6-Phenylpyrimidin-4-ones as Positive Allosteric Modulators at the M₁ mAChR: The Determinants of Allosteric Activity

Abstract

Targeting allosteric sites of the M₁ muscarinic acetylcholine receptor (mAChR) is an enticing approach to overcome the lack of receptor subtype selectivity observed with orthosteric ligands. This is a promising strategy for obtaining novel therapeutics to treat cognitive deficits observed in Alzheimer’s disease and schizophrenia, while reducing the peripheral side effects such as seen in the current treatment regimes, which are non-subtype selective. We previously described compound 2, the first positive allosteric modulator (PAM) of the M₁ mAChR based on a 6-phenylpyrimidin-4-one scaffold, which has been further developed in this study. Herein, we present the synthesis, characterization, and pharmacological evaluation of a series of 6-phenylpyrimidin-4-ones with modifications to the 4-(1-methylpyrazol-4-yl)benzyl pendant. Selected compounds, BQCA, 1, 2, 9i, 13, 14b, 15c, and 15d, were further profiled in terms of their allosteric affinity, cooperativity with acetylcholine (ACh), and intrinsic efficacy. Additionally, 2 and 9i were tested in mouse primary cortical neurons, displaying various degrees of intrinsic agonism and potentiation of the acetylcholine response. Overall, the results suggest that the pendant moiety is important for allosteric binding affinity and the direct agonistic efficacy of the 6-phenylpyrimidin-4-one based M₁ mAChR PAMs.

Introduction

The M₁ muscarinic acetylcholine receptor (M₁ mAChR) is a promising target for the treatment of the cognitive symptoms observed in patients with Alzheimer’s disease and schizophrenia.^1–5 Clinical trials with the M₁/M₄-preferring agonist, xanomeline, reduced the cognitive decline, behavioral disturbances and psychosis seen in both Alzheimer’s disease and schizophrenia^6,7 suggesting that the M₁ and M₄ mAChRs are relevant candidate targets for the treatment of these diseases. Unfortunately, the clinical trials with xanomeline failed due to intolerable gastrointestinal and cardiovascular side effects, thought to occur via activation of peripherally expressed M₂ and M₃ mAChRs.⁸

The five mAChRs have a highly conserved orthosteric ligand binding site, which makes it challenging to design selective compounds for this family of receptors.^9,10 Fortunately, the mAChRs also possess at least one topographically distinct allosteric binding site located above the orthosteric site in a vestibule formed by the top of the transmembrane helices and extracellular loops.^11,12 These allosteric sites show greater diversity, making them interesting regions for selectively targeting the different mAChR subtypes. Allosteric modulators act in complex and diverse ways at G protein-coupled receptors; they can alter the binding and signaling properties of orthosteric ligands, potentiating these properties (positive allosteric modulator, “PAM”), attenuate these properties (negative allosteric modulator; “NAM”), or have no effect on the orthosteric ligand at equilibrium (neutral allosteric ligand; “NAL”). In addition, many allosteric modulators can also activate the receptor in their own right (“allosteric agonists”).^13–16 Allosteric modulators are attractive therapeutic agents compared to orthosteric ligands because they may cause fewer side effects by selectively targeting different receptor subtypes, and offer the benefit of adjusting the orthosteric ligand binding affinity and/or efficacy in a more physiologically tailored and saturable manner; therefore, lower doses of allosteric modulators may achieve similar results to those observed with orthosteric site ligands.^13–16

With the development of the M₁ mAChR PAM, BQCA,¹⁸ came the proof that the mAChRs can be selectively targeted in this manner. Although BQCA was successfully used in initial studies, its progression into clinical development was hindered due to low receptor affinity and solubility as well as poor absorption, distribution, metabolism, and excretion (ADME) properties.¹⁹ BQCA is also likely to be exported from cellular locations, since its index of brain penetrability (K_p) and unbound fraction of the compound in the brain (K_puu) were 0.12 and 0.38, respectively, in C57Bl/6N mice (unpublished data). K_puu values < 1 indicate active efflux of BQCA at the blood-brain barrier level.²⁰ These, in turn, may be associated with the potential adverse effects (e.g., salivation, watery stools) observed with M₁ mAChR PAMs.¹⁴ Therefore, it is desirable to synthesize alternative M₁ PAMs with improved allosteric binding affinity (pK_B), cooperativity (αβ), and druglike properties for use in preclinical and clinical trials.

Several computational and structure−activity relationship (SAR) studies from our lab describe the efforts to create novel M₁ selective PAMs with the desired properties of high cooperativity with ACh and low allosteric agonism.^11,17,21,22 The 4-phenylpyridin-2-one derivatives (Figure 1) yielded several compounds with high binding (α > 100) and functional (αβ > 100) cooperativity values, and evolution of the 4-phenylpyridin-2-one scaffold into a 6-phenylpyrimidin-4-one scaffold (Figure 2) further improved the cooperativity parameters.¹⁷ However, many of the compounds also had the undesirable property of allosteric agonism, which can cause adverse side effects. This study aims to develop new 6-phenylpyrimidin-4-one-based M₁ PAMs with reduced intrinsic allosteric agonist properties, while maintaining or improving the cooperativity of the parent compound, MIPS1780 (compound 2), at the M₁ mAChR. We focus specifically on replacing the 4-(1-methylpyrazol-4-yl)benzyl pendant of 2 with a range of functionalities, selected on the basis of our previous studies with the 4-phenylpyridin-2-one analogues¹⁷ as well as other beneficial chemical modifications portrayed in other M₁ mAChR PAMs.^21,23–33 The results of this study will inform further M₁ PAM optimization studies and increase our understanding of the role of the pendant group of the 6-phenylpyrimidin-4-one scaffold.

Figure 1. Evolution from the prototype M₁ mAChR-selective allosteric modulator BQCA, to the novel 4-phenylpyridin-2-one derivatives.¹⁷.

Figure 2.

Structural evolvement from the 4-phenylpyridin-2-one to a 6-phenylpyrimidin-4-one scaffold represented as the N-methylpyrazole derivative. In this study, alterations to the 4-(1-methylpyrazol-4-yl)benzyl pendant (boxed in blue) were made in an effort to systematically determine the structure−activity relationship between the bottom pendant and the pharmacological effects at the M₁ mAChR.

Results and Discussion

Chemistry

The design strategy and reaction procedure for the synthesis of 2 from commercially available 6-chloropyrimidin-4(3H)-one (3) has previously been reported by our group.¹⁷ Due to low yields using this procedure, we optimized the reaction conditions and workup procedures to significantly improve the yields of the key steps. Furthermore, we also developed a one-step shorter reaction pathway, using 6-bromopyrimidin-4(3H)-one (7) as the starting material (Scheme 1). First, the 6-chloropyrimidin-4(3H)-one (3) was coupled with (2-methoxyphenyl)boronic acid in a Suzuki reaction with a catalytic amount of PdCl₂(PPh₃)₂ in a degassed solution of 1 M Na₂CO_3(aq)/THF (1:3). The reaction was stirred at 100 °C for 4 days, before it was adsorbed on Celite (without any prior workup) and purified by flash column chromatography. The extended reaction time and circumvention of the extractive workup improved the yield of intermediate 4 from 20% to 74%. The next step was an epoxide ring opening reaction with 1,2-cyclohexene oxide, which afforded a racemic mixture of the trans-isomers of lactam 5 in 69% yield. The LC-MS also indicated the formation of the corresponding O-alkylated byproduct as a minor product (~3:1 ratio); however, due to interfering impurities, the byproduct could not be isolated. The improved yield of lactam 5 (from 38% to 69%) can be explained by the higher purity of intermediate 4 compared to the previous procedure. Lastly, the 2-methoxyphenyl moiety of compound 5 was converted to the corresponding phenol using boron tribromide to give key intermediate 6 in good yield (83%). We previously reported that 6-chloropyrimidin-4(3H)-one (3) did not undergo an epoxide ring opening reaction with 1,2-cyclohexene oxide, therefore, it was essential to change the order of the reaction steps and perform the Suzuki reaction with (2-methoxyphenyl)boronic acid first.¹⁷ Alternatively, 6-bromopyrimidin-4(3H)-one (7) is able to undergo an epoxide ring opening reaction to form the desired pyrimidinone 8a and the O-alkylated byproduct 8b in 32% and 17%, respectively. Both products were isolated as the trans-isomer in a racemic mixture. A Suzuki reaction of 8a and 2-hydroxyphenylboronic under the previously mentioned conditions gave the key intermediate 6 in 61% yield. The new procedure significantly reduced the reaction times and resulted in more consistent reaction yields.

Scheme 1. Synthesis of Key Intermediate 6 and Final Analogues 9a−q^a.

^aReagents and conditions: (a) (2-methoxyphenyl)boronic acid, cat. PdCl₂(PPh₃)₂, 1 M Na₂CO_3(aq)/THF degassed, 100 °C, 74%; (b) 1,2-cyclohexene oxide, K₂CO₃, 120 °C, 69%; (c) 1 M BBr₃ in hexane, DCM, 0 °C to rt, 83%; (d) aryl methyl halide, K₂CO₃, KI, DMF, rt, 19−99%; (e) 1,2-cyclohexene oxide, K₂CO₃, 120 °C, 8a 32% and 8b 17%; (f) 2-hydroxyphenylboronic acid, cat. PdCl₂(PPh₃)₂, 1 M Na₂CO_3(aq)/THF degassed, 100 °C, 61%.

O-Alkylation of key intermediate 6 with a variety of benzyl bromides and benzyl chlorides was achieved using standard alkylation conditions with Finkelstein-type modification affording 9a−q in yields ranging between 19 and 99% (Scheme 1). Compounds 9a−q were tested for their allosteric properties at the M₁ mAChR. In addition, molecules 9a−d were also used for the synthesis of further analogues. It is worth pointing out that neither the reduction of the nitro analogue 9m using a range of methods (H₂ Pd/C, H₂ PtO, Zn, Fe, Na₂S₂O₄, SnCl₂) nor the acid-mediated Boc-deprotection of 9n gave the corresponding aniline. These reactions either did not progress or immediately formed the debenzylated intermediate 6 indicating that the aniline analogue is not stable.

Aryl bromide 9a was reacted with 1-Boc-pyrazole-4-boronic acid pinacol ester to furnish the pyrazole 10 via a Suzuki reaction using a previously reported protocol (Scheme 2).¹⁷ During the Suzuki reaction, the Boc group was conveniently removed, avoiding the need for an acid-mediated cleavage step. N-Alkylation of 10 with iodomethane offered an alternative route to afford the lead compound 2 in 48% yield. The same methodology was applied by using the appropriate alkyl bromides to furnish analogues 11a−d in good yields (59−80%).

Scheme 2. Synthesis of Analogues 2, 10, and 11a−d.

^aReagents and conditions: (a) 1-Boc-pyrazole-4-boronic acid pinacol ester, cat. PdCl₂(PPh₃)₂, 1 M Na₂CO_3(aq)/THF degassed, 100 °C, 32%; (b) alkyl halide, K₂CO₃, KI, DMF, rt to 40 °C, 48−80%.

Suzuki coupling reaction conditions were also used to react the aryl halides 9a−c with 1-methylpyrazole-4-boronic acid pinacol ester to afford 2 and the novel analogues 12a,b (Scheme 3). Appreciable amounts of dehalogenated side product 13 were formed during a scale up reaction of lead compound 2, allowing the isolation of 13 for pharmacological evaluation.

Scheme 3. Synthesis of Analogues 2, 12a,b, and 13^a.

^aReagents and conditions: (a) 1-methylpyrazole-4-boronic acid pinacol ester, cat. PdCl₂ (PPh₃)₂, 1 M Na₂CO_3(aq)/THF degassed, 100 °C, 12−66% (byproduct 13 was isolated in 16% when synthesizing 2).

The ester 9d was converted to the carboxylic acid 14a and carboxamide 14b, by basic hydrolysis with NaOH and direct aminolysis with aqueous ammonia (30%), respectively (Scheme 4). Finally, amides 15a−d were synthesized through BOP-mediated coupling of the carboxylic acid 14a and the respective amines in the presence of DIPEA and DMF in good yields (63−83%).

Scheme 4. Synthesis of Analogues 2, 14a,b, and 15a−e^a.

^aReagents and conditions: (a) NaOH, EtOH, water, 50 °C, 35% for 11a; (b) NH₄OH 30%, MeOH, 50 °C; (c) respective amine, BOP, DIPEA, DMF, 63−83%.

Crystallography

A small molecule X-ray structure was used to confirm the accurate NMR assignment of intermediates 5, 8a and 8b, as both the O- and N-alkylated isomers are formed during the epoxide ring opening reaction with 1,2-cyclohexane. Carboxamide 14b had the most favorable physical properties for this study, and crystals were grown successfully by evaporation of a solution of the compound in methanol. The small molecule X-ray structure of analogue 14b (Figure 3) unmistakably depicts a covalent bond between the atoms N1 and C2 consistent with the pyrimidinone scaffold. It further confirmed that the epoxide ring-opening reaction with 1,2-cyclohexene oxide afforded the corresponding trans-isomers.

Figure 3. Thermal ellipsoid plot for 14b; ellipsoids are at the 50% probability level.

Pharmacology

All compounds were tested as the racemic mixture and initially screened for allosteric potentiation of the endogenous agonist, ACh, using an IP₁ accumulation assay. Table 1 shows the changes in baseline activity (Δbaseline) and pEC₅₀ shift (ΔpEC₅₀) that were calculated for each modulator at 1 and 10 μM as initial surrogates for the degree of allosteric agonism (τ) and functional cooperativity (αβ) exerted by each concentration of modulator. Figure 4 shows the correlation between Δbaseline and ΔpEC₅₀ mediated by 1 μM or 10 μM of the tested compounds. When tested at 1 μM, the allosteric modulators showed a moderate and significant correlation (r = 0.6715; P < 0.0001) between allosteric agonism and allosteric potentiation of the ACh response. When tested at 10 μM, the correlation between allosteric agonism and potentiation was weaker (r = 0.3455; P = 0.0421) but still significant, suggesting that as the ability of an allosteric modulator for potentiating the ACh response improves, a concurrent increase in its intrinsic allosteric agonism is also observed.

Table 1. Allosteric Agonism and Allosteric Potentiation of 6-Phenylpyrimidin-4-one Analogues at the M₁ mAChR^a.

Compound	Y	X	R	ΔpEC50 (1 μM)	Δbaseline (1 μM)	ΔpEC50 (10 μM)	Δbaseline (10 μM)
BQCA				0.81±0.09	-9.3±2.7	1.17±0.15	50.0±2.8
1				0.88±0.09	15.8±2.2*	1.31±0.15	58.3±2.3
2	H	H	1-methylpyrazol-4-yl	1.52±0.14	55.1±2.4*	1.28±0.42	85.3±2.4*
9a	H	CH	Br	0.87±0.15	2.0±4.0	1.08±0.44	73.4±4.1*
9b	F	CH	Br	0.37±0.09	-7.4±2.5	1.33±0.12	36.0±2.8
9c	H	N	Cl	0.56±0.08	-6.4±2.3	1.04±0.12	41.1±2.4
9d	H	CH	COOMe	0.71±0.09	-12.5±2.5	1.38±0.13	40.8±2.8
9e	H	CH	oxazol-2-yl	0.96±0.13	5.5±3.4*	1.61±0.23	58.8±3.8
9f	H	CH	thiazol-2-yl	1.01±0.12	5.3±3.6*	1.62±0.35	75.0±3.8*
9g	H	CH	OCH3	0.78±0.14	-4.8±3.9	1.47±0.22	49.6±4.2
9h	H	CH	CH3	0.84±0.14	-6.4±3.9	1.32±0.20	45.5±4.2
9i	H	N	CH3	0.41±0.10	-13.4±2.9	1.35±0.16	42.6±3.2
9j	H	CH	SO2CH3	0.56±0.10	11.0±2.7*	1.27±0.17	52.4±2.9
9k	H	CH	SO2NH2	0.76±0.10	23.4±2.3*	1.52±0.18	64.2±2.5
9l	H	CH	SO2N(CH3)2	0.22±0.09	14.4±2.2*	0.65±0.09	20.9±2.2*
9m	H	CH	NO2	0.84±0.12	31.3±2.7*	1.52±0.22	67.4±2.8*
9n	H	CH	NHBoc	1.24±0.14	43.1±2.8*	1.57±0.30	77.8±2.9*
9o				0.88±0.10	46.3±1.8*	1.11±0.20	73.5±1.8*
9p				1.24±0.23	69.8±2.3*	0.95±1.04	90.6±2.3*
9q				1.21±0.10	58.1±1.5*	1.22±0.31	85.4±1.5*
10	H	CH	1H-pyrazo 1-4-yl	1.69±0.18*	54.5±3.1*	2.15±0.60	84.3±3.4*
11a	H	CH	1-ethylpyrazol-4-yl	1.81±0.13*	43.5±2.6*	2.60±0.39	82.0±3.0*
11b	H	CH	1-propylpyrazol-4-yl	1.49±0.16	52.6±2.7*	2.02±0.30	72.7±3.0*
11c	H	CH	1-isopropylpyrazol-4-yl	1.56±0.09	34.5±2.2*	1.71±0.33	81.0±2.7*
11d	H	CH	1-butylpyrazol-4-yl	1.49±0.10	24.7±2.4*	2.01±0.23	68.5±2.7*
12a	F	CH	1-methylpyrazol-4-yl	1.38±0.19	46.8±3.7*	0.69±0.62	82.9±3.4*
12b	H	N	1-methylpyrazol-4-yl	1.51±0.15	36.0±3.3*	1.65±0.43	78.7±3.4*
13	H	CH	H	0.75±0.06	-0.3±1.7	1.83±0.11	54.6±1.9
14a	H	CH	(C=O)OH	0.37±0.10	-6.7±2.8	1.10±0.14	35.1±3.0*
14b	H	CH	(C=O)NH2	1.71±0.15*	37.9±3.3*	2.77±0.49	80.5±4.1*
15a	H	CH	(C=O)N(CH3)2	0.32±0.09	21.5±2.3*	0.98±0.11	38.0±2.4
15b	H	CH	(C=O)NH(CH3)	1.22±0.69	88.3±2.9*	ND	101.7±4.0*
15c	H	CH	(C=O)N(CH2CH3)2	0.18±0.10	9.2±3.0*	0.74±0.13	27.9±3.1*
15d	H	CH	(C=O)NH(CH2CH3)	1.62±0.46	87.1±2.8*	ND	99.8±2.7*
15e	H	CH		0.08±0.12	11.1±3.0*	0.86±0.13	26.9±3.2*

^aData represent the mean ± SEM of 3 independent experiments performed in duplicate. Data were analyzed by one-way ANOVA and compared to the control PAM, BQCA with a posthoc Bonferroni test, where

^*p < 0.05 was considered to be significantly different. ND: not determined.

Figure 4.

Correlation between allosteric agonist activity (Δbaseline) of the 6-phenylpyrimidin-4-ones and the potentiation (ΔpEC₅₀) of ACh-mediated IP₁ accumulation in CHO-M₁ cells. The intrinsic activity of the 6-phenylpyrimidin-4-one allosteric modulators were plotted against their degree of potentiation of ACh-mediated IP₁ accumulation.

The results of the initial screening are summarized in Table 1, and show that all the analogues at 10 μM exhibit both allosteric agonist activity at the M₁ mAChR and positive allosteric modulation of the endogenous agonist, ACh. Plotting the data from Table 1 into two-point profiles, to compare the effects of 1 and 10 μM concentrations of the PAMs simultaneously, revealed that several compounds showed similar cooperativity and agonistic profiles. These similar profiles could be grouped into four main clusters (Figure 5). Figure 5A shows that the profiles for compounds 1, 9a−j, 13, and 14a were most similar to that of BQCA (cluster 1). The profile of compound 2 was different from that of BQCA, displaying stronger (but not significantly different) potentiation of ACh response and higher agonist activity, most similar to compounds 9k, 9m−q, 10, 11a−d, 12a,b, and 14b, as shown in Figure 5B (cluster 2). Several compounds (9l, 15a, 15c, and 15e), shown in Figure 5C, displayed less cooperativity with ACh when compared to the reference compound, BQCA (cluster 3), and compounds 15b and 15d were extremely efficacious PAM-agonists, as shown in Figure 5D (cluster 4).

Figure 5.

Two-point plots of the change in baseline versus the change in pEC₅₀ for the allosteric modulators. The endogenous agonist, ACh, at a range of concentrations (100 pM to 100 μM), was co-added with vehicle or allosteric modulator (either 1 or 10 μM) in CHO-M₁ cells for 60 min at 37 °C, and IP₁ accumulation was measured. Data were clustered into four main groups that were similar to BQCA (cluster 1) (A), greater than BQCA (cluster 2) (B), lower that BQCA (cluster 3) (C), and much greater than BQCA (cluster 4) (D). Data are mean ± SEM of three independent experiments with repeats in duplicate.

As shown in Figure 5A and Table 1, the BQCA profile was similar to those of compounds 1, 9a−j, 13, and 14a. Interestingly, there was no significant difference in the potentiation (ΔpEC₅₀) of ACh by BQCA when compared to the potentiation of ACh by compounds 1, 9c−j at 1 or 10 μM (one-way ANOVA, Bonferrroni posthoc test). However, the bromo (9a), thiazole (9f), and carboxylic acid (14a) derivatives had significantly greater allosteric agonism than BQCA (one-way ANOVA, Bonferroni posthoc test).

Figure 5B shows that the degree of allosteric agonism observed between BQCA and compound 2 was significantly different (one-way ANOVA, Bonferroni posthoc test) when tested at equipotent concentrations. A second cluster of compounds were identified, with greater allosteric agonism than BQCA (at 1 μM and 10 μM), which behaved most like compound 2. Of these compounds, significantly lower Δbaseline values were observed for 9k, 9m, 11c, 11d, 12b, and 14b when tested at 1 μM; however, these differences were lost when the compounds were tested at 10 μM. No significant differences were observed between 2 and 9k, 9m−q, 10, 11a–d, 12a,b, or 14b with respect to potentiation of ACh. In general, changes to the pyrazole moiety, such as in the nor-methyl analogue 10 or the introduction of prolonged or branched alkyl substituents (analogues 11a–d) improved potentiation of ACh while maintaining similar agonism to the parent compound 2. Pyrazole analogues 10 and 11a as well as carboxamide 14b exhibited the highest intrinsic efficacy (Δbaseline) and potentiation of the ACh response (ΔpEC₅₀), therefore could be considered to be good PAM-agonists. Analogues 9o, 9p, and 9q, all containing a bicyclic pendant, exhibited strong allosteric agonism at both 1 μM and 10 μM. Compound 9p with the 1-methyl-1H-indazol-5-yl pendant, showed a significant increase in allosteric agonism in comparison to 9o, with the 1-methyl-1H-indazol-6-yl pendant (one-way ANOVA, Bonferroni posthoc test, 1 μM P = 0.0003; 10 μM P = 0.001), suggesting that the methyl group interacts differently with proximal residues of the M₁ mAChR in this region. On the other hand, the 5-isoindolin-1-one analogue, 9q, did not significantly modify the behavior observed with 9p when tested at 10 μM (one-way ANOVA, Bonferroni posthoc test, 10 μM P = 0.105).

Figure 5C shows that several compounds (9l, 15a, 15c, and 15e) had low intrinsic efficacy while maintaining a moderate potentiation of the ACh response. These compounds all contain a dialkylated amine functionality, suggesting that this group is favorable for potentiating the ACh response, while minimizing their intrinsic efficacy. Interestingly, the N-alkylated benzamides 15b and 15d exhibited significantly greater agonism compared to the N,N-dialkylated benzamides 15a and 15c (Table 1). These results suggest that the existence of a hydrogen bond donor at the para position of the benzyl moiety might play a crucial role in allosteric intrinsic efficacy at the M₁ mAChR. This is further supported by the low agonism of the piperidine analogue 15e and the strong agonism of the carboxamide analogue 10. A similar tendency was observed with the N-methylsulfonamide 9k and the N,N-dimethylsulfonamide 9l, where the latter exhibited significantly lower allosteric agonism and cooperativity in comparison to 9k. However, this trend does not apply to the ester 9d and the carboxylic acid 14a, which both exhibited a comparable Δbaseline of 40.8 and 35.1, respectively at 10 μM. Interestingly, this drastic drop in intrinsic efficacy from the carboxamide to the carboxylic acid group observed for the 6-phenylpyrimidin-4-one series, was also observed for the 4-phenylpyridin-2-ones¹⁷ and the more recently published pyrrole-3-carboxamide-based³⁴ analogues.

BQCA, 1, 2, 9i, 13, 14b, 15c, and 15d, were selected for further quantitative pharmacological analysis as they displayed the full range of allosteric modulator profiles observed in our initial screening assay (9i and 13 represent cluster 1; 14b represents cluster 2, 15c represents cluster 3, and 15d represents cluster 4). Complete interaction experiments with ACh in IP₁ accumulation and radioligand equilibrium binding assays were performed, and the resulting parameters from these experiments are shown in Table 2.

Table 2. Binding and Functional Parameters for Selected Allosteric Modulators at the M₁ mAChR^a.

compd	pKBb	log αc	log αβd	log τBe
BQCA	5.78 ± 0.04	1.26 ± 0.13	1.33 ± 0.17	0.52 ± 0.04
1	5.74 ± 0.04	0.97 ± 0.14	0.98 ± 0.33	0.39 ± 0.08
2	6.49 ± 0.06*	1.07 ± 0.19	0.79 ± 0.38	0.45 ± 0.08
9i	5.26 ± 0.05*	1.30 ± 0.16	1.24 ± 0.18	0.37 ± 0.05
13	5.53 ± 0.04*	1.14 ± 0.13	1.30 ± 0.14	0.34 ± 0.05
14b	6.72 ± 0.03*	1.11 ± 0.09	1.14 ± 0.26	0.61 ± 0.06
15c	4.79 ± 0.03*	0.81 ± 0.12	0.85 ± 0.16	−0.09 ± 0.06*
15d	6.57 ± 0.04*	1.27 ± 0.13	1.05 ± 0.20	0.79 ± 0.04*

^aAll allosteric modulators showed high negative cooperativity with ³H-NMS, therefore for the purposes of model fitting, the logα parameter was constrainted to −3 (α = 0.001). Data were analyzed by one-way ANOVA and compared to the control PAM, BQCA, with a posthoc Bonferroni test, where *p < 0.05 was considered to be significantly different.

^bNegative logarithm of the binding affinity estimate of the allosteric modulator.

^cLogarithm of the binding cooperativity between ACh and allosteric modulator.

^dLogarithm of the functional cooperativity estimate between ACh and allosteric modulator.

^eLogarithm of the operational efficacy parameter of the allosteric modulator derived using eq 2. Where no intrinsic efficacy of the allosteric modulator was observed, the log τ_B values were constrained to −3 (τ_B = 0.001) to aid model convergence.

The 1-methylpyrazole analogue MIPS1780 (2) and the amides 14b and 15d exhibited similar allosteric binding affinities (pK_B range 6.49−6.72), which were significantly greater than the BQCA allosteric binding affinity (pK_B = 5.78 ± 0.04). Interestingly, both the carboxamide 14b and N-ethylamide 15d analogues showed high allosteric agonism. In contrast, the structurally related N,N-diethylamide analogue 15c had the weakest allosteric binding affinity and lower intrinsic efficacy and cooperativity with ACh when compared with 14b and 15d. This might be connected to the hydrogen donor missing in 15c compared to compounds 14b and 15d. Compounds 9i and 13 displayed a small but significant reduction in their allosteric binding affinities for the M₁ mAChR when compared to BQCA. When comparing compounds 1 and 2, analogues with the 1-methylprazol-4-yl pendant, it can be concluded that the additional nitrogen atom in the 6-pyrimidin-4-one scaffold of 2 improves the allosteric binding affinity compared to the 4-pyridin-2-one analogue 1, which might be attributed to an additional hydrogen bond interaction with the receptor. Equally, the functional group in the para-position of the benzyl pendant, seems to be important for the allosteric binding affinity of the M₁ mAChR PAMs. For instance, the more hydrophobic analogues 9i and 13, which lack of a hydrophilic group in para-position, showed weaker allosteric binding affinity compared to 2, 14b, and 15d that contain bulkier and more polar functionalities. Interestingly, there were no significant differences between the allosteric binding affinity or the functional cooperativity of the modulators tested, compared with BQCA.

Compound 9i was selected for further testing in mouse cortical neurons because it had lower intrinsic efficacy (log τ_B) relative to BQCA, although not statistically significantly different, in addition to favorable calculated clogP (Molinspiration Cheminformatices Software) compared to analogue 13. Figure 6 shows that BQCA, 1, 2, and 9i all potentiated the ACh response in the IP₁ accumulation assay, with the resulting parameter values shown in Table 3. Compound 9i however shows significantly less agonism compared to lead compound 2 in cortical neurons (one-way ANOVA, Bonferrroni posthoc test). Interestingly, while BQCA, 1, and 2 displayed higher functional cooperativity with ACh in cortical neurons compared with CHO-M₁ cells, the reverse was observed for 9i, although not statistically significant. Furthermore, the degrees of agonist activity were significantly lower in cortical neurons compared with CHO-M₁ cells for BQCA, 1, and 9i whereas they were similar for 2 (unpaired t test, Tables 2 and 3). This indicates the importance of studying allosteric properties of novel compounds in more physiologically relevant systems such as primary cortical neurons.

Figure 6.

IP₁ accumulation in cultured mouse primary cortical neurons. The prototypical M₁ mAChR PAM, BQCA, lead compounds 1 and 2, as well as 9i were tested in mouse cortical neuron cultures. Data are the mean + SEM of four independent experiments with repeats in duplicate. Parameter values for the four compounds are shown in Table 3.

Table 3. Parameter Values for Allosteric Modulation of ACh-Mediated IP₁ Accumulation in Mouse Cortical Neurons.

compd	pKBa	log αβb	(αβ)c	log τBd	(τB)e
BQCA	4.69 ± 0.40	1.73 ± 0.34	(54)	−0.18 ± 0.29	(0.67)
1	5.26 ± 0.11	1.26 ± 0.09	(18)	−0.15 ± 0.06	(0.71)
2	5.02 ± 0.18	2.12 ± 0.18	(132)	0.47 ± 0.13	(2.95)
9i	5.49 ± 0.18	0.98 ± 0.11	(9.5)	−0.52 ± 0.09	(0.30)

^aNegative logarithm of the functional binding affinity estimate for the allosteric modulator at the M₁ mAChR.

^bLogarithm of the functional cooperativity estimate between the orthosteric ligand and allosteric modulator.

^cAntilogarithm of the functional cooperativity parameter between the orthosteric ligand and the allosteric modulator

^dLogarithm of the operational efficacy estimate of the allosteric modulator.

^eAntilogarithm of the operational efficacy parameter of the allosteric modulator.

Additional pharmacokinetic and toxicology studies to determine K_p and K_puu values, elimination rate, as well as the half-life of 6-phenylpyrimidin-4-ones 2 and 9i will be important to decide on the suitability of these compounds to progress to further testing in animal models.

Conclusions

We have synthesized an extensive series of 6-phenylpyrimidin-4-ones with a range of modifications to the 4-(1-methylpyrazol-4-yl)benzyl pendant of our lead compound 2. All analogues at 10 μM had allosteric agonist activity and potentiated the actions of the endogenous agonist, ACh, in an IP₁ accumulation assay. This result demonstrates that changes to the pendant region of these analogues are tolerated without a detrimental effect on their allosteric activity. Analogs 9i, 13, 14b, 15c, and 15d, were further profiled by determining their binding affinities (pK_B), binding (log α) and functional cooperativity (log αβ) with ACh as well as intrinsic efficacy (log τ_B). The majority of modifications altered the allosteric binding affinity (pK_B) of the allosteric modulator to the allosteric site, without affecting the cooperativity (binding cooperativity, logα; or functional cooperativity, log αβ) between the allosteric and orthosteric ligands. With the exception of 15d and 15c, which exhibited more or less agonism, respectively, the PAMs also exhibited similar degrees of agonist activity. This study also demonstrated that the 6-phenylpyrimidin-4-ones 2 and 9i potentiate ACh-mediated IP₁ accumulation in a physiologically relevant system, the mouse cortical neurons. Overall, the pendant of the 6-phenylpyrimidin-4-ones is important for defining the allosteric binding affinity and direct allosteric agonism of the allosteric ligands for the M₁ mAChR.

Methods

Chemistry

Chemicals and solvents were purchased from standard suppliers and used without further purification. Davisil silica gel (40–63 μm), for flash column chromatography was supplied by Grace Davison Discovery Sciences (Victoria, Australia) and deuterated solvents were purchased from Cambridge Isotope Laboratories, Inc. (USA, distributed by Novachem PTY. Ltd., Victoria, Australia).

Reactions were monitored by thin layer chromatography on commercially available precoated aluminum-backed plates (Merck Kieselgel 60 F₂₅₄). Visualization was by examination under UV light (254 and 366 nm). Organic solvents were evaporated in vacuo at ≤40 °C (water bath temperature).

¹H NMR and ¹³C NMR spectra were recorded on a Bruker Avance Nanobay III 400 MHz Ultrashield Plus spectrometer at 400.13 and 100.62 MHz, respectively. Chemical shifts (δ) are recorded in parts per million (ppm) with reference to the chemical shift of the deuterated solvent. Coupling constants (J) and carbon−fluorine coupling constants (J_CF) are recorded in Hz and the significant multiplicities described by singlet (s), broad singlet (br s), doublet (d), triplet (t), quadruplet (q), broad (br), multiplet (m), doublet of doublets (dd), doublet of triplets (dt), and doublet of doublet of doublets (ddd). Spectra were assigned using appropriate COSY, distortionless enhanced polarization transfer (DEPT), HSQC and HMBC sequences. One tertiary carbon of the hydroxycyclohexane moiety has consistently not been observed in spectra with the solvents d₆-DMSO and CD₃OD.¹⁷

LCMS spectra were run to verify reaction outcome and purity using an Agilent 6120 Series Single Quad coupled to an Agilent 1260 Series HPLC. The following buffers were used; buffer A: 0.1% formic acid in H₂O; buffer B: 0.1% formic acid in MeCN. The following gradient was used with a Poroshell 120 EC-C18 50 × 3.0 mm 2.7 μm column, and a flow rate of 0.5 mL/min and total run time of 5 min; 0−1 min 95% buffer A and 5% buffer B, from 1 to 2.5 min up to 0% buffer A and 100% buffer B, held at this composition until 3.8 min, 3.8−4 min 95% buffer A and 5% buffer B, held until 5 min at this composition. Mass spectra were acquired in positive and negative ion mode with a scan range of 100−1000 m/z. UV detection was carried out at 214 and 254 nm. All retention times (t_R) are quoted in minutes. Preparative HPLC was performed using an Agilent 1260 infinity coupled with a binary preparative pump and Agilent 1260 FC-PS fraction collector, using Agilent OpenLAB CDS software (Rev C.01.04), and an Agilent 7 μM XDB-C8 21.2 × 250 mm column. The following buffers were used unless stated otherwise: buffer A was H₂O; buffer B was MeCN, with sample being run at a gradient of 5% or 30% buffer B to 100% buffer B over 10 min, at a flow rate of 20 mL/min. All compounds for screening were of >95% purity unless stated otherwise.

General Procedures A (N-Alkylation)

6-(2-((4-(1H-Pyrazol-4-yl)benzyl)oxy)phenyl)-3-(2-hydroxycyclohexyl)pyrimidin-4(3H)-one (8) (1.0 equiv), K₂CO₃ (1.1 equiv), KI (0.1 equiv), and the appropriate organohalide (1.1 equiv) were stirred in DMF (3 mL/100 mg) at rt for 3 days before the temperature was increased to 40 °C. Reaction progress was monitored through LC-MS analysis, with further additions of K₂CO₃ and organohalide until the reaction appeared complete or conversion remained stagnant. Then the reaction mixture was diluted with EtOAc and washed with water (2 × 20 mL) and brine (20 mL). The organic layer was dried with Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting crude product was purified by flash column chromatography (DCM 100% → DCM/MeOH 94:6).

General Procedures B (O-Alkylation)

3-(2-Hydroxycyclohexyl)-6-(2-hydroxyphenyl)pyrimidin-4(3H)-one (6) (1.0 equiv), K₂CO₃ (1.1 equiv), KI (0.1 equiv), and the appropriately substituted benzyl halide (1.1 equiv) were stirred in DMF (3 mL/100 mg) at rt until the reaction appeared complete (reaction progress was monitored via LC-MS analysis). The reaction mixture was diluted with EtOAc and washed with water (2 × 50 mL) and brine (50 mL). The organic layer was dried with Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting crude product was purified by flash column chromatography (PE/EtOAc 1:1 → EtOAc 100%).

General Procedure C (Suzuki Reaction with 1-Methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole)

A mixture of respective aryl halide (1.0 equiv) and 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1.5 equiv) in degassed THF/1 M Na₂CO_3(aq) (3 mL/100 mg) was flushed with nitrogen. PdCl₂(PPh₃)₂ (0.1 equiv) was added, and the reaction mixture was boiled under reflux for 6 h. The THF was evaporated under reduced pressure. The residue was dissolved in EtOAc and washed with water (2 × 50 mL) and brine (50 mL). Purification by flash column chromatography (DCM 100% → DCM/MeOH 9:1) and (ethyl acetate 100%) yielded the desired product.

General Procedure D (Coupling Reaction with BOP)

4-((2-(1-(2-Hydroxycyclohexyl)-6-oxo-1,6-dihydropyrimidin-4-yl)-phenoxy)methyl)benzoic acid (14a) (1.0 equiv), BOP (1.5 equiv), DIPEA (1.5 equiv), and respective amine (1.1 equiv) were stirred in DMF (3 mL/100 mg) at rt until the reaction appeared complete (reaction progress was monitored via LC-MS analysis), before EtOAc (150 mL) was added and the organic layer was washed with water (50 mL) and brine (50 mL). The organic layer was dried with Na₂SO₄ and filtered, and the solvent was removed under reduced pressure. The residue was purified by flash column chromatography (DCM 100% → DCM/MeOH 9:1) to give the desired product.

3-(2-Hydroxycyclohexyl)-6-(2-((4-(1-methyl-1H-pyrazol-4-yl)-benzyl)oxy)phenyl)pyrimidin-4(3H)-one (2)

General procedure A. The title compound was obtained as a white solid (20 mg, 48%). General procedure C. The title compound was obtained as a white solid (207 mg, 35%). ¹H NMR (CDCl₃) δ 8.21 (s, 1H), 7.96 (dd, J = 7.8, 1.7 Hz, 1H), 7.75 (s, 1H), 7.59 (s, 1H), 7.49−7.44 (m, 2H), 7.42−7.32 (m, 3H), 7.26 (s, 1H), 7.11−7.04 (m, 1H), 7.02 (d, J = 8.3 Hz, 1H), 5.18 (s, 2H), 4.59−4.43 (m, 1H), 3.93 (s, 3H), 3.92−3.82 (m, 1H), 2.98 (s, 1H), 2.25−2.16 (m, 1H), 2.08−1.97 (m, 1H), 1.91−1.75 (m, 3H), 1.56–1.35 (m, 3H); ¹³C NMR (CDCl₃) δ 162.9, 157.9, 156.9, 147.6, 136.8, 134.5, 132.3, 131.3, 130.7, 127.7, 127.0, 125.7, 125.6, 122.8, 121.1, 114.8, 113.2, 72.2, 70.4, 60.7, 39.1, 35.6, 30.9, 25.3, 24.4; resonance at δ 60.7 ppm was taken from the HSQC experiment; m/z MS (TOF ES⁺) 457.2 [M + H]⁺; LC-MS t_R: 3.21 min; HRMS C₂₇H₂₉N₄O₃ [M + H]⁺ calcd 457.2240; found 457.2246.

6-(2-Methoxyphenyl)pyrimidin-4(3H)-one (4)

A mixture of 6-chloropyrimidin-4(3H)-one (3) (2.00 g, 15.3 mmol, 1.0 equiv) and (2-methoxyphenyl)boronic acid (3.49 g, 23.0 mmol, 1.5 equiv) in degassed THF/1 M Na₂CO_3(aq) (3:1, 30 mL) was flushed with nitrogen. PdCl₂(PPh₃)₂ (1.08 g, 1.53 mmol, 0.1 equiv) was added and the reaction mixture was refluxed for 4 days. The THF was evaporated under reduced pressure, and the crude material was adsorbed on Celite. Purification by flash column chromatography (EtOAc 100%) yielded 2.32 g (74%) of the desired compound as a white solid. ¹H NMR (d₆-DMSO) δ 12.47 (br s, 1H), 8.24 (d, J = 1.0 Hz, 1H), 7.92 (dd, J = 7.8, 1.8 Hz, 1H), 7.45 (ddd, J = 8.4, 7.3, 1.8 Hz, 1H), 7.16 (dd, J = 8.4, 0.7 Hz, 1H),7.06 (td, J = 7.7, 1.0 Hz, 1H), 6.90 (d, J = 1.0 Hz, 1H), 3.87 (s, 3H); m/z MS (TOF ES⁺) 203.1 [M + H]⁺; LC-MS t_R: 3.25.

3-(2-Hydroxycyclohexyl)-6-(2-methoxyphenyl)pyrimidin-4(3H)-one (5)

A mixture of 6-(2-methoxyphenyl)pyrimidin-4(3H)-one (4) (1.80 g, 8.90 mmol), 1,2-cyclohexene oxide (9.00 mL, 49.0 mmol, 10 equiv), and K₂CO₃ (3.08 g, 22.2 mmol, 2.5 equiv) was heated at 120 °C for 5 h. The reaction mixture was cooled to rt and concentrated to dryness under reduced pressure. The remaining residue was take up in EtOAc (250 mL) and washed with water. The organic layer was dried with Na₂SO₄, filtered and the solvent was removed under reduced pressure. Purification by flash column chromatography (DCM 100% → DCM: MeOH 94:6) yielded the desired compound as a colorless oil (1.84 g, 69%). ¹H NMR (d₆-DMSO) δ 8.54 (s, 1H), 7.95 (dd, J = 7.8, 1.8 Hz, 1H), 7.45 (ddd, J = 8.4, 7.3, 1.8 Hz, 1H), 7.16 (dd, J = 8.4, 0.7 Hz, 1H), 7.07 (td, J = 7.7, 1.0 Hz, 1H), 6.96 (d, J = 0.4 Hz, 1H), 4.98 (d, J = 5.7 Hz, 1H), 4.48−4.16 (m, 1H), 3.97 (br s, 1H), 3.88 (s, 3H), 2.08−1.97 (m, 1H), 1.84−1.61 (m, 4H), 1.40−1.22 (m, 3H); m/z MS (TOF ES⁺) 301.2 [M + H]⁺; LC-MS t_R: 3.42.

3-(2-Hydroxycyclohexyl)-6-(2-hydroxyphenyl)pyrimidin-4(3H)-one (6)

Method A: Boron tribromide in hexane (1 M, 30.0 mL, 30.63 mmol) was added over 15 min at 0 °C to a solution of 3-(2-hydroxycyclohexyl)-6-(2-methoxyphenyl)pyrimidin-4(3H)-one (5) (1.84 g, 6.13 mmol) in dichloromethane (20 mL). The mixture was allowed to warm up to rt and was stirred for 7 h and then poured onto ice−water. The pH of the solution was adjusted to pH 6 by addition of sat. NaHCO₃. DCM (250 mL) was added and the layers were separated. The organic layer was washed with water (2 × 100 mL) and brine (100 mL) and then dried with Na₂SO₄ and filtered, and the solvent was evaporated under reduced pressure. The desired compound was obtained as a light yellow solid (4.46 g, 83%). Method B: A mixture of 6-bromo-3-(2-hydroxycyclohexyl)pyrimidin-4(3H)-one (8a) (70.0 mg, 256 μmol, 1.0 equiv) and 2-hydroxyphenylboronic acid (53.0 mg, 284 μmol, 1.5 equiv) in degassed THF/1 M Na₂CO_3(aq) (3:1, 8 mL) was flushed with nitrogen. PdCl₂(PPh₃)₂ (18.0 mg, 25.6 μmol, 0.1 equiv) was added and the reaction mixture boiled under reflux for 2 h. The THF was evaporated under reduced pressure and residue was taken up in EtOAc (50 mL). The organic layer was washed with water (2 × 50 mL) and brine (50 mL) before it was dried with Na₂SO₄, filtered and evaporated to dryness. The crude material was purified by flash column chromatography (EtOAc/PE 1:1 → EtOAc 100%) to give the desired compound as a yellow-white solid (45 mg, 61%). ¹H NMR (d₆-DMSO) δ 12.80−10.80 (br s, 1H), 8.61 (s, 1H), 7.84 (dd, J = 7.9, 1.6 Hz, 1H), 7.24 (ddd, J = 8.5, 7.2, 1.6 Hz, 1H), 6.99 (s, 1H), 6.87−6.77 (m, 2H), 4.78 (br s, 1H), 4.42−4.11 (m, 1H), 3.92−3.77 (br s, 1H), 1.96−1.88 (m, 1H), 1.79−1.53 (m, 4H), 1.31−1.16 (m, 3H); m/z MS (TOF ES⁺) 287.2 [M + H]⁺; LC-MS t_R: 3.46.

6-Bromo-3-(2-hydroxycyclohexyl)pyrimidin-4(3H)-one (8a) and 2-((6-bromopyrimidin-4-yl)oxy)cyclohexan-1-ol (8b)

A mixture of 6-bromopyrimidin-4(3H)-one (7) (500 mg, 2.86 mmol), 1,2-cyclohexene oxide (10.1 mL, 100 mmol, 35 equiv), and K₂CO₃ (987 mg, 22.2 mmol, 2.5 equiv) was heated at 120 °C for 24 h. The reaction mixture was cooled to rt and concentrated to dryness under reduced pressure. The remaining residue was take up in EtOAc (250 mL) and washed with water. The organic layer was dried with Na₂SO₄ and filtered, and the solvent was removed under reduced pressure. Purification by flash column chromatography (PE 100% → EtOAc 100%) yielded 8a as a light yellow solid (247 mg, 32%) and 8b as a colorless oil (130 mg, 17%).

6-Bromo-3-(2-hydroxycyclohexyl)pyrimidin-4(3H)-one (8a)

¹H NMR (d₆-DMSO) δ 8.46 (s, 1H), 6.75 (s, 1H), 5.01 (d, J = 5.4 Hz, 1H), 4.27 (br s, 1H), 3.88 (br s, 1H), 2.03−1.93 (m, 1H), 1.84−1.62 (m, 4H), 1.42−1.22 (m, 3H); m/z MS (TOF ES⁺) 274.9 [M + H]⁺; LC-MS t_R: 3.00.

2-((6-Bromopyrimidin-4-yl)oxy)cyclohexan-1-ol (8b)

¹H NMR (CDCl₃) δ 8.50 (d, J = 0.8 Hz, 1H), 6.98 (d, J = 0.8 Hz, 1H), 5.04−4.87 (m, 1H), 3.78−3.66 (m, 1H), 2.59 (s, 1H), 2.25−2.06 (m, 2H), 1.82−1.71 (m, 2H), 1.50−1.28 (m, 4H); m/z MS (TOF ES⁺) 274.9 [M + H]⁺; LC-MS t_R: 3.22.

6-(2-((4-Bromobenzyl)oxy)phenyl)-3-(2-hydroxycyclohexyl)-pyrimidin-4(3H)-one (9a)

General procedure B. The title compound was obtained as a colorless oil (942 mg, 99%). ¹H NMR (CDCl₃) δ 8.20 (s, 1H), 7.90 (dd, J = 7.8, 1.7 Hz, 1H), 7.52−7.47 (m, 2H), 7.37−7.31 (m, 1H), 7.30−7.24 (m, 2H), 7.15 (s, 1H), 7.08−7.02 (m, 1H), 6.94 (d, J = 8.3 Hz, 1H), 5.11 (s, 2H), 4.53−4.47 (br t, J = 9.3 Hz, 1H), 3.96−3.80 (m, 1H), 3.32 (br s, 1H), 2.22−2.11 (m, 1H), 2.04−1.94 (m, 1H), 1.90−1.68 (m, 3H), 1.52−1.33 (m, 3H); ¹³C NMR (CDCl₃) δ 162.8, 157.9, 156.6, 147.8, 135.6, 131.8, 131.3, 130.8, 128.7, 125.7, 121.9, 121.3, 114.7, 113.1, 71.9, 69.8, 60.8, 35.5, 31.1, 25.2, 24.4; resonance at δ 60.8 ppm was taken from the HSQC experiment; m/z MS (TOF ES⁺) 455.1 [M + H]⁺; LC-MS t_R: 3.34; HRMS C₂₃H₂₄BrN₂O₃ [M + H]⁺ calcd 455.0970; found 455.0975.

6-(2-((4-Bromo-2-fluorobenzyl)oxy) phenyl)-3-(2-hydroxycyclohexyl)pyrimidin-4(3H)-one (9b)

General procedure B. The title compound was obtained as a colorless oil (256 mg, 77%). ¹H NMR (CDCl₃) δ 8.19 (s, 1H), 7.86 (dd, J = 7.7, 1.6 Hz, 1H), 7.39−7.23 (m, 4H), 7.08 (s, 1H), 7.07−7.03 (m, 1H), 6.97 (d, J = 8.3 Hz, 1H), 5.13 (s, 2H), 4.56−4.41 (m, 1H), 3.93−3.79 (m, 1H), 3.59 (s, 1H), 2.21−2.10 (m, 1H), 2.03−1.93 (m, 1H), 1.88−1.66 (m, 3H), 1.51−1.31 (m, 3H); ¹³C NMR (CDCl₃) δ 162.7, 159.8 (d, J_CF = 251.2 Hz), 157.8, 156.3, 147.9, 131.4, 130.8, 130.4 (d, J_CF = 4.5 Hz), 127.9 (d, J_CF = 3.6 Hz), 125.8, 123.0 (d, J_CF = 14.2 Hz), 122.1 (d, J_CF = 9.5 Hz), 121.5, 119.0 (d, J_CF = 24.4 Hz), 114.7, 113.0, 71.6, 63.9 (d, J_CF = 4.1 Hz), 60.3, 35.5, 31.1, 25.2, 24.4, resonance at δ 60.9 ppm was taken from the HSQC experiment; m/z MS (TOF ES⁺) 473.2 [M + H]⁺; LC-MS t_R: 3.39; HRMS C₂₃H₂₃BrFN₂O₃ [M + H]⁺ calcd 473.0876; found 473.0874.

6-(2-((6-Chloropyridin-3-yl)methoxy)phenyl)-3-(2-hydroxycyclohexyl)pyrimidin-4(3H)-one (9c)

General procedure B. The title compound was obtained as a colorless oil (200 mg, 70%). ¹H NMR (CDCl₃) δ 8.31 (d, J = 2.2 Hz, 1H), 8.16 (s, 1H), 7.75 (dd, J = 7.7, 1.7 Hz, 1H), 7.62 (dd, J = 8.2, 2.4 Hz, 1H), 7.35−7.29 (m, 1H), 7.27 (d, J = 8.2 Hz, 1H), 7.5−6.98 (m, 1H), 6.95−6.90 (m, 2H), 5.05 (d, J = 12.5 Hz, 1H), 5.01 (d, J = 12.5 Hz, 1H), 4.54−4.35 (m, 1H), 4.09−4.03 (m, 1H), 3.89 (br s, 1H), 2.17−2.08 (m, 1H), 1.99−1.90 (m, 1H), 1.84−1.63 (m, 3H), 1.50−1.27 (m, 3H); ¹³C NMR (CDCl₃) δ 162.4, 157.9, 156.0, 151.0, 148.5, 148.1, 138.1, 131.3, 131.1, 130.7, 126.3, 124.4, 121.8, 114.7, 113.5, 71.2, 67.7, 61.0, 35.4, 31.1, 25.2, 24.4, resonance at δ 61.0 ppm was taken from the HSQC experiment; m/z MS (TOF ES⁺) 412.2 [M + H]⁺; LC-MS t_R: 3.18; HRMS C₂₂H₂₃ClN₃O₃ [M + H]⁺ calcd 412.1428; found 412.1431.

Methyl 4-((2-(1-(2-hydroxycyclohexyl)-6-oxo-1,6-dihydropyrimidin-4-yl)phenoxy)methyl)benzoate (9d)

General Procedure B. The title compound was obtained as a colorless oil (163 mg, 65%). ¹H NMR (CDCl₃) δ 8.22 (s, 1H), 8.07−8.02 (m, 2H), 7.93 (dd, J = 7.8, 1.8 Hz, 1H), 7.51−7.46 (m, 2H), 7.35 (ddd, J = 8.3, 7.4, 1.8 Hz, 1H), 7.20 (d, J = 0.7 Hz, 1H), 7.08 (td, J = 7.7, 1.0 Hz, 1H), 6.95 (dd, J = 8.3, 0.6 Hz, 1H), 5.25 (s, 2H), 4.63−4.45 (m, 1H), 3.93 (s, 3H), 3.91−3.84 (m, 1H), 2.80 (br s, 1H), 2.28−2.16 (m, 1H), 2.06−2.00 (m, 1H), 1.93−1.78 (m, 3H), 1.57−1.37 (m, 3H); ¹³C NMR (CDCl₃) δ 166.8, 162.9, 158.0, 156.6, 147.6, 141.8, 131.3, 130.8, 130.0, 129.7, 126.6, 125.8, 121.4, 114.8, 113.1, 72.3, 70.0, 60.8, 52.1, 35.6, 31.0, 25.3, 24.4; resonance at δ 60.8 ppm was taken from the HSQC experiment; m/z MS (TOF ES⁺) 435.2 [M + H]⁺; LC-MS t_R: 3.29; HRMS C₂₅H₂₇N₂O₅ [M + H]⁺ calcd 435.1920; found 435.1917.

3-(2-Hydroxycyclohexyl)-6-(2-((4-(oxazol-2-yl)benzyl)oxy)-phenyl)pyrimidin-4(3H)-one (9e)

General procedure B. The title compound was obtained as a colorless oil (69 mg, 59%). ¹H NMR (CDCl₃) δ 8.12 (s, 1H), 7.98−7.92 (m, 2H), 7.82 (dd, J = 7.8, 1.7 Hz, 1H), 7.61 (d, J = 0.6 Hz, 1H), 7.42−7.39 (m, 2H), 7.30−7.22 (m, 1H), 7.16−7.09 (m, 2H), 7.01−6.93 (m, 1H), 6.89 (d, J = 8.2 Hz, 1H), 5.13 (s, 2H), 4.51−4.36 (m, 1H), 3.90−3.75 (m, 1H), 3.18 (br s, 1H), 2.14−2.03 (m, 1H), 1.95−1.88 (m, 1H), 1.83−1.63 (m, 3H), 1.44−1.25 (m, 3H); ¹³C NMR (CDCl₃) δ 162.8, 161.7, 157.9, 156.6, 147.7, 139.0, 138.6, 131.3, 130.7, 128.4, 127.3, 127.0, 126.7, 125.9, 121.3, 114.8, 113.3, 71.9, 70.2, 61.0, 35.6, 31.0, 25.2, 24.4, resonance at δ 61.0 ppm was taken from the HSQC experiment; m/z MS (TOF ES⁺) 444.2 [M + H]⁺; LC-MS t_R: 3.23; HRMS C₂₆H₂₆N₃O₄ [M + H]⁺ calcd 444.1923; found 444.1923.

3-(2-Hydroxycyclohexyl)-6-(2-((4-(thiazol-2-yl)benzyl)oxy)-phenyl)pyrimidin-4(3H)-one (9f)

General procedure B. The title compound was obtained as a white solid (110 mg, 91%). ¹H NMR (CDCl₃) δ 8.22 (s, 1H), 7.99−7.96 (m, 2H), 7.94 (dd, J = 7.8, 1.7 Hz, 1H), 7.87 (d, J = 3.3 Hz, 1H), 7.5−7.45 (d, J = 8.3 Hz, 2H), 7.41−7.32 (m, 2H), 7.23 (s, 1H), 7.13−7.06 (m, 1H), 7.01 (d, J = 8.2 Hz, 1H), 5.25 (s, 2H), 4.63−4.48 (m, 1H), 3.97−3.83 (m, 1H), 2.75 (br s, 1H), 2.29−2.18 (m, 1H), 2.09−2.01 (m, 1H), 1.93−1.74 (m, 3H), 1.57−1.37 (m, 3H); ¹³C NMR (CDCl₃) δ 168.0, 162.9, 158.0, 156.7, 147.6, 143.7, 138.6, 133.2, 131.3, 130.8, 127.6, 126.9, 126.0, 121.4, 118.9, 114.8, 113.5, 72.4, 70.4, 60.7, 35.7, 31.0, 25.3, 24.4, resonance at δ 60.7 ppm was taken from the HSQC experiment; m/z MS (TOF ES⁺) 460.2 [M + H]⁺; LC-MS t_R: 3.30; HRMS C₂₆H₂₆N₃O₃S [M + H]⁺ calcd 460.1695; found 460.1695.

3-(2-Hydroxycyclohexyl)-6-(2-((4-methoxybenzyl)oxy)phenyl)-pyrimidin-4(3H)-one (9g)

General procedure B. The title compound was obtained as a colorless oil (105 mg, 74%). ¹H NMR (CDCl₃) δ 8.18 (s, 1H), 7.93 (dd, J = 7.7, 1.5 Hz, 1H), 7.38−7.27 (m, 3H), 7.21 (s, 1H), 7.07−6.98 (m, 2H), 6.94−6.87 (m, 2H), 5.11 (s, 2H), 4.57−4.40 (m, 1H), 3.91−3.84 (m, 1H), 3.80 (s, 3H), 3.34 (br s, 1H), 2.22−2.09 (m, 1H), 2.04−1.92 (m, 1H), 1.90−1.65 (m, 3H), 1.54−1.30 (m, 3H); ¹³C NMR (CDCl₃) δ 162.8, 159.4, 157.9, 157.0, 147.7, 131.3, 130.7, 128.7, 128.5, 125.6, 121.0, 114.7, 114.1, 113.3, 71.8, 70.4, 60.8, 55.3, 35.5, 31.0, 25.2, 24.4, resonance at δ 60.8 ppm was taken from the HSQC experiment; m/z MS (TOF ES⁺) 407.2 [M + H]⁺; LC-MS t_R: 3.29; HRMS C₂₄H₂₇N₂O₄ [M + H]⁺ calcd 407.1971; found 407.1949.

3-(2-Hydroxycyclohexyl)-6-(2-((4-methylbenzyl)oxy)phenyl)-pyrimidin-4(3H)-one (9h)

General procedure B. The title compound was obtained as a colorless oil (51 mg, 50%). ¹H NMR (CDCl₃) δ 8.10 (s, 1H), 7.84 (dd, J = 7.8, 1.7 Hz, 1H), 7.27−7.17 (m, 3H), 7.13 (s, 1H), 7.11−7.06 (m, 2H), 6.97−6.92 (m, 1H), 6.90 (d, J = 8.3 Hz, 1H), 5.05 (s, 2H), 4.46−4.32 (m, 1H), 3.84−3.69 (m, 1H), 3.18 (br s, 1H), 2.25 (s, 3H), 2.11−1.98 (m, 1H), 1.94−1.83 (m, 1H), 1.79−1.61 (m, 3H), 1.45−1.22 (m, 3H); ¹³C NMR (CDCl₃) δ 162.9, 157.9, 157.0, 147.7, 137.7, 133.5, 131.3, 130.7, 129.4, 127.1, 125.5, 121.0, 114.7, 113.2, 71.9, 70.5, 60.8, 35.5, 31.0, 25.2, 24.4, 21.2, resonance at δ 60.8 ppm was taken from the HSQC experiment; m/z MS (TOF ES⁺) 391.2 [M + H]⁺; LC-MS t_R: 3.34; HRMS C₂₄H₂₇N₂O₃ [M + H]⁺ calcd 391.2022; found 391.2020.

3-(2-Hydroxycyclohexyl)-6-(2-((6-methylpyridin-3-yl)methoxy)-phenyl)pyrimidin-4(3H)-one (9i)

General procedure B. An excess of K₂CO₃ (3.0 equiv) was used since the 5-(bromomethyl)-2-methylpyridine reagent was added as the hydrobromide salt. The compound was further purified by an additional column chromatography (EtOAc 100% → EtOAc/MeOH 9:1). The title compound was obtained as a colorless oil (21 mg, 20%). ¹H NMR (CDCl₃) δ 8.39 (d, J = 1.9 Hz, 1H), 8.15 (s, 1H), 7.73 (dd, J = 7.7, 1.7 Hz, 1H), 7.47−7.35 (m, 2H), 7.13−7.04 (m, 3H), 6.87 (s, 1H), 5.12 (d, J = 12.1 Hz, 1H), 5.03 (d, J = 12.1 Hz, 1H), 4.54−4.36 (m, 1H), 4.04−3.90 (m, 1H), 2.53 (s, 3H), 2.26−2.18 (m, 1H), 2.05−1.96 (m, 1H), 1.92−1.77 (m, 3H), 1.55−1.38 (m, 3H); ¹³C NMR (CDCl₃) δ 162.4, 158.4, 158.0, 156.2, 148.5, 148.0, 136.1, 131.3, 130.3, 128.9, 127.8, 123.1, 122.2, 115.6, 115.1, 71.4, 70.0, 61.8, 35.4, 30.7, 25.3, 24.4, 24.0; resonance at δ 61.8 ppm was taken from the HSQC experiment; m/z MS (TOF ES⁺) 392.2 [M + H]⁺; LC-MS t_R: 2.85; HRMS C₂₃H₂₆N₃O₃ [M + H]⁺ calcd 392.1974; found 392.1973.

3-(2-Hydroxycyclohexyl)-6-(2-((4-(methylsulfonyl)benzyl)oxy)-phenyl)pyrimidin-4(3H)-one (9j)

General procedure B. An additional purification by preparative HPLC (30−100%) was performed to afford the title compound as a colorless oil (30 mg, 38%). ¹H NMR (CDCl₃) δ 8.27 (s, 1H), 7.98−7.93 (m, 2H), 7.90 (dd, J = 7.8, 1.8 Hz, 1H), 7.63−7.57 (m, 2H), 7.37 (ddd, J = 8.3, 7.4, 1.8 Hz, 1H), 7.12−7.07 (m, 2H), 6.97−6.94 (m, 1H), 5.26 (s, 2H), 4.54 (br t, J = 9.5 Hz, 1H), 3.97−3.84 (m, 1H), 3.07 (s, 3H), 2.51 (br s, 1H), 2.27−2.19 (m, 1H), 2.09−2.01 (m, 1H), 1.93−1.73 (m, 3H), 1.55−1.37 (m, 3H); ¹³C NMR (CDCl₃) δ 162.6, 157.7, 156.3, 147.9, 142.9, 140.0, 131.5, 130.9, 127.9, 127.6, 125.7, 121.7, 114.7, 113.2, 72.1, 69.7, 60.8, 44.5, 35.6, 31.0, 25.2, 24.4; m/z MS (TOF ES⁺) 454.9 [M + H]⁺; LC-MS t_R: 3.18; HRMS C₂₄H₂₇N₂O₅S [M + H]⁺ calcd 455.1641; found 455.1640.

4-((2-(1-(2-Hydroxycyclohexyl)-6-oxo-1,6-dihydropyrimidin-4-yl)phenoxy)methyl) benzenesulfonamide (9k)

General procedure B. An additional purification by preparative HPLC (30−100%) was performed to afford the title compound as a colorless oil (25 mg, 31%). ¹H NMR (d₃-MeOD) δ 8.49 (s, 1H), 7.95−7.90 (m, 2H), 7.88 (dd, J = 7.8, 1.8 Hz, 1H), 7.64−7.57 (m, 2H), 7.43 (ddd, J = 8.4, 7.4, 1.8 Hz, 1H), 7.21−7.16 (m, 1H), 7.11 (dd, J = 7.5, 0.9 Hz, 1H), 7.08 (d, J = 0.7 Hz, 1H), 5.29 (s, 2H), 4.43 (br s, 1H), 4.11 (br s, 1H), 2.21−2.12 (m, 1H), 2.03−1.73 (m, 4H), 1.57−1.38 (m, 3H); ¹³C NMR (d₃-MeOD) δ 164.9, 160.7, 158.5, 145.1, 143.3, 133.3, 132.4, 129.4, 128.1, 127.7, 123.0, 116.0, 115.2, 71.5, 50.5, 37.0, 32.5, 27.0, 26.0; m/z MS (TOF ES⁺) 455.9 [M + H]⁺; LC-MS t_R: 3.12; HRMS C₂₃H₂₆N₃O₅S [M + H]⁺ calcd 456.1593; found 456.1588.

4-((2-(1-(2-Hydroxycyclohexyl)-6-oxo-1,6-dihydropyrimidin-4-yl)phenoxy)methyl)-N,N-dimethylbenzenesulfonamide (9l)

General procedure B. An additional preparative HPLC (30−100%) followed by recrystallization from methanol was performed to afford the title compound as a white solid (16 mg, 19%). ¹H NMR (CDCl₃) δ 8.35 (s, 1H), 7.93−7.86 (m, 1H), 7.79 (d, J = 7.9 Hz, 2H), 7.59 (d, J = 8.0 Hz, 2H), 7.44−7.36 (m, 1H), 7.16−7.07 (m, 2H), 6.99 (d, J = 8.2 Hz, 1H), 5.29 (s, 2H), 4.61−4.49 (m, 1H), 3.96−3.82 (m, 1H), 2.72 (s, 6H), 2.28−2.19 (m, 1H), 2.11−2.01 (m, 1H), 1.97−1.74 (m, 3H), 1.62−1.38 (m, 4H); ¹³C NMR (CDCl₃) δ 162.6, 157.8, 156.4, 148.0, 141.8, 134.9, 131.4, 130.9, 128.1, 127.2, 125.9, 121.5, 114.7, 113.1, 71.7, 37.9, 69.6, 35.5, 31.1, 25.2, 24.4; m/z MS (TOF ES⁺) 483.9 [M + H]⁺; LC-MS t_R: 3.29; HRMS C₂₅H₃₀N₃O₅S [M + H]⁺ calcd 484.1906; found 484.1902.

3-(2-Hydroxycyclohexyl)-6-(2-((4-nitrobenzyl)oxy)phenyl)-pyrimidin-4(3H)-one (9m)

General procedure B. The title compound was obtained as a white foam (299 mg, 63%). ¹H NMR (CDCl₃) δ 8.28 (s, 1H), 8.27−8.22 (m, 2H), 7.91 (dd, J = 7.8, 1.8 Hz, 1H), 7.61−7.55 (m, 2H), 7.38 (ddd, J = 8.3, 7.4, 1.8 Hz, 1H), 7.14−7.08 (m, 2H), 6.98−6.92 (m, 1H), 5.29 (s, 2H), 4.56 (br t, J = 9.5 Hz, 1H), 3.97−3.83 (m, 1H), 2.49 (br s, 1H), 2.30−2.18 (m, 1H), 2.06−1.99 (m, 1H), 1.95−1.72 (m, 3H), 1.58−1.38 (m, 3H); ¹³C NMR (CDCl₃) δ 162.7, 157.8, 156.2, 148.0, 147.6, 144.0, 131.4, 130.9, 127.4, 125.9, 124.0, 121.7, 114.7, 113.0, 71.9, 69.4, 60.8, 35.6, 31.1, 25.2, 24.4; resonance at δ 60.8 ppm was taken from the HSQC experiment; m/z MS (TOF ES⁺) 421.9 [M + H]⁺; LC-MS t_R: 3.36; HRMS C₂₃H₂₄N₃O₅ [M + H]⁺ calcd 422.1716; found 422.1721.

tert-Butyl (4-((2-(1-(2-hydroxycyclohexyl)-6-oxo-1,6-dihydropyrimidin-4-yl)phenoxy)methyl)phenyl)carbamate (9n)

General procedure B. The title compound was obtained as a colorless oil (191 mg, 37%). ¹H NMR (CDCl₃) δ 8.17 (s, 1H), 7.89 (dd, J = 7.8, 1.7 Hz, 1H), 7.35−7.26 (m, 5H), 7.16 (d, J = 0.5 Hz, 1H), 7.05−7.00 (m, 1H), 7.00−6.96 (m, 1H), 6.68 (s, 1H), 5.09 (s, 2H), 4.48 (t, J = 9.3 Hz, 1H), 3.93−3.80 (m, 1H), 2.97 (br s, 1H), 2.20−2.13 (m, 1H), 2.02−1.95 (m, 1H), 1.87−1.72 (m, 3H), 1.49 (s, 9H), 1.46−1.34 (m, 3H); ¹³C NMR (CDCl₃) δ 162.9, 158.0, 156.9, 152.8, 147.6, 138.2, 131.3, 130.9, 130.6, 128.1, 125.8, 121.1, 118.7, 114.7, 113.5, 80.5, 72.1, 70.5, 61.0, 28.3, 35.6, 31.0, 25.3, 24.4; m/z MS (TOF ES⁺) 491.9 [M + H]⁺; LC-MS t_R: 3.40; HRMS C₂₈H₃₄N₃O₅ [M + H]⁺ calcd 492.2498; found 364.0985.

3-(2-Hydroxycyclohexyl)-6-(2-((1-methyl-1H-indazol-6-yl)-methoxy)phenyl)pyrimidin-4(3H)-one (9o)

General procedure B. Additional purification steps by column chromatography (DCM 100% → DCM/MeOH 9:1) and preparative HPLC (5−100%) were performed to afford the title compound as a white solid (35 mg, 50%). ¹H NMR (CDCl₃) δ 8.10 (s, 1H), 7.86−7.81 (m, 2H), 7.59 (d, J = 8.3 Hz, 1H), 7.39 (s, 1H), 7.29−7.22 (m, 1H), 7.19−7.16 (m, 1H), 7.04−6.89 (m, 3H), 5.19 (s, 2H), 4.44−4.33 (m, 1H), 3.95 (s, 3H), 3.84−3.70 (m, 1H), 3.04 (br s, 1H), 2.09−1.99 (m, 1H), 1.94−1.85 (m, 1H), 1.77−1.54 (m, 3H), 1.42−1.16 (m, 3H); ¹³C NMR (CDCl₃) δ 162.7, 157.8, 156.8, 147.9, 140.1, 135.1, 132.5, 131.4, 130.8, 125.7, 123.5, 121.3, 121.2, 119.4, 114.8, 113.1, 107.1, 71.7, 70.7, 61.0, 35.6, 35.5, 31.0, 25.2, 24.4; m/z MS (TOF ES⁺) 430.9 [M + H]⁺; LC-MS t_R: 3.78; HRMS C₂₅H₂₇N₄O₃ [M + H]⁺ calcd 431.2083; found 431.2077.

3-(2-Hydroxycyclohexyl)-6-(2-((1-methyl-1H-indazol-5-yl)-methoxy)phenyl)pyrimidin-4(3H)-one (9p)

General procedure B was used without the addition of KI. The title compound was obtained as a white solid (12 mg, 20%). ¹H NMR (CDCl₃) δ 8.17 (s, 1H), 7.94 (d, J = 0.7 Hz, 1H), 7.92 (dd, J = 8.0, 1.8 Hz, 1H), 7.70 (s, 1H), 7.44 (dd, J = 8.7, 1.4 Hz, 1H), 7.40−7.36 (m, 1H), 7.36−7.29 (m, 1H), 7.22−7.18 (m, 1H), 7.06−6.99 (m, 2H), 5.23 (s, 2H), 4.52−4.41 (m, 1H), 4.03 (s, 3H), 3.90−3.76 (m, 1H), 3.22−3.06 (m, 1H), 2.20−2.08 (m, 1H), 2.00−1.92 (m, 1H), 1.88−1.67 (m, 3H), 1.50−1.30 (m, 3H); ¹³C NMR (CDCl₃) δ 162.9, 157.9, 157.0, 147.6, 139.7, 132.9, 131.3, 130.7, 128.7, 126.1, 125.6, 123.9, 121.1, 120.0, 114.7, 113.3, 109.5, 72.0, 71.0, 35.6 (2x), 31.0, 25.2, 24.4; m/z MS (TOF ES⁺) 430.9 [M + H]⁺; LC-MS t_R: 3.41; HRMS C₂₅H₂₇N₄O₃ [M + H]⁺ calcd 431.2083; found 431.2074.

3-(2-Hydroxycyclohexyl)-6-(2-((1-methyl-1H-indazol-5-yl)-methoxy)phenyl)pyrimidin-4(3H)-one (9q)

General procedure B. The title compound was obtained as a white solid (42 mg, 56%). ¹H NMR (CDCl₃) δ 8.26 (s, 1H), 7.93 (dd, J = 7.8, 1.8 Hz, 1H), 7.83 (d, J = 7.8 Hz, 1H), 7.54 (s, 1H), 7.44 (d, J = 8.0 Hz, 1H), 7.37 (ddd, J = 8.3, 7.4, 1.8 Hz, 1H), 7.20 (d, J = 3.9 Hz, 2H), 7.09 (td, J = 7.7, 0.9 Hz, 1H), 7.02−6.95 (m, 1H), 5.26 (s, 2H), 4.64−4.51 (m, 1H), 4.39 (s, 2H), 3.97−3.83 (m, 1H), 3.37 (s, 1H), 2.29−2.20 (m, 1H), 2.06−1.76 (m, 5H), 1.57−1.38 (m, 3H); ¹³C NMR (CDCl₃) δ 171.4, 162.9, 158.0, 156.6, 147.8, 144.4, 140.8, 131.8, 131.4, 130.8, 126.6, 125.9, 124.0, 121.7, 121.5, 114.8, 113.2, 72.2, 70.3, 45.6, 35.6, 31.1, 25.3, 24.5; m/z MS (TOF ES⁺) 431.9 [M + H]⁺; LC-MS t_R: 3.01; HRMS C₂₅H₂₆N₃O₄ [M + H]⁺ calcd 432.1923; found 432.1914.

6-(2-((4-(1H-Pyrazol-4-yl) benzyl)oxy)phenyl)-3-(2-hydroxycyclohexyl)pyrimidin-4(3H)-one (10)

A mixture of 6-(2-((4-bromobenzyl)oxy)phenyl)-3-(2-hydroxycyclohexyl)pyrimidin-4(3H)-one (7a) (879 mg, 1.93 mmol, 1.0 equiv) and tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1-carboxylate (852 mg, 2.90 mmol, 1.5 equiv) in degassed THF/1 M Na₂CO_3(aq) (3:1, 16 mL) was flushed with nitrogen. PdCl₂(PPh₃)₂ (135 mg, 193 μmol, 0.1 equiv) was added and the reaction mixture boiled under reflux for 2 days. The THF was evaporated under reduced pressure. The residue was dissolved in EtOAc and washed with water (2 × 50 mL) and brine (50 mL). Purification by flash column chromatography (DCM 100% → DCM/MeOH 9:1) and (ethyl acetate 100%) yielded the desired compound as a white solid (277 mg, 32%). ¹H NMR (d₆-DMSO) δ 12.96 (br s, 1H), 8.54 (s, 1H), 8.22 (s, 1H), 7.99 (dd, J = 7.8, 1.6 Hz, 1H), 7.95 (s, 1H), 7.68−7.60 (m, 2H), 7.50−7.39 (m, 3H), 7.26 (d, J = 8.3 Hz, 1H), 7.12−7.06 (m, 1H), 7.06 (s, 1H), 5.24 (s, 2H), 4.96 (d, J = 5.7 Hz, 1H), 4.47−4.14 (m, 1H), 3.95 (br s, 1H), 2.04−1.98 (m, 1H), 1.88−1.64 (m, 4H), 1.40−1.22 (m, 3H); ¹³C NMR (d₆-DMSO) δ 161.4, 157.2, 156.9, 150.1, 136.7, 134.5, 133.1, 131.8, 130.8, 128.7, 126.0, 125.6, 125.4, 121.2, 121.1, 113.9, 113.8, 70.2, 69.0, 35.7, 30.7, 25.4, 24.4; resonances at δ 150.1, 113.8, 69.0, and 30.7 ppm were taken from the HSQC experiment; m/z MS (TOF ES⁺) 443.2 [M + H]⁺; LC-MS t_R: 3.17; HRMS C₂₆H₂₇N₄O₃ [M + H]⁺ calcd 443.2083; found 443.2086.

6-(2-((4-(1-Ethyl-1H-pyrazol-4-yl)benzyl)oxy)phenyl)-3-(2-hydroxycyclohexyl)pyrimidin-4(3H)-one (11a)

General procedure A. The title compound was obtained as a white solid (32 mg, 75%). ¹H NMR (CDCl₃) δ 8.21 (s, 1H), 7.95 (dd, J = 7.8, 1.7 Hz, 1H), 7.77 (s, 1H), 7.64 (s, 1H), 7.59−4.43 (m, 2H), 7.42−7.32 (m, 3H), 7.26 (s, 1H), 7.09−7.04 (m, 1H), 7.02 (d, J = 8.3 Hz, 1H), 5.18 (s, 2H), 4.59−4.43 (m, 1H), 4.20 (q, J = 7.3 Hz, 2H), 3.98−3.80 (m, 1H), 3.08 (br s, 1H), 2.29−2.10 (m, 1H), 2.08−1.97 (m, 1H), 1.90−1.71 (m, 3H), 1.52 (t, J = 7.3 Hz, 3H), 1.49−1.32 (m, 3H); ¹³C NMR (CDCl₃) δ 162.9, 157.9, 156.9, 147.6, 136.5, 134.4, 132.5, 131.3, 130.7, 127.7, 125.7, 125.6, 125.4, 122.4, 121.1, 114.8, 113.2, 72.1, 70.4, 61.0, 47.2, 35.6, 31.0, 25.2, 24.40, 15.6; resonance at δ 61.0 ppm was taken from the HSQC experiment; m/z MS (TOF ES⁺) 471.2 [M + H]⁺; LC-MS t_R: 3.28; HRMS C₂₈H₃₁N₄O₃ [M + H]⁺ calcd 471.2396; found 471.2402.

3-(2-Hydroxycyclohexyl)-6-(2-((4-(1-propyl-1H-pyrazol-4-yl)-benzyl)oxy)phenyl)pyrimidin-4(3H)-one (11b)

General procedure A. The title compound was obtained as a white solid (35 mg, 80%). ¹H NMR (CDCl₃) δ 8.18 (s, 1H), 7.94 (dd, J = 7.8, 1.7 Hz, 1H), 7.75 (s, 1H), 7.60 (s, 1H), 7.49−7.44 (m, 2H), 7.40−7.30 (m, 3H), 7.24 (s, 1H), 7.08−7.01 (m, 1H), 6.99 (d, J = 8.3 Hz, 1H), 5.16 (s, 2H), 4.56−4.43 (m, 1H), 4.08 (t, J = 7.1 Hz, 2H), 3.93−3.79 (m, 1H), 2.96 (br s, 1H), 2.21−2.12 (m, 1H), 2.02−1.75 (m, 6H), 1.52−1.32 (m, 3H), 0.93 (t, J = 7.4 Hz, 3H); ¹³C NMR (CDCl₃) δ 162.9, 157.9, 156.9, 147.6, 136.6, 134.4, 132.5, 131.3, 130.7, 127.7, 126.1, 125.7, 125.6, 122.3, 121.1, 114.8, 113.2, 72.2, 70.4, 60.8, 54.0, 35.6, 31.0, 25.3, 24.4, 23.7, 11.2; resonance at δ 60.8 ppm was taken from the HSQC experiment; m/z MS (TOF ES⁺) 485.2 [M + H]⁺; LC-MS t_R: 3.33; HRMS C₂₉H₃₃N₄O₃ [M + H]⁺ calcd 485.2553; found 485.2558.

3-(2-Hydroxycyclohexyl)-6-(2-((4-(1-isopropyl-1H-pyrazol-4-yl)-benzyl)oxy)phenyl)pyrimidin-4(3H)-one (11c)

General procedure A. The title compound was obtained as a white solid (26 mg, 59%). ¹H NMR (CDCl₃) δ 8.20 (s, 1H), 7.96 (dd, J = 7.8, 1.8 Hz, 1H), 7.80–7.76 (m, 1H), 7.70−7.66 (m, 1H), 7.52−7.46 (m, 2H), 7.43−7.32 (m, 3H), 7.27−7.25 (m, 1H), 7.10−7.04 (m, 1H), 7.03−7.00 (m, 1H), 5.18 (s, 2H), 4.64−4.41 (m, 2H), 3.96−3.76 (m, 1H), 3.04 (brs, 1H), 2.29−2.15 (m, 1H), 2.07−1.97 (m, 1H), 1.91−1.74 (m, 3H), 1.55 (d, J = 6.7 Hz, 6H), 1.51−1.35 (m, 3H); ¹³C NMR (CDCl₃) δ 162.9, 157.9, 157.0, 147.6, 136.2, 134.3, 132.6, 131.3, 130.7, 127.7, 125.6, 125.5, 123.5, 122.1, 121.1, 114.8, 113.2, 72.1, 70.5, 61.0, 53.9, 35.6, 31.0, 25.2, 24.4, 23.0; resonance at δ 61.0 ppm was taken from the HSQC experiment; m/z MS (TOF ES⁺) 485.2 [M + H]⁺; LC-MS t_R: 3.32; HRMS C₂₉H₃₃N₄O₃ [M + H]⁺ calcd 485.2553; found 485.2557.

6-(2-((4-(1-Butyl-1H-pyrazol-4-yl)benzyl)oxy)phenyl)-3-(2-hydroxycyclohexyl)pyrimidin-4(3H)-one (11d)

General procedure A. The title compound was obtained as a white solid (27 mg, 60%). ¹H NMR (CDCl₃) δ 8.20 (s, 1H), 7.96 (dd, J = 7.8, 1.6 Hz, 1H), 7.77 (s, 1H), 7.62 (s, 1H), 7.51−7.45 (m, 2H), 7.42−7.31 (m, 3H), 7.26 (s, 1H), 7.09−7.03 (m, 1H), 7.01 (d, J = 8.3 Hz, 1H), 5.18 (s, 2H), 4.57−4.44 (m, 1H), 4.14 (t, J = 7.1 Hz, 2H), 3.98−3.81 (m, 1H), 3.10 (br s, 1H), 2.25−2.11 (m, 1H), 2.09−1.95 (m, 1H), 1.95−1.71 (m, 5H), 1.55−1.29 (m, 5H), 0.96 (t, J = 7.4 Hz, 3H); ¹³C NMR (CDCl₃) δ 162.9, 157.9, 156.9, 147.6, 136.5, 134.4, 132.5, 131.3, 130.7, 127.7, 126.0, 125.7, 122.3, 121.1, 114.8, 113.2, 72.1, 70.4, 60.8, 52.1, 35.6, 32.4, 31.0, 25.2, 24.4, 19.8, 13.6; resonance at δ 60.8 ppm was taken from the HSQC experiment; m/z MS (TOF ES⁺) 499.2 [M + H]⁺; LC-MS t_R: 3.39; HRMS C₃₀H₃₅N₄O₃ [M + H]⁺ calcd 499.2709; found 499.2712.

6-(2-((2-Fluoro-4-(1-methyl-1H-pyrazol-4-yl)benzyl)oxy)phenyl)-3-(2-hydroxycyclohexyl)pyrimidin-4(3H)-one (12a)

General proce-dure C. The title compound was obtained as a white solid (145 mg, 66%). ¹H NMR (d₆-DMSO) δ 8.52 (s, 1H), 8.24 (s, 1H), 7.99 (dd, J = 7.8, 1.6 Hz, 1H), 7.96 (s, 1H), 7.57−7.40 (m, 4H), 7.31 (d, J = 8.3 Hz, 1H), 7.14−7.07 (m, 1H), 6.98 (s, 1H), 5.25 (s, 2H), 4.94 (d, J = 5.7 Hz, 1H), 4.28 (br s, 1H), 3.91−3.88 (m, 1H), 3.87 (s, 3H), 2.06–1.93 (m, 1H), 1.88−1.58 (m, 4H), 1.39−1.19 (m, 3H); ¹³C NMR (CDCl₃) δ 161.5 (d, J_CF = 246.5 Hz), 161.4, 157.0, 156.7, 150.1, 136.9, 135.8 (d, J_CF = 8.9 Hz), 131.8, 131.7 (d, J_CF = 4.8 Hz), 130.8, 129.0, 125.4, 121.4, 121.2 (d, J_CF = 2.8 Hz), 121.0 (d, J_CF = 1.8 Hz), 120.9, 113.9, 113.8, 112.1 (d, J_CF = 22.3 Hz), 69.0, 64.7, 39.2, 38.7, 30.7, 25.4, 24.4; resonances at δ 150.1, 69.0, and 30.7 ppm was taken from the HSQC experiment (one tertiary carbon was not detected); m/z MS (TOF ES⁺) 475.2 [M + H]⁺; LC-MS t_R: 3.23; HRMS C₂₇H₂₈FN₄O₃ [M + H]⁺ calcd 475.2145; found 475.2153.

3-(2-Hydroxycyclohexyl)-6-(2-((6-(1-methyl-1H-pyrazol-4-yl)-pyridin-3-yl)methoxy)phenyl)pyrimidin-4(3H)-one (12b)

General procedure C. The title compound was obtained as a colorless oil (25 mg, 12%). ¹H NMR (CDCl₃) δ 8.43 (d, J = 1.7 Hz, 1H), 8.13 (s, 1H), 7.92 (m, 2H), 7.78 (dd, J = 7.7, 1.6 Hz, 1H), 7.57 (dd, J = 8.1, 2.2 Hz, 1H), 7.42−7.35 (m, 2H), 7.12−7.06 (m, 1H), 7.05 (d, J = 8.3 Hz, 1H), 6.99 (s, 1H), 5.12 (d, J = 12.2 Hz, 1H), 5.06 (d, J = 12.2 Hz, 1H), 4.58−4.45 (m, 1H), 4.33 (br s, 1H), 3.93 (s, 3H), 3.91−3.81 (m, 1H), 2.25−2.14 (m, 1H), 2.05–1.95 (m, 1H), 1.91−1.70 (m, 3H), 1.57−1.33 (m, 3H); ¹³C NMR (CDCl₃) δ 162.4, 158.2, 156.2, 151.5, 148.7, 147.9, 137.6, 136.2, 131.3, 130.4, 129.2, 129.1, 127.3, 122.9, 122.0, 119.4, 115.1, 114.7, 71.5, 69.3, 60.9, 39.2, 35.5, 30.9, 25.2, 24.4, resonance at δ 60.9 ppm was taken from the HSQC experiment; m/z MS (TOF ES⁺) 458.2 [M + H]⁺; LC-MS t_R: 2.98; HRMS C₂₆H₂₈N₅O₃ [M + H]⁺ calcd 458.2192; found 458.2198.

6-(2-(Benzyloxy)phenyl)-3-(2-hydroxycyclohexyl)pyrimidin-4(3H)-one (13)

The title compound 13 was observed in substantial quantities during the scale up reaction of compound 2. The compound was isolated via column chromatography (EtOAC 100%) followed by preparative HPLC (70:30 → 0:100% water/acetonitrile, spiked with 1% TFA). The combined product fractions were combined the acetonitrile removed under reduced pressure. The residue was basified with 1 M NaOH and extracted with chloroform. The organic layer was dried with Na₂SO₄, filtered and the solvent was removed under reduced pressure. The title compound was obtained as a white resin. ¹H NMR (CDCl₃) δ 8.21 (s, 1H), 7.94 (dd, J = 7.8, 1.8 Hz, 1H), 7.42−7.27 (m, 6H), 7.24−7.20 (m, 1H), 7.05 (td, J = 7.7, 1.0 Hz, 1H), 7.02−6.98 (m, 1H), 5.19 (s, 2H), 4.58−4.44 (m, 1H), 3.93−3.81 (m, 1H), 2.41 (br s, 1H), 2.24−2.14 (m, 1H), 2.08–1.96 (m, 1H), 1.92−1.72 (m, 3H), 1.55−1.32 (m, 3H); ¹³C NMR (CDCl₃) δ 162.9, 157.9, 157.0, 147.6, 136.6, 131.3, 130.7, 128.7, 128.0, 127.0, 125.6, 121.1, 114.8, 113.3, 72.4, 70.6, 60.5, 35.7, 30.9, 25.3, 24.4; resonances at δ 60.5 ppm was taken from the HSQC experiment; m/z MS (TOF ES⁺) 377.2 [M + H]⁺; LC-MS t_R: 3.40; HRMS C₂₃H₂₅N₂O₃ [M + H]⁺ calcd 377.1865; found 377.1860.

4-((2-(1-(2-Hydroxycyclohexyl)-6-oxo-1,6-dihydropyrimidin-4-yl)phenoxy)methyl)benzoic acid (14a)

NaOH (18.4 mg, 460 μmol, 4.0 equiv) was added to a solution of methyl 4-((2-(1-(2-hydroxycyclohexyl)-6-oxo-1,6-dihydropyrimidin-4-yl)phenoxy)-methyl)benzoate (50.0 mg, 115 μmol, 1.0 equiv) in EtOH/H₂O (1:1, 2 mL/0.1 mmol). The reaction mixture was heated at 50 °C for 2 h. The EtOH was evaporated under reduced pressure, before acidifying with 1 M HCl_(aq) to pH 2. The water layer was extracted with EtOAC (2 × 50 mL). The organic layer was dried with Na₂SO₄ and filtered, and the solvent was removed under reduced pressure. The title compound was obtained as a white solid (17 mg, 35%). ¹H NMR (d₆-DMSO) δ 12.99 (br s, 1H), 8.56 (s, 1H), 8.00−7.94 (m, 3H), 7.62–7.55 (m, 2H), 7.47−7.39 (m, 1H), 7.21 (d, J = 8.0 Hz, 1H), 7.13–7.06 (m, 1H), 7.02 (s, 1H), 5.35 (s, 2H), 4.32 (m, 1H), 4.09−3.89 (m, 1H), 3.55−3.15 (br s, 1H), 2.07−1.98 (m, 1H), 1.85−1.64 (m, 4H), 1.41−1.26 (m, 3H); ¹³C NMR (d₆-DMSO) δ 167.5, 161.3, 156.8, 156.8, 150.2, 142.3, 131.8, 130.8, 130.7, 130.0, 127.8, 125.4, 121.4, 114.1, 113.9, 69.8, 68.5, 35.7, 30.7, 25.4, 24.4; resonances at δ 150.2, 68.5, 30.7 ppm were taken from the HSQC experiment; m/z MS (TOF ES⁺) 421.2 [M + H]⁺; LC-MS t_R: 3.14; HRMS C₂₄H₂₅N₂O₅ [M + H]⁺ calcd 421.1763; found 421.1765.

4-((2-(1-(2-Hydroxycyclohexyl)-6-oxo-1,6-dihydropyrimidin-4-yl)phenoxy)methyl) benzamide (14b)

A mixture of 4-((2-(1-(2-hydroxycyclohexyl)-6-oxo-1,6-dihydropyrimidin-4-yl)phenoxy)-methyl)benzoate (50.0 mg, 115 μmol, 1.0 equiv) in MeOH/NH₄OH (1:1, 30 mL each) was stirred at rt for 9 days. The resulting precipitate was filtrated, and the filter cake was dissolved in MeOH. The solvent was removed under reduced pressure to give the title compound as a white solid (18 mg, 37%). ¹H NMR (CD₃OD) δ 8.49 (s, 1H), 7.93−7.86 (m, 3H), 7.57−7.52 (m, 2H), 7.43 (ddd, J = 8.4, 7.5, 1.8 Hz, 1H), 7.19 (d, J = 8.0 Hz, 1H), 7.14−7.05 (m, 2H), 5.29 (s, 2H), 4.58−4.29 (m, 1H), 4.23−4.02 (m, 1H), 2.22−2.11 (m, 1H), 2.02−1.77 (m, 4H), 1.55−1.39 (m, 3H); ¹³C NMR (CD₃OD) δ 170.6, 162.9, 158.7, 156.6, 149.2, 140.9, 133.1, 131.3, 130.3, 127.6, 127.0, 125.6, 120.9, 113.9, 113.2, 69.7, 69.3, 34.9, 29.1, 25.0, 24.0; resonances at δ 149.2, 69.3, and 29.1 ppm were taken from the HSQC experiment; m/z MS (TOF ES⁺) 420.2 [M + H; LC-MS t_R: 3.04; HRMS C₂₄H₂₆N₃O₄ [M + H]⁺ calcd 420.1923; found 420.1923.

4-((2-(1-(2-Hydroxycyclohexyl)-6-oxo-1,6-dihydropyrimidin-4-yl)phenoxy)methyl)-N,N-dimethylbenzamide (15a)

General procedure D. The title compound was obtained as a colorless oil (38 mg, 63%). ¹H NMR (CDCl₃) δ 8.19 (s, 1H), 7.87 (dd, J = 7.8, 1.7 Hz, 1H), 7.38 (s, 4H), 7.31 (ddd, J = 8.3, 7.4, 1.8 Hz, 1H), 7.12 (d, J = 0.6 Hz, 1H), 7.03 (td, J = 7.7, 0.9 Hz, 1H), 6.97−6.91 (m, 1H), 5.16 (s, 2H), 4.47 (br t, J = 9.5 Hz, 1H), 3.92−3.79 (m, 1H), 3.07 (br s, 3H), 2.94 (br s, 3H), 2.20−2.11 (m, 1H), 2.04−1.93 (m, 1H), 1.86−1.65 (m, 3H), 1.50−1.30 (m, 3H); ¹³C NMR (CDCl₃) δ 171.3, 162.7, 157.8, 156.7, 147.8, 138.1, 135.8, 131.3, 130.7, 127.5, 126.9, 125.9, 121.4, 114.8, 113.5, 71.8, 70.3, 60.5, 39.7, 35.6, 35.4, 31.1, 25.2, 24.4; resonance at δ 60.5 ppm was taken from the HSQC experiment; m/z MS (TOF ES⁺) 448.0 [M + H]⁺; LC-MS t_R: 3.12; HRMS C₂₆H₃₀N₃O₄ [M + H]⁺ calcd 448.2236; found 448.2237.

4-((2-(1-(2-Hydroxycyclohexyl)-6-oxo-1,6-dihydropyrimidin-4-yl)phenoxy)methyl)-N-methylbenzamide (15b)

General procedure D. The title compound was obtained as a colorless oil (41 mg, 70%). ¹H NMR (CDCl₃) δ 8.17 (s, 1H), 7.90 (dd, J = 7.8, 1.7 Hz, 1H), 7.73−7.68 (m, 2H), 7.40−7.31 (m, 3H), 7.11 (d, J = 2.0 Hz, 1H), 7.08−7.02 (m, 1H), 6.98−6.93 (m, 1H), 6.70 (br q, J = 4.4 Hz, 1H), 5.13 (d, J = 12.7 Hz, 1H), 5.10 (d, J = 12.6 Hz, 1H), 4.49 (br t, J = 9.3 Hz, 1H), 3.96−3.82 (m, 1H), 3.31−3.18 (m, 1H), 2.96 (d, J = 4.8 Hz, 3H), 2.25−2.15 (m, 1H), 2.03−1.95 (m, 1H), 1.88−1.79 (m, 3H), 1.51−1.34 (m, 3H); ¹³C NMR (CDCl₃) δ 168.3, 162.8, 157.9, 156.7, 147.9, 139.5, 134.3, 131.4, 130.7, 127.4, 127.0, 125.7, 121.3, 114.7, 113.2, 71.4, 70.2, 60.9, 35.5, 31.1, 26.8, 25.2, 24.4; resonance at δ 60.9 ppm was taken from the HSQC experiment; m/z MS (TOF ES⁺) 434.0 [M + H]⁺; LC-MS t_R: 3.26; HRMS C₂₅H₂₈N₃O₄ [M + H]⁺ calcd 434.2080; found 434.2079.

N,N-Diethyl-4-((2-(1-(2-hydroxycyclohexyl)-6-oxo-1,6-dihydropyrimidin-4-yl)phenoxy)methyl)benzamide (15c)

General procedure D. The title compound was obtained as a colorless oil (42 mg, 65%). ¹H NMR (CDCl₃) δ 8.20 (s, 1H), 7.87 (dd, J = 7.8, 1.7 Hz, 1H), 7.41−7.29 (m, 5H), 7.14 (s, 1H), 7.04 (td, J = 7.7, 0.9 Hz, 1H), 6.98−6.93 (m, 1H), 5.18 (s, 2H), 4.48 (br t, J = 9.5 Hz, 1H), 3.90–3.79 (m, 1H), 3.63−3.46 (m, 2H), 3.32−3.16 (m, 2H), 2.88 (br s, 1H), 2.21−2.11 (m, 1H), 2.04−1.94 (m, 1H), 1.87−1.67 (m, 3H), 1.50−1.31 (m, 3H), 1.28−1.16 (m, 3H), 1.16−1.02 (m, 3H); ¹³C NMR (CDCl₃) δ 171.0, 162.7, 157.8, 156.7, 147.8, 137.7, 136.7, 131.4, 130.7, 127.0, 126.7, 125.9, 121.4, 114.8, 113.6, 71.9, 70.4, 60.5, 43.4, 39.3, 35.6, 31.1, 25.2, 24.4, 14.2, 12.9; resonance at δ 60.5 ppm was taken from the HSQC experiment; m/z MS (TOF ES⁺) 475.9 [M + H]⁺; LC-MS t_R: 3.26; HRMS C₂₈H₃₄N₃O₄ [M + H]⁺ calcd 476.2549; found 476.2549.

N-Ethyl-4-((2-(1-(2-hydroxycyclohexyl)-6-oxo-1,6-dihydropyrimidin-4-yl)phenoxy)methyl)benzamide (15d)

General procedure D. The title compound was obtained as a colorless oil (46 mg, 76%). ¹H NMR (CDCl₃) δ 8.17 (s, 1H), 7.88 (dd, J = 7.7, 1.5 Hz, 1H), 7.74–7.66 (m, 2H), 7.40−7.30 (m, 3H), 7.09 (s, 1H), 7.04 (t, J = 7.4 Hz, 1H), 6.93 (d, J = 8.2 Hz, 1H), 6.67 (br t, J = 4.8 Hz, 1H), 5.19−5.01 (m, 2H), 4.55−4.39 (m, 1H), 3.94−3.82 (m, 1H), 3.49−3.39 (m, 2H), 2.22−2.13 (m, 1H), 2.04−1.93 (m, 1H), 1.89−1.68 (m, 3H), 1.52−1.32 (m, 3H), 1.22 (t, J = 7.3 Hz, 3H); ¹³C NMR (CDCl₃) δ 167.4, 162.8, 157.9, 156.7, 147.7, 139.6, 134.5, 131.4, 130.7, 127.3, 127.0, 125.8, 121.4, 114.7, 113.4, 71.8, 70.3, 60.7, 35.6, 34.9, 31.1, 25.2, 24.4, 14.8; resonance at δ 60.7 ppm was taken from the HSQC experiment; m/z MS (TOF ES⁺) 447.9 [M + H]⁺; LC-MS t_R: 3.14; HRMS C₂₆H₃₀N₃O₄ [M + H]⁺ calcd 448.2236; found 448.2237.

3-(2-Hydroxycyclohexyl)-6-(2-((4-(piperidine-1-carbonyl)benzyl)-oxy)phenyl)pyrimidin-4(3H)-one (15e)

General procedure D. The title compound was obtained as a colorless oil (55 mg, 83%). ¹H NMR (CDCl₃) δ 8.18 (s, 1H), 7.86 (dd, J = 7.8, 1.8 Hz, 1H), 7.41–7.27 (m, 5H), 7.12 (d, J = 0.6 Hz, 1H), 7.02 (td, J = 7.7, 0.9 Hz, 1H), 6.96−6.91 (m, 1H), 5.16 (s, 2H), 4.46 (br t, J = 9.4 Hz, 1H), 3.92–3.81 (m, 1H), 3.75−3.53 (m, 2H), 3.40−3.24 (m, 2H), 3.17−3.09 (m, 1H), 2.19−2.08 (m, 1H), 2.03−1.92 (m, 1H), 1.81−1.33 (m, 12H); ¹³C NMR (CDCl₃) δ 170.0, 162.6, 157.8, 156.7, 147.9, 138.0, 135.9, 131.3, 130.7, 127.2, 126.9, 125.9, 121.3, 114.8, 113.5, 71.6, 70.3, 60.7, 48.8, 47.9, 43.2, 35.5, 31.1, 26.5, 25.8, 25.6, 25.2, 24.6, 24.4; resonance at δ 60.7 ppm was taken from the HSQC experiment; m/z MS (TOF ES⁺) 488.0 [M + H]⁺; LC-MS t_R: 3.56; HRMS C₂₉H₃₄N₃O₄ [M + H]⁺ calcd 488.2549; found 488.2549.

Crystallography

Intensity data were collected with an Oxford Diffraction SuperNova CCD diffractometer using Mo Kα radiation, the temperature during data collection was maintained at 130.0(1) using an Oxford Cryosystems cooling device. The structure was solved by direct methods and difference Fourier synthesis.³⁵ Thermal ellipsoid plots were generated using the program ORTEP-3³⁶ integrated within the WINGX suite of programs.³⁷

Crystal data for 14b

C₂₄H₂₅N₃O₄, M = 419.47, T = 130.0 K, λ = 0.71073 Å, monoclinic, space group P2₁/c, a = 10.3922(3) b = 20.2113(4), c = 510.8629(3) Å, β = 114.712(3)° V = 2072.69(10) ų, Z = 4, D_c = 1.344 mg M⁻³ μ(Mo Kα) 0.093 mm⁻¹, θ_max = 30° F(000) = 888, crystal size 0.59 × 0.36 × 0.24 mm³, 19,695 reflections measured, 6033 independent reflections [R(int) = 0.0243], the final R was 0.0442, [I > 2σ(I), 5317 data] and wR(F²) was 0.1243 (all data), GOOF = 1.053.

Pharmacology

Cell Culture

FlpInCHO cells stably expressing hM₁ mAChR (37031 ± 3397 sites/cell) were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 5% (v/v) fetal bovine serum (FBS) (Thermo-Trace, Melbourne, Australia). Cells were lifted using trypsin (0.025%) and plated for binding and IP₁ accumulation assays as described below.

Radioligand Binding Assay

FlpInCHO-hM₁ cells were seeded into white 96 well isoplates (PerkinElmer) at a density of 50,000 cells/well and grown overnight at 37 °C. Cells were washed twice with binding buffer (150 mM NaCl, 20 mM HEPES, 10 mM MgCl₂), then ³H-NMS (0.5 nM), orthosteric and allosteric ligands diluted in binding buffer were added to the cells for 6 h at 23 °C. The binding reaction was terminated by rapid removal of the binding buffer and washing the cells twice with ice cold 0.9% NaCl. The plates were dried, 0.1 mL of Microscint O (PerkinElmer) was added to each well, and then the plates were sealed with TopSeal. Radioactivity was detected by counting the plates in the MicroBeta counter (PerkinElmer). Data were expressed as a percentage of the maximum amount of ³H-NMS binding in the absence of competing ligand.

IP₁ Accumulation Assay

FlpInCHO-hM₁ cells were seeded into clear 96-well plates at a density of 10,000 cells/well and grown overnight at 37 °C. Cells were washed twice with IP₁ stimulation buffer (10 mM HEPES, 1 mM CaCl₂, 0.5 mM MgCl₂, 4.2 mM KCl, 146 mM NaCl, 5.5 mM d-glucose, 50 mM LiCl, pH 7.4), then equilibrated in stimulation buffer for 1 h at 37 °C. Orthosteric and allosteric ligands were diluted in stimulation buffer and then added to the cells for a further 1 h at 37 °C. The cells were lysed in lysis buffer (50 mM HEPES-NaOH pH 7.0, 15 mM KF, 1.5% (v/v) Triton-X-100) and shaken on a plate shaker for 10 min, then 14 μL/well of cell lysates were transferred into 384 well Optiplate (PerkinElmer Life Sciences). The IP-one assay kit (Cisbio, France) was used for the direct quantitative measurement of myo-inositol 1 phosphate (IP₁). Homogenous time-resolved FRET (HTRF) reagents (cryptate-labeled anti-IP₁ antibody, the d₂-labeled IP₁ analogue; diluted 1:20 in lysis buffer) were added to each well, and the plates were incubated for 1 h at 37 °C. Samples were excited at 340 nm and emission was captured at 590 and 665 nm using the Envision multilabel plate reader (PerkinElmer Life Sciences). IP₁ concentrations were interpolated from the standard curve prepared in parallel for each experiment, and then the responses were normalized to the maximum ACh response.

IP1 Accumulation Assay in Cortical Neurons

The primary neuronal cell cultures were prepared as described previously.^38,39 Embryonic cortical tissue was dissected out from mouse pups (embryonic day 16) under HBSS (Gibco, Life Technology, Australia), triturated, centrifuged, and then resuspended in 37 °C B-27 supplemented Neurobasal medium (Gibco, Life Technology, Australia). Neuronal cells (50,000−60,000 cells/well) were then plated on poly-_D-lysine (Sigma, Australia) treated 96-well plates, and maintained for 6−7 days at 37 °C in a humidified cell culture incubator supplied with 5% CO₂. The cells were washed with stimulation buffer supplied with the IP-One HTRF assay kit (Cisbio Bioassays, Codolet, France), then treated with ACh in the presence or absence of 0.1 to 10 μM of the PAMs, in stimulation buffer in a total volume of 30 μL/well for 1 h at 37 °C. Cells were lysed by addition of 12 μL/well lysis buffer containing the cryptate-labeled anti-IP₁ antibody and the d₂-labeled IP₁ analogue, supplied with the IP-One HTRF assay kit (Cisbio Bioassays, Codolet, France), and incubated for 1 h at rt. Twenty μL/well of cell lysates were transferred into 384 well Optiplate (PerkinElmer Life Sciences), and the signal was measure using the Envision multilabel plate reader (PerkinElmer Life Sciences).

Data Analysis

All data were analyzed using GraphPad prism v 7.02 (Graphpad software, San Diego, CA). Radioligand binding experiments were globally fitted to the following allosteric ternary complex model.

$$Y=\frac{B_{\max }[A]}{[A]+\left(\frac{K_{\text{A}}{K}_{\text{B}}}{{\alpha }^{\prime }[B]+K_{\text{B}}}\right)\left(1+\frac{[I]}{K_{\text{I}}}+\frac{[B]}{K_{\text{B}}}+\frac{\alpha [I][B]}{K_{\text{I}}{K}_{\text{B}}}\right)}$$ (1)

where Y is the percentage of binding, B_max is the total number of binding sites. [A], [B], and [I] are the concentrations of ³H-NMS, allosteric modulator, and ACh, respectively. K_A K_B, and K_I are the equilibrium dissociation constants of ³H-NMS, the allosteric modulator, and ACh, respectively. α and α′ are the binding cooperativities between ACh and the allosteric modulator and ³H-NMS and the allosteric modulator, respectively. Saturation binding experiments determined the orthosteric antagonists binding affinity of ³H-NMS to be 0.26 nM (pK_D = 9.59 ± 0.02) at the M₁ mAChR expressed in this cell line, which was used to fix the log K_A value for the analysis.

The IP₁ accumulation experiments were globally fitted to the following operational model of allosterism and agonism.⁴⁰

$$E=\text{basal}+\frac{\left(E_{\text{m}}-\text{basal}\right)([A]{\left(K_{\text{B}}+\alpha \beta [B]+{\tau }_{\text{B}}[B]\left[{\text{EC}}_{50}\right]\right)}^n}{{\left[{\text{EC}}_{50}\right]}^n{\left(K_{\text{B}}+[B]\right)}^n+{\left([A]\left(K_{\text{B}}+\alpha \beta [B]\right)+{\tau }_{\text{B}}[B]\left[{\text{EC}}_{50}\right]\right)}^n}$$ (2)

where basal is the response in the presence of vehicle, and [A] and [B] are the concentrations of ACh and allosteric ligand, respectively. K_B represents the equilibrium dissociation constant of allosteric modulator. τ_B represents the relative operational measure of allosteric ligand efficacy, α denotes the binding cooperativity parameter between orthosteric and allosteric ligand respectively, whereas β denotes a scaling factor that quantifies the allosteric effect of the modulator on the orthosteric ligand efficacy. This model assumes that all the compounds are full agonists at the receptor and there is no efficacy modulation. n denotes the transducer slope that describes the stimulus-response coupling of the ligand-occupied receptor to the signaling pathway, which was constrained to 1. To aid model convergence, the allosteric binding affinity (pK_B) of each allosteric modulator was fixed to the value determined from analysis of the radioligand binding data.

Abbreviations Used

ACh

acetylchloline

AD

Alzheimer’s disease

Boc

tert-butyloxycarbonyl

cat

catalytic

DCM

dichloromethane

DMF

N,N-dimethylformamide

equiv

equivalent

EtOAc

ethyl acetate

NMS

N-methylscopolamine

M₁ mAChR

M₁ muscarinic acetylcholine receptor

PE

petroleum spirits 40−60

rt

room temperature

THF

tetrahydrofuran