Structure–Activity Relationships Comparing N-(6-Methylpyridin-yl)-Substituted Aryl Amides to 2-Methyl-6-(substituted-arylethynyl)pyridines or 2-Methyl-4-(substituted-arylethynyl)thiazoles as Novel Metabotropic Glutamate Receptor Subtype 5 Antagonists

Abstract

The metabotropic glutamate receptor subtype 5 (mGluR5) has been implicated in anxiety, depression, pain, mental retardation, and addiction. The potent and selective noncompetitive mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP, 1) has been a critically important tool used to further elucidate the role of mGluR5 in these CNS disorders. In an effort to provide novel and structurally diverse selective mGluR5 antagonists, we previously described a set of analogues with moderate activity wherein the alkyne bond was replaced with an amide group. In the present report, extended series of both amide and alkyne-based ligands were synthesized. MGluR5 binding and functional data were obtained that identified (1) several novel alkynes with comparable affinities to 1 at mGluR5 (e.g., 10 and 20–23), but (2) most structural variations to the amide template were not well tolerated, although a few potent amides were discovered (e.g., 55 and 56). Several of these novel analogues show drug-like physical properties (e.g., cLogP range) 2–5) that support their use for in vivo investigation into the role of mGluR5 in CNS disorders.

Introduction

Glutamate is the predominant excitatory neurotransmitter in the brain and mediates its effects through both the ionotropic, i.e., N-methyl-d-aspartate (NMDA), R-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainate receptors, and eight metabotropic receptors (mGluR 1–8). The metabotropic glutamate receptors are classified into three groups based on protein homology, signal transduction pathways, and pharmacology. Group I consists of the primarily postsynaptic mGluR1 and mGluR5 that are coupled via G_q to phospholipase C. The primarily presynaptically located mGluR2 and 3 in group II and mGluR4, 6, 7, and 8 in group III are coupled to G_i and associated signaling pathways, such as ion channels and inhibition of adenylyl cyclase (for reviews see refs ^1–3).^a

The metabotropic glutamate receptors are members of the family C G-protein coupled receptors (GPCR) that consists of a seven-transmembrane spanning domain protein with a large extracellular N-terminus wherein the orthosteric glutamate binding site resides. The metabotropic glutamate receptor allosteric binding site is located in the transmembrane region of the protein and has been described in detail,⁴ and specifically for mGluR5, using the high affinity and selective mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP, 1, see ref 5 for recent review).

The therapeutic potential of CNS drug targets is predicted, in part, based on brain localization. The mGluR5s are largely expressed in postsynaptic terminals of the limbic regions and especially in the limbic cortex, hippocampus, amygdala, and basal ganglia, involved in motivational, emotional, and memory processes.⁶ Over stimulation of mGluR5 has been implicated in numerous CNS disorders including anxiety, depression, neuropathic pain, drug addiction, and fragile X syndrome.⁷

Indeed, the identification of allosteric modulators of mGluR5 that exclusively access the allosteric binding site and hence are exquisitely selective for the mGluR5 subtype has led to an explosion of allosteric mGluR5-targeted drug discovery effort to potentially exploit preclinical discovery into medication development (see refs ^{5, 8–13} for recent reviews).

Specifically, the localization of mGluR5 in the nucleus accumbens places this receptor system in an area associated with the dopamine family of receptors and the dopamine transporter (DAT). As the dopaminergic system plays a direct and primary role in the reinforcing effects of drugs of abuse such as the psychostimulants cocaine and methamphetamine, significant interest in elucidating a role for mGluR5 in addiction has been ignited. Indeed, experiments with mutant mGluR5 knockout (KO) mice show similar baseline locomotion as compared to wildtype littermates but do not demonstrate dose-dependent locomotor stimulation induced by cocaine. Further, the mGluR5 KO mice do not self-administer cocaine at any dose tested.¹⁴ Likewise, the mGluR5 antagonist 1 dose-dependently decreases cocaine self-administration, but has no effect on food intake, under identical schedules of reinforcement.¹⁴ These studies have been followed with numerous reports investigating the effects of 1 on cocaine self-administration and brain reward function (e.g., refs ^15–18) and support further investigation into novel mGluR5 antagonists as molecular tools and as potential therapeutic agents for drug addiction.

Acetylenic analogues of the prototypic drug 1 have been largely mined, and the triple bond has proved to be a very important structural motif for high affinity mGluR5 antagonists.¹⁰ In fact, most published mGluR5 structure–activity relationship studies have used this motif as the basis to their drug design and have primarily investigated structural modifications to the appended aryl rings on either side of the alkyne linker.^19–23 Nevertheless, we and others,^24–28 in an attempt to design nonalkynyl mGluR5 antagonists, have discovered several moderately active diarylamides (e.g., 3 and 4 in Figure 1), supporting our hypothesis that the acetylenic linker could be replaced. To further explore SAR and potentially improve binding affinities in the amide series, we attempted to identify critical functional groups for high affinity mGluR5 antagonists in the alkyne series and then incorporate these into the amides. Specifically, a series of alkyne derivatives based on 1, and the thiazole analogue 2-methyl-4-(pyridin-3-ylethynyl)thiazole (MTEP, 2, Figure 1) were first prepared to establish optimal pharmacophoric elements on the “b”-ring. On the basis of this series of alkynes (Table 1), optimal “b”-side aryl ring substitutions were incorporated into amides and SAR was compared.

Figure 1.

mGluR5 antagonist templates.

Table 1.

In Vitro Data for Alkynyl mGluR5 Antagonists

compd	temp	R1	R2	R3	X1	X2	cLogPg	mGluR5 binding (Ki, nM)a	mGluR function IC50 (nM) (Ca+2flux)
1							3.8	13 ± 1b	3.54 ± 1.39
2							2.1	NT	13.6 ± 2.09
7d	A	Ph	H	H			5.5	10.76 ± 2.8b	3.05 ± 0.32
10	A	3-pyr	H	F			4.2	2.7 ± 0.7b	4.83 ± 0.60
11e	A	CN	H	F			3.2	0.9 ± 0.2b	0.813 ± 0.11
16d	B	H			N	C	4.2	5.49 ± 1.43b	1.21 ± 0.15
17d	B	H			N	N	2.8	5.65 ± 1.47b	5.24 ± 1.00
20	B	F			C	N	3.0	11.4 ± 3b	3.43 ± 0.51
9f	C		H				3.2	1.3 ± 0.09c	0.415 ± 0.10
21	C		Ph				5.1	4.0 ± 0.6c	3.08 ± 0.61
22	C		3′FPh				5.3	17.0 ± 2.2c	6.10 ± 1.95
23	C		4′FPh				5.3	3.0 ± 0.5c	7.19 ± 1.53

^aData provided by NIMH-PDSP.

^bCloned (ref ²¹).

^cRat brain (http://pdsp.med.unc.edu).

^dCompd. previously reported in ref 29.

^eCompd previously reported in ref 33.

^fCompd previously reported in ref 19.

^gDetermined using Sybyl 7.2.3, Tripos Inc.

Chemistry

Synthesis of the alkyne series of compounds followed the strategy outlined in Scheme 1. Compounds 7, 9, and 11 in this series were reported during the course of our investigation, and these are cited in Table 1. Synthons 2-methyl-4-((trimethylsilanyl)ethynyl)thiazole (5a) or 2-methyl-6-((trimethylsilanyl)ethynyl)pyridine (5b) were prepared according to literature procedures.^19,29 The phenols were protected as the methoxymethoxy (OMOM) ethers by treating with chlorom-ethylmethyl ether. Thus, compound 5a or 5b was coupled with 6(a, b, or c) or 12(a, b, or c) under Sonagashira reaction conditions to give the coupling products 7–9, or 13–15 in moderate to good yield. The aryl halides 8 and 15 were then converted to 10, 11, 16, and 17 through Suzuki coupling reactions or by palladium-catalyzed coupling with Zn(CN)₂. An alternative method to prepare compounds 10 and 11 was to replace the Br (R₁) of 1,3-dibromo-5-fluorobenzene (6b) with the pyridinyl or cyano group via Suzuki coupling or by Pdcatalyzed cyanation, respectively, followed by the Sonagashira reaction. However, there were side reactions that typically resulted when 6b was treated with either 3-pyridinylboronic acid or Zn(CN)₂ in the presence of palladium catalyst, especially for the latter reaction, wherein 1,3-dicyano-5-fluorobenzene was always the major product, with only very small quantities of the desired 3-bromo-5-fluorobenzonitrile obtained.

Scheme 1.

Synthetic Strategy toward Thiazole and Pyridyl Alkynesa

^a Reagents and conditions: (a) Pd(PPh₃)₄, CuI, TBAF, Et₃N; (b) arylboronic acid or (3-pyridin-yl)boronic acid, Na₂CO₃ or KF, DME/H₂O; (c) Zn(CN)₂, Pd(PPh₃)₄, DMF, 80 °C; (d) aq HCl (6 N), MeOH, 50 °C; (e) (CF₃SO₂)₂O, pyridine, CH₂Cl₂.

Compounds 13 and 14 were converted to their corresponding triflates 18 and 19 by deprotection of MOMO to the free hydroxyl group under acidic conditions, followed by treatment with trifluoromethanesulfonic anhydride. Noticeably, the deprotection was problematic when R₁) CN, resulting in hydrolysis under these strong acidic conditions. As a result, the yield of the triflate 19 was low (0–45%), with various amounts of byproduct amide or acid, which were insoluble in both aqueous and organic layers. It was observed that partial hydrolysis of the triflates 18 and 19 to the phenol occurred, in the Suzuki coupling reaction, when strong base such as Na₂CO₃ was employed, resulting in low yields. This problem could be circumvented by using mild base such as KF.

On the basis of previous SAR studies,²⁴ and concerns regarding in vivo toxicities with the thiazole “a” ring (ref ³⁰ and personal communications with Drs. S. Barak Caine and Roger Spealman), we elected to retain only the 2-methyl-6- pyridyl “a” ring in the amide series. Thus the initial 2-methyl-6-pyridyl amide analogues were prepared according to Schemes 2 and 3 starting with 2-methyl-6-aminopyridine (24) using standard amidation methods to give compounds 25–29, 32–36, 42, and 43. Addition of aryl substitution to the “b” ring was achieved using Suzuki coupling reactions with a variety of arylboronic acids and the Br-substituted amides (26, 35, 42, and 43) to give 30, 31, 37–41, and 44–47, respectively.

Scheme 2.

Synthetic Strategy toward Substituted-Phenylamidesa

^a Reagents and conditions: (a) ArCOOH, CDI, pyridine; (b) ArCOCl, pyridine/TEA, dichloromethane, rt, 1–2 h, 50–75%; (c) ArB(OH)₂ or 3-pyridyl boronic acid, Pd(PPh₃)₄, 2 M aq Na₂CO₃, toluene, 110 °C or DME/H₂O (3:1), 80 °C, overnight, 75–80%.

Scheme 3.

Synthetic Strategy toward Substituted-Pyridylamidesa

^a Reagents and conditions: (a) ArCOCl, TEA, dichloromethane, rt, 1–2 h, 85–100%; (b) ArB(OH)₂, Pd(OAc)₂, 2′-dicyclohexylphosphino-2,6- dimethoxy biphenyl, K₃PO₄, toluene/EtOH, 50% or 3-pyridyl boronic acid, Pd(PPlh₃)₄, 2 M aq Na₂CO₃, toluene, 110 °C or DME/H₂O (3:1), 80 °C, overnight, 75–80%.

As the 3-CN substitution on the “b”-ring in the alkyne series was determined to be important for potent activity at mGluR5, a series of similarly substituted amides were designed. To prepare the 3-CN-substituted amide analogues 55–59, a different synthetic strategy was taken starting with 3-bromo-4-hydroxy benzoic acid (48) as depicted in Scheme 4. Benzylic protection of both the carboxylic acid and the phenol was achieved using benzyl bromide to give 49. Displacement of the 3-Br with CN using Zn(CN)₂ and Pd(PPh₃)₄ gave excellent yields of intermediate 50. We could take advantage of selective deprotection of the carboxylic acid under basic conditions to give the benzyl protected phenolic carboxylate 51, which was readily converted to the amide 52 after making the acid chloride of 51 in SOCl₂ and then reacting with 24. Catalytic hydro- genolysis of the benzyl protected phenol was achieved with cyclohexene and 10% Pd/C to give 53. The triflate 54, prepared by treating the phenol with trifluoromethanesulfonic anhydride, was then reacted with arylboronic acids, under Suzuki cross- coupling reaction conditions, to give the 4-aryl substituted amides 55–59. Typically, all final products were purified by flash column chromatography, analytically characterized as the free base, and then converted to the HBr or HCl salts for biological testing, unless otherwise described in the Experi- mental Methods.

Scheme 4.

Synthesis of Substituted 3-Cyano-phenyl Amidesa

^a Reagents and conditions: (a) Benzyl bromide, anhydrous K₂CO₃, acetone, reflux, overnight, 99%. (b) Zn(CN)₂, Pd(PPh₃)₄, 80 °C overnight, DMF, 97%. (c) 4 N NaOH, MeOH, rt, 2 h, 92%. (d) (i) SOCl₂, dichloromethane, cat. DMF, reflux, 1 h; (ii) 2-amino-6-methyl pyridine, 24, TEA, dichloromethane, rt,1 h, 83%. (e) 10% Pd/C, cyclohexene, ethanol, reflux, 1 h, 87%. (f) trifluoromethanesulfonic anhydride, pyridine, dichloromethane, rt, overnight, 96%. (g) ArB(OH)₂, Pd(PPh₃)₄, 2 M aq Na₂CO₃, toluene, reflux overnight, 40–50%.

Structure–Activity Relationships

Radioligand binding assays for mGluR5 were performed using [³H]1 as the radioligand in either membranes from HEK 293-T cells transfected with cloned mGluR5 cDNA or rat brain membranes, as depicted in Table 1 and as previously described.²¹ A functional assay using calcium fluorescence was utilized to test compound activity by measuring receptor-induced intracellular release of calcium with a kinetic imaging plate reader that makes simultaneous measurements of calcium levels in each well of a 384-well plate. Cells expressing rat mGluR5 were preincubated with the test compounds for 140 s and then stimulated for 60 s with an EC₈₀ concentration of glutamate. In general, mGluR5 binding affinity (K_i) values were comparable to functional potency (IC₅₀) values in the calcium fluorescence assay. The results of these in vitro tests for the alkynes are in Table 1 and for the amides in Table 2, listed according to structural template.

Table 2.

In Vitro Data for Amide-Linked mGluR5 Antagonists

compd	temp	R1	R2/Ar	R3	cLogPe	mGluR5 binding (Ki, nM)a	mGluR5 function IC50(nM) (Ca+2flux)
3a	A	Cl	H	H	3.3	1730 ± 100c,d	1870 ± 320
3b	A	CN	H	H	2.1	330 ± 20c,d	490 ± 94
3c	A	Ph	H	H	4.4	7300 ± 390c,d	NT
4					3.8	43 ± 1.0f	98.1 ± 18.9
25	A	H	CN	H	2.1	>10000b	NT
26	A	H	Br	H	3.4	>10000b	NT
27	A	H	OPh	H	4.6	7290 ± 2190b	NT
28	A	H	OBz	H	4.4	>10000b	NT
29	A	H	Ph	H	4.4	7000 ± 2120b	NT
30	A	H	3′CNPh	H	3.8	>10000b	NT
31	A	H	3′ClPh	H	5.1	3330 ± 850b	NT
32	A	F	H	F	2.9	7580 ± 2280b	NT
33	A	F	F	H	2.3	>10000b	NT
34	A	CN	H	F	2.2	65.5 ± 20c	21.7 ± 5.41
37	A	3-Pyr	H	F	3.1	702 ± 76c	NT
38	A	2′OCH3Ph	H	F	4.0	>10000c	NT
39	A	2′CH3Ph	H	F	4.8	610 ± 8.0c	NT
40	A	F	3-Pyr	H	3.1	7720 ± 1000c	>3000
41	A	Cl	3-Pyr	H	3.4	2780 ± 750c	>1000
55	A	CN	Ph	H	40	9.8 ± 2.1b	13.7 ± 2.54
56	A	CN	3′FPh	H	4.1	22 ± 5.3b	25.3 ± 1.90
57	A	CN	4′FPh	H	4.1	134 ± 31c	4.57 ± 0.38
58	A	CN	1-naphth	H	5.1	72 ± 12c	640 ± 32.2
59	A	CN	3-Pyr	H	2.5	2040 ± 340c	>1000
44	B	3-Pyr	H		1.8	>10000c	NT
45	B	2′OCH3Ph	H		2.7	4920 ± 134c	NT
46	B	H	Ph		3.4	356 ± 41c	>3000
47	B	H	3-Pyr		2.0	2480 ± 520c	>3000

^aData provided by NIMH-PDSP.

^bCloned (ref ²¹).

^cRat brain (http://pdsp.med.unc.edu).

^dCompd previously reported in ref 24.

^eDetermined using Sybyl 7.2.3, Tripos Inc.

^fCompd and data reported in ref 25; NT = not tested.

As expected, both thiazole and pyridyl alkynes showed high affinity binding to mGluR5 and were potent antagonists in the functional assay. The compounds based on template A, as compared to their parent compound, 2, demonstrated tolerability at the 3-position (R₁) in the “b” ring, with the CN analogue, compound 11, showing subnanomolar affinity and functional potency across assays. Unfortunately, preliminary in vivo testing of this analogue in rats showed toxicity, which precluded further evaluation (personal communication with Dr. S. Barak Caine). However, replacing the thiazole ring with the 2-methylpyridyl pharmacophore of the parent compound 1 essentially retained the same pharmacological profile and thus this compound or analogues thereof may prove to be better in vivo tools as the 2-methylpyridines, as exemplified by 1 itself, have proven to be well tolerated in laboratory animals. The thiazole analogues with structural template B showed that R₁ need not be CN to have high affinity and functional potency at mGluR5, and further that a pyridyl “b” ring is well tolerated as is aryl substitution in the 62-position. By allowing the substitution of a bipyridyl system, the lipophilicity of the resulting compound 17 is reduced to give a cLogP) 2.8, which may prove desirable for both water-solubility and blood-brain barrier penetration.

Retaining the 3-CN substituted “b” ring of the compounds with template A and exploring the 4-position (R₂) of the 2-methylpyridyl analogues (template C) also demonstrated potent functional antagonism at mGluR5 and a tolerance for aryl ring substitution in the “b” ring. Although, predictably, this substitution serves to increase lipophilicity with cLogP values >5 for compounds 22 and 23, which may not be compatible with a “drug-like” profile.^31,32

On the basis of these data, we expanded our investigation of amide-linked mGluR5 analogues^24,25 to incorporate pharmacophores from the alkyne series that were deemed important for high affinity binding and functional potency at mGluR5, namely (1) exploring substitutions on the “b” ring that either include the 3-CN group (R₁) and investigate tolerability of substitution at the 4 and 5-positions of the “b” ring or (2) replace the 3-CN group with other substituents, and further (3) replace the aryl “b” ring of the parent compound 1 with the 3-pyridyl “b” ring (template B) to both explore tolerability and attempt to reduce lipophilicity of the analogues by incorporating this heterocycle. Previously we discovered that, in general, the amide linker replacement of the alkyne was poorly tolerated with only a few examples of moderately active mGluR5 antagonists (e.g., 3a–3c and 4^24,25). Compounds 25–33 were being prepared during the course of those earlier studies, and it was discovered that 4-substitution in the “b” ring was also poorly tolerated despite this substitution being well tolerated in the alkyne series (e.g., 16, 17, 20–23). Activity at mGluR5 was gained when the 3-CN and 5-F groups were incorporated into the amides (e.g. 11 vs 34; Figure 2). However, if the 3-CN group was replaced (37–39), activity was lost. Indeed, all other substitutions that were attempted proved fatal to mGluR5 activity if the 3-CN group was removed. For example, addition of pyridyl rings on the “b“-side of the alkynes was tolerated at mGluR5 (e.g. 16, 17, 20), whereas this substitution (template B) typically decreased affinity and antagonist potency in the amides (e.g., 59).

Figure 2.

3D-superimposition of11 (alkyne) and 34 (amide).

Hence, the final set of compounds all had a 3-CN-substitution in the “b” ring, and these were among the best mGluR5 antagonists in the amide series (55–58) with the exception of compound 59. Interestingly, the similarly substituted alkyne 11 and amide 34 align quite well (Figure 2), and yet the alkyne binds with >70-fold higher affinity at mGluR5 than the amide. However, addition of an aryl substitution at R₂ in the amides improved mGluR5 affinity and antagonist potency to give similar potency to the alkyne analogue (e.g., 22 (K_i) 17 nM) vs 56 (K_i) 22 nM); Figure 3).

Figure 3.

3D-superimposition of22 (alkyne) and 56 (amide).

These compounds are highly specific to mGluR5 as none of the compounds in Table 1 or 2 showed >70% inhibition of PI hydrolysis at a single concentration of 10 µM (data from NIMHPDSP) in any of the other mGluR subtype functional assays and further evaluation was abandoned. In addition, compounds 10, 20, 55, and 56 were evaluated for hDAT, hSERT, and hNET binding, using [¹²⁵I]RTI 55 as the radioligand, as well as inhibition of DA, 5-HT, and NE uptake, respectively, in HEK cells. None of the compounds showed significant activity in any of these assays, with K_i values in the 6 to >10 µM range (data from NIDA-ATDP).

X-ray Crystallographic Results and Discussion

Data presented in Tables 1 and 2 revealed that structural differences between the alkynyl- and amido-linked mGluR5 antagonists, although seemingly minor, can have significant impact on binding affinities and functional potencies at mGluR5. The divergence in SAR between the amides and alkynes suggests that the coplanarity of the alkynes is more favored, as described previously.^24–26 Thus, in an effort to collect additional conformational information, representative compounds 4, 10, and 57 were analyzed using single-crystal X-ray diffraction and compared (Figure 4A–C). All the bond lengths and angles were within the expected range of values, and as also seen in the energy-minimized molecular models of compound 11 compared to 34 and 22 compared to 56, respectively, in Figures 2 and 3, the X-ray data confirm similarities in the conformations of the alkynes and amides. However there are subtle differences in the orientation of the aromatic “b” rings, which may access different but overlapping binding sites, potentially affecting potency at mGluR5 that generally favors the alkynes.

Figure 4.

X-ray crystal structures of compounds 4 (A), 10 (B), and 57 (C). For all images, displacement ellipsoids are at the 50% level; for compound 4, solvent and the counterion are omitted for clarity; for compound 10, only one of the two molecules in the asymmetric unit is shown and solvent and counterions are omitted for clarity; for compound 57, the counterion is omitted for clarity.

Summary

A series of alkynyl analogues of the parent compounds 1 and 2 were synthesized in order to identify optimal substitution on the “b” ring of the molecule for mGluR5 activity. Several very high affinity (K_i) 0.9–11 nM) mGluR5 antagonists were identified in this series. A second series of amide analogues, based on previously described SAR^24,25 and templates 3b and 4, were synthesized and optimal “b” ring substitution, as identified in the alkynyl series, was incorporated. We discovered that only the 3-CN substituent in the “b” ring of both alkynes and amides served to consistently improve mGluR5 binding affinity and functional potency. Other substitutions that also gave high affinity ligands in the alkyne series were not tolerated in the amide series. Likewise, aryl substitutions in the 4-position of the “b” ring (R₂) served to either retain or improve mGluR5 affinity in the amide series, whereas this substitution had no additional positive effect on mGluR5 affinity for the alkynes. This SAR comparison, along with X-ray crystal data, demonstrate that the planar alkyne template is optimal for mGluR5 binding affinity and functional potency but that certain “b” ring substitutions (e.g., 3-CN) can also result in high affinity amide analogues. Likely due to the conformational differences between the alkynes and the amides, other substitutions on the “b” ring of the alkynes did not uniformly improve mGluR5 activity in the amides, and in fact, amides without the 3-CN substitution, in this series, were typically inactive. Nonetheless, cLogP values for most of the novel analogues reported herein are within the 2–5 range, suggesting that the more potent mGluR5 antagonists will be viable in vivo tools. And as several alkynes and amide analogues were identified to be both high affinity and selective mGluR5 antagonists, in vivo evaluation of selected compounds in models of anxiety and drug abuse will continue to provide needed information regarding the potential development of this class of agents as medications for the treatment of addiction and other neuropsychiatric disorders.

Experimental Methods

Reaction conditions and yields were not optimized, and spectroscopic data refer to the free base. Thin-layer chromatography was performed using analytical TLC plates (Analtech Uniplate, 250 µm), and flash chromatography was performed using silica gel (EMD Chemicals, Inc.; 230–400 mesh, 60 Å). ¹H and ¹³C NMR spectra were acquired using a Varian Mercury Plus 400 spectrom- eter. Chemical shifts are reported in parts-per-million (ppm) and referenced according to deuterated solvent for ¹H spectra (CDCl_3, 7.26) and ¹³C spectra (CDCl₃, 77.23). Infrared spectra were recorded as a neat film on NaCl plates using a Perkin-Elmer Spectrum RX I FT-IR spectrometer. Gas chromatography–mass spectrometry (GC/MS) data were acquired using an Agilent Technologies (Santa Clara, CA) 6890 GC equipped with an HP-5 MS column (cross- linked 5% PH ME siloxane, 30 m × 0.25 mm i.d. × 0.25 µm film thickness) and a 5973 mass-selective ion detector in electron-impact mode. Ultrapure grade helium was used as the carrier gas at a flow rate of 1.2 mL/min. The injection port and transfer line temperatures were 250 and 280 °C, respectively, and the oven temperature gradient used was as follows: the initial temperature (100 °C) was held for 3 min (0:00–3:00 min) and then increased to 295 at 15.0 °C/min over 13 min (3:00–16:00 min), and finally maintained at 295 °C for 10 min (16:00–26:00 min). Combustion analysis was performed by Atlantic Microlab, Inc. (Norcross, GA) and agrees within 0.4% of calculated values. Melting point determination was conducted using a Thomas–Hoover melting point apparatus and are uncorrected. Anhydrous solvents were purchased from Aldrich (pyridine, acetonitrile, dichloromethane, chloroform (CHCl₃), hydrazine) or J. T. Baker (diethyl ether) and were used without further purification, except for tetrahydrofuran, which was freshly distilled from sodium-benzophenone ketyl. All other chemicals and reagents were purchased from Aldrich Chemical Co., Lancaster Synthesis, Inc. (Alfa Aesar), and Combi-Blocks, and used without further purification. The final products were converted into the HBr salts, typically by treating the free base with methanolic HBr followed by precipitation with diethyl ether. The HCl salts were generally made by dissolving the free base in HCl ether solution and then recrystallizing from hot MeOH. On the basis of NMR, GC-MS (where obtainable), and combustion analysis data, all final com- pounds are >95% pure.

General Procedure A: Protection of Hydroxyl Group as Methoxymethoxy Ether (OMOM)

Chloromethyl methyl ether (6 mmol) in dry dichloromethane (2 mL) was added dropwise to an ice-cooled solution of phenol (5 mmol) and N-ethyldiisopropylamine (10 mmol) in dichloromethane (10 mL). The mixture was stirred at room temperature for 2 h after the addition. Water (20 mL) was then added, and the two phases were separated. The aqueous layer was extracted with dichloromethane (3 × 20 mL). The combined organic layer was dried (Na₂SO₄) and concentrated. The residue was purified by flash column chromatography.

General Procedure B: Sonagashira Reaction

To the mixture of 2-methyl-4-((trimethylsilanyl)ethynyl)thiazole (5a) or 2-methyl-6-((trimethylsilanyl)ethynyl)pyridine (5b) (1 equiv), aryl bromide 6a–c (1.2 equiv), CuI (20 mol %),and Pd(PPh₃)₄ (5 mol %) in DMF (5 mL DMF/1 mmol scale reaction) was added Bu₄NF (1 eq, 1.0 M solution in THF) dropwise at 80 °C under argon. After the addition, the mixture was stirred at this temperature overnight. The mixture was then filtered through celite, DMF was removed under reduced pressure, and the residue was diluted with H₂O and extracted with EtOAc. The combined organic layer was dried (MgSO₄) and concentrated. The residue was purified by flash column chromatography to afford the pure product.

General Procedure C: Suzuki Coupling Reaction for Alkynes

To the solution of aryl boronic acid (1.2 equiv), aryl halide, or triflate (1 equiv) and Na₂CO₃ (2 equiv, for halide) or KF · 2H₂O (2 equiv for triflate) in the mixture of solvents DME/H₂O (3/1, 4 mL solvents for 1 mmol scale reaction) was added Pd(PPh₃)₄ (5 mol%) under argon. The mixture was warmed to 80 °C for 3–5 h. The solvents were then removed under reduced pressure, and the residue was diluted with water and extracted with EtOAc. The organic layer was dried (MgSO₄) and concentrated. The residue was purified by flash column chromatography to afford the pure product.

3-Bromobiphenyl (6a)

Prepared from 1,3-dibromobenzene and phenylboronic acid according to general procedure C in 58% yield; purified by column chromatography eluting with hexane/EtOAc (3: 1) (white solid). ¹H NMR (CDCl₃) δ 7.2–7.8 (m, 9H), ppm; GC-MS (EI) m/z 232(M⁺), 234(M⁺).

4-Bromo-2-fluoro-1-(methoxymethoxy)benzene (12a)

Prepared from 4-bromo-2-fluorophenol according to the general procedure A in 97% yield; purified by column chromatography eluting with hexane/EtOAc (10:1)(clear oil). ¹H NMR (CDCl₃) δ 3.51 (s, 3H), 5.19 (s, 2H), 7.08 (dd, J = 8.8, 10 Hz, 1H), 7.16–7.20 (m, 1H), 7.25 (dd, J = 2.4, 10 Hz, 1H) ppm; GC-MS (EI) m/z 234 (M⁺), 236 (M⁺).

5-Bromo-2-(methoxymethoxy)benzonitrile (12b)

Prepared from 4-bromo-2-cyanophenol according to the general procedure A in 91% yield; purified by column chromatography using hexane/EtOAc (10:1) (light-brown solid); mp 67–69 °C. IR (KBr) 2223, 1583. 1256, cm⁻¹. ¹H NMR (CDCl₃) δ 3.52 (s, 3H), 5.28 (s, 2H), 7.14 (d, J = 9.2 Hz, 1H), 7.61 (dd, J = 2.4, 9.2 Hz, 1H), 7.68 (d, J = 2.4 Hz, 1H) ppm. GC-MS (EI) m/z 241 (M⁺), 243 (M⁺).

3-Phenyl-1-(2-methylthiazol-4-ylethynyl)benzene (7)

Prepared from 5a and 6a according to the general procedure B in 38% yield; purified by column chromatography eluting with hexane/EtOAc (2: 1) (brown solid); mp 84–86 °C. IR (KBr, cm⁻¹) 1598 cm⁻¹. ¹H NMR (CDCl₃) δ 2.76 (s, 3H), 7.40–7.54 (m, 4H), 7.45 (s, 1H), 7.58 (m, 1H), 7.60 (d, J = 1.6 Hz, 1H), 8.04 (t, J = 2.0 Hz, 1H), 8.76 (d, J = 2.0 Hz, 1H), 8.79 (d, J = 2.0 Hz, 1H) ppm. GC-MS (EI) m/z 275 (M⁺). Anal. (C₁₈H₁₃NS) for C, H, N.

1-Bromo-3-fluoro-5-(2-methylthiazol-4-ylethynyl)benzene(8)

Prepared from 5a and 6b according to general procedure B in 57% yield; purified by column chromatography eluting with hexane/EtOAc (2:1) (light-brown solid). ¹H NMR (CDCl₃) δ 2.74 (s, 3H), 7.19 (ddd, J) 1.6, 2.4, 8.8 Hz, 1H), 7.24 (ddd, J) 1.6, 2.4, 8.8 Hz, 1H), 7.42 (s, 1H), 7.49 (m, 1H) ppm. GC-MS (EI) m/z 295 (M⁺), 297 (M⁺).

3-[(6-Methylpyridin-2-yl)ethynyl]benzonitrile (9)

Prepared from 5b and 6c according to the general procedure B in 48% yield; purified by column chromatography eluting with hexane/EtOAc (2: 1). The free base was converted to its HBr salt and recrystallized from EtOH as a brown solid; mp 189–192 °C (dec). IR (KBr) 2223, 1604 cm⁻¹. ¹H NMR (CDCl₃) δ 2.60 (s, 3H), 7.17 (d, J = 8.0 Hz, 1H), 7.37 (d, J = 8.0 Hz), 7.49 (t, J) 8.0 Hz, 1H), 7.60 (d, J) 8.0 Hz, 1H), 7.64 (dt, J) 1.6, 8.0 Hz, 1H), 7.81 (dt, J) 1.6, 8.0 Hz, 1H), 7.87 (s, 1H) ppm. GC-MS (EI) m/z 218 (M⁺). Anal. (C₁₅H₁₀N₂ · HBr) for C, H, N.

1-Fluoro-3-(pyridin-3-yl)-5-(2-methylthiazol-4ylethynyl)benzene (10)

Prepared from 8 and 3-pyridineboronic acid according to the general procedure C in 95% yield; purified by column chromatography eluting with hexane/EtOAc (1:1). The free base was converted to the HBr salt and recrystallized from MeOH as a brown solid; mp 175–177 °C (dec). IR (KBr) 1604, 1592 cm⁻¹. ¹H NMR (CDCl₃) δ 2.76 (s, 3H), 7.29 (d, J) 1.2 Hz, 1H), 7.27 (s, 1H), 7.40 (m, 1H), 7.44 (s, 1H), 7.58 (t, J) 1.2 Hz, 1H), 7.86 (m, 1H), 8.64 (dd, J) 1.2, 5.0 Hz, 1H), 8.84 (d, J) 2.0 Hz, 1H) ppm. GC-MS (EI) m/z 294 (M⁺). Anal. (C₁₇H₁₁N₂FS · 2HBr) for C, H, N.

3-Fluoro-5-cyano-1-(2-methylthiazol-4-ylethynyl)benzene (11)

To a mixture of 8 (200 mg, 0.67 mmol) and Zn(CN)₂ (47.6 mg, 0.40 mmol) in DMF (5 mL) was added Pd(PPh₃)₄ (39 mg, 5 mol%), and the mixture was heated to 80 °C overnight. DMF was then removed under reduced pressure. The residue was then diluted with H₂O and extracted with EtOAc (3 × 10 mL). The combined organic layer was dried (MgSO₄) and concentrated. The residue was purified by column chromatography eluting with hexane/EtOAc (3:1) to give 11 (110 mg, 68%). The free base was converted to its HBr salt and recrystallized from EtOH as a dark-brown solid; mp 186–189°C (dec). IR (KBr): 2221, 1605, 1585 cm⁻¹. ¹H NMR (CDCl₃) δ 2.76 (s, 3H), 7.32–7.36 (m, 1H), 7.45–7.49 (m, 1H). 7.47 (s, 1H),7.62 (s, 1H) ppm. GC-MS (EI) m/z 242 (M⁺). Anal. (C₁₃H₇N₂FS ·HBr) for C, H, N.

2-Fluoro-1-(methoxymethoxy)-4-(2-methylthiazol-4-ylethynyl)-benzene (13)

Prepared from 5a and 12a according to the general procedure B in 47% yield; purified by column chromatography eluting with hexane/EtOAc (5:1) (clear oil). IR (neat): 1598, 1272, 1155 cm⁻¹. ¹H NMR (CDCl₃) δ 2.74 (s, 3H), 3.52 (s, 3H), 5.24 (s, 2H), 7.17 (m, 1H), 7.26–7.30 (m, 2H), 7.36 (s, 1H) ppm. GC-MS (EI) m/z 277 (M⁺).

2-(Methoxymethoxy)-5-(6-methylpyridin-2-ylethynyl)benzonitrile (14)

Prepared from 5a and 12b according to the general procedure B in 53% yield; purified by column chromatography eluting with hexane/EtOAc (3:1) (brown solid); mp 112–114 °C. IR (KBr): 2227, 1583, 1602 (w), 1255 cm⁻¹. ¹H NMR (CDCl₃) δ 2.59 (s, 3H), 3.54 (S, 3H), 5.32 (S, 2H), 7.14 (d, J = 8.0 Hz, 1H), 7.23 (d, J = 8.8 Hz,1H), 7.34 (d, J = 8.0 Hz, 1H), 7.59 (dd, J) 8.0, 8.0 Hz, 1H), 7.72 (dd, J = 1.6, 8.8 Hz, 1H), 7.78 (d, J = 1.6 Hz, 1H) ppm. GC-MS (EI) m/z 278 (M+).

2-Chloro-5-(2-methylthiazol-4-ylethynyl)pyridine (15)

Prepared from 5a and 12c according to the general procedure B in 73%; purified by column chromatography eluting with hexane/ EtOAc (3:1) (light-yellow solid); mp 139.5–141 °C. ¹H NMR (CDCl₃) δ 2.75 (s, 3H), 7.33 (dd, J = 0.8, 8 Hz, 1H), 7.44 (s, 1H),7.78 (dd, J = 2.4, 8 Hz, 1H), 8.56 (dd, J = 0.8, 2.4 Hz, 1H) ppm. GC-MS (EI) m/z 234 (M⁺).

2-Phenyl-5-(2-methylthiazol-4ylethynyl)pyridine (16)

Prepared from 15 and phenylboronic acid according to the general procedure C in 80% yield; purified by column chromatography eluting with hexane/EtOAc (7:3); mp 99.5–100.5 °C. The free base was converted to its HBr salt and recrystallized from EtOH as a light-brown solid; mp 188–191 °C (dec). IR (KBr) 1608, 1601 cm⁻¹. ¹H NMR (CDCl₃) δ 2.76 (s, 3H), 7.40–7.52 (m, 4H), 7.58 (m, 2H), 8.04 (m, 1H), 8.77 (dd, J) 2.2,14.4 Hz, 2H) ppm. ¹³C NMR (CDCl₃) δ 19.5, 85.7, 87.0, 119.9, 123.4, 127.4, 128.7, 129.4, 136.4, 137.1, 147.7, 151.0, 166.2 ppm. GC-MS (EI) m/z 276 (M⁺). Anal. (C₁₆H₁₂N₂S · HBr · H₂O) for C, H, N.

2-(Pyridin-3-yl)-5-(2-methylthiazol-4ylethynyl)pyridine (17)

Prepared from 15 and 3-pyridylboronic acid according to the general procedure C in 73% yield; purified by column chromatography eluting with hexane/EtOAc/Et₃N (2.5:7.5:0.1). The free base was converted to its HBr salt and recrystallized from EtOH as a light-brown solid; mp 224–226 °C (dec). IR (KBr) 1602, 1588 cm⁻¹. ¹H NMR (CDCl₃) δ 2.76 (s, 3H), 7.42 (m, 1H), 7.47 (s, 1H), 7.77 (d, J) 8.4 Hz, 1H), 7.94 (dd, J) 2.0, 8.4 Hz, 1H), 8.35(d, J) 8.0 Hz, 1H), 8.67 (d, J) 2.0 Hz, 1H), 8.88 (s, 1H), 9.22 (d, J) 1.6 Hz, 1H) ppm. GC-MS (EI) m/z 277 (M⁺). Anal. (C₁₆H₁₁N₃S · HBr · 0.5H₂O) for C, H, N.

2-Fluoro-4-((2-methylthiazol-4-yl)ethynyl)phenyl Trifluoromethanesulfonate (18)

To a solution of compound 13 (486 mg, 1.75 mmol) in MeOH (2 mL) was added aq HCl (6 N, 0.6 mL, 3.6 mmol), and the mixture was heated to 50 °C for 3 h. MeOH and H₂O were removed under reduced pressure, and the residue was further dried on high vacuum to afford 2-fluoro-4-[(2-methylthiazol-4-yl)ethynyl]phenol. This crude product was used in the next step without further purification. To a mixture of 2-fluoro-4-((2- methylthiazol-4-yl)ethynyl)phenol and pyridine (0.57 mL, 7.0 mmol) in dry CH₂Cl₂ (5 mL) was added trfluoromethanesulfonic anhydride (0.44 mL, 2.6 mmol) dropwise at 0 °C under argon, and the solution was further stirred at room temperature for 2 h after the addition. The mixture was then diluted with H₂O (10 mL). The two layers were separated, and the aqueous layer was extracted with EtOAc (3 × 10 mL). The combined organic layer was dried (MgSO₄) and concentrated. The residue was purified by column chromatography eluting with hexane/EtOAc (5:1) to provide the triflate 18 (607 mg) in 95% yield, as a light-yellow solid. ¹H NMR (CDCl₃) δ2.75 (s, 3H), 7.29–7.45 (m, 3H), 7.44 (s, 1H) ppm. ¹³C NMR (CDCl₃) δ 19.5, 86.0, 86.4, 117.3, 120.7, 120.9, 123.9, 124.8, 128.7, 136.1, 137.0, 152.2, 154.7, 166.4 ppm. GC-MS (EI) m/z 365 (M+).

2-Cyano-4-((6-methylpyridin-2-yl)ethynyl)phenyl Trifluo-romethanesulfonate (19)

Compound 14 (2.34 g, 8.41 mmol) was dissolved in MeOH (10 mL), and aq HCl (6 N) solution (3 mL, 18 mmol) was added. The mixture was heated to 50 °C. Heating was discontinued after TLC showed most of the starting material disappeared (less than 1 h). MeOH and H₂O were then removed under reduced pressure, and the residue was further dried on high vacuum to afford 2-cyano-4-((6-methylpyridin-2-yl)ethynyl)phenol, which was used in the next step without further purification. To a mixture of 2-cyano-4-((6-methylpyridin-2-yl)ethynyl)phenol and pyridine (2.67 mL, 33 mmol) in dry CH₂Cl₂ (20 mL) was added trfluoromethanesulfonic anhydride (2.12 mL, 12.61 mmol) drop-wise, at 0 °C under argon, and the solution was further stirred at room temperature for 2 h after the addition. The mixture was then diluted with H₂O (20 mL). The two layers were separated, and the aqueous layer was extracted with EtOAc (3 × 20 mL). The combined organic layer was dried (MgSO₄) and concentrated. The residue was purified by column chromatography eluting with hexane/EtOAc (5:1) to provide product 19 (1.31 g) in 43% yield, as a light-yellow solid. ¹H NMR (CDCl₃) δ 2.61 (s, 3H), 7.20 (d, J) 7.6 Hz, 1H), 7.38 (d, J) 7.6 Hz, 1H), 7.50 (d, J) 8.8 Hz, 1H), 7.62 (dd, J) 8.0, 8.0 Hz, 1H), 7.88 (dd, J) 2.4, 8.8 Hz, 1H), 7.95 (d, J) 2.4 Hz, 1H) ppm. GC-MS (EI) m/z 366 (M⁺).

1-(Pyridin-3-yl)-2-fluoro-4-(2-methylthiazol-4ylethynyl)benzene (20)

Prepared from 18 and 3-pyridylboronic acid according to the general procedure C in 68% yield; purified by column chromatography eluting with hexane/EtOAc (1:1). The free base was converted to its HBr salt and recrystallized from EtOH as a light-brown solid; mp 209.5–211 °C (dec). IR (KBr) 1605, 1599 cm⁻¹. ¹H NMR (CDCl₃) δ 2.76 (s, 3H), 7.38–7.44 (m, 5H), 7.89 (m, 1H), 8.63 (dd, J) 1.6, 4.8 Hz, 1H), 8.81 (s, 1H) ppm. ¹³C NMR (CDCl₃) δ 19.5, 85.4, 87.4, 119.4, 119.7, 123.3, 123.6, 124.6, 126.3, 128.4, 130.6, 131.2, 136.4, 136.6, 149.3, 149.7, 158.3, 160.8, 166.2 ppm. GC/MS (EI) m/z 294 (M⁺). Anal. (C₁₇H₁₁N₂SF · HBr · 0.25H₂O) for C, H, N.

2-Phenyl-5-(6-methylpyridin-2-ylethynyl)benzonitrile (21)

Prepared from 19 and phenylboronic acid according to the general procedure C in 65% yield; purified by column chromatography eluting with hexane/EtOAc (2:1)). The free base was converted to its HBr salt and recrystallized from 2-PrOH as a white solid; mp 188.5–190 °C (dec). IR (KBr) 2234, 1584 cm⁻¹. ¹H NMR (CDCl₃) δ 2.61 (s, 3H), 7.17(d, J) 8.0 Hz, 1H), 7.39 (d, J) 7.6 Hz, 1H), 7.45–7.65 (m, 7H), 7.83 (dd, J) 1.6, 8.0 Hz, 1H), 7.97 (d, J) 1.6 Hz) ppm. ¹³C NMR (CDCl₃) δ 24.8, 86.2, 91.4, 111.9, 118.1, 122.6, 123.5, 124.9, 128.9,129.0, 129.3, 130.5, 136.2, 136.8, 137.1, 137.6, 142.5, 145.5, 159.5 ppm. GC-MS (EI) m/z 294 (M⁺). Anal. (C₂₁H₁₄N₂ · HBr) for C, H, N.

2-(3-Fluorophenyl)-5-(6-methylpyridin-2-ylethynyl)benzonitrile (22)

Prepared from 19 and 3-fluorophenylboronic acid according to the general procedure C in 70% yield; purified by column chromatography eluting with hexane/EtOAc (2:1)). The free base was converted to its HBr salt and recrystallized from 2-PrOH as a light-yellow solid; mp 183–185 °C (dec). IR (KBr) 2250, 1593 cm⁻¹. ¹H NMR (CDCl₃) δ 2.61 (s, 3H), 7.17(m, 2H), 7.27 (m, 1H), 7.36–7.41 (m, 2H), 7.45–7.53 (m, 2H), 7.62 (d, J) 7.6 Hz, 1H), 7.85 (dd, J) 2.0, 8.4 Hz, 1H), 7.98 (d, J) 1.6 Hz, 1H) ppm. ¹³C NMR(CDCl₃) δ 24.6, 84.0, 92.7, 107.8, 112.7, 117.0, 120.2, 122.9, 123.7, 124.2, 124.8, 136.6, 137.3, 137.8, 141.2, 149.0, 159.5 ppm. GC-MS (EI) m/z 312 (M⁺). Anal. (C₂₁H₁₃N₂F · HBr) for C, H, N.

2-(4-Fluorophenyl)-5-(6-methylpyridin-ylethynyl)benzonitrile (23)

Prepared from 19 and 4-fluorophenylboronic acid according to the general procedure C in 78% yield; purified by column chromatography eluting with hexane/EtOAc (2:1). The free base was converted to its HBr salt and recrystallized from 2-PrOH as a light-brown solid; mp 197–198 °C (dec). IR (KBr): 2226, 1591, 1506 cm₋₁. ₁H NMR (CDCl₃) δ 2.61 (s, 3H), 7.15–7.25 (m, 3H), 7.39 (d, J =7.6 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.53–7.58 (m, 2H), 7.62 (dd, J = 8.0, 8.0 Hz, 1H), 7.81 (dd, J = 1.6, 8.0 Hz, 1H), 7.94 (d, J = 1.6 Hz, 1H) ppm. ₁₃C NMR (CDCl₃) δ 24.8, 86.1, 91.4, 111.9, 116.1, 116.4, 118.0, 122.8, 123.6, 124.9, 130.4, 130.8, 133.7, 136.2, 137.1, 142.0, 144.5, 159.5 ppm. GCMS (EI) m/z 312 (M+). Anal. (C₂₁H₁₃N₂F · HBr) for C, H, N. ppm. IR (KBr) 3423, 3053, 2232, 1678, 1601 cm⁻¹. GC-MS (EI) m/z 237 (M⁺). Anal. (C₁₄H₁₁N₃O · 0.75H₂O) C, H, N.

General Procedure D: Synthesis of the Amides

To a solution of aryl carboxylic acid (1 equiv) in anhydrous dichloromethane and catalytic DMF, thionyl chloride (1.1 equiv) was added at 0–5 °C. The reaction was stirred at reflux for 1 h and then evaporated under reduced pressure. The residue was triturated with dichloromethane and evaporated. The acid chloride was then dissolved in dichloromethane and added to a solution of 2-amino 6-methyl pyridine (0.9 equiv) and pyridine or triethylamine (4–8 equiv) in dichloromethane at 0–5 °C. The reaction was then monitored by TLC, and the product was isolated after washing the reaction mixture with saturated aq NaCl solution (×3), dried over anhydrous Na₂SO₄, and evaporated. The residue was further purified by column chromatography.

General Procedure E: Suzuki Coupling Reaction for Amides

To a solution of bromide or triflate (1 equiv) in toluene or DME/H₂O (3:1) was added a solution of aryl boronic acid (1.2 equiv) in EtOH and a 2 M aq Na₂CO₃ solution (2 equiv). The reaction was degassed with argon for 20 min, and Pd(PPh₃)₄ (0.03 equiv) was added under an argon atmosphere. The reaction was flushed with additional argon and quickly sealed airtight. The reaction was quenched by filtering through a pad of celite after stirring overnight at 110 or 80 °C, if DME/H₂O was used, until complete conversion of the starting material, as visualized by TLC. The filtrate was evaporated under reduced pressure, and the residue was further purified by column chromatography.

4-Cyano-N-(6-methylpyridin-2-yl)-benzamide (25)

To a solution of 4-cyano benzoic acid (0.50 g, 3.39 mmol) in pyridine (2 mL), carbonyldiimidazole (CDI, 0.55 g, 3.39 mmol) was added slowly and stirred at room temperature for 1 h. To this reaction mixture 2-amino-6-methyl pyridine (24, 0.30 g, 2.77 mmol) was added and stirred at room temperature for 48 h. The reaction mixture was then added to an ice–water mixture with vigorous stirring. The precipitated product was filtered and washed with water. The crude product was further purified by flash chromatography (CHCl₃) to give 25 as a white solid. Yield 0.37 g, 56%; mp 132–134 °C.¹HNMR (CDCl₃) δ 2.42 (s, 3H), 6.96–6.98 (d, J = 7.6 Hz, 1H), 7.66–7.70 (t, J = 8.0 Hz, 1H), 7.77–7.91 (dt, J = 8.8, 2.0 Hz, 2H), 8.00–8.03 (dt, J = 8.4, 2.0 Hz, 2H), 8.14–8.17 (d, J = 8.4 Hz, 1H), 8.82 (bs, 1H) ppm. ¹³CNMR (CDCl₃) δ 24.13, 111.42, 115.91, 118.1, 120.3, 128.2, 132.8, 138.5, 139.2, 150.5, 157.3, 164.1 ppm. IR (KBr) 3423, 3053, 2232, 1678, 1601 cm₋₁. GC-MS (EI) m/z 237 (M+). Anal. (C₁₄H₁₁N₃O· 0.75H₂O) C, H, N.

4-Bromo-N-(6-methylpyridin-2-yl)-benzamide hydrobromide (26)

Prepared as described in the general procedure D where commercially available 4-bromo benzoyl chloride (7.00 g, 31.89 mmol) was used and the product was converted to the HBr salt as white solid. Yield 7.1 g, 75%; mp 252–254 °C. 1H NMR (CDCl3) δ 2.46 (s, 1H), 6.93–6.95 (d, J) 7.2 Hz, 1H), 7.61–7.64 (dt, J) 8.8, 2.4 Hz, 2H), 7.63–7.67 (t, J) 7.6 Hz, 1H), 7.78–7.81 (dt, J) 6.4, 2.8, Hz, 2H), 8.15–8.17 (d, J)8.4 Hz, 1H), 8.54 (bs, 1H) ppm. 13C NMR (CDCl3) δ 24.2, 111.2, 119.9, 127.2, 129.0, 132.3, 133.4, 139.0, 150.8, 157.2, 164.9 ppm.IR (KBr) 3305, 3059, 1679, 1578, 1455 cm−1. GC-MS (EI) m/z 290 (M+). Anal. (C13H11BrN2O • HBr) C, H, N.

4-Phenoxy-N-(6-methylpyridin-2-yl)-benzamide Hydrobromide (27)

Prepared as described in the general procedure D using 4-phenoxy benzoic acid (1.00 g, 4.67 mmol). Yield 0.72 g, 51%; HBr salt: white solid; mp 95–96 C. 1H NMR (CDCI3) δ 2.45 (s, 3H), 6.91–6.93 (d, J = 7.6 Hz, 1H), 7.02–7.08 (m, 4H), 7.18–7.26 (m, 1H), 7.38–7.42 (m, 2H), 7.61–7.65 (t, J = 8.0 Hz, 1H), 7.88–7.92 (m, 2H), 8.17–8.19 (d, J = 8.0 Hz, 1H), 8.58 (bs, 1H) ppm. 13C NMR (CDCl3) δ 24,2, 111.2, 118.9, 119.6, 120.2, 124.7, 128.8, 129.7, 130.3, 139.0, 151.1, 155.9, 157.1, 161.4, 165.2 ppm. IR (KBr) 3316, 3063, 1677, 1601, 1455 cm−1. GC-MS (EI) mlz 304 (M+). Anal. (C13H11BrN2O HBr) C, H, N.

4-(Benzyloxy)-N-(6-methylpyridin-2-yl)-benzamide (28)

Prepared as described in the general procedure D using 4-benzyloxy benzoic acid (4.00 g, 17.5 mmol) as white solid. Yield 3.67 g, 66%, mp 125–126 °C. ¹H NMR (CDCl₃) δ 2.46 (s, 3H), 5.13 (s, 2H), 6.90–6.92 (d, J) 7.2 Hz, 1H), 7.03–7.05 (d, J) 8.8 Hz, 2H), 7.35–7.45 (m, 5H), 7.61–7.65 (t, J) 8.0 Hz, 1H), 7.88–7.90 (d, J) 9.2 Hz, 2H), 8.16–8.18 (d, J) 8.4 Hz, 1H), 8.48 (bs, 1H) ppm. ¹³C NMR (CDCl₃) δ 24.3, 70.4, 111.1, 115.1, 119.5, 127.0, 127.8, 128.5, 128.9, 129.4, 136.4, 138.9, 151.2, 157.1, 162.1, 165.3 ppm. IR (KBr) 3415, 3060, 1673, 1600, 1506 cm⁻¹. GC-MS (EI) m/z 318 (M⁺). Anal. (C₂₀H₁₈N₂O₂ · 0.25H₂O) C, H, N.

4-Phenyl-N-(6-methylpyridin-2-yl)-benzamide hydrobromide (29)

Prepared as described in the general procedure D where commercially available 4-biphenyl carbonyl chloride (2.00 g, 9.2 mmol) was used. Yield 2.38 g, 89%; mp 92–94 °C(white solid).¹H NMR (CDCl₃) δ 2.44 (s, 3H), 6.91–6.92 (d, J) 7.2 Hz, 1H), 7.37–7.41 (m, 1H), 7.44–7.48 (m, 2H), 7.60–7.70 (m,5H), 7.97–8.00 (dt, J) 8.2, 2, Hz, 2H), 8.21–8.23 (d, J) 8.0 Hz, 1H), 8.77 (bs, 1H) ppm. ¹³C NMR (CDCl₃) δ 24.2, 111.3,119.7, 127.5, 127.6, 128.0, 128.4, 129.2, 133.2, 139.0, 140.0, 145.2, 151.1, 157.2, 165.6 ppm. GC-MS (EI) m/z 288 (M⁺). Anal. (C₁₉H₁₆N₂O.HBr) C, H, N.

4-(3′-Cyano-phenyl)-N-(6-methylpyridin-2-yl)-benzamide Hydrobromide (30)

Prepared by following the general procedure E using 3-cyano benzene boronic acid (0.20 g, 1.36 mmol) and compound 26 (0.30 g, 1.13 mmol). Yield 0.27 g, 76%; HBr salt: white solid; mp 285 °C (dec). ¹H NMR (CDCl₃) δ 2.48 (s, 3H), 6.95–9.97 (d, J) 7.2 Hz, 1H), 7.58–7.61 (t, J) 7.6 Hz, 1H), 7.65–7.70 (m, 3H), 7.85–7.87 (dt, J) 8.8, 1.2 Hz, 1H), 7.90–7.91 (t, J) 1.6 Hz, 1H), 8.04–8.07 (dt, J) 8.4, 2.0 Hz, 2H), 8.20–8.22 (d, J) 8.4 Hz, 1H), 8.70 (bs, 1H) ppm. ¹³CNMR (CDCl₃) δ 24.3, 111.3, 113.5, 118.8, 119.9, 127.7, 128.33, 130.1, 131.0, 131.7, 131.8, 134.3, 141.3, 142.7, 150.9, 157.2, 165.2 ppm. IR (KBr) 3378, 3061, 2227, 1686, 1460 cm⁻¹. GC-MS (EI) m/z (M⁺) 313. Anal. (C₂₀H₁₅N₃O.HBr) C, H, N.

4-(3′-Chloro-phenyl)-N-(6-methylpyridin-2-yl)-benzamide Hydrobromide (31)

Prepared by following the general procedure E using 3-chloro benzene boronic acid (0.20 g, 1.28 mmol) and compound 26 (0.31 g, 1.06 mmol). Yield 0.27 g, 78%; HBr salt: white solid; mp 258–260 °C. ¹H NMR (CDCl₃) δ 2.47 (s, 3H), 6.93–6.95 (d, J) 7.6 Hz, 1H), 7.35–7.38 (m, 1H), 7.38–7.42 (t, J) 8.0 Hz, 1H), 7.49–7.52 (dt, J) 7.2, 2.0 Hz, 1H), 7.60–7.61 (m, 1H), 7.64–7.69 (m, 3H), 7.99–8.02 (dt, J) 8.0, 2.4, 2H), 8.20–8.22 (d, J) 8.4 Hz, 1H), 8.63 (bs, 1H) ppm. ¹³C NMR (CDCl₃) δ 24.3, 111.2, 119.8, 125.6, 127.6, 127.7, 128.1, 128.4, 130.4, 133.8, 135.1, 139.0, 141.9, 143.7, 151.0, 157.2, 165.3 ppm. IR (KBr) 3416, 3062, 1677, 1601, 1510 cm⁻¹. GC-MS (EI) m/z 322 (M⁺). Anal. (C₁₉H₁₅CIN₂O · HBr) C, H, N.

3,5-Difluoro-N-(6-methylpyridin-2-yl)-benzamide (32)

Prepared as described in the general procedure D using 3,5-difluoro benzoic acid (1.0 g, 5.66 mmol). Yield 1.0 g, 45%; mp 129–132 °C (white solid). ¹H NMR (CDCl₃) δ 2.45 (s, 3H), 6.95–7.03 (m, 2H), 7.41–7.46 (m, 2H), 7.64–7.68 (t, J) 8.0 Hz, 1H), 8.12–8.14 (d, J) 8.4 Hz, 1H), 8.56 (bs, 1H) ppm. ¹³CNMR (CDCl₃) δ 24.2, 107.5, 107.8, 108.0, 110.6, 110.7, 110.8, 110.9, 111.3, 120.2, 137.9, 138.0, 139.1, 150.5, 157.3, 162.0, 162.1, 164.5, 164.6 ppm. IR (KBr) 3338, 3074, 1696, 1599, 1580, 1561 cm⁻¹. GC-MS (EI) m/z 248 (M⁺). Anal. (C₁₃H₁₀F₂N₂O) C, H, N.

3,4-Difluoro-N-(6-methylpyridin-2-yl)-benzamide (33)

Prepared as described in the general procedure D using 3,4-difluoro benzoic acid (1.0 g, 5.66 mmol). Yield 1.10 g, 45%; mp 88–91 °C (white solid). ¹H NMR (CDCl₃) δ 2.45 (s, 3H), 6.94–6.96 (d, J) 7.2 Hz, 1H), 7.23–7.30 (m, 1H), 7.64–7.69 (m, 2H), 7.77–7.82 (m, 1H), 8.12–8.14 (d, J) 8.0 Hz, 1H), 8.55 (bs, 1H) ppm. ¹³C NMR (CDCl₃) δ 24.0, 111.1, 117.1, 117.1, 117.3, 117.6, 117,8, 119.8, 123.5, 123.6, 123.6, 123.6, 131.5, 138.9, 149.1, 149.3, 150.4, 151.6, 151.6, 151.7, 151.8, 154.1, 154.2, 157.1, 163.4 ppm IR (KBr) 3338, 3042, 1647, 1601, 1541 cm⁻¹. GC-MS (EI) m/z 248 (M⁺). Anal. (C₁₃H₁₀F₂N₂O) C, H, N.

3-Cyano-5-fluoro-N-(6-methylpyridin-2-yl)-benzamide Hydrochloride (34)

Prepared as described in the general procedure D using 3-cyano-5-fluorobenzoic acid (900 mg, 5.46 mmol). Yield 407 mg, 100%; HBr salt: white solid; mp 245–247 °C. ¹H NMR (CDCl₃) δ 2.49 (s, 3H), 6.98–7.00 (m, 1H), 7.55–7.57 (m, 1H), 7.67–7.71 (m, 2H), 7.91–7.93 (m, 1H), 8.04 (s, 1H), 8.12–8.14 (s, 1H) ppm. ¹³C NMR (CDCl₃) δ 24.2, 111.3, 120.0, 127.0, 139.3, 167.5 ppm. GC-MS (EI) m/z 255 (M⁺). Anal. (C₁₄H₁₀FN₃O · HCl), C, H, N.

4-Bromo-3-fluoro-N-(6-methylpyridin-2-yl)-benzamide (35)

Prepared as described in the general procedure D using 4-bromo-3-fluorobenzoic acid (0.87 g, 4.0 mmol) as a yellow solid. Yield 1.0 g, 81%. ¹H NMR (CDCl₃) δ 2.45–2.49 (s, 3H), 6.95–6.97 (m, 1H), 7.57–7.74 (m, 4H), 8.13–8.15 (m, 1H) ppm. GC-MS (EI) m/z 310 (M⁺).

3-Bromo-5-fluoro-N-(6-methylpyridin-2-yl)-benzamide (36)

Prepared as described in the general procedure D using 3-bromo-5-fluoro benzoic acid (2.0 g, 9.13 mmol) as a white solid. Yield 2.36 g, 83%; mp 189–190 °C. ¹H NMR (CDCl₃) δ 2.58 (s, 3H), 7.02–7.04 (d, J) 8.0 Hz, 1H), 7.45–7.48 (m, 1H), 7.73–7.80 (m, 2H), 8.08–8.11 (dt, J) 11.2, 1.2 Hz, 1H), 8.31–8.33 (d, J) 8.0 Hz, 1H) ppm. ¹³C NMR (CDCl₃) δ 23.5, 112.2, 114.2, 114.4, 120.4, 122.8, 123.1, 126.8, 126.8, 139.9, 150.8, 156.8 164.1 ppm. GC-MS (EI) m/z 309 (M⁺).

3-Fluoro-N-(6-methylpyridin-2-yl)-5-(pyridin-3-yl)-benzamide (37)

Prepared as described in the general procedure E using 3-pyridyl boronic acid (0.18 g, 1.45 mmol) and compound 36 (0.30 g, 0.97 mmol). Yield 0.11 g, 37%; HBr salt: white solid; mp >275 °C. ¹H NMR (CDCl₃) δ 2.47 (s, 3H), 6.96–6.98 (d, J) 7.6 Hz, 1H), 7.40–7.44 (m, 1H), 7.46–7.50 (m, 1H), 7.65–7.69 (m, 2H), 7.89–7.91 (m, 2H), 8.17–8.19 (d, J) 8.4 Hz, 1H), 8.67–8.68 (m, 1H), 8.69 (bs, 1H), 8.87–8.88 (m, 1H) ppm. ¹³C NMR (CDCl₃) δ 24.2, 111.3, 114.1, 114.4, 117.8, 118.0, 120.1, 121.9, 121.9, 124.0, 128.8, 132.3, 134.7, 137.7, 137.7, 139.1, 141.0, 141.1, 148.4, 149.9, 150.6, 157.3, 162.2, 164.2, 164.7 ppm. IR (KBr) 3382, 3063, 1686, 1597, 1554, 1461 cm⁻¹. GC-MS (EI) m/z 307 (M⁺). Anal. (C₁₈H₁₄FN₃O.2HBr · 1.5H₂O) C, H, N.

5-Fluoro-2′-methoxy-N-(6-methylpyridin-2-yl)biphenyl-3-carboxamide Hydrobromide (38)

To a clean and dry reaction tube, compound 36 (0.20 g, 0.64 mmol), 2-dicyclohexylphosphino-2′,6′-dimethoxy biphenyl (5 mg, 2 mol %), Pd(OAc)₂ (1.5 mg, 1 mol %), 2-methoxy benzene boronic acid (0.15 g, 0.9 mmol) powdered potassium phosphate (0.27 g, 1.2 mmol), and degassed toluene/EtOH (3 mL/0.5 mL) were added together and flushed with argon and quickly sealed airtight. The reaction was heated to 100 °C for 2 h and filtered through celite and the filtrate was evaporated. The crude product was passed through silica gel column eluting with chloroform. Yield 0.13 g, 60%; HBr salt: white solid; mp 206–208 °C. ¹H NMR (CDCl₃) δ 2.45 (s, 3H), 3.83 (s, 3H), 6.92–6.94 (d, J) 7.6 Hz, 1H), 7.00–7.02 (d, J) 8.4 Hz, 1H), 7.03–7.07 (td, J) 1.2, 7.2 Hz, 1H), 7.31–7.33 (dd, J) 1.6, 7.6 Hz, 1H), 7.36–7.40 (m, 1H), 7.44–4.48 (m, 1H), 7.58–7.62 (m, 1H), 7.62–7.66 (t, J) 8 Hz, 1H), 7.81–7.82 (t, J) 1.6 Hz, 1H), 8.17–8.19 (d, J) 8.4 Hz, 1H), 8.56 (bs, 1H) ppm. ¹³C NMR (CDCl₃) δ 24.2, 55.8, 111.2, 111.5, 113.1, 113.4, 119.9, 120.5, 120.7, 121.2, 124.0, 124.0, 128.4, 130.0, 130.9, 136.3, 136.4, 139.0, 141.6, 141.7, 150.8, 156.5, 157.2, 161.5, 164.0, 164.7, 164.8 ppm. IR (KBr) 3060, 1678, 1601, 1530, 1455 cm⁻¹. GC-MS (EI) m/z 336 (M⁺). Anal. (C₂₀H₁₇FN₂O₂ · HBr) C, H, N.

5-Fluoro-2′-methyl-N-(6-methylpyridin-2-yl)biphenyl-3-carboxamide hydrobromide (39)

Prepared from compound 36, as described above for compound 38, using 2-methyl benzene boronic acid (0.125 g, 0.9 mmol). Yield 0.14 g, 67%; HBr salt: white solid; mp 207–210 °C. ¹H NMR (CDCl₃) δ 2.28 (s, 3H), 2.46 (s, 3H), 6.94–6.96 (d, J) 7.6 Hz, 1H), 7.21–7.34 (m, 5H), 7.61–7.62 (d, J) 2.0 Hz, 1H), 7.63–7.67 (t, J) 7.6 Hz, 1H), 7.64–7.65 (m, 1H), 8.15–8.18 (d, J) 8.4 Hz, 1H), 8.48 (bs, 1H) ppm. ¹³C NMR (CDCl₃) δ 20.3, 24.0, 111.0, 113.0, 113.2, 119.7, 119.8, 120.0, 123.4, 123.4, 126.1, 128.3, 129.5, 130.6, 135.2, 136.4, 136.4, 138.8, 139.5, 144.9, 145.0, 150.5, 157.0, 161.3, 163.8, 164.3 ppm. IR (KBr) 3295, 3063, 1681, 1531, 1455 cm⁻¹. GC-MS (EI) m/z 320 (M⁺). Anal. (C₂₀H₁₇FN₂O · HBr) C, H, N.

3-Fluoro-N-(6-methylpyridin-2-yl)-4-(pyridine-3-yl)-benzamide hydrochloride (40)

Prepared as described in the general procedure E using pyridin-3-ylboronic acid (0.29 g, 2.41 mmol) and compound 35 (0.62 g, 2.0 mmol). Yield 0.40 g, 65%; HCl salt: white solid; mp 280 °C (dec). ¹H NMR (CDCl₃) δ 2.50 (s, 3H), 6.96–6.98 (m, 1H), 7.41–7.44 (m, 1H), 7.57–7.94 (m, 5H), 8.18–8.20 (m, 1H), 8.67 (m, 1H), 8.84 (s, 1H) ppm. ¹³C NMR (CDCl₃) δ 24.3, 111.3, 115.9, 116.1, 120.0, 123.3, 123.4, 123.7, 131.1, 136.6, 139.1, 149.7, 157.3 ppm. GC-MS (EI) m/z 307 (M+). Anal. (C₁₈H₁₄FN₃O · 2HCl · 0.25H₂O), C, H, N.

3-Chloro-N-(6-methylpyridin-2-yl)-4-(pyridine-3-yl)-benzamide Hydrochloride (41)

Prepared as described in the general procedure E using pyridin-3-ylboronic acid (431 mg, 3.53 mmol) and 4-bromo-3-chloro-N-(6-methylpyridin-2-yl) benzamide (1.04 g, 3.21 mmol), which was prepared using the general procedure D from 4-bromo-3-chlorobenzoic acid (942 mg, 4.0 mmol). Yield 775 mg, 60%; HCl salt: white solid; mp 252–254 °C (dec). ¹H NMR (CDCl₃) δ 2.50 (s, 3H), 6.97–6.99 (m, 1H), 7.41–7.50 (m, 2H), 7.66–7.70 (m, 1H), 7.82–7.92 (m, 2H), 8.11–8.20 (m, 2H), 8.70–8.72 (m, 2H) ppm. ¹³C NMR (CDCl₃) δ 24.3, 111.2, 120.1, 125.9, 129.5, 131.9, 133.8, 135.7, 137.0, 139.2, 149.6, 150.0, 157.3, 167.4 ppm. GC-MS (EI) m/z 323 (M⁺). Anal. (C₁₈H₁₄ClN₃O · 2HCl · 0.5H₂O), C, H, N.

5-Bromo-N-(6-methylpyridin-2-yl)nicotinamide (42)

Prepared as described in the general procedure D using 5-bromo nicotinic acid (3.0 g, 14.8 mmol). Yield 4.43 g, 100%; mp 140–142 °C. ¹H NMR (CDCl₃) δ 2.51 (s, 3H), 6.99–7.01 (m, 1H), 7.69–7.71 (m, 1H), 8.16–8.19 (m, 1H), 8.42–8.43 (m, 2H), 8.85–8.86 (m, 2H) ppm. ¹³C NMR (CDCl₃) δ 23.8, 111.9, 114.2, 120.4, 121.2, 131.6, 138.2, 139.7, 146.6, 154.2 ppm. Anal. (C₂₀H₁₈N₂O₂ · HBr · H₂O) C, H, N.

6-bromo-N-(6-methylpyridin-2-yl)-nicotinamide (43)

Prepared as described in the general procedure D using 6-bromonicotinic acid (1.01 g, 5 mmol). Yield 1.24 g, 85%. ¹H NMR (CDCl₃) δ2.47 (s, 3H), 6.97–6.99 (m, 1H), 7.46–7.48 (m, 1H), 7.67–7.69 (m, 1H), 8.11–8.13 (m, 1H), 8.19–8.20 (m, 1H), 8.94–8.95 (m,1H) ppm.

5-(3′-Pyridyl)-N-(6-methylpyridin-2-yl)-nicotinamide hydrobromide (44)

Prepared using the general procedure E using 3-pyridyl boronic acid (0.19 g, 1.54 mmol) and compound 42 (0.30 g, 1.0 mmol). Yield 0.19 g, 64%; HBr salt: light-orange solid; m>275 °C. ¹H NMR (CDCl₃) δ 2.48 (s, 3H), 6.97–6.99 (d, J) 7.6 Hz, 1H), 7.44–7.48 (m, 1H), 7.67–7.71 (t, J) 7.6 Hz, 1H),7.92–7.95 (m, 1H), 8.17–8.19 (d, J) 8.0 Hz, 1H), 8.44–8.45 (t,J) 2.4 Hz, 1H), 8.71–8.73 (dd, J) 1.6, 3.2 Hz, 1H), 8.83 (bs,1H), 8.90–8.91 (dd, J) 0.8, 1.6 Hz, 1H), 9.02–9.03 (d, J) 2.0 Hz, 1H), 9.18–9.19 (d, J) 2.0 Hz, 1H) ppm. ¹³CNMR (CDCl₃) δ 24.2, 111.5, 120.3, 124.2, 130.7, 132.6, 134.0, 134.8, 139.2, 147.7, 148.4, 150.2, 150.5, 151.3, 157.4, 163.7 ppm. IR (KBr) 3232, 3055, 1678, 1579, 1457 cm⁻¹. GC-MS (EI) m/z 289 (M⁺). Anal. (C₁₇H₁₄N₄O · 3HBr · 1.5H₂O) C, H, N.

5-(2-Methoxyphenyl)-N-(6-methylpyridin-2-yl)-nicotinamide Hydrobromide (45)

Prepared as described for the synthesis of compound 38 using 2-methoxy boronic acid (0.15 g, 1.0 mmol) and compound 42 (0.20 g, 0.68 mmol). Yield 0.11 g, 50%; HBr salt: white solid; mp 229–232 °C. ¹H NMR (CDCl₃) δ 2.47 (s, 3H), 3.84 (s, 3H), 6.95–6.97 (d, J) 7.2 Hz, 1H), 7.02–7.04 (dd, J) 0.8, 8.4 Hz, 1H), 7.07–7.11 (td, J) 7.6, 1.2 Hz, 1H), 7.33–7.36 (dd, J) 2, 7.2 Hz, 1H), 7.39–7.44 (m 1H), 7.65–7.69 (t, J) 7.6 Hz, 1H), 8.18–8.20 (d, J) 8.0 Hz, 1H), 8.37–8.39 (t, J) 2.4 Hz, 1H), 8.62 (bs, 1H), 8.93–8.94 (d, J) 2.4 Hz, 1H), 9.089.09 (d, J) 2.0 Hz, 1H) ppm. ¹³C NMR (CDCl₃) δ24.2, 55.8, 111.3, 111.5, 120.1, 121.4, 126.0, 129.7, 130.4, 130.9, 134.7, 135.9, 139.1, 146.6, 150.6, 153.5, 156.6, 156.8, 157.3, 164.2, 166.5 ppm. GC-MS (EI) m/z 319 (M⁺). Anal. (C₁₉H₁₇N₃O₂ · 2HBr · H₂O) C, H, N.

N-(6-Methylpyridin-2-yl)-6-phenylnicotinainide Hydrochloride (46)

Prepared as described in the general procedure E using phenyl boronic acid (146.3 mg, 1.2 mmol) and compound 43 (292 mg, 1 mmol). Yield 250 mg, 87%; HCl salt: white solid; mp 230 °C (dec). ¹H NMR (CDCl₃) δ 2.50 (s, 3H), 6.95–6.97 (m, 1H), 7.45–7.53 (m, 3H), 7.64–7.85 (m, 2H), 8.05–8.07 (m, 2H), 8.18–8.20 (m, 1H), 8.27–8.29 (m, 1H), 9.23 (m, 1H) ppm. ¹³C NMR (CDCl₃) δ 24.2, 111.5, 120.1, 127.5, 128.3, 129.2, 130.2,136.2, 138.4, 139.1, 148.7, 150.7, 157.3, 160.6, 164.1 ppm. GC-MS m/z 289 (M+). Anal. (C₁₈H₁₅N₃O · 2HCl · 0.25H₂O), C, H, N.

N-(6-Methylpyridin-2-yl)-2,3′-bipyridine-5-carboxainide Hydrochloride (47)

Prepared as described in the general procedure E using pyridine-3-ylboronic acid (148 mg, 1.2 mmol) and compound 43 (292 mg, 1 mmol). Yield 200 mg, 69%; HCl salt: white solid; mp 260 °C (dec). ¹H NMR (CDCl₃) δ 2.50 (3H,s, CH₃), 6.97–6.99 (1H, m), 7.44–7.47 (1H, m), 7.67–7.71 (1H,m), 7.89–7.91 (1H, m), 8.18–8.21 (1H, m), 8.34–8.42 (2H, m), 8.72 (1H, s), 9.27–9.28 (2H, m) ppm. ¹³C NMR (CDCl₃) δ 24.3, 11.4, 120.1, 120.4, 124.0, 129.0 135.0, 136.5, 139.2, 148.7, 149.0, 150.6,151.0, 157.3, 158.0, 163.7 ppm. Anal. (C₁₇H₁₄N₄O · 2HCl · 0.5H₂O), C, H, N.

Benzyl-4-(benzyloxy)-3-bromobenzoate (49)

3-Bromo, 4-hydroxy benzoic acid (48, 15.00 g, 69.0 mmol) was dissolved in 150 mL anhydrous acetone and then stirred at reflux overnight with anhydrous K₂CO₃ (24.0 g, 170 mmol) and benzyl bromide (24.8 g, 17.2 mL, 145 mmol). The reaction mixture was filtered hot, and the filtrate was evaporated under reduced pressure. The clear oil obtained solidified in nearly quantitative yield (29.0 g, 99%), which was used for next reaction without further purification. An analytical sample was purified by flash column chromatography eluting with CHCl₃; mp 74–76 °C. ¹H NMR (CDCl₃) δ 5.17 (s, 2H), 5.31 (s,2H), 6.89–6.91 (d, J) 8.8 Hz, 1H), 7.20–7.44 (m, 10H), 7.94–7.97 (m, 1H), 8.27–8.28 (m, 1H) ppm. ¹³CNMR (CDCl₃) δ66.9, 70.9, 112.3, 112.78, 124.0, 126.3, 127.1, 128.3, 128.4, 128.5,128.8, 128.8, 130.8, 133.2, 135.1, 135.2, 136.1, 158.8, 165.1 ppm. IR (KBr) 3033, 1717, 1598, 1497, 1268 cm⁻¹. GC-MS (EI) m/z 396 (M⁺).

Benzyl-4-(benzyloxy)-3-cyanobenzoate (50)

A solution of compound 49 (4.5 g, 11 mmol) and Zn(CN)₂ (0.8 g, 7 mmol) in DMF (20 mL) was stirred at room temperature for 20 min under an argon atmosphere. Pd(PPh₃)₄ (0.65 g, 5 mol %) was added, and the solution was heated to 80 °C overnight. The reaction was cooled to room temperature and filtered over celite. The clear solution was evaporated under vacuum to remove DMF. The residue was extracted with CHCl₃ (25 mL × 2) from H₂O (50 mL). The organic layer was washed with saturated aq NaCl solution (25 mL × 2), dried over anhydrous Na₂SO₄, and evaporated under reduced pressure. The residue obtained was purified by flash column chromatography eluting with n-hexanes/EtOAc (4:1) to obtain a white solid. Yield 3.8 g, 97%; mp 86–88 °C. ¹H NMR (CDCl₃) δ5.26 (s, 2H), 5.34 (s, 2H), 7.02–7.04 (d, J) 9.2 Hz, 1H), 7.31–7.45 (m, 10H), 8.17–8.20 (dd, J) 2.4, 6.4 Hz, 1H), 8.28–8.29 (d, J) 2.0, 1H) ppm. ¹³C NMR (CDCl₃) δ 67.3, 71.2, 102.8, 112.7, 115.6, 123.4, 127.2, 128.5, 128.7, 128.8, 129.0, 135.0,135.7, 135.9, 136.1, 163.5, 164.5 ppm. IR (KBr) 3090, 3034, 2231, 1716, 1607 cm⁻¹. GC-MS (EI) m/z 343 (M⁺).

4-(Benzyloxy)-3-cyanobenzoic Acid (51)

To a solution of compound 50 (3.5 g, 10 mmol) in MeOH (70 mL), aq solution of 4 N NaOH (1.2 g in 7.6 mL H₂O, 30 mmol) was added. The solution was evaporated after stirring at room temperature for 2 h. The residue was dissolved in H₂O, and the insolubles were removed by filtration. The filtrate was neutralized carefully with conc HCl (pH 2) and the precipitated white solid was filtered and washed with cold water and dried in vacuo. Yield 2.4 g, 93%; mp 203–207°C. ¹H NMR (MeOH-d₄) δ 5.27 (s, 2H), 7.25–7.39 (m, 4H), 7.45–7.46 (m, 2H), 8.16–8.19 (m, 2H) ppm. ¹³C NMR (MeOH-d₄) δ 70.9, 101.9, 112.8, 115.1, 123.9, 127.2, 128.1, 128.4, 135.3, 135.6, 136.0, 163.5, 166.2 ppm. IR (KBr) 3045, 2230, 1709, 1606,1502 cm⁻¹.

4-(Benzyloxy)-3-cyano-N-(6-methylpyridin-2-yl)-benzamide (52)

Prepared by following the general procedure D using compound 51 (2.2 g, 8.7 mmol). The residue was further purified by flash column chromatography eluting with n-hexanes/EtOAc (4:1). Yield 2.5 g, 83%; mp 158–160 °C (white solid). ¹H NMR (CDCl₃) δ 2.44 (s, 3H), 5.29 (s, 2H), 6.93–6.95 (d, J) 7.6 Hz, 1H), 7.07–7.09 (d, J) 8.8 Hz, 1H), 7.34–7.47 (m, 5H), 7.62–7.66 (t, J) 7.2 Hz, 1H), 8.04–8.06 (dd, J) 2.4, 6.0 Hz, 1H), 8.10–8.12 (d, J) 8.4 Hz, 1H), 8.16–8.17 (d, J) 2.4 Hz, 1H), 8.53 (bs, 1H) ppm. ¹³C NMR (CDCl₃) δ 24.2,71.3, 103.2, 111.2, 113.1, 115.5, 120.0, 127.3, 127.6, 128.8, 129.1, 133.5, 135.0, 139.1, 150.1, 157.3, 162.9, 163.3 ppm. IR (KBr) 3316, 3065, 2925, 2231, 1674, 1577, 1454 cm⁻¹. GC-MS (EI) m/z 343 (M⁺).

3-Cyano-4-hydroxy-N-(6-methylpyridin-2-yl)benzamide (53)

To a solution of compound 52 (2.4 g, 7.01 mmol) in anhydrous EtOH (40 mL) and cyclohexene (20 mL), 10% Pd/C (0.49 g) was added slowly. The suspension was refluxed for 1 h and carefully filtered hot through a celite pad. The filtrate was evaporated under reduced pressure to obtain the desired product as white solid. Yield 1.56 g, 87%; mp 268–270 °C. ¹H NMR (DMSO-d₆) δ 2.39 (s, 3H), 6.95–6.97 (d, J) 7.6 Hz, 1H), 7.03–7.05 (d, J) 8.8 Hz., 1H), 7.64–7.67 (t, J) 7.6 Hz, 1H), 7.91–7.93 (d, J) 8.0 Hz, 1H), 8.07–8.10 (dd, J) 2.0, 6.8 Hz, 1H), 8.32–8.33 (d, J) 2.4 Hz, 1H), 10.64 (bs, 1H), 11.81 (bs, 1H) ppm. ¹³C NMR (DMSO-d₆) δ 24.2, 99.3, 112.24, 116.2, 117.0, 119.7, 125.9, 134.4, 135.5, 139.1, 152.1, 157.2, 163.6, 164.6 ppm.

3-Cyano-4-trifluoromethylsulfonyl-N-(6-methylpyridin-2-yl)-benzamide (54)

To a solution of the compound 53 (0.95 g, 3.8 mmol) in anhydrous dichloromethane (20 mL) and pyridine (5 mL), trifluoromethanesulfonic anhydride (1.17 g, 0.7 mL, 4.1 mmol) was added at 0 °C. The reaction was further stirred at room temperature overnight, and additional trifluoromethanesulfonic anhydride (0.7 mL) was added and stirred at room temperature for 1 h. The reaction was then washed with brine (25 mL × 2), dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The yellowish oily product was used for next reaction without further purification. Crude yield 1.4 g (96%). GC-MS (EI) m/z 385 (M⁺).

3-Cyano-4-phenyl-N-(6-methylpyridin-2-yl)-benzamide Hydrobromide (55)

Prepared by following the general procedure E using phenyl boronic acid (2.02 g, 16.53 mmol) and compound 54 (5.0 g, 13 mmol). Yield 3.5 g, 86%; HBr salt: white solid; mp 238–242 °C. ¹H NMR (CDCl₃) δ 2.50 (s, 3H), 6.98–7.00 (d, J) 7.6 Hz, 1H), 7.50–7.56 (m, 3H), 7.60–7.62 (m, 2H), 7.66–7.68 (d, J) 8.4, 1H), 7.67–7.71 (t, J) 7.6 Hz, 1H), 8.16–8.18 (dd, J) 1.6, 6.4 Hz, 2H), 8.34–8.35 (d, J) 1.6 Hz, 1H), 8.50 (bs, 1H) ppm. ¹³C NMR (CDCl₃) δ 24.0, 111.1, 112.1, 117.7, 120.0, 128.7,129.0, 129.5, 130.7, 131.1, 132.8, 133.7, 137.0, 138.9, 148.7, 150.3,157.2, 163.2 ppm. IR (KBr) 3308, 3063, 2229, 1679, 1602, 1533,1455 cm⁻¹. GC-MS (EI) m/z 313 (M⁺); Anal. (C₂₀H₁₅N₃O · HBr) C, H, N.

3-Cyano-4-(3′-fluorophenyl)-N-(6-methylpyridin-2-yl)-benzamide Hydrobromide (56)

Prepared by following the general procedure E using 3-fluorophenyl boronic acid (2.31 g,16.53 mmol) and compound 54 (5.0 g, 13 mmol). Yield 3.8 g, 88%; HBr salt: white solid; mp 266–268 °C. ¹H NMR (CDCl₃) δ 2.50 (s, 3H), 6.98–7.00 (d, J) 7.2 Hz, 1H), 7.18–7.23 (m, 1H), 7.28–7.32 (dt, J) 2.4, 9.2 Hz, 1H), 7.39–7.42 (dt, J = 1.2, 8.4 Hz, 1H), 7.49–7.54 (m, 1H), 7.64–7.66 (d, J = 8.0, 1H), 7.67–7.71 (t, J = 7.2 Hz, 1H), 8.16–8.19 (t, J = 7.2 Hz, 1H), 8.17–8.19 (d, J = 8.0 Hz, 1H), 8.35–8.36 (d, J = 2.0 Hz, 1H), 8.51 (bs, 1H) ppm. ¹³C NMR (CDCl₃) 6 24.0, 111.1, 112.2, 115.8, 116.0, 116.4, 116.6, 117.4, 117.4, 120.1, 124.6, 124.6, 130.6, 130.6, 130.7, 131.2, 132.9, 134.3, 138.9, 139.0, 147.2, 147.2, 150.2, 157.2, 161.6, 163.0, 164.0 ppm. IR (KBr) 3378, 2231, 1685, 1585.96, 1459.5 60 cm⁻¹. GC-MS (EI) m/z 331 (M⁺). Anal. (C₂₀H₁₄FN₃O · HBr) C, H, N.

3-Cyano-4-(4′-fluorophenyl)-N-(6-methylpyridin-2-yl)-benzamide Hydrobromide (57)

Prepared by following the general procedure E using 4-fluorobenzene boronic acid (0.2 g, 1.4 mmol) and compound 54 (0.46 g, 1.10 mmol). The crude residue was purified by flash column chromatography, eluting with n-hexanes: EtOAc (4:1) to obtain a white solid. Yield 0.20 g, 50%; HBr salt: light-orange solid; mp >275 °C. ¹H NMR (CDCl₃) 6 2.50 (s, 3H), 6.98–7.00 (d, J = 7.2 Hz, 1H), 7.21–7.26 (m, 2H), 7.58–7.61 (m, 2H), 7.63–7.65 (d, J = 8.4 Hz, 1H), 7.67–7.71 (t, J = 7.6 Hz, 1H), 8.16–8.18 (dd, J = 2.0, 6.4 Hz, 2H), 8.33–8.34 (d, J = 1.6 Hz, 1H), 8.51 (bs, 1H) ppm. ¹³C NMR (CDCl₃) 6 24.0, 111.1, 112.1, 116.0, 116.2, 117.6, 120.1, 130.5, 130.6, 130.7, 131.2, 132.8,133.0, 133.0, 133.9, 139.0, 147.6, 150.2, 157.2, 162.3, 163.0, 164.8 ppm. IR (KBr) 3292, 3063, 2923, 2230, 1668, 1602.8, 1455 cm⁻¹. Anal. (C₂₀H₁₄FN₃O · HBr) C, H, N.

3-Cyano-N-(6-methylpyridin-2-yl)-4-(naphthalen-1-yl)-benzamide (58)

Prepared by following the general procedure E using 1-naphthyl boronic acid (0.25 g, 1.4 mmol) and compound 54 (0.46 g, 1.10 mmol). The crude residue was purified by flash column chromatography, eluting with n-hexanes:EtOAc (4:1) to obtain a white solid. Yield 0.20 g, 46%; mp158–162 °C. ¹H NMR (CDCl₃) 6 2.45 (s, 3H), 6.96–6.98 (d, J = 7.6 Hz, 1H), 7.44–7.59 (m, 5H), 7.63–7.65 (d, J = 8.4 Hz, 1H), 7.66–7.70 (t, J = 8.0 Hz, 1H), 7.93–9.95 (d, J = 8.4 Hz, 1H), 7.96–7.98 (d, J = 7.6 Hz, 1H), 8.18–8.20 (dd, J = 2.0, 5.6 Hz, 1H), 8.19–8.21 (d, J = 7.6 Hz, 1H), 8.40–8.41 (d, J = 2.0 Hz, 1H), 8.93 (bs, 1H) ppm. ¹³C NMR (CDCl₃) 6 24.0, 111.2, 114.3, 117.1, 120.0, 124,8, 125.2, 126.4, 126.9, 127.5, 128.7, 129.8, 130.6, 131.0, 132.1, 132.3, 133.7, 134.3, 134.7, 139.0, 147.8, 150.4, 157.1, 163.5 ppm. IR (neat, cm⁻¹) 3308, 3061, 2230, 1681, 1603, 1578, 15312 cm⁻¹. Anal. (C₂₄H₁₇N₃O · HBr) C, H, N.

3-Cyano-N-(6-methylpyridin-2-yl)-4-(pyridine-3-yl)-benzamide Hydrochloride (59)

Prepared by following the general procedure E using pyridine-3-ylboronic acid (204 mg, 1.66 mmol) and compound 54 (0.50 g, 1.3 mmol). Yield 74%; HBr salt: white solid; mp 258 °C (dec). ¹H NMR (CDCl₃) 6 2.50–2.52 (s, 3H), 6.99–7.01 (m, 1H), 7.48–7.51 (m, 1H), 7.68–7.72 (m, 2H), 7.99–8.02 (m, 1H), 8.17–8.26 (m, 2H), 8.41–8.42 (m, 1H), 8.76–8.82 (m, 2H), ppm. ¹³C NMR (CDCl₃) 6 24.3, 111.3, 112.6, 117.5, 120.3, 123.8, 130.8, 131.7, 133.2, 133.3, 134.9, 136.3, 139.3, 145.2, 149.4, 150.4, 150.8, 157.4, ppm. GC–MS (EI) m/z 314 (M⁺). Anal. (C₁₉H₁₄N₄O · 2HCl · 0.75H₂O), C, H, N.

X-ray Crystal Structures of compounds 4, 10, and 57

Single-crystal X-ray diffraction data on compound 4 was collected using Cu KR radiation and a Bruker Platinum-135 CCD area detector. Single-crystal X-ray diffraction data on compounds 10 and 57 were collected using Mo KR radiation and a Bruker APEX 2 CCD area detector. The structures was solved by direct methods and refined by full-matrix least-squares on F² values using the programs found in the SHELXTL suite (Bruker, SHELXTL v6.10, 2000, Bruker AXS Inc., Madison, WI). Parameters refined included atomic coordinates and anisotropic thermal parameters for all non-hydrogen atoms. Hydrogen atoms on carbons were included using a riding model [coordinate shifts of C applied to H atoms] with C–H distance set at 0.96 A. Atomic coordinates for these compounds have been deposited with the Cambridge Crystallographic Data Centre (deposition numbers 719936, 719937, and 719938 for compounds 4, 10, and 57 respectively). Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK [fax: +44(0)-1223–336033 or e-mail: deposit@ccdc.cam.ac.uk].

4-(3,5-Difluorophenyl)-N-(6-methylpyrindin-2-yl)picolinamide Hydrobromide (4)

A 0.20 × 0.01 × 0.005 mm³ crystal of 4 was prepared for data collection coating with high viscosity microscope oil (Paratone-N, Hampton Research). The oil-coated crystal was placed on a MicroMesh mount *MiTeGen, Ithaca, NY) and transferred immediately to the diffractometer. The crystal was orthorhombic in space group P2₁2₁2 with unit cell dimensions a = 14.2137(5) A, b = 34.1135(15) A, and c = 3.8411(2) A. Corrections were applied for Lorentz, polarization, and absorption effects. Data were 98.3% complete to 67.59° θ (approximately 0.83 A) with an average redundancy of 4.46.

1-Fluoro-3-(pyridin-3-yl)-5-(2-methylthiazol-4ylethynyl)benzene (10)

A 0.32 × 0.15 × 0.04 mm³ crystal of 10 was prepared for data collection coating with high viscosity microscope oil (Paratone-N, Hampton Research). The oil-coated crystal was placed on a MicroMesh mount *MiTeGen, Ithaca, NY) and transferred immediately to the cold stream on the diffractometer. The crystal was monoclininc in space group P2₁/c with unit cell dimensions a = 22.259(3) A, b = 16.020(2) A, c = 10.4700(15) A, and fi = 99.141(5)°. Corrections were applied for Lorentz, polarization, and absorption effects. Data were 98.4% complete to 24.74° θ (ap- proximately 0.75 A) with an average redundancy of 2.59.

3-Cyano-4-(4′-fluorophenyl)-N-(6-methylpyridin-2-yl)-benzamide Hydrobromide (57)

A 0.198 × 0.056 × 0.019 mm³ crystal of 57 was prepared for data collection coating with high viscosity microscope oil (Paratone-N, Hampton Research). The oil-coated crystal was placed on a MicroMesh mount (*MiTeGen, Ithaca, NY) and transferred immediately to the cold stream on the diffractometer. The crystal was monoclinic in space group P2₁/a with unit cell dimensions a = 7.2307(16) A, b = 16.994(4) A, c = 14.271(3) A, and fi = 90.278(4)°. Corrections were applied for Lorentz, polarization and absorption effects. Data were 99.9% complete to 25.00° θ (approximately 0.75 A) with an average redundancy of 5.2 for all data from the twinned crystal. Only nonoverlapped reflections from the strong component were used in the final refinement; these data were 82.5% complete to 25.00° θ.

Calcium Fluorescence Assay

HEK 293A cells stably expressing mGluR5 were plated in black-walled, clear-bottomed, poly-d-lysine coated 384-well plates (BD Biosciences, San Jose, CA) in 20 µL of assay medium (DMEM containing 10% dialyzed FBS, 20 mM HEPES, and 1 mM sodium pyruvate) at a density of 20K cells/well. The cells were grown overnight at 37 °C in the presence of 6% CO₂. The next day, medium was removed and the cells incubated with 20 µL of 2 µM Fluo-4, AM (Invitrogen, Carlsbad, CA) prepared as a 2.3 mM stock in DMSO and mixed in a 1:1 ratio with 10% (w/v) pluronic acid F-127 and diluted in assay buffer (Hank’s balanced salt solution, 20 mM HEPES, and 2.5 mM Probenecid (Sigma-Aldrich, St. Louis, MO)) for 45 m at 37 °C. Dye was removed, 20 µL ofassay buffer was added, and the plate was incubated for 10 m at room temperature. Ca²⁺ flux was measured using the Functional Drug Screening System (FDSS6000, Hamamatsu, Japan). Compounds were serially diluted 1:3 into 10 point concentration response curves and were transferred to daughter plates using the Echo acoustic plate reformatter (Labcyte, Sunnyvale, CA). Compounds were diluted into assay buffer to a 2× stock using a Thermo Fisher Combi (Thermo Fisher, Waltham, MA), which was applied to cells at t = 3 s. Cells were incubated with the test compounds for 140 s and then stimulated for 60 s with an EC₈₀ concentration of glutamate. Data were collected at 1 Hz. Concentration response curves were generated using a four point logistical equation in GraphPad Prism (GraphPad Software, Inc., La Jolla, CA). Subsequent confirmations of concentration response curves were performed using independent serial dilutions of source compounds, and data from experiments generated on multiple days were averaged to generate SEM.