Discovery of 4-Amino-8-quinoline Carboxamides as Novel

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Discovery of 4-Amino-8-quinoline carboxamides as novel, submicromolar inhibitors of the NAD hydrolyzing enzyme CD38 J. David Becherer, Eric E Boros, Tiffany Y. Carpenter, David J. Cowan, David Deaton, Curt D. Haffner, Michael R. Jeune, Istvan W. Kaldor, J. Chuck Poole, Frank Preugschat, Tara R Rheault, Christie A Schulte, Barry G Shearer, Lisa M. Shewchuk, Terrence L. Smalley, Eugene L. Stewart, J. Darren Stuart, and John C. Ulrich J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00992 • Publication Date (Web): 12 Aug 2015 Downloaded from http://pubs.acs.org on August 20, 2015

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Journal of Medicinal Chemistry

Discovery of 4-amino-8-quinoline carboxamides as novel, submicromolar inhibitors of the NAD hydrolyzing enzyme CD38

J. David Becherer, Eric E. Boros, Tiffany Y. Carpenter, David J. Cowan, David N. Deaton*, Curt D. Haffner, Michael R. Jeune, Istvan W. Kaldor, J. Chuck Poole, Frank Preugschat, Tara R. Rheault, Christie A. Schulte, Barry G. Shearer, Lisa M. Shewchuk, Terrence L. Smalley, Jr., Eugene L. Stewart, J. Darren Stuart, John C. Ulrich GlaxoSmithKline Research and Development, 5 Moore Drive, P.O. Box 13398, Research Triangle Park, North Carolina 27709 *

To whom correspondence should be addressed. Phone: 919-483-6270. E-mail: [email protected].

KEYWORDS: Nicotinamide adenine dinucleotide, NAD, CD38, NAD glycohydrolase

Abstract Starting from the micromolar 8-quinoline carboxamide high throughput screening hit 1a, a systematic exploration of the structure/activity relationships (SAR) of the 4-, 6-, and 8-substituents of the quinoline ring resulted in the identification of approximately 10-100-fold more potent human CD38 inhibitors. Several of these molecules also exhibited pharmacokinetic parameters suitable for in vivo animal studies, including low clearances and decent oral bioavailability. Two of these CD38 inhibitors, 1ah and 1ai were shown to elevate NAD tissue levels in liver and muscle in a diet-induced obese (DIO) C57BL6 mouse model. These inhibitor tool compounds will enable further biological studies of the CD38 enzyme as well as the investigation of the therapeutic implications of NAD enhancement in disease models of abnormally low NAD. Introduction In the ongoing study of immune cells, numerous laboratories have generated monoclonal antibodies (mAbs) against epitopes on the cell surface of leukocytes as a means to immunophenotype these white blood cells. In an effort to classify and harmonize the many recognition motifs, the International Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA) developed a nomenclature system designating cell surface molecules

ACS Paragon Plus Environment

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that are recognized by two specific mAbs as numbered Clusters of Differentiation (CD) (e.g. CD4, CD8, CD26). These CD proteins can have multiple and diverse functions including recognizing antigens, signaling as receptors, adhering to cell surfaces, and acting as enzymes. One such CD protein, CD38, was originally identified as T10 by a murine mAb obtained from ascitic fluid of CAF1 mice injected with human thymocytes.1 Subsequently, the protein was localized to chromosome 42 and cloned.3 CD38 is a type II membrane protein, containing a single, N-terminal transmembrane domain with multiple asparagine-linked glycosylation sites at its extracellular C-terminal end. In addition to its expression in immune cells, including T cells, B cells, and dendritic cells, CD38 is also expressed in multiple tissues, including bone, brain, intestine, kidney, liver, muscle, pancreas, and prostate.4 CD38 serves as a receptor for the immunoglobulin CD31 (PECAM-1) and this interaction is implicated in leukocyte migration.5 In addition to its receptor functions, CD38 can also act as an enzyme (EC 3.2.2.5). It catalyzes the transformation of the redox cofactor nicotinamide adenine dinucleotide (NAD) into the second messenger signaling molecule cyclic ADP-ribose (cADPR), which has been implicated in the regulation of cytosolic calcium fluxes, and cADPR’s hydrolysis to ADP-ribose.6,7 Furthermore, CD38 can function as a NAD glycohydrolase (NADase), reducing the pool size of this important enzyme cofactor, affecting its many dependent proteins, including the sirtuins, the poly(ADP-ribose) polymerases (PARPs), and oxidoreductases. NAD levels are decreased in the aged as well as the obese; however, exercise or caloric restriction can result in NAD elevation.8,9 One potential approach to increasing NAD levels is to inhibit NAD consuming enzymes like CD38. Support for this supposition is demonstrated in CD38 (-/-) mice, which exhibit elevated NAD levels relative to wild type mice.10 Moreover, these knockout mice have increased energy expenditure and are resistant to weight gain, when fed a high fat diet. Thus, a small molecule inhibitor of the enzyme function of CD38 that could increase NAD levels in conditions of abnormally low NAD, like obesity, might have therapeutic utility. Although there are a few articles disclosing CD38 inhibitors in the literature, most of these molecules exhibit only modest potency.11-17 Desiring more potent inhibitors, with drug properties suitable for oral dosing, a program was begun to discover novel classes of CD38 inhibitors suitable for probing NAD biology. A high throughput screen (HTS) of the GlaxoSmithKline compound collection was performed in an attempt to identify chemical starting


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points, excluding alternative substrate inhibition. This effort yielded a number of weakly active CD38 inhibitors with desirable kinetics against the human enzyme, including the recently published 6-thiazole quinoline hit series.18 Among these hits was a singleton quinoline carboxamide 1a, that database mining revealed had no known protein target associated with it in historical GlaxoSmithKline assays. As shown in Table 1, this inhibitor was a low micromolar inhibitor of human CD38 (IC50 = 7,200 ± 2,400 nM). Like the other inhibitors disclosed in this article, the quinoline carboxamide 1a exhibits mixed inhibition of CD38, binding in the catalytic active site (vide infra). Furthermore, preliminary pharmacokinetic experiments revealed that quinoline carboxamide 1a had a terminal i.v. clearance in mice of less than a third of hepatic blood flow (Cl = 29 mL/min/kg) coupled with a decent murine oral bioavailability (F = 32%), as shown in Table 2. With its positive attributes, inhibitor 1a was selected as a chemical small molecule lead for initiating the development of better CD38 inhibitors. This manuscript details the structure/activity relationships developed from screening hit 1a that resulted in the discovery of two molecules with potencies and pharmacokinetic properties sufficient to serve as robust tool molecules for the investigation of CD38 enzyme biology in vivo. Chemistry The quinoline CD38 inhibitors were prepared by one of several routes as depicted in Schemes 1-3. In one route, thermal ipso displacement of the 4-chloro substituent of commercially available quinoline 2a, with commercially available amines or thiols, afforded 4-amino- or 4-thioquinolines 3a-3k & 3m-3ac, as shown in Scheme 1. Bromoquinoline 3l was synthesized from commercially available 8-bromo-2-methyl-4-quinolinamine by alkylation with 3,6-dichloro-2-fluorobenzyl bromide. Subsequent palladium-mediated carbonylation of these quinolines at the 8-bromo moiety, with amine trapping, provided the desired 8-quinoline carboxamides 1a, 1f-1j, 1l, 1q, 1ah-1ai, 1al1bb, & 1be. Alternatively, Ullmann-like couplings of 8-bromoquinolines with copper(I) cyanide yielded 8-quinoline carbonitriles 4a, 4m, & 4r.

In lieu of this method, the Ullmann-like coupling could be carried out first, converting

the 8-bromoquinoline 5a to the 8-cyanoquinoline 6. Then, after transforming the hydroxyl substituent of 6 to the triflate 7 with trifluoromethanesulfonic anhydride, subsequent displacement, with commercially available amines, provided the 8-quinoline carbonitriles 4b-4g, 4j-4l, 4n-4q, & 4s-4ab. The 4-(2-cyanobenzylamino)quinoline 4i was prepared via alkylation, with 2-(bromomethyl)benzonitrile, of the 4-aminoquinoline 4h, synthesized from acid


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catalyzed hydrolysis of the 2,4-dimethoxybenzyl group of quinoline carbonitrile 4g.. The 4-amidoquinoline 4ac was prepared from 4-amino-2-methyl-8-quinoline carbonitrile 4h, via acylation, with 2,6-dichlorobenzoyl chloride. The 4-alkoxyquinoline 4ad was prepared by base-promoted alkylation of 4-hydroxyquinoline 6 with 1,3-dichlorobenzyl bromide. These cyanides could be hydrolyzed with basic hydrogen peroxide to give the 8-quinoline carboxamides 1c-1e, 1k,1m-1p, 1r-1ag, 1aj-1ak, & 1bc-1bd. Quinoline carboxamide 1b was synthesized from quinoline carboxamide 1l via acid catalyzed removal of the para-methoxybenzyl group. The 6-substituted quinolines were prepared as shown in Scheme 2. The 6-fluoroquinoline analog 1bf was synthesized from commercially available 2-bromo-4-fluoroaniline 8a. First, the aniline 8a was converted to the 4hydroxyquinoline 5b via thermal Michael addition/Friedel-Craft cyclization with a Meldrum’s acid derivative. Then, treatment of the hydroxyquinoline 5b with phosphorus oxychloride provided the 4-chloroquinoline 2b. Subsequent addition of 2,3,6-trichlorobenzylamine to chloroquinoline 2b afforded the 4-aminoquinoline 3ad. Finally, palladium-mediated carbonylation of bromoquinoline 3ad, with amine trapping, provided the desired 8quinoline carboxamides 1bf. Scheme 1. Synthesis of 4-substituted quinoline carboxamides.

Reagents and conditions: a) R1R2XH, iPr2NEt or K2CO3, DMSO, DMF or NMP, 130-160 °C, 11-82%; b) Pd2(dba)3 or Pd(OAc)2, 1,1’-bis(diphenylphosphino)ferrocene or Ph2P(CH2)3PPh2 (dppp), CO, iPr2NEt, (Me3Si)2NH, DMF, 110 °C, 13-81%; c) CuCN, DMF or DMA, 140-160 °C, 67-98%; d) Tf2O, 2,6-lutidine, CH2Cl2, -30 to 25 °C, 82%;


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e) R1R2NH, K2CO3, THF, 75-90 °C, 15-81%; f) R1Br, NaH or K2CO3, DMF, 70 °C 15-99%; g) TFA, 99%; h) 2,6dichlorobenzoic acid, SOCl2, 70 °C; 4h, LHMDS, THF, -75 °C, 51%; i) NaOH or KOH, 30% H2O2, DMSO, 8-82%. In an analogous manner to 5b, the 6-bromo-4-hydroxyquinoline 5c was synthesized from methyl 2-amino-5bromobenzoate 8b and methyl acetoacetate. Then, heating the hydroxyquinoline 5c with phosphorus oxychloride provided the 4-chloroquinoline 2c. A palladium-mediated cross coupling of 6-bromoquinoline 2c with potassium methyltrifluoroborate afforded the 6-methylquinoline 2d. Subsequently heating the ester 2d with ammonia provided the quinoline carboxamide 2e. Finally, thermal ipso displacement of the 4-chloro substituent of 4-chloroquinoline 2e with 2,6-dimethylbenzylamine gave the 6-methylquinoline carboxamide 1bg. Alternatively, aminolysis of the ester 2c provided the 6-bromoquinoline carboxamide 2f, which was converted to the CD38 analog 1bh via thermal ipso displacement of the 4-chloro substituent. The 6-phenylquinoline analog 1bi was made in a similar manner to the 6-methylquinoline 1bg via Suzuki cross coupling of the 6-bromoquinoline 2f with phenylboronic acid to provide the 6-phenylquinoline 2g, followed by displacement of the 4-chloro substituent with 2,6-dimethylbenzylamine. As shown in Scheme 3, the 8-bromoquinoline 1bk was prepared in a similar manner as above from 4chloroquinoline 2a and 2,3-dichlorobenzylamine. Subsequent palladium-mediated carbonylation of this quinoline at the 8-bromo moiety, followed by methylamine, dimethylamine, or methanol trapping, provided the desired 8quinoline carboxamides/carboxylate 1bl-bn. Base-catalyzed hydrolysis of quinoline carboxylate 1bn with lithium hydroxide afforded the 8-quinoline carboxylic acid 1bo. Alternatively, lithium borohydride mediated reduction of quinoline carboxylate 1bn provided the 8-quinoline alcohol 1bp. Furthermore, this primary alcohol could be converted to the primary amine 1bq via displacement of the in situ-generated mesylate with ammonia. Treatment of the 8-quinoline carboxamide 1q, derived from 1bk by carbonylation and amine trapping, with Belleau’s reagent gave the quinoline nitrile 1br instead of the expected quinoline thioamide. In a like manner, an attempt to produce the 8-quinoline boronic acid via palladium-mediated cross coupling with bis(pinacolato)diborane only gave the quinoline 1bj via hydropalladation of the arylbromide. Finally, the quinoline sulfonamide 1bs was prepared via amidation of the known sulfonyl chloride 2h19 with ammonia, giving the quinoline 2i, followed by ipso displacement of the 4-chloro moiety with 2,3-dichlorobenzylamine. Scheme 2. Synthesis of 6-substituted quinoline carboxamides.


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Reagents and conditions: a) 2,2-dimethyl-5-[1-(methylthio)ethylidene]-1,3-dioxane-4,6-dione, Ph2O, µW, 240 °C, 19%; b) CH3COCH2COOCH3, PPA, 160 °C, 16%; c) POCl3, 80-90 °C, 42-77%; d) KCH3BF3 or PhB(OH)2, Cs2CO3 or K2CO3, PdCl2(dppf) or (PPh3)2PdCl2, THF, 85 °C, 45-67%; e), NH3, MeOH, 100 °C, 37-39%; f) R1R2NH, 6N HCl (aq) or iPr2NEt, NMP or DMSO, µW, 150 °C, 5-65%; g) Pd(OAc)2, Ph2P(CH2)3PPh2 (dppp), CO, iPr2NEt, (Me3Si)2NH, 110 °C, 53%. Results and Discussion The structure–activity relationships of the quinoline analogs are depicted in Table 1. As the 4-aminoquinoline carboxamide 1b did not inhibit CD38 up to concentrations of 0.03 mM, the benzyl moiety in a compound such as 1a was clearly important for inhibitory activity. The unsubstituted benzyl analog 1c was a less potent CD38 inhibitor than the initial HTS hit 1a. With these results, it was decided to employ a Topliss-like approach on the phenyl moiety of the benzylic group in order to identify optimal substitutions that maximized binding interactions of this area of the ligand with the CD38 enzyme and provided improved potency.20 All mono-substituted chloro-, methyl, and methoxy-derivatives were prepared. The chloro-substituted inhibitor 1e, 1h, and 1k were equal or better inhibitors of CD38 than the methylated analogs 1d, 1g, and 1j or the methoxy derivatives 1f, 1i, and 1l, respectively, with the ortho-chloro analog 1e being the most potent inhibitor. These results suggest that the protein prefers electron withdrawing groups and/or hydrophobic groups in this region. With these results in mind, several more


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ortho derivatives were synthesized, with the trifluoromethyl moiety 1m and the bromo derivative 1n being equipotent to the chloro derivative 1e; however, the carboxamide derivative 1o was inactive. To further explore the phenyl ring SAR, all permutations of the dichloro- and dimethyl analogs were prepared. The starting 2,6-dimethyl lead 1a was equal or more potent than the 2,3- 1p, 2,4- 1r, 2,5- 1t, 3,4- 1w, and 3,5dimethylquinolines 1y. Similarly, the 2,6-dichloro inhibitor 1v was also more potent than the 2,4- 1s, 2,5- 1u, 3,41x, and 3,5-dichloroquinolines 1z, while the 2,3-dichloroquinoline 1q was slightly more potent than 1v. As previously demonstrated, chloro substitution was favored over methyl substitution in these analogs. Since ortho derivatives afforded greater activity, the favored binding mode of the ligand in the protein may be one in which the out of plane conformation of the benzylic moiety is preferred. Several 2,3-disubstituted benzyl analogs (1aa-1ad) were prepared in an attempt to increase the potency of the submicromolar inhibitor 1q. However, all four of the prepared compounds, the 2-fluoro-3-chloro derivative 1aa, the 2-methyl-3-chloro derivative 1ab, the 2-fluoro-3-trifluoromethyl derivative 1ac, and the α-naphthyl derivative 1ad, were less potent than the 2,3-dichloro derivative 1q. In a like manner, several 2,6-disubstituted benzyl analogs were synthesized to further explore the SAR of the submicromolar inhibitor 1v. This chemistry effort enriched the understanding of the SAR, as the 2-chloro-6-fluoro derivative 1af and the 2-trifluoromethyl-6-fluoro derivative 1ah exhibited similar activity to the 2,6-dichloro derivative 1v, while the 2,6-difluoro derivative 1ae and the 2-chloro-6methyl derivative 1ag were less active. Furthermore, in an attempt to enhance the potencies of the 2,3-dichloro compound 1q and 2,6-dichloro compound 1v, the 2,3,6-trichloro analog 1ai was prepared. This compound proved to be a more potent CD38 inhibitor than either 1q or 1v. Several more 2,3,6-trisubstituted benzyl analogs were synthesized. These efforts revealed three additional equipotent derivatives, the 2-chloro-3,6-difluoro analog 1ak, the 2,3-dichloro-6-fluoro analog 1am, and the 2-chloro-3-methyl-6-fluoro analog 1an. The 2-6-difluoro-3-chloro compound 1aj and 2-fluoro-3,6-dichloro compound 1al were also prepared; however, the potencies of these compounds were no better than their 2,6-comparators 1ae and 1af. Scheme 3. Synthesis of 8-substituted quinolines.


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Cl

Cl

Cl

Cl

NH

NH

Cl

N Br 2a R1 = Br 1 h 2h R = SO2Cl 1 2i R = SO2NH2

N

N

a

b

Br 1bk

c

a

R1 O 1bl R1 = NHMe 1bm R1 = NMe2 1bn R1 = OMe 1bo R1 = OH

b, f

g

d Cl

Cl Cl

Cl NH

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Cl

Cl

Cl

NH

Cl

NH

NH N

N O S NH2 O 1bs

N 1bj

N

N 2

1br

R 2 e 1bp R = OH 1bq R2 = NH2

Reagents and conditions: a) 2,3-dichlorobenzylamine, iPr2NEt, DMSO or NMP, 150 °C, 47-75%; b) Pd(OAc)2, Ph2P(CH2)3PPh2 (dppp), CO, iPr2NEt, (Me3Si)2NH, MeNH2, Me2NH, or MeOH, 110 °C, 47-66%; c) LiOH, THF, MeOH, H2O, rt, 57%; d) LiBH4, THF, rt, 64%; e) Ms2O, THF, rt; NH3, 29%; f) Belleau's reagent, 1,4-dioxane, 100 °C, 28%; g) PdCl2(dppf), (OCMe2CMe2O)2B2, KOAc, 1,4-dioxane, 100 °C, 45%; h) NH3 in dioxane, 60 °C, 82%. The most potent benzyl substituted inhibitors (e.g. 1ai) contain hydrophobic moieties, resulting in compounds with limited aqueous solubility. Therefore, heteroaryl isosteric replacements of the phenyl ring were explored with the aspiration of enhancing the drug properties, such as solubility, of the resulting compounds, while maintaining their CD38 activities. As such, four aza-analogs were prepared, the 2-trifluoromethyl-6-aza derivative 1ao, the 2trifluoromethyl-5-aza derivative 1ap, the 2-chloro-4-aza derivative 1aq, and the 2-trifluoromethyl-3-aza derivative 1ar. Not surprisingly, because these pyridine derivatives require a higher desolvation energy when binding to the enzyme than do their corresponding phenyl derivatives, many of these analogs were less active than their comparators 1e and 1m. The most potent of these pyridine compounds was 1ar, which likely has a lower desolvation energy than the other pyridine analogs due to the shielding of its nitrogen lone pair by the adjacent hydrophobic trifluoromethyl group. Additionally, two 5-membered ring heteroaryl derivatives were synthesized. In


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SAR similar to the pyridine series, both the thiophene 1as and the isoxazole 1at were poorer inhibitors when compared to the corresponding compounds 1e and 1a, respectively. It has been observed that compounds containing greater numbers of sp3 centers are more soluble than their more planar counterparts. One hypothesis that attempts to rationalize this phenomenon is that compounds containing sp3 centers do not pack in a crystaline lattice as well as compounds containing more planar sp2 centers. For less planar compounds, this situation results in lower crystallization energies and leads to faster dissolutions in bulk solvents per unit surface area. Under this hypothesis, the synthesis of saturated ring isosteres of the phenyl ring were undertaken, with the ultimate goal of enhancing solubility of the resulting compounds. The cyclohexyl analog 1au was prepared and found to be more potent than its corresponding phenyl analog 1c; however, introduction of acylated or basic nitrogen piperidine rings into compounds such as 1av or 1aw was detrimental to inhibitory potency, possibly due to increased desolvation energies. Table 1. Human CD38 enzyme inhibition data.

#

R1

R2

X

IC50 ± S.D.a nM

H

CONH2

7,200 ± 2,400

H

CONH2

>30,000

1c

H

CONH2

22,000 ± 1,900b

1d

H

CONH2

22,000 ± 2,700 b

1e

H

CONH2

3,300 ± 250

1f

H

CONH2

>30,000

1a

1b

H 2N


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1g

H

CONH2

>30,000

1h

H

CONH2

19,000 ± 2,700c

H

CONH2

>30,000

1j

H

CONH2

>30,000d

1k

H

CONH2

17,000 ± 3,500e

1l

H

CONH2

25,000 ± 2,000

1m

H

CONH2

3,300 ± 3,300

1n

H

CONH2

2,300 ± 320

1o

H

CONH2

>30,000

1p

H

CONH2

18,000 ± 3,400

1q

H

CONH2

620 ± 120

1r

H

CONH2

>30,000

1s

H

CONH2

4,500 ± 1,100

1t

H

CONH2

>30,000f

1u

H

CONH2

11,000 ± 1,600

1i

O

N H

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Cl

H

CONH2

990 ± 150

1w

H

CONH2

>30,000g

1x

H

CONH2

15,000 ± 4,200h

1y

H

CONH2

>30,000f

1z

H

CONH2

>30,000

1aa

H

CONH2

16,000, ± 940

1ab

H

CONH2

4,000 ± 2,500

1ac

H

CONH2

9,600 ± 2,800

1ad

H

CONH2

24,000 ± 3,800i

1ae

H

CONH2

6,200 ± 1,700

1af

H

CONH2

780 ± 300

1ag

H

CONH2

1,200 ± 370

1ah

H

CONH2

510 ± 100

1v

N H Cl


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1ai

H

CONH2

150 ± 32

1aj

H

CONH2

4,700 ± 2,400

1ak

H

CONH2

170 ± 75

1al

H

CONH2

3,600 ± 1,800

1am

H

CONH2

130 ± 34

1an

H

CONH2

280 ± 93

1ao

H

CONH2

8,500 ± 1,300

1ap

H

CONH2

7,300 ± 2,500

1aq

H

CONH2

>30,000

1ar

H

CONH2

2,100 ± 160

1as

H

CONH2

4,900 ± 590

1at

H

CONH2

>30,000

1au

H

CONH2

11,000 ± 2,500

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H

CONH2

>30,000

1aw

H

CONH2

>30,000

1ax

H

CONH2

>30,000

1ay

H

CONH2

>30,000

1az

H

CONH2

6,600 ± 1,800

1ba

H

CONH2

2,700 ± 780

H

CONH2

>30,000

1bc

H

CONH2

>30,000

1bd

H

CONH2

>30,000

H

CONH2

>30,000

1bf

F

CONH2

76 ± 13

1bg

Me

CONH2

24,000 ± 3,100 b

1bh

Br

CONH2

>10,000

1av O

1bb

N H

N

Cl N Cl

1be

Cl S Cl


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1bi

Ph

CONH2

>30,000

1bj

H

H

>30,000

1bk

H

Br

>30,000

1bl

H

CONMeH

>30,000

1bm

H

CONMe2

>30,000

1bn

H

CO2Me

>30,000

1bo

H

COOH

>30,000

1bp

H

CH2OH

>30,000

1bq

H

CH2NH2

>30,000

1br

H

CN

>30,000

1bs

H

SO2NH2

9,200 ± 2,200b

a

Page 14 of 107

S.D. = standard deviation; mean ± S.D. calculated from N ≥ 4, except where otherwise indicated; if the IC50 is higher than the highest test

concentration then the curve fitting software ActivityBase returns a value with a “>” modifier; in some cases the highest test concentration was removed from the curve analysis due to solubility or assay interference issues, hence the modified value may not be the same for every compound b

mean ± S.D. calculated from N = 3; one additional value not included in statistics was reported as >30,000 nM

c

mean ± S.D. calculated from N = 2; two additional values not included in statistics were reported as >30,000 nM

d

N = 3; 2 additional values exist with mean ± S.D. of 25,000 ± 5,300 nM


14

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e

mean ± S.D. calculated from N = 2

f

N = 3; one additional value not included was reported as >10,000 nM

g

one additional value was 14,000 nM

h

one additional value not included in statistics was reported as >3,300 nM

i

mean ± S.D. calculated from N = 4; one additional value not included in statistics was reported as >30,000 nM

Substitution at the benzylic position of the 4-amino moiety was also explored. Neither the (R)-chiral methyl analog 1ax nor the (S)-chiral methyl analog 1ay were as active as their comparators 1d and 1a, respectively. This potency loss likely results from an increased availability of potential rotamer populations as compared to the achiral inhibitor, rather than some inability of the protein to accommodate the methyl moiety, because the conformationally constrained tetrahydronaphthalene derivatives 1az and 1ba are better inhibitors than compound 1d. Methylation of the benzylic nitrogen to give derivative 1bb resulted in a large decrease in inhibitory activity relative to the 2,6-dichloro analog 1v. While a detrimental steric interaction between the methyl moiety and the protein could explain the significant decrease in potency, alternatively, the loss of the hydrogen could disrupt a key hydrogen bonding interaction between the protein and the amino group. Interesting, although the NH of the amide derivative 1bc could still act as a hydrogen bond donor, this compound is also a poor inhibitor of CD38. In this particular case, though, the sp2 benzylic center may bias the conformation of the inhibitor, precluding its ability to adopt a conformation more suitable for hydrogen bonding. Both the benzyl ether derivative 1bd and the thio derivative 1be are poor inhibitors of CD38, but should exist in conformations similar to the amine analog 1v. Therefore, one can infer from these results, that if a hydrogen bond exists between the protein and the ligand, that interaction requires a hydrogen bond donor, and not an acceptor group. In addition to probing the SAR of the 4-position of the quinoline ring, chemistry explorations were also focussed on the 6-position of the quinoline ring. Initially, three analogs were prepared, the 6-methyl derivative 1bg, the 6-bromo derivative 1bh, and the 6-phenyl derivative 1bi. All of these analogs proved to be less potent than the initial HTS hit 1a, with the 6-methyl analog 1bg being approximately 4-fold less potent. Subsequently, a 6-fluoro derivative 1bf was synthesized with the aim of improving metabolic stability. Surprisingly, this compound was slightly more active than its comparator 1ai.


15


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Page 16 of 107

As shown in Table 1, any isosteric replacement of the carboxamide at the 8-position of the quinoline ring provided compounds with little to no activity against CD38. Removal of the carboxamide, for example analog 1bj, resulted in compounds with no inhibitory activity. Furthermore, the 8-bromo 1bk, the 8-methyl ester 1bn, the carboxylate 1bo, the 8-methyl alcohol 1bp, the 8-methyl amine 1bq, and the 8-nitrile 1br derivatives exhibited no inhibitory activity at concentrations as high as 30,000 nM. Moreover, the N-methyl amide 1bl and the N,N-dimethyl amide 1bm provide no activity against the CD38 enzyme. Only the primary sulfonamide 1bs exhibited any inhibition, but was over 10-fold less potent that its congener 1q. Clearly, there is something special about the binding interaction of the carboxamide with CD38. During the chemical exploration of this series, co-crystal structures of quinoline carboxamides 1a and 1bf bound to human CD38 and ADPR or 5-phosphoribose, respectively, were obtained. Depictions of the binding complex of CD38 and these compounds are illustrated in Figures 1 and 2. These structures provide insight into the catalytic motif required for the enzyme’s recognition of CD38 substrates as well as reveal the binding modes of this class of CD38 inhibitors. As illustrated in both structures, the nitrogen of the carboxamide groups hydrogen bonds to the carboxylate side chains of both 146Glu and 155Asp. Furthermore, in each compound, the carboxamide is co-planar with the quinoline ring facilitating an internal hydrogen bond between the protonated basic quinoline nitrogen and a lone pair of the carbonyl oxygen of the carboxamide. Additionally, the quinoline ring forms a π-π face to face stacking interaction with the indole ring of 189Trp, providing increased binding affinity for the inhibitor. The coplanar 8-carboxamide group facilitates this interaction, withdrawing electron density from the quinoline ring and improving the interaction with the π-cloud of the indole ring. Moreover, the hydrogen of the 4-benzyl amine provides for a hydrogen bond to the side chain hydroxyl oxygen of 221Thr. The ortho substituents of the 4-benzyl ring induces and stabilizes a near perpendicular conformation of the phenyl ring and biases the ligand conformation resulting in a suitable binding affinity for CD38. Additionally, this conformation allows for the formation of a triple π- π -π stacking interaction between the 4-benzyl ring, the adenine ring of the substrate, and the indole ring of 176Trp. This conformation of the inhibitor also provides better hydrophobic interaction between the ortho- and metasubstituents of the 4-benzyl ring (e.g., 2,3-dichloro or 2-methyl moieties) and the lipophilic side chain of 190Lys. As observed in the 1bf structure, the small 6-fluoro group is easily accommodated in an equally small region between the ligand and the ribose substrate. Larger groups such as 6-phenyl and 6-bromo substituents cannot bind into this small region, thus explaining the poor activity of compounds containing these groups. Thus, these human CD38 co-


16

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crystal structures bound with ligand and substrate not only provide protein structural insight into the recognition, affinity, and catalytic mechanism of the enzyme, but help rationalize much of the SAR observed in the quinoline carboxamide inhibitor series.

Figure 1. Ligand binding domain of the X-ray co-crystal structure of 1a complexed with CD38 E226Q and ADPR. The CD38 carbons are colored green with inhibitor 1a carbons colored cyan and ADPR carbons colored magenta. Hydrogen bonds are depicted as yellow dashed lines. The coordinates have been deposited in the Brookhaven Protein Data Bank (PDB code 4XJT). This figure was generated using PyMOL version 1.7.4.0 (The PyMOL Molecular Graphics System, Version 1.7.4 Schrodinger, LLC).

In addition to evaluating the pharmacokinetic parameters of the HTS hit 1a, several of the more potent analogs also had their pharmacokinetic and physiochemical properties determined. These results are summarized in Table 2. The artificial membrane permeabilities of the analogs, providing similar information as Madin-Darby canine kidney (MDCK) cell permeabilities, were moderate to high, ranging from 80 to 780 nm/sec.21 In contrast and with the exception of the HTS hit 1a (FaSSIF = 0.63 mg/mL), the aqueous solubilities in fasted state-simulated intestinal fluid (FaSSIF) were low, ranging from 0.001-0.095 mg/mL.22 In general, these compounds displayed modest potency at inhibiting the cytochrome p450 3A4 enzyme, with pIC50’s ranging from 4.9 to 5.5, providing a small therapeutic window relative to the compound’s modest CD38 inhibitory potency. Furthermore, select inhibitors exhibited similar activity to that against CD38 in a human ether-a-go-go-related gene ion channel (hERG) binding assay, limiting their development potential.23 The in vitro half-lives of the analogs in murine S9 liver slices did not correlate with their in vivo parameters in mice, precluding useful predictions. However, all but two inhibitors exhibited clearances of less than one third of hepatic blood flow (Cl = 3.5 to 29 mL/min/kg) and had high steady state volumes of distribution (VSS = 3.1 to 19 L/kg), which lead to acceptable terminal half-lives in mice. Oral


17


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Page 18 of 107

bioavailabilities ranged from 17 to 64%. Several of the submicromolar CD38 analogs, including 1q, 1ah, 1ai, and 1am, provided oral dose normalized area under the curve exposures greater than 250 ng/hr/mL, making them acceptable for in vivo studies.

Figure 2. Ligand binding domain of the X-ray co-crystal structure of 1bf complexed with wild type CD38 and the oxonium remnant of (2S,3R,4R)-2-deoxy-2-fluoro-D-ribose 5-phosphate (FR5P). The CD38 carbons are colored green with inhibitor 1bf carbons colored blue and FR5P colored magenta. Hydrogen bonds are depicted as yellow dashed lines. The coordinates have been deposited in the Brookhaven Protein Data Bank (PDB code 4XJS). This figure was generated using PyMOL version 1.7.4.0 (The PyMOL Molecular Graphics System, Version 1.7.4 Schrodinger, LLC).

Two of these inhibitors 1ah (mouse CD38 IC50 = 115 ± 50 nM) and 1ai (mouse CD38 IC50 = 46 ± 28 nM) were selected as tool compounds to explore the pharmacodynamics of this class of CD38 inhibitor. Similar to GSK’s 6thiazole quinoline analogs, these inhibitors are more potent versus the mouse CD38 enzyme compared to the human version.18 Diet-induced obese (DIO) mice (60% high fat diet), which exhibit a deficit of NAD levels relative to lean controls, were dosed with inhibitor 1ah or 1ai (30 mg/kg) and changes in NAD levels were measured in liver and gastrocnemius muscle at two and six hours post dose and compared to control mice. As illustrated in Table 3, NAD levels were significantly increased at both the 2 and 6 hour time points for inhibitor 1ah in liver (1483% and 595% respectively) over control animals and for inhibitor 1ai in liver (515% and 224% respectively) over control animals. The NAD levels were also elevated in gastrocnemius muscle, although to a smaller extent for inhibitor 1ah (283% and 198% respectively, versus controls) and for inhibitor 1ai (219% and 200% respectively, versus controls) relative to liver. This study demonstrates that inhibition of CD38 in vivo with small molecule inhibitors elevates


18

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NAD tissue levels and these inhibitors could be utilized to explore the therapeutic potential of NAD enhancement in NAD deficient animal models of disease. Table 2. Measured physiochemical properties and mouse in vivo PK data.a Permb

FaSSIF

Cyp 3A4

Liver

hERG

t1/2

Cl

VSS

F

DNAUC

nm/sec

mg/mL

pIC50

t1/2 min

pIC50

h

mL/min/kg

L/kg

%

ng/hr/mL

1a

780

0.630

5.0

44

6.3B

3.1

29

6.6

32

194

1m

320

0.010

4.9

113

---

4.2

16

4.6

18

196

1q

80

90

6.4Q

4.1

24

6.4

35

260

1ai

170

0.0014

---

44

6.3Q

18

3.5

4.9

17

768

1am

280

---

5.4

33

---

4.0

24

4.2

64

437

a

Mice were dosed i.v. (2.0 mg/kg) or p.o. (10 mg/kg)

b

Artificial Membrane Permeability Assay: A 1.8% lipid (phosphatidyl choline, egg) in 1% cholesterol decane solution was applied to a Millicell

96-well, 0.4um, PCF culture plate. 250 mL and 100 mL 50 mM phosphate buffer pH 7.4 with 0.5% encapsin was applied to the donor and receiver compartments, respectively. 2.5 mL of a 10 mM stock solution of compound in DMSO was added to the donor compartment. The assay was incubated at room temperature for 3 hours. Samples from both donor and receiver compartments were analyzed by HPLC with UV detection at 215 and 254 nm and permeability was calculated.

Table 3. NAD tissue levels versus controls after treatment with CD38 inhibitor 1ah or 1ai. Tissue

Liver

Gastrocnemius

Time

1ah NAD Level

1ah NAD STDEV

1ai NAD Level

1ai NAD STDEV

hr

%

n=4

%

n=4

2

1483

186

515

213

6

595

150

224

75

2

283

4

219

24

6

198

31

200

18

% control calculated based on area ratio of NAD+ to nicotinamide 1,N6-ethenoadenine dinucleotide (Internal Standard) of control animal tissue taken at same time point

Conclusion


19


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Page 20 of 107

In summary, starting from a single, weakly active high throughput screening hit, a series of quinoline carboxamide human CD38 inhibitors were iteratively prepared, by employing feedback from structure/activity relationships and observing the binding conformations of inhibitors in co-crystal structures with CD38. These efforts led to the identification of several submicromolar, orally bioavailable CD38 tool compounds. Two of the inhibitors 1ah and 1ai were utilized in vivo to demonstrate pharmacodynamic elevation of tissue NAD levels in a DIO mouse model. Similar to 4-(((1r,4r)-4-(2-methoxyethoxy)cyclohexyl)amino)-1-methyl-6-(thiazol-5-yl)quinolin-2(1H)-one,18 these chemical tools will allow for the exploration of the therapeutic consequences of NAD modulation in NAD deficient animal models of disease, potentially revealing human diseases that could be treated with CD38 modulation. Experimental Section All commercial chemicals and solvents were reagent grade and were used without further purification unless otherwise specified. The following solvents and reagents have been abbreviated: tetrahydrofuran (THF), diethyl ether (Et2O), dimethyl sulfoxide (DMSO), ethyl acetate (EtOAc), dichloromethane (CH2Cl2), trifluoroacetic acid (TFA), N,N-dimethylformamide (DMF), methanol (MeOH), dimethoxyethane (DME), N-methylpyrrolidine (NMP), acetonitrile (MeCN), chloroform (CHCl3), phosphorous oxychloride (POCl3), magnesium sulfate (MgSO4), triethylamine (Et3N), 2-propanol (iPrOH), diisopropylethylamine (iPr2NEt), sodium hydroxide (NaOH), tbutylmethylether (TBME), acetic acid (AcOH or HOAc), ethanol (EtOH), di-t-butyldicarbonate (BOC2O), sodium sulfate (Na2SO4), N,N-dimethylacetamide (DMA), sodium bicarbonate (NaHCO3), potassium carbonate (K2CO3), 1[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), azobisisobutyronitrile (AIBN), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), dithiothreitol (DTT). All reactions except those in aqueous media were carried out with the use of standard techniques for the exclusion of moisture. Reactions were monitored by thin-layer chromatography (TLC) on 0.25 mm silica gel plates (60F-254, E. Merck) and visualized with UV light, iodine, iodoplatinate, potassium permanganate, cerium molybdate, or 5% phosphomolybdic acid in 95% ethanol. Final compounds were typically purified either by flash chromatography on silica gel (E. Merck 40-63 mm), radial chromatography on a Chromatotron using prepared silica gel plates, on a Biotage Horizon or ISCO Combiflash pump and fraction collection system utilizing prepacked silica gel. Analytical purity was assessed either by reversed phase high-performance liquid chromatography (RP-HPLC) using an Agilent 1100 system equipped with a diode array spectrometer (λ range 190-400 nm) or by the LC-MS method detailed


20

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below. The stationary phase was a Keystone Scientific BDS Hypersil C-18 column (5 µm, 4.6 mm x 200 mm). The mobile phase employed 0.1% aqueous TFA with MeCN as the organic modifier and a flow rate of 1.0 mL/min. Analytical data are reported as retention time (tR) in minutes and % purity. All compounds were found to be ≥ 95% pure unless otherwise indicated. Analytical data are reported as retention time (tR) in minutes and % purity. 1H NMR spectra were recorded on either a Varian UnityPlus-400 MHz or a Bruker Avance III 400 MHz NMR spectrometer. Chemical shifts are reported in parts per million (ppm, δ units). Coupling constants are reported in units of hertz (Hz). Splitting patterns are designated as s, singlet; d, doublet; t, triplet; q, quartet; p, pentet; h, hextet; m, multiplet; or br, broad. Low-resolution mass spectra (MS) were recorded on a Waters SQD. The UPLC analysis was conducted utilizing a Phenomenex Kinetex 1.7 µm 2.1 x 50mm XB-C18 column at 40 °C. The gradient employed was: mobile phase A: water + 0.2% v/v formic acid mobile phase B: MeCN + 0.15% v/v FA. Highresolution MS were recorded on a Waters (Micromass) LCT time-of-flight mass spectrometer. Low resolution mass spectra were obtained under electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), or fast atom bombardment (FAB) methods. Combustion analyses were performed by Robertson Microlit Laboratories, Inc. (Ledgewood, NJ). 8-Bromo-N-[(2,6-dimethylphenyl)methyl]-2-methyl-4-quinolinamine (3a)

A solution of N,N-dimethylformamide (80 mL) containing 8-bromo-4-chloro-2-methylquinoline 2a (5.02 g, 19.6 mmol), 2,6-dimethylbenzylamine (5.29 g, 39.1 mmol) and potassium carbonate (5.41 g, 39.1 mmol) was heated to 160 ºC in a sealed pressure vessel for 24 hours. The dark solution was dissolved in ethyl acetate and then washed with water (2×). The solvent was removed in vacuo, and the residual slurry was triturated with diethyl ether. The precipitated solid was collected via vacuum filtration to give 8-bromo-N-[(2,6-dimethylphenyl)methyl]-2-methyl-4quinolinamine 3a (3.14 g, 8.84 mmol, 45% yield, 80% pure by NMR) as a white solid. 1H NMR (400 MHz, CD3SOCD3) δ: 8.32 (d, 1H, J = 8 Hz), 7.92 (d, 1H, J = 7 Hz), 7.19-7.12 (m, 2H), 7.11-7.06 (m, 2H), 7.01-6.95 (m, 1H), 6.63 (s, 1H), 4.36 (d, 2H, J = 4 Hz), 2.55 (s, 3H), 2.33 (s, 6H); LC-MS (ES API) M+H = 355.


21


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Page 22 of 107

4-{[(2,6-Dimethylphenyl)methyl]amino}-2-methyl-8-quinolinecarboxamide (1a)

Palladium acetate (382 mg, 1.70 mmol) was added to 8-bromo-N-[(2,6-dimethylphenyl)methyl]-2-methyl-4quinolinamine 3a (3.02 g, 8.50 mmol), 1,3-bis(diphenylphosphino)propane (1.05 g, 2.55 mmol), N,Ndiisopropylethylamine (2.42 g, 18.7 mmol), and hexamethyldisilizane (9.60 g, 59.5 mmol) in N,Ndimethylformamide (70 mL) and the reaction mixture was fitted with a carbon monoxide balloon and evacuated and purged with carbon monoxide (3×). The reaction was then heated to 110 ºC overnight. The solution was allowed to cool and then the organics were dissolved in ethyl acetate. The organics were washed with 1M sodium hydroxide and water (2×), dried over magnesium sulfate, and filtered. The solvent was removed in vacuo and the residual dark oil was purified by silica gel chromatography, eluting with ethyl acetate:dichloromethane (2:3 to 9:1) to give an orange solid, which was further purified by reverse phase high performance liquid chromatography, eluting with acetonitrile:water with 0.05% trifluoroacetic acid (5:95:100:0) to give 4-{[(2,6-dimethylphenyl)methyl]amino}-2methyl-8-quinolinecarboxamide 1a (1.13 g, 3.54 mmol, 42% yield) as a light yellow solid. 1H NMR (400 MHz, CD3SOCD3) δ 11.21 (d, 1H, J = 4 Hz), 8.58-8.41 (m, 2H), 7.69 (d, 1H, J = 4 Hz), 7.38 (t, 1H J = 8 Hz), 7.19-7.12 (m, 2H), 7.12-7.06 (m, 2H), 6.66 (s, 1H), 4.39 (d, 2H, J = 4 Hz), 2.58 (s, 3H), 2.34 (s, 6H); HRMS: C20H21N3O requires M+H at m/z 320.1763; found, 320.1759; tR = 0.66 minutes, 97.7% purity. 4-Amino-2-methyl-8-quinolinecarboxamide (1b)

2-Methyl-4-({[4-(methyloxy)phenyl]methyl}amino)-8-quinolinecarboxamide 1l (0.16 g, 0.498 mmol) was dissolved in trifluoroacetic acid (1 mL) and stirred at room temperature overnight. The reaction was then concentrated under reduced pressure, the residue was purified by silica gel chromatography, eluting with methanol:dichloromethane


22

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(1:49 to 1:9) to give 4-amino-2-methyl-8-quinolinecarboxamide 1b (93 mg, 0.448 mmol, 90% yield) as a yellow solid. 1H NMR (400 MHz, CD3OD) δ 8.50 (d, 1H, J = 8 Hz), 8.46 (d, 1H, J = 8 Hz), 7.72 (t, 1H, J = 8 Hz), 6.74 (s, 1H), 2.70 (s, 3H); HRMS: C11H11N3O requires M+H at m/z 202.0980; found, 202.0971; tR = 0.37 minutes, 97.0% purity. N-Benzyl-8-bromo-2-methylquinolin-4-amine (3b)

Benzylamine (627 mg, 5.85 mmol) was added to a solution of 8-bromo-4-chloro-2-methylquinoline 2a (1.50 g, 5.85 mmol) in N, N-dimethylacetamide (10 mL) under nitrogen at room temperature. Then, potassium carbonate (1.616 g, 11.69 mmol) was added and the resulting mixture was heated in a microwave at 160 ºC for 5 hours. After cooling, the reaction mixture was filtered. Then, methanol was added and after stirring, the solid was collected by filtration to give N-benzyl-8-bromo-2-methylquinolin-4-amine 3b (850 mg, 2.60 mmol, 44% yield). LC-MS (ES API) M+H = 328. 4-(Benzylamino)-2-methylquinoline-8-carbonitrile (4a)

Copper(I) cyanide (465 mg, 5.20 mmol) was added to N-benzyl-8-bromo-2-methylquinolin-4-amine 3b (850 mg, 2.60 mmol) in N,N-dimethylacetamide (5 mL) and the reaction mixture was heated in a microwave at 160 ºC for 5 hours. After cooling, the reaction, mixture was diluted with methanol and the solid collected by filtration. The solid was purified by preparative thin layer chromatography (2:1 petroleum ether:ethyl acetate) to give 4-(benzylamino)2-methylquinoline-8-carbonitrile 4a (560 mg, 2.049 mmol, 79% yield). LC-MS (LC-ES) M+H = 274.


23


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Page 24 of 107

4-(Benzylamino)-2-methylquinoline-8-carboxamide (1c)

Potassium hydroxide (230 mg, 4.10 mmol) was added to 4-(benzylamino)-2-methylquinoline-8-carbonitrile 4a (560 mg, 2.049 mmol) in dimethyl sulfoxide (15 mL) at 0° C. Then, 30% hydrogen peroxide (697 mg, 20.49 mmol) was added dropwise over 5 minutes. The reaction mixture was stirred at room temperature overnight, then diluted with water, and the solid collected by filtration to give 4-(benzylamino)-2-methylquinoline-8-carboxamide 1c (490 mg, 1.682 mmol, 82% yield). 1H NMR (400 MHz, CD3SOCD3) δ 11.13 (br d, 1H, J = 4 Hz), 8.52-8.42 (m, 2H), 8.98 (br t, 1H, J = 6 Hz), 7.66 (br d, 1H, = 4 Hz), 7.48 (t, 1H, J = 8 Hz), 7.38 (d, 2H, J = 7 Hz), 7.33 (t, 2H, J = 7 Hz), 7.24 (t, 1H, J = 7 Hz), 6.38 (s, 1H), 4.57 (d, 2H, J = 6 Hz), 2.43 (s, 3H); HRMS: C18H17N3O requires M+H at m/z 292.1450; found, 292.1442; tR = 0.97 minutes, 97.5% purity. 4-Hydroxy-2-methylquinoline-8-carbonitrile (6)

8-Bromo-2-methylquinolin-4-ol 5a (37.4 g, 157 mmol) and copper(I) cyanide (42.2 g, 471 mmol) were stirred in N,N-dimethylformamide (300 mL) at 140 °C for twelve hours. After cooling to room temperature, the solvent was removed in vacuo to afford 4-hydroxy-2-methylquinoline-8-carbonitrile 6 (31 g, 109 mmol, 70% yield). LC-MS (ES API) M+H = 185. 8-Cyano-2-methylquinolin-4-yl trifluoromethanesulfonate (7)


24

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To a solution of 4-hydroxy-2-methylquinoline-8-carbonitrile 6 (3 g, 16.29 mmol) and 2,6-lutidine (4.36 g, 40.7 mmol) in dichloromethane (50 mL) under nitrogen at -30 °C was added a solution of trifluoromethanesulfonic anhydride (5.50 mL, 32.6 mmol) in dichloromethane (50 mL) dropwise during 15 minutes. The reaction mixture was stirred at room temperature overnight. The organic phase was washed with water (100 mL), 5% citric acid solution (100 mL) and saturated brine (100 mL). After drying over sodium sulfate and filtering, the solvent was evaporated in vacuo to give the crude product as an off-white solid. Petroleum ether was added to the solid and it was filtered and dried to afford 8-cyano-2-methylquinolin-4-yl trifluoromethanesulfonate 7 (4.2 g, 13.28 mmol, 82% yield) as a brown solid. LC-MS (ES API) M+H = 317. 2-Methyl-4-(2-methylbenzylamino)quinoline-8-carbonitrile (4b)

o-Tolylmethanamine

(380

mg,

3.14

mmol)

was

added

to

of

8-cyano-2-methylquinolin-4-yl

trifluoromethanesulfonate 7 (316 mg, 0.999 mmol) in tetrahydrofuran (3 mL) and the reaction mixture was heated at 80 °C overnight. After cooling, the reaction mixture was extracted by ethyl acetate, washed with brine (3×), dried over magnesium sulfate, filtered, and concentrated. The residue was purified by thin layer chromatography (5:2 petroleum ether:tetrahydrofuran) to give 2-methyl-4-(2-methylbenzylamino)quinoline-8-carbonitrile 4b (105 mg, 0.365 mmol, 37% yield) as yellow solid. LC-MS (LC-ES) M+H = 288. 2-Methyl-4-(2-methylbenzylamino)quinoline-8-carboxamide (1d)


25


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Page 26 of 107

Sodium hydroxide (121 mg, 3.03 mmol) was added to a solution of 2-methyl-4-(2-methylbenzylamino)quinoline-8carbonitrile 4b (105 mg, 0.365 mmol) in dimethyl sulfoxide (12 mL) at room temperature. Then, 30% hydrogen peroxide (3 mL, 98 mmol) was added and the reaction mixture was stirred for 2 hours. Water (20 mL) was added, the reaction mixture was filtered, washed with water, and crystallized from tetrahydrofuran and diethyl ether. Then, the crude product was purified by reverse phase high performance liquid chromatography, eluting with acetonitrile:water with 0.05% trifluoroacetic acid to give 2-methyl-4-(2-methylbenzylamino)quinoline-8carboxamide 1d (52 mg, 0.168 mmol, 46% yield) as white solid. 1H NMR (400 MHz, CD3SOCD3) δ 11.14 (br d, 1H, J = 4 Hz), 8.49 (d, 2H, J = 8 Hz), 7.89 (br t, 1H, J = 5 Hz), 7.67 (br d, 1H, = 4 Hz), 7.48 (t, 1H, J = 8 Hz), 7.247.08 (m, 4H), 6.33 (s, 1H), 4.52 (d, 2H, J = 5 Hz), 2.43 (s, 3H), 2.37 (s, 3H); HRMS: C19H19N3O requires M+H at m/z 306.1606; found, 306.1607; tR = 1.79 minutes, 98.5% purity. 4-(2-Chlorobenzylamino)-2-methylquinoline-8-carbonitrile (4c)

A mixture of 8-cyano-2-methylquinolin-4-yl trifluoromethanesulfonate 7 (316 mg, 1.0 mmol) and (2chlorophenyl)methanamine (424 mg, 2.99 mmol) in tetrahydrofuran (5 mL) was stirred at 80 °C overnight. LCMS monitoring of the reaction, showed that some starting material remained. The mixture was extracted by ethyl acetate and washed with brine (3×). The organic layers were dried over magnesium sulfate, filtered, and the solvent removed under reduced pressure. The residue was purified by thin layer chromatography (5:2 petroleum ether:tetrahydrofuran) to give of 4-(2-Chlorobenzylamino)-2-methylquinoline-8-carbonitrile 4c (180 mg, 0.58 mmol, 58% yield) as yellow solid. LC-MS (ES API) M+H = 308.


26

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2-Methyl-4-(2-methylbenzylamino)quinoline-8-carboxamide (1e)

To a solution of 4-(2-chlorobenzylamino)-2-methylquinoline-8-carbonitrile 4c (180 mg, 0.58 mmol) in dimethyl sulfoxide (10 mL) was added sodium hydroxide (180 mg, 4.50 mmol) at room temperature. Then, 30% hydrogen peroxide (2 mL, 19.6 mmol) was added after 15 minutes. The mixture was stirred for 2 hours, water (20 mL) was added, and the mixture was filtered. The residue was washed with plenty of water and recrystallized from tetrahydrofuran and diethyl ether. Then, the crude product was purified by reverse phase high performance liquid chromatography, eluting with acetonitrile:water with 0.05% trifluoroacetic acid to give 2-methyl-4-(2methylbenzylamino)quinoline-8-carboxamide 1e (57 mg, 0.17 mmol, 29% yield) as white solid. 1H NMR (400 MHz, CD3SOCD3) δ 11.10 (br d, 1H, J = 4 Hz), 8.51 (d, 1H, J = 8 Hz), 8.47 (d, 1H, J = 8 Hz), 8.04 (br t, 1H, J = 5 Hz), 7.70 (br d, 1H, J = 4 Hz), 7.61-7.44 (m, 2H), 7.42-7.21 (m, 3H), 6.32 (s, 1H), 4.63 (d, 2H, J = 5 Hz), 2.45 (s, 3H); HRMS: C18H16ClN3O requires M+H at m/z 326.1060; found, 326.1056; tR = 1.81 minutes, 98.7% purity. 8-Bromo-N-(2-methoxybenzyl)-2-methylquinolin-4-amine (3c)

N,N-Di-iso-propylethylamine (0.313 mL, 1.795 mmol) was added to 8-bromo-4-chloro-2-methylquinoline 2a (0.1535 g, 0.598 mmol) in dimethyl sulfoxide (1.682 mL) at room temperature, followed by (2methoxyphenyl)methanamine (0.123 g, 0.898 mmol) and the solution was heated at 150 °C for sixty-four hours. The reaction mixture was poured into ether and methanol, washed with water, dried over magnesium sulfate, filtered, and concentrated.

The residue was purified by silica gel chromatography, eluting with ethyl

acetate:hexanes (2:3) to give 8-bromo-N-(2-methoxybenzyl)-2-methylquinolin-4-amine 3c (0.1035 g, 0.275 mmol,


27


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Page 28 of 107

46% yield). 1H NMR (400 MHz, CD3SOCD3) δ 8.25 (d, 1H, J = 8 Hz), 7.94 (d, 1H, J = 7 Hz), 7.74 (t, 1H, J = 6 Hz), 7.30-7.20 (m, 2H), 7.14 (d, 1H, J = 6 Hz), 7.03 (d, 1H, J = 8 Hz), 6.85 (t, 1H, J = 7 Hz), 6.27 (s, 1H), 4.47 (d, 2H, J = 6 Hz), 3.87 (s, 3H), 2.39 (s, 3H); LC-MS (LC-ES) M+H = 357. 4-((2-Methoxybenzyl)amino)-2-methylquinoline-8-carboxamide (1f)

1,1'-Bis(diphenylphosphino)ferrocene (0.032 g, 0.058 mmol) and bis(trimethylsilyl)amine (0.917 mL, 4.35 mmol) were added to 8-bromo-N-(2-methoxybenzyl)-2-methylquinolin-4-amine 3c (0.1035 g, 0.290 mmol) in N,Ndimethylformamide (1.980 mL) at room temperature, followed by tris(dibenzylideneacetone)dipalladium(0) (0.027 g, 0.029 mmol) and the solution was fitted with a carbon monoxide balloon and heated to 100 °C and stirred for two hours. The reaction mixture was cooled, poured into ether, washed with 1 N sodium hydroxide and water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with methanol:ethyl acetate (1:24) to give 4-((2-methoxybenzyl)amino)-2-methylquinoline-8-carboxamide 1f (0.0442 g, 0.131 mmol, 45% yield). 1H NMR (400 MHz, CD3SOCD3) δ 11.14 (br d, 1H, J = 5 Hz), 8.48 (dd, 1H, J = 7, 1 Hz), 8.45 (dd, 1H, J = 8, 1 Hz), 7.92 (br t, 1H, J = 6 Hz), 7.68 (d, 1H, J = 5 Hz), 7.47 (t, 1H, J = 8 Hz), 7.24 (dt, 1H, J = 8, 1 Hz), 7.15 (d, 1H, J = 8 Hz), 7.04 (t, 1H, J = 8 Hz), 6.86 (t, 1H, J = 8 Hz), 6.30 (s, 1H), 4.50 (d, 2H, J = 5 Hz), 3.88 (s, 3H), 2.43 (s, 3H); HRMS: C19H19N3O2 requires M+H at m/z 322.1555; found, 322.1551; tR = 0.50 minutes, 100.0% purity. 8-Bromo-2-methyl-N-(3-methylbenzyl)quinolin-4-amine (3d)


28

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N,N-Di-iso-propylethylamine (0.314 mL, 1.804 mmol) was added to 8-bromo-4-chloro-2-methylquinoline 2a (0.1543 g, 0.601 mmol) in dimethyl sulfoxide (1.691 mL) at room temperature, followed by m-tolylmethanamine (0.109 g, 0.902 mmol) and the solution was heated at 150 °C for sixty-four hours. The reaction mixture was poured into ether and methanol, washed with water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (1:2) to give 8-bromo-2-methyl-N-(3methylbenzyl)quinolin-4-amine 3d (0.1398 g, 0.389 mmol, 65% yield). 1H NMR (400 MHz, CD3SOCD3) δ 8.25 (d, 1H, J = 8 Hz), 7.94 (d, 1H, J = 7 Hz), 7.88 (br t, 1H, J = 6 Hz), 7.26 (t, 1H, J = 8 Hz), 7.24-7.12 (m, 3H), 7.04 (d, 1H, J = 8 Hz), 6.34 (s, 1H), 4.50 (d, 2H, J = 6 Hz), 2.39 (s, 3H), 2.26 (s, 3H); LC-MS (LC-ES) M+H = 342. 2-Methyl-4-((3-methylbenzyl)amino)quinoline-8-carboxamide (1g)

1,1'-Bis(diphenylphosphino)ferrocene (0.045 g, 0.082 mmol) and bis(trimethylsilyl)amine (1.296 mL, 6.15 mmol) were added to 8-bromo-2-methyl-N-(3-methylbenzyl)quinolin-4-amine 3d (0.1398 g, 0.410 mmol) in N,Ndimethylformamide (2.80 mL) at room temperature, followed by tris(dibenzylideneacetone)dipalladium(0) (0.038 g, 0.041 mmol) and the solution was fitted with a carbon monoxide balloon and heated to 100 °C and stirred for four hours. The reaction mixture was cooled, poured into ether, washed with 1 N sodium hydroxide and water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with methanol:ethyl acetate (1:49) to give 2-methyl-4-((3-methylbenzyl)amino)quinoline-8-carboxamide 1g (0.0459 g, 0.143 mmol, 35% yield). 1H NMR (400 MHz, CD3SOCD3) δ 11.12 (br d, 1H, J = 5 Hz), 8.48 (d, 1H, J = 7 Hz), 8.45 (d, 1H, J = 8 Hz), 8.05 (br t, 1H, J = 6 Hz), 7.67 (d, 1H, J = 5 Hz), 7.48 (t, 1H, J = 8 Hz), 7.24-7.14 (m, 3H), 7.05 (d, 1H, J = 7 Hz), 6.37 (s, 1H), 4.52 (d, 2H, J = 6 Hz), 2.43 (s, 3H), 2.27 (s, 3H); HRMS: C19H19N3O requires M+H at m/z 306.1606; found, 306.1603; tR = 0.50 minutes, 99.0% purity. 8-Bromo-N-[(3-chlorophenyl)methyl]-2-methyl-4-quinolinamine (3e)


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Page 30 of 107

N,N-Diisopropylethylamine (1.038 mL, 5.94 mmol) was added to 8-bromo-4-chloro-2-methylquinoline 2a (0.5081 g, 1.981 mmol) in dimethyl sulfoxide (5.20 mL) at room temperature, followed by 3-chlorobenzylamine (0.363 mL, 2.97 mmol) and the solution was heated to 140 ºC and stirred for sixty-eight hours. The reaction mixture was cooled, poured into ether, washed with water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (1:3) to give 8-bromo-N-[(3chlorophenyl)methyl]-2-methyl-4-quinolinamine 3e (0.5460 g, 1.434 mmol, 72% yield).

1

H NMR (400 MHz,

CD3SOCD3) δ 8.24 (d, 1H, J = 8 Hz), 7.96 (d, 1H, J = 7 Hz), 7.91 (br t, 1H, J = 6 Hz), 7.42 (s, 1H), 7.39-7.24 (m, 4H), 6.37 (s, 1H), 4.56 (d, 2H, J = 6 Hz), 2.40 (s, 3H); LC-MS (LC-ES) M+H = 360. 4-{[(3-Chlorophenyl)methyl]amino}-2-methyl-8-quinolinecarboxamide

N,N-Diisopropylethylamine (0.221 mL, 1.264 mmol) and hexamethyldisilazane (0.843 mL, 4.02 mmol) were added to 8-bromo-N-[(3-chlorophenyl)methyl]-2-methyl-4-quinolinamine 3e (0.2078 g, 0.575 mmol) in N,Ndimethylformamide (4.68 mL) at room temperature, followed by 1,3-bis(diphenylphosphino)propane (0.071 g, 0.172 mmol) and palladium(II) acetate (0.026 g, 0.115 mmol) and the solution fitted with a carbon monoxide balloon and heated to 110 °C and stirred for four hours. The reaction mixture was cooled, poured into ether, washed with 1 N sodium hydroxide and water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with methanol:ethyl acetate (1:19) to give 4-{[(3-chlorophenyl)methyl]amino}-2methyl-8-quinolinecarboxamide 1h (0.0509 g, 0.148 mmol, 26% yield). 1H NMR (400 MHz, CD3SOCD3) δ 11.09 (br d, 1H, J = 4 Hz), 8.49 (d, 1H, J = 7 Hz), 8.44 (d, 1H, J = 8 Hz), 8.08 (br t, 1H, J = 6 Hz), 7.68 (br d, 1H, J = 5


30

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Hz), 7.49 (t, 1H, J = 8 Hz), 7.44 (s, 1H), 7.40-7.28 (m, 3H), 6.40 (s, 1H), 4.58 (d, 2H, J = 6 Hz), 2.44 (s, 3H); HRMS: C18H16ClN3O requires M+H at m/z 326.1060; found, 326.1061; tR = 0.58 minutes, 100.0% purity. 8-Bromo-N-(3-methoxybenzyl)-2-methylquinolin-4-amine (3f)

N,N-Di-iso-propylethylamine (0.316 mL, 1.814 mmol) was added to 8-bromo-4-chloro-2-methylquinoline 2a (0.1551 g, 0.605 mmol) in dimethyl sulfoxide (1.699 mL) at room temperature, followed by (3methoxyphenyl)methanamine (0.124 g, 0.907 mmol) and the solution was heated at 150 °C for sixty-four hours. The reaction mixture was poured into diethyl ether and methanol, washed with water, dried over magnesium sulfate, filtered, and concentrated.


acetate:hexanes (2:3) to give 8-bromo-N-(3-methoxybenzyl)-2-methylquinolin-4-amine 3f (0.1213 g, 0.323 mmol, 53% yield).. 1H NMR (400 MHz, CD3SOCD3) δ 8.25 (d, 1H, J = 8 Hz), 7.95 (d, 1H, J = 7 Hz), 7.91 (br s, 1H), 7.27 (t, 1H, J = 8 Hz), 7.23 (t, 1H, J = 8 Hz), 6.95 (s, 1H), 6.94 (d, 1H, J = 8 Hz), 6.80 (d, 1H, J = 8 Hz), 6.36 (s, 1H), 4.51 (d, 2H, J = 6 Hz), 3.70 (s, 3H), 2.40 (s, 3H); LC-MS (LC-ES) M+H = 358. 4-((3-Methoxybenzyl)amino)-2-methylquinoline-8-carboxamide (1i)

1,1'-Bis(diphenylphosphino)ferrocene (0.019 g, 0.034 mmol) and bis(trimethylsilyl)amine (0.544 mL, 2.58 mmol) were added to 8-bromo-N-(3-methoxybenzyl)-2-methylquinolin-4-amine 3f (0.0614 g, 0.172 mmol) in N,Ndimethylformamide (1.175 mL) at room temperature, followed by tris(dibenzylideneacetone)dipalladium(0) (0.016 g, 0.017 mmol) and the solution was fitted with a carbon monoxide balloon and heated to 100 °C and stirred for four


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Page 32 of 107

hours. The reaction mixture was cooled, poured into ether, washed with 1 N sodium hydroxide and water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with methanol:ethyl acetate (1:49) to give 4-((3-methoxybenzyl)amino)-2-methylquinoline-8-carboxamide 1i (0.0341 g, 0.101 mmol, 59% yield). 1H NMR (400 MHz, CD3SOCD3) δ 11.12 (br d, 1H, J = 5 Hz), 8.48 (d, 1H, J = 7 Hz), 8.44 (d, 1H, J = 8 Hz), 8.06 (br t, 1H, J = 6 Hz), 7.67 (d, 1H, J = 5 Hz), 7.48 (t, 1H, J = 8 Hz), 7.24 (t, 1H, J = 8 Hz), 6.96 (s, 1H), 6.95 (d, 1H, J = 6 Hz), 6.80 (dd, 1H, J = 7, 2 Hz), 6.38 (s, 1H), 4.53 (d, 2H, J = 6 Hz), 3.71 (s, 3H), 2.43 (s, 3H); HRMS: C19H19N3O2 requires M+H at m/z 322.1555; found, 322.1555; tR = 0.50 minutes, 100.0% purity. 8-Bromo-2-methyl-N-(4-methylbenzyl)quinolin-4-amine (3g)

N,N-Di-iso-propylethylamine (0.311 mL, 1.783 mmol) was added to 8-bromo-4-chloro-2-methylquinoline 2a (0.1525 g, 0.594 mmol) in dimethyl sulfoxide (1.671 mL) at room temperature, followed by p-tolylmethanamine (0.108 g, 0.892 mmol) and the solution was heated at 150 °C for eight hours. The reaction mixture was poured into ether and methanol, washed with water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (3:7) to give 8-bromo-2-methyl-N-(4methylbenzyl)quinolin-4-amine 3g (0.1371 g, 0.382 mmol, 64% yield). 1H NMR (400 MHz, CD3SOCD3) δ 8.24 (d, 1H, J = 8 Hz), 7.94 (d, 1H, J = 7 Hz), 7.88 (br t, 1H, J = 6 Hz), 7.26 (t, 1H, J = 8 Hz), 7.25 (d, 2H, J = 8 Hz), 7.12 (d, 2H, J = 8 Hz), 6.33 (s, 1H), 4.49 (d, 2H, J = 6 Hz), 2.38 (s, 3H), 2.25 (s, 3H); LC-MS (LC-ES) M+H = 342.

2-Methyl-4-((4-methylbenzyl)amino)quinoline-8-carboxamide (1j)


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1,1'-Bis(diphenylphosphino)ferrocene (0.045 g, 0.080 mmol) and bis(trimethylsilyl)amine (1.271 mL, 6.03 mmol) were added to 8-bromo-2-methyl-N-(4-methylbenzyl)quinolin-4-amine 3g (0.1371 g, 0.402 mmol) in N,Ndimethylformamide (2.75 mL) at room temperature, followed by tris(dibenzylideneacetone)dipalladium(0) (0.037 g, 0.040 mmol) and the solution was fitted with a carbon monoxide balloon and heated to 100 °C and stirred for four hours. The reaction mixture was cooled, poured into ether, washed with 1 N sodium hydroxide and water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with methanol:ethyl acetate (1:49) to give 2-methyl-4-((4-methylbenzyl)amino)quinoline-8-carboxamide 1j (0.0881 g, 0.274 mmol, 68% yield). 1H NMR (400 MHz, CD3SOCD3) δ 11.13 (br d, 1H, J = 5 Hz), 8.47 (d, 1H, J = 7 Hz), 8.44 (dd, 1H, J = 8, 1 Hz), 8.06 (br t, 1H, J = 6 Hz), 7.67 (d, 1H, J = 5 Hz), 7.47 (t, 1H, J = 8 Hz), 7.26 (d, 2H, J = 8 Hz), 7.13 (d, 2H, J = 8 Hz), 6.36 (s, 1H), 4.51 (d, 2H, J = 6 Hz), 2.42 (s, 3H), 2.25 (s, 3H); HRMS: C19H19N3O requires M+H at m/z 306.1606; found, 306.1605; tR = 0.53 minutes, 100.0% purity. 4-(4-Chlorobenzylamino)-2-methylquinoline-8-carbonitrile (4d)

(4-Chlorophenyl)methanamine

(340

mg,

2.401

mmol)

was

added

to


trifluoromethanesulfonate 7 (320 mg, 1.012 mmol) in dimethyl sulfoxide (2 mL) and the reaction mixture was heated at 80 °C overnight. The mixture was extracted by ethyl acetate, washed with brine (3×), dried over magnesium sulfate, filtered, and concentrated. The residue was purified by preparative thin layer chromatography


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(5:2 petroleum ether:tetrahydrofuran) to give 4-(4-chlorobenzylamino)-2-methylquinoline-8-carbonitrile 4d (110 mg, 0.357 mmol, 35% yield) as a yellow solid. LC-MS (LC-ES) M+H = 308. 4-(4-Chlorobenzylamino)-2-methylquinoline-8-carboxamide (1k)

Sodium hydroxide (167 mg, 4.18 mmol) was added to a solution of 4-(4-chlorobenzylamino)-2-methylquinoline-8carbonitrile 4d (110 mg, 0.357 mmol) in dimethyl sulfoxide (10 mL) at room temperature. Then, 30% hydrogen peroxide (2 mL, 19.58 mmol) was added and the reaction mixture was stirred for 2 hours. Water (20 mL) was added, and the mixture was filtered. The residue was washed with water and crystallized from tetrahydrofuran and diethyl ether. Then, the crude product was purified by reverse phase high performance liquid chromatography, eluting with acetonitrile:water with 0.05% trifluoroacetic acid to give 4-(4-chlorobenzylamino)-2-methylquinoline-8carboxamide 1k (18 mg, 0.055 mmol, 15% yield) as a white solid. 1H NMR (400 MHz, CD3SOCD3) δ 11.10 (br d, 1H, J = 4 Hz), 8.48 (d, 1H, J = 7 Hz), 8.43 (d, 1H, J = 8 Hz), 8.08 (br t, 1H, J = 6 Hz), 7.67 (br d, 1H, J = 4 Hz), 7.48 (t, 1H, J = 8 Hz), 7.46-7.34 (m, 4H), 6.36 (s, 1H), 4.56 (d, 2H, J = 6 Hz), 2.43 (s, 3H); HRMS: C18H16ClN3O requires M+H at m/z 326.1060; found, 326.1060; tR = 1.81 minutes, 98.9% purity. 8-Bromo-N-(4-methoxybenzyl)-2-methylquinolin-4-amine (3h)

N,N-Di-iso-propylethylamine (0.311 mL, 1.783 mmol) was added to 8-bromo-4-chloro-2-methylquinoline 2a (0.1525 g, 0.594 mmol) in dimethyl sulfoxide (1.671 mL) at room temperature, followed by (4-


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methoxyphenyl)methanamine (0.122 g, 0.892 mmol) and the solution was heated at 150 °C for seven hours. The reaction mixture was poured into ether and methanol, washed with water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (2:3) to give 8-bromo-N-(4-methoxybenzyl)-2-methylquinolin-4-amine 3h (0.1213 g, 0.323 mmol, 54% yield).

1

H NMR

(400 MHz, CD3SOCD3) δ 8.25 (d, 1H, J = 8 Hz), 7.94 (d, 1H, J = 7 Hz), 7.86 (br s, 1H), 7.30 (d, 2H, J = 9 Hz), 7.25 (t, 1H, J = 8 Hz), 6.88 (d, 2H, J = 9 Hz), 6.37 (s, 1H), 4.46 (d, 2H, J = 6 Hz), 3.70 (s, 3H), 2.40 (s, 3H); LC-MS (LCES) M+H = 358. 4-((4-Methoxybenzyl)amino)-2-methylquinoline-8-carboxamide (1l)

1,1'-Bis(diphenylphosphino)ferrocene (0.018 g, 0.032 mmol) and bis(trimethylsilyl)amine (0.509 mL, 2.414 mmol) were added to 8-bromo-N-(4-methoxybenzyl)-2-methylquinolin-4-amine 3h (0.0575 g, 0.161 mmol) in N,Ndimethylformamide (1.100 mL) at room temperature, followed by tris(dibenzylideneacetone)dipalladium(0) (0.015 g, 0.016 mmol) and the solution was fitted with a carbon monoxide balloon and heated to 100 °C and stirred for two hours. The reaction mixture was cooled, poured into ether, washed with 1 N sodium hydroxide and water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with methanol:ethyl acetate (1:24) to give 4-((4-methoxybenzyl)amino)-2-methylquinoline-8-carboxamide 1l (0.0439 g, 0.130 mmol, 81% yield). 1H NMR (400 MHz, CD3SOCD3) δ 11.13 (br d, 1H, J = 5 Hz), 8.47 (d, 1H, J = 7 Hz), 8.43 (d, 1H, J = 8 Hz), 8.02 (br t, 1H, J = 6 Hz), 7.67 (d, 1H, J = 5 Hz), 7.47 (t, 1H, J = 8 Hz), 7.31 (d, 2H, J = 9 Hz), 6.89 (d, 2H, J = 9 Hz), 6.39 (s, 1H), 4.48 (d, 2H, J = 6 Hz), 3.71 (s, 3H), 2.43 (s, 3H); HRMS: C19H19N3O2 requires M+H at m/z 322.1555; found, 322.1560; tR = 0.50 minutes, 98.3% purity. 2-Methyl-4-(2-(trifluoromethyl)benzylamino)quinoline-8-carbonitrile (4e)


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A solution of 8-cyano-2-methylquinolin-4-yl trifluoromethanesulfonate 7 (300 mg, 0.95 mmol) and (2(trifluoromethyl)phenyl)methanamine (332 mg, 1.90 mmol) in dimethyl sulfoxide (5 mL) was stirred under nitrogen at 90°C for 24 hours, cooled, and poured into water. The organics were extracted with ethyl acetate, washed with brine, concentrated, and purified by preparative thin layer chromatography (2:1 petroleum ether:ethyl acetate) to afford 2-methyl-4-(2-(trifluoromethyl)benzylamino)quinoline-8-carbonitrile 4e (190 mg, 0.56 mmol, 59% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ: 8.02 (br s, 1H), 8.01 (d, 1H, J = 8 Hz), 7.76 (d, 1H, J = 8 Hz), 7.56-7.50 (m, 2H), 7.48-7.40 (m, 2H), 6.36 (s, 1H), 4.78 (d, 2H, J = 5 Hz), 2.62 (s, 3H). 2-Methyl-4-(2-(trifluoromethyl)benzylamino)quinoline-8-carboxamide

To a mixture of 2-methyl-4-(2-(trifluoromethyl)benzylamino)quinoline-8-carbonitrile 4e (160 mg, 0.47 mmol) and sodium hydroxide (56.2 mg, 1.41 mmol) in dimethyl sulfoxide was added 30% hydrogen peroxide (0.14 mL, 4.69 mmol) dropwise. The reaction was then stirred at room temperature overnight. The reaction mixture was poured into water and filtered to give 2-methyl-4-(2-(trifluoromethyl)benzylamino)quinoline-8-carboxamide 1m (45 mg, 0.12 mmol, 26% yield) as a white solid. 1H NMR (400 MHz, CD3SOCD3) δ 11.08 (br d, 1H, J = 5 Hz), 8.53 (dd, 1H, J = 8, 1 Hz), 8.49 (dd, 1H, J = 8, 1 Hz), 8.14 (br t, 1H, J = 6 Hz), 7.81 (d, 1H, J = 8 Hz), 7.72 (br d, 1H, J = 5 Hz), 7.62 (t, 1H, J = 7 Hz), 7.58-7.47 (m, 3H), 6.24 (s, 1H), 4.74 (d, 2H, J = 5 Hz), 2.43 (s, 3H); HRMS: C19H16F3N3O requires M+H at m/z 360.1323; found, 360.1316; tR = 1.66 minutes, 99.8% purity. 4-(2-Bromobenzylamino)-2-methylquinoline-8-carbonitrile (4f)


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To a solution of 8-cyano-2-methylquinolin-4-yl trifluoromethanesulfonate 7 (316 mg, 1.0 mmoL) in tetrahydrofuran (20 mL) stirred under nitrogen at room temperature was added (2-bromophenyl)methanamine (930 mg, 5.0 mmol) dropwise during 1 minute. The reaction mixture was stirred at 80 °C overnight. The reaction mixture was partitioned between water (50 mL) and filtered and the filtrate was dissolved in ethyl acetate and tetrahydrofuran, purified by preparative thin layer chromatography (1:1 petroleum ether:tetrahydrofuran) to obtain 4-(2-bromobenzylamino)-2methylquinoline-8-carbonitrile 4f (190 mg, 0.49 mmol, 49% yield) as white solid. LC-MS (ES API) M+H = 352. 4-(2-Bromobenzylamino)-2-methylquinoline-8-carboxamide (1n)

To a solution of 4-(2-bromobenzylamino)-2-methylquinoline-8-carbonitrile 4f (150 mg, 0.43 mmol) and sodium hydroxide (68.1 mg, 1.70 mmol) in dimethyl sulfoxide (20 mL) under nitrogen at 0 °C was added hydrogen peroxide (5 mL, 163 mmol) dropwise during 5 minutes. The reaction mixture was stirred at room temperature overnight. Water (50 mL) was added to the mixture and the solution filtered. The filtrate was washed with water twice and then dried in vacuo to obtain 4-(2-bromobenzylamino)-2-methylquinoline-8-carboxamide 1n (75 mg, 0.20 mmol, 47% yield) as white solid. 1H NMR (400 MHz, CD3SOCD3) δ: 11.11 (br d, 1H, J = 4 Hz), 8.52 (dd, 1H, J = 8, 1 Hz), 8.47 (d, 1H, J = 9 Hz), 8.06 (br t, 1H, J = 6 Hz), 7.80-7.60 (m, 2H), 7.52 (t, 1H, J = 8 Hz), 7.43-7.15 (m, 3H), 6.30 (s, 1H), 4.59 (d, 2H, J = 6 Hz), 2.45 (s, 3H); HRMS: C18H16BrN3O requires M+H at m/z 370.0555; found, 370.0550; tR = 1.66 minutes, 98.3% purity. 4-({[2,4-Bis(methyloxy)phenyl]methyl}amino)-2-methyl-8-quinolinecarbonitrile (4g)


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1-[2,4-Bis(methyloxy)phenyl]methanamine (9.91 g, 59.3 mmol) was added to 8-cyano-2-methyl-4-quinolinyl trifluoromethanesulfonate 7 (7.5 g, 23.71 mmol) in tetrahydrofuran (200 mL) under nitrogen and the reaction mixture was heated at 75 °C twelve hours. The reaction mixture was concentrated and the residue was purified by silica

gel

chromatography,

eluting

with

acetone:petroleum

ether

(2:1)

to

give

4-({[2,4-

bis(methyloxy)phenyl]methyl}amino)-2-methyl-8-quinolinecarbonitrile 4g (3.0 g, 9.00 mmol, 38% yield) as yellow solid. LC-MS (ES API) M+H = 334. 4-Amino-2-methyl-8-quinolinecarbonitrile (4h)

Trifluoroacetic

acid

(50

mL)

was

added

to

4-({[2,4-bis(methyloxy)phenyl]methyl}amino)-2-methyl-8-

quinolinecarbonitrile 4g (6.6g, 19.80 mmol) and the reaction mixture was stirred at for 1 hour, then concentrated. The residue was washed with diethyl ether (100 mL), filtered, washed with saturated sodium bicarbonate, and filtered to give 4-amino-2-methyl-8-quinolinecarbonitrile 4h (4.0 g, 19.65 mmol, 99% yield) as yellow solid. LCMS (ES API) M+H = 184. 4-(2-Cyanobenzylamino)-2-methylquinoline-8-carbonitrile (4i)


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2-(Bromomethyl)benzonitrile (1070 mg, 5.46 mmol) was added to 4-amino-2-methylquinoline-8-carbonitrile 4h (500 mg, 2.73 mmol) in tetrahydrofuran (15 mL), followed by potassium carbonate (754 mg, 5.46 mmol) and the reaction mixture was heated under nitrogen at 85 ºC for overnight. The reaction mixture was filtered to remove the solid, the solid was washed with tetrahydrofuran, and the filtrate was concentrated. The residue was purified by preparative thin layer chromatography (1:1 petroleum ether:tetrahydrofuran) to give 4-(2-cyanobenzylamino)-2methylquinoline-8-carbonitrile 4i (270 mg, 0.905 mmol, 33% yield). LC-MS (ES API) M+H = 299. 4-(2-Carbamoylbenzylamino)-2-methylquinoline-8-carboxamide (1o)

Potassium hydroxide (203 mg, 3.62 mmol) was added to 4-(2-cyanobenzylamino)-2-methylquinoline-8-carbonitrile 4i (270 mg, 0.905 mmol) in dimethyl sulfoxide (3 mL) at room temperature. Then, 30% hydrogen peroxide (3 mL, 98 mmol) was added dropwise. The reaction mixture was stirred for 2 hours. The reaction mixture was diluted with water, extracted with ethyl acetate (3×), washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by preparative high performance liquid chromatography, eluting with acetonitrile:10 mM ammonium bicarbonate in water to give 4-(2-carbamoylbenzylamino)-2-methylquinoline-8carboxamide 1o (58 mg, 0.171 mmol, 19% yield). 1H NMR (400 MHz, CD3SOCD3) δ 11.12 (br d, 1H, J = 5 Hz), 8.49 (dd, 1H, J = 7, 1 Hz), 8.40 (dd, 1H, J = 7, 1 Hz), 8.03 (br t, 1H, J = 6 Hz), 7.98 (br s, 1H), 7.68 (br d, 1H, J = 5 Hz), 7.57 (br s, 1H), 7.52-7.46 (m, 2H), 7.40-7.28 (m, 3H), 6.36 (s, 1H), 4.72 (d, 2H, J = 6 Hz), 2.42 (s, 3H); HRMS: C19H18N4O2 requires M+H at m/z 335.1508; found, 335.1511; tR = 1.33 minutes, 98.4% purity. 4-(2,3-Dimethylbenzylamino)-2-methylquinoline-8-carbonitrile (4j)


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(2,3-Dimethylphenyl)methanamine (176 mg, 1.299 mmol) was added to 8-cyano-2-methylquinolin-4-yl trifluoromethanesulfonate 7 (316 mg, 1 mmol) in tetrahydrofuran (6 mL) under nitrogen.

Then, potassium

carbonate (276 mg, 1.998 mmol) was added and the reaction mixture was heated at 80 °C overnight. Water (10 mL) was added and the solid was collected by filtration to give 4-(2,3-dimethylbenzylamino)-2-methylquinoline-8carbonitrile 4j (100 mg, 0.285 mmol, 29% yield). LC-MS (ES API) M+H = 302. (2,3-Dimethylbenzylamino)-2-methylquinoline-8-carboxamide trifluoroacetate (1p)

Sodium hydroxide (26.5 mg, 0.664 mmol) was added to 4-(2,3-dimethylbenzylamino)-2-methylquinoline-8carbonitrile 4j (100 mg, 0.332 mmol) in dimethyl sulfoxide (5mL). Then, 30% hydrogen peroxide (1.881 mL, 16.59 mmol) was added and the reaction mixture was allowed to stir overnight. The reaction mixture was diluted with water (20mL) and the resulting precipitant was collected by filtration. The residue was purified by reverse phase preparative high performance liquid chromatography, eluting with acetonitrile:water with 0.01% trifluoroacetic acid (5:95 to 95:5) to give (2,3-dimethylbenzylamino)-2-methylquinoline-8-carboxamide trifluoroacetate 1p (50 mg, 0.115 mmol, 35% yield). 1H NMR (400 MHz, CD3SOCD3) δ 14.21 (br s, 1H), 9.66 (br s, 1H), 8.83 (br s, 1H), 8.78 (d, 1H, J = 8 Hz), 8.49 (d, 1H, J = 7 Hz), 8.27 (s, 1H), 7.76 (br s, 1H), 7.11 (s, 1H), 7.04 (s, 2H), 6.77 (s, 1H), 4.73 (d, 2H, J = 4 Hz), 2.63 (s, 3H), 2.27 (s, 3H), 2.23 (s, 3H); HRMS: C20H21N3O requires M+H at m/z 320.1763; found, 320.1759; tR = 1.68 minutes, 96.2% purity. 4-{[(2,3-Dichlorophenyl)methyl]amino}-2-methyl-8-quinolinecarboxamide (1q)


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N,N-Diisopropylethylamine (0.491 mL, 2.81 mmol) and hexamethyldisilazane (1.873 mL, 8.94 mmol) were added to 8-bromo-N-[(2,3-dichlorophenyl)methyl]-2-methyl-4-quinolinamine 1bk (0.5057 g, 1.277 mmol) in N,Ndimethylformamide (4.02 mL) at room temperature, followed by 1,3-bis(diphenylphosphino)propane (0.158 g, 0.383 mmol) and palladium(II) acetate (0.057 g, 0.255 mmol) and the solution fitted with a carbon monoxide balloon and heated to 110 °C and stirred for four hours. The reaction mixture was cooled, poured into ether, washed with 1 N sodium hydroxide and water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate to give 4-{[(2,3-dichlorophenyl)methyl]amino}-2-methyl-8quinolinecarboxamide 1q (0.1894 g, 0.499 mmol, 39% yield). 1H NMR (400 MHz, CD3SOCD3) δ 11.09 (br d, 1H, J = 4 Hz), 8.50 (d, 1H, J = 7 Hz), 8.45 (d, 1H, J = 8 Hz), 8.04 (br t, 1H, J = 6 Hz), 7.69 (br d, 1H, J = 4 Hz), 7.57 (d, 1H, J = 8 Hz), 7.51 (d, 1H, J = 8 Hz), 7.31 (t, 1H, J = 8 Hz), 7.26 (d, 1H, J = 7 Hz), 6.33 (s, 1H), 4.65 (d, 2H, J = 6 Hz), 2.44 (s, 3H); HRMS: C18H15Cl2N3O requires M+H at m/z 360.0670; found, 360.0671; tR = 0.70 minutes, 100.0% purity. 4-(2,4-Dimethylbenzylamino)-2-methylquinoline-8-carbonitrile (4k)

(2,4-Dimethylphenyl)methanamine (270 mg, 1.998 mmol) was added to a solution of 8-cyano-2-methylquinolin-4yl trifluoromethanesulfonate 7 (316 mg, 0.999 mmol) in tetrahydrofuran (10 mL) under nitrogen at room temperature. Then, potassium carbonate (276 mg, 1.998 mmol) was added and the reaction mixture was heated at 75 °C overnight. The reaction mixture was filtered, washed with tetrahydrofuran (3×), and purified by preparative


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Page 42 of 107

thin layer chromatography (1:2 tetrahydrofuran:petroleum ether) to give 4-(2,4-dimethylbenzylamino)-2methylquinoline-8-carbonitrile 4k (140 mg, 0.465 mmol, 46% yield). LC-MS (ES API) M+H = 302. 4-(2,4-Dimethylbenzylamino)-2-methylquinoline-8-carboxamide trifluoroacetate (1r)

Sodium hydroxide (37.2 mg, 0.929 mmol) was added to a solution of 4-(2,4-dimethylbenzylamino)-2methylquinoline-8-carbonitrile 4k (140 mg, 0.465 mmol) in dimethyl sulfoxide (3 mL) under nitrogen at room temperature. Then, 30% hydrogen peroxide (3 mL, 98 mmol) was added and the reaction mixture was stirred for 1 hour. The reaction mixture was diluted with water, filtered, the solid was washed with water (3×), and the solid was dried under reduced pressure.

The residue was further purified by preparative high performance liquid

chromatography, eluting with acetonitrile:water with 0.01% trifluoroacetic acid (5:95 to 95:5) to give 4-(2,4dimethylbenzylamino)-2-methylquinoline-8-carboxamide trifluoroacetate 1r (65 mg, 0.145 mmol, 31% yield).

1

H

NMR (400 MHz, CD3SOCD3) δ 14.21 (br s, 1H), 9.63 (br s, 1H), 8.83 (br s, 1H), 8.78 (d, 1H, J = 8 Hz), 8.50 (d, 1H, J = 8 Hz), 8.27 (br s, 1H), 7.78 (t, 1H, J = 8 Hz), 7.08 (d, 1H, J = 8 Hz), 7.07 (s, 1H), 6.96 (d, 1H, J = 8 Hz), 6.77 (s, 1H), 4.69 (d, 2H, J = 5 Hz), 2.64 (s, 3H), 2.33 (s, 3H), 2.25 (s, 3H); HRMS: C20H21N3O requires M+H at m/z 320.1763; found, 320.1756; tR = 1.37 minutes, 96.8% purity. 4-(2,4-Dichlorobenzylamino)-2-methylquinoline-8-carbonitrile (4l)


42

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To a solution of 8-cyano-2-methylquinolin-4-yl trifluoromethanesulfonate 7 (316 mg, 1.0 mmol) and (2,4dichlorophenyl)methanamine (264 mg, 1.50 mmol) in tetrahydrofuran (5 mL) stirred under nitrogen at room temperature was added solid potassium carbonate (276 mg, 2.0 mmol). The reaction mixture was stirred at 80 °C for overnight. After cooling, the reaction was filtered and the filter cake was rinsed with tetrahydrofuran (3×). The solution was combined and concentrated and the residue was purified by preparative thin layer chromatography (5:2 petroleum ether: tetrahydrofuran) to give 4-(2,4-dichlorobenzylamino)-2-methylquinoline-8-carbonitrile 4l (200 mg, 0.58 mmol, 58% yield) as a yellow solid. LCMS (ES API) M+H = 342. 4-(2,4-Dichlorobenzylamino)-2-methylquinoline-8-carboxamide (1s)

To a solution of 4-(2,4-dichlorobenzylamino)-2-methylquinoline-8-carbonitrile 4l (200 mg, 0.58 mmol) in dimethyl sulfoxide (5 mL) was added solid potassium hydroxide (98 mg, 1.75 mmol) at room temperature. The reaction mixture was stirred at room temperature for 2 minutes at which time 30% hydrogen peroxide (3 mL) was added dropwise over 2 minutes. The resulting mixture was stirred at room temperature for 30 minutes, then water (50 mL) was added. The precipitate was collected, washed with water, and dried under a vacuum to give a white solid. The solid was purified by reverse phase high performance liquid chromatography, eluting with acetonitrile:water with 0.5% trifluoroacetic acid (5:95 to 95:5) to give 4-(2,4-dichlorobenzylamino)-2-methylquinoline-8-carboxamide trifluoroacetate 1s (22 mg, 0.046 mmol, 8% yield) as a white solid. 1H NMR (400 MHz, CD3SOCD3) δ: 11.08 (br d, 1H, J = 4 Hz), 8.51 (d, 1H, J = 7 Hz), 8.46 (d, 1H, J = 8 Hz), 8.23 (br s, 1H), 8.03 (br t, 1H, J = 5 Hz), 7.76-7.65 (m, 2H), 7.52 (t, 1H, J = 8 Hz), 7.42-7.36 (dd, 1H, J = 8, 2 Hz), 7.31 (d, 1H, J = 8 Hz), 6.32 (s, 1H), 4.60 (d, 2H, J = 5 Hz), 2.46 (s, 3H); HRMS: C18H15Cl2N3O requires M+H at m/z 360.0670; found, 360.0669; tR = 1.91 minutes, 100.0% purity. 8-Bromo-N-(2,5-dimethylbenzyl)-2-methylquinolin-4-amine (3i)


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(2,5-Dimethylphenyl)methanamine (283 mg, 2.096 mmol) was added to a solution of 8-bromo-4-chloro-2methylquinoline 2a (512 mg, 1.996 mmol) in N,N-dimethylacetamide (10 mL) under nitrogen at room temperature. Then, potassium carbonate (414 mg, 2.99 mmol) was added and the resulting mixture was heated in a microwave at 160 ºC for 5 hours. After cooling, the reaction mixture was concentrated. Then, ethanol (4 mL) was added and after stirring, the solid was collected by filtration to give 8-bromo-N-(2,5-dimethylbenzyl)-2-methylquinolin-4-amine 3i (150 mg, 0.422 mmol, 21% yield) as a white solid. LC-MS (ES API) M+H = 355. 4-(2,5-Dimethylbenzylamino)-2-methylquinoline-8-carbonitrile (4m)

Copper(I) cyanide (76 mg, 0.844 mmol) was added to 8-bromo-N-(2,5-dimethylbenzyl)-2-methylquinolin-4-amine 3i (150 mg, 0.422 mmol) in N,N-dimethylacetamide (5 mL) and the reaction mixture was heated in a microwave at 160 ºC for 5 hours. After cooling, the reaction, mixture was concentrated. The residue was purified by preparative thin layer chromatography (petroleum ether) to give 4-(2,5-dimethylbenzylamino)-2-methylquinoline-8-carbonitrile 4m (85 mg, 0.282 mmol, 67% yield). LC-MS (ES API) M+H = 302. 4-(2,5-Dimethylbenzylamino)-2-methylquinoline-8-carboxamide (1t)


44

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Sodium hydroxide (47.8 mg, 1.194 mmol) was added to 4-(2,5-dimethylbenzylamino)-2-methylquinoline-8carbonitrile 4m (120 mg, 0.398 mmol) in dimethyl sulfoxide (30 mL). Then, 30% hydrogen peroxide (5 mL) was added dropwise and the reaction mixture was stirred overnight. The reaction was diluted with water and the resulting solid was collected by filtration to give 4-(2,5-dimethylbenzylamino)-2-methylquinoline-8-carboxamide 1t (55 mg, 0.172 mmol, 43% yield) as a white solid. 1H NMR (400 MHz, CD3SOCD3) δ 11.16 (br d, 1H, J = 5 Hz), 8.49 (d, 1H, J = 7 Hz), 8.48 (s, 1H), 7.85 (br t, 1H, J = 5 Hz), 7.69 (br d, 1H, J = 5 Hz), 7.47 (t, 1H, J = 8 Hz), 7.09 (d, 1H, J = 8 Hz), 7.00 (s, 1H), 6.97 (d, 1H, J = 8 Hz), 6.34 (s, 1H), 4.47 (d, 2H, J = 5 Hz), 2.45 (s, 3H), 2.31 (s, 3H), 2.17 (s, 3H); HRMS: C20H21N3O requires M+H at m/z 320.1763; found, 320.1764; tR = 1.16 minutes, 98.2% purity. 4-(2,5-Dichlorobenzylamino)-2-methylquinoline-8-carbonitrile (4n)

A solution of 8-cyano-2-methylquinolin-4-yl trifluoromethanesulfonate 7 (300 mg, 0.95 mmol) and (2,5dichlorophenyl)methanamine (167 mg, 0.95 mmol) in dimethyl sulfoxide (5 mL) was stirred under nitrogen at 90 °C for 24 hours. The solution was cooled, poured into water and extracted with ethyl acetate. The organics were washed with brine, concentrated and purified by preparative thin layer chromatography (2:1 petroleum ether:ethyl acetate) to afford 4-(2,5-dichlorobenzylamino)-2-methylquinoline-8-carbonitrile 4n (150 mg, 0.44 mmol, 46% yield) as a white solid. LC-MS (ES API) M+H = 342. 2-Methyl-4-(2-methylbenzylamino)quinoline-8-carboxamide (1u)


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30% Hydrogen peroxide (0.25 mL, 8.18 mmol) added, dropwise, to a mixture of 4-(2,5-dichlorobenzylamino)-2methylquinoline-8-carbonitrile 4n (140 mg, 0.41 mmol) and sodium hydroxide (65.4 mg, 1.64 mmol) in dimethyl sulfoxide (5 mL). The reaction mixture was then stirred at room temperature overnight. The reaction mixture was poured into water and filtered to give 4-(2,5-dichlorobenzylamino)-2-methylquinoline-8-carboxamide 1u (58 mg, 0.15 mmol, 37% yield) as a white solid. 1H NMR (400 MHz, CD3SOCD3) δ 11.07 (d, 1H, J = 4 Hz), 8.52 (d, 1H, J = 7 Hz), 8.45 (d, 1H, J = 8 Hz), 8.00 (t, 1H, J = 6 Hz), 7.71 (d, 1H, J = 4 Hz), 7.60-7.49 (m, 2H), 7.41 (dd, 1H, J = 8, 2 Hz), 7.35 (d, 1H, J = 2 Hz), 6.36 (s, 1H), 4.61 (d, 2H, J = 6 Hz), 2.48 (s, 3H); HRMS: C18H15Cl2N3O requires M+H at m/z 360.0670; found, 360.0669; tR = 1.88 minutes, 95.0% purity. 4-(2,6-Dichlorobenzylamino)-2-methylquinoline-8-carbonitrile (4o)

To a solution of 8-cyano-2-methylquinolin-4-yl trifluoromethanesulfonate 7 (0.3 g, 0.95 mmol) in tetrahydrofuran (10 mL), was added 1-(2,6-dichlorophenyl)methanamine (0.50 g, 2.85 mmol). The reaction mixture was stirred at 80 °C for 2 hours. After cooling, water (20 mL) was added and the precipitate was filtered to give 4-(2,6dichlorobenzylamino)-2-methylquinoline-8-carbonitrile 4o (0.25 g, 0.67 mmol, 71% yield) as pale solid. LC-MS (ES API) M+H = 342. 4-(2,6-Dichlorobenzylamino)-2-methylquinoline-8-carboxamide

To a solution of 4-(2,6-dichlorobenzylamino)-2-methylquinoline-8-carbonitrile 4o (0.25 g, 0.73 mmol) in dimethyl sulfoxide (10 mL), was added sodium hydroxide (58 mg, 1.46 mmol), followed by 30% hydrogen peroxide (2.48 g,


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73.1 mmol) which was added dropwise. The reaction mixture was stirred at room temperature overnight. Water (20 mL) was added and the resulting precipitate was collected via filtration. 100 mg of crude product was obtained which was combined with another experiment. The solid was recrystallized from dimethyl sulfoxide and water to give 4-(2,6-dichlorobenzylamino)-2-methylquinoline-8-carboxamide 1v (55 mg, 0.15 mmol, 21% yield) as a white solid. 1H NMR (400 MHz, CD3SOCD3) δ 11.15 (br d, 1H, J = 5 Hz), 8.52-8.46 (m, 2H), 7.69 (br d, 1H, J = 5 Hz), 7.56 (d, 2H, J = 8 Hz), 7.48-7.37 (m, 3H), 6.67 (s, 1H), 4.64 (d, 2H, J = 4 Hz), 2.57 (s, 3H); HRMS: C18H15Cl2N3O requires M+H at m/z 360.0670; found, 360.0660; tR = 1.88 minutes, 98.5% purity. 4-(3,4-Dimethylbenzylamino)-2-methylquinoline-8-carbonitrile (4p)

(3,4-Dimethylphenyl)methanamine (270 mg, 1.998 mmol) was added to 8-cyano-2-methylquinolin-4-yl trifluoromethanesulfonate 7 (316 mg, 0.999 mmol) in tetrahydrofuran (10 mL) under nitrogen. Then, potassium carbonate (276 mg, 1.998 mmol) was added and the reaction mixture was heated at 75°C overnight. The reaction mixture was diluted with water and extracted with ethyl acetate (3×). The combined extracts were dried over sodium sulfate, filtered, and concentrated to give 4-(3,4-dimethylbenzylamino)-2-methylquinoline-8-carbonitrile 4p (245 mg, 0.813 mmol, 81% yield) as a white solid. LC-MS (ES API) M+H = 302. 4-(3,4-Dimethylbenzylamino)-2-methylquinoline-8-carboxamide (1w)


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Sodium hydroxide (130 mg, 3.25 mmol) was added to 4-(3,4-dimethylbenzylamino)-2-methylquinoline-8carbonitrile 4p (245 mg, 0.813 mmol) in dimethyl sulfoxide (3 mL) under nitrogen at room temperature. Then, 30% hydrogen peroxide (4 mL, 131 mmol) was added dropwise and the reaction mixture was stirred at room temp for 1 hour. The reaction mixture was diluted with water and filtered to obtain a solid. The solid was further purified by preparative high performance liquid chromatography, eluting with acetonitrile:0.01 M ammonium bicarbonate in water (5:95 to 95:5) to give 4-(3,4-dimethylbenzylamino)-2-methylquinoline-8-carboxamide 1w (50 mg, 0.157 mmol, 19% yield). 1H NMR (400 MHz, CD3SOCD3) δ 11.14 (br d, 1H, J = 4 Hz), 8.48 (d, 1H, J = 7 Hz), 8.44 (d, 1H, J = 9 Hz), 8.03 (br t, 1H, 6 Hz), 7.68 (br d, 1H, J = 4 Hz), 7.47 (t, 1H, J = 7 Hz), 7.15 (s, 1H), 7.98 (s, 2H), 6.36 (s, 1H), 4.48 (d, 2H, J = 6 Hz), 2.43 (s, 3H), 2.18 (s, 3H), 2.17 (s, 3H); HRMS: C20H21N3O requires M+H at m/z 320.1763; found, 320.1766; tR = 1.66 minutes, 100.0% purity. 4-(3,4-Dichlorobenzylamino)-2-methylquinoline-8-carbonitrile (4q)

Potassium carbonate (415 mg, 3.00 mmol) was added to a solution of 8-cyano-2-methylquinolin-4-yl trifluoromethanesulfonate 7 (316 mg, 1.000 mmol) in tetrahydrofuran (30 mL) at room temperature. Then, (3,4dichlorophenyl)methanamine (528 mg, 3.00 mmol) was added dropwise over 1 minute. The reaction mixture was heated at 80 °C overnight. The reaction mixture was diluted with water (50 mL), filtered, and the filtrate was purified by preparative thin layer chromatography (1:1 petroleum ether:tetrahydrofuran) to obtain 4-(3,4dichlorobenzylamino)-2-methylquinoline-8-carbonitrile 4q (129 mg, 0.297 mmol, 30% yield) as white solid. LCMS (ES API) M+H = 342. 4-(3,4-Dichlorobenzylamino)-2-methylquinoline-8-carboxamide hydrochloride (1x)


48

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Sodium hydroxide (60.3 mg, 1.508 mmol) was added to a solution of 4-(3,4-dichlorobenzylamino)-2methylquinoline-8-carbonitrile 4q (129 mg, 0.377 mmol) in dimethyl sulfoxide (25 mL) under nitrogen at room temperature. Then, 30% hydrogen peroxide (2 mL, 65.3 mmol) was added dropwise over 5 minutes. The reaction mixture was stirred overnight. The reaction mixture was diluted with water (50 mL), filtered, and the filtrate washed with water (2×). The filtrate was purified by reverse phase high performance liquid chromatography, eluting with acetonitrile:water with 0.1% hydrochloric acid (30:70 to 65:35) to give 4-(3,4-dichlorobenzylamino)-2methylquinoline-8-carboxamide hydrochloride 1x (82 mg, 0.225 mmol, 60% yield) as white solid.

1

H NMR (400

MHz, CD3SOCD3) δ 14.25 (br s, 1H), 10.20 (br s, 1H), 8.91 (br s, 1H), 8.86 (s, 1H), 8.51 (d, 1H, J = 6 Hz), 8.28 (s, 1H), 7.80-7.70 (m, 2H), 7.62 (d, 1H, J = 6 Hz), 7.46 (d, 1H, J = 6 Hz), 6.86 (s, 1H), 4.78 (s, 2H), 2.44 (s, 3H); HRMS: C18H15Cl2N3O requires M+H at m/z 360.0670; found, 360.0668; tR = 1.69 minutes, 98.9% purity. 8-Bromo-N-(3,5-dimethylbenzyl)-2-methylquinolin-4-amine (3j)

(3,5-Dimethylphenyl)methanamine (135 mg, 0.998 mmol) was added to a solution of 8-bromo-4-chloro-2methylquinoline 2a (256 mg, 0.998 mmol) in N,N-dimethylacetamide (3 mL) under nitrogen at room temperature. Then, potassium carbonate (414 mg, 2.99 mmol) was added and the resulting mixture was heated in a microwave at 150 ºC for 1 hour. After cooling, the reaction mixture was concentrated. The residue was purified by thin layer chromatography (2:1 petroleum ether:ethyl acetate) to give 8-bromo-N-(3,5-dimethylbenzyl)-2-methylquinolin-4amine 3j (120 mg, 0.338 mmol, 34% yield). LC-MS (ES API) M+H = 355.


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4-(3,5-Dimethylbenzylamino)-2-methylquinoline-8-carbonitrile (4r)

HN

N N

4r

Copper(I) cyanide (91 mg, 1.013 mmol) was added to 8-bromo-N-(3,5-dimethylbenzyl)-2-methylquinolin-4-amine 3j (120 mg, 0.338 mmol) in N,N-dimethylacetamide (2 mL) and the reaction mixture was heated in a microwave at 150 ºC for 1 hour. After cooling, the reaction, mixture was filtered and the solution was concentrated. The residue was purified by preparative thin layer chromatography (1:1 petroleum ether:ethyl acetate) to give 4-(3,5dimethylbenzylamino)-2-methylquinoline-8-carbonitrile 4r (100 mg, 0.332 mmol, 98% yield) as a yellow solid. LC-MS (ES API) M+H = 302. 4-(3,5-Dimethylbenzylamino)-2-methylquinoline-8-carboxamide (1y)

Potassium hydroxide (93 mg, 1.659 mmol) in water (5.0 mL) was added to a solution of 4-(3,5dimethylbenzylamino)-2-methylquinoline-8-carbonitrile 4r (100 mg, 0.332 mmol) in dimethyl sulfoxide (5 mL) under nitrogen at 0°C. Then, 30% hydrogen peroxide (135 mg) was added and the reaction was stirred overnight. Water (10mL) was added and the resulting precipitate was collected by filtration, washed with water (2×), and concentrated to give 4-(3,5-dimethylbenzylamino)-2-methylquinoline-8-carboxamide 1y (45 mg, 0.137 mmol, 41% yield) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 11.80 (s, 1H), 8.74 (d, 1H, J = 8 Hz), 7.86 (d, 1H, J = 8 Hz), 7.44 (t, 1H, J = 8 Hz), 7.02-6.96 (m, 3H), 6.40 (s, 1H), 6.03 (s, 1H), 5.27 (br s, 1H), 4.41 (d, 2H, J = 5 Hz), 2.59 (s, 3H), 2.32 (s, 6H); HRMS: C20H21N3O requires M+H at m/z 320.1763; found, 320.1757; tR = 1.69 minutes, 98.5% purity.


50

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4-(3,5-Dichlorobenzylamino)-2-methylquinoline-8-carbonitrile (4s)

(3,5-Dichlorophenyl)methanamine (350 mg, 1.988 mmol) was added to 8-cyano-2-methylquinolin-4-yl trifluoromethanesulfonate 7 (316 mg, 0.999 mmol) in tetrahydrofuran (5 mL) and the reaction mixture was heated at 75 °C overnight. The reaction mixture was filtered and concentrated. The residue was purified by preparative thin layer

chromatography

(5:2

petroleum

ether:tetrahydrofuran)

to

give

4-(3,5-dichlorobenzylamino)-2-

methylquinoline-8-carbonitrile 4s (306 mg, 0.152 mmol, 15% yield) as yellow solid. LC-MS (ES API) M+H = 342. 4-(3,5-Dichlorobenzylamino)-2-methylquinoline-8-carboxamide (1z)

Sodium hydroxide (220 mg, 5.50 mmol) was added to a solution of 4-(3,5-dichlorobenzylamino)-2-methylquinoline8-carbonitrile 4s (306 mg, 0.894 mmol) in dimethyl sulfoxide (15 mL) at room temperature. Then, 30% hydrogen peroxide (3 mL, 29.4 mmol) was added and the mixture was stirred for 2 hours. Water (20 mL) was added, and the mixture was filtered. The residue was washed with water. Then, the crude product was purified by reverse phase high performance liquid chromatography, eluting with acetonitrile:water with 0.5% trifluoroacetic acid (5:95 to 95:5) to give 4-(3,5-dichlorobenzylamino)-2-methylquinoline-8-carboxamide trifluoroacetate 1z (25 mg, 0.069 mmol, 8% yield) as white solid. 1H NMR (400 MHz, CD3OD) δ 8.62 (d, 1H, J = 7 Hz), 8.50 (d, 1H, J = 6 Hz), 7.82 (t, 1H, J = 7 Hz), 7.44 (s, 3H), 6.79 (s, 1H), 4.85 (s, 2H), 2.73 (s, 3H); HRMS: C18H15Cl2N3O requires M+H at m/z 360.0670; found, 360.0669; tR = 1.39 minutes, 100.0% purity. 4-(3-Chloro-2-fluorobenzylamino)-2-methylquinoline-8-carbonitrile (4t)


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Cl F HN

N N

4t

(3-Chloro-2-fluorophenyl)methanamine (303 mg, 1.9 mmol) was added to a solution of 8-cyano-2-methylquinolin4-yl trifluoromethanesulfonate 7 (300 mg, 0.949 mmol) in tetrahydrofuran (8 mL) and the reaction mixture was heated under nitrogen at 80 °C overnight. After cooling, water (20 mL) was added, leading to the formation of a precipitate. The solids were collected by filtration, washed with water, and concentrated to give 4-(3-chloro-2fluorobenzylamino)-2-methylquinoline-8-carbonitrile 4t (200 mg, 0.614 mmol, 65% yield) as yellow solid. LC-MS (ES API) M+H = 326. 4-(3-Chloro-2-fluorobenzylamino)-2-methylquinoline-8-carboxamide (1aa)

Potassium hydroxide (68.9 mg, 1.228 mmol) in water (2 mL) was added dropwise to a solution of 4-(3-chloro-2fluorobenzylamino)-2-methylquinoline-8-carbonitrile 4t (200 mg, 0.614 mmol) in dimethyl sulfoxide (5 mL) at room temperature. Then, 30% hydrogen peroxide (4.5 mL, 147 mmol) was added and the reaction mixture was stirred overnight. The reaction mixture was diluted with water (15 mL) leading to the formation of a slurry. The precipitates were collected by filtration and purified by preparative high performance liquid chromatography, eluting with acetonitrile:water with 0.5% trifluoroacetic acid (5:95 to 95:5) to give 4-(3-chloro-2-fluorobenzylamino)-2methylquinoline-8-carboxamide trifluoroacetate 1aa (92 mg, 0.201 mmol, 33% yield).

1

H NMR (400 MHz,

CD3SOCD3) δ 14.30 (br s, 1H), 9.89 (br s, 1H), 8.88 (br s, 1H), 8.82 (d, 1H, J = 8 Hz), 8.52 (d, 1H, J = 7 Hz), 8.29 (br s, 1H), 7.79 (t, 1H, J = 8 Hz), 7.56 (t, 1H, J = 7 Hz), 7.37 (t, 1H, J = 7 Hz), 7.20 (t, 1H, J = 8 Hz), 6.91 (s, 1H),


52

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4.87 (d, 2H, J = 5 Hz), 2.66 (s, 3H). HRMS: C18H15ClFN3O requires M+H at m/z 344.0966; found, 344.0959; tR = 1.82 minutes, 100.0% purity. 4-(3-Chloro-2-methylbenzylamino)-2-methylquinoline-8-carbonitrile (4u)

To a flask was added 8-cyano-2-methylquinolin-4-yl trifluoromethanesulfonate 7 (316 mg, 1 mmol), (3-chloro-2methylphenyl)methanamine (202 mg, 1.30 mmol), and potassium carbonate (276 mg, 2.0 mmol) in tetrahydrofuran (5 mL) under nitrogen. The mixture was heated at 80 °C overnight and then water (10 mL) was added to it. The solid that precipitated was collected via filtration to give 4-(3-chloro-2-methylbenzylamino)-2-methylquinoline-8carbonitrile 4u (100 mg, 0.25 mmol, 25% yield). LC-MS (ES API) M+H = 322. 4-(3-Chloro-2-methylbenzylamino)-2-methylquinoline-8-carboxamide

4-(3-Chloro-2-methylbenzylamino)-2-methylquinoline-8-carbonitrile 4u (100 mg, 0.31 mmol) was dissolved in dimethyl sulfoxide (5 mL), sodium hydroxide (24.9 mg, 0.62 mmol) was added, followed by 30% hydrogen peroxide (2.64 mL, 31.1 mmol). The resulting homogeneous mixture was allowed to stir overnight. Then, water (20 mL) was added and the resulting solid was collected by filtration to give 4-(3-chloro-2-methylbenzylamino)-2methylquinoline-8-carboxamide 1ab (50 mg, 0.15 mmol, 47% yield). 1H NMR (400 MHz, CD3SOCD3) δ 11.13 (br d, 1H, J = 4 Hz), 8.50 (d, 1H, J = 7 Hz), 8.48 (d, 1H, J = 8 Hz), 7.91 (br t, 1H, J = 5 Hz), 7.68 (br d, 1H, J = 4 Hz), 7.49 (t, 1H, J = 8 Hz), 7.36 (dd, 1H, J = 8, 2 Hz), 7.20-7.10 (m, 2H), 6.36 (s, 1H), 4.57 (d, 2H, J = 5 Hz), 2.46 (s,


53


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3H), 2.42 (s, 3H); HRMS: C19H18ClN3O requires M+H at m/z 340.1216; found, 340.1212; tR = 1.24 minutes, 100.0% purity. 4-(2-Fluoro-3-(trifluoromethyl)benzylamino)-2-methylquinoline-8-carbonitrile (4v)

To

a

solution

of

potassium

carbonate

(0.262

g,

1.90

mmol)

and

8-cyano-2-methyl-4-quinolinyl

trifluoromethanesulfonate 7 (0.3 g, 0.95 mmol) in tetrahydrofuran (10 mL) was added {[2-fluoro-3(trifluoromethyl)phenyl]methyl}amine (0.366 g, 1.90 mmol). The reaction mixture was stirred at 82°C overnight. After cooling, water was added (30 mL) and the resulting precipitate was filtered to give 4-(2-fluoro-3(trifluoromethyl)benzylamino)-2-methylquinoline-8-carbonitrile 4v (0.4 g, 0.74 mmol, 77% yield) as a red solid. LC-MS (ES API) M+H = 360. 4-(2-Fluoro-3-(trifluoromethyl)benzylamino)-2-methylquinoline-8-carboxamide (1ac)

To a solution of 4-(2-fluoro-3-(trifluoromethyl)benzylamino)-2-methylquinoline-8-carbonitrile 4v (0.4 g, 1.11 mmol) and potassium hydroxide (0.250 g, 4.45 mmol) in dimethyl sulfoxide (8 mL) was added 30% hydrogen peroxide (3.41 mL, 111 mmol), dropwise. The reaction mixture was stirred at room temperature overnight at which time water (10 mL) was added. The resulting precipitate was filtered and further purified via reverse phase liquid chromatography, eluting with acetonitrile:water containing 0.05% trifluoroacetic acid (1:9 to 1:1) to give 4-(2fluoro-3-(trifluoromethyl)benzylamino)-2-methylquinoline-8-carboxamide 1ac (0.125 g, 0.33 mmol, 30% yield) as a


54

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white solid. 1H NMR (400 MHz, CD3OD) δ: 8.60 (d, 1H, J = 9 Hz), 8.48 (d, 1H, J = 7 Hz), 7.78 (t, 1H, J = 8 Hz), 7.74-7.66 (m, 2H), 7.37 (t, 1H, J = 8 Hz), 6.87 (s, 1H), 4.95 (s, 2H), 2.72 (s, 3H); HRMS: C19H15F4N3O requires M+H at m/z 378.1229; found, 378.1224; tR = 1.41 minutes, 100.0% purity. 2-Methyl-4-(naphthalen-1-ylmethylamino)quinoline-8-carbonitrile (4w)

Naphthalen-1-ylmethanamine

(314

mg,

1.998

mmol)

was

added

to


trifluoromethanesulfonate 7 (316 mg, 0.999 mmol) in tetrahydrofuran (10 mL) under nitrogen. Then, potassium carbonate (276 mg, 1.998 mmol) was added and the reaction mixture was heated at 80 °C overnight. Then, the reaction mixture was diluted with water and the resulting precipitate was collected by filtration to give 2-methyl-4(naphthalen-1-ylmethylamino)quinoline-8-carbonitrile 4w (180 mg, 0.557 mmol, 56% yield). LC-MS (ES API) M+H = 324. 2-Methyl-4-(naphthalen-1-ylmethylamino)quinoline-8-carboxamide (1ad)

Potassium hydroxide (1.026 g, 7.42 mmol) was added to 2-methyl-4-(naphthalen-1-ylmethylamino)quinoline-8carbonitrile 4w (120 mg, 0.371 mmol) in dimethyl sulfoxide (5 mL) at room temperature. Then, 30% hydrogen peroxide (4.28 mL, 41.9 mmol) was added dropwise over 15 minutes. The reaction mixture was stirred overnight. The reaction mixture was diluted with water, and the resulting precipitate was collected by filtration to give 2methyl-4-(naphthalen-1-ylmethylamino)quinoline-8-carboxamide 1ad (60 mg, 0.176 mmol, 47% yield).

1

H NMR

(400 MHz, CD3OD) δ: 11.14 (br d, 1H, J = 4 Hz), 8.54-8.50 (m, 2H), 8.23 (d, 1H, J = 8 Hz), 8.06 (br t, 1H, J = 5


55


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Page 56 of 107

Hz), 7.98 (d, 1H, J = 8 Hz), 7.86 (d, 1H, J = 6 Hz), 7.67 (d, 1H, J = 4 Hz), 7.64-7.56 (m, 2H), 7.54-7.44 (m, 3H), 6.45 (s, 1H), 5.04 (d, 2H, J = 5 Hz), 2.43 (s, 3H); HRMS: C22H19N3O requires M+H at m/z 342.1606; found, 342.1597; tR = 1.66 minutes, 99.2% purity. 4-(2,6-Difluorobenzylamino)-2-methylquinoline-8-carbonitrile (4x)

A mixture of 8-cyano-2-methylquinolin-4-yl trifluoromethanesulfonate 7 (316 mg, 1.0 mmol) and (2,6difluorophenyl)methanamine (280 mg, 1.96 mmol) in tetrahydrofuran (5 mL) was stirred at 75 °C overnight. The mixture was filtered and the solvent evaporated under reduced pressure. The residue was purified by preparative thin layer

chromatography

(5:2

petroleum

ether:tetrahydrofuran)

to

give

4-(2,6-difluorobenzylamino)-2-

methylquinoline-8-carbonitrile 4x (160 mg, 0.52 mmol, 23% yield) as a yellow solid. LCMS (ES API) M+H = 310. 4-(2-Chloro-3,6-difluorobenzylamino)-2-methylquinoline-8-carboxamide (1ae)

To a solution of 4-(2,6-difluorobenzylamino)-2-methylquinoline-8-carbonitrile 4x (160 mg, 0.52 mmol) in dimethyl sulfoxide (15 mL) was added sodium hydroxide (210 mg, 5.25 mmol) at room temperature. 30% Hydrogen peroxide (3 mL, 29.4 mmol) was added after 15 minutes. The mixture was stirred for 2 hours at which time water (20 mL) was added and the mixture was filtered. The residue was washed with water and the crude product was purified by reverse phase high performance liquid chromatography, eluting with acetonitrile:water with 0.05% trifluoroacetic acid to give 4-(2-chloro-3,6-difluorobenzylamino)-2-methylquinoline-8-carboxamide trifluoroacetate 1ae (20 mg, 0.061 mmol, 12% yield) as a white solid. 1H NMR (400 MHz, CD3SOCD3) δ: 14.34 (br s, 1H), 9.43 (br s, 1H), 8.83


56

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(br s, 1H), 8.72 (d, 1H, J = 7 Hz), 8.49 (d, 1H, J = 7 Hz), 8.30 (br s, 1H), 7.77 (br s, 1H), 7.60-7.46 (m, 1H), 7.21 (t, 2H, J = 8 Hz), 7.02 (br s, 1H), 4.80 (br s, 2H), 2.73 (s, 3H); HRMS: C18H15F2N3O requires M+H at m/z 328.1261; found, 328.1258; tR = 1.20 minutes, 100.0% purity. 4-(2-Chloro-6-fluorobenzylamino)-2-methylquinoline-8-carbonitrile (4y)

To a solution of 8-cyano-2-methylquinolin-4-yl trifluoromethanesulfonate 7 (316 mg, 1.0 mmol) in dimethyl sulfoxide

(10

mL)

stirred

under

nitrogen

at

room

temperature

was

added

neat

(2-chloro-6-

fluorophenyl)methanamine (798 mg, 5.0 mmol) dropwise during 1 minute. The reaction mixture was stirred at 80 °C overnight. The reaction mixture was partitioned between water (50 mL) and filtered and the filtrate washed with water twice. The filtrate was dried in vacuo to obtain 4-(2-chloro-6-fluorobenzylamino)-2-methylquinoline-8carbonitrile 4y (195 mg, 0.54 mmol, 54% yield) as a white solid. LC-MS (ES API) M+H = 326. 4-(2-Chloro-6-fluorobenzylamino)-2-methylquinoline-8-carboxamide (1af)

To a solution of 4-(2-chloro-6-fluorobenzylamino)-2-methylquinoline-8-carbonitrile 4y (195 mg, 0.60 mmol) and sodium hydroxide (96 mg, 2.39 mmol) in dimethyl sulfoxide (20 mL) under nitrogen at 0°C was added 30% hydrogen peroxide (5 mL, 163 mmol) dropwise during 5 minutes. The reaction mixture was stirred at room temperature overnight. Water (50 mL) was added to the mixture followed by filtration. The filtrate was washed with water twice, and then dried in vacuo to obtain 4-(2-chloro-6-fluorobenzylamino)-2-methylquinoline-8-carboxamide 1af (165 mg, 0.46 mmol, 77% yield) as white solid. 1H NMR (CDCl3) δ 11.70 (br s, 1H), 8.66 (dd, 1H, J = 8, 1 Hz),


57


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Page 58 of 107

7.78 (d, 1H, J = 9 Hz), 7.37 (t, 1H, J = 8 Hz), 7.26-7.14 (m, 2H), 7.01 (dt, 1H, J = 8, 2 Hz), 6.56 (s, 1H), 6.01 (br s, 1H), 5.43 (br s, 1H), 4.63 (d, 2H, J = 5 Hz), 2.56 (s, 3H); HRMS: C18H15ClFN3O requires M+H at m/z 344.0966; found, 344.0963; tR = 1.63 minutes, 95.6% purity. 4-(2-Chloro-6-methylbenzylamino)-2-methylquinoline-8-carbonitrile (4z)

A solution of 8-cyano-2-methylquinolin-4-yl trifluoromethanesulfonate 7 (335 mg, 1.06 mmol), (2-chloro-6methylphenyl)methanamine (150 mg, 0.96 mmol), and potassium carbonate (266 mg, 1.93 mmol) in tetrahydrofuran (5 mL) was stirred under nitrogen at 80 °C overnight. Water (15 mL) was added and the organics were extracted with tetrahydrofuran (2×). The solution was concentrated in vacuo to give 4-(2-chloro-6-methylbenzylamino)-2methylquinoline-8-carbonitrile 4z (90 mg, 0.28 mmol, 29% yield). LC-MS (ES API) MH+ = 322. 4-(2-Chloro-6-methylbenzylamino)-2-methylquinoline-8-carboxamide (1ag)

To a solution of 4-(2-chloro-6-methylbenzylamino)-2-methylquinoline-8-carbonitrile 4z (90 mg, 0.28 mmol) and sodium hydroxide (55.9 mg, 1.40 mmol) in dimethyl sulfoxide (15 mL) at 25°C was added 30% hydrogen peroxide (2 mL, 65.3 mmol) dropwise over 5 minutes. The reaction mixture was stirred at 25 °C overnight. Water was added to the reaction and the resulting solid collected via filtration. The solid was purified by reverse phase high performance liquid chromatography, eluting with acetonitrile:water with 0.05% trifluoroacetic acid (5:95 to 95:5) to give 4-(2-chloro-6-methylbenzylamino)-2-methylquinoline-8-carboxamide trifluoroacetate 1ag (35 mg, 0.10 mmol, 37% yield). 1H NMR (400 MHz, CD3SOCD3) δ: 11.15 (br s, 1H), 8.50 (dd, 1H, J = 7, 1 Hz), 8.48 (dd, 1H, J = 8, 2


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Hz), 8.16 (br s, 1H), 7.70 (d, 1H, J = 4 Hz), 7.41 (t, 1H, J = 8 Hz), 7.37 (d, 1H, J = 8 Hz), 7.33 (br s, 1H), 7.29 (t, 1H, J = 8 Hz), 7.27-7.22 (m, 1H), 6.66 (s, 1H), 4.54 (d, 2H, J = 4 Hz), 2.57 (s, 3H), 2.40 (s, 3H); HRMS: C19H18ClN3O requires M+H at m/z 340.1216; found, 340.1214; tR = 1.28 minutes, 94.6% purity. 8-Bromo-N-(2-fluoro-6-(trifluoromethyl)benzyl)-2-methylquinolin-4-amine (3j)

N,N-Di-iso-propylethylamine (1.034 mL, 5.94 mmol) was added to 8-bromo-4-chloro-2-methylquinoline 2a (0.5076 g, 1.979 mmol) in dimethyl sulfoxide (3.91 mL) at room temperature, followed by 2-fluoro-6trifluoromethylbenzylamine (0.573 g, 2.97 mmol) and the solution was heated to 140 °C and stirred for sixty-six hours. The reaction mixture was poured into ether, washed with water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (1:3) to give 8-bromo-N-(2-fluoro-6-(trifluoromethyl)benzyl)-2-methylquinolin-4-amine 3j (0.2838 g, 0.652 mmol, 33% yield). 1

H NMR (400 MHz, CD3SOCD3) δ 8.23 (d, 1H, J = 8 Hz), 7.93 (d, 1H, J = 7 Hz), 7.71-7.61 (m, 3H), 7.24 (br t, 1H,

J = 4 Hz), 7.19 (t, 1H, J = 8 Hz), 6.61 (s, 1H), 4.50 (d, 2H, J = 3 Hz), 2.53 (s, 3H); LC-MS (LC-ES) M+H = 413. 4-((2-Fluoro-6-(trifluoromethyl)benzyl)amino)-2-methylquinoline-8-carboxamide (1ah)

F F

F F HN

N O

NH2 1ah

1,1'-Bis(diphenylphosphino)ferrocene (0.076 g, 0.137 mmol) and bis(trimethylsilyl)amine (2.173 mL, 10.30 mmol) were added to 8-bromo-N-(2-fluoro-6-(trifluoromethyl)benzyl)-2-methylquinolin-4-amine 3j (0.2838 g, 0.687 mmol)

in

N,N-dimethylformamide

(4.69

mL)

at

room

temperature,

followed

by

tris(dibenzylideneacetone)dipalladium(0) (0.063 g, 0.069 mmol) and the solution fitted with a carbon monoxide


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(1.924 g, 68.7 mmol) balloon and heated to 100 °C and stirred for four hours. The reaction mixture was cooled, poured into ether, washed with 1 N sodium hydroxide and water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (4:1) to give 4-((2-fluoro-6-(trifluoromethyl)benzyl)amino)-2-methylquinoline-8-carboxamide 1ah (0.0617 g, 0.155 mmol, 23% yield). 1H NMR (400 MHz, CD3SOCD3) δ 11.12 (br d, 1H, J = 5 Hz), 8.47 (d, 1H, J = 7 Hz), 8.42 (d, 1H, J = 8 Hz), 7.72-7.62 (m, 4H), 7.44-7.37 (m, 2H), 6.64 (s, 1H), 4.52 (s, 2H), 2.56 (s, 3H); HRMS: C19H15F4N3O requires M+H at m/z 378.1229; found, 378.1226; tR = 0.55 minutes, 100.0% purity. 8-Bromo-2-methyl-N-[(2,3,6-trichlorophenyl)methyl]-4-quinolinamine (3k)

1-(2,3,6-Trichlorophenyl)methanamine hydrochloride (13.81 g, 55.9 mmol) was added to 8-bromo-4-chloro-2methylquinoline 2a (10.25 g, 10.25 mmol) in N-methyl-2-pyrrolidone (25 mL) and the reaction mixture was heated at 150 ºC overnight. The mixture was allowed to cool to room temperature and the solid was filtered. The cake was washed with small amount of N-methyl-2-pyrrolidone, followed by acetonitrile, and dried to obtain 8-bromo-2methyl-N-[(2,3,6-trichlorophenyl)methyl]-4-quinolinamine hydrochloride (15 g) as an off-white solid.

The 8-

bromo-2-methyl-N-[(2,3,6-trichlorophenyl)methyl]-4-quinolinamine hydrochloride was mixed with 10% aqueous NaOH solution (250 mL), then the suspension was kept at 100 ºC for 8 hours. After cooling to room temperature, the solid was filtered and washed with water until the last wash was neutral pH. The cake was then dried to obtain 8-bromo-2-methyl-N-[(2,3,6-trichlorophenyl)methyl]-4-quinolinamine 3k (13.2 g, 30.7 mmol, 77% yield). 1H NMR (400 MHz, CD3SOCD3) δ 8.23 (d, 1H, J = 6 Hz), 7.93 (d, 1H, J = 5 Hz), 7.71 (d, 1H, J = 8 Hz), 7.58 (d, 1H, J = 8 Hz), 7.27 (s, 1H), 7.20 (s, 1H), 6.63 (s, 1H), 4.62 (s, 2H), 2.53 (s, 3H). 2-Methyl-4-{[(2,3,6-trichlorophenyl)methyl]amino}-8-quinolinecarboxamide (1ai)


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A stirred mixture of 8-bromo-2-methyl-N-[(2,3,6-trichlorophenyl)methyl]-4-quinolinamine 3k (9.5 g, 22.06 mmol), 1,1'-bis(diphenylphosphino)ferrocene (1.22 g, 2.2 mmol), tris(dibenzylideneacetone)dipalladium(0) (1.01 g, 1.1 mmol), and bis(trimethylsilyl)amine (70 mL) in N,N-dimethylformamide (140 mL) was flushed with nitrogen, then carbon dioxide at room temperature, then the mixture was heated to 100 ºC, while carbon monoxide was passing through the reaction. The mixture was stirred at 100 ºC for 40 minutes under carbon monoxide blanket. The reaction mixture was cooled to room temperature, and water (70 mL) was added to convert the trimethylsilyl amide to the primary amide. After complete conversion, more water (1 L) was added, and the mixture was stirred at room temperature for 30 minutes, then, filtered. The solid was dissolved in tetrahydrofuran (300 mL) and a scavenger resin (10 g of PL-BnSH MP-resin, 2.47mol/g, Varian) was added. The mixture was stirred at room temperature overnight. The resin was filtered, washed with tetrahydrofuran (50 mL, 2×). The filtrate was concentrated to 100 mL, cooled to -78 ºC for 20 minutes, and filtered. The cake was dissolved in boiling ethanol (400 mL). Ethanol (100 mL) was distilled out to remove any residual tetrahydrofuran, and the solution was cooled to -75 ºC for 30 minutes, then, filtered. The cake was washed with cold ethanol (10 mL) and dried in vacuum oven at 100 ºC overnight to obtain 2-methyl-4-{[(2,3,6-trichlorophenyl)methyl]amino}-8-quinolinecarboxamide 1ai (6.3 g, 15.96 mmol, 72% yield). 1H NMR (400 MHz, CD3SOCD3) δ 11.11 (d, 1H, J = 5 Hz), 8.46 (d, 1H, J = 7 Hz), 8.41 (d, 1H, J = 9 Hz), 7.72 (d, 1H, J = 9 Hz), 7.71 (s, 1H), 7.59 (d, 1H, J = 9 Hz), 7.40 (t, 1H, J = 8 Hz), 7.36 (m, 1H), 6.64 (s, 1H), 4.63 (d, 2H, J = 4 Hz), 2.56 (s, 3H); HRMS: C18H14Cl3N3O requires M+H at m/z 394.0280; found, 394.0274; tR = 2.01 minutes, 100.0% purity. 4-{[(3-Chloro-2,6-difluorophenyl)methyl]amino}-2-methyl-8-quinolinecarbonitrile (4aa)


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To a solution of 8-cyano-2-methyl-4-quinolinyl trifluoromethanesulfonate 7 (300 mg, 0.95 mmol) in tetrahydrofuran (4 mL) under nitrogen was added 1-(3-chloro-2,6-difluorophenyl)methanamine (337 mg, 1.90 mmol). The mixture was stirred at 80 °C for 24 hours. After cooling, the reaction was taken up in ethyl acetate, followed by washing with brine. The crude compound was added purified by silica gel chromatography, eluting with ethyl acetate:hexanes (1:2) to give 4-{[(3-chloro2,6-difluorophenyl)methyl]amino}-2-methyl-8-quinolinecarbonitrile 4aa (126 mg, 0.35 mmol, 37% yield) as a white solid. LC-MS (ES API) M+H = 344. 4-{[(3-Chloro-2,6-difluorophenyl)methyl]amino}-2-methyl-8-quinolinecarboxamide (1aj)

To a dimethyl sulfoxide solution (20 mL) containing 4-{[(3-chloro-2,6-difluorophenyl)methyl]amino}-2-methyl-8quinolinecarbonitrile 4aa (126 mg, 0.37 mmol) was added sodium hydroxide (44.0 mg, 1.1 mmol), followed by slow addition of 30% hydrogen peroxide (249 mg, 7.33 mmol). The reaction mixture was stirred at temperature for 4 hours. After cooling, water was added and the solid was filtered. The crude product was washed with water (3×) to give 4-{[(3-chloro-2,6-difluorophenyl)methyl]amino}-2-methyl-8-quinolinecarboxamide 1aj (50 mg, 0.13 mmol, 37% yield) as a white solid. 1H NMR (400 MHz, CD3SOCD3) δ 11.11 (d, 1H, J = 5 Hz), 8.49 (dd, 1H, J = 8, 2 Hz), 8.40 (dd, 1H, J = 8, 2 Hz), 7.72-7.62 (m, 3H), 7.46 (t, 1H, J = 8 Hz), 7.25 (dt, 1H, J = 8, 2 Hz), 6.60 (s, 1H), 4.57 (d, 2H, J = 5 Hz), 2.55 (s, 3H); HRMS: C18H14ClF2N3O requires M+H at m/z 362.0871; found, 362.0873; tR = 1.82 minutes, 97.3% purity. 4-(2-Chloro-3,6-difluorobenzylamino)-2-methylquinoline-8-carbonitrile (4ab)


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To a solution of 8-cyano-2-methylquinolin-4-yl trifluoromethanesulfonate 7 (316 mg, 1.0 mmol), and (2-chloro-3,6difluorophenyl)methanamine (266 mg, 1.5 mmol) in tetrahydrofuran (5 mL) under nitrogen at room temperature was added solid potassium carbonate (276 mg, 2.0 mmol). The reaction mixture was stirred at 80 °C overnight. After cooling, the reaction was filtered and the filter cake was rinsed with tetrahydrofuran (3×). The solution was combined, concentrated, and the residue purified by thin layer chromatography (5:2 petroleum ether: tetrahydrofuran) to give 4-(2-chloro-3,6-difluorobenzylamino)-2-methylquinoline-8-carbonitrile 4ab (205 mg, 0.60 mmol, 60% yield) as a yellow solid. LC-MS (ES API) M+H = 344. 4-(2-Chloro-3,6-difluorobenzylamino)-2-methylquinoline-8-carboxamide (1ak)

To a solution of 4-(2-chloro-3,6-difluorobenzylamino)-2-methylquinoline-8-carbonitrile 4ab (205 mg, 0.60 mmol) in dimethyl sulfoxide (5 mL) was added solid potassium hydroxide (100 mg, 1.79 mmol). The reaction mixture was stirred at 25 °C for 2 minutes and then 30% hydrogen peroxide (3 mL) was added, dropwise, during 2 minutes. The resulting mixture was stirred at room temperature for 30 minutes. Water (50 mL) was added and the precipitated solid was collected, washed with water, and dried under vacuum to give 4-(2-chloro-3,6-difluorobenzylamino)-2methylquinoline-8-carboxamide 1ak (48 mg, 0.13 mmol, 21% yield) as a white solid. 1H NMR (400 MHz, CD3SOCD3) δ 11.11 (br s, 1H), 8.49 (d, 1H, J = 7 Hz), 8.42 (d, 1H, J = 8 Hz), 7.71 (br s, 1H), 7.64-7.48 (m, 2H), 7.48-7.34 (m, 2H), 6.63 (br s, 1H), 4.59 (br s, 2H), 2.56 (s, 3H); HRMS: C18H14ClF2N3O requires M+H at m/z 362.0871; found, 362.0865; tR = 0.97 minutes, 95.4% purity.


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8-Bromo-N-[(3,6-dichloro-2-fluorophenyl)methyl]-2-methyl-4-quinolinamine (3l)

Sodium hydride (60%, 0.095 g, 2.371 mmol) was added to 8-bromo-2-methyl-4-quinolinamine 3ae (0.5110 g, 2.155 mmol) in N,N-dimethylformamide (10.78 mL) and tetrahydrofuran (10.78 mL) at 0 ºC. Then, 2-(bromomethyl)-1,4dichloro-3-fluorobenzene (0.667 g, 2.59 mmol) was added and the reaction mixture was stirred for four hours at room temperature. The reaction mixture was diluted with water and extracted with diethyl ether, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (1:4) to give 8-bromo-N-[(3,6-dichloro-2-fluorophenyl)methyl]-2-methyl-4-quinolinamine 3l (0.1436 g, 0.329 mmol, 15% yield). 1H NMR (400 MHz, CD3SOCD3) δ 8.20 (d, 1H, J = 5 Hz), 7.94 (d, 1H, J = 7 Hz), 7.66 (t, 1H, J = 8 Hz), 7.45 (d, 1H, J = 9 Hz), 7.37 (br t, 1H, J = 4 Hz), 7.22 (t, 1H, J = 8 Hz), 6.60 (s, 1H), 4.56 (d, 2H, J = 4 Hz), 2.52 (s, 3H); LC-MS (LC-ES) M+H = 414. 4-{[(3,6-Dichloro-2-fluorophenyl)methyl]amino}-2-methyl-8-quinolinecarboxamide (1al)

N,N-Diisopropylethylamine (0.133 mL, 0.763 mmol) and hexamethyldisilazane (0.509 mL, 2.427 mmol) were added to 8-bromo-N-[(3,6-dichloro-2-fluorophenyl)methyl]-2-methyl-4-quinolinamine 3l (0.1436 g, 0.347 mmol) in N,N-dimethylformamide (3.47 mL) at room temperature, followed by 1,3-bis(diphenylphosphino)propane (0.043 g, 0.104 mmol) and palladium(II) acetate (0.016 g, 0.069 mmol) and the solution fitted with a carbon monoxide balloon and heated to 110 °C and stirred for four hours. The reaction mixture was cooled, poured into diethyl ether, washed with 1 N sodium hydroxide and water, dried over magnesium sulfate, filtered, and concentrated. The


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residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (4:1) to give 4-{[(3,6-dichloro2-fluorophenyl)methyl]amino}-2-methyl-8-quinolinecarboxamide 1al (0.0481 g, 0.121 mmol, 35% yield). 1H NMR (400 MHz, CD3SOCD3) δ 11.10 (br d, 1H, J = 5 Hz), 8.47 (d, 1H, J = 7 Hz), 8.39 (d, 1H, J = 8 Hz), 7.70 (br d, 1H, J = 5 Hz), 7.66 (t, 1H, J = 8 Hz), 7.52 (br t, 1H, J = 4 Hz), 7.45 (d, 1H, J = 9 Hz), 7.43 (t, 1H, J = 8 Hz), 6.63 (s, 1H), 4.58 (d, 2H, J = 4 Hz), 2.55 (s, 3H); HRMS: C18H14Cl2FN3O requires M+H at m/z 378.0576; found, 378.0573; tR = 0.60 minutes, 98.5% purity. 8-Bromo-N-[(2,3-dichloro-6-fluorophenyl)methyl]-2-methyl-4-quinolinamine (3m)

N,N-Diisopropylethylamine (1.036 mL, 5.93 mmol) was added to 8-bromo-4-chloro-2-methylquinoline 2a (0.5072 g, 1.977 mmol) in dimethyl sulfoxide (6.59 mL) at room temperature, followed by [(2,3-dichloro-6fluorophenyl)methyl]amine (0.575 g, 2.97 mmol) and the solution was heated to 150 °C and stirred for one hundred and twelve hours. The reaction mixture was cooled, poured into ether, washed with water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (3:7) to give 8-bromo-N-[(2,3-dichloro-6-fluorophenyl)methyl]-2-methyl-4-quinolinamine 3m (0.4665 g, 1.070 mmol, 54% yield). 1H NMR (400 MHz, CD3SOCD3) δ 8.21 (d, 1H, J = 8 Hz), 7.93 (d, 1H, J = 8 Hz), 7.74 (dd, 1H, J = 9, 5 Hz), 7.39 (t, 1H, J = 9 Hz), 7.35 (br t, 1H, J = 4 Hz), 7.21 (t, 1H, J = 8 Hz), 6.59 (s, 1H), 4.56 (d, 2H, J = 3 Hz), 2.51 (s, 3H); LC-MS (LC-ES) M+H = 414. 4-{[(2,3-Dichloro-6-fluorophenyl)methyl]amino}-2-methyl-8-quinolinecarboxamide (1am)


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N,N-Diisopropylethylamine (0.193 mL, 1.102 mmol) and hexamethyldisilazane (0.735 mL, 3.51 mmol) were added to 8-bromo-N-[(2,3-dichloro-6-fluorophenyl)methyl]-2-methyl-4-quinolinamine 3m (0.2075 g, 0.501 mmol) in N,Ndimethylformamide (10.02 mL) at room temperature, followed by 1,3-bis(diphenylphosphino)propane (0.062 g, 0.150 mmol) and palladium(II) acetate (0.022 g, 0.100 mmol) and the solution fitted with a carbon monoxide balloon and heated to 110 °C and stirred for two hours. The reaction mixture was cooled, poured into ether, washed with 1 N sodium hydroxide and water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (4:1) to give 4-{[(2,3-dichloro-6fluorophenyl)methyl]amino}-2-methyl-8-quinolinecarboxamide 1am (0.0525 g, 0.132 mmol, 26% yield). 1H NMR (400 MHz, CD3SOCD3) δ 11.10 (br d, 1H, J = 5 Hz), 8.47 (d, 1H, J = 8 Hz), 8.40 (d, 1H, J = 8 Hz), 7.74 (dd, 1H, J = 9, 5 Hz), 7.70 (br d, 1H, J = 5 Hz), 7.51 (br t, 1H, J = 4 Hz), 7.43 (t, 1H, J = 8 Hz), 7.40 (t, 1H, J = 9 Hz), 6.62 (s, 1H), 4.58 (d, 2H, J = 3 Hz), 2.55 (s, 3H); LC-MS (LC-ES) M-H = 379. HRMS: C18H14Cl2FN3O requires M+H at m/z 378.0576; found, 378.0572; tR = 0.66 minutes, 100.0% purity. 8-Bromo-N-[(2-chloro-6-fluoro-3-methylphenyl)methyl]-2-methyl-4-quinolinamine (3n)

N,N-Diisopropylethylamine (1.056 mL, 6.05 mmol) was added to 8-bromo-4-chloro-2-methylquinoline 2a (0.5172 g, 2.016 mmol) in dimethyl sulfoxide (5.66 mL) at room temperature, followed by [(2-chloro-6-fluoro-3methylphenyl)methyl]amine (0.525 g, 3.02 mmol) and the solution was heated to 150 ºC and stirred for sixteen hours. The reaction mixture was cooled, poured into ether, washed with water, dried over magnesium sulfate, filtered, and concentrated.


acetate:hexanes (3:7) to give 8-bromo-N-[(2-chloro-6-fluoro-3-methylphenyl)methyl]-2-methyl-4-quinolinamine 3n (0.6887 g, 1.662 mmol, 82% yield). 1H NMR (400 MHz, CD3SOCD3) δ 8.24 (d, 1H, J = 8 Hz), 7.92 (d, 1H, J = 7 Hz), 7.41 (dd, 1H, J = 8, 7 Hz), 7.36 (br t, 1H, J = 4 Hz), 7.24-7.16 (m, 2H), 6.59 (s, 1H), 4.53 (d, 2H, J = 4 Hz), 2.50 (s, 3H), 2.33 (s, 3H); LC-MS (LC-ES) M+H = 393.


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4-{[(2-Chloro-6-fluoro-3-methylphenyl)methyl]amino}-2-methyl-8-quinolinecarboxamide

N,N-Diisopropylethylamine (0.152 mL, 0.871 mmol) and hexamethyldisilazane (0.581 mL, 2.77 mmol) were added to 8-bromo-N-[(2-chloro-6-fluoro-3-methylphenyl)methyl]-2-methyl-4-quinolinamine 3n (0.1559 g, 0.396 mmol) in N,N-dimethylformamide (3.23 mL) at room temperature, followed by 1,3-bis(diphenylphosphino)propane (0.049 g, 0.119 mmol) and palladium(II) acetate (0.018 g, 0.079 mmol) and the solution fitted with a carbon monoxide balloon and heated to 110 °C and stirred for two hours. The reaction mixture was cooled, poured into ether, washed with 1 N sodium hydroxide and water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (4:1) to give 4-{[(2-chloro-6-fluoro-3methylphenyl)methyl]amino}-2-methyl-8-quinolinecarboxamide 1an (0.0477 g, 0.127 mmol, 32% yield). 1H NMR (400 MHz, CD3SOCD3) δ 11.12 (br d, 1H, J = 5 Hz), 8.46 (d, 1H, J = 8 Hz), 8.44 (d, 1H, J = 9 Hz), 7.68 (br d, 1H, J = 5 Hz), 7.52 (br t, 1H, J = 4 Hz), 7.46-7.38 (m, 2H), 7.21 (t, 1H, J = 9 Hz), 6.62 (s, 1H), 4.55 (d, 2H, J = 4 Hz), 2.54 (s, 3H), 2.34 (s, 3H); HRMS: C19H17ClFN3O requires M+H at m/z 358.1122; found, 358.1124; tR = 0.70 minutes, 96.0% purity. 8-Bromo-2-methyl-N-{[3-(trifluoromethyl)-2-pyridinyl]methyl}-4-quinolinamine (3o)

N,N-Diisopropylethylamine (1.378 mL, 7.89 mmol) was added to 8-bromo-4-chloro-2-methylquinoline 2a (0.5060 g, 1.973 mmol) in dimethyl sulfoxide (6.58 mL) at room temperature, followed by {[3-(trifluoromethyl)-2pyridinyl]methyl}amine (0.521 g, 2.96 mmol) and the solution was heated to 150 °C and stirred for eighty-eight hours. The reaction mixture was cooled, poured into ether, washed with water, dried over magnesium sulfate,


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filtered, and concentrated.

Page 68 of 107


acetate:hexanes (2:3) to give 8-bromo-2-methyl-N-{[3-(trifluoromethyl)-2-pyridinyl]methyl}-4-quinolinamine 3o (0.3020 g, 0.671 mmol, 34% yield). 1H NMR (400 MHz, CD3SOCD3) δ 8.78 (d, 1H, J = 5 Hz), 8.23 (d, 1H, J = 7 Hz), 8.21 (d, 1H, J = 8 Hz), 7.96 (d, 1H, J = 7 Hz), 7.78 (br t, 1H, J = 5 Hz), 7.57 (dd, 1H, J = 8, 5 Hz), 7.28 (t, 1H, J = 8 Hz), 6.35 (s, 1H), 4.77 (d, 2H, J = 5 Hz), 2.41 (s, 3H); LC-MS (LC-ES) M+H = 396. 2-Methyl-4-({[3-(trifluoromethyl)-2-pyridinyl]methyl}amino)-8-quinolinecarboxamide (1ao)

N,N-Diisopropylethylamine (0.152 mL, 0.871 mmol) and hexamethyldisilazane (0.581 mL, 2.77 mmol) were added to 8-bromo-2-methyl-N-{[3-(trifluoromethyl)-2-pyridinyl]methyl}-4-quinolinamine 3o (0.1569 g, 0.396 mmol) in N,N-dimethylformamide (7.92 mL) at room temperature, followed by 1,3-bis(diphenylphosphino)propane (0.049 g, 0.119 mmol) and palladium(II) acetate (0.018 g, 0.079 mmol) and the solution fitted with a carbon monoxide balloon and heated to 110 °C and stirred for two hours. The reaction mixture was cooled, poured into ether, washed with 1 N sodium hydroxide and water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with methanol:ethyl acetate (1:99) to give 2-methyl-4-({[3(trifluoromethyl)-2-pyridinyl]methyl}amino)-8-quinolinecarboxamide 1ao (0.0411 g, 0.108 mmol, 27% yield).

1

H

NMR (400 MHz, CD3SOCD3) δ 11.10 (br d, 1H, J = 5 Hz), 8.79 (d, 1H, J = 5 Hz), 8.49 (d, 1H, J = 7 Hz), 8.40 (d, 1H, J = 8 Hz), 8.23 (d, 1H, J = 8 Hz), 7.94 (br t, 1H, J = 4 Hz), 7.69 (br d, 1H, J = 4 Hz), 7.57 (dd, 1H, J = 8, 5 Hz), 7.49 (t, 1H, J = 8 Hz), 6.38 (s, 1H), 4.80 (d, 2H, J = 5 Hz), 2.44 (s, 3H); HRMS: C18H15F3N4O requires M+H at m/z 361.1276; found, 361.1276; tR = 0.57 minutes, 96.7% purity. 8-Bromo-2-methyl-N-{[4-(trifluoromethyl)-3-pyridinyl]methyl}-4-quinolinamine (3p)


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N,N-Diisopropylethylamine (0.696 mL, 3.99 mmol) was added to 8-bromo-4-chloro-2-methylquinoline 2a (0.5112 g, 1.993 mmol) in dimethyl sulfoxide (6.64 mL) at room temperature, followed by {[4-(trifluoromethyl)-3pyridinyl]methyl}amine (0.635 g, 2.99 mmol) and the solution was heated to 150 °C and stirred for eighty-six hours. The reaction mixture was cooled, poured into ether, washed with water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (2:3) to give 8-bromo-2-methyl-N-{[4-(trifluoromethyl)-3-pyridinyl]methyl}-4-quinolinamine 3p (0.1592 g, 0.382 mmol, 19% yield). 1H NMR (400 MHz, CD3SOCD3) δ 8.78 (d, 1H, J = 5 Hz), 8.75 (s, 1H), 8.24 (d, 1H, J = 7 Hz), 7.98 (d, 1H, J = 7 Hz), 7.83 (br t, 1H, J = 5 Hz), 7.79 (d, 1H, J = 5 Hz), 7.30 (t, 1H, J = 8 Hz), 6.36 (s, 1H), 4.71 (d, 2H, J = 5 Hz), 2.43 (s, 3H); LC-MS (LC-ES) M+H = 396. 2-Methyl-4-({[4-(trifluoromethyl)-3-pyridinyl]methyl}amino)-8-quinolinecarboxamide (1ap)

N,N-Diisopropylethylamine (0.154 mL, 0.884 mmol) and hexamethyldisilazane (0.590 mL, 2.81 mmol) were added to 8-bromo-2-methyl-N-{[4-(trifluoromethyl)-3-pyridinyl]methyl}-4-quinolinamine 3p (0.1592 g, 0.402 mmol) in N,N-dimethylformamide (8.04 mL) at room temperature, followed by 1,3-bis(diphenylphosphino)propane (0.050 g, 0.121 mmol) and palladium(II) acetate (0.018 g, 0.080 mmol) and the solution fitted with a carbon monoxide balloon and heated to 110 °C and stirred for two hours. The reaction mixture was cooled, poured into ether, washed with 1 N sodium hydroxide and water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with methanol:ethyl acetate (1:99) to give 2-methyl-4-({[4(trifluoromethyl)-3-pyridinyl]methyl}amino)-8-quinolinecarboxamide 1ap (0.0716 g, 0.189 mmol, 47% yield).

1

H


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NMR (400 MHz, CD3SOCD3) δ 11.05 (br d, 1H, J = 5 Hz), 8.79 (d, 1H, J = 5 Hz), 8.76 (s, 1H), 8.50 (d, 1H, J = 7 Hz), 8.43 (d, 1H, J = 8 Hz), 7.97 (br t, 1H, J = 5 Hz), 7.80 (d, 1H, J = 5 Hz), 7.71 (br d, 1H, J = 4 Hz), 7.51 (t, 1H, J = 8 Hz), 6.39 (s, 1H), 4.73 (d, 2H, J = 5 Hz), 2.32 (s, 3H); HRMS: C18H15F3N4O requires M+H at m/z 361.1276; found, 361.1273; tR = 0.54 minutes, 96.7% purity. 8-Bromo-N-[(3-chloro-4-pyridinyl)methyl]-2-methyl-4-quinolinamine (3q)

N,N-Diisopropylethylamine (1.397 mL, 8.00 mmol) was added to 8-bromo-4-chloro-2-methylquinoline 2a (0.5128 g, 1.999 mmol) in dimethyl sulfoxide (6.66 mL) at room temperature, followed by [(3-chloro-4pyridinyl)methyl]amine (0.646 g, 3.00 mmol) and the solution was heated to 150 °C and stirred for forty-four hours. The reaction mixture was cooled, poured into ether, washed with water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (1:1) to give 8-bromo-N-[(3-chloro-4-pyridinyl)methyl]-2-methyl-4-quinolinamine 3q (0.0927 g, 0.230 mmol, 12% yield). 1

H NMR (400 MHz, CD3SOCD3) δ 8.64 (s, 1H), 8.42 (d, 1H, J = 5 Hz), 8.25 (d, 1H, J = 8 Hz), 7.99 (d, 1H, J = 7

Hz), 7.90 (br t, 1H, J = 6 Hz), 7.31 (t, 1H, J = 8 Hz), 7.25 (d, 1H, J = 5 Hz), 6.28 (s, 1H), 4.63 (d, 2H, J = 6 Hz), 2.40 (s, 3H); LC-MS (LC-ES) M+H = 362. 4-{[(3-Chloro-4-pyridinyl)methyl]amino}-2-methyl-8-quinolinecarboxamide (1aq)

N,N-Diisopropylethylamine (0.098 mL, 0.562 mmol) and hexamethyldisilazane (0.375 mL, 1.789 mmol) were added to 8-bromo-N-[(3-chloro-4-pyridinyl)methyl]-2-methyl-4-quinolinamine 3q (0.0927 g, 0.256 mmol) in N,N-


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dimethylformamide (5.11 mL) at room temperature, followed by 1,3-bis(diphenylphosphino)propane (0.032 g, 0.077 mmol) and palladium(II) acetate (0.011 g, 0.051 mmol) and the solution fitted with a carbon monoxide balloon and heated to 110 °C and stirred for two hours. The reaction mixture was cooled, poured into ether, washed with 1 N sodium hydroxide and water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica

gel

chromatography,

eluting

with

methanol:ethyl

acetate

(1:99)

to

give

4-{[(3-chloro-4-

pyridinyl)methyl]amino}-2-methyl-8-quinolinecarboxamide 1aq (0.0244 g, 0.071 mmol, 28% yield). 1H NMR (400 MHz, CD3SOCD3) δ 11.05 (br d, 1H, J = 5 Hz), 8.65 (s, 1H), 8.51 (d, 1H, J = 7 Hz), 8.44 (d, 1H, J = 8 Hz), 8.42 (d, 1H, J = 5 Hz), 8.06 (br t, 1H, J = 6 Hz), 7.70 (br d, 1H, J = 5 Hz), 7.53 (t, 1H, J = 8 Hz), 7.27 (d, 1H, J = 5 Hz), 6.31 (s, 1H), 4.66 (d, 2H, J = 6 Hz), 2.44 (s, 3H); HRMS: C17H15ClN4O requires M+H at m/z 327.1012; found, 327.1010; tR = 0.47 minutes, 95.9% purity. 8-Bromo-2-methyl-N-{[2-(trifluoromethyl)-3-pyridinyl]methyl}-4-quinolinamine (3r)

N,N-Diisopropylethylamine (1.370 mL, 7.84 mmol) was added to 8-bromo-4-chloro-2-methylquinoline 2a (0.5029 g, 1.960 mmol) in dimethyl sulfoxide (6.53 mL) at room temperature, followed by {[2-(trifluoromethyl)-3pyridinyl]methyl}amine (0.518 g, 2.94 mmol) and the solution was heated to 150 °C and stirred for eighty-eight hours. The reaction mixture was cooled, poured into ether, washed with water, dried over magnesium sulfate, filtered, and concentrated.


acetate:hexanes (2:3) to give 8-bromo-2-methyl-N-{[2-(trifluoromethyl)-3-pyridinyl]methyl}-4-quinolinamine 3r (0.3018 g, 0.724 mmol, 37% yield). 1H NMR (400 MHz, CD3SOCD3) δ 8.64 (d, 1H, J = 5 Hz), 8.24 (d, 1H, J = 8 Hz), 7.98 (d, 1H, J = 7 Hz), 7.91 (br t, 1H, J = 5 Hz), 7.90 (d, 1H, J = 8 Hz), 6.65 (dd, 1H, J = 8, 5 Hz), 7.31 (t, 1H, J = 8 Hz), 6.27 (s, 1H), 4.73 (d, 2H, J = 5 Hz), 2.40 (s, 3H); LC-MS (LC-ES) M+H = 396. 2-Methyl-4-({[2-(trifluoromethyl)-3-pyridinyl]methyl}amino)-8-quinolinecarboxamide (1ar)


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N,N-Diisopropylethylamine (0.141 mL, 0.808 mmol) and hexamethyldisilazane (0.539 mL, 2.57 mmol) were added to 8-bromo-2-methyl-N-{[2-(trifluoromethyl)-3-pyridinyl]methyl}-4-quinolinamine 3r (0.1455 g, 0.367 mmol) in N,N-dimethylformamide (3.67 mL) at room temperature, followed by 1,3-bis(diphenylphosphino)propane (0.045 g, 0.110 mmol) and palladium(II) acetate (0.016 g, 0.073 mmol) and the solution fitted with a carbon monoxide balloon and heated to 110 °C and stirred for two hours. The reaction mixture was cooled, poured into ether, washed with 1 N sodium hydroxide and water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate to give 2-methyl-4-({[2-(trifluoromethyl)-3pyridinyl]methyl}amino)-8-quinolinecarboxamide 1ar (0.0387 g, 0.102 mmol, 28% yield).

1

H NMR (400 MHz,

CD3SOCD3) δ 11.05 (br d, 1H, J = 5 Hz), 8.65 (d, 1H, J = 5 Hz), 8.51 (d, 1H, J = 7 Hz), 8.44 (d, 1H, J = 8 Hz), 8.06 (br t, 1H, J = 5 Hz), 7.92 (d, 1H, J = 8 Hz), 7.71 (br d, 1H, J = 5 Hz), 7.66 (dd, 1H, J = 8, 5 Hz), 7.52 (t, 1H, J = 8 Hz), 6.31 (s, 1H), 4.75 (d, 2H, J = 5 Hz), 2.44 (s, 3H); HRMS: C18H15F3N4O requires M+H at m/z 361.1276; found, 361.1272; tR = 0.54 minutes, 100.0% purity. 8-Bromo-N-[(3-chloro-2-thienyl)methyl]-2-methyl-4-quinolinamine (3s)

N,N-Diisopropylethylamine (0.684 mL, 3.92 mmol) was added to 8-bromo-4-chloro-2-methylquinoline 2a (0.5026 g, 1.959 mmol) in dimethyl sulfoxide (6.53 mL) at room temperature, followed by [(3-chloro-2thienyl)methyl]amine (0.434 g, 2.94 mmol) and the solution was heated to 150 °C and stirred for eighty-eight hours. The reaction mixture was cooled, poured into ether, washed with water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (1:4) to


72

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give 8-bromo-N-[(3-chloro-2-thienyl)methyl]-2-methyl-4-quinolinamine 3s (0.1573 g, 0.406 mmol, 21% yield). 1H NMR (400 MHz, CD3SOCD3) δ 8.18 (d, 1H, J = 8 Hz), 7.96 (d, 1H, J = 7 Hz), 7.95 (br t, 1H, J = 5 Hz), 7.53 (d, 1H, J = 5 Hz), 7.28 (t, 1H, J = 8 Hz), 7.05 (d, 1H, J = 5 Hz), 6.45 (s, 1H), 4.66 (d, 2H, J = 6 Hz), 2.45 (s, 3H); LC-MS (LC-ES) M+H = 367. 4-{[(3-Chloro-2-thienyl)methyl]amino}-2-methyl-8-quinolinecarboxamide (1as)

N,N-Diisopropylethylamine (0.164 mL, 0.941 mmol) and hexamethyldisilazane (0.628 mL, 2.99 mmol) were added to 8-bromo-N-[(3-chloro-2-thienyl)methyl]-2-methyl-4-quinolinamine 3s (0.1573 g, 0.428 mmol) in N,Ndimethylformamide (8.56 mL) at room temperature, followed by 1,3-bis(diphenylphosphino)propane (0.053 g, 0.128 mmol) and palladium(II) acetate (0.019 g, 0.086 mmol) and the solution fitted with a carbon monoxide balloon and heated to 110 °C and stirred for one hour. The reaction mixture was cooled, poured into ether, washed with 1 N sodium hydroxide and water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (4:1) to give 4-{[(3-chloro-2-thienyl)methyl]amino}2-methyl-8-quinolinecarboxamide 1as (0.0648 g, 0.186 mmol, 43% yield).

1

H NMR (400 MHz, CD3SOCD3) δ

11.05 (br d, 1H, J = 5 Hz), 8.49 (d, 1H, J = 7 Hz), 8.38 (d, 1H, J = 7 Hz), 8.11 (br t, 1H, J = 6 Hz), 7.70 (br d, 1H, J = 5 Hz), 7.54 (d, 1H, J = 5 Hz), 7.49 (t, 1H, J = 8 Hz), 7.06 (d, 1H, J = 5 Hz), 6.48 (s, 1H), 4.69 (d, 2H, J = 6 Hz), 2.49 (s, 3H); HRMS: C16H14ClN3OS requires M+H at m/z 332.0624; found, 332.0623; tR = 0.54 minutes, 100.0% purity. 8-Bromo-N-[(3,5-dimethyl-4-isoxazolyl)methyl]-2-methyl-4-quinolinamine (3t)


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Page 74 of 107

N,N-Diisopropylethylamine (1.034 mL, 5.92 mmol) was added to 8-bromo-4-chloro-2-methylquinoline 2a (0.5062 g, 1.973 mmol) in dimethyl sulfoxide (6.58 mL) at room temperature, followed by [(3,5-dimethyl-4isoxazolyl)methyl]amine (0.373 g, 2.96 mmol) and the solution was heated to 150 ºC and stirred for sixteen hours. The reaction mixture was cooled, poured into ether, washed with water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (2:3) to give 8-bromo-N-[(3,5-dimethyl-4-isoxazolyl)methyl]-2-methyl-4-quinolinamine 3t (0.4127 g, 1.132 mmol, 57% yield). 1H NMR (400 MHz, CD3SOCD3) δ 8.22 (d, 1H, J = 8 Hz), 7.93 (d, 1H, J = 7 Hz), 7.34 (br t, 1H, J = 5 Hz), 7.34 (t, 1H, J = 8 Hz), 6.45 (s, 1H), 4.23 (d, 2H, J = 5 Hz), 2.49 (s, 3H), 2.39 (s, 3H), 2.18 (s, 3H); LC-MS (LC-ES) M+H = 346. 4-{[(3,5-Dimethyl-4-isoxazolyl)methyl]amino}-2-methyl-8-quinolinecarboxamide (1at)

N,N-Diisopropylethylamine (0.242 mL, 1.385 mmol) and hexamethyldisilazane (0.923 mL, 4.41 mmol) were added to 8-bromo-N-[(3,5-dimethyl-4-isoxazolyl)methyl]-2-methyl-4-quinolinamine 3t (0.2179 g, 0.629 mmol) in N,Ndimethylformamide (12.59 mL) at room temperature, followed by 1,3-bis(diphenylphosphino)propane (0.078 g, 0.189 mmol) and palladium(II) acetate (0.028 g, 0.126 mmol) and the solution fitted with a carbon monoxide balloon and heated to 110 °C and stirred for two hours. The reaction mixture was cooled, poured into ether, washed with 1 N sodium hydroxide and water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with methanol:ethyl acetate (1:99) to give 4-{[(3,5-dimethyl-4isoxazolyl)methyl]amino}-2-methyl-8-quinolinecarboxamide 1at (0.0337 g, 0.103 mmol, 16% yield).

1

H NMR

(400 MHz, CD3SOCD3) δ 11.12 (br d, 1H, J = 5 Hz), 8.47 (d, 1H, J = 7 Hz), 8.41 (d, 1H, J = 8 Hz), 7.69 (br d, 1H, J = 5 Hz), 7.50 (br t, 1H, J = 5 Hz), 7.44 (t, 1H, J = 8 Hz), 6.48 (s, 1H), 4.28 (d, 2H, J = 5 Hz), 2.53 (s, 3H), 2.40 (s, 3H), 2.19 (s, 3H); HRMS: C17H18N4O2 requires M+H at m/z 311.1508; found, 311.1502; tR = 0.43 minutes, 97.0% purity.


74

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8-Bromo-N-(cyclohexylmethyl)-2-methyl-4-quinolinamine (3u)

(Cyclohexylmethyl)amine (88 mg, 0.780 mmol) was added to 8-bromo-4-chloro-2-methylquinoline 2a (100 mg, 0.390 mmol) in N-methyl-2-pyrrolidone (2 mL). Then, N, N-diisopropylethylamine (0.2 mL, 1.145 mmol) was added and the reaction mixture was heated at 150 °C overnight. The reaction mixture was diluted with ethyl acetate and washed with brine (2×), dried over magnesium sulfate, and filtered. The solution was evaporated onto silica gel and purified by silica gel chromatography, eluting with ethyl acetate:hexane (1:9 to 1:0) to give 8-bromo-N(cyclohexylmethyl)-2-methyl-4-quinolinamine 3u (90 mg, 0.243 mmol, 62% yield).

1

H NMR (400 MHz,

CD3SOCD3) δ 8.22 (d, 1H, J = 8 Hz), 7.92 (d, 1H, J = 7 Hz), 7.22 (t, 1H, J = 8 Hz), 7.17 (br t, 1H, J = 6 Hz), 6.42 (s, 1H), 3.10 (t, 2H, J = 6 Hz), 2.49 (s, 3H), 1.82 (br d, 2H, J = 13 Hz), 1.76-1.58 (m, 4H), 1.28-1.10 (m, 3H), 1.04-0.90 (m, 2H); LC-MS (LC-ES) M+H = 333. 4-[(Cyclohexylmethyl)amino]-2-methyl-8-quinolinecarboxamide (1au)

Hexamethyldisilazane (0.849 mL, 4.05 mmol) was added to a solution of 8-bromo-N-(cyclohexylmethyl)-2-methyl4-quinolinamine 3u (90 mg, 0.270 mmol) in N,N-dimethylformamide (2 mL), followed by 1,1'bis(diphenylphosphino)ferrocene (14.97 mg, 0.027 mmol) and tris(dibenzylideneacetone)dipalladium(0) (12.36 mg, 0.014 mmol). This solution was purged with nitrogen, and then evacuated and treated with carbon monoxide. The reaction mixture was heated to 100 ºC and the reaction mixture was again evacuated and purged with carbon monoxide. The reaction was then stirred under carbon monoxide for 2 hours. The solution was cooled and extracted into ethyl acetate (2×), washed with water (2×), dried over magnesium sulfate, and filtered. The solution was


75


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Page 76 of 107

evaporated onto silica gel and purified by silica gel chromatography, eluting with ethyl acetate:hexane (4:1 to 1:0) to give 4-[(cyclohexylmethyl)amino]-2-methyl-8-quinolinecarboxamide 1au (65 mg, 0.208 mmol, 77% yield) as a orange solid. 1H NMR (400 MHz, CD3SOCD3) δ 11.22 (br d, 1H, J = 5 Hz), 8.46 (dd, 1H, J = 8, 1 Hz), 8.41 (dd, 1H, J = 8, 1 Hz), 7.66 (br d, 1H, J = 5 Hz), 7.43 (t, 1H, J = 8 Hz), 7.36 (br t, 1H, J = 6 Hz), 6.45 (s, 1H), 3.13 (t, 2H, J = 6 Hz), 2.52 (s, 3H), 1.81 (br d, 2H, J = 12 Hz), 1.78-1.58 (m, 4H), 1.28-1.12 (m, 3H), 1.04-0.92 (m, 2H); HRMS: C18H23N3O requires M+H at m/z 298.1919; found, 298.1917; tR = 0.65 minutes, 100.0% purity. N-[(1-Acetyl-4-piperidinyl)methyl]-8-bromo-2-methyl-4-quinolinamine (3v)

8-Bromo-4-chloro-2-methylquinoline 2a (0.15 g, 0.585 mmol), methanamine-1-acetyl-4-methylpiperidine (1:1) (0.119 g, 0.760 mmol), and N,N-diisopropylethylamine (0.15 mL, 0.859 mmol) in N-methyl-2-pyrrolidone (1.8 mL) were heated to 130 °C for 3 days. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate, washed with water, then brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was dried onto Celite® and purified by reverse phase liquid chromatography, eluting with acetonitrile:water with 0.1% trifluoroacetic acid (1:9 to 1:0). The combined fractions were diluted with ethyl acetate and washed with 1.0 N aqueous sodium hydroxide. The organics were dried over sodium sulfate, filtered, then concentrated under reduced pressure to give N-[(1-Acetyl-4-piperidinyl)methyl]-8-bromo-2-methyl-4-quinolinamine 3v (161 mg, 0.428 mmol, 73% yield) as a pale solid. LC-MS (LC-ES) M+H = 376. 4-{[(1-Acetyl-4-piperidinyl)methyl]amino}-2-methyl-8-quinolinecarboxamide (1av)


76

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N-[(1-Acetyl-4-piperidinyl)methyl]-8-bromo-2-methyl-4-quinolinamine

3v

(0.16g,

0.425

mmol),

hexamethyldisilazane (1.337 mL, 6.38 mmol), tris(dibenzylideneacetone)dipalladium(0) (0.019 g, 0.021 mmol), and 1,1'-bis(diphenylphosphino)ferrocene (0.024 g, 0.043 mmol) in N,N-dimethylformamide (2.5 mL) were stirred under nitrogen, then fitted with a balloon of carbon monoxide. The vessel was evacuated and filled with carbon monoxide, heated to 100 °C and stirred for 2 hours. After cooling to room temperature, the reaction mixture was filtered through a pad of Celite® and washed with ethyl acetate. The filtrate was washed with water, then brine and the combined organics were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was taken up in dichloromethane, dried onto Celite®, purified by reverse phase liquid chromatography, eluting with acetonitrile:water with 0.1% trifluoroacetic acid (1:9 to 1:0). The combined fractions were diluted with ethyl acetate and washed with 1.0 N aqueous sodium hydroxide and brine. magnesium

sulfate,

filtered,

then

concentrated

under

reduced

The organics were dried over

pressure

to

give

4-{[(1-acetyl-4-

piperidinyl)methyl]amino}-2-methyl-8-quinolinecarboxamide 1av (63 mg, 0.178 mmol, 42% yield). 1H NMR (400 MHz, CD3OD) δ 8.54 (dd, 1H, J = 8, 1 Hz), 8.26 (dd, 1H, J = 8, 1 Hz), 7.43 (t, 1H, J = 8 Hz), 6.47 (s, 1H), 4.55 (d, 1H, J = 13 Hz), 3.94 (d, 1H, J = 14 Hz), 3.27 (d, 2H, J = 7 Hz), 3.11 (dt, 1H, J = 13, 2 Hz), 2.70-2.54 (m, 2H), 2.57 (s, 3H), 2.09 (s, 3H), 1.90 (t, 2H, J = 16 Hz), 1.36-1.12 (m, 2H); HRMS: C19H24N4O2 requires M+H at m/z 341.1977; found, 341.1978; tR = 0.35 minutes, 95.9% purity. 8-Bromo-2-methyl-N-[(1-methyl-4-piperidinyl)methyl]-4-quinolinamine (3w)


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Page 78 of 107

8-Bromo-4-chloro-2-methylquinoline 2a (0.15 g, 0.585 mmol), 1-(1-methyl-4-piperidinyl)methanamine (97 mg, 0.760 mmol), and N,N-diisopropylethylamine (0.15 mL, 0.859 mmol) in N-methyl-2-pyrrolidone (1.8 mL) were heated to 130 °C for 24 hours. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate, washed with water, then brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was dried onto Celite® and purified by reverse phase liquid chromatography, eluting with acetonitrile:water with 0.1% trifluoroacetic acid (1:9 to 1:0). The combined fractions were diluted with ethyl acetate and washed with 1.0 N aqueous sodium hydroxide. The organics were dried over sodium sulfate, filtered, then concentrated

under

reduced

pressure

to

give

8-Bromo-2-methyl-N-[(1-methyl-4-piperidinyl)methyl]-4-

quinolinamine 3w (143 mg, 0.411 mmol, 70% yield) as a pale solid. 1H NMR (400 MHz, CD3SOCD3) δ 8.22 (d, 1H, J = 8 Hz), 7.93 (dd, 1H, J = 8, 1 Hz), 7.22 (t, 1H, J = 8 Hz), 7.17 (br t, 1H, J = 5 Hz), 6.44 (s, 1H), 3.15 (br t, 2H, J = 6 Hz), 2.76 (br d, 2H, J = 11 Hz), 2.49 (s, 3H), 2.14 (s, 3H), 1.81 (br t, 2H, J = 12 Hz), 1.77 (br d, 2H, J = 12 Hz), 1.62-1.78 (m, 1H), 1.25 (dq, 2H, J = 12, 3 Hz); LC-MS (LC-ES) M+H = 350. 2-Methyl-4-{[(1-methyl-4-piperidinyl)methyl]amino}-8-quinolinecarboxamide (1aw)

8-Bromo-2-methyl-N-[(1-methyl-4-piperidinyl)methyl]-4-quinolinamine

3w

(0.14

g,

0.402

mmol),

hexamethyldisilazane (1.264 mL, 6.03 mmol), tris(dibenzylideneacetone)dipalladium(0) (0.018 g, 0.020 mmol), and 1,1'-bis(diphenylphosphino)ferrocene (0.022 g, 0.040 mmol) in N,N-dimethylformamide (2.5 mL) were stirred under nitrogen, then fitted with a balloon of carbon monoxide. The vessel was evacuated and filled with carbon monoxide, heated to 100 °C and stirred for 2 hours. After cooling to room temperature, the reaction mixture was filtered through a pad of Celite®, and washed with ethyl acetate. The filtrate was washed with water, then brine and the combined organics were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was taken up in dichloromethane, dried onto Celite®, purified by reverse phase liquid chromatography, eluting with acetonitrile:water with 0.1% trifluoroacetic acid (1:9 to 1:0). The combined fractions were diluted with


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ethyl acetate and washed with 1.0 N aqueous sodium hydroxide. The organics were dried over magnesium sulfate, filtered, then concentrated under reduced pressure to give 2-methyl-4-{[(1-methyl-4-piperidinyl)methyl]amino}-8quinolinecarboxamide 1aw (17.6 mg, 0.054 mmol, 13% yield) as a yellow solid. 1H NMR (400 MHz, CD3OD) δ 8.54 (dd, 1H, J = 8, 1 Hz), 8.27 (d, 1H, J = 8 Hz), 7.43 (t, 1H, J = 8 Hz), 6.47 (s, 1H), 3.27 (br d, 2H, J = 6 Hz), 2.91 (br d, 2H, J = 12 Hz), 2.58 (s, 3H), 2.28 (s, 3H), 2.03 (br t, 2H, J = 6 Hz), 1.86(br d, 2H, J = 6 Hz), 1.88-1.76 (m, 1H), 1.38 (br q, 2H, J = 12 Hz); HRMS: C18H24N4O requires M+H at m/z 313.2028; found, 313.2029; tR = 0.26 minutes, 100.0% purity. 8-Bromo-2-methyl-N-[(1R)-1-(2-methylphenyl)ethyl]-4-quinolinamine (3x)

N,N-Diisopropylethylamine (0.693 mL, 3.97 mmol) was added to 8-bromo-4-chloro-2-methylquinoline 2a (0.5092 g, 1.985 mmol) in dimethyl sulfoxide (6.62 mL) at room temperature, followed by [(1R)-1-(2methylphenyl)ethyl]amine (0.403 g, 2.98 mmol) and the solution was heated to 150 ºC and stirred for sixteen hours. The reaction mixture was cooled, poured into ether, washed with water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (1:3) to give 8-bromo-2-methyl-N-[(1R)-1-(2-methylphenyl)ethyl]-4-quinolinamine 3x (0.1476 g, 0.395 mmol, 20% yield). 1

H NMR (400 MHz, CD3SOCD3) δ 8.49 (d, 1H, J = 8 Hz), 7.95 (d, 1H, J = 7 Hz), 7.43 (d, 1H, J = 7 Hz), 7.33 (dd,

1H, J = 7, 2 Hz), 7.28 (t, 1H, J = 8 Hz), 7.18 (dd, 1H, J = 7, 2 Hz), 7.13-7.05 (m, 2H), 6.00 (s, 1H), 4.87 (p, 1H, J = 7 Hz), 2.45 (s, 3H), 2.32 (s, 3H), 1.54 (d, 3H, J = 7 Hz); LC-MS (LC-ES) M+H = 355. 2-Methyl-4-{[(1R)-1-(2-methylphenyl)ethyl]amino}-8-quinolinecarboxamide (1ax)


79


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N,N-Diisopropylethylamine (0.157 mL, 0.900 mmol) and hexamethyldisilazane (0.600 mL, 2.86 mmol) were added to 8-bromo-2-methyl-N-[(1R)-1-(2-methylphenyl)ethyl]-4-quinolinamine 3x (0.1453 g, 0.409 mmol) in N,Ndimethylformamide (8.18 mL) at room temperature, followed by 1,3-bis(diphenylphosphino)propane (0.051 g, 0.123 mmol) and palladium(II) acetate (0.018 g, 0.082 mmol) and the solution fitted with a carbon monoxide balloon and heated to 110 °C and stirred for eighteen hours. The reaction mixture was cooled, poured into ether, washed with 1 N sodium hydroxide and water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate to give 2-methyl-4-{[(1R)-1-(2-methylphenyl)ethyl]amino}8-quinolinecarboxamide 1ax (0.0417 g, 0.124 mmol, 30% yield). 1H NMR (400 MHz, CD3SOCD3) δ 11.07 (br d, 1H, J = 5 Hz), 8.68 (d, 1H, J = 8 Hz), 8.48 (d, 1H, J = 6 Hz), 7.65 (br d, 1H, J = 5 Hz), 7.59 (d, 1H, J = 7 Hz), 7.50 (t, 1H, J = 8 Hz), 7.34 (dd, 1H, J = 7, 3 Hz), 7.18 (dd, 1H, J = 6, 3 Hz), 7.14-7.06 (m, 2H), 6.03 (s, 1H), 4.91 (p, 1H, J = 7 Hz), 2.46 (s, 3H), 2.36 (s, 3H), 1.56 (d, 3H, J = 7 Hz); HRMS: C20H21N3O requires M+H at m/z 320.1763; found, 320.1763; tR = 0.64 minutes, 96.6% purity. 8-Bromo-N-[(1S)-1-(2,6-dimethylphenyl)ethyl]-2-methyl-4-quinolinamine (3y)

N,N-Diisopropylethylamine (0.684 mL, 3.92 mmol) was added to 8-bromo-4-chloro-2-methylquinoline 2a (0.5022 g, 1.958 mmol) in dimethyl sulfoxide (6.53 mL) at room temperature, followed by [(1S)-1-(2,6dimethylphenyl)ethyl]amine (0.438 g, 2.94 mmol) and the solution was heated to 150 ºC and stirred for sixteen hours. The reaction mixture was cooled, poured into ether, washed with water, dried over magnesium sulfate, filtered, and concentrated.


acetate:hexanes (3:7) to give 8-bromo-N-[(1S)-1-(2,6-dimethylphenyl)ethyl]-2-methyl-4-quinolinamine 3y (0.1275 g, 0.311 mmol, 16% yield). 1H NMR (400 MHz, CD3SOCD3) δ 8.52 (d, 1H, J = 8 Hz), 7.94 (d, 1H, J = 7 Hz), 7.30 (br d, 1H, J = 5 Hz), 7.27 (t, 1H, J = 8 Hz), 7.02-6.92 (m, 3H), 5.77 (s, 1H), 4.08 (p, 1H, J = 5 Hz), 2.39 (s, 6H), 2.26 (s, 3H), 1.63 (d, 3H, J = 7 Hz); LC-MS (LC-ES) M+H = 369.


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4-{[(1S)-1-(2,6-Dimethylphenyl)ethyl]amino}-2-methyl-8-quinolinecarboxamide (1ay)

N,N-Diisopropylethylamine (0.133 mL, 0.760 mmol) and hexamethyldisilazane (0.507 mL, 2.417 mmol) were added to 8-bromo-N-[(1S)-1-(2,6-dimethylphenyl)ethyl]-2-methyl-4-quinolinamine 3y (0.1275 g, 0.345 mmol) in N,N-dimethylformamide (6.90 mL) at room temperature, followed by 1,3-bis(diphenylphosphino)propane (0.043 g, 0.104 mmol) and palladium(II) acetate (0.016 g, 0.069 mmol) and the solution fitted with a carbon monoxide balloon and heated to 110 °C and stirred for eighteen hours. The reaction mixture was cooled, poured into ether, washed with 1 N sodium hydroxide and water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate to give 4-{[(1S)-1-(2,6dimethylphenyl)ethyl]amino}-2-methyl-8-quinolinecarboxamide 1ay (0.0418 g, 0.119 mmol, 35% yield). 1H NMR (400 MHz, CD3SOCD3) δ 11.06 (br d, 1H, J = 5 Hz), 8.72 (d, 1H, J = 8 Hz), 8.47 (d, 1H, J = 7 Hz), 7.63 (br d, 1H, J = 5 Hz), 7.49 (t, 1H, J = 8 Hz), 7.47 (br d, 1H, J = 5 Hz), 7.02-6.92 (m, 3H), 5.78 (s, 1H), 4.98 (p, 1H, J = 6 Hz), 2.40 (s, 6H), 2.30 (s, 3H), 1.65 (d, 3H, J = 7 Hz); HRMS: C21H23N3O requires M+H at m/z 334.1919; found, 334.1919; tR = 0.72 minutes, 94.8% purity. 8-Bromo-2-methyl-N-[(1R)-1,2,3,4-tetrahydro-1-naphthalenyl]-4-quinolinamine (3z)

N,N-Diisopropylethylamine (0.694 mL, 3.97 mmol) was added to 8-bromo-4-chloro-2-methylquinoline 2a (0.5096 g, 1.987 mmol) in dimethyl sulfoxide (6.62 mL) at room temperature, followed by (1R)-1,2,3,4-tetrahydro-1naphthalenamine (0.439 g, 2.98 mmol) and the solution was heated to 160 ºC and stirred for forty hours. The reaction mixture was cooled, poured into ether, washed with water, dried over magnesium sulfate, filtered, and


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concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (1:4) to give 8-bromo-2-methyl-N-[(1R)-1,2,3,4-tetrahydro-1-naphthalenyl]-4-quinolinamine 3z (0.0849 g, 0.220 mmol, 11% yield). 1H NMR (400 MHz, CD3SOCD3) δ 8.31 (d, 1H, J = 8 Hz), 7.93 (d, 1H, J = 7 Hz), 7.35 (d, 1H, J = 9 Hz), 7.22-7.08 (m, 5H), 6.72 (s, 1H), 5.04-4.94 (s, 1H), 2.88-2.74 (m, 2H), 2.49 (s, 3H), 2.10-1.76 (m, 4H); LC-MS (LC-ES) M+H = 369. 2-Methyl-4-[(1R)-1,2,3,4-tetrahydro-1-naphthalenylamino]-8-quinolinecarboxamide (1az)

N,N-diisopropylethylamine (0.213 mL, 1.217 mmol) and hexamethyldisilazane (0.812 mL, 3.87 mmol) were added to 8-bromo-2-methyl-N-[(1R)-1,2,3,4-tetrahydro-1-naphthalenyl]-4-quinolinamine 3z (0.2032 g, 0.553 mmol, from multiple

batches)

in

N,N-dimethylformamide

(10.04

mL)

at

room temperature,

followed

by 1,3-

bis(diphenylphosphino)propane (0.068 g, 0.166 mmol) and palladium(II) acetate (0.025 g, 0.111 mmol) and the solution was fitted with a carbon monoxide (1.661 g, 55.3 mmol) balloon and heated to 110 °C and stirred for sixteen hours. The reaction mixture was cooled, poured into ether, washed with 1 N sodium hydroxide and water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting

with

ethyl

acetate

to

give

2-methyl-4-[(1R)-1,2,3,4-tetrahydro-1-naphthalenylamino]-8-

quinolinecarboxamide 1az (0.0277 g, 0.079 mmol, 14% yield). 1H NMR (400 MHz, CD3SOCD3) δ 11.22 (d, 1H, J = 5 Hz), 8.49 (d, 1H, J = 8 Hz), 8.47 (d, 1H, J = 8 Hz), 7.67 (d, 1H, J = 5 Hz), 7.53 (d, 1H, J = 9 Hz), 7.39 (t, 1H, J = 8 Hz), 7.22-7.08 (m, 4H), 6.75 (s, 1H), 5.07-4.94 (s, 1H), 2.90-2.74 (m, 2H), 2.53 (s, 3H), 2.10-1.74 (m, 4H); HRMS: C21H21N3O requires M+H at m/z 332.1763; found, 332.1755; tR = 0.67 minutes, 100.0% purity. 8-Bromo-2-methyl-N-[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]-4-quinolinamine (3aa)


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N,N-Diisopropylethylamine (0.695 mL, 3.98 mmol) was added to 8-bromo-4-chloro-2-methylquinoline 2a (0.5104 g, 1.990 mmol) in dimethyl sulfoxide (6.63 mL) at room temperature, followed by (1S)-1,2,3,4-tetrahydro-1naphthalenamine (0.439 g, 2.98 mmol) and the solution was heated to 160 ºC and stirred for sixty-six hours. The reaction mixture was cooled, poured into ether, washed with water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (1:4) to give 8-bromo-2-methyl-N-[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]-4-quinolinamine 3aa (0.1043 g, 0.270 mmol, 14% yield). 1H NMR (400 MHz, CD3SOCD3) δ 8.31 (d, 1H, J = 8 Hz), 7.93 (d, 1H, J = 7 Hz), 7.35 (d, 1H, J = 9 Hz), 7.22-7.08 (m, 5H), 6.72 (s, 1H), 5.04-4.94 (s, 1H), 2.88-2.74 (m, 2H), 2.49 (s, 3H), 2.10-1.76 (m, 4H); LC-MS (LC-ES) M+H = 367. 2-Methyl-4-[(1S)-1,2,3,4-tetrahydro-1-naphthalenylamino]-8-quinolinecarboxamide (1ba)

N,N-Diisopropylethylamine (0.109 mL, 0.625 mmol) and hexamethyldisilazane (0.417 mL, 1.988 mmol) were added to 8-bromo-2-methyl-N-[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]-4-quinolinamine 3aa (0.1043 g, 0.284 mmol) in N,N-dimethylformamide (5.68 mL) at room temperature, followed by 1,3-bis(diphenylphosphino)propane (0.035 g, 0.085 mmol) and palladium(II) acetate (0.013 g, 0.057 mmol) and the solution fitted with a carbon monoxide balloon and heated to 110 ºC and stirred for sixteen hours. The reaction mixture was cooled, poured into ether, washed with 1 N sodium hydroxide and water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate to give 2-methyl-4-[(1S)-1,2,3,4tetrahydro-1-naphthalenylamino]-8-quinolinecarboxamide 1ba (0.0512 g, 0.147 mmol, 52% yield). 1H NMR (400


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MHz, CD3SOCD3) δ 11.22 (d, 1H, J = 5 Hz), 8.49 (d, 1H, J = 8 Hz), 8.47 (d, 1H, J = 8 Hz), 7.67 (d, 1H, J = 5 Hz), 7.53 (d, 1H, J = 8 Hz), 7.39 (t, 1H, J = 8 Hz), 7.22-7.08 (m, 4H), 6.75 (s, 1H), 5.07-4.94 (s, 1H), 2.90-2.74 (m, 2H), 2.53 (s, 3H), 2.10-1.74 (m, 4H); HRMS: C21H21N3O requires M+H at m/z 332.1763; found, 332.1759; tR = 0.67 minutes, 88.2% purity. 8-Bromo-N-(2,6-dichlorobenzyl)-N,2-dimethylquinolin-4-amine (3ab)

N,N-Di-iso-propylethylamine (0.317 mL, 1.822 mmol) was added to 8-bromo-4-chloro-2-methylquinoline 2a (0.1558 g, 0.607 mmol) in dimethyl sulfoxide (1.707 mL) at room temperature, followed by 1-(2,6-dichlorophenyl)N-methylmethanamine (0.173 g, 0.911 mmol) and the solution was heated at 150 °C for sixty-four hours. The reaction mixture was poured into ether and methanol, washed with water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (1:4) to give 8-bromo-N-(2,6-dichlorobenzyl)-N,2-dimethylquinolin-4-amine 3ab (0.0888 g, 0.206 mmol, 34% yield).

1

H

NMR (400 MHz, CD3SOCD3) δ 7.99 (d, 1H, J = 4 Hz), 7.97 (d, 1H, J = 4 Hz), 7.53 (s, 1H), 7.51 (s, 1H), 7.39 (dd, 1H, J = 8, 7 Hz), 7.31 (t, 1H, J = 8 Hz), 7.13 (s, 1H), 4.72 (br s, 2H), 2.82 (s, 3H), 2.61 (s, 3H); LC-MS (LC-ES) M+H = 410. 4-((2,6-Dichlorobenzyl)(methyl)amino)-2-methylquinoline-8-carboxamide (1bb)

1,1'-Bis(diphenylphosphino)ferrocene (0.024 g, 0.043 mmol) and bis(trimethylsilyl)amine (0.685 mL, 3.25 mmol) were added to 8-bromo-N-(2,6-dichlorobenzyl)-N,2-dimethylquinolin-4-amine 3ab (0.0888 g, 0.217 mmol) in N,N-


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dimethylformamide (1.480 mL) at room temperature, followed by tris(dibenzylideneacetone)dipalladium(0) (0.020 g, 0.022 mmol) and the solution was fitted with a carbon monoxide balloon and heated to 100 °C and stirred for two hours. The reaction mixture was cooled, poured into ether, washed with 1 N sodium hydroxide and water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (4:1) to give 4-((2,6-dichlorobenzyl)(methyl)amino)-2-methylquinoline-8-carboxamide 1bb (0.0571 g, 0.145 mmol, 67% yield). 1H NMR (400 MHz, CD3SOCD3) δ 10.74 (br d, 1H, J = 4 Hz), 8.48 (dd, 1H, J = 7, 1 Hz), 8.16 (dd, 1H, J = 8, 1 Hz), 7.78 (br d, 1H, J = 4 Hz), 7.56-7.48 (m, 3H), 7.40 (dd, 1H, J = 8, 7 Hz), 7.15 (s, 1H), 4.76 (s, 2H), 2.85 (s, 3H), 2.64 (s, 3H); HRMS: C19H17Cl2N3O requires M+H at m/z 374.0827; found, 374.0820; tR = 0.56 minutes, 98.9% purity. 2,6-Dichloro-N-(8-cyano-2-methyl-4-quinolinyl)benzamide (4ac)

Thionyl chloride (15 mL, 1.092 mmol) was added to 2,6-dichlorobenzoic acid (1043 mg, 5.46 mmol) and the reaction mixture was stirred under nitrogen at 70°C for 1 hour, then concentrated. The residue was dissolved in tetrahydrofuran (5 mL) and added to a solution of 4-amino-2-methyl-8-quinolinecarbonitrile 4h (200 mg, 1.092 mmol) and lithium hexamethyldisilazide (0.458 mL, 2.183 mmol) in tetrahydrofuran (20 mL) dropwise, during 15 minutes at -75 °C. The reaction mixture was stirred at -75 °C for 10 hours, then water (30 mL) was added, and the reaction mixture was extracted with ethyl acetate and concentrated. The residue was washed with diethyl ether to give 2,6-dichloro-N-(8-cyano-2-methyl-4-quinolinyl)benzamide 4ac (200 mg, 0.561 mmol, 51% yield) as a mixture with the bis-acylated 2,6-dichloro-N-(8-cyano-2-methylquinolin-4-yl)-N-(2,6-dichlorobenzoyl)benzamide. LC-MS (LC-ES) M+H = 356. 4-(2,6-Dichlorobenzamido)-2-methylquinoline-8-carboxamide (1bc)


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Sodium hydroxide (300 mg, 7.50 mmol) was added to 2,6-dichloro-N-(8-cyano-2-methyl-4-quinolinyl)benzamide 4ac containing 2,6-dichloro-N-(8-cyano-2-methylquinolin-4-yl)-N-(2,6-dichlorobenzoyl)benzamide (300 mg, 0.567 mmol combined) in dimethyl sulfoxide (10 mL). Then 30% hydrogen peroxide (3 mL, 29.4 mmol) was added. Once the reaction was complete, the reaction mixture was extracted with ethyl acetate and washed with water. The organic layer was dried over magnesium sulfate, filtered, and concentrated. The residue was purified by preparative high performance chromatography, eluting with acetonitrile:water with 10 mM ammonium bicarbonate (5:95 to 95:5) to give 4-(2,6-dichlorobenzamido)-2-methylquinoline-8-carboxamide 1bc (80 mg, 0.214 mmol, 38% yield) as white solid. 1H NMR (400 MHz, CD3SOCD3) δ 11.26 (br s, 1H), 10.43 (br d, 1H, J = 4 Hz), 8.57 (dd, 1H, J = 7, 1 Hz), 8.44 (dd, 1H, J = 8, 2 Hz), 8.08 (s, 1H), 7.89 (br d, 1H, J = 3 Hz), 7.72-7.52 (m, 4H), 2.76 (s, 3H); HRMS: C18H13Cl2N3O2 requires M+H at m/z 374.0463; found, 374.0452; tR = 1.61 minutes, 100.0% purity. 4-(2,6-Dichlorobenzyloxy)-2-methylquinoline-8-carbonitrile (4ad)

2-(Bromomethyl)-1,3-dichlorobenzene (86 mg, 0.358 mmol) was added to 4-hydroxy-2-methylquinoline-8carbonitrile 6 (55 mg, 0.289 mmol) in dry N,N-dimethylformamide (3 mL). Then, potassium carbonate (83 mg, 0.597 mmol) was added and the reaction mixture was stirred at 70 °C for 15 minutes. After cooling, the solid was collected by filtration and dried to give 4-(2,6-dichlorobenzyloxy)-2-methylquinoline-8-carbonitrile 4ad (102 mg, 0.299 mmol, 99% yield) as a white solid which was used directly in next reaction without further purification. LCMS (LC-ES) M+H = 342.


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4-((2,6-Dichlorobenzyl)oxy)-2-methylquinoline-8-carboxamide (1bd)

Cl

Cl O

N O

NH2 1bd

Potassium hydroxide (50 mg, 0.892 mmol) was added to 4-(2,6-dichlorobenzyloxy)-2-methylquinoline-8carbonitrile 4ad (102 mg, 0.297 mmol) in dimethyl sulfoxide (8 mL). Then, 30% hydrogen peroxide (3 mL) was added dropwise with stirring. The reaction mixture turned yellow and upon cooling a white solid appeared. The solid was collected by filtration, washed with methanol (5 mL), and dried to give 4-((2,6-dichlorobenzyl)oxy)-2methylquinoline-8-carboxamide (1bd) (75 mg, 0.206 mmol, 69% yield) as a white solid.

1

H NMR (400 MHz,

CD3SOCD3) δ 10.61 (br d, 1H, J = 3 Hz), 8.46 (dd, 1H, J = 7, 1 Hz), 8.11 (d, 1H, J = 4 Hz), 7.85 (br d, 1H, J = 4 Hz), 7.86-7.60 (m, 2H), 7.58-52 (m, 2H), 7.38 (d, 1H, J = 2 Hz), 5.54 (s, 2H), 2.73 (s, 3H); HRMS: C18H14Cl2N2O2 requires M+H at m/z 361.0510; found, 361.0510; tR = 1.75 minutes, 99.1% purity. 8-Bromo-4-((2,6-dichlorobenzyl)thio)-2-methylquinoline (3ac)

Sodium hydride (0.039 g, 0.969 mmol) was added to (2,6-dichlorophenyl)methanethiol (0.203 g, 1.050 mmol) in dimethyl sulfoxide (2.69 mL) at room temperature, followed by 8-bromo-4-chloro-2-methylquinoline 2a (0.2072 g, 0.808 mmol) and the solution was heated at 100 °C for four hours. The reaction mixture was poured into ether and methanol, washed with water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (1:6) to give 8-bromo-4-((2,6-dichlorobenzyl)thio)-2methylquinoline 3ac (0.1487 g, 0.342 mmol, 42% yield).

1

H NMR (400 MHz, CD3SOCD3) δ 8.11 (d, 1H, J = 7

Hz), 8.00 (d, 1H, J = 8 Hz), 7.66 (s, 1H), 7.57 (s, 1H), 7.55 (s, 1H), 7.46-7.38 (m, 2H), 4.65 (br s, 2H), 2.70 (s, 3H); LC-MS (LC-ES) M+H = 413.


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4-((2,6-Dichlorobenzyl)thio)-2-methylquinoline-8-carboxamide (1be)

1,1'-Bis(diphenylphosphino)ferrocene (0.040 g, 0.072 mmol) and bis(trimethylsilyl)amine (1.139 mL, 5.40 mmol) were added to 8-bromo-4-((2,6-dichlorobenzyl)thio)-2-methylquinoline 3ac (0.1487 g, 0.360 mmol) in N,Ndimethylformamide (2.460 mL) at room temperature, followed by tris(dibenzylideneacetone)dipalladium(0) (0.033 g, 0.036 mmol) and the solution was fitted with a carbon monoxide balloon and heated to 100 °C and stirred for two hours. The reaction mixture was cooled, poured into ether, washed with 1 N sodium hydroxide and water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (7:3) to give 4-((2,6-dichlorobenzyl)thio)-2-methylquinoline-8-carboxamide 1be (0.0602 g, 0.152 mmol, 42% yield). 1H NMR (400 MHz, CD3SOCD3) δ 10.39 (br d, 1H, J = 4 Hz), 8.54 (dd, 1H, J = 7, 1 Hz), 8.18 (dd, 1H, J = 8, 1 Hz), 7.89 (br d, 1H, J = 4 Hz), 7.70 (s, 1H), 7.63 (t, 1H, J = 8 Hz), 7.59 (s, 1H), 7.57 (s, 1H), 7.44 (t, 1H, J = 8 Hz), 4.67 (s, 2H), 2.74 (s, 3H); HRMS: C18H14Cl2N2OS requires M+H at m/z 377.0282; found, 377.0278; tR = 0.88 minutes, 100.0% purity. 8-Bromo-6-fluoro-2-methylquinolin-4-ol (5b)

2,2-Dimethyl-5-[1-(methylthio)ethylidene]-1,3-dioxane-4,6-dione (991.5 mg, 4.58 mmol) and 2-bromo-4fluoroaniline 8a (871 mg, 4.58 mmol) were suspended in diphenyl ether (20 mL) and heated in a microwave to 240 ºC for 30 minutes. The reaction was cooled to room temperature and the mixture was allowed to stand overnight. The precipitated solid was collected by filtration and washed with diethyl ether to give 8-bromo-6-fluoro-2-methyl4-quinolinol 5b (0.2263 g, 0.884 mmol, 19% yield) as a red solid. 1H NMR (400 MHz, CD3SOCD3) δ 10.59 (br s, 1H), 8.04 (dd, 1H, J = 9, 3 Hz), 7.76 (dd, 1H, J = 9, 3 Hz), 6.04 (br s, 1H), 2.46 (s, 3H).


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8-Bromo-4-chloro-6-fluoro-2-methylquinoline (2b)

8-Bromo-6-fluoro-2-methylquinolin-4-ol 5b (0.1g, 0.391 mmol) was added to phosphorus oxychloride (0.5 mL, 5.36 mmol) and heated to 90 °C for 2 hours. The reaction was cooled to room temperature and then poured over ice and let sit overnight. A brown precipitate formed and was collected, then washed with ethyl acetate. The organics were dried with magnesium sulfate, filtered, and concentrated under reduced pressure to give 8-bromo-4-chloro-6fluoro-2-methylquinoline 2b (83 mg, 0.302 mmol, 77% yield) as a brown solid. 1H NMR (400 MHz, CDCl3) δ 7.88 (dd, 1H, J = 8, 3 Hz), 7.82 (dd, 1H, J = 9, 3 Hz), 7.47 (s, 1H), 2.78 (s, 3H); LC-MS (LC-ES) M+H = 276. 8-Bromo-6-fluoro-2-methyl-N-(2,3,6-trichlorobenzyl)quinolin-4-amine (3ad)

8-Bromo-4-chloro-6-fluoro-2-methylquinoline 2b (0.083 g, 0.302 mmol), (2,3,6-trichlorophenyl)methanamine (0.046 mL, 0.363 mmol), and a drop of 6N aqueous hydrochloric acid (0.050 mL, 0.302 mmol) in N-methyl-2pyrrolidone (0.5 mL) were heated in a microwave at 150 °C for 6 hours. After cooling to room temperature, a precipitate formed, which was collected by filtration and washed with ethyl acetate to give 8-bromo-6-fluoro-2methyl-N-(2,3,6-trichlorobenzyl)quinolin-4-amine 3ad (40 mg, 0.089 mmol, 29%) as a pale solid.

1

H NMR (400

MHz, CD3OD) δ 8.30 (dd, 1H, J = 10, 3 Hz), 8.15 (dd, 1H, J = 7, 3 Hz), 7.80 (s, 1H), 7.62 (d, 1H, J = 9 Hz), 7.51 (d, 1H, J = 9 Hz), 7.03 (s, 1H), 5.02 (s, 2H), 2.85 (s, 3H); LC-MS (LC-ES) M+H = 449. 6-Fluoro-2-methyl-4-((2,3,6-trichlorobenzyl)amino)quinoline-8-carboxamide (1bf)


89


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A flask containing 8-bromo-6-fluoro-2-methyl-N-(2,3,6-trichlorobenzyl)quinolin-4-amine 3ad (0.04 g, 0.089 mmol), N,N-di-iso-propylethylamine (0.04 mL, 0.229 mmol), bis(trimethylsilyl)amine (0.13 mL, 0.620 mmol), palladium(II) acetate (4.00 mg, 0.018 mmol), and 1,3-bis(diphenylphosphino)propane (0.015 g, 0.036 mmol) in N,N-dimethylformamide (0.8 mL) was evacuated and filled with carbon monoxide (3×) via balloon. The reaction was heated to 120 °C for 3 hours, cooled to room temperature and poured into water. The reaction mixture was extracted with ethyl acetate (2×), the combined organics were washed with brine, dried over magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (1:1) to give 6-fluoro-2-methyl-4-((2,3,6-trichlorobenzyl)amino)quinoline-8-carboxamide 1bf (20 mg, 0.047 mmol, 53% yield) as a yellow solid. 1H NMR (400 MHz, CD3SOCD3) δ 11.09 (br d, 1H, J = 4 Hz), 8.32 (dd, 1H, J = 10, 3 Hz), 8.23 (dd, 1H, J = 10, 3 Hz), 7.91 (br d, 1H, J = 4 Hz), 7.75 (d, 1H, J = 9 Hz), 7.62 (d, 1H, J = 9 Hz), 7.25 (br t, 1H, J = 4 Hz), 6.71 (s, 1H), 4.66 (s, 2H), 2.59 (s, 3H); HRMS: C18H13Cl3FN3O requires M+H at m/z 412.0186; found, 412.0184; tR = 0.61 minutes, 97.4% purity. Methyl 6-bromo-4-hydroxy-2-methylquinoline-8-carboxylate (5c)

A mixture of methyl 2-amino-5-bromobenzoate (25.03 g, 109 mmol), methyl acetoacetate (23.5 mL, 218 mmol) and polyphosphoric acid (76.20 g) was heated to 160 ºC overnight and was cooled to room temperature. Water (400 mL) was added and the mixture was swirled vigorously until the solid lump had dissolved. The resulting mixture was extracted with methylene chloride (5×).

The combined organics were dried over sodium sulfate and

concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (3:2 to 9:1) to provide methyl 6-bromo-4-hydroxy-2-methylquinoline-8-carboxylate 5c (5.28 g, 17.83 mmol, 16% yield) as a


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yellow solid. 1H NMR (400 MHz, CD3SOCD3) δ 11.16 (br s, 1H), 8.39 (d, 1H, J = 2 Hz), 8.32 (d, 1H, J = 2 Hz), 6.13-6.04 (m, 1H), 3.97 (s, 3H), 2.43 (s, 3H).

Methyl 6-bromo-4-chloro-2-methyl-8-quinolinecarboxylate (2c)

Methyl 6-bromo-4-hydroxy-2-methylquinoline-8-carboxylate 5c (5.28 g, 17.28 mmol) and phosphorous oxychloride (17.0 mL, 182 mmol) were heated to 80 ºC for 60 minutes and cooled to room temperature. Ice was carefully added with external cooling. After all of the phosphorous oxychloride had been hydrolyzed, water (50 mL) was added. The mixture was basicified with 5N aqueous sodium hydroxide to pH = 8 and extracted with ethyl acetate (4×). The combined organics were dried over magnesium sulfate and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (1:19 to 1:4) to provide methyl 6-bromo-4-chloro-2-methyl-8quinolinecarboxylate 2c (2.38 g, 7.57 mmol, 42% yield) as a tan solid. 1H NMR (400 MHz, CDCl3) δ 8.45 (d, 1H, J = 2 Hz), 8.04 (d, 1H, J = 2 Hz), 7.45 (s, 1H), 4.04 (s, 3H), 2.72 (s, 3H).

Methyl 4-chloro-2,6-dimethyl-8-quinolinecarboxylate (2d)

Methyl 6-bromo-4-chloro-2-methyl-8-quinolinecarboxylate

2c

(0.2497

g,

0.794

mmol)

and

potassium

methyltrifluoroborate (0.1059 g, 0.868 mmol) were dissolved in tetrahydrofuran (8 mL) in a pressure tube (at this point, cesium fluoride was mistakenly added). Cesium carbonate (0.7888 g, 2.421 mmol) was added followed by [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane adduct (0.0638 g, 0.078 mmol). The tube was sealed and the mixture was heated to 85 ºC overnight and was cooled to room temperature. Water (50 mL) and brine (25 mL) were added and the mixture was extracted with diethyl ether (3×). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (1:19 to 1:4) to provide methyl 4-chloro-2,6-dimethyl-8-quinolinecarboxylate 2d


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(0.1320 g, 0.529 mmol, 67% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ 8.11-8.04 (m, 1H), 7.83 (d, 1H, J = 2 Hz), 7.39 (s, 1H), 4.04 (s, 3H), 2.71 (s, 3H), 2.57 (s, 3H).

4-Chloro-2,6-dimethyl-8-quinolinecarboxamide (2e)

Methyl 4-chloro-2,6-dimethyl-8-quinolinecarboxylate 2d (0.1320 g, 0.529 mmol) was suspended in 2M ammonia in methanol (5.0 mL) in a sealed tube. The mixture was heated to 100 ºC overnight and cooled to room temperature. A solid began to precipitate out of solution and the mixture was cooled further to 0 ºC. The solid was collected by filtration and washed with cold methanol to provide 4-chloro-2,6-dimethyl-8-quinolinecarboxamide 2e (0.0464 g, 0.198 mmol, 37% yield) as a white solid. 1H NMR (400 MHz, CD3SOCD3) δ 10.12 (br s, 1H), 8.44 (d, 1H, J = 2 Hz), 8.14 (dd, 1H, J = 2, 1 Hz), 7.92 (br s, 1H), 7.80 (s, 1H), 2.71 (s, 3H), 2.58 (s, 3H). 4-{[(2,6-Dimethylphenyl)methyl]amino}-2,6-dimethyl-8-quinolinecarboxamide (1bg)

4-Chloro-2,6-dimethyl-8-quinolinecarboxamide 2e (0.0464 g, 0.198 mmol) was suspended in dimethyl sulfoxide (1 mL) in a pressure tube.

2,6-Dimethylbenzylamine (0.040 mL, 0.287 mmol) was added, followed by N,N-

diisopropylethylamine (0.070 mL, 0.401 mmol). The tube was sealed and the mixture was heated to 140 ºC, then it was cooled to room temperature. Water (25 mL) was added and the solid was collected by filtration. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (1:6 to 4:1) to provide 4-{[(2,6dimethylphenyl)methyl]amino}-2,6-dimethyl-8-quinolinecarboxamide 1bg (0.0239 g, 0.072 mmol, 36% yield) as a yellow solid. 1H NMR (400 MHz, CD3SOCD3) δ 11.22 (br s, 1H), 8.37-8.26 (m, 2H), 7.66 (br s, 1H), 7.21-7.13 (m,


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1H), 7.11 (s, 2H), 6.98 (br s, 1H), 6.63 (s, 1H), 4.36 (d, 2H, J = 4 Hz), 2.56 (s, 3H), 2.42 (s, 3H), 2.34 (s, 6H); HRMS: C21H23N3O requires M+H at m/z 334.1919; found, 334.1909; tR = 0.69 minutes, 79.1% purity. 6-Bromo-4-chloro-2-methylquinoline-8-carboxamide (2f)

Methyl 6-bromo-4-chloro-2-methyl-8-quinolinecarboxylate 2c (0.6031 g, 1.917 mmol) was dissolved in 2M ammonia in methanol (30 mL) in a pressure flask. The flask was sealed and the mixture was heated to 100 ºC for 3 hours and cooled to room temperature. The precipitated solid was collected by filtration to provide 6-bromo-4chloro-2-methylquinoline-8-carboxamide 2f (0.2293 g, 0.765 mmol, 40% yield) as a yellow solid.

1

H NMR (400

MHz, CD3SOCD3) δ 9.88 (br s, 1H), 8.55 (d, 1H, J = 2 Hz), 8.48 (d, 1H, J = 2 Hz), 8.09 (br s, 1H), 7.91 (s, 1H), 2.72 (s, 3H). 6-Bromo-4-((2,6-dimethylbenzyl)amino)-2-methylquinoline-8-carboxamide (1bh)

6-Bromo-4-chloro-2-methyl-8-quinolinecarboxamide 2f (0.2293 g, 0.765 mmol) was suspended in dimethyl sulfoxide (4 mL) in a pressure tube.

2,6-Dimethylbenzyl amine (0.150 mL, 1.076 mmol) and N,N-

diisopropylethylamine (0.270 mL, 1.546 mmol) were added, the tube was sealed, and the mixture was heated to 140 ºC for 20 hours then cooled to room temperature. Water (100 mL) was added and the solid was collected by filtration. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (1:9 to 1:3) to provide 6-bromo-4-((2,6-dimethylbenzyl)amino)-2-methylquinoline-8-carboxamide 1bh (0.1986 g, 0.499 mmol, 65% yield) as a pale yellow solid. 1H NMR (400 MHz, CDC13) δ 11.71 (br s, 1H), 8.80 (d, 1H, J = 2 Hz), 7.86 (d, 1H, J = 2 Hz), 7.26-7.20 (m, 1H), 7.19-7.08 (m, 2H), 6.55 (s, 1H), 6.17-6.05 (m, 1H), 4.66 (br s, 1H), 4.40 (d, 2H, J


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= 4 Hz), 2.67 (s, 3H), 2.41 (s, 6H); HRMS: C20H20BrN3O requires M+H at m/z 398.0868; found, 398.0870; tR = 0.66 minutes, 92.8% purity. 4-Chloro-2-methyl-6-phenyl-8-quinolinecarboxamide (2g)

6-Bromo-4-chloro-2-methyl-8-quinolinecarboxamide 2f (0.2024 g, 0.676 mmol) was dissolved in tetrahydrofuran (1.5 mL). Phenylboronic acid (0.0896 g, 0.735 mmol) was added, followed by 2M aqueous potassium carbonate (0.670 mL, 1.34 mmol), and bis(triphenylphosphine)palladium(II) chloride (0.0266 g, 0.038 mmol). The resulting mixture was heated to 80 ºC for 60 minutes, then cooled to room temperature and stirred overnight. Brine (25 mL) and water (25 mL) were added and the mixture was extracted with ethyl acetate (3×). The combined organics were dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (3:7 to 9:11) to provide 4-chloro-2-methyl-6-phenyl-8-quinolinecarboxamide 2g (0.0907 g, 0.306 mmol, 45% yield) as a pale yellow solid. 1H NMR (400 MHz, CD3SOCD3) δ 10.10 (d, 1H, J = 3 Hz), 8.87 (d, 1H J = 2 Hz), 8.51 (d, 1H, J = 2 Hz), 8.05 (d, 1H, J = 3 Hz), 7.92-7.81 (m, 3H), 7.67-7.53 (m, 2H), 7.53-7.44 (m, 1H), 2.76 (s, 3H). 4-{[(2,6-Dimethylphenyl)methyl]amino}-2-methyl-6-phenyl-8-quinolinecarboxamide (1bi)

4-Chloro-2-methyl-6-phenyl-8-quinolinecarboxamide 2g (0.1611 g, 0.543 mmol) was suspended in dimethyl sulfoxide (4 mL) in a pressure tube. 2,6-Dimethylbenzylamine (0.110 mL, 0.789 mmol) was added, followed by N,N-diisopropylethylamine (0.190 mL, 1.086 mmol). The tube was sealed and the mixture was heated to 140 ºC overnight, then cooled to room temperature. Water (25 mL) was added and the solid was collected by filtration and


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dried. The material was triturated with ethanol and filtered. The solid was purified by silica gel chromatography (2:3 to 7:3 ethyl acetate:hexanes. The resulting residue was recrystallized from ethyl acetate to provide 4-{[(2,6dimethylphenyl)methyl]amino}-2-methyl-6-phenyl-8-quinolinecarboxamide 1bi (0.0092 g, 0.023 mmol, 4.3% yield) as a white solid. 1H NMR (400 MHz, CD3SOCD3) δ 11.23 (br s, 1H), 8.76 (d, 2H, J = 9 Hz), 7.84-7.76 (m, 3H), 7.49 (t, 2H, J = 8 Hz), 7.44-7.34 (m, 2H), 7.16 (d, 1H, J = 7 Hz), 7.14-7.07 (m, 2H), 6.71 (s, 1H), 4.41 (d, 2H, J = 5 Hz), 2.60 (s, 3H), 2.36 (s, 6H); HRMS: C26H25N3O requires M+H at m/z 396.2076; found, 396.2072; tR = 0.82 minutes, 91.4% purity. N-[(2,3-Dichlorophenyl)methyl]-2-methyl-4-quinolinamine (1bj)

4,4,4',4',5,5,5',5'-Octamethyl-2,2'-bi-1,3,2-dioxaborolane (0.160 g, 0.629 mmol) was added to 8-bromo-N-[(2,3dichlorophenyl)methyl]-2-methyl-4-quinolinamine 1bk (0.1661 g, 0.419 mmol) in 1,4-dioxane (8.39 mL) under nitrogen at room temperature. Then, potassium acetate (0.185 g, 1.887 mmol) was added, followed by PdCl2(dppf) (0.015 g, 0.021 mmol) and the reaction was evacuated and purged with nitrogen, then heated to 80 °C and stirred for sixteen hours. The reaction mixture was filtered through Celite®, concentrated, and the crude residue was purified via column chromatography eluting with methanol:ethyl acetate (1:4) to give the debromonated product N-[(2,3dichlorophenyl)methyl]-2-methyl-4-quinolinamine 1bj (0.0624 g, 0.187 mmol, 45% yield), instead of the desired boronate ester. 1H NMR (400 MHz, CD3SOCD3) δ 8.22 (d, 1H, J = 8 Hz), 7.77 (br t, 1H, J = 6 Hz), 7.71 (d, 1H, J = 8 Hz), 7.59 (t, 1H, J = 7 Hz), 7.56 (d, 1H, J = 8 Hz), 7.40 (t, 1H, J = 8 Hz), 7.30 (t, 1H, J = 8 Hz), 7.25 (d, 1H, J = 7 Hz), 6.18 (s, 1H), 4.61 (d, 2H, J = 6 Hz), 2.36 (s, 3H); HRMS: C17H14Cl2N2 requires M+H at m/z 317.0612; found, 317.0593; tR = 0.63 minutes, 100.0% purity. 8-Bromo-N-[(2,3-dichlorophenyl)methyl]-2-methyl-4-quinolinamine (1bk)


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N,N-Diisopropylethylamine (2.104 mL, 12.05 mmol) was added to 8-bromo-4-chloro-2-methylquinoline 2a (1.03 g, 4.02 mmol) in dimethyl sulfoxide (8.03 mL) at room temperature, followed by [(2,3-dichlorophenyl)methyl]amine (0.803 mL, 6.02 mmol) and the solution was heated to 140 °C and stirred for sixty-eight hours. The reaction mixture was cooled, poured into ether, washed with water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (1:4) to give 8-bromo-N[(2,3-dichlorophenyl)methyl]-2-methyl-4-quinolinamine 1bk (1.26 g, 3.02 mmol, 75% yield). 1H NMR (400 MHz, CD3SOCD3) δ 8.26 (d, 1H, J = 8 Hz), 7.97 (d, 1H, J = 7 Hz), 7.89 (br t, 1H, J = 6 Hz), 7.57 (dd, 1H, J = 8, 2 Hz), 7.31 (dd, 1H, J = 8, 2 Hz), 7.29 (dd, 1H, J = 8, 2 Hz), 7.24 (dd, 1H, J = 7, 1 Hz), 6.29 (s, 1H), 4.62 (d, 2H, J = 6 Hz), 2.41 (s, 3H); HRMS: C17H13BrCl2N2 requires M+H at m/z 394.9717; found, 394.9703; tR = 0.77 minutes, 92.0% purity. 4-{[(2,3-Dichlorophenyl)methyl]amino}-N,2-dimethyl-8-quinolinecarboxamide (1bl)

N,N-Diisopropylethylamine (0.076 mL, 0.434 mmol) and methylamine (0.690 mL, 1.380 mmol) were added to 8bromo-N-[(2,3-dichlorophenyl)methyl]-2-methyl-4-quinolinamine

1bk (0.0781

g,

0.197

mmol)

in

N,N-

dimethylformamide (3.18 mL) at room temperature, followed by 1,3-bis(diphenylphosphino)propane (0.024 g, 0.059 mmol) and palladium(II) acetate (8.85 mg, 0.039 mmol) and the solution fitted with a carbon monoxide balloon and heated to 110 °C and stirred for four hours. The reaction mixture was cooled, poured into ether, washed with 1 N sodium hydroxide and water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate to give 4-{[(2,3-dichlorophenyl)methyl]amino}-N,2-dimethyl-


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8-quinolinecarboxamide 1bl (0.0466 g, 0.118 mmol, 60% yield). 1H NMR (400 MHz, CD3SOCD3) δ 11.69 (br q, 1H, J = 5 Hz), 8.51 (d, 1H, J = 7 Hz), 8.44 (d, 1H, J = 8 Hz), 8.06 (br t, 1H, J = 6 Hz), 7.58 (d, 1H, J = 8 Hz), 7.51 (t, 1H, J = 8 Hz), 7.31 (t, 1H, J = 8 Hz), 7.26 (d, 1H, J = 7 Hz), 6.33 (s, 1H), 4.65 (d, 2H, J = 6 Hz), 2.93 (d, 3H, J = 5 Hz), 2.47 (s, 3H); HRMS: C19H17Cl2N3O requires M+H at m/z 374.0827; found, 374.0821; tR = 0.65 minutes, 100.0% purity. 4-{[(2,3-Dichlorophenyl)methyl]amino}-N,N,2-trimethyl-8-quinolinecarboxamide (1bm)

N,N-Diisopropylethylamine (0.080 mL, 0.460 mmol) and dimethylamine (0.733 mL, 1.465 mmol) were added to 8bromo-N-[(2,3-dichlorophenyl)methyl]-2-methyl-4-quinolinamine

1bk (0.0829

g,

0.209

mmol)

in

N,N-

dimethylformamide (3.37 mL) at room temperature, followed by 1,3-bis(diphenylphosphino)propane (0.026 g, 0.063 mmol) and palladium(II) acetate (9.40 mg, 0.042 mmol) and the solution fitted with a carbon monoxide balloon and heated to 110 °C and stirred for four hours. The reaction mixture was cooled, poured into ether, washed with 1 N sodium hydroxide and water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica

gel

chromatography,

eluting

with

methanol:ethyl

acetate

(1:19)

to

give

4-{[(2,3-

dichlorophenyl)methyl]amino}-N,N,2-trimethyl-8-quinolinecarboxamide 1bm (0.0399 g, 0.098 mmol, 47% yield). 1

H NMR (400 MHz, CD3SOCD3) δ 8.25 (d, 1H, J = 8 Hz), 7.82 (br t, 1H, J = 6 Hz), 7.57 (d, 1H, J = 7 Hz), 7.45 (d,

1H, J = 7 Hz), 7.41 (t, 1H, J = 8 Hz), 7.31 (t, 1H, J = 8 Hz), 7.27 (t, 1H, J = 7 Hz), 6.22 (s, 1H), 4.62 (d, 2H, J = 6 Hz), 2.88 (s, 3H), 2.63 (s, 3H), 2.34 (s, 3H); HRMS: C20H19Cl2N3O requires M+H at m/z 388.0983; found, 388.0970; tR = 0.60 minutes, 100.0% purity. Methyl 4-{[(2,3-dichlorophenyl)methyl]amino}-2-methyl-8-quinolinecarboxylate (1bn)


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N,N-Diisopropylethylamine (0.212 mL, 1.215 mmol) and methanol (0.447 mL, 11.04 mmol) were added to 8bromo-N-[(2,3-dichlorophenyl)methyl]-2-methyl-4-quinolinamine 1bk ( 0.2187 g, 0.552 mmol) in N,Ndimethylformamide (10.38 mL) at room temperature, followed by 1,3-bis(diphenylphosphino)propane (0.068 g, 0.166 mmol) and palladium(II) acetate (0.025 g, 0.110 mmol) and the solution fitted with a carbon monoxide balloon and heated to 110 °C and stirred for two hours. The reaction mixture was cooled, poured into ether, washed with saturated sodium bicarbonate and water, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with methanol:ethyl acetate (1:4) to give methyl 4-{[(2,3dichlorophenyl)methyl]amino}-2-methyl-8-quinolinecarboxylate 1bn (0.1446 g, 0.366 mmol, 66% yield). 1H NMR (400 MHz, CD3SOCD3) δ 8.35 (d, 1H, J = 8 Hz), 7.86 (br s, 1H), 7.69 (d, 1H, J = 7 Hz), 7.57 (d, 1H, J = 8 Hz), 7.43 (t, 1H, J = 8 Hz), 7.30 (t, 1H, J = 8 Hz), 7.25 (d, 1H, J = 7 Hz), 6.25 (s, 1H), 4.62 (d, 2H, J = 5 Hz), 3.84 (s, 3H), 2.35 (s, 3H); HRMS: C19H16Cl2N2O2 requires M+H at m/z 375.0667; found, 375.0667; tR = 0.68 minutes, 100.0% purity. 4-{[(2,3-Dichlorophenyl)methyl]amino}-2-methyl-8-quinolinecarboxylic acid (1bo)

Lithium hydroxide (3.91 mg, 0.163 mmol) was added to methyl 4-{[(2,3-dichlorophenyl)methyl]amino}-2-methyl8-quinolinecarboxylate 1bn ( 0.0438 g, 0.117 mmol) in tetrahydrofuran (0.778 mL), methanol (0.778 mL), and water (0.778 mL) and the reaction mixture was stirred for eighteen hours at room temperature. After concentration, the residue was purified by reverse phase column chromatography eluting with acetonitrile:water (25:75 to 100:0) with 0.1% trifluoroacetic acid to give 4-{[(2,3-dichlorophenyl)methyl]amino}-2-methyl-8-quinolinecarboxylic acid


98

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1bo (0.0331 g, 0.066 mmol, 57% yield). 1H NMR (400 MHz, CD3SOCD3) δ 9.45 (br s, 1H), 8.74 (d, 1H, J = 8 Hz), 8.57 (d, 1H, J = 8 Hz), 7.77 (t, 1H, J = 8 Hz), 7.64 (dd, 1H, J = 8, 4 Hz), 7.40-7.32 (m, 2H), 6.82 (s, 1H), 4.84 (d, 2H, J = 6 Hz), 2.66 (s, 3H); HRMS: C18H14Cl2N2O2 requires M+H at m/z 361.0510; found, 361.0507; tR = 0.66 minutes, 100.0% purity. (4-{[(2,3-Dichlorophenyl)methyl]amino}-2-methyl-8-quinolinyl)methanol (1bp)

Lithium borohydride (0.121 mL, 0.241 mmol) was added to methyl 4-{[(2,3-dichlorophenyl)methyl]amino}-2methyl-8-quinolinecarboxylate 1bn ( 0.0905 g, 0.241 mmol) in tetrahydrofuran (2.291 mL) at 0 ºC and the solution was allowed to warm to room temperature and stirred for two hours. The reaction mixture was quenched with methanol, then water, and the reaction mixture was extracted with diethyl ether, dried over magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography, eluting with ethyl acetate to give (4-{[(2,3-dichlorophenyl)methyl]amino}-2-methyl-8-quinolinyl)methanol 1bp (0.0564 g, 0.154 mmol, 64% yield). 1

H NMR (400 MHz, CD3SOCD3) δ 8.11 (d, 1H, J = 8 Hz), 7.75 (br t, 1H, J = 6 Hz), 7.64 (d, 1H, J = 7 Hz), 7.56 (d,

1H, J = 8 Hz), 7.38 (t, 1H, J = 8 Hz), 7.29 (t, 1H, J = 8 Hz), 7.24 (d, 1H, J = 8 Hz), 6.20 (s, 1H), 5.32 (br s, 1H), 5.00 (s, 2H), 4.61 (d, 2H, J = 6 Hz), 2.37 (s, 3H); HRMS: C18H16Cl2N2O requires M+H at m/z 347.0718; found, 347.0716; tR = 0.72 minutes, 96.6% purity. 8-(Aminomethyl)-N-[(2,3-dichlorophenyl)methyl]-2-methyl-4-quinolinamine (1bq)


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Methane sulfonic anhydride (0.024 g, 0.137 mmol) was added to (4-{[(2,3-dichlorophenyl)methyl]amino}-2methyl-8-quinolinyl)methanol 1bp (0.0397 g, 0.114 mmol) in tetrahydrofuran (3.48 mL) at room temperature. Then, the reaction mixture was stirred for forty-one hours at room temperature. Then, ammonia (0.327 mL, 2.287 mmol) was added and the reaction was stirred for seventeen hours at room temperature. The reaction mixture was concentrated. The residue was purified by reverse phase high performance liquid chromatography, eluting with water:acetonitrile

with

0.05%

trifluoroacetic

acid

(1:3

to

0:1)

to

give

8-(aminomethyl)-N-[(2,3-

dichlorophenyl)methyl]-2-methyl-4-quinolinamine bis(trifluoroacetate) 1bq (0.0197 g, 0.033 mmol, 29% yield). 1H NMR (400 MHz, CD3SOCD3) δ 12.70 (br s, 1H), 9.64 (br s, 1H), 8.57 (d, 1H, J = 6 Hz), 8.41 (br s, 3H), 8.02 (s, 1H), 7.76 (s, 1H), 7.64 (d, 1H, J = 7 Hz), 7.35 (t, 1H, J = 8 Hz), 7.29 (d, 1H, J = 7 Hz), 6.85 (s, 1H), 4.86 (s, 2H), 4.61 (s, 2H), 2.67 (s, 3H); HRMS: C18H17Cl2N3 requires M+H at m/z 346.0878; found, 346.0870; tR = 0.46 minutes, 100.0% purity. 4-{[(2,3-Dichlorophenyl)methyl]amino}-2-methyl-8-quinolinecarbonitrile (1br)

Belleau's Reagent (2,4-bis[4-(phenyloxy)phenyl]-1,3,2,4-dithiadiphosphetane 2,4-disulfide) (0.366 g, 0.693 mmol) was added to 4-{[(2,3-dichlorophenyl)methyl]amino}-2-methyl-8-quinolinecarboxamide 1q (0.0832 g, 0.231 mmol) in 1,4-dioxane (4.62 mL) at room temperature, and the reaction mixture was heated at reflux for sixteen hours. The reaction mixture was cooled and the solution was purified by silica gel chromatography, eluting with ethyl acetate:hexanes (2:3) to give an impure mixture that was further purified by reverse phase high performance liquid chromatography, eluting with acetonitrile:water (1:3 to 3:1) to give 4-{[(2,3-dichlorophenyl)methyl]amino}-2methyl-8-quinolinecarbonitrile 1br (0.0237 g, 0.066 mmol, 28% yield), instead of the thioamide.

1

H NMR (400

MHz, CD3SOCD3) δ 8.56 (d, 1H, J = 8 Hz), 8.16 (d, 1H, J = 8 Hz), 8.07 (br t, 1H, J = 6 Hz), 7.58 (d, 1H, J = 8 Hz), 7.52 (t, 1H, J = 8 Hz), 7.31 (t, 1H, J = 6 Hz), 7.27 (d, 1H, J = 7 Hz), 6.37 (s, 1H), 4.63 (d, 2H, J = 6 Hz), 2.44 (s, 3H); HRMS: C18H13Cl2N3 requires M+H at m/z 342.0565; found, 342.0554; tR = 0.74 minutes, 94.6% purity.


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4-Chloro-2-methyl-8-quinolinesulfonamide (2h)

4-Chloro-2-methyl-8-quinolinesulfonyl chloride 2h19 (34 mg, 0.123 mmol) was placed in a reaction vial and dissolved in 0.5M ammonia in dioxane (3 mL, 1.50 mmol). The reaction vial was sealed and the mixture was heated at 60 ºC while stirring for 1 hour. A white precipitate formed during the reaction. After cooling to room temperature, the mixture was evaporated to dryness. The remaining solid was triturated with hexane, collected by vacuum filtration, washed with water and dried in vacuo to give 4-chloro-2-methyl-8-quinolinesulfonamide 2i (26 mg, 0.101 mmol, 82%) as a white solid. 1H NMR (400 MHz, CD3SOCD3) δ 8.42 (dd, 1H, J = 8, 1 Hz), 8.36 (dd, 1H, J = 7, 1 Hz), 7.89 (s, 1H), 7.81 (t, 1H, J = 8 Hz), 7.24 (s, 2H), 2.77 (s, 3H); LC-MS (LC-ES) M+H = 257. 4-{[(2,3-Dichlorophenyl)methyl]amino}-2-methyl-8-quinolinesulfonamide (1bs)

4-Chloro-2-methyl-8-quinolinesulfonamide 2i (23.5 mg, 0.092 mmol) was placed in a reaction vial and dissolved in N-methyl pyrrolidinone (0.5 mL). 2,3-Dichlorobenzylamine (33 mg, 0.188 mmol) was added to the mixture. The vial was sealed and the mixture was heated at 150 ºC while stirring overnight. After cooling to room temperature, the mixture was diluted with ethyl acetate, washed with saturated aqueous sodium bicarbonate, dried over sodium sulfate and filtered. The filtrate was evaporated onto silica gel and chromatographed on silica gel, eluting with ethyl acetate:hexanes (1:4 to 9:1) to give impure product. This material was further purified by reverse phase high performance liquid chromatography, eluting with a acetonitrile:water with 0.1% trifluoroacetic acid (3:7 to 1:0) and concentrated. The remaining material was dissolved in ethyl acetate, washed with 1N aqueous sodium hydroxide, brine, dried over sodium sulfate, filtered, and concentrated to give 4-{[(2,3-dichlorophenyl)methyl]amino}-2-


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methyl-8-quinolinesulfonamide 1bs (17 mg, 0.0429 mmol, 47%) as a white solid. 1H NMR (400 MHz, CD3SOCD3) δ 8.53 (d, 1H, J = 8 Hz), 8.16 (d, 1H, J = 7 Hz), 8.11 (t, 1H, J = 6 Hz), 7.58 (dd, 1H, J = 8, 1 Hz), 7.54 (t, 1H, J = 8 Hz), 7.35-7.29 (m, 1H), 7.27-7.22 (m, 3H), 6.38 (s, 1H), 4.66 (d, 2H, J = 5.5 Hz), 2.47 (s, 3H); HRMS: C17H15Cl2N3O2S requires M+H at m/z 396.0340; found, 396.0343; tR = 0.59 minutes, 95.3% purity. Biochemical assay details for the pIC50 determinations against the human CD38 enzyme. CD38 inhibitors were tested for their capacity to inhibit human CD38 enzyme activity in a colorimetric based assay.24 The extracellular domain of human CD38 was expressed in pichia pastoris and purified to homogeneity. The enzyme activity assay was performed in a low-volume 384-well plate in a total volume of 20 µL. A range of concentrations of test compound in 200 nL of dimethyl sulfoxide was delivered into the assay plate wells. Columns 6 and 18 of the plate contained dimethyl sulfoxide with no compound and served as the high signal and low signal controls (no CD38 added), respectively. All additions of assay reagents to the plate were done using a Multidrop Combi, and the plate was shaken 3-5 seconds after each addition. CD38 (0.8 nM) was incubated with test compound in 10 µL containing 100 mM HEPES, pH 7.4, 4 mM EDTA, and 1 mM CHAPS for 30 minutes prior to initiation of the reaction. The reaction was initiated by a 10 µL addition containing 5 mM sodium acetate, pH 4.5, 1 mM CHAPS, 200 µM NAD and 500 µM GW323424X. The solutions for each of the two additions were prepared fresh each day from concentrated stocks of the individual components. The final concentrations in the assay were 50 mM HEPES, 2 mM EDTA, 1 mM CHAPS, and 2.5 mM sodium acetate, 100 µM NAD, 250 µM GW323434X, and 0.4 nM CD38. GW323434X is a 4-pyridynal compound that acts as a nucleophile that participates in the base exchange reaction with the nicotinamide on NAD to form a novel dinucleotide that absorbs at 405 nm. Catalytic formation of this novel chromophore was followed in an Envision microplate reader by reading absorbance at two time points, typically 30 minutes apart within the first 45 minutes of the reaction. These time points were established empirically to ensure the rates determined were in a linear range of product formation. Data analysis was performed in the following way using Abase XE. The data from the 15 and 45 minute reads was processed by performing a subtraction function of 45 minute read value minus 15 minute read value for each plate well. The resulting values for non-control wells were converted to % inhibition using the formula 100*((U-C1)/(C2-C1)) where U is the value of the test well, C1 is the average of the values of the high signal (column 6) control wells, and C2 is the average of the values of the low signal (column 18) control wells. Percent inhibition (y) was plotted versus inhibitor concentration (x), and curve fitting was performed with the following four parameter equation: y = A+((B-


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A)/(1+(10^x/10^C)^D)), where A is the minimum response, B is the maximum response, C is the log10IC50, and D is the Hill slope. The results for each compound were recorded as pIC50 values (-C in the above equation). Crystallization conditions for mutated human CD38 (E226Q) complexed with 4-{[(2,6dimethylphenyl)methyl]amino}-2-methyl-8-quinolinecarboxamide (1a) and ADP-ribose (ADPR) (PDP Code #4XJT). Apo human CD38 (E226Q) protein was concentrated to 7mg/mL. 5mM NADP was added 1 hour prior to setting up trays. Crystals were grown by hanging drop vapor diffusion in 24 well Linbro trays from 23% PEG3350, 0.1 M BTP pH 8.5 at 22 ºC (2+2µL drops over a 500 µL well). After allowing the crystals to grow for several days, they were soaked with 5mM 4-{[(2,6-dimethylphenyl)methyl]amino}-2-methyl-8-quinolinecarboxamide 1a for 1 day. Prior to data collection, crystals were flash frozen in paraffin oil. Crystallization conditions for wild-type human CD38 complexed with 6-fluoro-2-methyl-4-((2,3,6trichlorobenzyl)amino)quinoline-8-carboxamide (1bf) and the oxonium remnant of (2S,3R,4R)-2-deoxy-2fluoro-D-ribose 5-phosphate (FR5P) (PDP Code #4XJS). Apo human CD38 protein was concentrated to ~7mg/mL. Crystals were grown in 24 well hanging drop Linbro plates from 23% PEG3350, 0.1mM BTP pH 8.5 at 22 ºC (2+2µL drops over a 500 µL well). Crystals were soaked with ~5mM 6-fluoro-2-methyl-4-((2,3,6trichlorobenzyl)amino)quinoline-8-carboxamide 1bf and ~5mM FR5P for 1 day. Prior to data collection, crystals were flash frozen in paraffin oil. NAD tissue extraction and analysis procedure. All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed by the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed. All samples were snap frozen in liquid nitrogen to minimize the degradation on NAD before sample analysis. Samples were diluted 1:4 with 80:20 acetonitrile:water that contained 18O labeled NAD and a CD38 inhibitor to be used as an internal standard. Tissue were then homogenized in a bead beater with metal beads and then centrifuged. The sample extract was diluted 1:10 with water and injected on Zorbax Hillic Plus C18 column monitoring the 664-428 transition for NAD+ and 668-136 for 18O NAD internal standard. The samples were injected on a C18 using reverse phase chromatography to obtain the relevant drug concentration and understand drug distribution in different tissues. Corresponding Author


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David N. Deaton. Phone: 919-483-6270. E-mail: [email protected]. Notes The authors declare no competing financial interest. Acknowledgements The authors would like to thank the following chemists at ChemPartners CRO for synthetic assistance: C. Chuantao, L. Huifeng, W. Lu, D. Qiu, S. Ran, Z. Tao, Y. Wei, H. Xiao, C. Yanyan, L. Yuanxin, Z. Yunpeng, H. Zeng, L. Zhichao. The authors would also like to thank Iris V. Paulus for determination of high resolution mass determinations and Yingnian Shen, David Taylor, Jamey Christie, Scott Sigethy, Angela Rauer, George Barrett, Tim Broderick, Luke Carter, and Kurt Weaver for the cloning, expression, and purification of the CD38 protein that was used in the biochemical assays. References (1)

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