of 68 in mice produced plasma concentrations that remained above the plasma-protein binding corrected cell based HT1080 IC50 for ~20 hours (Figure 6). This level of oral exposure provided confidence that 68 would likely provide robust efficacy in a mouse tumor xenograft experiment. Additionally, dog PK of compound 68 was more promising than in rodents. An improved IVIVC was observed in the dog with in vitro liver microsome and hepatocyte clearance (moderate to low) being in reasonable agreement with in vivo clearance (bordering moderate and low). Finally, consistent with reduced clearance, oral bioavailability was significantly improved in the dog (86%) relative to the rat (29%).

Table 11. In Vivo PK Parameters of Compound 68.

Cmpd.

Species

Cla (mL/min/kg)

t1/2a (h)

Vssa (L/kg)

AUCb (µM·h)

Cmaxb (µM)

Fb (%)

Rat

110

1.4

9.0

0.55

0.64

29

31



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Page 32 of 92

68 Dog

10.8

1.2

0.8

10.5

4.8

86

Compounds were formulated in EtOH/PEG400/H2O (10:60:30) prior to dosing. a

IV dosing (1 mg/kg for rat, 0.5 mg/kg for dog).

b

PO dosing (5 mg/kg for rat, 3 mg/kg for dog).

Figure 5. Plasma concentration-time profiles of compound 68 after intravenous and oral administration in male Sprague Dawley rats and male beagle dogs. Each time point shows the mean plasma concentration (± standard deviation) from three separate animals.

32


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Figure 6. Plasma concentration-time profile of compound 68 after oral administration (25 mg/kg BID) in female NCR nude mice (black line). Each time point shows the mean plasma concentration (± standard deviation) from three separate animals.

Compound 68 was formulated in

EtOH/PEG400/H2O (10:60:30) prior to dosing. The HT1080 cell based potency is included for reference (blue lines).

As shown in Figure 7, compound 68 was then advanced to a mouse tumor xenograft efficacy study in an HT1080 human fibrosarcoma model. The inhibitor was dosed twice daily for five days at both 25 and 50 mg/kg. Consistent with the plasma exposure relative to unbound cell potency relationship depicted in Figure 6, robust tumor growth inhibition and tumor regressions were observed at both dose levels (Figure 7). Based on body weights of treated animals, the NAMPT inhibitor appeared to be well tolerated. Indeed, body weights of untreated mice tended to decline over the course of the study due to tumor-induced cachexia which was reversed upon treatment with compound 68. However, additional toxicity studies were not undertaken to further characterize the safety of compound 68.29 33



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Page 34 of 92

Figure 7. In vivo efficacy and body weight change after treatment with vehicle (60%PEG400/30% H2O/10% EtOH) or compound 68 (25 and 50 mg/kg BID, PO) for 5 consecutive days in the HT1080 human fibrosarcoma xenograft model. 10 female NCR tumor-bearing mice were used per group. Data reflects the mean tumor volume or % body weight change (± standard error of mean). Rx denotes treatment period of 5 days. ***p95% for all final compounds as assessed by LCMS.30 Further details on the analytical conditions used for individual compounds may be found in the Supporting Information.

6-Amino-N-(4-((3,5-difluorophenyl)sulfonyl)benzyl)-1H-pyrrolo[3,2-c]pyridine-2-carboxamide (16). To a mixture of 79 (60 mg, 0.126 mmol) and potassium acetate (0.50 g, 5.10 mmol) in MeOH (20 mL) was added 10% palladium on carbon (60 mg). The mixture was stirred under 1 atmosphere of H2 for 20 h at 25 °C. The catalyst was removed by filtration, then the filtrate was concentrated under vacuum and the residue was purified on a silica gel column eluting with DCM/MeOH (5/1). The semi pure product was further purified by Prep-HPLC (Column: Gemini 10 µm, 25*200 mm; mobile phase, 18 to 38 % CH3CN:H2O with 0.4% NH4HCO3 in 20 min; Detector, UV 254 nm) to give 7.4 mg (13%) of 16 as an off-white solid. 1H NMR (300 MHz, DMSO-d6) δ 11.26 (s, 1H), 9.04 (s, 1H), 8.38 (s, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.75 – 7.55 (m, 5H), 7.08 (s, 1H), 6.37 (s, 1H), 5.64 (br, 2H), 4.55 (d, J = 5.7 Hz, 2H). LCMS (Method S, ESI) RT = 1.58 min, m/z = 443.2 [M+H]+. 45



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Page 46 of 92

6-Amino-N-(4-(phenylsulfonyl)benzyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide (18) A mixture of 90 (60 mg, 0.44 mmol) and 92 (200 mg, 0.49 mmol) in EtOH (20 mL) was stirred for 1 h at 90 °C. The reaction mixture was cooled to 25 °C and concentrated under vacuum. The residue was purified on a silica gel column eluting with EtOAc/petroleum ether (10:1) to give 74.1 mg (44%, over two steps) of 18 as an off-white solid. 1H NMR (300 MHz, DMSO-d6) δ: 7.95 – 7.87 (m, 5H), 7.72 – 7.60 (m, 3H), 7.58 – 7.46 (m, 2H), 7.02 (t, J = 6.0 Hz, 1H), 6.36 (s, 1H), 5.84 (s, 2H), 4.45 (s, 4H), 4.31 (d, J = 5.7 Hz, 2H). LCMS (Method G, ESI) RT= 1.46 min, m/z = 409.0 [M+H]+. 5-Amino-N-(4-(phenylsulfonyl)benzyl)furo[2,3-c]pyridine-2-carboxamide (20) A solution of 18% aqeuous HCl (3 mL) was added to 101 (300 mg, 0.52 mmol) in THF (5 mL) and the reaction mixture was stirred for 30 min at 25 °C. The pH value was adjusted to 7 with 5% aqueous sodium carbonate solution, then the mixture was extracted with 100 mL of EtOAc. The organic layer was separated, dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was purified on a silica gel column eluting with DCM/MeOH (100/4) to give 100 mg (47%, over two steps) of 20 as a light yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 9.41 (t, J = 6.0 Hz, 1H), 8.36 (s, 1H), 7.94 – 7.91 (m, 4H), 7.68 – 7.53 (m, 5H), 7.32 (s, 1H), 6.64 (d, J = 0.9 Hz, 1H), 5.68 (s, 2H), 4.50 (d, J = 5.7 Hz, 2H). LCMS (Method V, ESI) RT = 1.87 min, m/z = 408.0 [M+H]+. N-(4-(Methylsulfonyl)benzyl)-1H-pyrrolo[3,2-c]pyridine-2-carboxamide (21). Prepared by a method similar to that used for (±)-36, substituting the appropriate carboxylic acid and amine coupling partners. 1H NMR (400 MHz, DMSO-d6) δ 12.41 (br, 1H), 9.43 (t, J = 6.1 Hz, 1H), 9.10 (s, 1H), 8.29 (d, J = 6.1 Hz, 1H), 7.91 (d, J = 8.1 Hz, 2H), 7.61 (d, J = 8.1 Hz, 2H), 7.52 (d, J = 6.1

46


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Hz, 1H), 7.45 (s, 1H), 4.63 (d, J = 6.0 Hz, 2H), 3.19 (s, 3H). LCMS (Method X, ESI) RT = 2.63 min, m/z = 330.0 [M + H]+. N-(4-Sulfamoylbenzyl)-1H-pyrrolo[3,2-c]pyridine-2-carboxamide (22). Prepared by a method similar to that used for (±)-36, substituting the appropriate carboxylic acid and amine coupling partners. 1H NMR (400 MHz, DMSO-d6) δ 12.68 (br, 1H), 9.49 (t, J = 6.0 Hz, 1H), 9.21 (s, 1H), 8.34 (d, J = 6.3 Hz, 1H), 7.84 – 7.76 (m, 2H), 7.64 (d, J = 6.2 Hz, 1H), 7.56 – 7.49 (m, 3H), 7.31 (s, 2H), 4.61 (d, J = 5.9 Hz, 2H). LCMS (Method X, ESI) RT = 2.68 min, m/z = 331.0 [M + H]+. N-Benzyl-1H-pyrrolo[3,2-c]pyridine-2-carboxamide (23). Prepared by a method similar to that used for (±)-36, substituting the appropriate carboxylic acid and amine coupling partners. 1H NMR (400 MHz, DMSO-d6) δ 12.01 (br, 1H), 9.19 (t, J = 6.1 Hz, 1H), 8.92 (s, 1H), 8.21 (d, J = 5.8 Hz, 1H), 7.40 – 7.20 (m, 7H), 4.53 (d, J = 5.9 Hz, 2H). LCMS (Method X, ESI) RT = 2.36 min, m/z = 252.1 [M + H]+. N-(4-((3-(Dimethylcarbamoyl)benzyl)oxy)benzyl)-1H-pyrrolo[3,2-c]pyridine-2-carboxamide (24). Prepared by a method similar to that used for (±)-36, substituting the appropriate carboxylic acid and amine coupling partners. 1H NMR (300 MHz, DMSO-d6) δ 12.04 (br, 1H), 9.13 (t, J = 5.7 Hz, 1H), 8.91 (s, 1H), 8.21 (d, J = 5.7 Hz, 1H), 7.53 – 7.24 (m, 8H), 7.02 – 6.96 (m, 2H), 5.14 (s, 2H), 4.45 (d, J = 5.7 Hz, 2H), 2.99 – 2.86 (m, 6H). LCMS (Method W, ESI) RT = 1.13 min, m/z = 429.3 [M + H]+. N-(4-(Cyclopentanesulfonamido)benzyl)-1H-pyrrolo[3,2-c]pyridine-2-carboxamide

(25).

Prepared by a method similar to that used for (±)-36, substituting the appropriate carboxylic acid and amine coupling partners. 1H NMR (300 MHz, DMSO-d6) δ 12.01 (br, 1H), 9.66 (s, 1H), 9.14 (t, J = 5.7 Hz, 1H), 8.92 (s, 1H), 8.22 (d, J = 5.7 Hz, 1H), 7.41 – 7.12 (m, 6H), 4.47 (d, J = 6.0 Hz, 2H),

47



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3.58 – 3.41 (m, 1H), 1.92 – 1.48 (m, 8H). LCMS (Method A, ESI) RT = 1.32 min, m/z = 399.1 [M + H]+. N-(4-(Pyrrolidine-1-carbonyl)benzyl)-1H-pyrrolo[3,2-c]pyridine-2-carboxamide (26). Prepared by a method similar to that used for (±)-36, substituting the appropriate carboxylic acid and amine coupling partners. 1H NMR (400 MHz, DMSO-d6) δ 12.05 (s, 1H), 9.25 (t, J = 6.0 Hz, 1H), 8.94 (s, 1H), 8.23 (d, J = 5.6 Hz, 1H), 7.56 – 7.31 (m, 6H), 4.57 (d, J = 6.0 Hz, 2H), 3.48 – 3.32 (m, 4H), 2.89 – 2.78 (m, 4H). LCMS (Method A, ESI) RT = 1.36 min, m/z = 349.2 [M + H]+. N-(Cyclohexylmethyl)-1H-pyrrolo[3,2-c]pyridine-2-carboxamide (27). Prepared by a method similar to that used for (±)-36, substituting the appropriate carboxylic acid and amine coupling partners. 1H NMR (400 MHz, DMSO-d6) δ 11.98 (br, 1H), 8.90 (s, 1H), 8.53 (t, J = 5.6 Hz, 1H), 8.20 (d, J = 5.8 Hz, 1H), 7.35 (d, J = 5.8 Hz, 1H), 7.25 (s, 1H), 3.15 (t, J = 6.4 Hz, 2H), 1.77 – 1.65 (m, 4H), 1.65 – 1.49 (m, 2H), 1.28 – 1.11 (m, 3H), 1.02 – 0.88 (m, 2H). LCMS (Method Y, ESI) RT = 2.35 min, m/z = 258.2 [M + H]+. N-Isobutyl-1H-pyrrolo[3,2-c]pyridine-2-carboxamide (28). Prepared by a method similar to that used for (±)-36, substituting the appropriate carboxylic acid and amine coupling partners. 1H NMR (400 MHz, DMSO-d6) δ 11.91 (br, 1H), 8.91 (s, 1H), 8.56 (t, J = 5.9 Hz, 1H), 8.20 (d, J = 5.8 Hz, 1H), 7.35 (d, J = 5.8 Hz, 1H), 7.26 (s, 1H), 3.12 (t, J = 6.4 Hz, 2H), 1.94 – 1.79 (m, 1H), 0.91 (d, J = 6.7 Hz, 6H). LCMS (Method Y, ESI) RT = 1.77 min, m/z = 218.1 [M + H]+. The parallel synthesis library (including compounds (±)-30, (±)-31, 32, and (±)-33) was synthesized by the following general procedure. The amine coupling partners (0.15 mmol) were weighed into individual vials. Stock solutions of carboxylic acid 102 (2.50 g, 15.3 mmol), and HBTU (9.03 g, 23.7 mmol) in DMF (75 mL each) were then prepared. 0.5 mL of the carboxylic acid 102 stock 48


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solution (0.10 mmol), followed by TEA (42 µL, 0.30 mmol), then 0.5 mL of the HBTU stock solution (0.16 mmol) was added to each vial containing amine, and the vials were capped and shaken for 16 h at 25 °C. The crude reaction mixtures were evaporated to dryness using a Genevac evaporator, then re-diluted in 1 mL of DMF. The mixtures were transferred to 96-well plates, then purified by a mass-triggered Prep-HPLC instrument to afford the desired products in 8 – 73% yield. (±)-tert-Butyl

3-((1H-pyrrolo[3,2-c]pyridine-2-carboxamido)methyl)-2-oxa-9-

azaspiro[5.5]undecane-9-carboxylate ((±)-30). 1H NMR (400 MHz, DMSO-d6) δ 11.91 (br, 1H), 8.91 (s, 1H), 8.71 – 8.63 (m, 1H), 8.20 (d, J = 5.8 Hz, 1H), 7.35 (d, J = 5.8 Hz, 1H), 7.28 (s, 1H), 3.76 (d, J = 11.3 Hz, 1H), 3.47 – 3.19 (m, 6H), 3.07 (d, J = 11.5 Hz, 1H), 1.77 (d, J = 13.0 Hz, 1H), 1.62 – 1.33 (m, 5H), 1.38 (s, 9H), 1.32 – 1.20 (m, 1H), 1.20 – 1.12 (m, 2H). LCMS (Method Y, ESI) RT = 3.25 min, m/z = 429.2 [M + H]+. (±)-tert-Butyl

1-((1H-pyrrolo[3,2-c]pyridine-2-carboxamido)methyl)-6-azaspiro[2.5]octane-6-

carboxylate ((±)-31). 1H NMR (400 MHz, DMSO-d6) δ 11.94 (br, 1H), 8.91 (s, 1H), 8.64 (t, J = 5.6 Hz, 1H), 8.20 (d, J = 5.8 Hz, 1H), 7.35 (d, J = 5.8, 1H), 7.26 (s, 1H), 3.52 – 3.20 (m, 5H), 1.62 – 1.51 (m, 1H), 1.45 – 1.31 (m, 3H), 1.38 (s, 9H), 1.22 – 1.11 (m, 1H), 1.06 – 0.94 (m, 1H), 0.58 – 0.50 (m, 1H), 0.31 – 0.25 (m, 1H). LCMS (Method Y, ESI) RT = 3.19 min, m/z = 385.2 [M + H]+. tert-Butyl 4-(2-(1H-pyrrolo[3,2-c]pyridine-2-carboxamido)ethyl)piperidine-1-carboxylate (32). 1

H NMR (400 MHz, DMSO-d6) δ 11.93 (br, 1H), 8.91 (s, 1H), 8.55 (t, J = 5.4 Hz, 1H), 8.20 (d, J =

5.8 Hz, 1H), 7.35 (d, J = 5.8 Hz, 1H), 7.22 (s, 1H), 3.92 (d, J = 13.1 Hz, 2H), 3.39 – 3.30 (m, 2H), 2.70 – 2.65 (m, 2H), 1.69 (d, J = 13.3 Hz, 2H), 1.53 – 1.45 (m, 3H), 1.39 (s, 9H), 1.07 – 0.97 (m, 2H). LCMS (Method Y, ESI) RT = 2.58 min, m/z = 373.2 [M + H]+.

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3-(2-(1H-pyrrolo[3,2-c]pyridine-2-carboxamido)ethyl)piperidine-1-carboxylate

((±)-33). 1H NMR (400 MHz, DMSO-d6) δ 11.92 (br, 1H), 8.90 (s, 1H), 8.56 (t, J = 5.9 Hz, 1H), 8.20 (d, J = 5.7 Hz, 1H), 7.35 (dt, J = 5.8, 1.2 Hz, 1H), 7.23 (s, 1H), 3.87 – 3.59 (m, 2H), 3.39 – 3.29 (m, 2H), 2.91 – 2.78 (m, 1H), 1.87 – 1.79 (m, 1H), 1.64 – 1.55 (m, 1H), 1.55 – 1.39 (m, 3H), 1.37 (s, 9H), 1.38 – 1.25 (m, 2H), 1.20 – 1.07 (m, 1H). LCMS (Method Y, ESI) RT = 2.62 min, m/z = 373.2 [M + H]+. (±)-tert-Butyl

1-((3-(pyridin-3-ylmethyl)ureido)methyl)-6-azaspiro[2.5]octane-6-carboxylate

((±)-34). TEA (551 uL, 3.96 mmol) was added dropwise to a mixture of 4-nitrophenyl chloroformate (810 mg, 4.02 mmol) and pyridin-3-ylmethanamine (430 mg, 3.98 mmol) in DCM (12 mL) at 0 °C. The reaction mixture was stirred for 30 min, then a solution of (±)-tert-butyl 1-(aminomethyl)-6azaspiro[2.5]octane-6-carboxylate ((±)-104), (960 mg, 3.99 mmol) in DCM (5.0 mL) was added, followed by dropwise addition of additional TEA (1.10 mL, 7.92 mmol). The reaction mixture was warmed to 25 °C and stirred for 2 h, then washed with H2O (3 x 50 mL) and brine (3 x 50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The crude product was purified by Prep-HPLC with the following conditions: Column: XBridge Shield RP18 OBD, 5 µm, 19*150 mm; Mobile phase: H2O with 10 mM NH4HCO3 and CH3CN (25% CH3CN up to 40% in 10 min, up to 95% in 1 min, hold 95% for 1 min, down to 25% in 2 min); Detector: UV 254/220 nm. 0.5 g (34%) of (±)-34 was obtained as colorless oil. 1HNMR (400MHz, DMSO-d6) δ 8.47 – 8.43 (m, 2H), 7.63 (d, J = 8.0 Hz, 1H), 7.32 (dd, J = 7.6, 4.8 Hz, 1H), 6.39 (t, J = 6.0 Hz, 1H), 5.95 (t, J = 5.0 Hz,1H), 4.22 (d, J = 6.0 Hz, 2H), 3.48 – 3.40 (m, 2H), 3.20 – 3.14 (m, 3H), 2.95 – 2.88 (m, 1H), 1.53 – 1.48 (m, 1H), 1.40 (s, 9H), 1.33 – 1.30 (m, 2H), 1.15 – 1.14 (m,

50


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1H), 0.85 – 0.78 (m, 1H), 0.48 – 0.43 (m, 1H), 0.21 – 0.17 (m, 1H). LCMS (Method F, ESI) RT = 2.17 min, m/z = 375.3 [M+H]+. (±)-tert-Butyl 1-((1H-pyrazolo[3,4-b]pyridine-5-carboxamido)methyl)-6-azaspiro[2.5]octane-6carboxylate ((±)-35). Prepared as described for compound (±)-36, substituting the appropriate carboxylic acid. 1H NMR (400 MHz, DMSO-d6) δ 8.97 (s, 1H), 8.69 (s, 1H), 8.66 (t, J = 4.0 Hz, 1H), 8.27 (s, 1H), 3.53 – 3.40 (m, 2H), 3.27 – 3.16 (m, 2H), 1.64 – 1.51 (m, 1H), 1.44 – 1.32 (m, 4H), 1.38 (s, 9H), 1.20 – 1.09 (m, 1H), 1.07 – 0.95 (m, 1H), 0.58 – 0.50 (m, 1H), 0.31 – 0.21 (m, 1H). LCMS (Method X, ESI) RT = 4.25 min, m/z = 286.1 [M - Boc + H]+. (±)-tert-Butyl

1-((imidazo[1,2-a]pyrimidine-6-carboxamido)methyl)-6-azaspiro[2.5]octane-6-

carboxylate ((±)-36). EDCI (79.6 mg, 0.415 mmol) was added to a mixure of (±)-104 (50.0 mg, 0.208 mmol), imidazo[1,2-a]pyrimidine-6-carboxylic acid (37.4 mg, 0.229 mmol), HOBt (33.7 mg, 0.249 mmol) and TEA (116 uL, 0.834 mmol) in DMF (1.0 mL). The resulting solution was stirred for 16 h at 25 °C. 50 mL of H2O was added and the mixture was extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with brine (3 x 50 mL), then the organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified by preparative HPLC with the following conditions: Column: Gemini 10 µm, 25*200 mm; Mobile phase: H2O with 0.4% NH4HCO3, CH3CN (12% CH3CN up to 39% in 15 min, up to 95% in 17 min); Detector: UV 254 nm. 50 mg (62%) of (±)-36 was obtained as a white solid. 1HNMR (300MHz, CDCl3) δ 9.39 (s, 1H), 9.18 (s, 1H), 7.94 (s, 1H), 7.77 – 7.62 (m, 2H), 3.60 – 3.45 (m, 4H), 3.22 – 3.12 (m, 2H ), 1.68 – 1.64 (m, 1H), 1.58 – 1.54 (m, 1H), 1.43 (s, 9H), 1.28 – 1.21 (m, 1H), 1.18 – 1.05 (m, 2H), 0.65 – 0.62 (m, 1H), 0.33 – 0.28 (m, 1H). LCMS (Method B, ESI) RT = 3.43 min, m/z = 386.1 [M+H]+. 51



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1-((imidazo[1,2-a]pyridine-6-carboxamido)methyl)-6-azaspiro[2.5]octane-6-

carboxylate ((±)-37). Prepared as described for compound (±)-36, substituting the appropriate carboxylic acid. 1H NMR (300 MHz, CDCl3) δ 8.80 (s, 1H), 7.69 – 7.61 (m, 3H), 7.36 (d, J = 9.3 Hz, 1H), 6.16 (br, 1H), 3.71 – 3.54 (m, 3H), 3.45 – 3.41 (m, 1H), 3.27 – 3.17 (m, 2H), 1.69 – 1.49 (m, 2H), 1.43 (s, 9H), 1.40 – 1.37 (m, 1H), 1.21 – 1.11 (m, 1H), 0.99 – 0.92 (m, 1H), 0.69 – 0.56 (m, 1H), 0.34 – 0.28 (m, 1H). LCMS (Method B, ESI) RT = 3.60 min, m/z = 385.1 [M+H]+. (±)-tert-Butyl

1-((thieno[2,3-c]pyridine-2-carboxamidgo)methyl)-6-azaspiro[2.5]octane-6-

carboxylate ((±)-38). Prepared as described for compound (±)-36, substituting the appropriate carboxylic acid. 1H NMR (300 MHz, CDCl3) δ 9.18 (s, 1H), 8.53 (d, J = 6.0 Hz, 1H), 7.75 (d, J = 6.0 Hz, 1H), 7.73 (s, 1H), 6.29 (br, 1H), 3.64 – 3.40 (m, 4H), 3.28 – 3.17 (m, 2H), 1.66 – 1.63 (m, 1H), 1.57 – 1.49 (m, 1H), 1.44 (s, 9H), 1.38 – 1.22 (m, 1H), 1.17 – 1.12 (m, 1H), 1.02 – 0.94 (m, 1H), 0.63 – 0.58 (m, 1H), 0.33 – 0.28 (m, 1H). LCMS (Method H, ESI) RT = 1.63 min, m/z = 402.0 [M+H]+. (±)-tert-Butyl

1-((furo[2,3-c]pyridine-2-carboxamido)methyl)-6-azaspiro[2.5]octane-6-

carboxylate ((±)-39). Prepared as described for compound (±)-36, substituting the appropriate carboxylic acid. 1H NMR (300 MHz, CDCl3) δ 8.99 (br, 1H), 8.50 (s, 1H), 7.69 (d, J = 4.5 Hz, 1H), 7.50 (s, 1H), 6.72 (br, 1H), 3.65 – 3.57 (m, 3H), 3.48 – 3.42 (m, 1H), 3.25 – 3.19 (m, 2H), 1.71 – 1.53 (m, 2H), 1.44 (s, 9H), 1.38 – 1.32 (m, 1H), 1.15 – 1.11 (m, 1H), 1.00 – 0.92 (m, 1H), 0.66 – 0.63 (m, 1H), 0.35 – 0.30 (m, 1H). LCMS (Method H, ESI) RT = 1.60 min, m/z = 386.0 [M+H]+.

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(±)-tert-butyl


1-((2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-2-carboxamido)methyl)-6-

azaspiro[2.5]octane-6-carboxylate ((±)-40). TEA (2.6 mL, 18.3 mmol) was added dropwise to a mixture of (±)-104 (4.00 g, 16.6 mmol), 4nitrophenyl chloroformate (3.35 g, 16.6 mmol) in DCM (80 mL) at 0 °C. The reaction mixture was warmed to 25 °C and stirred for 2 h, monitoring for completion by TLC. The reaction mixture was used in the next step without further purification. TLC: EtOAc/petroleum ether = 1/1, Rf = 0.2. The solution prepared above was cooled to 0 °C, then 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine dihydrochloride (3.20 g, 16.6 mmol) was added in portions, followed by dropwise addtion of TEA (4.7 mL, 33.7 mmol). The reaction mixture was stirred for 5 min at 0 °C – 5 °C, then 100 mL of DCM was added. The organic layer was washed with brine (3 x 20 mL), then dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was purified on a silica gel column eluting with DCM/MeOH (60/40) to afford 3.5 g (54%) of (±)-40 as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 8.56 (s, 1H), 8.46 (d, J = 5.1 Hz, 1H), 7.39 (d, J = 5.1 Hz, 1H), 6.42 (t, J = 5.4 Hz, 1H), 4.63 (s, 2H), 4.62 (s, 2H), 3.50 – 3.38 (m, 2H), 3.26 – 3.07 (m, 4H), 1.62 – 1.54 (m, 1H), 1.39 (s, 9H), 1.40 – 1.30 (m, 2H), 1.18 – 1.09 (m, 1H), 0.96 – 0.88 (m, 1H), 0.47 (dd, J = 8.6, 4.5 Hz, 1H), 0.20 (t, J = 4.7 Hz, 1H). LCMS (Method N, ESI) RT = 1.40 min, m/z = 387.3 [M + H]+. tert-Butyl

(S)-1-((2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-2-carboxamido)methyl)-6-

azaspiro[2.5]octane-6-carboxylate (41). Prepared as described for compound (±)-40, substituting enantiopure 119 in place of racemic (±)104. Analytical data consistent with (±)-40. tert-Butyl

(R)-1-((2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-2-carboxamido)methyl)-6-

azaspiro[2.5]octane-6-carboxylate (42). 53



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Prepared as described for compound (±)-40, substituting enantiopure 122 in place of racemic (±)104. Analytical data consistent with (±)-40. (±)-N-((6-(3,3-Dimethylbutanoyl)-6-azaspiro[2.5]octan-1-yl)methyl)-1,3-dihydro-2Hpyrrolo[3,4-c]pyridine-2-carboxamide ((±)-43). EDCI (52.0 mg, 0.271 mmol) was added to a mixture of (±)-111 (80.0 mg, 0.179 mmol, based on 81% LCMS purity), 3,3-dimethylbutanoic acid (42.0 mg, 0.362 mmol), HOBt (36.5 mg, 0.270 mmol), and DIPEA (119 uL, 0.721 mmol) in DMF (2.0 mL). The reaction mixture was stirred for 16 h at 25 °C, then 50 mL of water was added and the mixture was extracted with DCM (2 x 50 mL). The combined organic layers were washed with brine (2 x 50 mL), dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was purified on a silica gel column eluting with DCM/MeOH (96/4) to afford 22.2 mg (32%) of (±)-43 as a white solid. 1H NMR (300 MHz, CD3OD) δ 8.56 (s, 1H), 8.47 (d, J = 5.2 Hz, 1H), 7.45 (d, J = 5.2 Hz, 1H), 4.76 (s, 4H), 3.89 – 3.76 (m, 1H), 3.73 – 3.60 (m, 1H), 3.58 – 3.45 (m, 2H), 3.29 – 3.24 (m, 2H), 2.40 – 2.31 (m, 2H), 1.75 – 1.70 (m, 1H), 1.58 – 1.46 (m, 2H), 1.33 – 1.30 (m, 1H), 1.11 – 1.06 (m, 1H), 1.05 (s, 9H), 0.65 – 0.62 (m, 1H), 0.37 – 0.34 (m, 1H). LCMS (LCMS 19, ESI) RT = 1.35 min, m/z = 385.0 [M + H]+. (±)-N-((6-(3-Methylbutanoyl)-6-azaspiro[2.5]octan-1-yl)methyl)-1,3-dihydro-2H-pyrrolo[3,4c]pyridine-2-carboxamide ((±)-44). Prepared as described for compound (±)-43, substituting the appropriate carboxylic acid. 1H NMR (300 MHz, DMSO-d6) δ 8.56 (s, 1H), 8.45 (d, J = 4.8 Hz, 1H), 7.37 (d, J = 5.1 Hz, 1H), 6.41 (t, J = 5.3 Hz, 1H), 4.61 (s, 4H), 3.62 – 3.49 (m, 2H), 3.38 – 3.33 (m, 1H), 3.29 – 3.25 (m, 1H), 3.15 – 3.09 (m, 2H), 2.16 (d, J = 7.2 Hz, 2H), 2.00 – 1.94 (m, 1H), 1.58 – 1.52 (m, 1H), 1.41 – 1.15 (m, 3H),

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0.94 – 0.93 (m, 1H), 0.86 (d, J = 6.6 Hz, 6H), 0.50 – 0.45 (m, 1H), 0.24 – 0.19 (m, 1H). LCMS (Method I, ESI) RT = 1.23 min, m/z = 371.3 [M+H]+. (±)-N-((6-(2-(4-Methyltetrahydro-2H-pyran-4-yl)acetyl)-6-azaspiro[2.5]octan-1-yl)methyl)-1,3dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide ((±)-45). Prepared as described for compound (±)-43, substituting the appropriate carboxylic acid. 1H NMR (300 MHz, DMSO-d6) δ 8.55 (s, 1H), 8.46 (d, J = 5.1 Hz, 1H), 7.38 (d, J = 5.1 Hz, 1H), 6.42 (t, J = 4.9 Hz, 1H), 4.61 (s, 4H), 3.69 – 3.31 (m, 8H), 3.15 – 3.08 (m, 2H), 2.31 (s, 2H), 1.62 – 1.49 (m, 3H), 1.45 – 1.25 (m, 4H), 1.21 – 1.05 (m, 1H), 1.04 – 0.89 (m, 4H), 0.55 – 0.51 (m, 1H), 0.25 – 0.15 (m, 1H). LCMS (Method I, ESI) RT = 1.19 min, m/z = 428.0 [M+H]+. (±)-N-((6-(3-(Tetrahydro-2H-pyran-4-yl)propanoyl)-6-azaspiro[2.5]octan-1-yl)methyl)-1,3dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide ((±)-46). Prepared as described for compound (±)-43, substituting the appropriate carboxylic acid. 1H NMR (300 MHz, DMSO-d6) δ 8.56 (s, 1H), 8.46 (d, J = 4.8 Hz,1H), 7.38 (d, J = 5.4 Hz, 1H), 6.41 (t, J = 4.7 Hz, 1H), 4.63 (s, 4H), 3.83 – 3.78 (m, 2H), 3.73 – 3.42 (m, 2H), 3.37 – 3.23 (m, 4H), 3.20 – 3.10 (m, 2H), 2.29 (t, J = 6.5 Hz, 2H), 1.59 – 1.40 (m, 8H), 1.37 – 1.31 (m, 3H), 1.17 – 1.14 (m, 1H), 0.55 – 0.40 (m, 1H), 0.30 – 0.15 (m, 1H). LCMS (Method D, ESI) RT = 1.16 min, m/z = 427.3 [M+H]+. (±)-N-((6-(2-(Tetrahydro-2H-pyran-4-yl)acetyl)-6-azaspiro[2.5]octan-1-yl)methyl)-1,3-dihydro2H-pyrrolo[3,4-c]pyridine-2-carboxamide ((±)-47). Prepared as described for compound (±)-43, substituting the appropriate carboxylic acid. 1H NMR (300 MHz, CDCl3) δ 8.70 (s, 1H), 8.60 (d, J = 4.5 Hz, 1H), 7.50 (s, 1H), 4.85 (s, 4H), 4.44 (s, 1H), 3.97 – 3.92 (m, 3H), 3.58 – 3.49 (m, 1H), 3.46 – 3.26 (m, 5H), 2.26 (d, J = 6.9 Hz, 2H), 2.09 – 2.07

55



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(m, 1H), 1.70 – 1.66 (m, 3H), 1.57 – 1.21 (m, 5H), 1.01 – 0.99 (m, 1H), 0.71 – 0.58 (m, 1H), 0.38 – 0.29 (m, 1H). LCMS (Method I, ESI) RT = 1.09 min, m/z = 413.4 [M+H]+. (±)-N-((6-(2-(3-Hydroxy-3-methylcyclobutyl)acetyl)-6-azaspiro[2.5]octan-1-yl)methyl)-1,3dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide ((±)-48). Prepared as described for compound (±)-43, substituting the appropriate carboxylic acid. 1H NMR (300 MHz, DMSO-d6) δ 8.56 (s, 1H), 8.47 (d, J = 5.1 Hz, 1H), 7.39 (d, J = 5.1 Hz, 1H), 6.42 (t, J = 4.8 Hz, 1H), 4.75 (s, 1H), 4.62 (s, 4H), 3.68 – 3.42 (m, 2H), 3.32 – 3.24 (m, 2H), 3.13 – 3.11 (m, 2H), 2.41 (d, J = 4.5 Hz, 2H), 2.04 – 2.02 (m, 3H), 1.68 – 1.50 (m, 3H), 1.48 – 1.30 (m, 2H), 1.19 (s, 3H), 1.18 – 1.10 (m, 1H), 0.99 – 0.87 (m, 1H), 0.55 – 0.41 (m, 1H), 0.27 – 0.19 (m, 1H). LCMS (Method P, ESI) RT = 7.80 min, m/z = 413.0 [M+H]+. (±)-N-((6-(2-(3-Methyloxetan-3-yl)acetyl)-6-azaspiro[2.5]octan-1-yl)methyl)-1H-pyrrolo[3,4c]pyridine-2(3H)-carboxamide ((±)-49). Prepared as described for compound (±)-43, substituting the appropriate carboxylic acid.1H NMR (400 MHz, DMSO-d6) δ 8.57 (s, 1H), 8.47 (d, J = 4.8 Hz, 1H), 7.39 (d, J = 5.2 Hz, 1H), 6.44 (t, J = 4.8 Hz, 1H), 4.64 (s, 2H), 4.63 (s, 2H), 4.41 (d, J = 5.6 Hz, 2H), 4.20 (d, J = 6.0 Hz, 2H), 3.66 – 3.41 (m, 4H), 3.14 – 3.12 (m, 2H), 2.71 – 2.68 (m, 2H), 1.68 – 1.52 (m, 1H), 1.48 – 1.34 (m, 2H), 1.32 (s, 3H), 1.26 – 1.14 (m, 1H), 0.93 – 0.86 (m, 1H), 0.57 – 0.45 (m, 1H), 0.29 – 0.19 (m, 1H). LCMS (Method B, ESI): RT = 2.61 min, m/z = 399.1 [M+1]+. (±)-N-((6-(4,4,4-Trifluorobutanoyl)-6-azaspiro[2.5]octan-1-yl)methyl)-1H-pyrrolo[3,4c]pyridine-2(3H)-carboxamide ((±)-50). Prepared as described for compound (±)-43, substituting the appropriate carboxylic acid. 1H NMR (300 MHz, DMSO-d6) δ 8.56 (s, 1H), 8.46 (d, J = 5.1 Hz, 1H), 7.38 (d, J = 5.1 Hz, 1H), 6.41 (t, J = 56


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4.8 Hz, 1H), 4.62 (s, 4H), 3.68 – 3.42 (m, 2H), 3.40 – 3.37 (m, 2H), 3.14 – 3.12 (m, 2H), 2.59 – 2.57 (m, 3H), 1.69 – 1.54 (m, 1H), 1.48 – 1.32 (m, 2H), 1.28 – 1.12 (m, 2H), 0.96 – 0.87 (m, 1H), 0.55 – 0.45 (m, 1H), 0.28 – 0.19 (m, 1H). LCMS (Method Y, ESI) RT = 0.90 min, m/z = 411.2 [M+H]+. (±)-N-((6-Benzoyl-6-azaspiro[2.5]octan-1-yl)methyl)-1H-pyrrolo[3,4-c]pyridine-2(3H)carboxamide ((±)-51). Prepared as described for compound (±)-43, substituting the appropriate carboxylic acid. 1H NMR (300 MHz, DMSO-d6) δ 8.58 (s, 1H), 8.47 (d, J = 4.8 Hz, 1H), 7.45 – 7.36 (m, 6H), 6.43 (t, J = 5.1 Hz, 1H), 4.65 (s, 4H), 3.85 – 3.78 (m, 1H), 3.48 – 3.32 (m, 3H), 3.13 – 3.10 (m, 2H), 1.68 – 1.62 (m, 1H), 1.58 – 1.42 (m, 2H), 1.38 – 1.14 (m, 1H), 0.97 – 0.87 (m, 1H), 0.52 – 0.50 (m, 1H), 0.26 – 0.22 (m, 1H). LCMS (Method Q, ESI) RT= 1.44 min, m/z = 391.1[M+H]+. (±)-N-[[6-[4-(1-Methyl-4-piperidyl)benzoyl]-6-azaspiro[2.5]octan-2-yl]methyl]-1,3dihydropyrrolo[3,4-c]pyridine-2-carboxamide ((±)-52). Prepared as described for compound (±)-43, substituting the appropriate carboxylic acid. 1H NMR (300 MHz, DMSO-d6) δ 8.56 (s, 1H), 8.46 (d, J = 4.5 Hz, 1H), 7.38 (d, J = 3.6 Hz, 1H), 7.32 – 7.21 (m, 4H), 6.43 (t, J = 4.8 Hz, 1H), 4.63 (s, 4H), 3.76 – 3.74 (m, 1H), 3.51 – 3.33 (m, 3H), 3.20 – 3.12 (m, 2H), 2.87 –2.84 (m, 2H), 2.44 – 2.39 (m, 1H), 2.18 (s, 3H), 1.98 – 1.91 (m, 2H), 1.70 – 1.62 (m, 5H), 1.54 – 1.24 (m, 3H), 1.01 – 0.96 (m, 1H), 0.54 – 0.49 (m, 1H), 0.24 – 0.21 (m, 1H). LCMS (Method R, ESI) RT = 1.25 min, m/z = 488.0 [M+H]+. (±)-N-((6-(2-(Pyrimidin-2-yl)acetyl)-6-azaspiro[2.5]octan-1-yl)methyl)-1H-pyrrolo[3,4c]pyridine-2(3H)-carboxamide ((±)-53). Prepared as described for compound (±)-43, substituting the appropriate carboxylic acid. 1H NMR (300 MHz, CD3OD-d4) δ 8.62 (t, J = 4.8 Hz, 2H), 8.45 (s, 1H), 8.35 (dd, J = 5.0, 2.3 Hz, 1H), 7.34 57



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(d, J = 3.9 Hz, 1H), 7.29 – 7.23 (m, 1H), 4.65 (s, 4H), 4.02 (d, J = 2.7 Hz, 2H), 3.72 – 3.70 (m, 1H), 3.57 – 3.53 (m, 1H), 3.47 – 3.40 (m, 2H), 3.22 – 3.15 (m, 2H), 1.61 – 1.58 (m, 1H), 1.45 – 1.38 (m, 2H), 1.22 – 1.20 (m, 1H), 0.98 – 0.88 (m, 1H), 0.51 (dd, J = 8.7, 4.2 Hz, 1H), 0.24 (t, J = 4.8 Hz, 1H). LCMS (Method W, ESI) RT = 0.92 min, m/z = 407.2 [M+H]+. (±)-N-((6-(2,4-Dimethylthiazole-5-carbonyl)-6-azaspiro[2.5]octan-1-yl)methyl)-1H-pyrrolo[3,4c]pyridine-2(3H)-carboxamide ((±)-54). Prepared as described for compound (±)-43, substituting the appropriate carboxylic acid. 1H NMR (300 MHz, DMSO-d6) δ 8.57 (s, 1H), 8.46 (d, J = 4.8 Hz, 1H), 7.39 (d, J = 4.8 Hz, 1H), 6.44 (t, J = 5.3 Hz, 1H), 4.64 (s, 2H), 4.63 (s, 2H), 3.72 – 3.60 (m, 2H), 3.59 – 3.41 (m, 2H), 3.14 – 3.10 (m, 2H), 2.61 (s, 3H), 2.26 (s, 3H), 1.63 – 1.60 (m, 1H), 1.45 – 1.39 (m, 2H), 1.25 – 1.18 (m, 1H), 0.99 – 0.95 (m, 1H), 0.52 (dd, J = 8.4, 4.4 Hz, 1H), 0.26 (t, J = 4.4 Hz, 1H). LCMS (Method O, ESI): RT = 1.16 min, m/z = 426.0 [M+H]+. (±)-N-((6-(4-Methylnicotinoyl)-6-azaspiro[2.5]octan-1-yl)methyl)-1H-pyrrolo[3,4-c]pyridine2(3H)-carboxamide ((±)-55). Prepared as described for compound (±)-43, substituting the appropriate carboxylic acid. 1H NMR (300MHz, DMSO-d6) δ 8.47 (s, 1H), 8.45 – 8.43 (m, 2H), 8.35 (s ,1H), 7.38 (d, J = 4.8 Hz, 1H), 7.29 (d, J = 4.8 Hz, 1H), 6.43 (t, J = 5.1 Hz, 1H), 4.62 (s, 4H), 3.83 – 3.72 (m, 1H), 3.64 – 3.55 (m, 1H), 3.09 – 2.99 (m, 4H), 2.26 (s, 3H), 1.79 – 1.62 (m, 1H), 1.51 – 1.32 (m, 2H), 1.15 – 1.00 (m, 1H), 0.98 – 0.92 (m, 1H), 0.54 – 0.45 (m, 1H), 0.27 – 0.21 (m, 1H). LCMS (Method I, ESI) RT = 0.94 min, m/z = 406.3 [M+H]+. (±)-N-((6-(1,5-Dimethyl-1H-pyrazole-4-carbonyl)-6-azaspiro[2.5]octan-1-yl)methyl)-1Hpyrrolo[3,4-c]pyridine-2(3H)-carboxamide ((±)-56). 58


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Prepared as described for compound (±)-43, substituting the appropriate carboxylic acid. 1H NMR (400 MHz, DMSO-d6) δ 8.57 (s, 1H), 8.47 (d, J = 5.2 Hz, 1H), 7.44 (s, 1H), 7.39 (d, J = 4.8 Hz, 1H), 6.43 (t, J = 5.0 Hz, 1H), 4.65 (s, 2H), 4.63 (s, 2H), 3.76 (s, 3H), 3.73 – 3.55 (m, 2H), 3.45 – 3.38 (m, 2H), 3.15 – 3.12 (m, 2H), 2.28 (s, 3H), 1.65 – 1.61 (m, 1H), 1.44 – 1.40 (m, 2H), 1.24 – 1.20 (m, 1H), 1.00 – 0.93 (m, 1H), 0.52 – 0.45 (m, 1H), 0.27 – 0.21 (m, 1H). LCMS (Method B, ESI) RT = 2.51 min, m/z = 409.2 [M+H]+. (±)-N-((6-(1,3,5-Trimethyl-1H-pyrazole-4-carbonyl)-6-azaspiro[2.5]octan-1-yl)methyl)-1Hpyrrolo[3,4-c]pyridine-2(3H)-carboxamide ((±)-57). Prepared as described for compound (±)-43, substituting the appropriate carboxylic acid. 1H NMR (300 MHz, DMSO-d6) δ 8.55 (s, 1H), 8.45 (d, J = 4.8 Hz, 1H), 7.37 (d, J = 4.8 Hz, 1H), 6.42 (t, J = 5.1 Hz, 1H), 4.62 (s, 2H), 4.61 (s, 2H), 3.62 (s, 3H), 3.52 – 3.13 (m, 4H), 3.11 – 3.05 (m, 2H), 2.14 (s, 3H), 2.03 (s, 3H), 1.68 – 1.56 (m, 1H), 1.38 – 1.35 (m, 2H), 1.19 – 1.12 (m, 1H), 0.99 – 0.92 (m, 1H), 0.48 (dd, J = 8.4, 4.2 Hz, 1H), 0.21 (t, J = 4.2 Hz, 1H). LCMS (Method I, ESI) RT = 1.06 min, m/z = 423.3 [M+H]+. (±)-N-((6-(1,3-Dimethyl-1H-pyrazole-4-carbonyl)-6-azaspiro[2.5]octan-1-yl)methyl)-1Hpyrrolo[3,4-c]pyridine-2(3H)-carboxamide ((±)-58). Prepared as described for compound (±)-43, substituting the appropriate carboxylic acid. 1H NMR (300 MHz, DMSO-d6) δ 8.57 (s, 1H), 8.46 (d, J = 5.1 Hz, 1H), 7.79 (s, 1H), 7.38 (d, J = 4.8 Hz, 1H), 6.43 (t, J = 5.1 Hz, 1H), 4.64 (s, 2H), 4.63 (s, 2H), 3.75 (s, 3H), 3.70 – 3.61 (m, 2H), 3.44 –3.38 (m, 2H), 3.13 – 3.11 (m, 2H), 2.14 (s, 3H), 1.64 – 1.58 (m, 1H), 1.44 – 1.38 (m, 2H), 1.23 – 1.18 (m, 1H), 1.00 – 0.92 (m, 1H), 0.50 (dd, J = 8.6, 4.5 Hz, 1H), 0.23 (t, J = 4.4 Hz, 1H). LCMS (Method C, ESI) RT = 1.62 min, m/z = 409.2 [M+H]+. 59



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(±)-N-((6-(2,4-Dimethyloxazole-5-carbonyl)-6-azaspiro[2.5]octan-1-yl)methyl)-1H-pyrrolo[3,4c]pyridine-2(3H)-carboxamide ((±)-59). Prepared as described for compound (±)-43, substituting the appropriate carboxylic acid. 1H NMR (300 MHz, DMSO-d6) δ 8.57 (s, 1H), 8.46 (d, J = 5.1 Hz, 1H), 7.38 (d, J = 4.8 Hz, 1H), 6.44 (t, J = 5.4 Hz, 1H), 4.64 (s, 2H), 4.63 (s, 2H), 3.73 – 3.64 (m, 2H), 3.49 – 3.43 (m, 2H), 3.16 – 3.12 (m, 2H), 2.40 (s, 3H), 2.17 (s, 3H), 1.70 – 1.63 (m, 1H), 1.47 – 1.42 (m, 2H), 1.27 – 1.22 (m, 1H), 1.01 – 0.92 (m, 1H), 0.52 (dd, J = 8.6, 4.6 Hz, 1H), 0.26 (t, J = 4.5 Hz, 1H). LCMS (Method I, ESI) RT = 1.14 min, m/z = 410.2 [M+H]+. (±)-N-((6-(tert-Butylcarbamoyl)-6-azaspiro[2.5]octan-1-yl)methyl)-1H-pyrrolo[3,4-c]pyridine2(3H)-carboxamide ((±)-60). A solution of (±)-111 (80.0 mg, 0.223 mmol) in DCM (10 mL) was cooled to 0 °C, then 2isocyanato-2-methylpropane (27.0 mg, 0.272 mmol), followed by TEA (62 uL, 0.446 mmol) was added. The resulting solution was allowed to warm to 25 °C, then stirred for 3 h. The resulting mixture was concentrated under vacuum. The residue was applied onto a silica gel column eluting with DCM/MeOH (94/6) to provide 12.5 mg (15%) of (±)-60 as a white solid. 1H NMR (300MHz, DMSO-d6) δ 8.56 (s, 1H), 8.46 (d, J = 5.1 Hz, 1H), 7.38 (d, J = 4.8 Hz, 1H), 6.41 (t, J = 5.1 Hz, 1H), 5.67 (s, 1H), 4.63 (s, 2H), 4.62 (s, 2H), 3.39 – 3.32 (m, 2H), 3.17 – 3.08 (m, 4H), 1.55 – 1.49 (m, 1H), 1.34 – 1.30 (m, 2H), 1.23 (s, 9H), 1.13 – 1.09 (m, 1H), 0.92 – 0.87 (m, 1H), 0.43 (dd, J = 8.7, 4.2 Hz, 1H), 0.19 (t, J = 4.5 Hz, 1H). LCMS (Method J, ESI) RT = 2.04 min, m/z = 386.3 [M+H]+. (±)-N-((6-Phenethyl-6-azaspiro[2.5]octan-1-yl)methyl)-1H-pyrrolo[3,4-c]pyridine-2(3H)carboxamide ((±)-61).

60


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To a solution of (±)-111 (100 mg, 0.279 mmol) in DCM (2.0 mL) was added 2-phenylacetaldehyde (110 mg, 0.917 mmol), followed by acetic acid (100 uL, 1.75 mmol). The mixture was stirred for 2 h at 25 °C. NaBH(OAc)3 (660 mg, 3.06 mmol) was added in several portions, then the reaction mixture was stirred for 16 h. 10 mL of water was added and the layers separated. The aqueous layer was further extracted with EtOAc (3 x 20 mL) and the combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The crude product was purified by Prep-HPLC with the following conditions Column, XBridge Shield RP18 OBD Column, 5µm, 19*150 mm; mobile phase, H2O with 10 mM NH4HCO3 and CH3CN (10% to 50% CH3CN in 10 min, up to 95% in 1 min, hold for 1 min, down to 5% in 2 min); Detector, UV 254nm. 11.4 mg (10%) of (±)-61 was obtained as a yellow solid. 1H NMR (300 MHz, CD3OD-d4) δ 8.59 (s, 1H), 8.45 (d, J = 4.8 Hz, 1H), 7.44 (d, J = 5.1 Hz, 1H), 7.29 – 7.17 (m, 5H), 4.67 (s, 2H), 4.66 (s, 2H), 3.26 – 3.08 (m, 2H), 2.86 – 2.81 (m, 2H), 2.76 – 2.48 (m, 6H), 1.83 – 1.78 (m, 1H), 1.69 – 1.58 (m, 2H), 1.41 – 1.36 (m, 1H), 1.10 – 0.99 (m, 1H), 0.57 (dd, J = 8.7, 4.8 Hz, 1H), 0.28 (t, J = 4.8 Hz, 1H). LCMS (Method D, ESI) RT = 1.17 min, m/z = 390.9 [M+H]+. (±)-Isopropyl


azaspiro[2.5]octane-6-carboxylate ((±)-62). To a solution of (±)-111 (120 mg, 0.334 mmol) in DCM (10 mL) was added TEA (140 uL, 1.01 mmol) at 0 °C. Isopropyl chloroformate (49.5 mg, 0.404 mmol) was then added dropwise at 0 – 5 °C. The reaction mixture was warmed to 25 °C and stirred for 3 h, then diluted with 80 mL of DCM and washed with brine (3 x 100 mL). The organic layers was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The crude product was purified by Prep-HPLC with the following conditions: Column, SunfireC18 19*150 mm; mobile phase, H2O with 0.2% NH4OH and CH3CN (25% CH3CN up to 95% in 12 min, up to 100% in 15 min); Detector, UV 254 nm. 31.6 mg 61



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(25%) of (±)-62 was obtained as a white solid. 1H NMR (300 MHz, CD3OD-d4) δ 8.54 (s, 1H), 8.45 (d, J = 5 .1 Hz, 1H), 7.44 (d, J = 5.1 Hz, 1H), 4.90 – 4.80 (m, 1H), 4.76 (s, 4H), 3.68 – 3.55 (m, 2H), 3.40 – 3.36 (m, 2H), 3.29 – 3.20 (m, 2H), 1.72 – 1.64 (m, 1H), 1.53 – 1.44 (m, 2H), 1.25 – 1.23 (m, 7H), 1.10 – 1.02 (m, 1H), 0.62 (dd, J = 8.8, 4.3 Hz, 1H), 0.31 (t, J = 4.6 Hz, 1H). LCMS (Method A, ESI) RT = 1.46 min, m/z = 373.0 [M+H]+. (±)-Ethyl


azaspiro[2.5]octane-6-carboxylate ((±)-63). Prepared as described for compound (±)-62, substituting ethyl chloroformate. 1H NMR (300 MHz, DMSO-d6) δ 8.56 (s, 1H), 8.46 (d, J = 4.2 Hz, 1H), 7.38 (d, J = 4.2 Hz, 1H), 6.41 (t, J = 4.5 Hz, 1H), 4.63 (s, 4H), 3.98 (q, J = 6.9 Hz, 2H), 3.49 – 3.32 (m, 2H), 3.26 – 3.20 (m, 2H), 3.11 – 3.06 (m, 2H), 1.62 – 1.57 (m, 1H), 1.42 – 1.36 (m, 2H), 1.22 – 1.15 (m, 1H), 1.14 (t, J = 6.9 Hz, 3H), 0.93 – 0.86 (m, 1H), 0.52 – 0.46 (m, 1H), 0.25 – 0.20 (m, 1H). LCMS (Method K, ESI) RT = 1.22 min, m/z = 359.2 [M+H]+. (±)-1-Methylcyclobutyl 1-((2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-2-carboxamido) methyl)-6azaspiro[2.5]octane-6-carboxylate ((±)-64). To a solution of

(±)-111 (430 mg, 1.20 mmol) in EtOH (5 mL) was added TEA (500 uL, 3.56

mmol), followed by 1-methylcyclobutyl 4-nitrophenyl carbonate (107, 300 mg, 1.19 mmol). The reaction mixture was stirred for 3 h at 80 °C, then cooled to room temperature and concentrated under vacuum. 5 mL of H2O was added and extracted with DCM (3 x 10 mL). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The crude product was purified by Prep-HPLC with the following conditions: Column, XBridge Shield RP18 OBD, 5µm, 19*150 mm; mobile phase, H2O with 10 mM NH4HCO3 and CH3CN (5% CH3CN 62


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up to 30.5% in 10 min, up to 100% in 1 min, down to 5% in 2 min); Detector, UV 254 nm. 11.1 mg (2%, over two steps) of (±)-64 was obtained as an off-white solid. 1HNMR (300 MHz, CD3OD-d4) δ 8.55 (s, 1H), 8.47 (d, J = 5.4 Hz, 1H), 7.45 (d, J = 5.1 Hz, 1H), 4.77 (s, 4H), 3.65 – 3.58 (m, 2H), 3.34 – 3.26 (m, 4H), 2.34 – 2.26 (m, 2H), 2.15 – 2.08 (m, 2H), 1.81 – 1.65 (m, 3H), 1.60 – 1.41 (m, 5H), 1.35 – 1.15 (m, 1H), 1.15 – 0.90 (m, 1H), 0.60 (dd, J = 8.7, 4.2 Hz, 1H), 0.32 (t, J = 4.8 Hz, 1H). LCMS (Method T, ESI) RT = 1.42 min, m/z = 399.3 [M+H]+. (±)-1-Methoxy-2-methylpropan-2-yl

1-((2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-2-

carboxamido)methyl)-6-azaspiro[2.5]octane-6-carboxylate ((±)-65). To a solution of (±)-111 (60.0 mg, 0.167 mmol) in THF (15 mL) was added bis(1-methoxy-2methylpropan-2-yl) dicarbonate (110, 93.1 mg, 0.335 mmol), then sodium hydroxide (1.0 mL, 2.0 M aqueous solution). The reaction mixture was warmed to 35 °C and stirred for 5 h at that temperature, then concentrated under vacuum. The residue was dissolved in 50 mL of DCM and washed with H2O (1 x 50 mL) and brine (1 x 50 mL), then drived over anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was purified with a silica gel column eluting with DCM/MeOH (96/4). The product (60 mg) was further purified by Prep-HPLC with the following conditions: Column, XBridge Shield RP18 OBD Column, 5 µm, 19*150 mm, mobile phase, H2O with 10 mM NH4HCO3 and CH3CN (18% CH3CN up to 43% in 10 min, up to 95% in 1 min, hold 95% for 1 min, down to 18% in 2 min); Detector, UV 254/220 nm. 22.1 mg (32%) of (±)-65 was obtained as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 8.57 (s, 1H), 8.46 (d, J = 4.8 Hz, 1H), 7.38 (d, J = 4.8 Hz, 1H), 6.41 (t, J = 5.1 Hz, 1H), 4.63 (s, 2H), 4.62 (s, 2H), 3.48 – 3.40 (m, 4H), 3.33 – 3.23 (m, 5H), 3.13 – 3.09 (m, 2H), 1.58 – 1.52 (m, 1H), 1.35 – 1.31 (m, 8H), 1.16 – 1.11 (m, 1H), 0.95 – 0.90 (m, 1H), 0.49 – 0.42 (m, 1H) , 0.24 – 0.19 (m, 1H). LCMS (Method E, ESI) RT = 2.12 min, m/z = 439.2 [M+Na]+. 63



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(±)-3-Methyloxetan-3-yl 1-((2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-2-carboxamido) methyl)-6azaspiro[2.5]octane-6-carboxylate ((±)-66). A solution of (±)-111 (9.50 g, 26.4 mmol) in DCM (400 mL) was cooled to 0 °C. TEA (19.6 mL, 140.3 mmol) was added dropwise, followed by 3-methyloxetan-3-yl 4-nitrophenyl carbonate (108, 7.08 g, 28.0 mmol). The reaction mixture was warmed to 25 °C and stirred for 2 h. 200 mL of DCM was then added and the mixture was washed with brine (3 x 100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was purified with a silica gel column eluting with DCM/MeOH (95/5) to give 4.62 g (44%) of (±)-66 as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 8.56 (d, J = 1.0 Hz, 1H), 8.47 (d, J = 5.0 Hz, 1H), 7.39 (dd, J = 5.1, 1.1 Hz, 1H), 6.41 (t, J = 5.4 Hz, 1H), 4.64 (s, 2H), 4.63 (s, 2H), 4.60 (d, J = 7.5 Hz, 2H), 4.37 (d, J = 7.5 Hz, 2H), 3.58 – 3.43 (m, 2H), 3.32 – 3.22 (m, 2H), 3.14 – 3.10 (m, 2H), 1.62 (s, 3H), 1.62 – 1.53 (m, 1H), 1.42 – 1.34 (m, 2H), 1.21 – 1.17 (m, 1H), 0.95 – 0.88 (m, 1H), 0.48 (dd, J = 8.6, 4.4 Hz, 1H), 0.22 (t, J = 4.8 Hz, 1H). LCMS (Method L, ESI) RT = 2.11 min, m/z = 401.3 [M+H]+. (±)-3-Methyloxetan-3-yl

1-((2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-2-carboxamido-1,1,3,3-

d4)methyl)-6-azaspiro[2.5]octane-6-carboxylate ((±)-67). Prepared as described for compound (±)-66, substituting amine 127. 1H NMR (300 MHz, DMSO-d6) δ 8.57 (s, 1H), 8.46 (d, J = 4.8 Hz, 1H), 7.38 (dd, J = 5.1, 0.9 Hz, 1H), 6.42 (t, J = 5.3 Hz, 1H), 4.59 (d, J = 6.9 Hz, 2H), 4.37 (d, J = 7.5 Hz, 2H), 3.50 – 3.47 (m, 2H), 3.31 – 3.28 (m, 2H), 3.16 – 3.06 (m, 2H), 1.62 (s, 3H), 1.64 – 1.56 (m, 1H), 1.40 – 1.36 (m, 2H), 1.20 – 1.17 (m, 1H), 1.00 – 0.96 (m, 1H) , 0.48 (dd, J = 8.5, 4.2 Hz, 1H), 0.22 (t, J = 4.4 Hz, 1H). LCMS (Method Q, ESI) RT = 1.39 min, m/z = 405.0 [M+H]+.

64


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(S)-3-methyloxetan-3-yl 1-((2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-2-carboxamido)methyl)-6azaspiro[2.5]octane-6-carboxylate (68). Prepared as described for compound (±)-66, substituting enantiopure 119 in place of racemic (±)104. 1H NMR and LCMS data consistent with (±)-66.

13

C NMR (101 MHz, DMSO-d6) δ 156.26,

152.84, 147.77, 146.34, 144.31, 133.58, 118.19, 80.88, 76.07, 51.29, 49.62, 43.88, 43.22, 39.71 (overlaps with DMSO-d6), 35.87, 29.42, 23.60, 21.86, 21.41, 15.79. HRMS (ESI) [M + H]+ expected = 401.2183, found = 401.2176. Chiral SFC (Method SFC1) RT = 1.37 min, >98% e.e. [α]20D –7.6 (c 0.00340, MeOH). IR (KBr pellet) νmax 3480, 2997, 2933, 2878, 1694, 1632, 1542, 1432, 1388, 1245, 1194, 1122, 966, 829, 765, 630 cm-1. 3-Methyloxetan-3-yl (R)-1-((2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-2-carboxamido)methyl)-6azaspiro[2.5]octane-6-carboxylate (69). Prepared as described for compound (±)-66, substituting enantiopure 122 in place of racemic (±)-104. 1H NMR and LCMS data consistent with (±)-66.

13

C

NMR consistent with 68. HRMS (ESI) [M + H]+ expected = 401.2183, found = 401.2174. Chiral SFC (Method SFC1) RT = 1.48 min, >98% e.e. [α]20D 5.0 (c 0.00343, MeOH). IR (film) consistent with 68. 6-Chloro-N,N-bis(4-methoxybenzyl)pyridin-2-amine (71). To a solution of 6-chloropyridin-2-amine (70) (20 g, 156 mmol) in dimethylacetamide (200 mL) was added sodium hydride (33.3 g, 60% dispersion in mineral oil, 833 mmol) at 0 °C. The reaction mixture was stirred at 0 °C for 20 min then 1-(chloromethyl)-4-methoxybenzene (48 g, 307 mmol) was added dropwise with stirring at 0 °C. The resulting solution was allowed to warm to 25 °C and stirring was continued for 20 h. The reaction was quenched by the dropwise addition of H2O (100 mL), then 400 mL of EtOAc was added and the layers separated. The organic layer was further washed with H2O (2 x 100 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was 65



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concentrated under vacuum to give 50.1 g of crude 71 as a yellow oil. TLC: EtOAc/petroleum ether = 1/2, Rf = 0.5. 6-Chloro-5-iodo-N,N-bis(4-methoxybenzyl)pyridin-2-amine (72). N-iodosuccinimide (24.0 g, 107 mmol) was added to a solution of 71 (10.0 g, 27.2 mmol) in CH3CN (200 mL), and the reaction mixture was stirred at 25 °C for 20 h. A solution containing K2CO3 (20.0 g) and Na2S2O3 (20.0 g) in H2O (30 mL) was then added. After stirring for 5 min, the solids were filtered off, the filtrate was concentrated under vacuum, and the residue was diluted with 400 mL of EtOAc. The organic layer was washed with H2O (2 x 50 mL) then dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was purified on a silica gel column eluting with EtOAc/petroleum ether (1/5) to give 13.1 g (85%, over two steps) of 72 as a light yellow solid. TLC: EtOAc/petroleum ether = 1/2, Rf = 0.6. 6-(bis(4-Methoxybenzyl)amino)-2-chloronicotinaldehyde (73). n-BuLi (8 mL of a 2.5 M solution in hexanes) was added to a solution of 72 (8.00 g, 16.2 mmol) in THF (100 mL) at –78 °C. The reaction mixture was stirred for 5 min at –78 °C then ethyl formate (5.0 mL) was added. The reaction mixture was allowed to stir for an additional 10 min at –78 °C before being quenched with 10 mL of H2O. The resulting mixture was concentrated under vacuum. The residue was diluted with 100 mL of DCM, then the organic layer was washed with H2O (1 x 30 mL), dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was purified on a silica gel column eluting with EtOAc/petroleum ether (1/5) to afford 2.41 g (37%) of 73 as a white solid. TLC: EtOAc/petroleum ether = 1/2, Rf = 0.5. Ethyl-2-azido-3-(6-(bis(4-methoxybenzyl)amino)-2-chloropyridin-3-yl)acrylate (74). To a solution of 73 (640 mg, 1.62 mmol) and ethyl 2-azidoacetate (800 mg, 6.20 mmol) in EtOH (100 mL) at 0 °C was added sodium ethoxide (400 mg, 5.88 mmol) in several portions. The reaction 66


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mixture was stirred at 0 °C for 2 h, then quenched with 10 mL of H2O and concentrated under vacuum. The residue was purified on a silica gel column eluting with EtOAc/petroleum ether (1/2) to give 320 mg of crude 74 as a light yellow solid. TLC: EtOAc/petroleum ether = 1/2, Rf = 0.3. Ethyl 6-(bis(4-methoxybenzyl)amino)-4-chloro-1H-pyrrolo[3,2-c]pyridine-2-carboxylate (75). A solution of 74 (250 mg, 0.49 mmol) in xylene (20 mL) was stirred for 3 h at 130 °C. The reaction mixture was cooled to 25 °C then concentrated under vacuum. The residue was purified on a silica gel column eluting with EtOAc/petroleum ether (1/2) to give 101 mg (17%, over two steps) of 75 as a yellow oil. TLC: EtOAc/ petroleum ether = 1/1, Rf = 0.3. 6-(bis(4-Methoxybenzyl)amino)-4-chloro-1H-pyrrolo[3,2-c]pyridine-2-carboxylic acid (76). To a solution of 75 (400 mg, 0.833 mmol) in EtOH (10 mL) was added a solution of potassium hydroxide (400 mg, 7.14 mmol) in H2O (10 mL). The reaction mixture was stirred for 20 h at 25 °C, then the pH value was adjusted to 6 with 1 M aqueous HCl solution. The mixture was concentrated under vacuum to give 1.01 g of crude 76 as a yellow solid. TLC: DCM/MeOH=5/1, Rf = 0.3. 6-(bis(4-Methoxybenzyl)amino)-4-chloro-N-(4-((3,5-difluorophenyl)sulfonyl)benzyl)-1Hpyrrolo[3,2-c]pyridine-2-carboxamide (78). A mixture of 76 (300 mg, 0.664 mmol), (4-((3,5-difluorophenyl)sulfonyl)phenyl)methanamine (77) (400 mg, 1.41 mmol), EDCI (300 mg, 1.57 mmol), HOBt (200 mg, 1.48 mmol) and DIPEA (0.77 mL, 4.6 mmol) in DMF (20 mL) was stirred for 1 h at 50 °C. The resulting mixture was concentrated under vacuum and the residue was purified on a silica gel column eluting with EtOAc/petroleum ether (1/1) to give 401 mg (67%, over two steps) of 78 as a red oil. TLC: EtOAc/petroleum ether = 1/1, Rf = 0.2. 6-Amino-4-chloro-N-(4-((3,5-difluorophenyl)sulfonyl)benzyl)-1H-pyrrolo[3,2-c]pyridine-2carboxamide (79). 67



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A solution of 78 (200 mg, 0.279 mmol) and TFA (1.0 mL) in DCM (10 mL) was stirred for 2 h at 25 °C. The resulting mixture was concentrated under vacuum and the residue was purified on a silica gel column eluting with EtOAc/petroleum ether(1/1) followed by EtOAc to give 60.1 mg (45%) of 79 as a red oil. TLC: DCM/MeOH=5/1, Rf = 0.6. 6-Chloronicotinoyl chloride (81). DMF (1.06 mL, 13.7 mmol) and thionyl chloride (20 mL, 275 mmol) were added to a mixture of 6chloronicotinic acid (80) (20.0 g, 127 mmol) in toluene (200 mL), and the reaction mixture was stirred under nitrogen for 3 h at 80 °C. The resulting solution was cooled to 25 °C and concentrated under vacuum to give 25 g of crude 81 as a light yellow solid. 6-Chloro-N,N-diisopropylnicotinamide (82). Diisopropylamine (69.3 mL, 495 mmol) was added dropwise to a solution of 81 (25 g, 142 mmol) in DCM (500 mL) at 0 °C. The reaction mixture was warmed to 25 °C and stirred for 50 min, then quenched with H2O (300 mL). The organic layer was collected and the aqueous layer was extracted with DCM (2 x 300 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under vacuum to give 25.1 g (82%, over two steps) of 82 as a light yellow solid. TLC: EtOAc/petroleum ether=1:2, Rf = 0.4. 1H NMR (400 MHz, CDCl3) δ 8.39 (s, 1H), 7.65 (dd, J = 8.0, 2.4 Hz, 1H), 7.39 (d, J = 8.0 Hz, 1H), 3.67 – 3.41 (m, 2H), 1.63 – 1.29 (m, 12H). 6-Chloro-4-formyl-N,N-diisopropylnicotinamide (83). n-BuLi (5.0 mL, 2.5 M solution in hexanes) was added dropwse to a solution of diisopropylamine (1.39 mL, 9.90 mmol) in diethyl ether (30 mL) at –50 °C. The reaction mixture was stirred for 30 min at –50 °C, then solid 82 (500 mg, 2.08 mmol) was added in a single portion. The reaction mixture was stirred for 30 min at –50 °C, then DMF (1.0 mL) was added dropwise. The reaction mixture was stirred at –50 °C for a further 3 h then warmed to 25 °C and stirred for 16 h. The 68


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reaction mixture was quenched with 10% aqueous citric acid solution (30 mL) and then extracted with diethyl ether (2 x 50 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under vacuum to give 501 mg of crude 83 as a yellow solid. LCMS (Method M, ESI) RT = 1.40 min, m/z = 269.0 [M+H]+. 1H NMR (300 MHz, CDCl3) δ 10.07 (s, 1H), 8.43 (s, 1H), 7.67 (s, 1H), 3.67 – 3.54 (m, 2H), 1.64 – 1.10 (m, 12H). 6-Chloro-4-(hydroxymethyl)-N,N-diisopropylnicotinamide (84). A mixture of 83 (500 mg, 1.87 mmol) and NaBH4 (500 mg, 13.2 mmol) in EtOH (50 mL) was stirred for 50 min at 30 °C. The reaction mixture was then quenched with 1 M aqueous HCl. The solids were removed by filtration and the filtrate was concentrated to provide 502 mg of crude 84 as a light yellow solid. LCMS (Method I, ESI) RT = 1.25 min, m/z = 271.0 [M+H]+. 6-Chlorofuro[3,4-c]pyridin-3(1H)-one (85). A mixture of 84 (2.00 g, 7.41 mmol) in 6 M aqueous HCl (40 mL) was stirred for 30 min at 100 °C. The reaction mixture was cooled to 25 °C and the pH value of the reaction mixture was adjusted to 8 with a saturated aqueous solution of sodium carbonate. The mixture was extracted with 200 mL of DCM, then the organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum to yield 1.01 g of crude 85 as a light yellow solid. LCMS (Method W, ESI) RT= 1.13 min, m/z = 170.0 [M+H]+. 1H NMR (300 MHz, CDCl3) δ 8.93 (s, 1H), 7.49 (s, 1H), 5.31 (s, 2H). (6-Chloropyridine-3,4-diyl)dimethanol (86). A mixture of 85 (1.01 g, 5.94 mmol) and NaBH4 (500 mg, 13.2 mmol) in EtOH (50 mL) was stirred for 1 h at 25 °C. The pH value of the reaction mixture was adjusted to 1 with 6 M aqueous HCl. The solids were removed by filtation and the filtrate was concentrated under vacuum. The residue was purified on a silica gel column eluting with DCM/MeOH (20/1) to give 402 mg (27%, over four steps) of 86 as a light yellow solid. LCMS (Method W, ESI) RT = 0.95 min, m/z = 174.0 [M+H]+. 69



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1

Page 70 of 92

H NMR (300 MHz, DMSO-d6) δ 8.24 (s, 1H), 7.42 (s, 1H), 5.47 (t, J = 5.6 Hz, 1H), 5.29 (t, J = 5.3

Hz, 1H), 4.56 (d, J = 5.4 Hz, 2H), 4.44 (d, J = 5.1 Hz, 2H). 2-Chloro-4,5-bis(chloromethyl)pyridine hydrochloride (87). A mixture of 86 (100 mg, 0.575 mmol) and thionyl chloride (2.0 mL, 27.5 mmol) in DCM (20 mL) was stirred at 25 °C for 1 h. The resulting mixture was concentrated under vacuum to give 101 mg of crude 87 as a dark red solid. 6-Chloro-2-(2,4-dimethoxybenzyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridine (88). DIPEA (1.28 mL, 7.75 mmol) was added to a mixture of 87 (1.00 g, 4.05 mmol) and (2,4dimethoxyphenyl)methanamine (1.00 g, 5.99 mmol) in DCM (60 mL), and the reaction mixture was stirred for 16 h at 25 °C, then concentrated under vacuum. The residue was purified on a silica gel column eluting with EtOAc/petroleum ether (2/1) to give 902 mg (52%, over two steps) of 88 as a light red oil. LCMS (Method W, ESI) RT= 0.94 min, m/z = 305.0 [M+H]+. N,2-bis(2,4-Dimethoxybenzyl)-2,3-dihydro-1H-pyrrolo[3,4-c]pyridin-6-amine (89). A mixture of 88 (200 mg, 0.655 mmol), Pd2(dba)3•CHCl3 (100 mg, 0.097 mmol), sodium tertbutoxide

(200

mg,

2.08

mmol),

BINAP

(100

mg,

0.161

mmol)

and

(2,4-

dimethoxyphenyl)methanamine (400 mg, 2.4 mmol) in toluene (20 mL) was stirred under nitrogen for 16 h at 80 °C. The reaction mixture was cooled to 25 °C, then 20 mL of H2O was added. The layers were separated and the organic layer was extracted with EtOAc (2 x 50 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified on a silica gel column eluting with EtOAc/petroleum ether (1/1) to give 202 mg (70%) of 89 as a dark red solid. LCMS (Method W, ESI) RT= 0.92 min, m/z = 436.0 [M+H]+. 2,3-Dihydro-1H-pyrrolo[3,4-c]pyridin-6-amine (90).

70


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TFA (20 mL) was added to 89 (300 mg, 0.689 mmol), and the reaction mixture was stirred under nitrogen for 16 h at 90 °C. The resulting mixture was cooled to 25 °C and concentrated under vacuum to remove most of the TFA. The pH value of the residue was adjusted to 8 with a saturated aqueous solution of sodium carbonate. The mixture was concentrated under vacuum and the residue was dissolved in hot EtOAc and filtered. The filtrate was concentrated under reduced pressure to provide 151 mg of crude 90 as a red oil. LCMS (Method W, ESI): RT= 0.18 min, m/z = 136.0 [M+H]+. 4-Nitrophenyl (4-(phenylsulfonyl)benzyl)carbamate (92). 4-nitrophenyl

chloroformate

(500

mg,

2.5

mmol)

was

added

to

a

mixture

of

4-

(phenylsulfonyl)phenyl)methanamine (91) (500 mg, 2.0 mmol) in toluene (30 mL). The reaction mixture was stirred under nitrogen at 120 °C for 30 min, then cooled to 25 °C. A solid precipitated and was collected by filtration, then washed with toluene (5 mL) and dried under vacuum to give 798 mg (96%) of 92 as a light yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 8.67 (t, J = 6.2 Hz, 1H), 8.22 (d, J = 9.0 Hz, 2H), 7.97 – 7.92 (m, 4H), 7.72 – 7.56 (m, 5H), 7.43 (d, J = 9.3 Hz, 2H), 4.38 (d, J = 6.0 Hz, 2H). 2-Chloro-5-(methoxymethoxy)pyridine (94). To a solution of 6-chloropyridin-3-ol (93, 10.0 g, 77.5 mmol) in DMF (120 mL) at 0 °C was added NaH (3.8 g , 60% in mineral oil, 95.0 mmol) in small portions. The reaction mixture was warmed to 25 °C and stirred for 1 h, then re-cooled to 0 °C. MOM-Br (8.5 mL, 104 mmol) was then added dropwise while maintaining the reaction temperature at 95% at the time of their biological assessments as determined by LCMS analysis with UV detection at 220 and/or 254 nm. We also noted, however, that some molecules containing the 2,3-dihydro-1Hpyrrolo[3,4-c]pyridine-derived urea moiety experienced oxidation upon prolonged storage (detected via LCMS analysis of stored materials). No attempts were made to study or suppress this process and the oxidized compounds were neither isolated nor biologically characterized. LCMS fragmentation patterns suggested the oxidation products depicted below, but no additional analyses were conducted to confirm these assignments.

31 Djakovitch, L.; Wagner, M.; Hartung, C. G.; Beller, M.; Koehler, K. Pd-catalyzed Heck arylation of cycloalkenes—studies on selectivity comparing homogeneous and heterogeneous catalysts. J. Mol. Catal. A: Chem. 2004, 219, 121–130.

91



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TABLE OF CONTENTS GRAPHIC:

Fsp3: 0.19 CYP2C9 IC50: 0.08 µM NAMPT Cell IC50: 11 nM Efficacious in xenograft model

Fsp3: 0.67 CYP2C9 IC50: >10 µM NAMPT Cell IC50: 0.6 nM Efficacious in xenograft model

92


Minimizing CYP2C9 Inhibition of Exposed-Pyridine NAMPT

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