Discovery of novel 6,6-heterocycles as transient receptor potential vanilloid (TRPV1) antagonists

3330 J. Med. Chem. 2010, 53, 3330–3348

DOI: 10.1021/jm100051g

Discovery of Novel 6,6-Heterocycles as Transient Receptor Potential Vanilloid (TRPV1) Antagonists

Charles A. Blum,^†Timothy Caldwell,^†Xiaozhang Zheng,^†Rajagopal Bakthavatchalam,^†Scott Capitosti,^†Harry Brielmann,^†

†

Stephane De Lombaert, Mark T. Kershaw, David Matson, James E. Krause, Daniel Cortright, Marci Crandall,

†

ꢀ

William J. Martin,^‡Beth Ann Murphy,^‡Susan Boyce,^‡A. Brian Jones,^‡Glenn Mason,^‡Wayne Rycroft,^‡Helen Perrett,^‡

Rachael Conley,^‡Nicola Burnaby-Davies,^‡Bertrand L. Chenard,^†and Kevin J. Hodgetts*^,†

‡

^†Neurogen Corporation, 35 Northeast Industrial Road, Branford, Connecticut 06405, and Merck Sharp and Dohme Limited,

Hertford Road, Hoddesdon Herts EN11 9BU, U.K.

Received January 14, 2010

The transient receptor potential cation channel, subfamily V, member 1 (TRPV1) is a nonselective

cation channel that can be activated by a wide range of noxious stimuli, including capsaicin, acid, and

heat. Blockade of TRPV1 activation by selective antagonists is under investigation in an attempt to

identify novel agents for pain treatment. The design and synthesis of a series of novel TRPV1

antagonists with a variety of different 6,6-heterocyclic cores is described, and an extensive evaluation

of the pharmacological and pharmacokinetic properties of a number of these compounds is reported.

For example, the 1,8-naphthyridine 52 was characterized as an orally bioavailable and brain penetrant

TRPV1 antagonist. In vivo, 52 fully reversed carrageenan-induced thermal hyperalgesia (CITH) in rats

and dose-dependently potently reduced complete Freund’s adjuvant (CFA) induced chronic inflam-

matory pain after oral administration.

Introduction

greater than 50 °C.⁷In addition to the treatment of inflam-

matory pain, evidence suggests that a number of other

disorders, including urinary urge incontinence, cough, and

irritable bowel syndrome, may be treatable through modula-

tion of TRPV1 signaling.⁹

The cloning of TRPV1^a(transient receptor potential cation

channel, subfamily V, member 1) by Julius and colleagues in

1997 represented a landmark in the understanding of the

molecular mechanisms that underpin the transduction of

noxious thermal and chemical stimuli by sensory neurons.¹

TRPV1 is a nonselective cation channel, predominantly ex-

pressed by peripheral nociceptors, that is activated by a

diverse range of stimuli including protons (pH < 6.0), heat

(> 43 °C), and noxious chemicals such as capsaicin, the

ingredient responsible for the pungency of chili peppers, and

resiniferatoxin, isolated from the latex of the cactus-like

perennial Euphorbia resinifera. TRPV1 is also activated or

potentiated by anandamide,²other endovanilloids, notably

N-arachidonyldopamine and N-oleoyldopamine,³lipoxyge-

nase products such as 12-hydroperoxyeicosatetraenoic acid,⁴

and toxins from spiders⁵and jellyfish.⁶These diverse stimuli

not only directly activate TRPV1 but also sensitize and reduce

theactivation thresholdsof thechannel toother stimuli, which

leads to an influx of calcium and sodium ions through the

channel, causing depolarization of the cell and the transmis-

sion of painful stimuli. TRPV1 knockout mice have shown

reduced thermal hyperalgesia in several models of inflamma-

tory pain⁸and also exhibited no change in physiological heat

sensation in the hot-plate test at 50 °C.⁷However, TRPV1-/-

mice did exhibit significantly longer (1.7- to 2.5-fold) res-

ponse latencies than wild-type mice at hot-plate temperatures

The identification of selective and potent antagonists of

TRPV1 has become a focus of attention within the pharma-

ceutical industry,¹⁰and prominent examples of extensively

characterized TRPV1 antagonists are depicted in Figure 1.

The urea 1 (SB-705498) and pyrimidine 2 (AMG 517) were

among the first TRPV1 antagonists reported to have reached

clinical trials. The potent and selective pyrrolidine urea 1¹¹

was discovered following optimization of the activity and

pharmacokinetics of a simple 1,3-disubstituted arylurea series

identified from screening of the GSK library.¹²In healthy

human volunteers, 1 was reported to significantly reduce both

(i) capsaicin-evoked flare and heat-evoked pain on nonsensi-

tized skin and (ii) heat-evoked pain after ultraviolet B-evoked

inflammation.¹³The pyrimidine 2 was identified by Amgen

scientists following a sequence of modifications from a simple

cinnamide lead.¹⁴In rats, 2 blocked capsaicin-induced flinch-

ing and reversed thermal hyperalgesia in the complete

Freund’s adjuvant (CFA) induced pain model. In humans, 2

has a long half-life although other analogues with improved

physicochemical and pharmacokinetic properties have been

reported bythe samegroup.^14dIn addition toa long half-life, 2

was reported to elicit marked but reversible hyperthermia in

humans in a plasma concentration-dependent manner.¹⁵Re-

cently, researchers at Abbott have described the discovery¹⁶

and characterization¹⁷of the clinical candidate ABT-102 (3),

identified following optimization of an original 7-hydroxy-

naphthalene urea lead. Compound 3 exhibited potent oral

analgesic efficacy in a number of rodent pain models including

*To whom correspondence should be addressed. Phone: 1-203-675-

7540. Fax: 617-374-1320. E-mail: kjhodgetts@aol.com. Present address:

Galenea, 300 Technology Square, Cambridge, Massachusetts.

^aAbbreviations: TRPV1, transient receptor potential vanilloid 1;

CFA, complete Freund’s adjuvant; CITH, carrageenan-induced ther-

mal hyperalgesia; TFMA, 4-trifluoromethylaniline.

r

pubs.acs.org/jmc

Published on Web 03/22/2010

2010 American Chemical Society

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3331

Figure 1. Examples of extensively characterized TRPV1 antagonists.

Figure 2. Evolution of TRPV1 antagonists.

carrageenan-induced thermal hyperalgesia (CITH). In addi-

tion to these prominent examples, a number of other TRPV1

antagonists with undisclosed structures are thought to be

currently in various stages of clinical study. In addition to

efficacy in pain trials the effects of other TRPV1 antagonists

on body temperature in humans are awaited.

an MC homogeneous suspension. Unfortunately, significant

hERG inhibition with 6b (44% at 3 μM)²²was deemed

undesirable, as it could translate into cardiac QT interval

prolongation in vivo. A second concern with compounds such

as 6a and 6b was the presence of the 4-trifluoromethylaniline

(TFMA) group at the 4-position of the quinazoline. TFMA is

known to be mutagenic in the Ames assay and clastogenic in

the in vitro assay for chromosome aberrations in Chinese

hamster ovary cells.²³Although TFMA is not clastogenic in

the in vivo mouse micronucleus assay or in the in vivo

cytogenetic test in Chinese hamster bone marrow cells, its

potential release in vivo was considered a liability.²³Third, the

4-aminoquinazoline skeleton has an extensive history of use in

the pharmaceutical industry;²⁴therefore, enhancing the struc-

tural novelty of this class was another important factor.

Finally, improving aqueous solubility while maintaining po-

tency and oral exposure remained critical considerations.

With these concerns in mind, we initially chose to probe the

effects of incorporating a nitrogen atom into the D-ring of the

generalstructure 7 in anattempt toincrease aqueoussolubility

and to lower the potential for oxidation to possibly toxic

metabolites. Second, we investigated the importance of the

location of the nitrogen atoms in the B- and C-rings of the

general structure 7 on activity and “druglike” properties. For

comparison, the 3-trifluoromethylpyridyl A-ring was kept

constant throughout, since an earlier study had identified it

In a previous communication we described the discovery of

a novel TRPV1 antagonist, the 4-aminoquinazoline 6a (R =

H), which was derived from the conformational restriction of

the biarylamide 5.¹⁸The biarylamide 5 in turn resulted from

replacement of the piperazine in the original arylurea lead 4

with a phenyl ring (Figure 2).^19,20The prototypical compound

6a was superior to its forerunners in a number of respects,

most notably in vitro potency. Furthermore, excellent oral

exposure of 6a was achieved using a dosing vehicle supple-

mented with vitamin E TPGS (^D-R-tocopheryl polyethylene

glycol 1000 succinate). Under these conditions, 6a demon-

strated good in vivo efficacy, fully reversing CITH in rats

(MED of 0.1 mg/kg). Plasma levels of 6a, however, were

significantly diminished upon dosing as a standard methyl-

cellulose (MC) suspension. Efforts to improve the solubility

and oral bioavailability of the series initially focused on the

introduction of polar substituents at the C-2 position.²¹From

this effort the 2-methoxymethyl analogue 6b (R = CH₂OMe)

exhibited good in vitro and in vivo potency (MED of 0.3 mg/

kg in CITH) and was well absorbed following oral dosing as

3332 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8

Scheme 1. Synthesis of Quinazolines 6a, 17, and 18^a

Blum et al.

^aReaction conditions: (i) 4-MePhB(OH)₂, 2 M Na₂CO₃, Pd(PPh₃)₄, DME, 85 °C (83%); (ii) H₂SO₄, HNO₃, 0 °C (100%);(iii) KMnO₄, pyridine, H₂O,

110 °C (63%); (iv) 10% Pd/C, H₂, EtOH (92%); (v) HCONH₂, 145 °C (63%); (vi) OPCl₃, 2,6-lutidine, CHCl₃, 90 °C; (vii) 14, IPA, 80 °C (68% over two

steps); (viii) 15, DMA, 125 °C, (21% over two steps); (ix) 16, CH₃CN, 80 °C (81% over two steps).

Scheme 2. Synthesis of Pyrido[3,2-d]pyrimidine 26^a

^aReaction conditions: (i) 2-methoxypyridine-5-boronic acid, 2 M Na₂CO₃, Pd(PPh₃)₄, PhMe, 90 °C (95%); (ii) HBr, AcOH, reflux (93%); (iii) HNO₃,

H₂SO₄, 0 °C, then 70 °C; (iv) SOCl₂, DMF (cat.), reflux (86% over two steps); (v) Fe, CaCl₂, EtOH, H₂O, reflux (85%); (vi) Zn(CN)₂, Pd₂dba₃, DPPF,

DMF, H₂O, 120 °C (90%); (vii) HC(OEt)₃, 130 °C (91%); (viii) SOCl₂, DMF (cat.), reflux (87%); (ix) 14, IPA, 80 °C (88%).

as a preferred substituent.¹⁸Herein we describe the synthesis,

in vitro SAR, and in vivo characterization of a series of 6,6-

heterocycles as replacements for the original quinazoline core.

The synthesis of the pyrido[3,2-d]pyrimidine 26 is outlined

in Scheme 2. Suzuki coupling of 8 and 2-methoxypyridine-

5-boronic acid produced 19 which was demethylated with

HBr in refluxing acetic acid to give the pyridone 20. Nitration

of 20 and chlorination of the product afforded 2-chloro-

4-nitropyridine 21 in excellent yield over the two steps. The

selective reduction of the nitro group of 21 in the presence of

the 2-chloro group was achieved with iron and calcium

chloride in refluxing ethanol, affording the 3-aminopyridine

22. Cyanation of 22 was achieved using conditions described

by Maligres;²⁵however, analysis of an aliquot of the reaction

mixture indicated thatpartial hydrolysis of the cyano group to

the corresponding amide had occurred. Therefore, with addi-

tional water and further heating, the nitrile hydrolysis was

brought to completion, providing an excellent yield of the

amide 23. Cyclization of 23 with triethyl orthoformate fol-

lowed by chlorination with thionyl chloride and thermal

Chemistry

The synthesis of the 4-aminoquinazolines 6a, 17, and 18 is

outlined in Scheme 1. Suzuki coupling of the commer-

cially available 2-chloro-3-(trifluoromethyl)pyridine (8) with

p-tolylboronic acid gave the biaryl 9. Subsequent nitration

and benzylic oxidation with permanganate gave the acid 11 in

good overall yield. Hydrogenation of the nitro group of 11

provided the aniline 12, which was cyclized with formamide to

the quinazoline 13. Conversion of the quinazoline 13 to the

corresponding chloride was achieved with phosphorus oxy-

chloride and 2,6-lutidine. Since the product was sensitive to

hydrolysis, itwas usedimmediately in the final step toyield the

desired targets 6a, 17, and 18.

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3333

Scheme 3. Synthesis of Pyrido[2,3-d]pyrimidine 32^a

^aReaction conditions: (i) MeMgI, THF, 0 °C, 83%; (ii) Me₂NCH(OMe)₂, 105 °C, 100%; (iii) 3-amino-3-methoxyacrylonitrile, NH₄OAc, EtOH,

80 °C, 41%; (iv) Me₂NCH(OMe)₂, 110 °C, 100%; (v) 14, AcOH, H₂O, reflux, 57%.

Scheme 4. Synthesis of Pteridine 34^a

^aReaction conditions: (i) AcOH, HBr, Br₂, 0 °C, 100%; (ii) 4,5,6-triaminopyrimidine, NaHCO₃, H₂O, 100 °C, air-oxidation, 23%;

(iii) 4-trifluoromethylbromobenzene, Pd₂dba₃, Cs₂CO₃, xantphos, dioxane, 100 °C (39%).

Scheme 5. Synthesis of Pyrimido[4,5-d]pyrimidine 36^a

^aReaction conditions: (i) LiN(SiMe₃)₂, Et₂O, 0 °C, then AcOH, H₂O, room temp (66%); (ii) Et₃N, MeOH, EtOCHC(CN)₂, reflux (69%);

(iii) (MeO)₂CHNMe₂, 110 °C (100%); (iv) 14, AcOH, H₂O, reflux (49%).

displacement of the chloride with TFMA gave the pyrido-

[3,2-d]pyrimidine 26.

was achieved by a palladium-coupling reaction and using

xantphos as the catalyst²⁶to afford the target 34.

The synthesis of the pyrido[2,3-d]pyrimidine 32 is depicted

in Scheme 3. Addition of methyl Grignard reagent to 2-cyano-

3-trifluoromethylpyridine (27) gave the ketone 28 which was

condensed with DMF dimethylacetal to yield the dimethyla-

minopropenone 29 quantitatively. Cyclization of 29 with

3-amino-3-methoxyacrylonitrile produced the 2-amino-3-

cyanopyridine 30 in 41% yield. Condensation of the 2-ami-

nopyridine 30 with DMF dimethylacetal afforded the forma-

midine 31 which was treated with TFMA in acetic acid at

100 °C to provide the desired pyrido[2,3-d]pyrimidine 32.

The concise, albeit low yielding synthesis of the pteridine 34

is illustrated in Scheme 4. The methyl ketone 28 was treated

with bromine and HBr in acetic acid. The resulting R-bromo-

ketone was condensed with 4,5,6-triaminopyrimidine, then

air-oxidized and purified to give the 4-aminopteridine 33 in

moderate yield. Introduction of TFMA to the pteridine core

The synthesis of the pyrimido[4,5-d]pyrimidine 36 is shown

in Scheme 5. 2-Cyano-3-trifluoromethylpyridine (27) was

converted to the corresponding amidine which was thermally

cyclized with (ethoxymethylene)malononitrile to give the suit-

ably functionalized pyrimidine 35. Condensation of the ami-

nopyrimidine 35 with DMF dimethylacetal gave the corres-

ponding formamidine which was treated with TFMA in acetic

acid at 100 °C to afford the desired target compound 36.

The route to the quinoline 43 is outlined in Scheme 6. The

previously described acid 12 was converted to the methyl ester

37, and by use of conditions similar to those described by Cai,²⁷

cyclization followed by chlorination gave the 2,4-dichloroqui-

noline 38. Treatment of 38 with sodium methoxide produced a

mixture of the 2-methoxyquinoline 39 and the 4-methoxyqui-

noline 40 which were separated by chromatography. Dechlo-

rination of 40 using phase-transfer hydrogenation conditions²⁸

3334 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8

Scheme 6. Synthesis of Quinoline 43^a

Blum et al.

^aReaction conditions: (i) MeOH, HCl(g), reflux (92%); (ii) AcOH, dioxane, 60 °C, then KN(SiMe₃)₂, THF, PhMe, -78 °C to room temp; (iii) OPCl₃,

reflux (77% over two steps); (iv) NaOMe, THF, room temp (59% for 39 and 30% for 40); (v) 40, CO₂NH₄, 10% Pd/C, MeOH, room temp (96%);

(vi) 33% HBr in AcOH, 100 °C (100%); (vii) OPCl₃, reflux (89%); (viii) 15, Pd₂dba₃, Cs₂CO₃, xantphos, dioxane, 100 °C (68%).

Scheme 7. Synthesis of [1,5]Naphthyridine 47^a

^aReaction conditions: (i) 15, Pd₂dba₃, Cs₂CO₃, xantphos, dioxane, 100 °C (85%); (ii) 33% HBr in AcOH, 100 ^oC; (iii) OPCl₃, reflux (63% over two

steps); (iv) HCO₂NH₄, 10% Pd/C, MeOH, 50 °C (71%).

Scheme 8. Synthesis of [1,8]Naphthyridines 51 and 52^a

^aReaction conditions: (i) ⁿBuLi, THF, -78 °C, then DMF (73%); (ii) TFA, CH₂Cl₂, room temp (94%); (iii) ^tBuOK, THF, -20 °C (87%); (iv) 14, 2 M

HCl, ⁱPrOH, 80 °C (78%); (v) 15, 180 °C (62%).

gave 41 in excellent yield. Subsequent demethylation and

chlorination furnished the 4-chloroquinoline 42. The 2-

amino-5-trifluoromethyl pyridine D-ring was introduced in

good yield using a xantphos-mediated palladium-catalyzed

coupling²⁶reaction with 42 to yield the quinoline 43.

In a fashion analogous to the synthesis of the quinoline 41,

the 1,5-naphthyridine 44 was prepared from the previously

described pyridine 23. The 2-amino-5-trifluoromethylpyridyl

group was introduced using the previously described xant-

phos/Pd₂dba₃system²⁶to provide 45. The 2-methoxy sub-

stituent was removed following a three-step demethylation,

chlorination, and hydrogenation sequence to afford the

1,5-naphthyridine 47 (Scheme 7).

The preparation of the [1,8]naphthyridines 51 and 52 is

illustrated in Scheme 8. Ortho-lithiation of the commercially

available 2-BOC-amino-4-chloropyridine (48), quenching of

the aryllithium with DMF, and cleavage of the BOC group

gave the functionalized pyridine 49. Friedlander²⁹reaction

between the aldehyde 49 and the ketone 28 directly gave the

5-chloro-1,8-naphthyridine 50 in excellent yield. Introduction

of the TFMA moiety under standard conditions gave 51.

A melt between 50 and 2-amino-5-trifluoromethylpyridine

furnished 52.

The route employed for the synthesis of the pyrido[2,3-b]-

pyrazine 55 and the pyrido[3,4-b]pyrazine 56 is shown in

Scheme 9. The methyl ketone 28 was brominated as described

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3335

Scheme 9. Synthesis of Pyrido[2,3-b]pyrazine 55 and Pyrido[3,4-b]pyrazine 56^a

^aReaction conditions: (i) AcOH, HBr, Br₂, 0 °C, 100%; (ii) 2,3,4-triaminopyridine, NaHCO₃, H₂O, 100 °C (32% yield of a mixture of 53 and 54);

(iii) 2-chloro-5-trifluoromethylpyridine, Pd₂dba₃, Cs₂CO₃, xantphos, dioxane, 100 °C (28% yield of 55 and 23% yield of 56).

Scheme 10. Synthesis of Cinnoline 60^a

a

€

Reaction conditions: (i) HN(OMe)Me HCl, EDCI, Hunig’s base, CH₂Cl₂, room temp (34%); (ii) MeMgI, THF, 0 °C (43%); (iii) NaNO₂, HCl,

3

H₂O, 0 °C (62%); (iv) OPCl₃, reflux (87%); (v) 15, Pd₂dba₃, Cs₂CO₃, xantphos, dioxane, 100 °C (76%).

previously and treated with 2,3,4-triaminopyridine to afford

an inseparable mixture of the two regioisomers, the pyrido-

[2,3-b]pyrazin-8-amine 53 and the pyrido[3,4-b]pyrazin-

5-amine 54. Palladium-catalyzed coupling²⁶was carried out

on this mixture, using 2-chloro-5-trifluoromethylpyridine to

give 55 and 56 which were successfully separated in moderate

yield by flash chromatography.

61 and subsequent cyclization with methyl 2-(4-methylphe-

nylsulfonamido)acetate and sodium ethoxide in refluxing

ethanol gave the [1,7]naphthyridine 62 in 61% yield over the

two steps. Hydrolysis and decarboxylation of the ester 62 was

followed by conversion to the triflate to afford the final

intermediate 63. Palladium-catalyzed coupling of 63 with

the aminopyridine 15 gave the desired [1,7]naphthyridine 64.

The synthesis of the cinnoline 60 is depicted in Scheme 10. The

acid 12 described previously was converted, via the Weinreb

amide, to the ketone 57 in moderate overall yield. Diazotization

of the 2-aminoacetophenone 57 and ensuing cyclization³⁰fur-

nished the 4-cinnoline 58 in moderated yield. The cinnoline 58

was converted to the chloride 59 with phosphorus oxychloride,

and then palladium-catalyzed coupling of 59 with the amino-

pyridine 15 gave the desired cinnoline 60.

Results and Discussion

The compounds were evaluated for their ability to block

capsaicin activation of human TRPV1 channels using fluoro-

metric imaging plate reader (FLIPR) technology, and the

results areshownin Table 1.³¹In the first three compounds6a,

17, and 18, the aminoquinazoline core was kept constant

and the three p-trifluoromethylaryl D-rings were compared.

The potency of 17 (2-amino-5-trifluoromethylpyridine) was

The synthesis of the [1,7]naphthyridine analogue 64 is

outlined in Scheme 11. Bromination of the methylpyridine

3336 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8

Scheme 11. Synthesis of [1,7]Naphthyridine 64^a

Blum et al.

^aReaction conditions: (i) AIBN, NBS, CH₂Cl₂, H₂O; (ii) TosNHCH₂CO₂Et, NaOEt, EtOH, (61% over two steps); (iii) 6 M HCl, 95 °C; (iv) Tf₂O,

€

Hunig’s base, CH₂Cl₂, (59% over two steps); (v) 15, Pd₂dba₃, Cs₂CO₃, xantphos, dioxane, 110 °C (61%).

similar to the benchmark 6a (TFMA), while that of 18 (3-

amino-6-trifluoromethylpyridine) was slightly lower. Because

of the poor solubility of 6a (below the limit of detection at pH

7.4)²¹and to allow comparisons following modification, the

solubility of compounds was measured in 0.1 N HCl. The

solubility of 6a was measurable under these conditions (16 μg/

mL), and the addition of a nitrogen atom into the D-ring

essentially doubled the solubility when measured in 0.1 N

HCl.

The effect of the introduction of nitrogen atoms into the

B-ring was investigated next, while the TFMA D-ring was

retained for each of the four analogues 26, 32, 34, and 36.

Introduction of a nitrogen at the 5-position of the quinazoline

(pyrido[3,2-d]pyrimidine 26) or at the 8-position (pyrido[2,3-

d]pyrimidine 32) gave compounds that were of similar activity

to 6a. Incorporation of nitrogen at both the 5- and 8-positions

gave the pteridine 34 (IC₅₀= 0.21 nM) which was the most

active compound prepared in the series. The compound

derived from introduction of a single nitrogen atom at the

6-position of the aminoquinazoline was not prepared in this

series; however, introduction of nitrogen at both the 6- and

8-positions, as in the pyrimido[4,5-d]pyrimidine 36, resulted in

a dramatic loss in activity (IC₅₀= 665 nM). This result was in

accord with the introduction of nitrogen at positions equiva-

lent to the 6-position in other templates which also resulted in

a significant loss in activity.¹⁸In terms of solubility, the

addition of nitrogen atoms into the B-ring had a detrimental

effect and all four analogues had very poor solubility in 0.1 N

HCl (<10 μg/mL).

The effect on TRPV1 activity of the nitrogen atoms in the

C-ring was investigated next, with the 2-amino-5-trifluoro-

methylpyridine D-ring kept constant. Removal of the nitro-

gen at the 3-position of the quinazoline core gave quinoline 43

and only a small loss in the ability of the compound to block

activation of TVRP1 (compare 43 IC₅₀= 1.65 nM vs 17

IC₅₀= 0.85 nM). Surprisingly, introduction of nitrogen at the

5-position, a change that had given a boost in activity in the

quinazoline series (26 IC₅₀= 0.63 nM), gave a significant

loss in activity for the corresponding [1,5]naphthyridine 47

(IC₅₀= 308 ( 190 nM); it is unclear why this led to such a loss

in activity. Addition of nitrogen at the 8-position in the

quinazoline series had given an increase in activity. However,

in the quinoline series, this modification, exemplified with

[1,8]naphthyridine 52, resulted in a minor loss in activity

(compare 32 IC₅₀= 0.83 nM vs 52 IC₅₀= 2.2 nM). The

TFMA analogue 51 was also made with the [1,8]naphthy-

ridine core, and consistent with the activity observed for

D-rings in the quinazoline series, the activity was slightly

reduced. The introduction of nitrogen at both the 5- and

8-positions in the quinazoline template gave the most active

compound in the study, pteridine 34 (IC₅₀= 0.21 nM), and

the same modification in the quinoline series afforded the

similarly potent pyrido[2,3-b]pyrazine 55 (IC₅₀= 0.23 nM).

The importance of the nitrogen at the 1-position of the C-ring

was established by the loss in activity observed for pyrido-

[3,4-b]pyrazine 56 in which the nitrogen at the 1-position had

been deleted (compare 34 IC₅₀= 0.21 nM vs 56 IC₅₀= 79

nM). The effect of the introduction of a nitrogen atom at the

2-position was investigated with the cinnoline 60 and the

[1,7]naphthyridine 64 analogues. In both examples, the in-

hibitory potency was greatly diminished (compare 60 IC₅₀

=

29 nM vs 43 IC₅₀= 1.65 nM and 64 IC₅₀= 460 nM vs 52

IC₅₀= 2.2 nM). In terms of solubility the quinoline 43 and

aza-quinoline analogues 47, 51, 52, and 64 had measurable

solubility in 0.1 N HCl, but the addition of a third nitrogen

atom into the core (e.g., 55 and 56) resulted in a significant loss

in solubility.

The aminoquinazolines 6a, 17, and 18 and the [1,8]naph-

thyridine 52 demonstrated acceptable potency, solubility in

0.1 N HCl, and stability in rat liver microsomes. They were

advanced into rat pharmacokinetic experiments (Table 2).

Upon oral dosing as an MC suspension, the bioavailabilty of

6a (TFMA D-ring) was very low (F = 8%). To determine if

the low bioavailability of 6a was related to poor permeability,

the rate of basolateral to apical (B to A) movement and the

rate of apical to basolateral (A to B) movement across a

monolayer of MDR1-transfected LLC-PK1 cells were deter-

mined for 6a.³²Adequate permeability (P_app= 11.0 ꢀ 10^-6

cm/s) and a Pgp efflux ratio (B to A)/(A to B) of close to unity

(1.1) implied that 6a was not a Pgp substrate, and the excellent

stability in rat liver microsomes³³suggested that the poor oral

exposure was more likely due to low solubility rather than

poor permeability or metabolic stability. Indeed, the levels of

6a were below the limits of quantification (BLQ) when

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3337

Table 1. In Vitro Ability of Compounds To Inhibit Activation of hTRPV1 Receptors^aand Solubility in 0.1 N HCl^b

^aHuman TRPV1 receptor activated by capsaicin. Unless otherwise stated, all values are the mean ( SEM of at least three separate experiments.

^bDetermined by HPLC/MS analysis after 4 h in 0.1 N HCl. ^cND = not determined.

Table 2. Rat Pharmacokinetic Data for Selected Compounds^a

iv/po dose

(mg/kg)

Vd

T_max(h) T_1/2(h) CL ((mL/min)/kg) (L/kg) F (%)

solubility, 0.1 N HCl solubility, pH 7.4 rat liver microsomes,

(μg/mL)^b

(μg/mL)^c

% remaining^d

6a

17

18

52

2.7/2

1.5/2

1.5/10

1/1

3.0

5.7

4.7

1.1

8

38

4

23

4.2

13

12

8

89

65

52

16

33

35

40

0

2

93

91

22

34

5.5

3.9

100

1.4

^aCompounds dosed in 50% PEG-400/water (iv) and 0.5% methylcellulose, 0.1% triacetin (po). ^bDetermined in 0.1 N HCl. ^cEquilibrium solubility

determined at pH 7.4. ^dPercentage of compound remaining after 10 min in rat liver microsomes.³³

assessed at pH 7.4 in an equilibrium solubility study. Replace-

ment of the TFMA with the 2-amino-5-trifluoromethylpyri-

dine group, as in compound 17, resulted in higher solubility

than 6a in 0.1 N HCl but not at pH 7.4. Nevertheless, when

dosed in the same vehicle, 17 achieved a remarkably higher

oral bioavailability (F = 89%) than 6a (F = 8%). The very

low clearance (4.2 (mL/min)/kg) and large volume of distribu-

tion (12 L/kg) exhibited by 17 accounted for its very long

half-life (38 h). In fact, a significant improvement in pharma-

cokinetic properties upon replacing a TFMA group by

2-amino-5-trifluoromethylpyridine had previously been de-

scribed by Carruthers in the arylurea series 4 of TRPV1

antagonists.^20cThe 3-amino-6-trifluoromethylpyridine ana-

logue 18, which displayed a similar solubilityprofile to17, also

had goodoral bioavailability(F = 65%) but a greatlyreduced

half-life (4 h) and volume of distribution (5.5 L/kg) compared

to 17 and consequently a higher but still moderate clearance.

The [1,8]naphthyridine 52 was rapidly absorbed compared to

3338 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8

Blum et al.

Figure 3. Stability of selected compounds in 0.1 N HCl at 40 °C.

Table 3. Key in Vitro and in Vivo Data for Compound 52

the three quinazolines (T_max= 1.1 h) and had good oral bio-

availability (F = 52%). Compound 52 had a relatively high

clearance (∼45% liver blood flow) and lower volume of

distributionthan6a, 17, and 18, resulting ina shorterobserved

half-life (1.4 h).

parameter

hTRPV1-cap IC₅₀(nM)^a

rTRPV1-pH IC₅₀(nM)^b

plasma protein binding, rat (%)^c

plasma protein binding, human (%)^c

Pgp efflux ratio^d

2.2

3.25

89.3

93.5

1.7

Because of concerns about the stability of the compounds

during transit through the stomach (pH between 1 and 2), the

acid stability of the three quinazolines 6a, 17, and 18 and the

[1,8]naphthyridine 52 was studied in 0.1 N HCl at 40 °C over

6 h. Aliquots were removed and assayed at 1 h intervals

(Figure 3). Compound 6a (TFMA D-ring) degraded by 3%

after 6 h, while 18 (3-aminopyridine D-ring) was more stable

and had degraded by only 1% over 6 h. In contrast, the

2-amino-5-trifluoromethylpyridine D-ring analogue 17 was

hydrolytically labile and rapidly degraded by ∼60% after 4 h

(approximate stomach transit time in humans) and by ∼73%

after 6 h. In principle, hydrolysis could have occurred to

produce quinazoline 13 or 5-trifluoromethylpyridin-2-one.

However, analysis was highly selective, yielding quinazoline

13 and 2-amino-5-trifluoromethylpyridine (15) exclusively.

The lability of 17 was disappointing, since 2-amino-5-trifluoro-

methylpyridine (15) was inactive in an in-house Ames muta-

genicity assay and had emerged as a preferred D-ring

and TFMA alternative in this series. Presumably, protona-

tion of the activated pyridinylaminoquinazoline rende-

red it susceptible to the hydrolysis. Accordingly, reten-

tion of the 2-aminopyridine D-ring but deletion of one of the

activating nitrogen’s from the core, as in the [1,8]naphthyri-

dine 52, afforded a hydrolytically stable compound (0.06%

degradation after 6 h).

P_app(10^-6cm/s)^e

29.8

0.3

brain/plasma ratio^f

hERG, % inhibition at 10 μM^g

47

0.98

hERK1, IC₅₀(μM)^h

^aHuman TRPV1 receptor activated by capsaicin. ^bRat TRPV1

receptor activated by low pH (5.0-5.5). ^cPercentage plasma protein

bound to fresh rat or human plasma (centrifugation method using ¹⁴C

radiolabeled 52). ^dMDR1 directional transport ratio (B to A)/(A to B).

^ePassive permeability (10^-6cm/s). ^fBrain to plasma ratio at 1 h

following a 3 mg/kg oral dose in rats (0.5% methylcellulose, 0.1%

triacetin vehicle). ^gBlockade of hERG potassium channel in whole cell

(Cos7) electrophysiological assay. ^hHuman protein serine/threonine

kinase, ERK1.

activation at low pH (5.0-5.5) was consistent with the activity

at the human receptor (capsaicin activation), demonstrating

the effectiveness of 52 to block the TRPV1 channel irrespec-

tive of the species or the mode of activation. Measurement of

the plasma protein binding in fresh rat and human plasma

indicated relatively high free-fractionsfor 52: 10.7%free in rat

plasma and 6.5% free in human plasma. The high perme-

ability (P_app= 29.8 ꢀ 10^-6cm/s) and Pgp efflux ratio of 1.7

indicated that 52 was not a Pgp substrate, although an in vivo

brain to plasma ratio of less than unity (B/P = 0.3) indicated

that there was resistance to crossing the blood-brain barrier.

hERG inhibition had beenidentified as a potential liabilityfor

some analogues in the quinazoline series.²¹SAR suggested

that a substituent at the 2-position contributed to this un-

desirable activity (compare 6a R = H, 8% inhibition at 3 μM

but 6b R = CH₂OMe, 44% inhibition at 3 μM in a whole cell

On the basis of a number of factors including hTRPV1

potency, solubility, oral bioavailability, and acid stability, the

[1,8]naphthyridine 52 was profiled further. Key in vitro and

in vivo parameters obtained for 52 are compiled in Table 3.

The ability of 52 to inhibit the rat TRPV1 receptor upon

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3339

Table 4. Pharmacokinetic Data for Ascending Doses of 52 in Rat^a

species po dose (mg/kg) AUC_0-24h(μM h) C_max(μM) T_max(μM)

3

rat

1

10

0.6

22.6

0.24

2.87

1.1

3.33

6.00

100

203.9

12.87

^aCompounds dosed in 0.5% methylcellulose, 0.1% triacetin (po).

(Cos7) electrophysiological assay). We were therefore grati-

fied to observe a moderate but acceptable level of hERG

inhibition for 52 (47% at 10 μM) which lacks a C-2 substi-

tuent. Compound 52 was highly selective and showed few

effects at 10 μM against a panel of more than 160 receptors,

ion channels, transporters, and kinases (MDS Pharma

Services). The one exception among the kinases was the pro-

tein serine/threonine kinase ERK1 with IC₅₀= 0.98 μM.

Compound 52 was devoid of activity at other protein serine/

threonine kinases including p38R, PKA, PKC, PKCR, and the

tyrosine kinase EGF and insulin receptors. The compound

didinhibit themonoaminetransporter (rabbit, IC₅₀≈0.5μM)

and L-type calcium channels (rat, IC₅₀≈ 3-4 μM).

Systemic exposure was high in rats following oral adminis-

tration of 52 at 1, 10, and 100 mg/kg as a 0.5% methylcellulose

suspension. In terms of proportionality, there was a

37-fold increase in AUC_0-24hbetween the 1 and 10 mg/kg

doses (12-fold difference in C_max) and a 9-fold increase in

AUC_0-24hbetween the 10 and 100 mg/kg doses (4- to 5-fold

difference in C_max) (Table 4).

On the basis of the excellent in vitro potency, good selecti-

vity, and oral exposure, compound 52 was selected for further

in vivo evaluation in a number of pain models. Compound 52

was tested in the rat carrageenan-induced thermal hyperalge-

sia model of acute inflammatory pain.³⁴Rats received an

intraplantar injection of λ-carrageenan (0.1 mL of 1% solu-

tion made up in saline) or saline(0.1mL of 0.9% solution) into

one hind paw, and thermal hyperalgesia was determined using

a Hargreaves apparatus by measuring the latency to withdraw

the paw from a radiant heat stimulus 3 h later. Thermal

hyperalgesia was defined as the difference in paw withdrawal

latencies determined for rats injected with saline or carragee-

nan. Compound 52 (0.3, 1, or 3 mg/kg) or vehicle (5 mL/kg

0.5% methylcellulose) was administered orally 2 h after

carrageenan (i.e., when the thermal hyperalgesia had already

developed). Immediately following testing, blood was col-

lected for the determination of plasma drug levels. The

[1,8]naphthyridine 52 significantly reversed thermal hyperal-

gesia induced by carrageenan injection (Figure 4). The effects

of 52 were dose-dependent. A complete reversal of the effects

of carrageenan was observed at 3 mg/kg po with associated

compound plasma concentration of 1.40 μM. Taking into

account plasma protein binding in rat (89.3%, Table 3), this

corresponds to a free plasma concentration of 150 nM.

Compound 52 reversed mechanical hyperalgesia in a rat

model of persistent inflammation. Rats received an intraplan-

tar complete Freund’s adjuvant (CFA, 200 μL) in the left hind

paw. The next day the rats were dosed orally once a day for 3

consecutive days with compound 52 (1, 3, or 10 mg/kg),

vehicle, or naproxen (20 mg/kg) as the positive control.

Mechanical hypersensitivity was recorded as a differential

weight-bearing response, measured 0, 1, 2, 4, and 24 h

postdosing. In a separate cohort plasma samples were also

taken each day, 2 h after dosing compound 52. Figure 5 shows

the difference in weight bearing between ipsilateral (CFA-

injected) and contralateral hind paws over the course of the

Figure 4. Effect of compound 52 on carrageenan-induced thermal

hyperalgesia in rats and plasma concentrations at 3.0, 1.0, and

0.3 mg/kg. Plasma samples were taken immediately after testing (t ≈

1 h), and the associated plasma concentrations are denoted in blue.

study. Compound 52 significantly attenuated the favoring

response compared to vehicle at all doses tested, and by day 3,

compound 52 (10 mg/kg) had completely reversed the effects

of CFA. The magnitude of the effect was equivalent to that

observed for rats treated with naproxen (20 mg/kg). Plasma

concentrations at 2 h postdose ranged between 2.3 and 5.2 μM

for the 10 mg/kg dose. On the basis of an in vitro rat plasma

protein binding of 89.3% (Table 3), this corresponds to free

plasma concentrations of 0.25 and 0.56 μM, respectively.

Recent reports have indicated that acute administration of

brain penetrant TRPV1 antagonists, at doses producing max-

imal inhibition in acute pain models, causes hyperthermia (∼0.5

to 1.5 °C) in rats.^15,35To investigate this phenomenon with a

moderately brain penetrant compound from the azaquinazo-

line series, compound 52 was dosed at 3 mg/kg po once a day for

4 days to rats fitted with telemetry devices and core body

temperature readings were taken (see Supporting Information).

On day 1, 52 caused a significant increase (∼1.5 °C) in core

body temperature above vehicle controls 1 h after dosing and

remained elevated for greater than 10 h. The hyperthermic

effect was tolerated upon repeat dosing such that by day 4 the

core body temperature of rats treated with compound 52 was

indistinguishable from that observed for vehicle treated rats.

Conclusion

In summary, we have investigated the importance of insert-

ing nitrogens into the B-, C-, and D-rings of quinazoline 6a

with the goal of addressing issues related to pharmacokinetics

(e.g., low oral exposure), druglike properties (e.g., aqueous

solubility), and potential metabolic/toxicological risks

(e.g., release of TFMA and hERG channel inhibition). To this

3340 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8

Blum et al.

were obtained for all new compounds and were within 0.4% of

theoretical C, H, and N. H NMR spectra were recorded in

1

deuteriochloroform (unless otherwise noted) with tetramethyl-

silane as the internal standard at 400 MHz. Coupling constants

(J values) are quoted to the nearest 0.5 Hz. ¹³C NMR spectra

were recorded in deuteriochloroform (unless otherwise noted) at

100 MHz. Mass spectra were recorded on a VG 70SE magnetic

sector mass spectrometer. Organic solutions were dried using

anhydrous magnesium sulfate and concentrated by rotary eva-

poration. Analytical thin layer chromatography (TLC) was

carried out on Camlab Polygram SIL G/UV₂₅₄plates. Unless

otherwise stated, preparative column chromatography was

carried out on 60H silica gel (Merck 9385). Compositions of

solvent mixtures are quoted as ratios of volume. Known com-

pounds gave spectral and analytical data consistent with litera-

ture values. Analytical HPLC used to determine the purity of

target compounds was conducted using two different methods.

The results are expressed as retention times in minutes, and the

relative content is expressed as a percentage. All final com-

pounds had purity of >95% by both methods (see Supporting

Information for conditions and chromatograms).

2-p-Tolyl-3-trifluoromethylpyridine (9). To a degassed mix-

ture of 2-chloro-3-(trifluoromethyl)pyridine (8) (12.7 g, 70.1

mmol), p-tolylboronic acid (9.6 g, 70.6 mmol), and 2 M Na₂CO₃

(175 mmol) in DME (200 mL) under a nitrogen atmosphere was

added Pd(PPh₃)₄(3.23 g, 2.8 mmol). The mixture was heated at

80 °C overnight, concentrated, and extracted with ethyl acetate

(3 ꢀ 400 mL). The combined extracts were dried (Na₂SO₄), con-

centrated, filtered through a silica gel pad (elution with ethyl

acetate), and gave the title compound 9 (13.8 g, 83%). **MS 238

(M þ 1), ¹H NMR δ (CDCl₃) 8.83 (1H, d, J = 4 Hz), 8.06 (1H, d,

J = 8 Hz), 7.42-7.37 (3H, m), 7.25 (2H, d, J = 7.5 Hz), 2.42

(3H, s).

2-(4-Methyl-3-nitrophenyl)-3-(trifluoromethyl)pyridine (10).

To a solution of 2-p-tolyl-3-trifluoromethylpyridine (9) (1.99

g, 8.4 mmol) in H₂SO₄(6 mL) was cautiously added fuming

HNO₃(2 mL). The mixture was stirred for 1 h at RT and poured

onto ice-water (30 mL). The mixture was extracted with ethyl

acetate (3 ꢀ 75 mL) and the combined extracts washed with 1 N

NaOH (50 mL), dried (Na₂SO₄), concentrated and afforded the

title compound 10 (2.37 g, 100%). MS 283 (M þ 1). ¹H NMR δ

(CDCl₃) 8.87 (1H, d, J = 4 Hz), 8.20 (1H, s), 8.13 (1H, d, J = 7.5

Hz), 7.67 (1H, d, J = 7 Hz), 7.52-7.43 (2H, m), 2.69 (3H, s).

2-Nitro-4-(3-trifluoromethylpyridin-2-yl)benzoic Acid (11). To

a solution of 2-(4-methyl-3-nitrophenyl)-3-(trifluoromethyl)-

pyridine (10) (2.01 g, 7.1 mmol) in a mixture of pyridine

(10 mL) and water (5 mL) was added potassium permanganate

(3.99 g, 25.3 mmol) in portions. The mixture was heated at

110 °C for 4 h and cooled to room temperature. Additional

potassium permanganate (3.99 g, 25.3 mmol) and water (10 mL)

were added. The mixture was reheated at 110 °C for 16 h. The

mixture was cooled to room temperature and filtered through

Celite. The filtrate was concentrated, diluted with water

(50 mL), and the aqueous layer was washed with ethyl acetate

(50 mL). The aqueous layer was neutralized with 2 N HCl and

the precipitate collected and gave the title compound 11 (1.4 g,

63%). MS 313 (M þ 1). ¹H NMR δ (DMSO) 8.96 (1H, d, J =

4.5 Hz), 8.38 (1H, d, J = 8 Hz), 8.09 (1H, s), 7.99 (1H, d, J = 8.5

Hz), 7.86 (1H, d, J = 7 Hz), 7.86-7.73 (1H, m).

Figure 5. Effect of compound 52 at 1, 3, and 10 mg/kg, po, in the

CFA model of inflammation and spontaneous nociception in rats

and plasma levels 2 h after each daily dose (V).

end, chemistry was developed to synthesize a number of

6,6-heterocyclic cores. Of particular note was the concise

synthesis of the 1,8-napthyridine core (e.g., 52) which utilized

a Friedlander cyclization of two readily available fragments.

Acceptable on-target potency was maintained with a number

of core examples, while compound 52 demonstrated marked

improvements in oral exposure and aqueous solubility while

achieving a lowered inhibition of the hERG channel. Impor-

tantly, compound 52 was devoid of a TFMA moiety. The 1,8-

napthyridine 52 also displayed a good selectivity profile in an

off-target screen of over 160 receptors, ion channels, and

enzymes. The overall profile of 52 made it an excellent candi-

date for additional in vivo preclinical studies to assess the

pharmacological effects of a TRPV1 antagonist. In rats, the

compound significantly attenuated inflammatory pain in both

an acute (carrageenan) and a subchronic (CFA) setting, further

supporting the utility of a TRPV1 antagonist for the treatment

of inflammatory pain states (e.g., osteoarthritis). Oral admin-

istration of 52 to rats produced an increase in core body

temperature of ∼1.5 °C, a finding consistently observed with

a number of other TRPV1 antagonists.³⁵The observed hyper-

thermic effect tolerated after repeated doses of 52.

2-Amino-4-(3-trifluoromethylpyridin-2-yl)benzoic Acid (12).

A mixture of 2-nitro-4-(3-trifluoromethylpyridin-2-yl)benzoic

acid (11) (1.2 g, 3.84 mmol) and 10% palladium on carbon (150

mg) in 95% ethanol was hydrogenated (1 atm) for 16 h. The

mixture was filtered through Celite and evaporated to yield the

title compound 12 (1.01 g, 92%). MS 283 (M þ 1). ¹H NMR δ

(DMSO) 8.87 (1H, d, J = 4 Hz), 8.28 (1H, d, J = 8 Hz), 7.73

(1H, d, J = 9 Hz), 7.67-7.62 (1H, m), 6.78 (1H, s), 6.53 (1H, d,

J = 8.5 Hz).

Experimental Section

General. Melting points were determined using a capillary

melting apparatus and were uncorrected. Elemental analyses

7-(3-Trifluoromethylpyridin-2-yl)quinazolin-4-ol (13). A solu-

tion of 2-amino-4-(3-trifluoromethylpyridin-2-yl)benzoic acid

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3341

(12) (552 mg, 1.95 mmol) in formamide (10 mL) was heated at

145 °C for 4 h. The mixture was cooled to room temperature and

water (20 mL) added. The precipitate was collected by filtration,

air-dried and afforded the title compound 13 (359 mg, 63%). MS

292 (M þ 1). ¹H NMR δ (CDCl₃) 8.93 (1H, d, J = 4 Hz), 8.41

(1H, d, J = 8.5 Hz), 8.19-8.17 (2H, m, J = 8 Hz), 7.95 (1H, s),

7.68 (1H, d J = 7 Hz), 7.58-7.52 (1H, m).

layer was extracted with ethyl acetate (2 ꢀ 250 mL), and the

combined extracts were washed with 4 M sodium hydroxide

(250 mL), water (250 mL), brine (250 mL), dried (MgSO₄), and

concentrated under reduced pressure. The oil was purified by

flash chromatography (elution with ether/hexane, 1:1) and gave

the title compound 19 (48.2 g, 95%), a colorless oil. MS 255

1

(M þ 1). H NMR δ (CDCl₃) 8.85 (1H, d, J = 5 Hz), 8.34

(4-Trifluoromethylphenyl)-[7-(3-trifluoromethylpyridin-2-yl)-

quinazolin-4-yl]amine (6a). A mixture of7-(3-trifluoromethylpyr-

idin-2-yl)quinazolin-4-ol (13) (111 mg, 0.38 mmol), 2,6-lutidine

(102 mg, 0.95 mmol), OPCl₃(145 mg, 0.95 mmol), and chloro-

form (HPLC grade, 5 mL) was refluxed for 24 h. The mixture was

cooled to room temperature, and the volatiles were removed

under reduced pressure. The residue was redissolved in dichloro-

methane and filtered through a short pad ofsilicagel (elution with

ethyl acetate/hexane, 1:1). The resulting solid was dissolved in

IPA (5 mL), and 4-(trifluoromethyl)aniline (14) (106 mg,

0.66 mmol) was added. The mixture was heated at 80 °C for

4 h, cooled to room temperature, and ethyl acetate (5 mL) was

added. The precipitate was collected by filtration and partitioned

between ethyl acetate (10 mL) and saturated sodium bicarbonate

solution (10 mL). The layers were separated, and the aqueous

portion was extracted with ethyl acetate (10 mL). The combined

organic extracts were washed with brine (10 mL), dried (MgSO₄),

evaporated and gave the title compound 6a (112 mg, 68% over

two steps). MS 435 (M þ 1). ¹H NMR δ (CDCl₃) 11.75 (1H, br s),

9.03-8.99 (3H, m), 8.42 (1H, d, J = 7 Hz), 8.07-8.04 (3H, m),

7.96 (1H, d, J = 8.5 Hz), 7.87 (2H, d, J = 7.5 Hz), 7.79 (1H, dd,

J = 2.5 and 7.5 Hz). Analytical HPLC: method 1, 12.29 (94.9%);

method 2, 5.62 (99.4%).

(5-Trifluoromethylpyridin-2-yl)-[7-(3-trifluoromethylpyridin-

2-yl)quinazolin-4-yl]amine (17). An identical procedure as de-

scribed above was followed. The intermediate 4-chloro-7-

(3-trifluoromethylpyridin-2-yl)quinazoline was dissolved in

DMA (5 mL), and 2-amino-5-trifluoromethyl pyridine (15)

(105 mg, 0.65 mmol) was added. The mixture was heated at 125

°C for 6 h, cooled to room temperature, and partitioned between

ethyl acetate (10 mL) and saturated sodium bicarbonate solution

(10 mL). The layers were separated, and the aqueous layer was

extracted with ethyl acetate (2 ꢀ 10 mL). The combined organic

extracts were washed with water (10 mL), brine (10 mL), dried

(MgSO₄), and evaporated. The residue was purified by prepara-

tive thin layer chromatography (elution with ethyl acetate/hex-

ane, 1:1) and afforded the title compound 17 (34 mg, 21% over

two steps). MS 436 (M þ 1). ¹H NMR δ (DMSO) 10.99 (1H, s),

8.97 (1H, d, J = 5 Hz), 8.86-8.81 (3H, m), 8.68 (1H, d, J =8 Hz),

8.39 (1H, d, J = 8.5 Hz), 8.26 (1H, d, J = 8 Hz), 7.90 (1H, s),

7.77-7.72 (2H, m). Analytical HPLC: method 1, 12.51 (99.1%);

method 2, 5.00 (98.3%).

(6-Trifluoromethylpyridin-3-yl)-[7-(3-trifluoromethylpyridin-

2-yl)quinazolin-4-yl]amine Hydrochloride (18). An identical pro-

cedure as described above was followed. The intermediate

4-chloro-7-(3-trifluoromethylpyridin-2-yl)quinazoline was dis-

solved in acetonitrile (5 mL), and 3-amino-6-trifluoromethyl-

pyridine (16) (105 mg, 0.65 mmol) was added. The mixture was

heated at 80 °C for 8 h and then cooled to room temperature.

The resulting precipitate was collected by filtration, washed with

ether, air-dried and gave the title compound 18 as the mono-

hydrochloride salt (145 mg, 81% over two steps). MS 436 (M þ

1). ¹H NMR δ (DMSO) 12.28 (1H, br s), 9.24 (1H, s), 9.17 (1H,

d, J = 8.5 Hz), 9.07 (1H, s), 8.99 (1H, d, J = 5 Hz), 8.55 (1H, d,

J = 8 Hz), 8.43 (1H, d, J = 8 Hz), 8.12-8.08 (2H, m), 7.99 (1H,

d, J = 7.5 Hz), 7.79 (1H, dd, J = 2.5 and 7.5 Hz).

6⁰-Methoxy-3-trifluoromethyl[2,3⁰]bipyridinyl (19). A mixture

of 2-chloro-3-trifluoromethylpyridine (8) (37 g, 0.2 mol),

2-methoxypyridine-5-boronic acid (32 g, 0.21 mol), tetrakis-

(triphenylphosphine)palladium(0) (9 g, 7 mmol), and 2 M

potassium carbonate (150 mL) in toluene (500 mL) was heated,

under a nitrogen atmosphere, at 90 °C for 8 h. The reaction

mixture was cooled, and the layers were separated. The aqueous

(1H, s), 8.09 (1H, d, J = 7.5 Hz), 7.76 (1H, dd, J = 2.5 and 8.5

Hz), 7.46-7.41 (1H, m), 6.82 (1H, d, J = 8 Hz), 3.76 (3H, s).

3-Trifluoromethyl-1⁰H-[2,3⁰]bipyridinyl-6⁰-one (20). 6⁰-Meth-

oxy-3-trifluoromethyl[2,3⁰]bipyridinyl (19) (41 g, 0.16 mol) in

HBr (30% in acetic acid, 100 mL) was heated to reflux for 1 h.

The mixture was cooled, filtered and the precipitate washed with

ether (100 mL). The precipitate was transferred into 10 M sodium

hydroxide (500 mL) and stirred for 1 h. The solution was then

treated with hydrochloric acid until the solution was approxi-

mately pH 7. The white solid was then collected by filtration, air-

dried to yield the title compound 20 (36 g, 93%) as a white solid.

MS 241 (M þ 1). ¹H NMR δ (DMSO) 8.87 (1H, d, J = 5 Hz), 8.29

(1H, d, J = 7.5 Hz), 7.68-7.54 (3H, m), 6.46 (1H, d, J = 8 Hz).

6⁰-Chloro-5⁰-nitro-3-trifluoromethyl[2,3⁰]bipyridinyl (21). To a

solution of 3-trifluoromethyl-1⁰H-[2,3⁰]bipyridinyl-6⁰-one (20)

(25 g, 0.1 mol) in concentrated sulfuric acid (100 mL) at 0 °C was

added dropwise a solution of fuming nitric acid (35 mL) and

concentrated sulfuric acid (10 mL). The reaction mixture was

then heated to 70 °C for 1 h, cooled, and poured onto ice

(500 mL). The mixture was filtered and the filtrate treated with

10 M sodium hydroxide until the solution was at pH 4-5. The

precipitate was collected by filtration and air-dried. The white

solid (26.2 g, 92%) was dissolved in thionyl chloride (300 mL)

and DMF (3 mL) and heated to reflux for 4 h. The volatiles were

removed by rotary evaporation, and the residue was partitioned

between ethyl acetate (350 mL) and saturated sodium bicarbo-

nate solution (250 mL). The aqueous layer was extracted with

further ethyl acetate (250 mL), and the combined organics were

washed with brine (250 mL), dried (MgSO₄), concentrated and

gave the title compound 21 (25 g, 93%), a yellow oil. MS 304

(M þ 1). ¹H NMR δ (CDCl₃) 8.94 (1H, d, J = 5 Hz), 8.79 (1H, d,

J = 1.5 Hz), 8.45 (1H, d, J = 1.5 Hz), 8.18 (1H, d, J = 8 Hz),

7.60 (1H, dd, J = 2.5 and 8.5 Hz).

6⁰-Chloro-3-trifluoromethyl[2,3⁰]bipyridinyl-5⁰-ylamine (22).

To a solution of 6⁰-chloro-5⁰-nitro-3-trifluoromethyl[2,3⁰]bi-

pyridinyl (21) (25 g, 0.082 mol) and calcium chloride (11 g, 0.1

mol) in ethanol (300 mL) and water (50 mL) was added iron

powder (45 g, 0.82 mol). The solution was heated at reflux for

1.5 h, cooled, and filtered through Celite. The mixture was

concentrated under reduced pressure, redissolved in ethyl acet-

ate (300 mL), and washed with brine (200 mL). The solution was

concentrated under reduced pressure, purified by flash chroma-

tography (elution with ether/hexane, 1:1), and gave the title

compound 22 (19 g, 85%), a pale-yellow solid. MS 274 (M þ 1).

¹H NMR δ (CDCl₃) 8.85 (1H, d, J = 4 Hz), 8.10 (1H, d, J = 8

Hz), 7.93 (1H, s), 7.49-7.46 (1H, m), 7.18 (1H, s).

3-Amino-5-[3-(trifluoromethyl)(2-pyridyl)]pyridine-2-carbox-

amide (23). A solution of 6⁰-chloro-3-trifluoromethyl[2,3⁰]bi-

pyridinyl-5⁰-ylamine 22 (25 g, 0.091 mol), zinc cyanide (6.75 g,

0.058 mol), Pd₂(dba)₃(2.63 g, 2.86 mmol), DPPF (3.16 g, 5.72

mmol) in DMF (250 mL), and water (2.5 mL), under a nitrogen

atmosphere, was heated at 120 °C for 1 h. Water (30 mL) was

then added and the solution reheated at 120 °C for a further 4 h

to complete the hydrolysis. The mixture was cooled to 0 °C, and

a solution of saturated ammonium chloride (200 mL), water

(200 mL), and ammonium hydroxide (50 mL) was added. After

the mixture was stirred at 0 °C for 1 h, the yellow precipitate was

filtered and washed with water (200 mL) and a 1:1 mixture of

ether/hexane (200 mL). The solid was air-dried and then in a

vacuum oven to yield (23 g, 90%) the title compound 23. MS 283

(M þ 1). ¹H NMR δ (CDCl₃) 8.90 (1H, d, J = 4 Hz), 8.12 (1H, d,

J = 7 Hz), 7.99 (1H, s), 7.96-7.88 (1H, m), 7.55-7.48 (1H, m),

7.15 (1H, s), 6.06 (2H, brs), 5.48 (1H, brs).

3342 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8

Blum et al.

7-(3-Trifluoromethylpyridin-2-yl)pyrido[3,2-d]pyrimidin-4-ol (24).

A solution of 3-amino-5-[3-(trifluoromethyl)-(2-pyridyl)]pyri-

dine-2-carboxamide (23) (283 mg, 1.0 mmol) and triethyl ortho-

formate (5 mL) was heated at 130 ^oC for 16 h. The mixture was

cooled to room temperature, and the volatiles were removed by

rotary evaporation. The residue was triturated with ether/

hexane and the white solid collected by filtration, air-dried

(267 mg, 91%) and yielded the title compound 24. MS 293

(M þ 1). ¹H NMR δ (DMSO) 8.98 (1H, d, J = 5 Hz), 8.80 (1H,

s), 8.41 (1H, J = 9 Hz), 8.22 (1H, s), 8.18 (1H, s) 7.77 (1H, dd,

J = 2.5 and 8.5 Hz).

mixture was stirred at 0 °C for a further 1 h, and the resulting

yellow solid was collected by filtration. The solid was washed

with HCO₂Me (2 ꢀ 75 mL) and air-dried for 0.5 h. The solid was

dried under vacuum at 25 °C for 1 h and gave 3-amino-

3-methoxyacrylonitrile hydrochloride as a white solid (96.4 g,

60%) which was used in the next step without further purifica-

tion. 3-Dimethylamino-1-(3-trifluoromethylpyridin-2-yl)pro-

penone (29) (191.7 g, 0.79 mol), ethanol (2.0 L), NH₄OAc

(302.6 g, 9.93 mol), and 3-amino-3-methoxyacrylonitrile hydro-

chloride (211.2 g, 1.57 mol) were heated at 80 °C for 7 h. The

mixture was cooled to room temperature and the solvent

removed under reduced pressure. The solid that separated was

collected by filtration and washed with a small amount of cold

ethanol. The solid was dissolved in ethyl acetate (750 mL) and

the mixture washed with aqueous NaHCO₃(500 mL). The

organic extract was dried (Na₂SO₄), concentrated and afforded

6-amino-3⁰-trifluoromethyl-[2, 2⁰]bipyridinyl-5-carbonitrile (30)

as a light-brick-colored solid (85.5 g, 41%). MS 265 (M þ 1). ¹H

NMR δ (CDCl₃) 8.85 (1H, d, J = 5 Hz), 8.12 (1H, d, J = 8 Hz),

7.84 (1H, d, J = 7.5 Hz), 7.52 (1H, dd, J = 5 and 8 Hz), 7.09 (1H,

d, J = 7 Hz), 5.30 (1H, brs).

N⁰-(5-Cyano-3⁰-trifluoromethyl[2,2⁰]bipyridinyl-6-yl)-N,N-di-

methylformamidine (31). A mixture of 6-amino-3⁰-trifluoro-

methyl[2, 2⁰] bipyridinyl-5-carbonitrile (30) (2.64 g, 10 mmol)

and N,N-dimethylformamide dimethyl acetal (50 mL) was

heated at 110 °C for 1 h. The mixture was cooled to room

temperature and the excess reagent removed by evaporation to

yield the title compound 31 as a light-brown solid (3.2 g, 100%).

MS 320 (M þ 1). ¹H NMR δ (CDCl₃) 8.84 (1H, d, J = 5 Hz),

8.70 (1H, s), 8.15 (1H, d, J = 8 Hz), 7.92 (1H, d, J = 7.5 Hz),

7.51 (1H, dd, J = 5 and 8 Hz), 7.35 (1H, d, J = 7 Hz), 3.19 (3H,

s), 3.16 (3H, s).

4-Chloro-7-(3-trifluoromethylpyridin-2-yl)pyrido[3,2-d]pyrimi-

dine (25). A mixture of 7-(3-trifluoromethylpyridin-2-yl)pyrido-

[3,2-d]pyrimidin-4-ol (24) (220 mg, 0.75 mmol), thionyl chloride

(5 mL), and DMF (3 drops) was heated at reflux for 3 h. The

mixture was evaporated to dryness and the residue partitioned

between ethyl acetate (10 mL) and saturated aqueous sodium

bicarbonate (10 mL). The layers were separated, and the aqu-

eous layer was further extracted with ethyl acetate (15 mL). The

combined organic extracts were washed with brine, dried

(MgSO₄), evaporated and afforded the title compound 25

1

(202 mg, 87%). MS 311 (M þ 1). H NMR δ (DMSO) 9.29

(1H, d, J = 2 Hz), 9.27 (1H, s), 9.04 (1H, d, J = 7 Hz), 8.64 (1H,

s), 8.47 (1H, d, J = 8.5 Hz), 7.83 (1H, dd, J = 2 and 8.5 Hz).

(4-Trifluoromethylphenyl)-[7-(3-trifluoromethylpyridin-2-yl)-

pyrido[3,2-d]pyrimidin-4-yl]amine (26). A mixture of 4-chloro-

7-(3-trifluoromethylpyridin-2-yl)pyrido[3,2-d]pyrimidine (25)

(622 mg, 2.0 mmol) and 4-(trifluoromethyl)aniline (14) (480

mg, 3.0 mmol) in IPA (20 mL) was heated at 80 ^oC for 2 h. The

mixture was cooled to room temperature and the precipitate

collected by filtration. The precipitate was partitioned between

ethyl acetate (50 mL) and saturated sodium bicarbonate solu-

tion (50 mL). The layers were separated, and the aqueous layer

was extracted with further ethyl acetate (50 mL). The com-

bined organic extracts were washed with brine (40 mL), dried

(MgSO₄), evaporated and gave the title compound 26 (765 mg,

88%). MS 436 (M þ 1). ¹H NMR δ (CDCl₃) 11.75 (1H, br s),

9.03-8.99 (3H, m), 8.42 (1H, d, J = 7 Hz), 8.07-8.04 (2H, m),

7.96 (1H, d, J = 8.5 Hz), 7.87 (2H, d, J = 7.5 Hz), 7.79 (1H, dd,

J = 2.5 and 7.5 Hz). Analytical HPLC: method 1, 13.02

(99.3%); method 2, 5.84 (99.4%).

(4-Trifluoromethylphenyl)-[7-(3-trifluoromethylpyridin-2-yl)-

pyrido[2,3-d]pyrimidin-4-yl]amine (32). A mixture of N⁰-(5-cya-

no-3⁰-trifluoromethyl[2,2⁰]bipyridinyl-6-yl)-N,N-dimethylforma-

midine (31) (160 mg, 0.5 mmol) and 4-trifluoromethylaniline

(14) (80 mg, 0.5 mmol) in a solution of AcOH (1 mL) and water

(6 mL) was heated at reflux for 6 h. The mixture was cooled to

room temperature and basified by dropwise addition of 1 M

sodium hydroxide. The mixture was extracted with ethyl acetate

(3 ꢀ 10 mL), and the combined extracts were washed with brine

(10 mL), dried (Na₂SO₄), and concentrated. The residue was

purified by preparative TLC (elution with hexanes/ethyl acetate,

1:2) and gave the title compound 32 as a pale-yellow solid

2-Acetyl-3-trifluoromethylpyridine (28). A stirred solution of

2-cyano-3-trifluoromethylpyridine (27) (179 g, 1.04 mol) and

THF (1.2 L) was cooled to 0 °C, and methylmagnesium iodide

(3.0 M in ether (694 mL, 2.08 mol) was added dropwise over a

period of 1.5 h. The reaction mixture was stirred at 0 °C for a

further 0.5 h and then slowly poured over a stirred mixture of

ice-water (3 kg). The resulting mixture was acidified with 6 M

HCl to pH ∼2 and stirred for a further 0.5 h. The mixture was

extracted with ethyl acetate (5 ꢀ 1.0 L), and the combined

extracts were washed with brine (1.5 L) and dried (Na₂SO₄). The

extract was concentrated and gave a dark-brown oil. The oil was

distilled under vacuum (45-50 °C at 0.1 Torr) and gave the title

compound 28 as a pale-yellow liquid (163.7 g, 83%). ¹H NMR δ

(CDCl₃) 8.78 (1H, d, J = 5 Hz), 8.07 (1H, d, J = 8 Hz), 7.54 (1H,

dd, J = 5 and 8 Hz), 2.09 (3H, s).

3-Dimethylamino-1-(3-trifluoromethylpyridin-2-yl)propenone (29).

2-Acetyl-3-trifluoromethylpyridine (28) (150 g, 0.79 mol) and

(Me)₂NCH(OMe)₂(236 g, 2.0 mol) were combined, and the

mixture was heated at 105 °C for 5 h. The mixture was

concentrated by rotary evaporation and then under a high

vacuum (0.1 Torr) for an additional 1 h and gave the title

compound 29 as a brown oil (193 g, 100%). MS 245 (M þ 1).

¹H NMR δ (CDCl₃) 8.72 (1H, d, J = 5 Hz), 8.03 (1H, J = 8 Hz),

7.80 (1H, brs), 7.41 (1H, dd, J = 5 and 8 Hz), 5.52 (1H, br d),

3.10 (3H, br s), 2.88 (3H, s).

1

(124 mg, 57%). MS 436 (M þ 1). H NMR δ (DMSO) 9.75

(1H, d, J = 7.5 Hz), 9.05-9.03 (2H, m), 8.72 (1H, brs), 8.48 (1H,

d, J = 7.5 Hz), 8.25 (1H, d, J = 7 Hz), 8.07 (2H, d, J = 7 Hz),

7.90-7.87 (3H, m). Analytical HPLC: method 1, 12.03 (95.4%);

method 2, 5.13 (99.1%).

7-(3-Trifluoromethylpyridin-2-yl)pteridin-4-ylamine (33). 2-

Acetyl-3-trifluoromethylpyridine (28) (2.10 g, 11.1 mmol) was

dissolved in HBr (30% by weight in AcOH) (14 mL). The

mixture was cooled to 0 °C, and bromine (0.619 mL) was added

dropwise. The resulting solution was allowed to warm to room

temperature and stirred for 3 h. The mixture was concentrated

under reduced pressure and gave 2-bromo-1-(3-trifluoromethyl-

pyridin-2-yl)ethanone hydrobromide (3.85 g, 100%) which was

used without further purification in the following step. 4,5,6-

Triaminopyrimidine sulfate (1.12 g, 5.0 mmol) was dissolved in a

solution of water (45 mL) and barium chloride hydrate (1.77 g,

5.0 mmol). The mixture was heated to 80 °C for 4 h and cooled to

room temperature. The solids were removed by filtration and

NaHCO₃(1.26 g, 15 mmol), dioxane (20 mL), and 2-bromo-

1-(3-trifluoromethylpyridin-2-yl)ethanone hydrobromide (1.00

g, 2.86 mmol) were added to the filtrate. The resulting mixture

was stirred for 1 h at room temperature and 1 h at 100 °C. The

mixture was cooled, filtered through Celite and the Celite bed

washed with ethyl acetate (20 mL). The two phases were

separated, and the aqueous layer was extracted with ethyl

acetate (4 ꢀ 100 mL). The combined organic extracts were dried

6-Amino-3⁰-trifluoromethyl[2, 2⁰]bipyridinyl-5-carbonitrile (30).

Malononitrile (198 g, 3 mol), HCO₂Me (1.0 L), and MeOH

(240 mL) were cooled to 0 °C, and thionyl chloride (176 mL, 2.4

mol) was added dropwise over a period of 1 h. The reaction

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3343

(Na₂SO₄), and the solvent was removed under reduced pressure.

The residue was dissolved in methanol, and air was bubbled

through the solution for 16 h. The methanol was removed

under reduced pressure and the residue was purified by column

chromatography (elution with hexanes/acetone, 1:1) and af-

forded the title compound 33 as an off-white solid (192 mg,

23%). MS 293 (M þ 1).

reflux for 4 days and then evaporated to dryness. The mixture

was partitioned between ethyl acetate (150 mL) and saturated

aqueous sodium bicarbonate (100 mL). The layers were sepa-

rated, and the aqueous layer was extracted with ethyl acetate

(2 ꢀ 100 mL). The combined organic extracts were washed with

brine, dried (MgSO₄), evaporated and afforded the title com-

pound 37 (4.9 g, 92%) which was used without further purifica-

tion. MS 297 (M þ 1). ¹H NMR δ (CDCl₃) 8.82 (1H, d, J = 5

Hz), 8.08 (1H, d, J = 8.5 Hz), 7.92 (1H, d, J = 8 Hz), 7.45 (1H,

dd, J = 5 and 8.5 Hz), 6.77 (2H, m), 3.86 (3H, s).

(4-Trifluoromethylphenyl)-[7-(3-trifluoromethylpyridin-2-yl)-

pteridin-4-yl]amine (34). To a degassed mixture of 7-(3-trifluoro-

methylpyridin-2-yl)pteridin-4-ylamine (33) (73 mg, 0.25 mmol),

Cs₂CO₃(162 mg, 0.5 mmol), 4-(trifluoromethyl)bromobenzene

(67 mg, 0.3 mmol) in dioxane (5 mL) under nitrogen were added

Pd₂dba₃(11 mg, 0.0125 mmol) and xantphos (7 mg, 0.0125

mmol). The mixture was heated at 100 °C for 8 h, cooled, water

(8 mL) added and extracted with ethyl acetate (3 ꢀ 15 mL). The

combined extracts were dried (Na₂SO₄) and concentrated. The

residue was purified by preparative TLC (elution with hexanes/

acetone, 3:1) and gave the title compound 34 as a yellow solid

(42 mg, 39%). MS 437 (M þ 1). ¹H NMR δ (CDCl₃) 9.29 (1H, s),

9.24 (1H, brs), 9.08 (1H, s), 8.98 (1H, d, J = 5 Hz), 8.28 (1H, d,

J = 7 Hz), 8.14 (2H, d, J = 7.5 Hz), 7.74 (2H, d, J = 7.5 Hz),

7.32 (1H, m). Analytical HPLC: method 1, 12.55 (100%);

method 2, 5.05 (98.2%).

2,4-Dichloro-7-(3-trifluoromethylpyridin-2-yl)quinoline (38).

A solution of 2-amino-4-(3-trifluoromethylpyridin-2-yl)benzoic

acid methyl ester (37) (296 mg, 1.0 mmol) and AcOH (1 mL) in

dioxane (2 mL) was heated at 60 °C for 3 h. The solution was

cooled, water (1 mL) was added, and the mixture was evapo-

rated to dryness. Toluene (5 mL) was added to the residue which

was evaporated to dryness. The solid was dissolved in THF

(4 mL) and added dropwise to a stirred solution of potassium

bis(trimethylsilyl)amide (600 mg, 3.0 mmol) in toluene (6 mL) at

-78 °C. The mixture was allowed to return to room temperature

overnight, and water (10 mL) was added. The mixture was

washed with ethyl acetate (20 mL) and the aqueous layer

acidified with 4 M hydrochloric acid. The precipitate that

formed was collected by filtration, air-dried and gave a white

solid. The solid was added in portions to a stirred solution of

OPCl₃(5 mL) and the mixture heated at reflux for 18 h. The

excess reagent was evaporated and the residue carefully neutra-

lized with saturated aqueous NaHCO₃(10 mL). The mixture

was extracted with ethyl acetate (3 ꢀ 25 mL), and the combine

extracts were dried (Na₂SO₄), concentrated and gave the title

4-Amino-2-(3-trifluoromethylpyridin-2-yl)pyrimidine-5-carbo-

nitrile (35). 2-Cyano-3-trifluoromethylpyridine (27) (2.0 g, 11.5

mmol) in diethyl ether (40 mL) was added dropwise to a solution

of lithium bis(trimethylsilyl)amide (3.88 g, 23 mmol) in diethyl

ether (40 mL) at 0 °C. The mixture was allowed to return to

room temperature and stirred for 2 h. A solution of AcOH

(4 mL) and water (16 mL) was added to the mixture which was

stirred for a further 1 h. The layers were separated, and the

aqueous layer was basified by addition of 4 M sodium hydroxide

solution. The resulting solution was extracted with dichloro-

methane (3 ꢀ 50 mL) and the combined extracts were dried

(MgSO₄) and evaporated to give a white solid (1.43 g, 66%). The

solid (1.43 g, 7.56 mmol) was dissolved in a mixture of methanol

(40 mL) and triethylamine (4 mL), and (ethoxymethylene)-

malononitrile (1.06 g, 8.69 mmol) was added dropwise. The

mixture was heated at reflux for 18 h and evaporated. The

residue was purified by flash chromatography (elution with 3:1

hexane/acetone) and yielded the title compound 35 as a yellow

solid (1.38 g, 69%). MS 266 (M þ 1). ¹H NMR δ (CDCl₃) 8.90

(1H, d, J = 1.5 Hz), 8.70 (1H, s), 8.14 (1H, J = 7.5 Hz), 7.56 (1H,

dd, J = 2.5 and 8.5 Hz), 5.86 (2H, brs).

(4-Trifluoromethylphenyl)-[7-(3-trifluoromethylpyridin-2-yl)-

pyrimido[4,5-d]pyrimidin-4-yl]amine (36). A mixture of 4-amino-

2-(3-trifluoromethylpyridin-2-yl)pyrimidine-5-carbonitrile (35)

(200 mg, 0.75 mmol) and N,N-dimethylformamide dimethyl

acetal (5 mL) was heated at 110 °C for 1 h. The mixture was

cooled to room temperature and the excess reagent removed by

evaporation. The residue was dissolved in a solution of AcOH

(2 mL) and water (10 mL). 4-Trifluoromethylaniline (14) (120

mg, 0.75 mmol) was added, and the mixture was heated at reflux

for 3 h. The mixture was cooled to room temperature and

basified by dropwise addition of 1 M sodium hydroxide. The

mixture was extracted with ethyl acetate (3 ꢀ 10 mL), and the

combined extracts were washed with brine (10 mL), dried

(Na₂SO₄), and concentrated. The residue was purified by pre-

parative TLC (elution with 0.5% ammonium hydroxide and 5%

methanol in dichloromethane) and gave the title compound 36

as a pale-yellow solid (160 mg, 49%). MS 437 (M þ 1). ¹H NMR

δ (CDCl₃) 9.88 (1H, s), 9.10 (1H, brs), 9.04 (1H, s), 8.84 (1H, d,

J = 1.5 Hz), 8.16 (1H, d, J = 7.5 Hz), 7.88 (2H, d, J = 8.5 Hz),

7.65 (2H, J = 8.5 Hz), 7.57 (1H, dd, J = 2.5 and 8.5 Hz).

Analytical HPLC: method 1, 11.93 (97.2%); method 2, 3.61

(95.3%).

1

compound 38 (264 mg, 77%). MS 343 (M þ 1). H NMR δ

(CDCl₃) 8.91 (1H, d, J = 5 Hz), 8.30 (1H, d, J = 8.5 Hz), 8.19

(1H, s), 8.16 (1H, d, J = 8 Hz), 7.81 (1H, d, J = 8.5 Hz), 7.58

(1H, s), 7.53 (1H, dd, J = 5 and 8.5 Hz).

4-Chloro-2-methoxy-7-[3-(trifluoromethyl)pyridin-2-yl]quino-

line (39) and 2-Chloro-4-methoxy-7-[3-(trifluoromethyl)pyridin-

2-yl]quinoline (40). Sodium methoxide (4 M, 0.27 mL, 1.1 mmol)

was added dropwise to a solution of 2,4-dichloro-7-(3-trifluoro-

methylpyridin-2-yl)quinoline (38) (342 mg, 1.0 mmol) in THF

(10 mL). The mixture was stirred at room temperature for 1 h,

water (15 mL) was added, and the mixture was extracted with

ethyl acetate (3 ꢀ 20 mL). The combined organic extracts were

washed with brine (25 mL), dried (MgSO₄), and evaporated.

The mixture was purified by flash chromatography (elution

with 2:1 hexane/ether) and afforded first 4-chloro-2-methoxy-

7-[3-(trifluoromethyl)pyridin-2-yl]quinoline (39) (199 mg, 59%).

MS 339 (M þ 1). ¹H NMR δ (CDCl₃) 8.89 (1H, d, J = 5 Hz), 8.19

(1H, d, J = 8.5 Hz), 8.13 (1H, d, J = 8.5 Hz), 8.02 (1H, s), 7.60

(1H, d, J = 8.5 Hz), 7.49 (1H, dd, 5 and 8 Hz), 7.08 (1H, s), 4.06

(3H, s). Later fractions contained 2-chloro-4-methoxy-7-[3-

(trifluoromethyl)pyridin-2-yl]quinoline (40) (101 mg, 30%). MS

339 (M þ 1). ¹H NMR δ (CDCl₃) 8.88 (1H, d, J = 5 Hz), 8.23

(1H, d, J = 8.5 Hz), 8.13 (1H, d, J = 8.5 Hz), 8.08 (1H, s), 7.64

(1H, d, J = 8.5 Hz), 7.49 (1H, dd, 5 and 8 Hz), 6.79 (1H, s), 4.09

(3H, s).

4-Methoxy-7-[3-(trifluoromethyl)pyridin-2-yl]quinoline (41).

A mixture of 2-chloro-4-methoxy-7-[3-(trifluoromethyl)pyri-

din-2-yl]quinoline (40) (111 mg, 0.32 mmol), ammonium for-

mate (190 mg, 3 mmol), and 10% palladium on carbon (30 mg)

in methanol (10 mL) was stirred at room temperature for 2 h.

The mixture was filtered through Celite and evaporated to give

the title compound 41 (94 mg, 96%) which was used without

further purification. MS 305 (M þ 1). ¹H NMR δ (CDCl₃) 8.86

(1H, d, J = 5 Hz), 8.79 (1H, d, J = 5 Hz), 8.28 (1H, d, J = 8.5

Hz), 8.18 (1H, s), 8.10 (1H, d, J = 8 Hz), 7.64 (1H, d, 8.5 Hz),

7.45 (1H, dd, J = 5 and 8 Hz), 6.76 (1H, d, J = 5 Hz), 4.04

(3H, s).

2-Amino-4-(3-trifluoromethylpyridin-2-yl)benzoic Acid Methyl

Ester (37). A solution of 2-amino-4-(3-trifluoromethylpyridin-

2-yl)benzoic acid (12) (5 g, 18 mmol) in methanol (100 mL) was

saturated with hydrogen chloride gas. The mixture was heated at

4-Chloro-7-[3-(trifluoromethyl)pyridin-2-yl]quinoline (42). A

solution of 4-methoxy-7-[3-(trifluoromethyl)pyridin-2-yl]quino-

line (41) (100 mg, 0.33 mmol) and 33% hydrogen bromide in

3344 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8

Blum et al.

1

AcOH (5 mL) was heated at 100 °C for 18 h. The mixture was

evaporated to dryness and the resulting solid added to a stirred

solution of OPCl₃(2 mL). The mixture was heated at reflux for

2 h, cooled to room temperature and the excess reagent removed

under reduced pressure. The residue was carefully neutralized

with saturated NaHCO₃and the mixture extracted with ethyl

acetate (3 ꢀ 20 mL), dried (Na₂SO₄), concentrated and afforded

the title compound 42 (91 mg, 89%). MS 309 (M þ 1). ¹H NMR

δ (CDCl₃) 8.90 (1H, d, J = 5 Hz), 8.84 (1H, d, J = 5 Hz), 8.33

(1H, d, J = 8.5 Hz), 8.29 (1H, s), 8.14 (1H, d, J = 8 Hz), 7.80

(1H, d, 8.5 Hz), 7.56 (1H, d, J = 5 Hz), 7.50 (1H, dd, J = 5 and

8 Hz).

7-[3-(Trifluoromethyl)pyridin-2-yl]-N-[5-(trifluoromethyl)-

pyridin-2-yl]quinolin-4-amine (43). To a degassed mixture of

4-chloro-7-[3-(trifluoromethyl)pyridin-2-yl]quinoline (42) (76

mg, 0.25 mmol), Cs₂CO₃(162 mg, 0.5 mmol), 2-amino-5-

trifluoromethylpyridine (43 mg, 0.3 mmol) in dioxane (5 mL)

under nitrogen were added Pd₂dba₃(11 mg, 0.0125 mmol) and

xantphos (7 mg, 0.0125 mmol). The mixture was heated at

100 °C for 3 h, cooled, and water (8 mL) was added. The layer

was extracted with ethyl acetate (3 ꢀ 15 mL). The combined

extracts were dried (Na₂SO₄) and concentrated. Purification of

the residue by flash chromatography (elution with 0.5% ammo-

nium hydroxide and 5% methanol in dichloromethane) yielded

the title compound 43 (74 mg, 68%). MS 435 (M þ 1). ¹H NMR

δ (CDCl₃) 8.73 (1H, d, J = 5 Hz), 8.56 (1H, s), 8.51 (1H, s), 8.40

(2H, d, J = 8 Hz), 8.11-8.09 (3H, m), 7.85 (1H, d, J = 8.5 Hz),

7.67 (1H, d, J = 9 Hz), 7.53 (1H, d, J = 8.5 Hz), 7.47 (1H, dd,

J = 5 and 8 Hz). Analytical HPLC: method 1, 10.80 (100%);

method 2, 4.86 (100%).

(191 mg, 63%). MS 470 (M þ 1). H NMR δ (CDCl₃) 9.02

(1H, s), 8.98 (1H, d, J = 2 Hz), 8.61-8.57 (2H, m), 8.39-8.34

(2H, m), 8.18 (1H, d, J = 8 Hz), 8.01-7.90 (2H, m), 7.61-7.57

(1H, m).

7-[3-(Trifluoromethyl)pyridin-2-yl]-N-[5-(trifluoromethyl)-

pyridin-2-yl]-1,5-naphthyridin-4-amine (47).

A mixture of

2-chloro-7-[3-(trifluoromethyl)pyridin-2-yl]-N-[5-(trifluoro-

methyl)pyridin-2-yl]-1,5-naphthyridin-4-amine (46) (94 mg,

0.20 mmol), ammonium formate (126 mg, 2.0 mmol), and

10% palladium on carbon (25 mg) in methanol (10 mL) was

heated at 50 °C for 2 h. The mixture was cooled, filtered through

Celite, and evaporated to dryness. The residue was partitioned

between ethyl acetate (10 mL) and saturated aqueous NaHCO₃

(10 mL). The layers were separated, and the aqueous layer was

extracted with ethyl acetate (2 ꢀ 10 mL). The combined extracts

were washed with brine (10 mL), dried (MgSO₄), and concen-

trated. The residue was purified by preparative thin layer

chromatography (elution with 0.5% ammonium hydroxide

and 5% methanol in dichloromethane) and yielded the title

compound 47 (62 mg, 71%). MS 437 (M þ 1). ¹H NMR δ

(CDCl₃) 8.95-8.93 (2H, m), 8.63-8.57 (2H, m), 8.34-8.32 (2H,

m), 8.26 (1H, s), 8.18 (1H, d, J = 8 Hz), 7.91 (1H, dd, J = 2 and 9

Hz), 7.57-7.52 (2H, m). Analytical HPLC: method 1, 12.63

(97.9%); method 2, 5.48 (98.6%).

tert-Butyl-4-chloro-3-formylpyridin-2-yl Carbamate. To a

stirred solution of tert-butyl 4-chloropyridin-2-yl carbamate

(48) (19.5 g, 0.085 mol) in THF (400 mL) at -78 °C was added

dropwise n-butyllithium (2.5 M in hexanes, 80 mL, 0.20 mol).

The solution was stirred at -78 °C for 1 h, and DMF (33 mL, 0.4

mol) was then added dropwise. After an additional 1 h at -78 °C

the reaction was quenched by the dropwise addition of saturated

ammonium chloride solution. The solution was extracted with

ethyl acetate (3 ꢀ 500 mL), and the combined extracts were dried

(MgSO₄) and evaporated. Purification by column chromato-

graphy (elution with ethyl acetate/hexane, 1:4) gave the title

4-Chloro-2-methoxy-7-[3-(trifluoromethyl)pyridin-2-yl]-1,5-

naphthyridine (44). Following analogous procedures to those

described above, the title compound 44 was prepared. MS 340

(M þ 1). ¹H NMR δ (CDCl₃) 9.07 (1H, s), 8.95 (1H, d, J = 5

Hz), 8.41 (1H, s), 8.18 (1H, d, J = 8.5 Hz), 7.57 (1H, dd, 5 and

8 Hz), 7.02 (1H, s), 4.19 (3H, s).

1

compound, a white solid (15.9 g, 73%). H NMR δ (CDCl₃)

10.73 (1H, brs), 10.53 (1H, s), 8.52 (1H, d, J = 1.5 Hz), 7.07 (1H,

d, J = 1.5 Hz), 1.55 (9H, s).

2-Methoxy-7-[3-(trifluoromethyl)pyridin-2-yl]-N-[5-(trifluo-

romethyl)pyridin-2-yl]-1,5-naphthyridin-4-amine (45). To a de-

gassed mixture of 4-chloro-2-methoxy-7-[3-(trifluoromethyl)-

pyridin-2-yl]-1,5-naphthyridine (44) (339 mg, 1.0 mmol),

Cs₂CO₃(650 mg, 2.0 mmol), and 2-amino-5-trifluoromethyl-

pyridine (176 mg, 1.2 mmol) in dioxane (20 mL) under nitro-

gen were added Pd₂dba₃(46 mg, 0.05 mmol) and xantphos

(29 mg, 0.05 mmol). The mixture was stirred at 100 °C for 3 h,

cooled, water (10 mL) was added, and the layer was extracted

with ethyl acetate (3 ꢀ 10 mL). The combined extracts were

dried (Na₂SO₄) and evaporated. Purification by column chro-

matography (elution with 0.5% ammonium hydroxide and

5% methanol in dichloromethane) gave the title compound 45

2-Amino-4-chloronicotinaldehyde (49). Trifluoroacetic acid

(42.7 mL, 551 mmol) was added dropwise to a mixture of tert-

butyl-4-chloro-3-formylpyridin-2-yl carbamate (28.5 g, 110

mmol) in dichloromethane (500 mL), and the resulting mixture

was stirred for 18 h. A saturated solution of sodium bicarbonate

(400 mL) was added, and the layers were separated. The aqueous

layer was further extracted with dichloromethane (2 ꢀ 250 mL)

and the combined organic extracts were dried (MgSO₄), evapo-

rated and gave the title compound 49, a yellow solid (16.1 g,

94%). ¹H NMR δ (CDCl₃) 10.45 (1H, s), 8.12 (1H, d, J = 1.5

Hz), 6.69 (1H, d, J = 1.5 Hz), 6.05 (2H, brs).

5-Chloro-2-[3-(trifluoromethyl)pyridin-2-yl]-1,8-naphthyri-

dine (50). Potassium tert-butoxide (8.4 g, 75 mmol) was added in

portions to a stirred solution of 2-amino-4-chloronicotinalde-

hyde (49) (7.8 g, 50 mmol) and 2-acetyl-3-trifluoromethylpyr-

idine (28) (9.45 g, 50 mmol) in THF (200 mL) at -20 °C. The

solution was allowed to reach 10 °C and stirred for 2 h. The

reaction mixture was evaporated under reduced pressure and

the residue diluted with saturated aqueous ammonium chloride

(150 mL). The solid was collected by filtration, air-dried and

afforded the title compound (50) (13.4 g, 87%), a white solid.

MS 310 (M þ 1). ¹H NMR δ (CDCl₃) 9.09 (1H, d, J = 2 Hz),

8.91 (1H, d, J = 2 Hz), 8.77 (1H, d, J = 8.5 Hz), 8.22 (1H, d, J =

7 Hz), 8.09 (1H, d, J = 8.5 Hz), 7.64 (1H, d, J = 4 Hz), 7.58 (1H,

dd, J = 5 and 8 Hz).

1

(397 mg, 85%) as a pale-yellow solid. MS 466 (M þ 1). H

NMR δ (CDCl₃) 8.95 (1H, d, J = 2 Hz), 8.90 (1H, s), 8.58 (1H,

s), 8.37 (1H, d, J = 8 Hz), 8.29 (1H, s), 8.19 (1H, d, J = 8 Hz),

8.07 (1H, br s), 7.88 (1H, dd, J = 2 and 8.5 Hz), 7.57-7.54 (1H,

m), 7.07 (1H, s), 4.16 (s, 3H).

2-Chloro-7-[3-(trifluoromethyl)pyridin-2-yl]-N-[5-(trifluoro-

methyl)pyridin-2-yl]-1,5-naphthyridin-4-amine (46). A solution

of 2-methoxy-7-[3-(trifluoromethyl)pyridin-2-yl]-N-[5-(trifluo-

romethyl)pyridin-2-yl]-1,5-naphthyridin-4-amine (45) (300 mg,

0.64 mmol) and 33% hydrogen bromide in AcOH (10 mL) was

heated at 100 °C for 18 h. The mixture was concentrated to

dryness, saturated aqueous sodium bicarbonate (10 mL) added,

and the mixture extracted with ethyl acetate (3 ꢀ 20 mL). The

combined extracts were dried (Na₂SO₄), and concentrated. The

residue was added to a stirred solution of phosphorus oxychlo-

ride (3 mL) and the mixture heated at reflux for 0.5 h. The

mixture was cooled to room temperature and evaporated.

Saturated aqueous NaHCO₃(20 mL) was added and the

mixture extracted with ethyl acetate (3 ꢀ 15 mL). The combined

organic extracts were washed with brine (20 mL), dried

(Na₂SO₄), evaporated and afforded the title compound 46

(4-Trifluoromethylphenyl)-[7-(3-trifluoromethylpyridin-2-yl)-

[1,8]naphthyridin-4-ylamine (51). Hydrochloric acid (2 M in

ether, 0.5 mL, 1.0 mmol) was added to a solution of 5-chloro-

2-[3-(trifluoromethyl)pyridin-2-yl]-1,8-naphthyridine (50) (309

mg, 1.0 mmol) and 4-trifluoromethylaniline (14) (161 mg, 1.0

mmol) in IPA (5 mL). The mixture was heated at 80 °C for 16 h.

The solid was collected by filtration and partitioned between

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3345

ethyl acetate (10 mL) and saturated aqueous sodium bicarbo-

nate (10 mL). The layers were separated, and the aqueous layer

was extracted with ethyl acetate (10 mL). The combined extracts

were washed with brine (10 mL), dried (MgSO₄), evaporated

and gave the title compound 51, a white solid (340 mg, 78%).

MS 435 (M þ 1). ¹H NMR δ (CDCl₃) 8.98 (1H, d, J = 8.5 Hz),

8.74 (1H, s), 8.62 (1H, m), 8.18 (1H, d, J = 7.5 Hz), 7.78 (1H, d,

J = 8.5 Hz), 7.62 (2H, d, J = 7.5 Hz), 7.50 (3H, m), 6.80 (1H, m).

Analytical HPLC: method 1, 10.38 (95.0%); method 2, 5.15

(95.0%).

J = 8.5 Hz), 7.61 (1H, dd, J = 5 and 8 Hz), 7.13 (1H, d, J = 8.5

Hz). Analytical HPLC: method 1, 12.37 (100%); method 2, 4.95

(100%).

Later fractions contained, 2-[3-(trifluoromethyl)pyridin-

2-yl]-N-[5-(trifluoromethyl)pyridin-2-yl]pyrido[3,4-b]pyrazin-

5-amine (56) (25 mg, 23%). MS 437 (M þ 1). ¹H NMR δ

(CDCl₃) 9.26 (1H, s), 9.00-8.97 (2H, m), 8.61 (1H, s), 8.48 (1H,

d, J = 5.5 Hz), 8.25 (1H, d, J = 8.5 Hz), 7.99 (1H, d, J = 8.5 Hz),

7.65 (1H, dd, J = 5 and 8 Hz), 7.50 (1H, d, J = 5.5 Hz).

Analytical HPLC: method 1, 13.84 (98.6%); method 2, 6.50

(100%).

7-[3-(Trifluoromethyl)pyridin-2-yl]-N-[5-(trifluoromethyl)-

pyridin-2-yl]-1,8-naphthyridin-4-amine (52). A mixture of 5-

chloro-2-(3-(trifluoromethyl)pyrid-2-yl)-1,8-naphthyridine (50)

(6.72 g, 21.7 mmol) and 2-amino-5-trifluoromethylpyridine (15)

(7.13 g, 44 mmol) was heated in a sealed tube at 180 °C for 2 h.

The mixture was allowed to cool to room temperature and the

resulting solid was dissolved in a small amount of methanol.

Sodium hydroxide (1 M, 200 mL) was added and the mixture

extracted with ethyl acetate (500 mL). The extract was washed

with brine (200 mL), dried (MgSO₄), and evaporated. Purifica-

tion by column chromatography (elution with ethyl acetate/

hexane, 4:1, and then ethyl acetate) afforded the title compound

52 as a yellow solid (5.85 g, 62%). MS 436 (M þ 1). ¹H NMR δ

(CDCl₃) 8.99 (1H, s), 8.84 (1H, d, J = 5 Hz), 8.65 (1H, d, J = 8.5

Hz), 8.60 (1H, s), 8.32 (1H, brs), 8.14 (1H, d, J = 8 Hz), 8.06

(1H, s), 7.85-7.79 (2H, m), 7.51 (1H, dd, J = 8 and 5 Hz), 7.22

(1H, d, J = 8.5 Hz). Analytical HPLC: method 1, 10.65 (100%);

method 2, 4.36 (100%).

3-[3-(Trifluoromethyl)pyridin-2-yl]pyrido[2,3-b]pyrazin-8-amine

(53) and 2-(3-Trifluoromethylpyridin-2-yl)pyrido[3,4-b]pyrazin-

5-amine (54). 1-(3-Trifluoromethylpyridin-2-yl)ethanone (28)

(2.65 g, 14 mmol) was dissolved in HBr (30% by weight in

AcOH) (14 mL). The mixture was cooled to 0 °C, and bromine

(0.8 mL) was added dropwise. The resulting solution was

allowed to warm to room temperature and stirred for 3 h. The

mixture was concentrated under reduced pressure and gave

2-bromo-1-(3-trifluoromethylpyridin-2-yl)ethanone hydrobro-

mide (4.85 g, 100%) which was used without further purification

in the following step. To a solution of 2,3,4-triaminopyridine

(3.10 g, 25 mmol) in water (200 mL) was added NaHCO₃(6.3 g,

75 mmol), dioxane (100 mL), and 2-bromo-1-(3-trifluoro-

methylpyridin-2-yl)ethanone hydrobromide (4.85 g). The mix-

ture was heated at 100 °C for 2 h, cooled to room temperature,

and extracted with EtOAc (4 ꢀ 100 mL). The combined organic

extracts were washed with brine (100 mL) and dried (Na₂SO₄).

Purification of the residue by column chromatography (elution

with 0.5% ammonium hydroxide and 5% methanol in dichlo-

romethane) gave a mixture containing the two title compounds

53 and 54 (1.3 g, 32%) which were used in the next step without

isolation of the two individual products.

3-[3-(Trifluoromethyl)pyridin-2-yl]-N-[5-(trifluoromethyl)-

pyridin-2-yl]pyrido[2,3-b]pyrazin-8-amine (55) and 2-[3-(Trifluo-

romethyl)pyridin-2-yl]-N-[5-(trifluoromethyl)pyridin-2-yl]pyrido-

[3,4-b]pyrazin-5-amine (56). To a degassed mixture of 3-[3-(tri-

fluoromethyl)pyridin-2-yl]pyrido[2,3-b]pyrazin-8-amine (53),

2-(3-trifluoromethylpyridin-2-yl)pyrido[3,4-b]pyrazin-5-amine

(54) (72 mg, 0.25 mmol), Cs₂CO₃(162 mg, 0.5 mmol), and

2-chloro-5-trifluoromethylpyridine (45 mg, 0.25 mmol) in diox-

ane (5 mL) under nitrogen were added Pd₂dba₃(11 mg, 0.0125

mmol) and xantphos (7 mg, 0.0125 mmol). The mixture was

stirred at 100 °C for 3 h, cooled, water (10 mL) was added, and

the layer was extracted with ethyl acetate (3 ꢀ 10 mL). The

combined organic layers were dried (Na₂SO₄) and evaporated.

Purification by column chromatography (elution with 0.5%

ammonium hydroxide and 5% methanol in dichloromethane)

gave first 3-[3-(trifluoromethyl)pyridin-2-yl]-N-[5-(trifluoro-

methyl)pyridin-2-yl]pyrido[2,3-b]pyrazin-8-amine (55) (31 mg,

28%). MS 437 (M þ 1). ¹H NMR δ (CDCl₃) 9.42 (1H, s), 9.28

(1H, s), 9.11 (1H, d, J = 5 Hz), 8.95 (1H, d, J = 5 Hz), 8.90 (1H,

d, J = 5 Hz), 8.72 (1H, s), 8.25 (1H, d, J = 8 Hz), 7.89 (1H, d,

1-[2-Amino-4-(3-trifluoromethylpyridin-2-yl)phenyl]ethanone

(57). A solution of 2-amino-4-(3-trifluoromethylpyridin-2-yl)-

benzoic acid (12) (2.8 g, 10 mmol), N,O-dimethylhydroxylamine

hydrochloride (2.14 g, 22 mmol), EDCI (2.1 g, 11 mmol), and

€

Hunig’s base (5.1 g, 40 mmol) in dichloromethane (100 mL) was

stirred at room temperature for 24 h. The reaction mixture was

poured into saturated aqueous sodium bicarbonate (100 mL)

and extracted with dichloromethane (2 ꢀ 100 mL). The com-

bined organics were washed with brine (200 mL), dried

(Na₂SO₄), and evaporated. The residue was purified by flash

chromatography (elution with 10% hexane in ethyl acetate and

then ethyl acetate) and gave N-methoxy-N-methyl-2-amino-

4-(3-trifluoromethylpyridin-2-yl)benzamide as a yellow oil

1

(1.1 g, 34%). MS 326 (M þ 1). H NMR δ (CDCl₃) 8.82 (1H,

s), 8.05 (1H, d, J = 9 Hz), 7.48-7.40 (2H, m), 6.82-6.78 (2H,

m). To a solution of N-methoxy-N-methyl-2-amino-4-(3-tri-

fluoromethylpyridin-2-yl)benzamide (1.1 g, 3.4 mmol) in THF

(30 mL) at 0 °C was added methylmagnesium iodide (3 M in

toluene, 3.3 mL, 10 mmol). The mixture was stirred at room

temperature for 4 h, poured into 2 M hydrochloric acid (25 mL),

and stirred for 1 h. The mixture was neutralized by portionwise

addition of sodium bicarbonate and extracted with ethyl acetate

(3 ꢀ 50 mL). The combined organics were washed with brine

(100 mL), dried (Na₂SO₄), and evaporated. The residue was

purified by flash chromatography (elution with ethyl acetate)

and afforded the title compound 57 as a colorless oil (412 mg,

43%). MS 281 (M þ 1). ¹H NMR δ (CDCl₃) 8.88 (1H, s), 8.18

(1H, d, J = 6 Hz), 7.80 (1H, d, J = 7.5 Hz), 7.57-7.50 (1H, m),

6.82-6.78 (2H, m), 2.80 (3H, s).

7-(3-Trifluoromethylpyridin-2-yl)cinnolin-4-ol (58). To 1-[2-

amino-4-(3-trifluoromethylpyridin-2-yl)phenyl]ethanone (57)

(220 mg, 0.8 mmol) in concentrated hydrochloric acid (2 mL)

at 0 °C was added sodium nitrite (70 mg, 1.0 mmol) in water

(0.2 mL). After being stirred for 4 h, the solution was evaporated

to dryness and water (5 mL) and sodium acetate (328 mg,

4.0 mmol) were added. The mixture was refluxed for 2 h, cooled,

and extracted with ethyl acetate (3 ꢀ 25 mL). The combined

organics were washed with brine (30 mL), dried (Na₂SO₄), and

evaporated. The residue was purified by flash chromatography

(elution with 0.5% ammonium hydroxide and 5% methanol in

dichloromethane) and gave the title compound 58 (145 mg,

62%). MS 292 (M þ 1). ¹H NMR δ (CDCl₃) 11.42 (1H, s), 8.86

(1H, d, J = 5 Hz), 8.34 (1H, d, J = 9 Hz), 8.15 (1H, d, J = 7 Hz),

7.88 (1H, s), 7.58 (1H, s), 7.56-7.45 (2H, m).

4-Chloro-7-(3-trifluoromethylpyridin-2-yl)cinnoline (59). A solu-

tion of 7-(3-trifluoromethylpyridin-2-yl)cinnolin-4-ol (58) (292

mg, 1.0 mmol) in phosphorus oxychloride (10 mL) was heated at

reflux for 4 h. The mixture was evaporated to dryness and

partitioned between ethyl acetate (75 mL) and saturated sodium

bicarbonate solution (50 mL). The aqueous layer was extracted

with ethyl acetate (2 ꢀ 50 mL) and the combined organics were

washed with brine, dried (Na₂SO₄) and gave the title compound

59 (256 mg, 87%) which was used without further purification.

MS 310 (M þ 1). ¹H NMR δ (CDCl₃) 9.38 (1H, s), 8.45 (1H, d,

J = 5 Hz), 8.64 (1H, s), 8.22 (1H, d, J = 9 Hz), 8.18 (1H, d, J = 7

Hz), 7.98 (1H, d, J = 8.5 Hz), 7.50-7.46 (1H, m).

(5-Trifluoromethylpyridin-2-yl)-[7-(3-trifluoromethylpyridin-

2-yl)cinnolin-4-yl]amine (60). To a degassed mixture of 4-chloro-

7-(3-trifluoromethylpyridin-2-yl)cinnoline (59) (310 mg, 1.0 mmol),

3346 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8

Blum et al.

Cs₂CO₃(650 mg, 2.0 mmol), and 2-amino-5-trifluoromethylpyr-

idine (15) (162 mg, 1.0 mmol) in dioxane (10 mL) under nitrogen

were added Pd₂dba₃(44 mg, 0.05 mmol) and xantphos (28 mg, 0.05

mol). The mixture was stirred at 90 °C for 16 h, concentrated, and

extracted with ethyl acetate (3 ꢀ 20 mL). The combined extracts

were dried (Na₂SO₄) and concentrated. Purification by column

chromatography (elution with 0.5% ammonium hydroxide and

5% methanol in dichloromethane) yielded the title compound 60 as

8.93 (1H, d, J = 5 Hz), 8.73 (1H, s), 8.54 (1H, d, J = 8.0 Hz),

8.29 (1H, d, J = 8.5 Hz), 8.25 (1H, J = 7.5 Hz), 7.61 (1H, dd,

J = 5 and 8 Hz).

(5-Trifluoromethylpyridin-2-yl)[2-(3-trifluoromethylpyridin-

2-yl)[1,7]naphthyridin-5-yl]amine (64). To a degassed mixture

of trifluoromethanesulfonic acid 2-(3-trifluoromethylpyridin-

2-yl)[1,7]naphthyridin-5-yl ester (63) (96 mg, 0.23 mmol),

Cs₂CO₃(244 mg, 0.68 mmol), and 2-amino-5-trifluoromethyl-

pyridine (15) (74 mg, 0.45 mmol) in dioxane (3 mL) under

nitrogen were added Pd₂dba₃(21 mg, 0.023 mmol) and xantphos

(13 mg, 0.023 mol). The mixture was stirred at 110 °C for 2 h,

cooled and ethyl acetate (20 mL) added. The mixture was

filtered through Celite, concentrated, and purified by prepara-

tive thin layer chromatography (elution with 0.5% ammonium

hydroxide and 5% methanol in dichloromethane) and afforded

the title compound 64 (61 mg, 61%). MS 436 (M þ 1). ¹H NMR

δ (CDCl₃) 9.42 (1H, s), 8.92 (1H, d, J = 5 Hz), 8.65 (1H, s), 8.39

(1H, d, J = 8.5 Hz), 8.24 (1H, d, J = 7.5 Hz), 8.04 (1H, J = 8.0

Hz), 7.58 (1H, dd, J = 5 and 8 Hz), 7.49 (1H, J = 8.5 Hz), 6.92

(1H, J = 8.5 Hz), 6.17 (1H, s). Analytical HPLC: method 1,

11.85 (97.6%); method 2, 5.17 (100%).

1

a yellow solid (235 mg, 76%). MS 436 (M þ 1). H NMR δ

(DMSO) 10.46 (1H, s), 10.22 (1H, s), 8.99 (1H, d, J = 5 Hz), 8.76

(1H, s), 8.65 (1H, d, J = 9 Hz), 8.42 (1H, s), 8.38 (1H, d, J = 7.5

Hz), 8.14 (1H, d, J = 9 Hz), 7.95 (1H, d, J = 8.5 Hz), 7.78-7.74

(1H, m), 7.54 (1H, d, J = 8.5 Hz). Analytical HPLC: method 1,

11.92 (96.1%); method 2, 5.06 (100%).

5-Hydroxy-2-(3-trifluoromethylpyridin-2-yl)[1,7]naphthyri-

dine-6-carboxylic Acid Ethyl Ester (62). To a solution of

6-methyl-3⁰-trifluoromethyl[2,2⁰]bipyridinyl-5-carboxylic acid

ethyl ester (61) (720 mg, 2.32 mmol) in dichloromethane

(25 mL) and water (25 mL) were added 2,2⁰-azobis(2-methyl-

propionitrile) (10 mg) and N-bromosuccinimide (826 mg, 4.64

mmol). The mixture was heated at 70 °C for 4 h, cooled to room

temperature, and diluted with dichloromethane (50 mL). The

layers were separated and the organic layer was washed with

saturated aqueous NaHCO₃(30 mL), brine (40 mL), dried

(MgSO₄), and concentrated to give 6-bromomethyl-3⁰-

trifluoromethyl[2,2⁰]bipyridinyl-5-carboxylic acid ethyl ester

(905 mg) which was used in the next step without further

purification. A mixture of 6-bromomethyl-3⁰-trifluoromethyl-

[2,2⁰]bipyridinyl-5-carboxylic acid ethyl ester (905 mg, 2.32

mmol) and methyl 2-(4-methylphenylsulfonamido)acetate

(581 mg, 2.35 mmol) in ethanol (15 mL) was heated to 75 °C,

and sodium ethoxide (2.68 M in ethanol, 0.98 mL, 2.63 mmol)

was added dropwise. The mixture was heated for 1 h, and

additional sodium ethoxide (2.68 M in ethanol, 0.98 mL, 2.63

mmol) was added dropwise. The mixture was heated an addi-

tional 1 h and cooled to room temperature. The volatiles were

removed by evaporation, and the residue was partitioned

between ethyl acetate (20 mL) and sodium hydroxide (1 M,

20 mL). The aqueous layer was extracted with additional ethyl

acetate (2 ꢀ 25 mL), and the organic layers were combined. The

aqueous layer was acidified to pH ∼5-6 with 6 M hydrochloric

acid and extracted with additional ethyl acetate (3 ꢀ 25 mL).

The combined extracts were dried (Na₂SO₄) and concentrated

Supporting Information Available: Purity methods, chroma-

tograms, and ESI spectra for all final compounds; experimental

procedure for the complete Freund’s adjuvant in vivo assay;

graphs of core body changes in rats following daily dosing

(3 mg/kg) of compound 52. This material is available free of

charge via the Internet at http://pubs.acs.org.

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1

and gave the title compound 62 (513 mg, 61%). H NMR δ

(CDCl₃) 11.96 (1H, s), 9.15 (1H, s), 8.93 (1H, d, J = 5 Hz), 8.87

(1H, d, J = 8.5 Hz), 8.23 (1H, d, J = 7.5 Hz), 8.11 (1H, d, J =

8.5 Hz), 7.60 (1H, dd, J = 5 and 8 Hz), 4.62 (2H, d), 1.54 (3H, t).

Trifluoromethanesulfonic Acid 2-(3-Trifluoromethylpyridin-2-

yl)[1,7]naphthyridin-5-yl Ester (63). A mixture of 5-hydroxy-

2-(3-trifluoromethylpyridin-2-yl)[1,7]naphthyridine-6-carboxy-

lic acid ethyl ester (62) (147 mg, 0.4 mmol) and 6 M hydrochloric

acid (20 mL) was heated at 95 °C for 6 h. Concentrated

hydrochloric acid (6 mL) was added and the mixture heated

for an additional 12 h. The mixture was poured onto ice-water

(25 mL) and treated with 6 M sodium hydroxide until pH ∼4-5

was obtained. The solution was extracted with ethyl acetate (3 ꢀ

50 mL) and the combined extracts were dried (MgSO₄) and

evaporated to give 2-(3-trifluoromethylpyridin-2-yl)[1,7]naph-

thyridin-5-ol (80 mg, 69%) which was used in the next step

without further purification. To a mixture of 2-(3-trifluoro-

methylpyridin-2-yl)[1,7]naphthyridin-5-ol (80 mg, 0.27 mmol)

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€

in dichloromethane (2 mL) was added Hunig’s base (78 mg,

0.6 mmol). The mixture was cooled to 0 °C, and triflic anhydride

(100 mg, 0.35 mmol) in dichloromethane (1 mL) was added

dropwise. The solution was stirred for 2 h, diluted with dichlo-

romethane (10 mL), and washed with saturated aqueous sodium

bicarbonate (10 mL). The layers were separated, and the organic

layer was washed with brine (10 mL), dried (MgSO₄), evapo-

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Article Doi

Discovery of novel 6,6-heterocycles as transient receptor potential vanilloid (TRPV1) antagonists

DOI: 10.1021/jm100051g

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