Isoquinolone Derivatives as Lysophosphatidic Acid Receptor 5 (LPA5) Antagonists: Investigation of Structure-Activity Relationships, ADME Properties and Analgesic Effects

Abstract

Blockade of lysophosphatidic acid receptor 5 (LPA5) by a recently reported antagonist AS2717638 (2) attenuated inflammatory and neuropathic pains, although it showed moderate in vivo efficacy and its structure-activity relationships and the ADME properties are little studied. We therefore designed and synthesized a series of isoquinolone derivatives and evaluated their potency in LPA5 calcium mobilization and cAMP assays. Our results show that substituted phenyl groups or bicyclic aromatic rings such as benzothiophenes or benzofurans are tolerated at the 2-position, 4-substituted piperidines are favored at the 4-position, and methoxy groups at the 6- and 7-positions are essential for activity. Compounds 65 and 66 showed comparable in vitro potency, excellent selectivity against LPA1-LPA4 and >50 other GPCRs, moderate metabolic stability, and high aqueous solubility and brain permeability. Both 65 and 66 significantly attenuated nociceptive hypersensitivity at lower doses than 2 and had longer-lasting effects in an inflammatory pain model, and 66 also dose-dependently reduced mechanical allodynia in the chronic constriction injury model and opioid-induced hyperalgesia at doses that had no effect on the locomotion in rats. These results suggest that these isoquinolone derivatives as LPA5 antagonists are of promise as potential analgesics.

1. Introduction

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid that plays a critical role in diverse cellular processes including cell proliferation, migration, and survival [1–5]. LPA activates at least six G protein-coupled receptors (GPCRs), LPA1–LPA6, which belong to either the endothelial differentiation gene (EDG) subfamily (LPA1–LPA3) or the phylogenetically distant non-EDG subfamily (LPA4–LPA6). Among them, LPA5 was discovered in 2001 as an orphan GPCR GPR92 [6], and was subsequently named the fifth LPA receptor in 2006 [7, 8]. The LPA5 protein sequence demonstrates 80% identity between human and rodents [7]. Primarily coupled to Gα_12/13 and Gα_q/11 proteins, LPA5 mediates neurite retraction and intracellular calcium, respectively, but the receptor also increases cAMP accumulation and evokes eletrophysiological currents [7]. LPA5 is expressed in various tissues throughout the body including brain, spleen, heart, stomach, small intestine, kidney, and platelets [5, 7–10], and is involved in multiple pathological conditions such as platelet activation [11] and cancer [12]. In particular, LPA5 is highly expressed in the spinal cord and dorsal root ganglion (DRG), two major pain-associated regions [13, 14], suggesting a role of LPA5 in pain modulation.

In response to injury, levels of LPA [15–20], as well as the key LPA synthesis enzymes [17], increase significantly in the brain and cerebrospinal fluid in rodents and humans. Studies suggest that the LPA signaling is mediated, at least in part, by the LPA1 receptor, which is involved in the initiation of nerve injury-induced neuropathic pain [14, 15, 21, 22]. Now emerging evidence supports that LPA5 is also critically involved in pain processing. Activation of LPA5 was observed under the conditions of nerve injury- and multiple sclerosis-induced neuropathic pain [13, 22, 23]. LPA5 was also found to upregulate in the spinal cord in a rat model of neuropathic pain [13]. Compared to wild-type mice, Lpar5 knockout (KO) mice showed an attenuation of cold allodynia induced by nerve injury and faster recovery from complete Freund’s adjuvant (CFA)-induced inflammatory pain [24]. LPA5 blockade prevented LPA from activating microglia and mast cells [25, 26], a key step in acute and/or chronic inflammation [27]. Finally, LPA5 appears to modulate pain through different mechanisms from those of LPA1, as LPA1-deficient mice show normal expression of phosphorylated cAMP-responsive element-binding protein (pCREB), an increase of which is associated with neuropathic pain after nerve injury [28, 29]; however, in LPA5-deficient mice pCREB is significantly reduced. Together, these results support that LPA5 plays an important role in the modulation of multiple painful conditions.

The first LPA5 antagonist, a pyrazole carboxylic acid (1, IC₅₀ = 800 nM, Figure 1), was identified by Kozian and colleagues in 2012 via high-throughput screening and was examined for its effect on inhibiting platelet activation [30]. Kawamoto and colleagues later reported that an LPA5 antagonist AS2717638 (2, IC₅₀ = 38 nM in cAMP assay) improved weight bearing in a rat model of adjuvant-induced inflammatory pain and also ameliorated static mechanical allodynia in a rat model of chronic constriction injury (CCI)-induced neuropathic pain [31]. When resynthesized and tested in our lab, intraperitoneally (i.p.) administered 2 produced clear dose-response effects in both chronic constriction injury (CCI) and Complete Freund’s adjuvant (CFA) rat models; however, high doses (32 mg/kg) were required to achieve full antinociceptive effects in the CCI model and this dose did not fully reverse nociceptive hypersensitivity in the CFA model (see Results and Discussion). In the subsequent and only structure-activity relationship (SAR) study on 2 conducted by the same group, a total of 11 analogs with substituents at the 2- and 4-positions were reported, although they all showed lower potencies than 2 (e.g., 3; IC₅₀ = 120 nM, Figure 1) [32]. The drug-like properties of these compounds were not examined. In an effort to better understand the SARs and to start to assess the pharmacokinetic properties of this promising series of molecules, we have designed and synthesized a series of isoquinolone derivatives bearing diverse substitutions at the 4-position (4–33), the 6- and 7-postions (34–45), or the 2-postion (46–66, Figure 2). The in vitro potency of these LPA5 antagonists was evaluated in calcium mobilization and cAMP assays. The target selectivity and the ADME (absorption, distribution, metabolism, and excretion) properties of two promising compounds from the series (65 and 66), as well as the in vivo brain permeability of 66, were determined. Finally, the in vivo analgesic effects of these two LPA5 antagonists were assessed in well-established pain models including CFA-induced inflammatory pain, chronic constriction injury (CCI) pain, and opioid-induced hyperalgesia (OIH).

Figure 1.

LPA5 antagonists reported in the literature

Figure 2.

Structure-activity relationship plan

2. Results and Discussion

2.1. Synthesis

Target compounds were synthesized following procedures outlined in Schemes 1–4. Compounds 4–33 with differential substituents at the 4-position of the isoquinolone were synthesized following procedures described by Kawamoto and colleagues [32]. Thus, starting material 67 was coupled with dimethylmalonate in the presence of sodium hydride and catalytic cupric (I) bromide to afford intermediate 68, which was then hydrolyzed with 2N sodium hydroxide followed by treatment with 6N HCl to give intermediate 69. Esterification of 69 using SOCl₂ in MeOH provided diester 70 in 52% yield over three steps. Compound 70 was then transformed to carboxylic acid 71 following a three-step sequence of formylation, imination, and cycloamidation and basic hydrolysis. Finally, amide coupling of 71 with various amines afforded target compounds 4–33 in moderate to good yields over four steps. Compound 7 was obtained by treatment of 12 with cyclohexylmagnesium chloride in THF.

Scheme 1.

Syntheses of isoquinolones 4–33 with different 4-position substitutions

Scheme 4.

Syntheses of isoquinolones 46–66 with different 2-position substitutions

The SAR on the 6- and 7-positions of the isoquinoline core was realized by the synthetic route as shown in Scheme 2 and 3. Following a 7-step sequence similar to that for compounds 4–33, target compounds 34–36 were obtained in reasonable overall yield starting from substituted benzoic acids (72) and through intermediates 73–76 (Scheme 2). For compounds 37–45 (Scheme 3), the phenol of the commercially available 77 was first protected by a benzyl group and the aldehyde was then oxidized by sodium chlorite in the presence of hydrogen peroxide to afford key intermediate carboxylic acid 79. Subsequent CuBr-catalyzed coupling of 79, followed by decarboxylation, esterification, cyclization and amide coupling afforded intermediate 84. Palladium on carbon-catalyzed debenzylation of 84 gave phenol 37, which was converted to target compounds 38–45 by O-alkylation using various alkyl bromide or iodide.

Scheme 2.

Syntheses of isoquinolones 34–36 with different 6- and 7-position substitutions

Scheme 3.

Syntheses of isoquinolones 37–45 with different 5- and 6-position substitutions

The synthesis described by Kawamoto et. al. required the construction of the isoquinolone scaffold early on using anilines and the installation of the carboxamide last [32]. In order to rapidly assess the SAR at the 2-position of the isoquinolone, we attempted to design a general synthetic route that installs the 4-carboxamide first and enables the introduction of a variety of aromatic groups at the 2-position at the last step (Scheme 4). We have thus developed a different synthetic route that introduces 2-substituents via transition metal-catalyzed cross-coupling reactions using aromatic halides and boronic acids or boronic esters (Scheme 4). These bromides are readily available and are generally less expensive compared to the corresponding anilines. The treatment of 70 with 1,3,5-triazine in the presence of sodium methoxide led to 85 in quantitative yield, which was then subjected to ester hydrolysis under basic conditons and then amide coupling to afford key intermediate 86.

With 86 in hand, we examined different coupling conditions to introduce aromatic rings to the 2-position. Ulmann coupling catalyzed by CuI using aryl halides afforded the desired products, but only in modest yields as determined by liquid chromatography-mass spectrometry (LC-MS). In addition, the purification of the crude reaction mixture as a dark mixture was difficult and little product was obtained. In addition, we also found that the indoline as present in 3 was oxidized under these conditions to the correponding indoles. Therefore, we next explored Chan-Lam coupling conditions using boronic acids or esters and cupric salt as catalysts, which provided significantly improved results. For boronic acids, CuOTf as the catalyst in the presence of 1,10-Phen-anthroline as the ligand in DMSO afforded good yields (70%–80%), whereas Cu(OAc)₂ together with pyridine in DMSO proved to be the most efficient conditions for boronic pinacol esters (80%–90%). For the reactions using boronic ester as reactants, we found that the temperature was essential in these reactions. While room temperature, as commonly used for these reactions [33–35], only gave trace amount of product in 24 hours, the reactions went to completion at 80 °C in 12 hours. Finally, it should be noted that the Chan-Lam coupling reactions were conducted open to the air, providing good reproducibility and scalability.

Finally, for 66, as the acetylenyl group will likely have side reactions in the presence of the cupric salt [36, 37], the reaction sequence was altered with the Chan-Lam coupling conducted before introduction of the acetylenyl group. Therefore, 85 was transformed to 87 in 75% yield with treatement with the corresponding aromatic boronic pinacol ester in the presence of Cu(OAc)₂ and pyridine in DMSO at 80°C overnight. Hydrolysis of the methyl ester followed by amide coupling with 4-ethynylpiperidine finalized the synthesis of 66 in 89% yield over two steps.

2.2. Biological Evaluations

All compounds were first evaluated for LPA5 antagonist and agonist activity in an in vitro FLIPR-based calcium mobilization assay using RH7777 cells stably expressing hLPA5. In this assay, hexadecyl LPA 16:0 has an EC₅₀ value (± SEM) of 15 ± 1 nM, which is similar to the IC₅₀ (7.5 nM, binding to [³H]LPA) or pEC₅₀ (6.40) values reported in the literature for this agonist receptor pairing [8]. The antagonist potency (IC₅₀) of all target compounds was determined by measuring the inhibition of the mobilization of intracellular calcium levels stimulated by the agonist hexadecyl LPA 16:0 and agonist activity was measured via a single concentration screen at 10 μM. None of the compounds had any significant agonist effects (<10% of hexadecyl LPA 16:0 signal at 10 μM). Select compounds were then evaluated in a TR-FRET cAMP assay for their ability to reverse the inhibitory effect of hexadecyl LPA 16:0 on forskolin-mediated cAMP production using the same RH7777-hLPA5 cells. Hexadecyl LPA 16:0 has an EC₅₀ value (± SEM) of 95 ± 16 nM in this cAMP assay and 2 had an IC₅₀ of 207 nM, lower than the reported IC₅₀ value of 38 nM in cAMP [31], suggesting slight sensitivity differences between the two cAMP assays.

Activities in Calcium Mobilization Assay.

We first examined the SARs at the 4-amide position of the isoquinolone using the calcium mobilization assay. To facilitate a rapid exploration of the 4-position, instead of a 1,2-benzoxazole as present in 2 at the 2-position, we chose to employ a 3-dimethylaminophenyl group, which was examined by Kawamoto and colleagues in the SAR studies and showed potency (IC₅₀ = 0.11 μM) similar to 3 and slightly lower than 2 [32]. The synthetic conditions to 3-dimethylaminophenyl analogs were described in the study, whereas no synthesis of 2 has been reported. Kawamoto and colleagues only reported two analogs at the 4-position, which were both cyclic secondary amides (an azepane and a 4-fluropiperidine, 3) [32]. We first investigated primary and secondary amides at this position. All primary amides bearing either aliphatic or aromatic groups were inactive (IC₅₀ > 10,000 nM, compound 4–9), suggesting primary amides, likely because of the hydrogen-bond donor, are not tolerated here. A dimethyl amide (10) was also inactive, possibly because of the smaller size. Interestingly, while the methylpyridylmethyl amide (11) was also inactive, the Weinreb–Nahm amide (12) displayed moderate potency (IC₅₀ = 320 nM), suggesting possible hydrogen-bond interactions with the receptor, but had minimal inhibitory activity (33%). We then turned our attention to cyclic amides and examined the impact of ring size and substitutions on potency. Kawamoto reported that the 7-membered azepane was ~15-fold less potent than the 6-membered 4-fluopiperidine [32], suggesting limited steric tolerance. While the azetidine (13) and pyrrolidine (14) were both inactive at concentrations up to 10 μM, the piperidine (15) had an IC₅₀ of 87 nM, similar to 2 [31]. However, larger ring sizes (16–18) resulted in a drop in potency, consistent with earlier observations [32]. These results suggest that the 4-amide position has a strict size requirement, and a six-membered aliphatic ring is preferred.

Given the good potency of the piperidine ring at the 4-position, different substituents on this 6-membered ring were examined. Among the methyl substituted analogs, the 4-methylpiperidyl analog (21, IC₅₀ = 160 nM) showed greater potency than the 2- and 3-methylpiperidyl analogs (19, IC₅₀ = 990 nM and 20, IC₅₀ = 260 nM). However, the 4-dimethylpiperidyl (22, IC₅₀ = 1100 nM) demonstrated a considerable potency drop, suggesting limited steric tolerance at this position. Additional substitutions were then investigated with groups of different electronic and steric properties. Small substituents, either electron withdrawing or donating, such as F (23, IC₅₀ = 79 nM) and CN (24, IC₅₀ = 102 nM) all gave similar good potency. However, larger groups such as CF₃ (25, IC₅₀ = 1560 nM) and methoxy (28, IC₅₀ = 640 nM) led to a considerable drop in potency, consistent with the 4-methyl and 4-dimethyl analogs (21 vs. 22). Both acetylenyl (26, IC₅₀ = 46 nM) and methylacetylenyl (27, IC₅₀ = 91 nM) led to good potency. The low potency of the primary amide at the 4-postion of the piperidine (29) and the morpholine (30) suggests polar groups are not tolerated at this position. Finally, an aromatic ring is not favored, either at the 4-position (31: IC₅₀ = 1190 nM, 32: IC₅₀ > 10 μM) or within a bicyclic system (33: IC₅₀ = 4320 nM). These results suggest that a small substitution at the 4-position of the piperidine is well tolerated, although not required, for activity at LPA5. The acetylenyl analog (26) had the greatest potency within this series.

To gain more information on the SARs at the isoquinolone core, we next investigated substitutions at the 6- and 7-positions. In order to compare with the earlier series, we kept the same 3-dimethylaminophenyl group at the 2-postion and a 4-fluoropiperidinyl amide, as present in 2, at the 4-postion. Removal of both methoxy group (34) or the 7-methoxy (35) resulted in total loss of potency, demonstrating their importance for activity. Removal of the 6-methoxy (36, IC₅₀ = 1060 nM) or demethylation of the 6-methoxy (37, IC₅₀ = 1370 nM) also led to considerable drop, although some potency remained. We therefore retained the 7-methoxy group and examined different substituents at the 6-position. The lower potency and inhibitory activity of the trifluoroethyl (39, IC₅₀ = 2500 nM, 74% inhibition) compared to the propyl analog (38, IC₅₀ = 480 nM, 109% inhibition) suggests that electron-withdrawing groups are not favored at this position. All the other alkyl analogs (40–43), either straight chain or cyclic, all showed lower potency than the methoxy analog 23. Finally, naphthalene (44) and pyridylmethyl (45) resulted in total loss of activity. These results suggest that both methoxy groups, particularly the 7-methoxy, are important for LPA5 antagonist activity.

Finally, we examined the SARs at the 2-position of the isoquinolone. The good potency of the 3-dimethylamino phenyl group (e.g. 23) as shown in Table 1 suggests a single aromatic group is sufficient for activity, and we therefore explored several 3-substituted phenyl groups. The 3-isopropyl analog (46, IC₅₀ = 63 nM), in which the nitrogen is removed in the dimethylamino group (15, IC₅₀ = 87 nM), showed almost identical potency, suggesting the amino group is not required for activity. However, the larger tert-butyl (47, IC₅₀ = 173 nM) and the isopropoxy (48, IC₅₀ = 630 nM) both had reduced potency. The introduction of an acetyl group showed a further decrease in potency and inhibition (49, IC₅₀ = 1760 nM, 74% inhibition), and amides (50, 51) were completely inactive, as were the pyridyl analogs (52–54). These results suggest that a non-polar substituent at the 3-position of the phenyl group is preferred. As 2, which showed similar potency in our calcium assay (IC₅₀ = 36 nM) as the literature (IC₅₀ = 38 nM) [31], has a bicyclic system at the 2-position of the isoquinolone, we next examined a series of bicyclic aromatic rings. Interestingly, the reversal of the 1,2-benzoxazole group (55, IC₅₀ = 3900 nM) led to significant loss of potency as well as inhibitory activity (56%). However, the benzofuran (56, IC₅₀ = 48 nM) and benzothiophene (57, IC₅₀ = 32 nM) showed excellent potency. These results demonstrate that the shape of the substituents at this position was well tolerated but the nitrogen in 1,2-benzoxazole (vs. benzofuran) may have unfavorable interactions with the receptor and is therefore detrimental to LPA5 activity. Similarly, the indole analog (58, IC₅₀ = 64 nM) was also potent. The naphthalene analog had an IC₅₀ of 209 nM. Interestingly, throughout this bicyclic series, the methyl group was clearly important for the LPA5 antagonistic activity, as evidenced by the dramatic decrease in potency in the unsubstituted benzofuran and benzothiophene analogs (60 vs. 56; 61 vs. 57; 62 vs 64).

Table 1.

Examination of 4-position analogs and their antagonist potencies in the hLPA5 calcium mobilization assay

No.	R	IC50, nM (% Inhibition)a	No.	R	IC50, nM (% Inhibition)a
4		>10,000b	19		990 ± 190 (98 ± 4%)
5		>10,000b	20		260 ± 50 (104 ± 3%)
6		>10,000b	21		160 ± 30 (105 ± 3%)
7		>10,000b	22		1100 ±60 (90 ± 9%)
8		>10,000b	23		79 ± 10 (102 ± 2%)
9		>10,000b	24		102 ± 9 (89 ± 14%)
10		>10,000b	25		1560 ± 210 (87 ± 5%)
11		>10,000b	26		46 ± 8 (105 ± 2%)
12		320 ± 40 (33 ± 7%)	27		91 ± 6 (106 ± 1%)
13		>10,000b	28		640 ± 160 (89 ± 4%)
14		>10,000b	29		>10,000b
15		87 ± 16 (104 ± 2%)	30		2200 ± 500 (71 ± 2%)
16		301 ± 19 (109 ± 6%)	31		1190 ± 270 (108 ± 3%)
17		>10,000b	32		>10,000b
18		230 ± 50 (103 ± 2%)	33		4320 ± 220 (86 ± 7%)

^aCompounds tested against EC₈₀ (30 nM) of hexadecyl LPA 16:0. Values are the mean IC₅₀ ± SEM of at least three independent experiments performed in duplicate. % Inhibition is calculated with the equation: % Inhibition = (1 − (cmpd signal/hexadecyl LPA 16:0 EC₈₀)) × 100.

^bValues are the mean IC₅₀ ± SEM of two independent experiments performed in duplicate.

Finally, we attempted to combine several favored substituents from both the 2- and 4-positions. In the 2-(3-dimethylaminophenyl) series (Table 1), the 4-fluoropiperidinyl and the unsubstituted piperidinyl at the 4-position provided almost identical potency (15 vs. 23), whereas the 4-acetylenyl analog was ~2-fold more potent than the unsubstituted piperidine (26 vs. 15). However, in the 2-position bicyclic ring series, the 4-fluoropiperidinyl analogs appeared to have lower potency than the corresponding unsubstituted piperidinyl analogs (63 vs. 46; 64 vs. 56; 65 vs. 57). These results suggest that, while both the substituted phenyl and bicyclic rings at the 2-position provided good LPA5 antagonistic potency, they may have slightly differential binding orientation which rendered the 4-fluoro on the piperidinyl ring less favorable in the interactions with the LPA5 receptor. Similar to the 2-(3-dimethylaminophenyl) series (26 vs. 23), the 4-acetylene analog (66, IC₅₀ = 32 nM) was 2-fold more potent than the 4-fluoro analog (65) and was the most potent compound of the entire series.

Activity in LPA5 cAMP Assay.

We then selected several compounds that showed good potency in the calcium mobilization assay and tested them in a TR-FRET Lance^™ Ultra cAMP assay (PerkinElmer), an assay platform that our group uses to measure cAMP accumulation for several other GPCR targets [38–42]. In this cAMP assay using the same stable RH7777-hLPA5 cells, hexadecyl LPA 16:0 has an EC₅₀ value (± SEM) of 95 ± 16 nM, which is about 6-fold less potent compared to the calcium mobilization assay (EC₅₀ = 15 nM). This trend seems to carry over to the antagonists as the cAMP potencies are generally lower compared to the calcium mobilization potencies, although the potency differences varied among the compounds tested. For example, compound 2 has IC₅₀ values of 210 nM and 36 nM in the cAMP and calcium mobilization assays, respectively, a 5.8-fold difference similar to hexadecyl LPA 16:0. Compounds 56 (3.9-fold), 58 (7.2-fold), and 65 (4.9-fold) had similar potency differences in the 4-fold to 7-fold range between the two assays. Despite these potency differences, SAR trends were consistent between the two assays for some of the compounds. For example, compound 3 was 2–3-fold less potent than 2, whereas 65 was ~1.5–2-fold less potent than 2 in both assays. Compound 66, the most potent LPA5 antagonist in the calcium mobilization assay, was only 1.7-fold less potent than 2 in the cAMP assay (210 vs. 350 nM).

However, several other compounds have differential ranking orders between the two assays. For instance, 64 was ~4-fold less potent than 3 and 66 in the calcium assay, but had similar potency in the cAMP assay. In contrast, compounds 23, 24, and 26 were similar or slightly less potent than 2 in the calcium assay, but they were 4 – 10-fold less potent in the cAMP assay. LPA5 is known to couple to both Gα_q/11 proteins, which stimulate intracellular calcium release, and Gα_12/13 proteins [7], which have been shown to mediate cAMP synthesis [43, 44]. It is possible that the potency discrepancies resulted from potential G protein signaling bias with some compounds preferentially signaling through Gα_q/11 proteins and others through Gα_12/13 proteins; however, more work is needed to fully investigate this potential trend. It is interesting to note, however, that compounds 23, 24, and 26 are from the 3-methylphenyl series, whereas 64 is from the bicyclic ring series. This is consistent with the potency differences at the 4-position observed in the calcium assay between the 3-methylphenyl and bicyclic ring series (4-fluopiperidine vs. unsubstituted piperidine, such as 15 vs. 23 and 56 vs. 64), suggesting different binding conformations.

2.3. ADME and PK studies.

Compounds 65 and 66 were two of the most potent compounds from the series with potencies similar or slightly less than 2 and were then further assessed for their preliminary ADME properties in vitro. As shown in Table 5, both compounds showed good aqueous solubility, particularly 65 (96.4 μM). Both compounds had moderate metabolic stability against rat liver microsomes (RLM) in vitro, with intrinsic clearance (C_L) of 48.4 and 74.7 mL/min/kg, respectively. In the MDCK-MDR1 Transwell assay measuring CNS permeability, both 65 and 66 showed excellent permeability, particularly 66. The apparent permeability P_app(A-to-B) of 65 and 66 were 22.2 × 10⁻⁶ cm/s and 30.4 × 10⁻⁶ cm/s and P_app(B-to-A) of 31.1 × 10⁻⁶ cm/s and 38.7 × 10⁻⁶ cm/s, respectively. Neither of the compounds were P-glycoprotein substrates (efflux ratio < 2.5). Compound 66 was highly protein bound against rat plasma with 99.0% protein bound. Finally, in a preliminary snapshot PK study, 30 minutes after male Sprague-Dawley rats were injected i.p. with 66 (17.8 mg/kg), brain concentrations were 652 ng/mL, confirming good brain exposure in vivo.

Table 5.

Selectivity, Solubility, metabolic stability, and CNS permeability of 65 and 66

Property	65	66
LPA1–LPA4 inhibitiona	<50%	<50%
Solubility (μM, pH 7.4)	96.4	19.5
RLM CL (mL/min/kg)	48.4	74.7
Papp (A-to-B) (10−6 cm/s)	22.2	31.1
Papp (B-to-A) (10−6 cm/s)	30.4	38.7
Efflux ratio	1.4	1.2
Rat Plasma Protein Binding		99.0
PK (brain conc.)		652 ng/mL

^a10 μM in Ca²⁺ for LPA1–LPA3 and cAMP for LPA4.

2.4. Receptor Selectivity of 65 and 66

The target selectivity of 65 and 66 for LPA5 was determined first against LPA1-LPA4 receptors and then against a panel of >50 GPCRs offered by the NIMH Psychoactive Drug Screening Program (PDSP). These GPCRs include adrenergic (α_1A, α_1B, α_1D, α_2A, α_2B, α_2C, β1, β2, β3), dopamine (D₁–D₅), GABA_A, histamine (H1–H4), muscarinic (M1–M5), opioid (μ, κ, δ), serotonin (5-HT_1A, 5-HT_1B, 5-HT_1D, 5-HT_1E, 5-HT_2A, 5-HT_2B, 5-HT_2C, 5-HT₃, 5-HT_5A, 5-HT₆, 5-HT_7A), sigma (σ1, σ2), dopamine transporter (DAT), norepinephrine transporter (NET), serotonin transporter (SERT), and Benzodiazepine Rat Brain Site. Both compounds were first assayed at a single concentration of 10 μM against the respective radioligand for each receptor, and for any receptor with 50% or more inhibition, the binding affinity pKi was obtained (see Supplementary Data). Compounds 65 and 66 showed no significant affinity for most of the receptors (< 50% inhibition at 10 μM or pKi ≤ 6) except D3 (66, pKi = 6.42), suggesting good target selectivity for LPA5.

2.5. Antinociceptive Effects of 65 and 66

Compound 2 was reported to attenuate both CCI-induced pain and CFA-induced pain in rats [31]. These two models are well established pain models and represent chronic neuropathic pain and inflammatory pain, respectively, both significant clinical pain challenges. In our hands, 2 produced dose- and time-dependent antiallodynic effects in both pain models in male Sprague-Dawley rats, although its effect appeared less potent and effective in CFA rats than in CCI rats (Figure 4). In the CFA model, a high dose of 32 mg/kg did not fully reverse the nociceptive hypersensitivity, suggesting limited analgesic effects. We therefore tested 65 and 66 in the CFA models, in comparison to 2, for potential improvement in the analgesic potency or efficacy. We found that both 65 and 66 markedly alleviated nociceptive hypersensitivity at doses lower than that for 2 (17.8 vs. 32 mg/kg) and were longer lasting than 2 (~5 h vs ~2 h) in CFA-induced inflammatory pain model (Figure 4), demonstrating improved in vivo effects.

Figure 4.

Antiallodynic effects of LPA5 antagonists in rats. Compound 2 (i.p.) produced dose-dependent antiallodynic effect after CCI surgery (top left) or after CFA injection (top right) in rats (n = 6, p < 0.01, two-way ANOVA). Both 65 and 66 attenuated CFA-induced nociceptive hypersensitivity (bottom panels) at lower doses (17.8 mg/kg) with longer duration than AS2717638 in rats (n = 6/group, p < 0.01).

We then tested 66, which showed slightly greater efficacy than 65 in the CFA model, in the CCI-induced neuropathic pain model. In the CCI model, CCI surgery induced significant mechanical allodynia consistent with our previous studies [45, 46]. Compound 66 dose-dependently and significantly reduced mechanical allodynia and completely reversed this hypersensitivity condition at the dose of 17.8 mg/kg, revealing strong antinociceptive efficacy in this model (left panel, Fig. 5). In addition, we also tested 66 in opioid-induced hyperalgesia (OIH) to explore whether LPA5 antagonists have broad spectrum analgesic activities. In the OIH model, four consecutive injections of fentanyl significantly led to mechanical hypersensitivity evidenced by the reduction in mechanical thresholds, consistent with our previous findings [41]. Compound 66 dose-dependently and significantly mitigated this mechanical hypersensitivity and achieved statistical significance at a small dose of 5.6 mg/kg (i.p.), clearly demonstrating antinociceptive efficacy of 66 (middle panel, Figure 5). Importantly, at the dose of 17.8 mg/kg that fully reversed several painful conditions (Figs 4 and 5), 66 did not have any measurable effect on the locomotor activity in rats (P > 0.05, right panel, Fig. 5), suggesting that the observed antinociceptive effects of 66 were truly due to pain relief and not due to other non-specific behavioral impairment. These results suggest that LPA5 antagonists as described here are effective for a diverse spectrum of clinical painful conditions.

Figure 5.

66 reversed mechanical allodynia in CCI model and fentanyl-induced hyperalgesia in female rats (n = 6/group, p < 0.01). 66 had no significant effect on the locomotion in rats (n = 6/group, p > 0.05).

3. Conclusion

LPA5 plays an important role in chronic pain, particularly neuropathic and inflammatory pain, and KO or blockade of LPA5 has been demonstrated to be effective in several pain models. The present study aims to better understand the SAR on a previously reported LPA5 antagonist 2 and assess these preliminary drug-like properties and in vivo potency and efficacy of these isoquinolone derivatives. To this end, we have designed and synthesized a series of isoquinolones with a variety of substituents at the 2-, 4-, 6- and 7-positions. Among the 2-position substituents, either single aromatic rings (e.g. substituted phenyl) or bicyclic aromatic rings such as benzofuran and benzothiophene provided good biological activities that are comparable to 2 in both the calcium mobilization and cAMP assays. In particular, the methyl group in these bicyclic rings is important for activity, removal of which resulted in considerable drop in potency. 4-Position substituted piperidines were the preferred substituents at the 4-position of the isoquinolone. Methoxy groups at the 6- and 7-position are essential for LPA5 antagonistic activity, removal of which resulted in significant or total loss of activity. Interestingly, potency discrepancies between two biological assays (calcium vs. cAMP) were observed with several compounds (e.g., 23, 24 and 26), suggesting potential biased signaling. Compounds 65 (RLPA-84) and 66 (RLPA-86), as two of the most potent LPA5 antagonists from the series, showed good selectivity against LPA1-LPA4 and >50 other GPCRs. ADME assessment suggests that both 65 and 66 had moderate metabolic stability but excellent aqueous solubility and brain permeability. Both 65 and 66 significantly attenuated nociceptive hypersensitivity in the CFA model at doses lower than that for 2 (17.8 vs. 32 mg/kg) and were longer lasting (~5 h vs ~2 h). In addition, 66 dose-dependently blocked CCI-induced neuropathic pain and opioid-induced hyperalgesia (OIH) at 5.6 mg/kg (i.p.) and at higher doses without affecting the general locomotion in rats, clearly demonstrating antinociception. These results suggest that these isoquinolone derivatives as LPA5 antagonists have promising potency and efficacy as potential analgesics and warrant further investigation.

4. Experimental Section

4.1. Synthesis

All solvents and chemicals were reagent grade. Unless otherwise mentioned, all reagents and solvents were purchased from commercial vendors and used as received. Flash column chromatography was carried out on a Teledyne ISCO CombiFlash Rf system using prepacked columns. Solvents used include hexane, ethyl acetate (EtOAc), dichloromethane, methanol, and Chloroform/methanol/ammonium hydroxide (80:18:2) (CMA-80). Purity and characterization of compounds were established by a combination of HPLC, TLC, mass spectrometry, and NMR analyses. Melting point was recorded by the Mel-Temp II instrument (Laboratory Devices Inc., U.S.). ¹H and ¹³C NMR spectra were recorded on a Bruker Avance DPX-300 (300 MHz) spectrometer and were determined in Chloroform-d, DMSO-d6, or methanol-d4 with tetramethylsilane (TMS) (0.00 ppm) or solvent peaks as the internal reference. Chemical shifts are reported in ppm relative to the reference signal, and coupling constant (J) values are reported in hertz (Hz). Thin layer chromatography (TLC) was performed on EMD precoated silica gel 60 F254 plates, and spots were visualized with UV light or iodine staining. Low resolution mass spectra were obtained using a Waters Alliance HT/Micromass ZQ system (ESI). All test compounds were greater than 95% pure as determined by HPLC on an Agilent 1100 system using an Agilent Zorbax SB-Phenyl, 2.1 mm × 150 mm, 5 μm column using a 15 minute gradient elution of 5–95% solvent B at 1 mL/min followed by 10 minutes at 95% solvent B (solvent A, water with 0.1% TFA; solvent B, acetonitrile with 0.1% TFA and 5% water; absorbance monitored at 220 and 280 nm).

General procedure A: Preparation of dimethyl esters 70, 75 and 82:

To a 2-necks 250 mL flask were introduced brominated benzoic acid (67, 72, or 79, 1 equiv.), diethylmalonate (15 equiv.) and copper bromide (0.1 equiv.). The suspension was degassed and placed under nitrogen at 0 °C. Sodium hydride (2.4 equiv.) was added over 20–25 min. The mixture was then heated to 90 °C and stirred for 2h. After cooling to RT, 300 mL water were added and the mixture was extracted twice with 150 mL dimethyl ether. The brown solid present in the mixture was removed by filtration. The aqueous layer was then acidified with 6N HCl and extracted 4 times with ethyl acetate. Combined ethyl acetate layers were concentrated under vacuum to give crude product 68, 73 or 80 which was used in the next step without further purification.

Intermediate 68 (or 73, 80) was dissolved in THF (0.2 M) and then 2 N NaOH (10 equiv.) was added. The mixture was stirred at 60°C for 3 hours. After the reaction was completed as indicated by TLC, the reaction was cooled to 0°C and acidified with 4 N HCl (12 equiv.). Then the reaction was again stirred at 60°C for another 3 hours, monitored by TLC. THF was evaporated, and then the resulting aqueous layer was extracted with 100 ml ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated to afford intermediate 69 (or 74, 81).

Diacid 69, (or 74, 81, 1 equiv.) was dissolved in MeOH (0.2 M). The solution was cooled to 0°C and SOCl₂ (10 equiv.) was added dropwise. Until completion of addition, the reaction mixture was heated to 65°C and stirred overnight. The volatile was then removed by reduced pressure. The residue was redissolved in DCM and washed with saturated NaHCO₃. The solvent was then removed to give compound dimethyl ester 70 (or 75, 82).

General procedure B: Construction of the ring of 2H-isoquinolin-1-one-4-carboxylic acid 71, 76, or 83:

The dimethyl ester 70 (or 75, 82, 1 equiv.) was mixed with Bredereck’s reagent (3.28 equiv.) and heated to 100°C for 3 hours. Then AcOH (0.25 M) was added followed by the addition of 3-dimethylaminoaniline (3 equiv.). The reaction was then stirred at room temperature for 2 hours. AcOH was then removed under reduced pressure and the residue was dissolved in EtOH (2.5 × Volume of AcOH) followed by the addition of NaOH (8 equiv.). The reaction mixture was then heated to 80°C and stirred for 1 hour. The volatile was then removed by reduced pressure and the residue was suspended in minimum amount of EtOH followed by the addition of water. The precipitate was then collected by filtration and dried by vacuum at 40 °C to afford crude solid 71 (or 76, 83) that is used in the next step without further purification.

General procedure C: Amide coupling:

To a solution of acid 71 (or 76, 83, 1 eqiuv.), amine (1.6 eqiuv.) and HATU (1.3 eqiuv.) in DMF (0.1 M) was added DIPEA (2.2 eqiuv.). The reaction mixture was then stirred overnight and the solvent was removed by reduced pressure. DCM (30 mL) and sat. NaHCO₃ (30 mL) were added. The organic layer was separated and dried by anhydrous magnesium sulfate. The solvent was then removed under reduced pressure and the residue was purified by ISCO to afford pure desired product.

Procedure D: Synthesis of compound 7 from 12:

A solution of 12 (83 mg, 0.2 mmol) in DCM was cooled to −78°C and DIBAL-H (0.3 mL, 0.3 mmol) was added. After 1 hour, the reaction was quenched by saturated aqueous NH₄Cl. DCM (20 mL) was added, and the organic layer was separated and dried. The solvent was then removed and the residue was used in the next step without further purification.

The residue from last step was dissolved in THF (3 mL) and the reaction mixture was cooled to 0°C. Cyclohexylmagnesium chloride (0.12 mL, 1.3 M, 0.15 mmol) was added dropwise. The reaction was then allowed to warm up to room temperature and stirred for 2 hours. The reaction was quenched by saturated NH₄Cl. DCM (20 mL) was added, and the organic layer was separated and dried. The solvent was then removed and the residue was used in the next step without further purification.

The residue from last step was dissolved in CHCl₃ (3 mL) and Dess-Martin reagent (64 mg, 0.15 mmol) was added. The reaction was then stirred for 2 hours at room temperature and quenched by saturated sodium thiosulfate. DCM (20 mL) was added, and the organic layer was separated and dried. The solvent was then removed and the residue was purified by ISCO to give the desired product 12.

For Scheme 1, 3,4-dimethoxy-6-bromobenzoic acid (10 mmol) and dimethyl malonate were used as starting material. General procedure A was followed to afford 70.

Methyl 4,5-dimethoxy-2-(2-methoxy-2-oxoethyl)benzoate (70):

1.40 g pale white solid, yield: 52% for four steps. ¹H NMR (300 MHz, Chloroform-d) δ 7.56 (s, 1H), 6.72 (s, 1H), 3.97 (s, 2H), 3.93 (s, 3H), 3.92 (s, 3H), 3.85 (s, 3H), 3.71 (s, 3H). MS (ESI) m/z: 269.2. [M+H]⁺.

General procedure B was followed starting from 70 (0.1 mmol) to afford 2H-isoquinolin-1-one-4-carboxylic acid 71, which was then subjected to General procedure C to give the final products 4 – 6 and 8–33. For compound 7, procedure D was followed starting from 12.

2-(3-(Dimethylamino)phenyl)-N-ethyl-6,7-dimethoxy-1-oxo-1,2-dihydroisoquinoline-4-carboxam-ide (4):

Yield: 61% for four steps. ¹H NMR (300 MHz, Chloroform-d) δ 7.85 (s, 1H), 7.73 (s, 1H), 7.48 (s, 1H), 7.29 – 7.39 (m, 1H), 6.78 (dd, J = 1.70, 8.48 Hz, 1H), 6.65 – 6.73 (m, 2H), 5.89 (m, 1H), 4.02 (d, J = 8.10 Hz, 6H), 3.49 (dd, J = 5.65, 7.16 Hz, 2H), 2.99 (s, 6H), 1.25 (t, J = 7.25 Hz, 3H). MS (ESI) m/z: 396.2. [M+H]⁺.

2-(3-(Dimethylamino)phenyl)-6,7-dimethoxy-1-oxo-N-pentyl-1,2-dihydroisoquinoline-4-carboxa-mide (5):

Yield: 63% for four steps. ¹H NMR (300 MHz, Chloroform-d) δ 7.86 (s, 1H), 7.71 (s, 1H), 7.47 (s, 1H), 7.34 (t, J = 8.29 Hz, 1H), 6.78 (dd, J = 2.07, 8.29 Hz, 1H), 6.65 – 6.73 (m, 2H), 5.88 (m, 1H), 4.02 (d, J = 6.59 Hz, 6H), 3.39 – 3.51 (m, 2H), 2.99 (s, 6H), 1.58 – 1.67 (m, 2H), 1.37 (qd, J = 3.58, 7.16 Hz, 4H), 0.86 – 0.96 (m, 3H). MS (ESI) m/z: 438.2. [M+H]⁺.

N-Cyclopentyl-2-(3-(dimethylamino)phenyl)-6,7-dimethoxy-1-oxo-1,2-dihydroisoquinoline-4-ca-rboxamide (6):

Yield: 57% for four steps. ¹H NMR (300 MHz, Chloroform-d) δ 7.86 (s, 1H), 7.70 (s, 1H), 7.45 (s, 1H), 7.35 (t, J = 8.29 Hz, 1H), 6.78 (dd, J = 1.98, 8.19 Hz, 1H), 6.65 – 6.73 (m, 2H), 5.84 (d, J = 7.16 Hz, 1H), 4.40 (d, J = 6.78 Hz, 1H), 4.02 (d, J = 7.16 Hz, 6H), 2.99 (s, 6H), 2.11 (dd, J = 5.75, 12.72 Hz, 2H), 1.62 – 1.77 (m, 4H), 1.47 (dd, J = 6.59, 13.37 Hz, 2H). MS (ESI) m/z: 436.2. [M+H]⁺.

4-(Cyclohexanecarbonyl)-2-(3-(dimethylamino)phenyl)-6,7-dimethoxyisoquinolin-1(2H)-one (7):

Yield: 47% for the three steps from compound 12. ¹H NMR (300 MHz, Chloroform-d) δ 8.47 (s, 1H), 7.99 (s, 1H), 7.86 (s, 1H), 7.34 – 7.45 (m, 1H), 6.78 – 6.89 (m, 1H), 6.67 – 6.77 (m, 2H), 4.06 (s, 3H), 4.00 (s, 3H), 3.02 (s, 6H), 1.77 – 1.92 (m, 4H), 1.73 (m, 1H), 1.64 (m, 6H). MS (ESI) m/z: 435.2. [M+H]⁺.

2-(3-(Dimethylamino)phenyl)-6,7-dimethoxy-1-oxo-N-phenyl-1,2-dihydroisoquinoline-4-carbox-amide (8):

Yield: 65% for four steps. ¹H NMR (300 MHz, Chloroform-d) δ 7.86 (s, 1H), 7.69 – 7.76 (m, 2H), 7.67 (s, 1H), 7.60 (s, 1H), 7.57 (s, 1H), 7.35 – 7.42 (m, 2H), 7.33 (s, 1H), 7.17 (s, 1H), 6.74 – 6.80 (m, 1H), 6.67 – 6.73 (m, 2H), 4.00 (d, J = 1.70 Hz, 6H), 2.98 (s, 6H). MS (ESI) m/z: 444.2. [M+H]⁺.

N-Benzyl-2-(3-(dimethylamino)phenyl)-6,7-dimethoxy-1-oxo-1,2-dihydroisoquinoline-4-carboxa-mide (9):

Yield: 67% for four steps. ¹H NMR (300 MHz, Chloroform-d) δ 7.86 (s, 1H), 7.71 (s, 1H), 7.51 (s, 1H), 7.35 – 7.39 (m, 4H), 7.33 (m, 2H), 6.76 (d, J = 7.91 Hz, 1H), 6.63 – 6.72 (m, 2H), 6.16 (m, 1H), 4.64 (d, J = 5.65 Hz, 2H), 3.99 (d, J = 8.10 Hz, 6H), 2.98 (s, 6H). MS (ESI) m/z: 458.2. [M+H]⁺.

2-(3-(Dimethylamino)phenyl)-6,7-dimethoxy-N,N-dimethyl-1-oxo-1,2-dihydroisoquinoline-4-car-boxamide (10):

Yield: 62% for four steps. ¹H NMR (300 MHz, Chloroform-d) δ 7.89 (s, 1H), 7.29 – 7.38 (m, 1H), 7.25 (s, 1H), 6.98 (s, 1H), 6.76 (d, J = 9.42 Hz, 1H), 6.66 – 6.73 (m, 2H), 4.00 (d, J = 5.27 Hz, 6H), 3.12 (m, 6H), 2.98 (s, 6H). MS (ESI) m/z: 396.2. [M+H]⁺.

2-(3-(Dimethylamino)phenyl)-6,7-dimethoxy-N-methyl-1-oxo-N-(pyridin-4-ylmethyl)-1,2-dihydr-oisoquinoline-4-carboxamide (11):

Yield: 70% for four steps. ¹H NMR (300 MHz, Chloroform-d) δ 8.61 (d, J = 5.84 Hz, 2H), 7.89 (s, 1H), 7.31 (m, 4H), 6.91 (m, 1H), 6.75 (d, J = 8.10 Hz, 1H), 6.66 (m, 2H), 4.74 (m, 2H), 4.00 (s, 3H), 3.91 (m, 3H), 3.05 (m, 3H), 2.97 (s, 6H). MS (ESI) m/z: 473.2. [M+H]⁺.

2-(3-(Dimethylamino)phenyl)-N,6,7-trimethoxy-N-methyl-1-oxo-1,2-dihydroisoquinoline-4-carb-oxamide (12):

Yield: 51% for four steps. ¹H NMR (300 MHz, Chloroform-d) δ 7.88 (s, 1H), 7.51 (s, 1H), 7.34 (t, J = 8.29 Hz, 1H), 7.26 (s, 1H), 6.74 – 6.81 (m, 1H), 6.67 – 6.73 (m, 2H), 4.01 (s, 6H), 3.63 (s, 3H), 3.40 (s, 3H), 2.99 (s, 6H). MS (ESI) m/z: 412.2. [M+H]⁺.

4-(Azetidine-1-carbonyl)-2-(3-(dimethylamino)phenyl)-6,7-dimethoxyisoquinolin-1(2H)-one (13):

Yield: 69% for four steps. ¹H NMR (300 MHz, Chloroform-d) δ 7.87 (s, 1H), 7.58 (s, 1H), 7.29 – 7.38 (m, 2H), 6.77 (dd, J = 1.98, 7.82 Hz, 1H), 6.64 – 6.72 (m, 2H), 4.22 (t, J = 7.72 Hz, 4H), 4.04 (s, 3H), 4.01 (s, 3H), 2.99 (s, 6H), 2.33 (t, J = 7.82 Hz, 2H). MS (ESI) m/z: 408.2. [M+H]⁺.

2-(3-(Dimethylamino)phenyl)-6,7-dimethoxy-4-(pyrrolidine-1-carbonyl)isoquinolin-1(2H)-one (14):

Yield: 71% for four steps. ¹H NMR (300 MHz, Chloroform-d) δ 7.88 (s, 1H), 7.29 – 7.36 (m, 2H), 7.14 (s, 1H), 6.73 – 6.79 (m, 1H), 6.65 – 6.73 (m, 2H), 4.00 (d, J = 4.52 Hz, 6H), 3.70 (m, 2H), 3.45 (m, 2H), 2.98 (s, 6H), 1.95 (m, 4H). MS (ESI) m/z: 422.2. [M+H]⁺.

2-(3-(Dimethylamino)phenyl)-6,7-dimethoxy-4-(piperidine-1-carbonyl)isoquinolin-1(2H)-one (15):

Yield: 65% for four steps. ¹H NMR (300 MHz, Chloroform-d) δ 7.89 (s, 1H), 7.28 – 7.38 (m, 1H), 7.23 (s, 1H), 6.99 (s, 1H), 6.74 – 6.80 (m, 1H), 6.67 – 6.73 (m, 2H), 4.00 (d, J = 4.71 Hz, 6H), 3.34 – 3.82 (m, 4H), 2.98 (s, 6H), 1.57 – 1.78 (m, 6H). MS (ESI) m/z: 436.2. ¹³C NMR (75 MHz, Chloroform-d) δ 166.60, 160.80, 153.81, 151.30, 149.74, 142.00, 130.25, 129.74, 129.58, 120.43, 114.37, 113.36, 112.26, 110.76, 108.68, 104.38, 56.20, 56.16, 40.45, 26.57, 26.40, 24.47.

[M+H]⁺.

4-(4-(Azocan-1-yl)piperidine-1-carbonyl)-2-(3-(dimethylamino)phenyl)-6,7-dimethoxyisoquinoli-n-1(2H)-one (16):

Yield: 54% for four steps. ¹H NMR (300 MHz, Chloroform-d) δ 7.89 (s, 1H), 7.30 – 7.39 (m, 1H), 7.18 (s, 1H), 6.93 (s, 1H), 6.76 (d, J = 9.80 Hz, 1H), 6.67 – 6.74 (m, 2H), 3.99 (d, J = 10.36 Hz, 6H), 3.70 (m, 2H), 3.42 (m, 2H), 2.98 (s, 6H), 1.92 (m, 2H), 1.59 – 1.67 (m, 8H). MS (ESI) m/z: 464.2. [M+H]⁺.

4-(4-(7-Azabicyclo[2.2.1]heptan-7-yl)piperidine-1-carbonyl)-2-(3-(dimethylamino)phen-yl)-6,7-dimethoxyisoquinolin-1(2H)-one (17):

Yield: 66% for four steps. ¹H NMR (300 MHz, Chloroform-d) d 7.88 (s, 1H), 7.39 (d, J = 0.57 Hz, 1H), 7.29 – 7.37 (m, 2H), 6.74 – 6.80 (m, 1H), 6.68 – 6.73 (m, 2H), 4.55 – 4.94 (m, 1H), 4.05 – 4.35 (m, 1H), 4.00 (d, J = 9.04 Hz, 6H), 2.99 (s, 6H), 1.86 (m, 4H), 1.53 (d, J = 7.72 Hz, 4H). MS (ESI) m/z: 448.2. [M+H]⁺.

4-(4-(8-Azabicyclo[3.2.1]octan-8-yl)piperidine-1-carbonyl)-2-(3-(dimethylamino)pheny-l)-6,7-dimethoxyisoquinolin-1(2H)-one (18):

Yield: 68% for four steps. ¹H NMR (300 MHz, Chloroform-d) δ 7.89 (s, 1H), 7.29 – 7.40 (m, 2H), 7.08 (s, 1H), 6.67 – 6.80 (m, 3H), 3.90–4.10 (m, 8H), 2.98 (s, 6H), 2.06–1.54 (m, 10H). MS (ESI) m/z: 462.2. [M+H]⁺.

(±)-2-(3-(Dimethylamino)phenyl)-6,7-dimethoxy-4-(2-methylpiperidine-1-carbonyl)isoquinolin-1(2-H)-one (19):

Yield: 59% for four steps. ¹H NMR (300 MHz, Chloroform-d) d 7.89 (s, 1H), 7.29 – 7.37 (m, 1H), 7.19 (m, 1H), 6.89 – 7.08 (m, 1H), 6.67 – 6.79 (m, 3H), 4.15 – 4.62 (m, 1H), 3.93 – 4.06 (m, 6H), 3.05 – 3.69 (m, 2H), 2.98 (s, 6H), 1.68 (m, 5H), 1.25 (m, 4H). MS (ESI) m/z: 450.2. [M+H]⁺.

(±)-2-(3-(Dimethylamino)phenyl)-6,7-dimethoxy-4-(3-methylpiperidine-1-carbonyl)isoquinolin-1(2-H)-one (20):

Yield: 61% for four steps. ¹H NMR (300 MHz, Chloroform-d) δ 7.89 (s, 1H), 7.35 (t, J = 8.01 Hz, 1H), 7.22 (m, 1H), 6.98 (m, 1H), 6.77 (m, 3H), 4.00 (d, J = 7.16 Hz, 6H), 3.32 – 3.76 (m, 2H), 2.99 (s, 6H), 2.80 (s, 2H), 1.87 (m, 1H), 1.10 – 1.52 (m, 4H), 0.80 – 1.00 (m, 3H). MS (ESI) m/z: 450.2. [M+H]⁺.

2-(3-(Dimethylamino)phenyl)-6,7-dimethoxy-4-(4-methylpiperidine-1-carbonyl)isoquinolin-1(2-H)-one (21):

Yield: 65% for four steps. ¹H NMR (300 MHz, Chloroform-d) δ 7.89 (s, 1H), 7.29 – 7.38 (m, 1H), 7.23 (m, 1H), 6.98 (m, 1H), 6.76 (d, J = 9.80 Hz, 1H), 6.67 – 6.73 (m, 2H), 4.00 (d, J = 5.65 Hz, 6H), 3.27 – 3.82 (m, 2H), 2.84 – 3.05 (m, 8H), 1.67 (m, 3H), 1.05 – 1.26 (m, 2H), 0.97 (d, J = 6.22 Hz, 3H). MS (ESI) m/z: 450.2. [M+H]⁺.

2-(3-(Dimethylamino)phenyl)-4-(4,4-dimethylpiperidine-1-carbonyl)-6,7-dimethoxyisoquinolin-1-(2H)-one (22):

Yield: 62% for four steps. ¹H NMR (300 MHz, Chloroform-d) d 7.89 (s, 1H), 7.28 – 7.38 (m, 1H), 7.23 (s, 1H), 6.98 (s, 1H), 6.76 (d, J = 8.48 Hz, 1H), 6.67 – 6.73 (m, 2H), 4.00 (d, J = 3.96 Hz, 6H), 3.30 – 3.85 (m, 4H), 2.98 (s, 6H), 1.40 (m, 4H), 1.00 (s, 6H). MS (ESI) m/z: 464.2 [M+H]⁺.

2-(3-(Dimethylamino)phenyl)-4-(4-fluoropiperidine-1-carbonyl)-6,7-dimethoxyisoquinolin-1(2H-)-one (23):

Yield: 68% for four steps. ¹H NMR (300 MHz, Chloroform) d 7.89 (s, 1H), 7.32 (d, J = 8.48 Hz, 1H), 7.26 – 7.28 (m, 1H), 6.97 (s, 1H), 6.74 – 6.80 (m, 1H), 6.67 – 6.72 (m, 2H), 4.75 – 5.06 (m, 1H), 4.00 (d, J = 7.16 Hz, 6H), 3.66 (m, 4H), 2.98 (s, 6H), 1.92 (m, 4H). MS (ESI) m/z: 454.2 [M+H]⁺.

1-(2-(3-(Dimethylamino)phenyl)-6,7-dimethoxy-1-oxo-1,2-dihydroisoquinoline-4-carbonyl)piper-idine-4-carbonitrile (24):

Yield: 62% for four steps. ¹H NMR (300 MHz, Chloroform-d) δ 7.89 (s, 1H), 7.33 (t, J = 8.29 Hz, 1H), 7.26 – 7.28 (m, 1H), 6.95 (s, 1H), 6.74 – 6.82 (m, 1H), 6.63 – 6.73 (m, 2H), 3.95 – 4.06 (m, 6H), 3.82 (m, 2H), 3.68 (m, 2H), 2.89 – 3.03 (m, 7H), 1.92 (m, 4H). ¹³C NMR (75 MHz, Chloroform-d) δ 166.99, 160.71, 153.99, 151.32, 149.92, 141.81, 130.97, 129.85, 129.21, 120.48, 120.37, 114.20, 112.37, 112.11, 110.62, 108.83, 104.11, 77.22, 56.23 (2×OCH₃), 40.43 (NMe₂), 28.95, 26.31. MS (ESI) m/z: 461.2 [M+H]⁺.

2-(3-(Dimethylamino)phenyl)-6,7-dimethoxy-4-(4-(trifluoromethyl)piperidine-1-carbonyl)isoqui-nolin-1(2H)-one (25):

Yield: 57% for four steps. ¹H NMR (300 MHz, Chloroform-d) δ 7.89 (s, 1H), 7.29 – 7.38 (m, 1H), 7.25 (s, 1H), 6.95 (m, 1H), 6.76 (d, J = 8.48 Hz, 1H), 6.65 – 6.72 (m, 2H), 4.00 (d, J = 8.48 Hz, 7H), 2.58 – 3.23 (m, 9H), 2.23 – 2.42 (m, 1H), 1.74 – 2.18 (m, 2H), 1.36 – 1.55 (m, 2H). MS (ESI) m/z: 504.2 [M+H]⁺.

2-(3-(Dimethylamino)phenyl)-4-(4-ethynylpiperidine-1-carbonyl)-6,7-dimethoxyisoquinolin-1(2-H)-one (26):

Yield: 69% for four steps. ¹H NMR (300 MHz, Chloroform-d) δ 7.89 (s, 1H), 7.31 (d, J = 8.29 Hz, 1H), 7.24 (s, 1H), 6.97 (s, 1H), 6.76 (d, J = 9.42 Hz, 1H), 6.67 – 6.72 (m, 2H), 3.97 – 4.05 (m, 6H), 3.67 – 3.96 (m, 2H), 3.40 – 3.61 (m, 2H), 2.98 (s, 6H), 2.80 (s, 1H), 2.65 – 2.78 (m, 1H), 1.79 – 1.94 (m, 2H), 1.62 – 1.76 (m, 2H). MS (ESI) m/z: 460.2 [M+H]⁺.

2-(3-(Dimethylamino)phenyl)-6,7-dimethoxy-4-(4-(prop-1-yn-1-yl)piperidine-1-carbonyl)isoquin-olin-1(2H)-one (27):

Yield: 61% for four steps. ¹H NMR (300 MHz, Chloroform-d) δ 7.89 (s, 1H), 7.29 – 7.37 (m, 1H), 7.23 (s, 1H), 6.98 (s, 1H), 6.77 (s, 1H), 6.65 – 6.72 (m, 2H), 3.96 – 4.04 (m, 6H), 3.13 – 3.94 (m, 4H), 2.98 (s, 6H), 2.61 – 2.72 (m, 1H), 1.80 (d, J = 2.26 Hz, 7H). MS (ESI) m/z: 474.2 [M+H]⁺.

2-(3-(Dimethylamino)phenyl)-6,7-dimethoxy-4-(4-methoxypiperidine-1-carbonyl)isoquinolin-1(2-H)-one (28):

Yield: 60% for four steps. ¹H NMR (300 MHz, Chloroform-d) δ 7.89 (s, 1H), 7.31 (d, J = 8.29 Hz, 1H), 7.24 (s, 1H), 6.97 (s, 1H), 6.76 (d, J = 9.42 Hz, 1H), 6.66 – 6.72 (m, 2H), 4.00 (d, J = 6.97 Hz, 8H), 3.49 (m, 3H), 3.36 (s, 3H), 2.98 (s, 6H), 1.78 – 1.98 (m, 2H), 1.60 – 1.71 (m, 2H). MS (ESI) m/z: 466.2 [M+H]⁺.

1-(2-(3-(Dimethylamino)phenyl)-6,7-dimethoxy-1-oxo-1,2-dihydroisoquinoline-4-carbonyl)pipe-ridine-4-carboxamide (29):

Yield: 54% for four steps. ¹H NMR (300 MHz, Chloroform-d) d 7.89 (s, 1H), 7.29 – 7.37 (m, 1H), 7.23 – 7.25 (m, 1H), 6.96 – 7.03 (m, 1H), 6.73 – 6.80 (m, 1H), 6.66 – 6.72 (m, 2H), 5.17 – 5.56 (m, 2H), 4.23 – 4.98 (m, 1H), 4.01 (s, 7H), 2.98 (s, 8H), 2.38 – 2.50 (m, 1H), 1.65 – 2.09 (m, 4H). MS (ESI) m/z: 479.2 [M+H]⁺.

2-(3-(Dimethylamino)phenyl)-6,7-dimethoxy-4-(morpholine-4-carbonyl)isoquinolin-1(2H)-one (30):

Yield: 60% for four steps. ¹H NMR (300 MHz, Chloroform-d) δ 7.89 (s, 1H), 7.29 – 7.39 (m, 1H), 7.27 (s, 1H), 7.00 (s, 1H), 6.73 – 6.80 (m, 1H), 6.66 – 6.72 (m, 2H), 4.01 (s, 6H), 3.71 (m, 8H), 2.98 (s, 6H). MS (ESI) m/z: 438.2 [M+H]⁺.

2-(3-(Dimethylamino)phenyl)-6,7-dimethoxy-4-(4-phenylpiperidine-1-carbonyl)isoquinolin-1(2-H)-one (31):

Yield: 52% for four steps. ¹H NMR (300 MHz, Chloroform-d) δ 7.90 (s, 1H), 7.31 (m, 4H), 7.14 – 7.25 (m, 4H), 6.74 – 6.79 (m, 1H), 6.68 – 6.74 (m, 2H), 4.01 (m, 8H), 2.98 (m, 8H), 2.76 – 2.87 (m, 1H), 1.84 – 2.11 (m, 2H), 1.61 – 1.82 (m, 2H). MS (ESI) m/z: 512.2 [M+H]⁺.

4-(4-(2,4-Difluorophenyl)piperidine-1-carbonyl)-2-(3-(dimethylamino)phenyl)-6,7-dimethoxyiso-quinolin-1(2H)-one (32):

Yield: 58% for four steps. ¹H NMR (300 MHz, Chloroform-d) δ 7.90 (s, 1H), 7.32 (m, 2H), 7.07 – 7.15 (m, 1H), 6.68 – 6.89 (m, 6H), 4.01 (m, 8H), 3.03 – 3.17 (m, 2H), 2.98 (m, 7H), 1.79 – 2.05 (m, 2H), 1.59 – 1.73 (m, 2H). MS (ESI) m/z: 548.2 [M+H]⁺.

2-(3-(Dimethylamino)phenyl)-6,7-dimethoxy-4-(1,2,3,4-tetrahydroquinoline-1-carbonyl)isoquin-olin-1(2H)-one (33):

Yield: 51% for four steps. ¹H NMR (300 MHz, Chloroform-d) d 7.87 (s, 1H), 7.41 (s, 1H), 7.21 (t, J = 8.01 Hz, 1H), 7.13 (d, J = 6.78 Hz, 1H), 6.99 – 7.06 (m, 1H), 6.95 (s, 1H), 6.92 (s, 1H), 6.80 – 6.86 (m, 1H), 6.68 (dd, J = 2.35, 8.19 Hz, 1H), 6.25 – 6.39 (m, 2H), 4.02 (d, J = 5.84 Hz, 6H), 3.92 – 3.99 (m, 1H), 2.94 – 3.01 (m, 1H), 2.90 (s, 6H), 2.80 (t, J = 6.59 Hz, 2H), 2.06 (t, J = 6.31 Hz, 2H). MS (ESI) m/z: 484.2 [M+H]⁺.

General procedure

A was followed to afford 75a, 75b, and 75c, starting from dimethyl malonate and 6-bromobenzoic acid (10 mmol), 3-methoxy-6-bromobenzoic acid (10 mmol), and 4-methoxy-6-bromobenzoic acid (10 mmol), respectively.

Methyl 2-(2-methoxy-2-oxoethyl)benzoate (75a):

1.02 g white solid, yield: 49% for four steps. ¹H NMR (300 MHz, Chloroform-d) δ 8.02 (dd, J = 1.22, 7.82 Hz, 1H), 7.44 – 7.55 (m, 1H), 7.32 – 7.42 (m, 1H), 7.26 (dd, J = 1.51, 6.03 Hz, 1H), 4.01 (s, 2H), 3.87 (s, 3H), 3.70 (s, 3H). MS (ESI) m/z: 209.2. [M+H]⁺.

Methyl 5-methoxy-2-(2-methoxy-2-oxoethyl)benzoate (75b):

1.07 g white solid, yield: 45% for four steps. ¹H NMR (300 MHz, Chloroform-d) δ 7.53 (dd, J = 2.83, 5.27 Hz, 1H), 7.34 (d, J = 8.67 Hz, 0.5 H), 7.17 (d, J = 8.29 Hz, 0.5 H), 6.99 – 7.11 (m, 1H), 3.69 – 3.94 (m, 11H). MS (ESI) m/z: 239.2. [M+H]⁺.

Methyl 4-methoxy-2-(2-methoxy-2-oxoethyl)benzoate (75c):

1.33 g white solid, yield: 56% for four steps. ¹H NMR (300 MHz, Chloroform-d) δ 7.95 – 8.08 (m, 1H), 6.86 – 6.96 (m, 1H), 6.74 – 6.85 (m, 1H), 3.70 – 3.99 (m, 11H). MS (ESI) m/z: 239.2. [M+H]⁺.

Final compounds 34, 35 and 36 were obtained following General procedure B using dimethyl esters 75a, 75b and 75c (3 mmol), respectively, to afford the corresponding 2H-isoquinolin-1-one-4-carboxylic acids, followed by General procedure C.

2-(3-(Dimethylamino)phenyl)-4-(4-fluoropiperidine-1-carbonyl)isoquinolin-1(2H)-one (34):

Yield: 62% for four steps. ¹H NMR (300 MHz, Chloroform-d) δ 8.53 (d, J = 7.72 Hz, 1H), 7.68 – 7.79 (m, 1H), 7.52 – 7.63 (m, 2H), 7.29 – 7.39 (m, 2H), 6.76 (d, J = 8.48 Hz, 1H), 6.67 – 6.74 (m, 2H), 5.00 (m, 1H), 3.64 (m, 4H), 2.98 (s, 6H), 1.92 (m, 4H). MS (ESI) m/z: 394.2. [M+H]⁺.

2-(3-(Dimethylamino)phenyl)-4-(4-fluoropiperidine-1-carbonyl)-7-methoxyisoquinolin-1(2H)-on-e (35):

Yield: 58% for four steps. ¹H NMR (300 MHz, Chloroform-d) δ 7.93 (d, J = 2.83 Hz, 1H), 7.52 (d, J = 8.85 Hz, 1H), 7.29 – 7.37 (m, 2H), 7.24 (s, 1H), 6.76 (d, J = 8.10 Hz, 1H), 6.67 – 6.73 (m, 2H), 4.78 – 5.03 (m, 1H), 3.94 (s, 3H), 3.62 (m, 4H), 2.98 (s, 6H), 1.91 (m, 4H). MS (ESI) m/z: 424.2. [M+H]⁺.

2-(3-(Dimethylamino)phenyl)-4-(4-fluoropiperidine-1-carbonyl)-6-methoxyisoquinolin-1(2H)-on-e (36):

Yield: 65% for four steps. ¹H NMR (300 MHz, Chloroform-d) δ 8.44 (d, J = 8.85 Hz, 1H), 7.28 – 7.37 (m, 2H), 7.12 (dd, J = 2.45, 9.04 Hz, 1H), 6.95 (m, 1H), 6.75 (dd, J = 2.26, 8.48 Hz, 1H), 6.69 (dd, J = 1.60, 3.86 Hz, 2H), 4.99 (m, 1H), 3.91 (s, 3H), 3.65 (m, 4H), 2.98 (s, 6H), 1.93 (m, 4H). MS (ESI) m/z: 424.2. [M+H]⁺.

The synthesis of intermediate 79: To a solution of commercially available 77 (2.31 g, 10.0 mmol) in DMF (10 mL), benzyl bromide (1.97 g, 11.5 mmol) was added followed by the addition of potassium carbonate (2.07 g, 15.0 mmol). The reaction was heated at 60°C for 2 hours. Water (50 mL) and MTBT (80 mL) were then added. The organic layer was separated and dried. The solvent was then removed under reduced pressure and the residue (compound 78) was used in the next step without further purification.

At 0°C, compound 78 from last step (3.21 g, 10.0 mmol) was dissolved in the mixture of t-BuOH (5 mL), THF (20 mL) and H₂O (10 mL). Then NaH₂PO₄ (9.60 g, 80.0 mmol) was added followed by the addition of H₂O₂ (6.2 mL, 200 mmol). Then NaClO₂ (3.62 g, 40.0 mmol) in H₂O (10 mL) was added. The reaction was then monitored by TLC. After completion, the organic layer was separated and the aqueous layer was then acidified by 1 N HCl and the solid was then collected by filtration to afford compound 79.

4-(Benzyloxy)-2-bromo-5-methoxybenzoic acid (79):

3.17 g as a white solid. Yield: 94% over two steps. ¹H NMR (300 MHz, DMSO-d₆) δ 10.93 – 14.85 (m, 1H), 7.27 – 7.50 (m, 7H), 5.18 (s, 2H), 3.80 (s, 3H). MS (ESI) m/z: 337.0 [M+H]⁺.

6-(Benzyloxy)-2-(3-(dimethylamino)phenyl)-4-(4-fluoropiperidine-1-carbonyl)-7-methoxyisoqui-nolin-1(2H)-one (84).

Compound 84 was obtained from 79 (3.17 g 0.94 mmol) following General procedure A and then General procedure B. 3.1 g brown solid, yield: 46.5% over seven steps. ¹H NMR (300 MHz, Chloroform-d) δ 7.88 (s, 1H), 7.38 (dd, J = 7.91, 9.04 Hz, 1H), 7.25 (s, 1H), 6.92 – 6.99 (m, 3H), 6.87 (d, J = 7.91 Hz, 1H), 4.77 – 5.07 (m, 1H), 4.00 (d, J = 6.78 Hz, 6H), 3.83 – 3.88 (m, 4H), 3.65 (m, 4H), 3.16 – 3.24 (m, 4H), 1.93 (m, 4H). MS (ESI) m/z: 530.2. [M+H]⁺.

2-(3-(Dimethylamino)phenyl)-4-(4-fluoropiperidine-1-carbonyl)-6-hydroxy-7-methoxyisoquinoli-n-1(2H)-one (37).

Compound 84 (3.0 g, 5.625 mmol) was dissolved in the mixture of DCM (100 mL) and MeOH (20 mL) and Pd/C (300 mg) was added. The reaction was then subjected to hydrogen atmosphere (40 psi) for 12 hours. The reaction mixture was filtered and the filtrate was concentrated to afford pure compound 37 (2.37 g) as a yellow foam, yield: 96%. ¹H NMR (300 MHz, Chloroform-d) δ 7.91 (s, 1H), 7.29 – 7.37 (m, 1H), 7.26 – 7.28 (m, 1H), 7.05 (s, 1H), 6.75 (dd, J = 1.51, 7.35 Hz, 1H), 6.67 – 6.72 (m, 2H), 6.27 (s, 1H), 4.75 – 5.05 (m, 1H), 4.03 (s, 3H), 3.62 (m, 4H), 2.98 (s, 6H), 1.91 (m, 4H). MS (ESI) m/z: 440.2. [M+H]⁺.

Compound 37 (1 equiv.) was then treated with the corresponding alkyl bromide or alkyl iodide (0.12 equiv.) in the presence of potassium carbonate (0.15 equiv.) and catalytic 18-crown-6 (0.01 equiv.) in acetonitrile (0.1 M) in sealed tube at 100°C for 12 hours. Water (5 mL) and DCM (10 mL) were then added. The organic layer was separated and dried. The solvent was then removed under reduced pressure and the residue was purified by ISCO to afford pure desired product 38 –45.

2-(3-(Dimethylamino)phenyl)-4-(4-fluoropiperidine-1-carbonyl)-7-methoxy-6-propoxyisoquinoli-n-1(2H)-one (38):

Yield: 78%. ¹H NMR (300 MHz, Chloroform-d) d 7.86 (s, 1H), 7.37 – 7.49 (m, 1H), 6.94 (s, 5H), 4.79 – 5.06 (m, 1H), 4.03 – 4.12 (m, 2H), 3.99 (s, 3H), 3.58 – 3.92 (m, 4H), 3.06 (s, 6H), 1.86 – 2.02 (m, 6H), 1.09 (t, J = 7.44 Hz, 3H). ¹³C NMR (75 MHz, Chloroform-d) δ 166.85, 160.77, 153.63, 150.52, 150.20, 141.99, 130.30, 129.94, 129.32, 120.15, 115.72, 113.08, 112.95, 111.63, 108.88, 105.18, 88.43, 86.14, 70.63, 56.21, 41.06, 31.73, 31.47, 22.30, 10.41. MS (ESI) m/z: 482.2. [M+H]⁺.

2-(3-(Dimethylamino)phenyl)-4-(4-fluoropiperidine-1-carbonyl)-7-methoxy-6-(2,2,2-trifluoroeth-oxy)isoquinolin-1(2H)-one (39):

Yield: 70%. ¹H NMR (300 MHz, Chloroform-d) δ 7.95 (s, 1H), 7.30 – 7.40 (m, 1H), 7.25 – 7.26 (m, 1H), 7.10 (s, 1H), 6.77 (dd, J = 1.98, 8.01 Hz, 1H), 6.63 – 6.72 (m, 2H), 4.78 – 5.06 (m, 1H), 4.52 (q, J = 8.10 Hz, 2H), 4.00 (s, 3H), 3.64 (d, J = 8.85 Hz, 4H), 2.98 (s, 6H), 1.92 (m, 4H). MS (ESI) m/z: 522.2. [M+H]⁺.

2-(3-(Dimethylamino)phenyl)-4-(4-fluoropiperidine-1-carbonyl)-7-methoxy-6-(pentyloxy)isoquin-olin-1(2H)-one (40):

40 mg off-white solid, yield: 78%. ¹H NMR (300 MHz, Chloroform-d) d 7.88 (s, 1H), 7.28 – 7.37 (m, 1H), 7.25 (s, 1H), 6.94 (s, 1H), 6.73 – 6.79 (m, 1H), 6.65 – 6.72 (m, 2H), 4.76 – 5.05 (m, 1H), 4.09 (t, J = 6.88 Hz, 2H), 3.99 (s, 3H), 3.56 – 3.81 (m, 4H), 2.98 (s, 6H), 1.76 – 2.00 (m, 6H), 1.36 – 1.51 (m, 4H), 0.92 – 0.98 (m, 3H). MS (ESI) m/z: 510.2. [M+H]⁺.

2-(3-(Dimethylamino)phenyl)-4-(4-fluoropiperidine-1-carbonyl)-6-isopropoxy-7-methoxyisoquin-olin-1(2H)-one (41):

41 mg off-white solid, yield: 85%. ¹H NMR (300 MHz, Chloroform-d) d 7.88 (s, 1H), 7.31 (d, J = 8.29 Hz, 1H), 7.24 (m, 1H), 6.96 (s, 1H), 6.77 (s, 1H), 6.66 – 6.72 (m, 2H), 4.79 – 5.07 (m, 1H), 4.63 – 4.75 (m, 1H), 3.98 (s, 3H), 3.65 (m, 4H), 2.98 (s, 6H), 1.92 (m, 4H), 1.44 (d, J = 6.03 Hz, 6H). MS (ESI) m/z: 482.2. [M+H]⁺.

6-(Cyclopentyloxy)-2-(3-(dimethylamino)phenyl)-4-(4-fluoropiperidine-1-carbonyl)-7-methoxyis-oquinolin-1(2H)-one (42):

Yield: 82%. ¹H NMR (300 MHz, Chloroform-d) δ 7.87 (s, 1H), 7.29 – 7.37 (m, 1H), 7.25 (s, 1H), 6.93 (s, 1H), 6.73 – 6.79 (m, 1H), 6.66 – 6.72 (m, 2H), 4.86 (td, J = 3.01, 6.03 Hz, 2H), 3.97 (s, 3H), 3.50 – 3.93 (m, 4H), 2.98 (s, 6H), 1.74 – 2.15 (m, 10H), 1.62 – 1.72 (m, 2H). MS (ESI) m/z: 508.2. [M+H]⁺.

6-(Cyclohexyloxy)-2-(3-(dimethylamino)phenyl)-4-(4-fluoropiperidine-1-carbonyl)-7-methoxyiso-quinolin-1(2H)-one (43):

Yield: 84%. ¹H NMR (300 MHz, Chloroform-d) δ 7.88 (s, 1H), 7.28 – 7.39 (m, 1H), 7.24 (s, 1H), 6.95 (s, 1H), 6.75 (d, J = 9.42 Hz, 1H), 6.67 – 6.72 (m, 2H), 4.75 – 5.06 (m, 1H), 4.27 – 4.43 (m, 1H), 3.98 (s, 3H), 3.54 – 3.89 (m, 4H), 2.98 (s, 6H), 2.05 (m, 2H), 1.87 (d, J = 10.36 Hz, 6H), 1.64 (d, J = 9.42 Hz, 3H), 1.36 (m, 3H). MS (ESI) m/z: 522.2. [M+H]⁺.

2-(3-(Dimethylamino)phenyl)-4-(4-fluoropiperidine-1-carbonyl)-7-methoxy-6-(naphthalen-1-ylm-ethoxy)isoquinolin-1(2H)-one (44):

Yield: 78%. ¹H NMR (300 MHz, Chloroform-d) d 8.07 (d, J = 8.29 Hz, 1H), 7.89 – 7.98 (m, 2H), 7.84 (d, J = 8.10 Hz, 1H), 7.50 – 7.68 (m, 3H), 7.37 – 7.47 (m, 1H), 7.30 (t, J = 8.01 Hz, 1H), 7.19 (s, 1H), 6.78 – 6.92 (m, 1H), 6.70 – 6.78 (m, 1H), 6.62 – 6.70 (m, 2H), 5.81 (m, 2H), 4.04 (s, 3H), 3.53 – 3.77 (m, 1H), 3.06 – 3.51 (m, 2H), 2.96 (s, 6H), 2.83 (s, 2H), 1.61 (m, 4H) MS (ESI) m/z: 580.2. [M+H]⁺.

2-(3-(Dimethylamino)phenyl)-4-(4-fluoropiperidine-1-carbonyl)-7-methoxy-6-(pyridin-2-ylmeth-oxy)isoquinolin-1(2H)-one (45):

Yield: 68%. ¹H NMR (300 MHz, Chloroform-d) d 8.63 (d, J = 3.77 Hz, 1H), 7.92 (s, 1H), 7.70 (dd, J = 1.70, 7.72 Hz, 1H), 7.53 (d, J = 7.91 Hz, 1H), 7.29 – 7.36 (m, 1H), 7.28 (d, J = 2.83 Hz, 1H), 7.24 (m, 1H), 6.94 (s, 1H), 6.72 – 6.78 (m, 1H), 6.64 – 6.71 (m, 2H), 5.40 (s, 2H), 4.68 – 5.03 (m, 1H), 4.04 (s, 3H), 3.08 – 3.87 (m, 4H), 2.97 (s, 6H), 1.64 – 2.03 (m, 4H). MS (ESI) m/z: 531.2. [M+H]⁺.

Methyl 6,7-dimethoxy-1-oxo-1,2-dihydroisoquinoline-4-carboxylate (85).

To a solution of compound 70 (2.68 g, 10 mmol) in MeOH (50 mL) was added 1, 3, 5-triazine (973 mg, 12 mmol). MeONa (2.7 g, 50 mmol) was added and the reaction was stirred for 1 hour at room temperature. The reaction was then quenched by water and extracted by DCM (50 mL *3). The organic layer was then separated and dried. The solvent was then removed under reduced pressure and the off-white solid collected compound 85 was used in the next step without further purification. 1.6 g, yield: 61%. ¹H NMR (300 MHz, DMSO-d₆) δ 11.63 – 11.87 (m, 1H), 8.30 (s, 1H), 7.96 (d, J = 6.59 Hz, 1H), 7.61 (s, 1H), 3.90 (s, 3H), 3.88 (s, 3H), 3.82 (s, 3H). MS (ESI) m/z: 264.2. [M+H]⁺.

Compound 85 (1.32 g, 5 mmol) was dissolved in MeOH (10 mL) and NaOH (10 mL, 2 N, 20 mmol) was added. The reaction was then heated to 50°C for 1 hour. After cooling to room temperature, 1 N HCl (45 mL) was added and the solvent was then removed to give crude product that was then used in the next step following General procedure C with piperidine or 4-fluoro-piperidine to give desired product.

6,7-Dimethoxy-4-(piperidine-1-carbonyl)isoquinolin-1(2H)-one (86a):

1.34 g off-white solid, yield: 80%. ¹H NMR (300 MHz, Chloroform-d) δ 11.20 (m, 1H), 7.80 (s, 1H), 7.27 (s, 1H), 7.00 (s, 1H), 4.03 (s, 3H), 3.99 (s, 3H), 3.44 – 3.76 (m, 4H), 1.40 – 1.48 (m, 6H). MS (ESI) m/z: 317.2. [M+H]⁺.

4-(4-Fluoropiperidine-1-carbonyl)-6,7-dimethoxyisoquinolin-1(2H)-one (86b):

1.63 g off-white solid, yield: 86%. ¹H NMR (300 MHz, Chloroform-d) δ 7.80 (s, 1H), 7.28 – 7.36 (m, 1H), 7.13 (s, 1H), 6.94 (s, 1H), 4.80 – 5.07 (m, 1H), 4.02 (s, 3H), 3.93 – 3.99 (m, 3H), 3.37 – 3.90 (m, 4H), 1.94 (m, 4H). MS (ESI) m/z: 335.2. [M+H]⁺.

General procedure D:

CuOTf catalyzed coupling of RB(OH)₂ and 86a or 86b.

A round-bottom flask charged with compound 86a or compound 86b (1 equiv.), RB(OH)₂ (1.5 equiv.), CuOTf (0.2 equiv.), 1,10-Phen (0.2 equiv.) and DMSO (0.1 M), the reaction mixture was stirred at room temperature under open air overnight. The progress of the reaction was monitored by TLC and after completion of the reaction, the crude reaction mixture was diluted with 20 mL ice cold water and extracted with ethyl acetate (3 × 15 mL). The combined Organic layers were dried over Na₂SO₄ and concentrated under vacuum. The crude product was purified by column chromatography (petroleum ether/ethyl acetate) to provide the desired product.

General procedure E:

Cu(OAc)₂ catalyzed coupling of RBpin and compound 86a, 86b or 85.

A round-bottom flask charged with compound 86a, 86b or 85 (1.0 equiv.), RBpin (1.5 equiv.), pyridine (10.0 equiv.), Cu(OAc)₂ (5.0 equiv.) and DMSO (0.4 M), the reaction mixture was heated to 80°C open to the air overnight. The progress of the reaction was monitored by TLC and after completion of the reaction, the crude reaction mixture was diluted with 20 mL ice cold water and extracted with ethyl acetate (3 × 15 mL). The combined Organic layers were dried over Na₂SO₄ and concentrated under vacuum. The crude product was purified by column chromatography (petroleum ether/ethyl acetate) to provide the desired product.

Compounds 46–65 were synthesized following General procedures D and E.

2-(3-Isopropylphenyl)-6,7-dimethoxy-4-(piperidine-1-carbonyl)isoquinolin-1(2H)-one (46):

Yield: 75%. ¹H NMR (300 MHz, Chloroform-d) δ 7.89 (s, 1H), 7.38 – 7.48 (m, 1H), 7.27 – 7.31 (m, 2H), 7.20 – 7.26 (m, 2H), 6.98 (s, 1H), 4.00 (d, J = 3.77 Hz, 6H), 3.34 – 3.91 (m, 4H), 2.91 – 3.04 (m, 1H), 1.58 – 1.81 (m, 6H), 1.29 (d, J = 6.78 Hz, 6H). ¹³C NMR (75 MHz, Chloroform-d) δ 166.49, 160.78, 153.91, 150.34, 149.83, 140.97, 130.03, 129.12, 126.44, 124.92, 124.09, 120.35, 113.81, 108.66, 104.39. 56.19, 56.18, 34.04, 26.49, 26.37, 24.45, 23.85. MS (ESI) m/z: 435.2. [M+H]⁺.

2-(3-(tert-Butyl)phenyl)-6,7-dimethoxy-4-(piperidine-1-carbonyl)isoquinolin-1(2H)-one (47):

Yield: 78%. ¹H NMR (300 MHz, Chloroform-d) d 7.89 (s, 1H), 7.43 (d, J = 7.35 Hz, 3H), 7.20 – 7.25 (m, 2H), 6.99 (s, 1H), 4.00 (d, J = 3.20 Hz, 6H), 3.36 – 3.82 (m, 4H), 1.67 (d, J = 15.07 Hz, 6H), 1.35 (d, J = 0.57 Hz, 9H). MS (ESI) m/z: 449.2. [M+H]⁺.

2-(3-Isopropoxyphenyl)-6,7-dimethoxy-4-(piperidine-1-carbonyl)isoquinolin-1(2H)-one (48):

Yield: 78%. ¹H NMR (300 MHz, Chloroform-d) δ 7.88 (s, 1H), 7.37 (t, J = 8.29 Hz, 1H), 7.22 (s, 1H), 6.91 – 7.01 (m, 4H), 4.51 – 4.64 (m, 1H), 4.00 (d, J = 5.09 Hz, 6H), 3.36 – 3.86 (m, 4H), 1.67 (d, J = 14.32 Hz, 6H), 1.35 (d, J = 6.03 Hz, 6H). MS (ESI) m/z: 451.2. [M+H]⁺.

2-(3-Acetylphenyl)-6,7-dimethoxy-4-(piperidine-1-carbonyl)isoquinolin-1(2H)-one (49):

Yield: 73%. ¹H NMR (300 MHz, Chloroform-d) δ 8.03 (td, J = 1.79, 3.96 Hz, 2H), 7.87 (s, 1H), 7.58 – 7.71 (m, 2H), 7.24 (s, 1H), 6.97 (s, 1H), 4.01 (d, J = 3.58 Hz, 6H), 3.24 – 3.88 (m, 4H), 2.65 (s, 3H), 1.49 – 1.78 (m, 6H). MS (ESI) m/z: 435.2. [M+H]⁺.

3-(6,7-Dimethoxy-1-oxo-4-(piperidine-1-carbonyl)isoquinolin-2(1H)-yl)benzamide (50):

Yield: 76%. ¹H NMR (300 MHz, Chloroform-d) δ 7.87 – 7.92 (m, 1H), 7.84 (s, 1H), 7.81 (s, 1H), 7.51 – 7.61 (m, 2H), 7.21 (s, 1H), 6.92 (s, 1H), 3.97 (d, J = 3.58 Hz, 6H), 3.21 – 3.88 (m, 6H), 1.66 (m, 6H). MS (ESI) m/z: 436.2. [M+H]⁺.

3-(6,7-Dimethoxy-1-oxo-4-(piperidine-1-carbonyl)isoquinolin-2(1H)-yl)-N,N-dimethylbenzamide (51):

Yield: 69%. ¹H NMR (300 MHz, Chloroform-d) δ 7.86 (s, 1H), 7.46 – 7.63 (m, 4H), 7.24 (s, 1H), 6.97 (s, 1H), 4.01 (d, J = 2.83 Hz, 6H), 3.31 – 3.87 (m, 4H), 3.08 (m, 6H), 1.45 – 1.79 (m, 6H). MS (ESI) m/z: 464.2. [M+H]⁺.

6,7-Dimethoxy-4-(piperidine-1-carbonyl)-2-(pyridin-2-yl)isoquinolin-1(2H)-one (52):

27 mg off-white solid, yield: 68%. ¹H NMR (300 MHz, Chloroform-d) δ 8.53 – 8.63 (m, 1H), 7.99 (d, J = 8.29 Hz, 1H), 7.90 (s, 1H), 7.81 – 7.88 (m, 2H), 7.31 (dd, J = 4.71, 6.97 Hz, 1H), 7.01 (s, 1H), 4.03 (s, 3H), 4.00 (s, 3H), 3.60 (m, 4H), 1.67 (d, J = 17.71 Hz, 6H). MS (ESI) m/z: 394.2. [M+H]⁺.

6,7-Dimethoxy-4-(piperidine-1-carbonyl)-2-(pyridin-3-yl)isoquinolin-1(2H)-one (53):

Yield: 62%. ¹H NMR (300 MHz, Chloroform-d) δ 8.65 (d, J = 2.45 Hz, 1H), 8.61 (dd, J = 1.32, 4.71 Hz, 1H), 7.76 – 7.83 (m, 2H), 7.36 – 7.44 (m, 1H), 7.12 (s, 1H), 6.91 (s, 1H), 3.94 (d, J = 4.52 Hz, 6H), 3.54 (m, 4H), 1.64 (m, 6H). MS (ESI) m/z: 394.2. [M+H]⁺.

6,7-Dimethoxy-4-(piperidine-1-carbonyl)-2-(pyridin-4-yl)isoquinolin-1(2H)-one (54):

Yield: 62%. ¹H NMR (300 MHz, Chloroform-d) δ 8.78 (d, J = 6.03 Hz, 2H), 7.87 (s, 1H), 7.44 – 7.51 (m, 2H), 7.20 (s, 1H), 6.96 (s, 1H), 4.01 (d, J = 6.40 Hz, 6H), 3.33 – 3.84 (m, 4H), 1.59 – 1.80 (m, 6H). MS (ESI) m/z: 394.2. [M+H]⁺.

6,7-Dimethoxy-2-(5-methylbenzo[d]isoxazol-3-yl)-4-(piperidine-1-carbonyl)isoquinolin-1(2H)-o-ne (2):

Yield: 70%. ¹H NMR (300 MHz, Chloroform-d) δ 7.91 (s, 1H), 7.56 (s, 1H), 7.48 – 7.54 (m, 2H), 7.40 – 7.47 (m, 1H), 7.02 (s, 1H), 4.05 (s, 3H), 4.02 (s, 3H), 3.42 – 3.84 (m, 4H), 2.48 (s, 3H), 1.60 – 1.79 (m, 6H). MS (ESI) m/z: 448.2. [M+H]⁺.

6,7-Dimethoxy-2-(3-methylbenzo[d]isoxazol-5-yl)-4-(piperidine-1-carbonyl)isoquinolin-1(2H)-o-ne (55):

Yield: 62%. ¹H NMR (300 MHz, Chloroform-d) δ 7.88 (s, 1H), 7.74 (d, J = 1.32 Hz, 1H), 7.64 – 7.70 (m, 1H), 7.57 – 7.64 (m, 1H), 7.26 (s, 1H), 6.99 (s, 1H), 4.02 (d, J = 3.01 Hz, 6H), 3.57 (m, 4H), 2.60 (s, 3H), 1.58 – 1.82 (m, 6H). MS (ESI) m/z: 448.2. [M+H]⁺.

6,7-Dimethoxy-2-(3-methylbenzofuran-5-yl)-4-(piperidine-1-carbonyl)isoquinolin-1(2H)-one (56):

Yield: 71%. ¹H NMR (300 MHz, Chloroform-d) δ 7.90 (s, 1H), 7.59 (d, J = 2.07 Hz, 1H), 7.54 (d, J = 8.67 Hz, 1H), 7.49 (d, J = 1.13 Hz, 1H), 7.28 – 7.34 (m, 2H), 7.00 (s, 1H), 4.01 (d, J = 3.01 Hz, 6H), 3.38 – 3.83 (m, 4H), 2.25 (d, J = 1.32 Hz, 3H), 1.62 (m, 6H). MS (ESI) m/z: 447.2. [M+H]⁺.

6,7-Dimethoxy-2-(3-methylbenzo[b]thiophen-5-yl)-4-(piperidine-1-carbonyl)isoquinolin-1(2H)-one (57):

Yield: 78%. ¹H NMR (300 MHz, Chloroform-d) δ 7.94 (d, J = 8.48 Hz, 1H), 7.90 (s, 1H), 7.76 (d, J = 1.88 Hz, 1H), 7.40 (dd, J = 1.98, 8.57 Hz, 1H), 7.30 (s, 1H), 7.19 (d, J = 0.94 Hz, 1H), 7.00 (s, 1H), 4.02 (d, J = 3.20 Hz, 6H), 3.62 (m, 4H), 2.44 (d, J = 1.13 Hz, 3H), 1.57 – 1.79 (m, 6H). ¹³C NMR (75 MHz, Chloroform-d) δ 165.46, 160.07, 152.97, 148.90, 139.39, 139.11, 136.59, 131.30, 129.23, 128.61, 122.51, 122.46, 121.88, 119.34, 119.04, 112.94, 107.69, 103.46, 55.22, 55.19, 41.66, 25.40, 23.45, 12.87. MS (ESI) m/z: 463.2. [M+H]⁺.

6,7-Dimethoxy-2-(1-methyl-1H-indol-6-yl)-4-(piperidine-1-carbonyl)isoquinolin-1(2H)-one (58):

Yield: 80%. ¹H NMR (300 MHz, Chloroform-d) δ 7.91 (s, 1H), 7.70 (d, J = 8.29 Hz, 1H), 7.42 (s, 1H), 7.33 (s, 1H), 7.15 (d, J = 3.20 Hz, 1H), 7.11 (dd, J = 1.70, 8.48 Hz, 1H), 7.01 (s, 1H), 6.54 (d, J = 3.01 Hz, 1H), 4.01 (d, J = 3.39 Hz, 6H), 3.81 (s, 3H), 3.45 – 3.76 (m, 4H), 1.66 (d, J = 18.27 Hz, 6H). MS (ESI) m/z: 446.2. [M+H]⁺.

6,7-Dimethoxy-2-(8-methylnaphthalen-2-yl)-4-(piperidine-1-carbonyl)isoquinolin-1(2H)-one (59):

Yield: 72%. ¹H NMR (300 MHz, Chloroform-d) δ 8.01 (d, J = 1.70 Hz, 1H), 7.96 (d, J = 8.67 Hz, 1H), 7.91 (s, 1H), 7.77 (d, J = 7.72 Hz, 1H), 7.57 (dd, J = 2.07, 8.67 Hz, 1H), 7.42 – 7.49 (m, 1H), 7.36 – 7.41 (m, 1H), 7.34 (s, 1H), 7.02 (s, 1H), 3.96 – 4.07 (m, 6H), 3.34 – 3.86 (m, 4H), 2.69 (s, 3H), 1.59 – 1.76 (m, 6H). MS (ESI) m/z: 457.2. [M+H]⁺.

2-(Benzofuran-5-yl)-6,7-dimethoxy-4-(piperidine-1-carbonyl)isoquinolin-1(2H)-one (60):

Yield: 76%. ¹H NMR (300 MHz, Chloroform-d) δ 7.89 (s, 1H), 7.72 (d, J = 2.26 Hz, 1H), 7.65 (d, J = 1.88 Hz, 1H), 7.61 (d, J = 8.67 Hz, 1H), 7.34 (dd, J = 2.07, 8.67 Hz, 1H), 7.27 (s, 1H), 7.00 (s, 1H), 6.82 (d, J = 2.07 Hz, 1H), 4.01 (d, J = 3.77 Hz, 6H), 3.59 (m, 4H), 1.70 (m, 6H). MS (ESI) m/z: 433.2. [M+H]⁺.

2-(Benzo[b]thiophen-5-yl)-6,7-dimethoxy-4-(piperidine-1-carbonyl)isoquinolin-1(2H)-one (61):

Yield: 78%. ¹H NMR (300 MHz, Chloroform-d) δ 7.98 (d, J = 8.67 Hz, 1H), 7.90 (s, 1H), 7.87 (d, J = 1.88 Hz, 1H), 7.56 (d, J = 5.46 Hz, 1H), 7.41 (dd, J = 1.98, 8.57 Hz, 1H), 7.38 (d, J = 5.46 Hz, 1H), 7.29 (s, 1H), 7.00 (s, 1H), 4.01 (d, J = 3.96 Hz, 6H), 3.61 (m, 4H), 1.67 (d, J = 14.51 Hz, 6H). MS (ESI) m/z: 449.2. [M+H]⁺.

2-(Benzofuran-5-yl)-4-(4-fluoropiperidine-1-carbonyl)-6,7-dimethoxyisoquinolin-1(2H)-one (62):

Yield: 76%. ¹H NMR (300 MHz, Chloroform-d) δ 7.89 (s, 1H), 7.72 (d, J = 2.26 Hz, 1H), 7.65 (d, J = 2.07 Hz, 1H), 7.61 (d, J = 8.67 Hz, 1H), 7.29 – 7.36 (m, 2H), 6.98 (s, 1H), 6.83 (d, J = 2.07 Hz, 1H), 4.78 – 5.07 (m, 1H), 4.01 (d, J = 6.40 Hz, 6H), 3.51 – 3.94 (m, 4H), 1.94 (m, 4H). MS (ESI) m/z: 451.2. [M+H]⁺.

2-(1-Ethylindolin-6-yl)-4-(4-fluoropiperidine-1-carbonyl)-6,7-dimethoxyisoquinolin-1(2H)-one (3):

Yield: 64%. ¹H NMR (300 MHz, Chloroform-d) δ 7.89 (s, 1H), 7.25 (s, 1H), 7.11 (d, J = 7.54 Hz, 1H), 6.97 (s, 1H), 6.58 (dd, J = 1.79, 7.63 Hz, 1H), 6.44 (s, 1H), 4.78 – 5.04 (m, 1H), 4.00 (d, J = 7.72 Hz, 6H), 3.65 (m, 4H), 3.44 (t, J = 8.29 Hz, 2H), 3.14 (q, J = 7.28 Hz, 2H), 3.00 (t, J = 8.48 Hz, 2H), 1.92 (m, 4H), 1.18 (t, J = 7.25 Hz, 3H). MS (ESI) m/z: 480.2. [M+H]⁺.

4-(4-Fluoropiperidine-1-carbonyl)-2-(3-isopropylphenyl)-6,7-dimethoxyisoquinolin-1(2H)-one (63):

Yield: 81%. ¹H NMR (300 MHz, Chloroform-d) δ 7.89 (s, 1H), 7.37 – 7.48 (m, 1H), 7.27 – 7.33 (m, 2H), 7.18 – 7.26 (m, 2H), 6.96 (s, 1H), 4.77 – 5.06 (m, 1H), 4.00 (d, J = 6.40 Hz, 6H), 3.66 (m, 4H), 2.97 – 3.01 (m, 1H), 1.94 (m, 4H), 1.29 (d, J = 6.97 Hz, 6H). MS (ESI) m/z: 453.2. [M+H]⁺.

4-(4-Fluoropiperidine-1-carbonyl)-6,7-dimethoxy-2-(3-methylbenzofuran-5-yl)isoquinolin-1(2H)-one (64):

Yield: 79%. ¹H NMR (300 MHz, Chloroform-d) δ 7.90 (s, 1H), 7.59 (d, J = 2.07 Hz, 1H), 7.55 (d, J = 8.67 Hz, 1H), 7.50 (d, J = 1.13 Hz, 1H), 7.28 – 7.34 (m, 2H), 6.98 (s, 1H), 4.79 – 5.05 (m, 1H), 4.01 (d, J = 6.03 Hz, 6H), 3.67 (m, 4H), 2.25 (d, J = 1.13 Hz, 3H), 1.94 (m, 4H). MS (ESI) m/z: 465.2. [M+H]⁺.

4-(4-Fluoropiperidine-1-carbonyl)-6,7-dimethoxy-2-(3-methylbenzo[b]thiophen-5-yl)isoquinolin-1(2H)-one (65):

Yield: 88%. ¹H NMR (300 MHz, Chloroform-d) δ 7.95 (d, J = 8.48 Hz, 1H), 7.91 (s, 1H), 7.76 (d, J = 1.88 Hz, 1H), 7.39 (dd, J = 1.98, 8.57 Hz, 1H), 7.33 (s, 1H), 7.19 (d, J = 0.94 Hz, 1H), 6.99 (s, 1H), 4.79 – 5.05 (m, 1H), 4.01 (d, J = 5.84 Hz, 6H), 3.67 (m, 4H), 2.45 (d, J = 1.13 Hz, 3H), 1.65 – 2.03 (m, 4H). ¹³C NMR (75 MHz, Chloroform-d) δ 166.74, 161.05, 154.08, 150.01, 140.43, 140.23, 137.48, 132.31, 130.65, 129.42, 123.64, 123.54, 122.82, 120.39, 120.03, 113.25, 108.79, 104.26, 88.36, 86.08, 56.23, 31.74 (m), 22.65, 13.91. MS (ESI) m/z: 481.2. [M+H]⁺.

Methyl 6,7-dimethoxy-2-(3-methylbenzo[b]thiophen-5-yl)-1-oxo-1,2-dihydroisoquinoline-4-carb-oxylate (87).

General procedure E was followed starting from compound 85 (0.2 mmol) to afford compound 87 (62 mg) as an off-white solid, yield: 75%. ¹H NMR (300 MHz, Chloroform-d) d 8.41 – 8.52 (m, 1H), 8.17 – 8.31 (m, 1H), 7.97 (d, J = 8.48 Hz, 1H), 7.82 – 7.90 (m, 1H), 7.76 (d, J = 1.70 Hz, 1H), 7.37 – 7.46 (m, 1H), 7.09 – 7.24 (m, 1H), 4.09 (s, 3H), 4.02 (s, 3H), 3.89 (s, 3H), 2.46 (s, 3H). MS (ESI) m/z: 410.2. [M+H]⁺.

4-(4-Ethynylpiperidine-1-carbonyl)-6,7-dimethoxy-2-(3-methylbenzo[b]thiophen-5-yl)isoquinoli-n-1(2H)-one (66):

Compound 87 (62 mg, 0.15 mmol) was dissolved in MeOH (1 mL) and NaOH (1 mL, 2 N, 2 mmol) was added. The reaction was then heated to 50°C for 1 hour. After cooling to room temperature, 1 N HCl (2.5 mL) was added and the solvent was then removed to give crude acid. The acid was then rected with 4-ethylnyl piperidine hydrogen chloride following General procedure C to give compound 66 (65 mg) as a white solid, yield: 89% over two steps. ¹H NMR (300 MHz, Chloroform-d) δ 7.95 (d, J = 8.48 Hz, 1H), 7.90 (s, 1H), 7.75 (s, 1H), 7.40 (d, J = 8.85 Hz, 1H), 7.31 (s, 1H), 7.19 (s, 1H), 6.99 (s, 1H), 4.83–5.02 (m, 1 H), 4.02 (s, 3H), 4.00 (s, 3H), 3.33 – 3.89 (m, 4H), 2.45 (s, 3H), 1.86–1.70 (m, 4H). ¹³C NMR (75 MHz, Chloroform-d) δ 166.61, 161.08, 154.05, 150.00, 140.43, 140.21, 137.54, 132.32, 130.48, 129.49, 123.59, 123.53, 122.86, 120.06, 113.49, 110.41, 108.77, 104.37, 89.64, 85.40, 77.23, 70.37, 56.27, 31.76, 26.66, 13.91. MS (ESI) m/z: 487.2. [M+H]⁺.

4.2. Pharmacological Evaluation

The RH7777-hLPA5 cells were purchased from MultiSpan, Inc. and validated for activity in our laboratory using the known LPA5 agonist, hexadecyl LPA 16:0. Cell culture materials, assay consumables, and assay reagents were purchased from Fisher SSI. The Calcium 5 dye was purchased from Molecular Devices and the LanceUltra kit (TRF0262) was purchased from PerkinElmer.

LPA5 Calcium mobilization assay.

Stable RH7777-hLPA5 cells (Multispan, Inc., Hayward, CA, USA) were maintained in Dulbecco’s Modified Eagles Medium – High Glucose (DMEM-HG) supplemented with 10% fetal bovine serum (FBS), 100 units of penicillin/streptomycin (P/S), and 5 ug/mL puromycin. Cells were plated into 96-well black-walled assay plates pre-coated with poly-D-lysine at 20,000 cells/well in 100 μL of DMEM-HG (supplemented with 0.2% FBS and 100 units of P/S) and incubated for 18–20 hours at 37 °C, 5% CO₂. Calcium 5 dye (Molecular Devices, CA) was reconstituted according to the manufacturer’s instructions. The reconstituted dye was diluted 1:40 in prewarmed (37 °C) assay buffer (1x HBSS, 20 mM HEPES, 2.5 mM probenecid, pH 7.4 at 37 °C). Growth medium was removed, and the cells were gently washed with 100 μL of prewarmed (37 °C) assay buffer. The cells were incubated for 45 min at 37 °C, 5% CO₂ in 200 μL of the diluted Calcium 5 dye solution. For antagonist assays to determine IC₅₀ values, the EC₈₀ concentration of hexadecyl LPA 16:0 (30 nM) was prepared at 10x the desired final concentration in 0.25% BSA/1% DMSO/0.5% EtOH/assay buffer, aliquoted into 96-well polypropylene plates, and warmed to 37 °C. Serial dilutions (8 concentrations) of the test compounds were prepared at 9x the desired final concentration in 2.25% BSA/9% DMSO/5% EtOH/assay buffer. After the dye loading incubation period, the cells were pretreated with 25 μL of the test compound serial dilutions and incubated for 15 min at 37 °C. After the pretreatment incubation period, the plate was read with a FLIPR Tetra (Molecular Devices, CA, USA). Calcium-mediated changes in fluorescence were monitored every 1 s over a 90 s time period, with the Tetra adding 25 μL of the hexadecyl LPA 16:0 EC₈₀ concentration at the 10s time point (excitation/emission: 485/525 nm). Relative fluorescence units (RFU) were plotted against the log of compound concentrations. Data were fit to a three-parameter logistic curve to generate IC₅₀ values (GraphPad Prism, GraphPad Software, Inc., San Diego, CA). For agonist screens, the above procedure was followed except that cells were pretreated with 2.25% BSA/9% DMSO/5% EtOH/assay buffer and the Tetra added single concentration dilutions of the test compounds prepared at 10x the desired final concentration in 0.25% BSA/1% DMSO/0.5% EtOH/assay buffer. Test compound RFUs were compared to the hexadecyl LPA 16:0 10 μM RFUs to generate % E_max values. All assays were conducted with duplicate determinations.

LPA5 cAMP assay.

Assays were conducted using the Lance^™Ultra cAMP assay kit (PerkinElmer). Stable RH7777-hLPA5 cells (Multispan, Inc., Hayward, CA, USA) were maintained as described for the calcium mobilization assays. Twenty hours prior to the assay, the maintenance media was changed to DMEM-HG (supplemented with 0.2% FBS and 100 units of P/S) and the cells were incubated at 37 °C, 5% CO₂. On the day of the assay, stimulation buffer containing 1X Hank’s Balanced Salt Solution (HBSS), 5 mM HEPES, 0.1% BSA stabilizer, and 0.5 mM final IBMX was prepared and titrated to pH 7.4 at room temperature. Serial dilutions of the test compounds (5 μL) and the EC₈₀ concentration (300 nM) of hexadecyl LPA 16:0 (5 μL), both prepared at 4x the desired final concentration in 2% DMSO/stimulation buffer, were added to a 96-well white ½ area microplate (PerkinElmer). A cAMP standard curve was prepared at 4x the desired final concentration in stimulation buffer and 10 μL was added to the assay plate. Stable RH7777-hLPA5 cells were lifted with versene and spun at 270g for 10 minutes. The cell pellet was resuspended in stimulation buffer and 2,000 cells (10 μL) were added to each well except wells containing the cAMP standard curve. After incubating for 30 min at room temperature, Eu-cAMP tracer and uLIGHT-anti-cAMP working solutions were added per the manufacturer’s instructions. After incubation at room temperature for 1 hour, the TR-FRET signal (ex 337 nm) was read on a CLARIOstar multimode plate reader (BMG Biotech, Cary, NC). The TR-FRET signal (665 nm) was converted to fmol cAMP by interpolating from the standard cAMP curve. Fmol cAMP was plotted against the log of compound concentration and data were fit to a three-parameter logistic curve to generate IC₅₀ values (GraphPad Prism, GraphPad Software, Inc., San Diego, CA).

LPA1–LPA4 assays.

LPA1–LPA3 calcium mobilization assays were conducted by Eurofins using CHO cells over-expressing LPA1–LPA3 receptors, respectively, following their established protocols. The LPA4 cAMP assay was conducted in our laboratory using CHO cells over-expressing the LPA4 receptor, following procedures described above for the LPA5 cAMP assay except that compounds were run against 250 nM hexadecyl LPA 16:0 (EC₈₀).

4.3. ADME and PK Profiling

Kinetic Solubility.

The kinetic solubility of the test compound was measured in commercial PBS, which consisted of potassium phosphate monobasic 1 mM, sodium phosphate dibasic 3 mM and sodium chloride 155 mM. A 10 μL of test compound stock solution (20 mM DMSO) was combined with 490 μL of PBS buffer to reach a targeted concentration of 400 μM. The solution was agitated on a VX-2500 multi-tube vortexer (VWR) for 2 hours at room temperature. Following agitation, the sample was filtrated on a glass-fiber filter (1 μm) and the eluate was diluted 400-fold with a mixture of acetonitrile: water (1:1). On each experimental occasion, nicardipine and imipramine were assessed as reference compounds for low and high solubility, respectively. All samples were assessed in triplicate and analyzed by LC-MS/MS using electrospray ionization against standards prepared in the same matrix.

Metabolic stability.

Compounds were incubated with rat liver microsomes at 37 °C for a total of 45 minutes. The reaction was performed at pH 7.4 in 100 mM potassium phosphate buffer containing 0.5 mg/mL of rat liver microsomal protein. Phase I metabolism was assessed by adding NADPH to a final concentration of 1 mM and collecting samples at time points 0, 5, 15, 30 and 45 minutes. All collected samples were quenched 1:1 with ice-cold stop solution (1 μM labetalol and 1 μM glyburide in acetonitrile), and centrifuged to remove precipitated protein. Resulting supernatants were further diluted 1:4 with acetonitrile:water (1:1). Samples were analyzed by LC/MS/MS and calculations for half-life, and in vitro clearance were accomplished using Microsoft Excel (2007). Half-life and clearance were determined from two independent experiments in duplicate.

Bidirectional MDCK-MDR1 permeability.

Assays were performed by Paraza Pharma Inc. (Montreal, Canada). MDCK-mdr1 cells at passage 5 were seeded onto permeable polycarbonate supports in 12-well Costar Transwell plates and allowed to grow and differentiate for 3 days. On day 3, culture medium (DMEM supplemented with 10% FBS) was removed from both sides of the transwell inserts and cells were rinsed with warm HBSS. After the rinse step, the chambers were filled with warm transport buffer (HBSS containing 10 mM HEPES, 0.25% BSA, pH 7.4) and the plates were incubated at 37 °C for 30 min prior to TEER (Trans Epithelial Electric Resistance) measurements.

The buffer in the donor chamber (apical side for A-to-B assay, basolateral side for B-to-A assay) was removed and replaced with the working solution (10 μM test article in transport buffer). The plates were then placed at 37 °C under light agitation. At designated time points (30, 60 and 90 min), an aliquot of transport buffer from the receiver chamber was removed and replenished with fresh transport buffer. Samples were quenched with ice-cold ACN containing internal standard and then centrifuged to pellet protein. Resulting supernatants are further diluted with 50/50 ACN/H₂O (H₂O only for Atenolol) and submitted for LC-MS/MS analysis. Reported apparent permeability (Papp) values were calculated from single determination. Atenolol and propranolol were tested as low and moderate permeability references. Bidirectional transport of digoxin was assessed to demonstrate Pgp activity/expression.

The apparent permeability (P_app, measured in cm/s) of a compound is determined according to the following formula from two independent experiments in duplicate.

$$\mathrm{P}\mathrm{a}\mathrm{p}\mathrm{p}=\frac{\left(\mathrm{d}\mathrm{Q}\right)/\left(\mathrm{d}\mathrm{t}\right)}{\mathrm{A}\mathrm{*}\mathrm{C}\mathrm{i}\mathrm{*}60}$$

dQ/dt is the net rate of appearance in the receiver compartment

A is the area of the Transwell measured in cm² (1.12 cm²)

Ci is the initial concentration of compound added to the donor chamber

60 is the conversion factor for minute to second

Plasma Protein Binding.

Test compound and controls (1 μM) were spiked in rat plasma (BioreclamationIVT, Westbury, NY, USA) and aliquoted in triplicate in a high throughput dialysis (HTD) 96-well plate, where the plasma and dialysate buffer were separated by a semi-permeable cellulose membrane (12–14K MWCO). Once sealed, the HTD plate was incubated at 37 °C and kept under light agitation for 6 hours, until equilibrium was reached. Plasma and buffer samples were then extracted along with their corresponding standard curve samples using ice-cold acetonitrile in methanol (1:1). After centrifugation, supernatants from both plasma- and buffer-containing samples were further diluted prior to being submitted to bioanalysis by LC-MS/MS. Acebutolol and warfarin served as low-bound and highly-bound controls, respectively. Determination of the percentage of plasma protein binding and recovery was achieved in Microsoft Excel.

In vivo pharmacokinetic assay was performed by Paraza Pharma Inc. (Montreal, Canada). On the morning of the PK study, male Sprague-Dawley rats weighing 258–277 g were dosed with either vehicle (5% Cremorphor, 5% ethanol in saline) or 66 (17.8 mg/kg, i.p.). At 30 minutes post dose, 2 rats were anesthetized with isoflurane gas to perform a cardiac puncture to collect blood (for plasma analysis), followed by whole body perfusion with phosphate saline buffer (PBS, pH 7.4) to wash out any remaining blood from the organs. Brains were harvested and homogenized by mechanical sheering with a polytron with 1:4 (w/v) 25% isopropanol in water. Brain homogenates were extracted for drug quantification by LC-MS/MS.

4.4. Behavioral testing

Animals:

Adult male Sprague-Dawley rats (Harlan, Indianapolis, IN, n=6 per group) weighing 230–280 gram were individually housed on a 12/12-h light/dark cycle with behavioral experiments conducted during the light period. Rats had free access to food and water except during test sessions, and animals were maintained and experiments were conducted in accordance with guidelines of the International Association for the Study of Pain (Zimmermann, 1983) and with the 2011 Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources on Life Sciences, National Research Council, National Academy of Sciences, Washington, DC) and were approved by the Institutional Animal Care and Use Committee, University at Buffalo, the State University of New York (Buffalo, NY).

Drugs:

Compounds were dissolved in a vehicle of 20% dimethyl sulfoxide (Fisher Chemical, Fair Lawn, NJ), and 10% emulphor (Solvay, Cranbury, NJ) in 0.9% saline. AS2717638 was dissolved in 30% propylene gycol solvent [Tween 80:emulphor:propylene glycol in a 1:2:4 ratio] in distilled water. All aforementioned drugs were administered intraperitoneally in a volume of 1–2 ml/kg. Fentanyl was purchased from Sigma-Aldrich (St. Louis, MO), dissolved in 0.9% saline, and injected subcutaneously in a volume of 1 ml/kg.

Complete Freund’s Adjuvant:

Inflammatory pain was induced by Complete Freund’s Adjuvant (CFA) inoculation. In brief, the right food paw (hind paw) of rats were injected with 0.1 ml of CFA (Sigma-Aldrich, St. Louis, MO) containing approximately 0.05 mg of Mycobacterium butyricum dissolved in paraffin oil under isoflurane anesthesia (2% isoflurane mixed with 100% oxygen). The level of anesthesia was assessed by the loss of righting reflex. Mechanical and thermal nociception tests were conducted beginning 1–2 days after CFA inoculation and were thereafter conducted every other day.

Chronic Constriction Injury:

Neuropathic pain was induced by chronic constriction injury (CCI) procedure [47]. Briefly, rats were anesthetized with a mixture of ketamine (75 mg/kg) and xylazine (5 mg/kg) intraperitoneally (i.p.) prior to surgery. The right sciatic nerve was exposed, and four ligatures (4.0 chromic gut suture, Patterson Veterinary, Devens, MA) were placed around the nerve (approximately 1 mm apart) proximal to the trifurcation. Ligatures were loosely tied so that circulation through the epineural vasculature was uninterrupted. The incisions were closed with surgical clips. Mechanical and thermal nociception tests were conducted beginning 10 days after CCI surgery and were thereafter conducted every other day.

Fentanyl-Induced Hyperalgesia:

Rats were used for studying opioid-induced hyperalgesia with the following established protocol. After a nociceptive baseline was established for each rat on the day prior to and the day of fentanyl treatment (D−1 and D0), four subcutaneous injections of 0.06 mg/kg fentanyl each were injected at 15 min intervals for a total dose of 0.24 mg/kg. PWT measurements were taken following drug administration on days 1–2 post-fentanyl treatment.

Mechanical Nociception:

Nociceptive thresholds were measured using calibrated von Frey filaments (4–26 g; North Coast Medical, Morgan Hill, CA). Rats (n = 6 per group) were placed in elevated plastic chambers with a wire mesh floor (IITC Life Science Inc., Woodland Hills, CA) and allowed to habituate prior to testing. Filaments were applied perpendicularly to the medial plantar surface of the hind paw from below the mesh floor in an ascending order of filament force, beginning with the lowest filament. Filaments were applied until buckling occurred for approximately 2 s. Mechanical paw withdrawal thresholds (PWTs) correspond to the lowest force that elicited a withdrawal of the hind paw in at least two out of three applications. Forces larger than 26 g would physically elevate the paw and did not reflect pain-like behavior. For time course procedures, rats received a single injection of a drug or vehicle immediately following the t=0 measurement, and were assessed every 30 minutes. In experiments where a cumulative dosing procedure was used, measurements were recorded every 30 minutes, and immediately after each measurement, rats received the next dose of drug.

Locomotion:

Locomotor activity was measured using an infrared motion-sensor system (AccuScan Instruments, Inc. Columbus, OH) surrounding plexiglas cages (40 × 40 × 30 cm). Versa Max software (Omnitech Electronics, Inc., Columbus, OH) was used to monitor the distance the animal travelled for a total of 60 min. Prior to test sessions, rats were exposed to at least three days of handling by the experimenter. On the test day, rats received a 60 min pretreatment of either vehicle or 17.8 mg/kg 66 (i.p.), then rats were placed in the chambers and the locomotor activity was recorded for 60 min. The 60 min pretreatment time was chosen because 66 had a relatively slow onset of action and the duration of 1–2 hours pot-injection included the period that the compound exerted the maximal antinociceptive effect.

Data Analysis:

All graphs and statistical analyses were performed with the GraphPad Prism 7.0 program (GraphPad Software, San Diego, CA, USA). The mean values (± SEM) were calculated from individual animals for mechanical and thermal nociception assays. P < 0.05 was considered statistically significant.

Figure 3.

Antagonist activity for selected LPA5 antagonists in calcium mobilization (A) and cAMP (B) assays. Each data point is the mean ± SEM of three independent experiments conducted in duplicate. The control hexadecyl LPA 16:0 is included for each assay.

Table 2.

Examination of 6,7-position analogs and their antagonist potencies in the hLPA5 calcium mobilization assay.

No.	R1	R2	IC50, nM (% Inhibition)a	No.	R1	R2	IC50, nM (% Inhibition)a
34	H	H	>10,000b	40		OCH3	230 ± 30 (111 ± 3%)
35	OCH3	H	>10,000b	41		OCH3	369 ± 29 (96 ± 0%)
36	H	OCH3	1060 ± 250 (96 ± 6%)	42		OCH3	890 ± 100 (103 ± 0%)
37	OH	OCH3	1370 ± 140 (93 ± 3%)	43		OCH3	510 ± 80 (96 ± 10%)
38		OCH3	480 ± 70 (109 ± 2%)	44		OCH3	>10,000b
39		OCH3	2500 ± 400 (74 ± 2%)	45		OCH3	>10,000b

^aCompounds tested against EC₈₀ (30 nM) of hexadecyl LPA 16:0. Values are the mean IC₅₀ ± SEM of at least three independent experiments performed in duplicate. % Inhibition is calculated with the equation: % Inhibition = (1−(cmpd signal/hexadecyl LPA 16:0 EC₈₀)) × 100.

^bValues are the mean IC₅₀ ± SEM of two independent experiments performed in duplicate.

Table 3.

Examination of 2-position analogs and their antagonist potencies in the hLPA5 calcium mobilization assay

No.	R1	R2	IC50, nM (% Inhibition)a	No.	R1	R2	IC50, nM (% Inhibition)a
46	H		63 ± 6 (105 ± 1%)	57	H		32 ± 5 (105 ± 1%)
47	H		173 ± 14 (107 ± 0%)	58	H		64 ± 5 (104 ± 0%)
48	H		630 ± 100 (100 ± 1%)	59	H		210 ± 40 (108 ± 0%)
49	H		1760 ± 30 (74 ± 2%)	60	H		1100 ± 60 (93 ± 1%)
50	H		>10,000b	61	H		640 ± 60 (101 ± 0%)
51	H		>10,000b	62	F		1490 ± 180 (88 ± 1%)
52	H		>10,000b	3	F		58 ± 9 (104 ± 1%)
53	H		>10,000b	63	F		200 ± 30 (103 ± 1%)
54	H		>10,000b	64	F		144 ± 14 (102 ± 1%)
2	H		36 ± 5 (108 ± 3%)	65	F		69 ± 7 (107 ± 1%)
55	H		3900 ± 600 (56 ± 3%)	66			32 ± 5 (107 ± 3%)
56	H		48 ± 6 (105 ± 1%)

^aCompounds tested against EC₈₀ (30 nM) of hexadecyl LPA 16:0. Values are the mean IC₅₀ ± SEM of at least three independent experiments performed in duplicate. % Inhibition is calculated with the equation: % Inhibition = (1 − (cmpd signal/hexadecyl LPA 16:0 EC₈₀)) × 100.

^bValues are the mean IC₅₀ ± SEM of two independent experiments performed in duplicate.

Table 4.

Potency of select LPA5 antagonists in the hLPA5 cAMP assay.

No.	IC50, nM (% Inhibition)a	No.	IC50, nM (% Inhibition)a
2	210 ± 40 (102 ± 4%)	56	190 ± 20 (88 ± 3%)
3	570 ± 70 (94 ± 15%)	58	460 ± 60 (109 ± 3%)
15	790± 190 (89 ± 5%)	63	394 ± 26 (98 ± 1%)
23	950 ± 240 (94 ± 4%)	64	203 ± 21 (100 ± 9%)
24	2100 ± 300 (92 ± 11%)	65	340 ± 30 (104 ± 5%)
26	890 ± 100 (101 ± 4%)	66	350 ± 50 (100 ± 1%)

^aCompounds tested against EC₈₀ (300 nM) of hexadecyl LPA 16:0. Values are the mean IC₅₀ ± SEM of at least three independent experiments performed in duplicate. % Inhibition is calculated with the equation: % Inhibition = (cmpd signal/hexadecyl LPA 16:0 EC₈₀) × 100.