Discovery of Benzamidine- and 1-Aminoisoquinoline-Based Human MAS-Related G Protein-Coupled Receptor X1 (MRGPRX1) Agonists

Abstract

Mas-related G-protein-coupled receptor X1 (MRGPRX1) is a human sensory neuron-specific receptor and has been actively investigated as a therapeutic target for the treatment of pain. By use of two HTS screening hit compounds, 4-(4-(benzyloxy)-3-methoxybenzylamino)benzimidamide (5a) and 4-(2-(butylsulfonamido)-4-methylphenoxy)benzimidamide (11a), as molecular templates, a series of human MRGPRX1 agonists were synthesized and evaluated for their agonist activity using HEK293 cells stably transfected with human MrgprX1. Conversion of the benzamidine moiety into a 1-aminoisoquinoline moiety carried out in the later stage of structural optimization led to the discovery of a highly potent MRGPRX1 agonist, N-(2-(1-aminoisoquinolin-6-yloxy)-4-methylphenyl)-2-methoxybenzenesulfonamide (16), not only devoid of positively charged amidinium group but also with superior selectivity over opioid receptors. In mice, compound 16 displayed favorable distribution to the spinal cord, the presumed site of action for the MRGPRX1-mediated analgesic effects.

Graphical Abstract

Introduction

A subset of mas-related G protein-coupled receptors (Mrgprs) is expressed specifically in the small diameter DRG sensory neurons and is implicated in the modulation of nociception.¹ Among these, human homologue MRGPRX1 has gained increased interest as a promising therapeutic target partly owing to the analgesic effects of BAM8-22 (Figure 1),² a proteolytic product derived from proenkephalin A with full agonist activity at human MRGPRX1 and its closest rodent orthologues, mouse MRGPRC11 and rat MRGPRC.³ For example, intrathecal (spinal cord) injection of BAM8-22 was found to attenuate both mechanical and thermal hyperalgesia in mice⁴ and rats.⁵ We have also found that JHU-58 (Figure 1), an Arg-Phe-NH₂ peptidomimetic with full agonist activity at mouse MRGPRC11 and rat MRGPRC,⁶ exhibits analgesic effects in rodent models of neuropathic pain.⁵ JHU-58, however, displayed negligible agonist activity at human MRGPRX1, hindering its ability to serve as a molecular template for further structural optimization efforts aimed at clinical translation. Human MRGPRX1 agonists 1 and 2 (Figure 1) reported by GSK⁷ and ACADIA,⁸ respectively, may have a potential to be explored as new leads though their high molecular weight (>500) likely contributes to unfavorable CNS drug-like molecular properties and solubility. Indeed, compound 1 could not be tested in vitro at concentrations above 2 μM due to its limited solubility.⁶

Figure 1.

Representative Mrgpr agonists.

This prompted us to conduct high throughput screening of Eisai’s compound library in HEK293 cells stably transfected with human MRGPRX1 by using FLIPR calcium 4 Assay Kit (Molecular Devices) to measure intracellular calcium responses.⁹ We identified two submicromolar hits 5a and 11a (Figure 2) as full agonists with EC₅₀ values of 0.51 μM and 0.92 μM, respectively. The common structural feature of the two lead compounds is obviously the presence of benzamidine moiety, which is known as a bioisostere of a guanidyl group of arginine. Thus, it is conceivable that the benzamidine portion of these lead compounds shares a binding site with the Arg residue of BAM8-22. While compound 1 is more potent than 5a and 11a, what differentiates these HTS hits from the existing human MRGPRX1 agonists 1 and 2 is their higher ligand efficiency¹⁰ (LE = 0.32–0.33) values owing to their lower molecular weight (MW = 361) as compared to those of 1 (LE = 0.27) and 2 (LE = 0.19).

Figure 2.

New lead human MRGPRX1 agonists 5a and 11a identified by HTS.

Here, we describe the design, synthesis, and structure–activity relationships (SAR) of a new series of human MRGPRX1 agonists using the new leads 5a and 11a as molecular templates. Our optimization efforts aimed not only at improving agonist potency but also replacing the benzamidine group with other functional groups with reduced basicity in an attempt to improve CNS permeability. These efforts led to the discovery of a highly potent and CNS-penetrant agonist potentially valuable in exploring the therapeutic utility of human MRGPRX1 activation in neuropathic pain.

CHEMISTRY

Compounds 5a-h were synthesized by reductive amination of substituted benzaldehydes 3a-f with aminobenzamidines 4a-c mediated by NaCNBH₃ (Scheme 1).

Scheme 1.

Synthesis of compounds 5a-h^a

^aReagents and conditions: (a) NaCNBH₃, DIEA, AcOH, EtOH, rt.

Compounds 11a–i were synthesized in four steps starting with nucleophilic aromatic substitution of 1-fluoro-2-nitrobenzene derivatives 6a–c with hydroxybenzonitriles 7a–c in the presence of potassium carbonate as a base (Scheme 2).¹¹ The nitro group of resulting 2-nitrodiphenyl ethers 8a–e underwent catalytic hydrogenation, yielding the corresponding amines 9a–e. Reaction of 9a-e with sulfonyl chlorides afforded sulfonamides 10a–i. Conversion of the nitrile group of 10a–i into amidine group via Pinner reaction followed by treatment with ammonia¹² gave the desired compounds 11a–i.

Scheme 2.

Synthesis of compounds 11a-i^a

^aReagents and conditions: (a) K₂CO₃, DMF, 50 °C; (b) H₂, 10% Pd/C, MeOH, 20–30 psi, 30 min; (c) R₂-SO₂Cl, pyridine, 50 °C, 1.5 h; (d) (i) HCl (gas), MeOH, 0 °C - rt, (ii) 7 N NH₃ in MeOH, 65 °C, 1h.

As shown in Scheme 3, we also synthesized analogs of compounds 5a and 11a, in which the highly basic amidine moiety is replaced by less basic bioisosteres. Compound 13 containing an 1-aminoisoquinoline was synthesized by reductive amination of benzaldehyde 3a with isoquinoline-1,6-diamine 12 mediated by NaCNBH₃. Sulfonamide-based agonists 16 and 19 were obtained from 6b, which was first coupled with 14 or 17 in the presence of potassium carbonate to give 15 or 18, respectively. Catalytic hydrogenation of the nitro group of 15 and 18 followed by reaction with 2-methoxybenzene-1-sulfonyl chloride in pyridine provided the desired compounds 16 and 19.

Scheme 3.

Synthesis of compounds 13, 16, and 19^a

^aReagents and conditions: (a) NaCNBH₃, DIEA, AcOH, EtOH, rt. (b) ArOH, K₂CO₃, DMF, 50 °C; (c) (i) H₂, Pd/C, MeOH, 20–30 psi, 30 min; (ii) 2-methoxybenzene-1-sulfonyl chloride, pyridine, 50 °C, 1.5 h.

Results and Discussion

In vitro experiments measuring agonist activity at MRGPRX1 were performed in a FLIPR assay using HEK293 cells stably transfected with human MrgprX1 The results are summarized in Table 1. All of the compounds acted as full MRGPRX1 agonists (E_max >95% of BAM8-22) except for compounds 5c and 19, which showed negligible agonist activity at concentrations up to 100 μM. It appears that the position of the amidine group has significant impact on the agonist activity. Meta- (compound 5b) and ortho-substitution (compound 5c) resulted in 6-fold and >100-fold decreases in potency, respectively, as compared to 5a. This suggests the critical role played by the para-amidine group of compound 5a in strong interaction with MRGPRX1. Replacement of the terminal benzyl group with a 3-pyridinylmethyl group led to 10-fold loss of potency (compound 5d). In contrast, substitution with 3-thiophenylmethyl group (compound 5e) and [4-(1H-pyrrol-1-yl)phenyl]methyl (compound 5f) resulted in similar potency to 5a. Meanwhile, both compound 5g containing an aliphatic group instead of the benzyl group and compound 5h devoid of the methoxy group from the linker region exhibited decreased agonist potency.

Table 1.

In vitro MRGPRX1 Agonist Potency of Compounds 5a-h, 11a-i, 13, 16, and 19

Cmpd	Structure	EC50 (μM)a	Cmpd	Structure	EC50 (μM) a
5a		0.51 ± 0.49	11c		2.2 ± 1.0
5b		3.3 ± 1.8	11d		0.16 ± 0.08
5c		>100	11e		0.15 ±0.04
5d		5.2 ± 0.9	11f		2.1 ± 0.4
5e		0.29 ±0.06	11g		0.10 ±0.09
5f		0.68 ± 0.20	11h		3.6 ± 1.5
5g		1.9 ± 0.9	11i		23 ± 7
5h		5.2 ± 1.1	13		3.7 ± 0.6
11a		0.92 ± 0.47	16		0.05 ± 0.02
11b		0.30 ± 0.12	19		>100

^aValues are mean ± SD of at least four experiments.

Given the preference for para-amidine substitution observed in analogs of 5a, SAR studies on lead compound 11a focused on its non-benzamidine portion. Structural changes in the internal linker and/or the terminal alkylsulfonamide group had various impact in MRGPRX1 agonist potency as seen in compounds 11a–g. The most potent agonists within this series, compounds 11e and 11g, exhibited EC₅₀ values of 150 and 100 nM, respectively. Using compound 11g as a template, we moved the amidine group to the meta (compound 11h) and the ortho (compound 11i) position. Both compounds exhibited decreased agonist potency compared to 11g, confirming the critical role played by the para-amidine group in MRGPRX1 agonist potency.

Compounds 13, 16, and 19 possess a potential bioisostere of the benzamidine group, an effective strategy often used in inhibitors of serine proteases involved in the coagulation cascade.^13–16 Compound 13 contains a 1-aminoisoquinoline instead of the benzamidine group of compound 5a. This modification resulted in decline of agonist potency from 0.51 μM (compound 3a) to 3.7 μM (compound 13). In contrast, similar modification to compound 11g resulted in the most potent agonist 16 with an EC₅₀ value of 0.05 μM. Compound 19 containing a 2-aminobenzothiazole, however, showed no agonist activity at concentrations up to 100 μM. The discovery of agonist 16 is significant not only because of its high agonist potency but also because of its substantially lower pK_a value for the 1-amino group of the aminoisoquinoline (pK_a ~ 7.5) as compared to that of the benzamidine moiety (pKa ~ 12.5) of compound 11g.^13–16

Given the complete loss of agonist activity for ortho-amidine analog 5c, structurally close compounds 5a and 5c served as ideal positive and negative controls in target engagement studies. Activation of MRGPRX1 is known to inhibit high voltage-activated (HVA) Ca²⁺ currents, which reduces neurotransmitter release and attenuates spinal nociceptive transmission.¹⁷ To this end, we assessed BAM8-22, potent agonist 5a, and compound 5c devoid of MRGPRX1 agonist activity for their inhibitory effect on HVA Ca²⁺ currents in cultured human MRGPRX1-expressing DRG neurons obtained from humanized MrgprX1 mice.¹⁷ As shown in Figure 3, both BAM8-22 and compound 5a inhibited HVA Ca²⁺ currents while compound 5c showed no inhibitory effects, indicating the MRGPRX1-dependent mechanism involved in inhibition of HVA Ca²⁺ currents.

Figure 3.

Traces of HVA Ca²⁺ currents evoked by depolarization before (black), after (red) bath application of 5 μM BAM8-22 (A), 10 μM of 5a (B), and 10 μM of 5c (C). Currents were recorded by whole-cell patch clamp recording of DRG neurons from MrgprX1 mice. HVA Ca²⁺ currents are inward currents (negative nA) evoked by −10 mV depolarization.

Given the close relation between opioid and MrgC receptors in regard to ligand pharmacology^{2, 3} and function,^{18, 19} we assessed the selectivity of the two most potent MRGPRX1 agonists 11g and 16 over δ-, μ- and κ-opioid receptors by radioligand binding assays. As shown in Table 2, benzamidine-based agonist 11g exhibited substantial affinity to δ and μ-opioid receptors. Compound 16, however, exhibited 50-fold or greater selectivity over all three receptors, unmasking additional advantage brought by the 1-aminoisoquinoline moiety. Compound 16 was also found to be inactive against mouse MRGPRC11 even at concentration of 40 μM (see the Supporting Information).

Table 2.

Binding affinities (K_i) of 11g and 16 to opioid receptors

	Ki (nM)
cmpd	δ ([3H]DADLE)	μ ([3H]DAMGO)	κ ([3H]U-69,593)
11g	220 nM	72.7 nM	1430 nM
16	2470 nM	3730 nM	12100 nM

Given its reduced basicity, potent MRGPRX1 agonist activity, and good selectivity over opioid receptors, we selected compound 16 for in vivo pharmacokinetics studies in mice. In addition to plasma levels, we measured drug levels in spinal cord, which is the site of action for MRGPRX1 agonists to attenuate spinal nociceptive transmission. As shown in Figure 4, substantial levels of 16 were detected in plasma and spinal cord (AUC: 0.90 ± 0.09 nmol*h/mL plasma vs 5.66 ± 0.33 nmol*h/g spinal cord) following intravenous administration of 16 at 10 mg/kg dose in male CD1 mice (n = 3 for each time point), resulting in a spinal cord to plasma ratio of 6.3. While this is encouraging, 16 had a half-life of approximately 0.33 h. Subsequent studies revealed that 16 is extensively metabolized (t_1/2 < 15 min) in mouse liver microsomes (results not shown), which likely contributed to the short half-life observed in mice after intravenous administration.

Figure 4.

Pharmacokinetics of 16 in mice (10 mg/kg, iv).

Conclusions

Chronic pain remains one of the most prevalent yet undertreated health problems. Furthermore, given the ongoing opioid crisis, there is a critical unmet medical need for new chronic pain therapies devoid of abuse potential and adverse effects. Restricted expression of human MRGPRX1 in primary nociceptive neurons could provide a substantial advantage to its agonists over opioid-based analgesics in treating pain. By use of two amidine-based hit compounds 5a and 11a as molecular templates, systematic lead optimization was carried out in an attempt to improve potency and drug-like molecular properties. In particular, successful conversion of the benzamidine moiety of 11g into a 1-aminoisoquinoline moiety without loss of agonist potency led to a highly potent MRGPRX1 agonist 16 not only devoid of positively charged amidinium group but also with superior selectively over opioid receptors. Compound 16 displayed favorable distribution to the spinal cord, presumably due to the reduced pK_a value for the 1-amino group of the aminoisoquinoline as compared to that of the benzamidine moiety. Although compound 16 showed high degree of clearance, the preferential distribution to the spinal cord over the circulatory system is particularly appealing since it will minimize the itch side effect caused by peripheral MRGPRX1 activation. Given its high selectivity over opioid receptors, compound 16 appears to be a promising agent that merits further investigation. Compound 16, however, failed to act as an agonist at mouse MrgprC11, limiting its utility in conventional mouse models of pain. It should be noted, however, that humanized MrgprX1 mice have been developed to overcome the limited cross-species reactivity of human MRGPRX1 agonists on the murine receptors.¹⁷ These mice lack a cluster containing most MrgprA and MrgprC genes including MrgprC11 and are well suited for studying the pharmacology of human MRGPRX1 modulation. Indeed, intrathecal injection of BAM8-22 was found to inhibit heat hypersensitivity and spontaneous pain in MrgprX1 mice after peripheral nerve injury,¹⁷ demonstrating the utility of this strain in evaluating human MRGPRX1 agonists. Studies are underway to evaluate our human MRGPRX1 agonists in the humanized mice and improve the metabolic stability in liver microsomes to achieve sustained exposure. Collectively, these efforts may lead to the development of viable treatment options for chronic pain.

Experimental Section

General.

All solvents were reagent grade or HPLC grade. Unless otherwise noted, all materials were obtained from commercial suppliers and used without further purification. Syntheses of compound 3,²⁰ 4c²¹ and 14²² were previously reported. Compounds 3c, 3d and 3e were obtained by Mitsunobu reactions of 4-hydroxy-3-methoxybenzaldehyde with the corresponding alcohols (ROH) by following the method previously reported for similar reactions.²³ Melting points were obtained on a Mel-Temp apparatus and are uncorrected. ¹H NMR spectra were recorded at 400 or 500 MHz. ¹³C NMR spectra were recorded at 101 or 125 MHz. The HPLC solvent system consisted of deionized water and acetonitrile, both containing 0.1% formic acid. Preparative HPLC purification was performed on an Agilent 1200 series HPLC system equipped with an Agilent G1315D DAD detector using a Phenomenex Luna 5 μm C18 column (21.2 mm × 250 mm, 5 μm). Analytical HPLC was performed on an Agilent 1200 series HPLC system equipped with an Agilent G1315D DAD detector (detection at 220 nm) and an Agilent 6120 quadrupole MS detector. Unless otherwise specified, the analytical HPLC conditions involve: (A) for nonpolar compounds 20% acetonitrile/80% water for 0.25 min followed by gradient to 85% acetonitrile/15% water over 1.5 min and continuation of 85% acetonitrile/15% water for 2.25 min with a Luna C18 column (2.1 mm × 50 mm, 3.5 μm) at a flow rate of 1.25 mL/min; (B) for polar compounds 5% acetonitrile/95% water for 0.25 min followed by gradient to 40% acetonitrile/60% water over 1.5 min and continuation of 85% acetonitrile/15% water for 2.25 min with a Luna C18 column (2.1 mm × 50 mm, 3.5 μm) at a flow rate of 1.25 mL/min. Unless otherwise noted, all final compounds biologically tested were confirmed to be of ≥ 95% purity by the HPLC methods described above.

4-(4-(Benzyloxy)-3-methoxybenzylamino)benzimidamide (5a).

4-(Benzyloxy)-3-methoxybenzaldehyde 3a (300 mg, 1.24 mmol), 4-aminobenzimidamide dihydrochloride 4a (184 mg, 1.36 mmol), DIEA (2.48 mmol, 0.43 mL) and a trace of bromocresol green were dissolved in ethanol (5 mL) at rt and the mixture was stirred for 15 min. Acetic acid was added dropwise until the blue solution turned slightly green and the resulting mixture was stirred at rt for 15 min. Sodium cyanoborohydride (311 mg, 4.95 mmol) was added portionwise at rt and the reaction was stirred overnight. Volatiles were removed under reduced pressure and the residue was dissolved in EtOAc, washed with 2 N NaOH, water and brine. The organic layer was dried over magnesium sulfate, filtered and solvents evaporated to give a white solid which was purified using preparative HPLC (Method 20–70%: flowrate of 25 mL/min; gradient of 20% acetonitrile/80% H₂O for 5.0 minutes followed by an increase to 70% acetonitrile/30% H₂O over 40 minutes and continuation of 70% acetonitrile/30% H₂O until 50 minutes) to give 94 mg (21% yield) of title compound as white solid; mp 181 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 3.75 (s, 3H), 4.28 (d, J = 6.1 Hz, 2H), 5.04 (s, 2H), 6.69 (d, J = 9.1 Hz, 2H), 6.82 (dd, J = 1.8, 8.2 Hz, 2H), 6.96 (d, J = 8.1 Hz, 1H), 7.00 (d, J = 1.8, 1H), 7.32 (m, 1H), 7.36–7.43 (m, 5H), 7.61 (d, J = 9.1 Hz, 2H), 8.74 (bs, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ 45.6, 55.5, 69.9, 111.5, 113.5, 113.7, 119.2, 127.7, 128.4, 129.2, 131.9, 137.2, 146.7, 149.1, 153.0, 164.9. LCMS: (A) retention time 1.53 min, m/z = 362 [M + H]⁺.

3-(4-(Benzyloxy)-3-methoxybenzylamino)benzimidamide (5b).

Compound 5b was prepared as described for the preparation of 5a except 3-aminobenzimidamide 4b was used in place of 4-aminobenzimidamide 4a and the compound was obtained by recrystallization in EtOAc/hexanes. Light blue solid (34% yield); mp 153 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 3.75 (s, 3H), 4.22 (d, J = 6.1 Hz, 2H), 5.04 (s, 2H), 6.49 (m, 1H), 6.78–6.88 (m, 3H), 6.94–7.00 (m, 3H), 7.17 (m, 1H), 7.32–7.41 (m, 4H), 8.24 (bs, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ 46.1, 55.5, 69.9, 110.5, 111.6, 113.5, 114.5, 116.1, 119.3, 127.8, 128.4, 129.1, 132.5, 137.3, 146.7, 148.9, 149.1, 165.7. LCMS: (A) retention time 1.55 min, m/z = 362 [M + H]⁺.

2-(4-(Benzyloxy)-3-methoxybenzylamino)benzimidamide (5c).

Compound 5c was prepared as described for the preparation of 5a except 2-aminobenzimidamide dihydrochloride 4c was used in place of 4-aminobenzimidamide dihydrochloride 4a and the residue was purified using a Biotage Isolera One flash purification system with a silica gel cartridge (EtOAc/hexanes, 2% Et₃N), followed by a second purification by preparative HPLC (Method 20–70%: flowrate of 25 mL/min; gradient of 20% acetonitrile/80% H₂O for 5.0 minutes followed by an increase to 70% acetonitrile/30% H₂O over 40 minutes and continuation of 70% acetonitrile/30% H₂O until 50 minutes). Off-white solid foam (4% yield); mp 172 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 3.76 (s, 3H), 4.28 (bs, 2H), 5.04 (s, 2H), 6.59 (dd, J = 8.8, 17.2 Hz, 2H), 6.89 (dd, J = 8.3, 19.7 Hz, 2H), 7.05 (d, J = 1.5, 1H), 7.23 (m, 2H), 7.31 (m, 1H), 7.36–7.43 (m, 4H), 8.44 (s, 1H). ¹³C NMR (125 MHz, DMSO-d₆) δ 46.2, 55.5, 69.9, 111.4, 111.7, 113.4, 115.2, 115.5, 119.0, 127.8, 128.4, 129.0, 132.4, 137.3, 145.8, 146.6, 149.1, 166.4, 167.9. LCMS: (B) retention time 2.50 min, m/z = 362 [M + H]⁺.

4-(3-Methoxy-4-(pyridin-3-ylmethoxy)benzylamino)benzimidamide (5d).

Compound 5d was prepared as described for the preparation of 5a except 3-methoxy-4-(pyridin-3-ylmethoxy)benzaldehyde 3b was used in place of 4-(benzyloxy)-3-methoxybenzaldehyde 3a and the compound was purified using preparative HPLC (Method 20–70%: flowrate of 25 mL/min; gradient of 20% acetonitrile/80% H₂O for 5.0 minutes followed by an increase to 70% acetonitrile/30% H₂O over 40 minutes and continuation of 70% acetonitrile/30% H₂O until 50 minutes). White fluffy solid (35% yield); mp 87 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 3.75 (s, 3H), 4.29 (d, J = 6.1 Hz, 2H), 5.09 (s, 2H), 6.69 (d, J = 9.1 Hz, 2H), 6.83 (dd, J = 2.0, 7.8 Hz, 1H), 7.00 (dd, J = 2.5, 4.3 Hz, 2H), 7.32 (d, J = 6.3 Hz, 1H), 7.41 (dd, J = 4.8, 7.8 Hz, 1H), 7.58 (d, J = 8.8 Hz, 2H), 7.83 (d, J = 8.1 Hz, 1H), 8.32 (bs, 2H), 8.53 (dd, J = 1.5, 4.8 Hz, 1H), 8.63 (d, J = 2.0 Hz, 1H), 8.74 (bs, 1H). ¹³C NMR (125 MHz, DMSO-d₆) δ 45.5, 55.6, 67.8, 111.6, 113.0, 113.9, 119.2, 123.6, 129.6, 132.2, 132.8, 135.7, 146.5, 149.1, 149.2, 153.3, 164.3. LCMS: (C) retention time 1.63 min, m/z = 363 [M + H]⁺.

4-(3-Methoxy-4-(thiophen-3-ylmethoxy)benzylamino)benzimidamide (5e).

Compound 5e was prepared as described for the preparation of 5a except 3-methoxy-4-(thiophen-3-ylmethoxy)benzaldehyde 3c was used in place of 4-(benzyloxy)-3-methoxybenzaldehyde 3a and the compound was purified using preparative HPLC (Method 20–70%: flowrate of 25 mL/min; gradient of 20% acetonitrile/80% H₂O for 5.0 minutes followed by an increase to 70% acetonitrile/30% H₂O over 40 minutes and continuation of 70% acetonitrile/30% H₂O until 50 minutes). White fluffy solid (28% yield); mp 112 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 3.74 (s, 3H), 4.29 (d, J = 5.6 Hz, 2H), 5.02 (s, 2H), 6.69 (d, J = 8.8 Hz, 2H), 6.82 (dd, J = 2.3, 5.6 Hz, 1H), 6.98 (m, 2H), 7.14 (dd, J = 2.0, 3.8 Hz, 1H), 7.31 (t, J = 5.8 Hz, 1H), 7.53 (m, 2H), 7.58 (d, J = 8.8 Hz, 2H), 8.41 (bs, 2H), 8.72 (bs, 2H). ¹³C NMR (125 MHz, DMSO-d₆) δ 45.8, 55.7, 65.7, 111.8, 113.0, 113.8, 119.4, 124.3, 126.8, 128.0, 129.9, 132.0, 138.3, 146.9, 149.4, 153.6, 164.5. LCMS: (A) retention time 1.40 min, m/z = 368 [M + H]⁺.

4-(4-(4-(1H-Pyrrol-1-yl)benzyloxy)-3-methoxybenzylamino)benzimidamide (5f).

Compound 5f was prepared as described for the preparation of 5a except 4-(4-(1H-pyrrol-1-yl)benzyloxy)-3-methoxybenzaldehyde 3d was used in place of 4-(benzyloxy)-3-methoxybenzaldehyde 3a and the compound was purified using preparative HPLC (Method 20–70%: flowrate of 25 mL/min; gradient of 20% acetonitrile/80% H₂O for 5.0 minutes followed by an increase to 70% acetonitrile/30% H₂O over 40 minutes and continuation of 70% acetonitrile/30% H₂O until 50 minutes). White solid (45%); mp 225 °C (decomp). ¹H NMR (400 MHz, DMSO-d₆) δ 3.76 (s, 3H), 4.28 (d, J = 5.8 Hz, 2H), 5.06 (s, 2H), 6.26 (t, J = 2.3 Hz, 2H), 6.67 (d, J = 8.8 Hz, 2H), 6.83 (dd, J = 1.5, 7.8 Hz, 1H), 6.98 (m, 2H), 7.25 (t, J = 6.1 Hz, 1H), 7.37 (t, J = 2.3 Hz, 2H), 7.48 (m, 2H), 7.57–7.60 (m, 4H), 8.43 (s, 1H), 8.52 (bs, 2H), 10.22 (bs, 2H). ¹³C NMR (125 MHz, DMSO-d₆) δ 45.6, 55.6, 69.4, 110.5, 111.5, 113.6, 119.0, 119.2, 129.2, 131.9, 134.1, 139.5, 146.7, 149.2, 153.1, 164.8, 167.6. LCMS: (A) retention time 1.73 min, m/z = 427 [M + H]⁺.

4-(4-(Isopentyloxy)-3-methoxybenzylamino)benzimidamide (5g).

Compound 5g was prepared as described for the preparation of 5a except 4-(isopentyloxy)-3-methoxybenzaldehyde 3e was used in place of 4-(benzyloxy)-3-methoxybenzaldehyde 3a and the compound was purified using preparative HPLC (Method 20–70%: flowrate of 25 mL/min; gradient of 20% acetonitrile/80% H₂O for 5.0 minutes followed by an increase to 70% acetonitrile/30% H₂O over 40 minutes and continuation of 70% acetonitrile/30% H₂O until 50 minutes). White hygroscopic solid (37% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 0.90 (d, J = 6.6 Hz, 6H), 1.55 (qt, J =6.8, 13.4 Hz, 2H), 1.76 (m, 1H), 3.73 (s, 3H), 3.90 (t, J = 6.8 Hz, 2H), 4.27 (d, J = 5.8 Hz, 2H), 6.68 (d, J = 8.8 Hz, 2H), 6.82 (m, 1H), 6.89 (d, J = 8.3 Hz, 1H), 6.96 (d, J = 1.8 Hz, 1H), 7.28 (t, J = 6.1 Hz, 1H), 7.57 (d, J = 9.1 Hz, 2H), 8.45 (s, 1H), 8.71 (bs, 1H), 9.33 (bs, 2H). ¹³C NMR (125 MHz, DMSO-d₆) δ 22.5, 24.5, 37.6, 46.0, 55.5, 66.7, 111.5, 113.0, 113.5, 119.3, 129.3, 131.4, 147.2, 149.0, 153.1, 164.6, 168.0. LCMS: (A) retention time 1.57 min, m/z = 342 [M + H]⁺.

4-(4-(Benzyloxy)benzylamino)benzimidamide (5h).

Compound 5h was prepared as described for the preparation of 5a except 4-(benzyloxy)benzaldehyde 3f was used in place of 4-(benzyloxy)-3-methoxybenzaldehyde 3a and the compound was purified using preparative HPLC (Method 20–70%: flowrate of 25 mL/min; gradient of 20% acetonitrile/80% H₂O for 5.0 minutes followed by an increase to 70% acetonitrile/30% H₂O over 40 minutes and continuation of 70% acetonitrile/30% H₂O until 50 minutes). White solid (57% yield). mp 222 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 4.28 (d, J = 5.8 Hz, 2H), 5.07 (s, 2H), 6.85 (d, J = 8.8 Hz, 2H), 6.96 (d, J = 8.6 Hz, 2H), 7.25 (m, 3H), 7.32 (m, 1H), 7.36 (m, 2H), 7.42 (m, 2H), 7.56 (d, J = 8.8 Hz, 2H), 8.47 (s, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 45.6, 69.6, 114.1, 115.2, 128.1, 128.3, 128.9, 128.9, 129.7, 131.6, 137.6, 153.4, 157.8, 165.3, 168.0. LCMS: (A) retention time 1.76 min, m/z = 332 [M + H]⁺.

4-(4-Methyl-2-nitrophenoxy)benzonitrile (8a).

A mixture of 1-fluoro-4-methyl-2-nitrobenzene 6a (7.5 g, 48.4 mmol), 4-hydroxybenzonitrile 7a (5.76 g, 48.4 mmol) and potassium carbonate (20.0 g, 145.0 mmol) in DMF (100 mL) was heated at 50 °C overnight. After cooling, solvent was removed under reduced pressure, 10% KHSO₄ solution in water and EtOAc were added and the layers were separated. The organic layer was washed with brine, dried over magnesium sulfate and concentrated. The residue was triturated in a EtOAc-hexanes mixture to give 11 g of product as a yellow solid (89% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 2.43 (s, 3H), 7.12 (d, J = 8.8 Hz, 2H), 7.33 (d, J = 8.3 Hz, 1H), 7.63 (dd, J = 0.8, 8.3 Hz, 1H), 7.85 (d, J = 9.1 Hz, 2H), 8.00 (d, J = 1.0 Hz, 1H).

4-(2-Nitrophenoxy)benzonitrile (8b).

Compound 8b was prepared as described for the preparation of 8a except 1-fluoro-2-nitrobenzene 6b was used in place of 1-fluoro-4-methyl-2-nitrobenzene 6a. Yellow solid (85% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 7.16 (d, J = 8.8 Hz, 2H), 7.40 (d, J = 8.3 Hz, 1H), 7.51 (m, 1H), 7.79 (m, 1H), 7.87 (d, J = 9.1 Hz, 2H), 8.15 (m, 1H).

4-(5-Methyl-2-nitrophenoxy)benzonitrile (8c).

Compound 8c was prepared as described for the preparation of 8a except 2-fluoro-4-methyl-1-nitrobenzene 6c was used in place of 1-fluoro-4-methyl-2-nitrobenzene 6a. Tan solid (76% yield). ¹H NMR (400 MHz, CDCl₃) δ 2.45 (s, 3H), 6.99–7.04 (m, 3H), 7.18 (dd, J = 1.8, 8.3 Hz, 1H), 7.64 (m, 2H), 7.98 (d, J = 8.6 Hz, 1H).

3-(5-Methyl-2-nitrophenoxy)benzonitrile (8d).

Compound 8d was prepared as described for the preparation of 8a except 2-fluoro-4-methyl-1-nitrobenzene 6c was used in place of 1-fluoro-4-methyl-2-nitrobenzene 6a and 3-hydroxybenzonitrile 7b in place of 4-hydroxybenzonitrile 7a. Light yellow solid (80% yield). ¹H NMR (400 MHz, CDCl₃) δ 2.44 (s, 3H), 6.93 (s, 1H), 7.15 (m, 1H), 7.20 (m, 1H), 7.24 (m, 1H), 7.43 (m, 1H), 7.48 (m, 1H), 7.97 (d, J = 8.6 Hz, 1H).

2-(5-Methyl-2-nitrophenoxy)benzonitrile (8e).

Compound 8e was prepared as described for the preparation of 8a except 2-fluoro-4-methyl-1-nitrobenzene 6c was used in place of 1-fluoro-4-methyl-2-nitrobenzene 6a and 2-hydroxybenzonitrile 7c in place of 4-hydroxybenzonitrile 7a. Light tan solid (65% yield). ¹H NMR (400 MHz, CDCl3) δ 2.47 (s, 3H), 6.77 (d, J = 8.3 Hz, 1H), 7.09 (d, J = 8.3 Hz, 1H), 7.17 (td, J = 1.0, 7.8 Hz, 1H). 7.45 (m, 2H), 7.88 (dd, J = 1.8, 7.8 Hz, 1H), 7.88 (d, J = 2.3 Hz, 1H).

4-(2-Amino-4-methylphenoxy)benzonitrile (9a).

To a solution of 8a in methanol was added a spatula tip of 5% Pd/C and the mixture was hydrogenated for 40 min at 30 psi. The reaction was filtered through celite and the filtrate was concentrated in vacuo. Beige solid (90% yield). ¹H NMR (400 MHz, CDCl3) δ 2.19 (s, 3H), 4.94 (s, 2H), 6.38 (d, J = 7.8 Hz, 1H), 6.64 (s, 1H), 6.76 (d, J = 7.8 Hz, 1H). 6.95 (d, J = 8.1 Hz, 2H), 7.76 (d, J = 8.1 Hz, 2H).

4-(2-Aminophenoxy)benzonitrile (9b).

The title compound was prepared as described for the preparation of 9a except compound 8b was used in place of 8a. Light tan solid (quantitative yield). ¹H NMR (400 MHz, DMSO-d₆) δ 5.04 (s, 2H), 6.58 (m, 1H), 6.84 (m, 2H), 6.98 (m, 3H), 7.79 (m, 2H).

4-(2-Amino-5-methylphenoxy)benzonitrile (9c).

The title compound was prepared as described for the preparation of 9a except compound 8c was used in place of 8a. Yellow solid (quantitative yield). ¹H NMR (400 MHz, DMSO-d₆) δ 2.15 (s, 3H), 4.80 (s, 2H), 6.72 (m, 2H), 6.81 (m, 1H), 6.96 (m, 2H), 7.77 (m, 2H).

3-(2-Amino-5-methylphenoxy)benzonitrile (9d).

The title compound was prepared as described for the preparation of 9a except compound 8d was used in place of 8a. Beige solid (75% yield);. ¹H NMR (400 MHz, CDCl3) δ 2.25 (s, 3H), 3.62 (s, 2H), 6.72 (d, J = 2.0 Hz, 1H), 6.76 (d, J = 8.1 Hz, 1H), 6.87 (m, 1H), 7.18 (m, 1H), 7.20 (m, 1H), 7.32 (m, 1H), 7.39 (m, 1H).

2-(2-Amino-5-methylphenoxy)benzonitrile (9e).

The title compound was prepared as described for the preparation of 9a except compound 8e was used in place of 8a. Light tan solid (87% yield). ¹H NMR (400 MHz DMSO-d₆) δ 2.20 (s, 3H), 5.01 (s, 2H), 6.38 (m, 1H), 6.65 (m, 2H), 6.77 (d, J = 8.1 Hz, 1H), 7.13 (td, J = 1.0, 7.6 Hz, 1H), 7.56 (m, 1H), 7.80 (dd, J = 1.5, 7.3 Hz, 1H).

N-(2-(4-Cyanophenoxy)-5-methylphenyl)butane-1-sulfonamide (10a).

To a solution of Compound 9a (298 mg, 1.33 mmol) in pyridine (10 mL) was added butane-1-sulfonyl chloride (208 mg, 1.61 mmol) at rt. The reaction was stirred at rt for 10 min, then heated at 50 °C for 1.5 h. Pyridine was removed in vacuo and the crude material was partitioned between water and EtOAc. The organic layer was washed with brine, dried over sodium sulfate and concentrated in vacuo. The residue was purified using a Biotage Isolera One flash purification system with a silica gel cartridge (EtOAc/hexanes) to give 256 mg (56% yield) of title compound as light brown solid. ¹H NMR (500 MHz, CDCl₃) δ 0.87 (dd, J = 7.8, 6.9 Hz, 3H), 1.38 (q, J = 7.4 Hz, 2H), 1.74 (td, J = 7.6, 7.0, 3.0 Hz, 2H), 2.38 (s, 3H), 3.16 – 2.92 (m, 2H), 6.56 (s, 1H), 6.88 (d, J = 8.3 Hz, 1H), 6.95 (dd, J = 8.3, 2.0 Hz, 1H), 7.03 (d, J = 8.6 Hz, 2H), 7.50 (d, J = 2.0 Hz, 1H), 7.64 (d, J = 8.7 Hz, 2H).

N-(2-(4-Cyanophenoxy)phenyl)butane-1-sulfonamide (10b).

The title compound was prepared as described for the preparation of 10a except compound 9b was used in place of 9a and the crude material was purified using DCM as mobile phase. Brown oil (46% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 0.76 (t, J = 7.3 Hz, 3H), 1.25 (m, 2H), 1.59 (m, 2H), 2.98 (t, J = 7.8 Hz, 2H), 7.03 (dd, J = 1.5, 7.8 Hz, 1H), 7.09 (d, J = 8.8 Hz, 2H), 7.15 (m, 1H), 7.21 (m, 1H), 7.49 (dd, J = 1.8, 8.1 Hz, 1H), 7.83 (d, J = 8.8 Hz, 2H), 8.42 (s, 1H).

N-(2-(4-Cyanophenoxy)-4-methylphenyl)butane-1-sulfonamide (10c).

The title compound was prepared as described for the preparation of 10a except compound 9c was used in place of 9a. Dark orange oil (57% yield). ¹H NMR (500 MHz, CDCl₃) δ 0.87 (td, J = 7.4, 1.1 Hz, 3H), 1.37 (q, J = 7.5 Hz, 2H), 1.79 – 1.69 (m, 2H), 2.30 (s, 3H), 3.08 – 3.02 (m, 2H), 6.50 (s, 1H), 6.78 (d, J = 1.8 Hz, 1H), 7.08 – 7.00 (m, 3H), 7.56 (d, J = 8.3 Hz, 1H), 7.66 (d, J = 8.8 Hz, 1H).

N-(2-(4-Cyanophenoxy)-4-methylphenyl)-4-methylbenzenesulfonamide (10d).

The title compound was prepared as described for the preparation of 10a except compound 9c was used in place of 9a and 4-tolylsulfonyl chloride in place of butane-1-sulfonyl chloride. Beige solid (98% yield). ¹H NMR (400 MHz, CDCl₃) δ 2.26 (s, 3H), 2.39 (s, 3H), 6.61 (m, 3H), 6.75 (s, 1H), 7.00 (dd, J = 1.5, 8.3 Hz, 1H), 7.10 (d, J = 7.8 Hz, 2H), 7.45 (m, 2H), 7.52 (m, 2H), 7.63 (d, J = 8.1 Hz, 1H).

N-(2-(4-Cyanophenoxy)-4-methylphenyl)-2-(trifluoromethoxy)benzenesulfonamide (10e).

The title compound was prepared as described for the preparation of 10a except compound 9c was used in place of 9a and 2-(trifluoromethoxy)benzene-1-sulfonyl chloride in place of butane-1-sulfonyl chloride. Off white solid (82% yield). ¹H NMR (400 MHz, CDCl₃) δ 2.23 (s, 3H), 6.65 (d, J = 1.8 Hz, 1H), 6.78 (m, 2H), 6.94 (m, 1H), 7.09 (s, 1H), 7.11 (m, 1H), 7.31 (m, 1H), 7.53 (m, 4H), 7.93 (dd, J = 1.8, 7.8 Hz, 1H).

N-(2-(4-Cyanophenoxy)-4-methylphenyl)-4-fluorobenzenesulfonamide (10f).

The title compound was prepared as described for the preparation of 10a except compound 9c was used in place of 9a and 4-fluorobenzene-1-sulfonyl chloride in place of butane-1-sulfonyl chloride. White solid (85% yield). ¹H NMR (500 MHz, CDCl₃) δ 2.26 (s, 3H), 6.62 (s, 1H), 6.71–6.64 (m, 3H), 6.99 – 7.04 (m, 3H), 7.53 (dd, J = 7.8, 1.7 Hz, 2H), 7.60 (d, J = 8.4 Hz, 1H), 7.72 – 7.64 (m, 2H).

N-(2-(4-Cyanophenoxy)-4-methylphenyl)-2-methoxybenzenesulfonamide (10g).

The title compound was prepared as described for the preparation of 10a except compound 9c was used in place of 9a and 2-methoxybenzene-1-sulfonyl chloride in place of butane-1-sulfonyl chloride. Light tan solid (quantitative yield). ¹H NMR (400 MHz, DMSO-d₆) δ 2.18 (s, 3H), 3.65 (s, 3H), 6.73 (m, 3H), 6.96 (m, 3H), 7.25 (d, J = 8.1 Hz, 1H), 7.47 (m, 1H), 7.57 (m, 1H), 7.71 (m, 2H), 9.31 (s, 1H).

N-(2-(3-Cyanophenoxy)-4-methylphenyl)-2-methoxybenzenesulfonamide (10h).

The title compound was prepared as described for the preparation of 10a except compound 9d was used in place of 9a and 2-methoxybenzene-1-sulfonyl chloride in place of butane-1-sulfonyl chloride. Tan solid (83% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 2.17 (s, 3H), 2.28 (s, 3H), 3.61 (s, 3H), 6.68 (m, 2H), 6.75 (m, 2H), 6.94 (m, 1H), 6.98 (m, 1H), 7.27 (d, J = 8.1 Hz, 1H), 7.43 (dd, J = 17.8, 16.9 Hz, 2H), 7.51 (m, 1H), 9.15 (s, 1H).

N-(2-(2-Cyanophenoxy)-4-methylphenyl)-2-methoxybenzenesulfonamide (10i).

The title compound was prepared as described for the preparation of 10a except compound 9e was used in place of 9a and 2-methoxybenzene-1-sulfonyl chloride in place of butane-1-sulfonyl chloride. Tan solid (quantitative yield). ¹H NMR (400 MHz, DMSO-d₆) δ 2.23 (s, 3H), 3.73 (s, 3H), 6.37 (d, J = 8.6 Hz, 1H), 6.83 (d, J = 8.3 Hz, 1H), 6.97 (m, 3H), 7.17 (td, J = 0.8, 7.6 Hz, 1H), 7.22 (d, J = 2.0 Hz, 1H), 7.48 (m, 2H), 7.66 (dd, J = 1.8, 7.8 Hz, 1H), 7.78 (dd, J = 1.8, 7.8 Hz, 1H), 9.55 (s, 1H).

4-(2-(Butylsulfonamido)-4-methylphenoxy)benzimidamide (11a).

A solution of 10a (230 mg, 0.668 mmol) in mixture of anhydrous methanol (10 mL) and anhydrous dioxane (1 mL) was subjected to a stream of bubbling HCl (gas) at 0 °C for 30 min. The flask was capped with a septum and the mixture was stirred at rt overnight. Methanol was removed in vacuo and the residue was dried under high vacuum. The obtained material was re-dissolved in a 7 N solution of ammonia in methanol (20 mL) and the mixture was heated at 65 °C for 1 h before cooling to rt and stirring overnight. The solvent was removed in vacuo. The resulting residue was purified using reverse phase preparative HPLC (20% MeCN/80 % water followed by an increase to 70 % MeCN over 40 min and an increase to 100 % MeCN over 10 min; flow rate 15 mL/min) to give title compound (15% yield) as a white solid; mp 82 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 0.77 (t, J = 7.3 Hz, 3H), 1.24 (h, J = 7.4 Hz, 2H), 1.54 – 1.36 (m, 2H), 2.23 (s, 3H), 2.73 – 2.65 (m, 2H), 6.54 (dd, J = 8.2, 2.2 Hz, 1H), 6.78 (d, J = 7.9 Hz, 1H), 6.92 – 6.83 (m, 2H), 7.24 (d, J = 2.1 Hz, 1H), 7.77 – 7.68 (m, 2H), 9.18 (s, 4H). LCMS: (A) retention time 1.40 min, m/z 362 [M + H]⁺. ¹³C NMR (101 MHz, DMSO-d₆) δ 14.0, 21.1, 21.3, 25.8, 52.2, 117.4, 121.3, 123.3, 126.9, 127.1, 128.5, 129.3, 130.4, 135.4, 162.3, 165.4. LCMS: (A) retention time 1.40 min, m/z 362 [M + H]⁺.

4-(2-(Butylsulfonamido)phenoxy)benzimidamide (11b).

The title compound was prepared as described for the preparation of 11a except compound 10b was used in place of 10a. White solid (25% yield); mp 50 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 0.75 (t, J = 7.3 Hz, 3H), 1.26 (m, 2H), 1.58 (m, 2H), 2.97 (t, J = 7.8 Hz, 2H), 7.02 (dd, J = 1.5, 7.8 Hz, 1H), 7.08 (d, J = 8.8 Hz, 2H), 7.15 (m, 1H), 7.20 (m, 1H), 7.48 (dd, J = 1.8, 8.1 Hz, 1H), 7.82 (d, J = 8.8 Hz, 2H), 8.41 (s, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ 13.5, 20.9, 24.9, 117.1, 119.4, 122.9, 124.6, 126.0, 128.8, 130.4, 132.8, 146.3, 165.5. LCMS: (A) retention time 1.45 min, m/z 348 [M + H]⁺.

4-(2-(Butylsulfonamido)-5-methylphenoxy)benzimidamide (11c).

The title compound was prepared as described for the preparation of 11a except compound 10c was used in place of 10a. White solid (57% yield); mp 178 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 0.77 (t, J = 7.6 Hz, 3H), 1.25 (m, 2H), 1.58 (m, 2H), 2.24 (s, 3H), 2.10 (m, 2H), 6.85 (d, J = 1.3 Hz, 1H), 7.01 (dd, J = 1.3, 8.5 Hz, 1H), 7.08 (d, J = 8.8 Hz, 2H), 7.34 (d, J = 8.2 Hz, 1H), 8.44 (s, 1H). ¹³C NMR (125 MHz, DMSO-d₆) δ 13.5, 20.4, 20.8, 25.3, 51.6, 117.4, 121.0, 122.7, 126.0, 128.7, 129.9, 165.1, 167.3. LCMS: (A) retention time 1.60 min, m/z 362 [M + H]⁺.

4-(5-Methyl-2-(4-methylphenylsulfonamido)phenoxy)benzimidamide (11d).

The title compound was prepared as described for the preparation of 11a except compound 10d was used in place of 10a. White powder (22% yield); mp 140 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 2.19 (s, 3H), 2.29 (s, 3H), 6.75 (m, 3H), 6.96 (dd, J = 2.0, 8.3 Hz, 1H), 7.20 (dd, J = 6.3, 8.6 Hz, 3H), 7.51 (d, J = 8.1 Hz, 2H), 7.73 (d, J = 8.8 Hz, 2H), 9.17 (bs, 2H), 9.71 (bs, 2H). ¹³C NMR (125 MHz, DMSO-d₆) δ 20.3, 20.9, 116.9, 121.1, 121.9, 125.5, 126.0, 126.6, 129.3, 129.7, 138.3, 142.4, 147.2, 161.4, 165.0, 167.7. LCMS: (A) retention time 1.44 min, m/z 396 [M + H]⁺.

4-(5-Methyl-2-(2-(trifluoromethoxy)phenylsulfonamido)phenoxy)benzimidamide (11e).

The title compound was prepared as described for the preparation of 11a except compound 10e was used in place of 10a. White fluffy solid (80% yield); mp 110 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 2.17 (s, 3H), 6.71 (s, 1H), 6.81 (dd, J = 1.5, 8.6 Hz, 2H), 6.86 (d, J = 8.6 Hz, 1H), 7.15 (d, J = 8.1 Hz, 1H), 7.33 (m, 2H), 7.54 (t, J = 7.8 Hz, 1H), 7.72 (m, 3H), 8.28 (s, 1H), 9.07 (bs, 2H), 9.61 (bs, 2H). ¹³C NMR (125 MHz, DMSO-d₆) δ 20.3, 116.7, 118.6 120.7 121.1, 121.2, 121.4, 125.9, 126.8, 129.8, 130.1, 133.4, 145.2, 161.8, 164.9, 166.4. LCMS: (A) retention time 1.60 min, m/z 466 [M + H]⁺.

4-(2-(4-Fluorophenylsulfonamido)-5-methylphenoxy)benzimidamide (11f).

The title compound was prepared as described for the preparation of 11a except compound 10f was used in place of 10a. White fluffy solid (43% yield); mp 185 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 2.10 (s, 3H), 6.61 (d, J = 2.3 Hz, 1H), 6.65 (m, 1H), 6.80 (m, 2H), 7.10 (m, 3H), 7.56 (dd, J = 5.7, 8.8 Hz, 2H), 7.71 (m, 2H), 8.91 (bs, 3H). ¹³C NMR (125 MHz, DMSO-d₆) 20.4, 115.8, 116.1, 116.8, 121.3, 122.1, 125.9, 126.1, 128.3, 129.4, 129.5, 129.8, 135.4, 137.8, 137.8, 147.3, 161.4, 162.7, 165.2, 165.2. LCMS: (A) retention time 1.35 min, m/z 400 [M + H]⁺.

4-(2-(2-Methoxyphenylsulfonamido)-5-methylphenoxy)benzimidamide (11g).

The title compound was prepared as described for the preparation of 11a except compound 10g was used in place of 10a. White needles (58% yield); mp 149 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 2.17 (s, 3H), 3.67 (s, 3H), 6.72 (d, J = 1.3 Hz, 1H), 6.79 (d, J = 8.8 Hz, 2H), 6.94–7.01 (m, 3H), 7.23 (d, J = 8.3 Hz, 1H), 7.47 (m, 1H), 7.58 (dd, J = 1.5, 7.8 Hz, 1H), 7.74 (d, J = 8.8 Hz, 2H), 9.23 (bs, 2H), 9.78 (bs, 2H). ¹³C NMR (125 MHz, DMSO-d₆) δ 20.3, 55.8, 112.7, 117.2, 119.8, 120.8 122.9, 125.8, 129.4, 130.0, 134.7, 147.5, 156.3, 161.5, 164.8. LCMS: (A) retention time 1.34 min, m/z 412 [M + H]⁺.

3-(2-(2-Methoxyphenylsulfonamido)-5-methylphenoxy)benzimidamide (11h).

The title compound was prepared as described for the preparation of 11a except compound 10h was used in place of 10a. Brown solid (14% yield); mp 117 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 2.16 (s, 3H), 3.66 (s, 3H), 6.65 (d, J = 1.3 Hz, 1H), 6.88 (m, 2H), 6.97 (m, 2H), 7.15 (s, 1H), 7.18 (d, J = 8.3 Hz, 1H), 7.48 (m, 3H), 7.62 (m, 1H), 8.40 (bs, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 20.4, 55.8, 112.6, 117.1, 119.7, 120.2, 122.1, 125.3, 129.5, 130.4, 130.5, 134.5, 156.3, 157.0, 165.4. LCMS: (A) retention time 1.62 min, m/z 412 [M + H]⁺.

2-(2-(2-Methoxyphenylsulfonamido)-5-methylphenoxy)benzimidamide (11i).

The title compound was prepared as described for the preparation of 11a except compound 10i was used in place of 10a. Light tan solid (21% yield); mp 232 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 2.04 (s, 3H), 3.56 (s, 3H), 6.36 (d, J = 7.1 Hz, 1H), 6.81 (m, 1H), 6.92 (m, 4H), 7.16 (m, 1H), 7.29 (m, 1H), 7.47 (m, 1H), 7.54 (d, J = 7.1 Hz, 1H), 7.58 (d, J = 6.8 Hz, 1H), 8.49 (bs, 2H), 10.98 (bs, 1H). ¹³C NMR (125 MHz, DMSO-d₆) δ 21.2, 55.6, 99.5, 112.3, 116.4, 116.6, 119.0, 120.0 122.0, 122.7, 129.7, 129.8, 131.5, 133.6, 134.1, 143.7, 156.6, 157.5, 163.7. LCMS: (A) retention time 1.37 min, m/z 412 [M + H]⁺.

N⁶-(4-(Benzyloxy)-3-methoxybenzyl)isoquinoline-1,6-diamine (13).

The title compound was prepared as described for the preparation of 5a except isoquinoline-1,6-diamine 12 was used in place of 4-aminobenzimidamide 4a. Crude material was purified using a Biotage Isolera One flash purification system with a silica gel cartridge (CHCl₃/MeOH, 0.5% Et₃N). Tan powder (10% yield); mp 155 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 3.76 (s, 3H), 4.26 (d, J = 5.6 Hz, 2H), 5.04 (s, 2H), 6.39 (s, 2H), 6.50 (d, J = 2.0 Hz, 1H), 6.55 (d, J = 5.8 Hz, 1H), 6.71 (t, J = 5.8 Hz, 1H), 6.87 (m, 2H), 6.96 (d, J = 8.1 Hz, 1H), 7.04 (d, J = 1.5 Hz, 1H), 7.31 (m, 1H), 7.36–7.43 (m, 4H), 7.52 (d, J = 5.8 Hz, 1H), 7.84 (d, J = 9.1 Hz, 1H). ¹³C NMR (125 MHz, DMSO-d₆) δ 46.0, 55.5, 69.9, 102.0, 109.3, 109.4, 111.6, 113.5, 116.1, 119.3, 125.0, 127.8, 128.4, 132.3, 137.3, 139.1, 140.9, 146.7, 149.1, 149.7, 156.7. LCMS: (A) retention time 1.65 min, m/z 386 [M + H]⁺.

6-(5-Methyl-2-nitrophenoxy)isoquinolin-1-amine (15).

The title compound was prepared as described for the preparation of 8a except 1-fluoro-2-nitrobenzene 6c was used in place of 1-fluoro-4-methyl-2-nitrobenzene 6a and 1-aminoisoquinolin-6-ol 14 in place of 4-hydroxybenzonitrile 4a. Crude material was purified using a Biotage Isolera One flash purification system with a silica gel cartridge (DCM/EtOAc, 0.5% Et₃N). Yellow powder (92% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 2.37 (s, 3H), 6.81 (m, 3H), 7.12 (d, J = 2.5 Hz, 1H), 7.15 (s, 1H), 7.21 (dd, J = 2.8, 9.1 Hz, 1H), 7.26 (m, 1H), 7.74 (d, J = 5.8 Hz, 1H), 8.04 (d, J = 8.3 Hz, 1H), 8.24 (d, J = 9.1 Hz, 1H).

N-(2-(1-Aminoisoquinolin-6-yloxy)-4-methylphenyl)-2-methoxybenzenesulfonamide (16).

To a solution of compound 15 (219 mg, 0.825 mmol) in pyridine (10 mL) was added 2-methoxybenzenesulfonyl chloride (341 mg, 1.65 mmol) at rt. The reaction was stirred at rt for 10 min, then heated at 65 °C overnight. Pyridine was removed in vacuo and the crude material was partitioned between water and EtOAc. The organic layer was washed with brine, dried over magnesium sulfate and concentrated in vacuo. The residue was purified using a Biotage Isolera One flash purification system with a silica gel cartridge (CHCl₃/MeOH) to give 16 as yellow solid (98% yield); mp 238 °C (decomp). ¹H NMR (400 MHz, DMSO-d₆) δ 2.26 (s, 3H), 3.61 (s, 3H), 6.83 (d, J = 2.5 Hz, 1H), 6.85 (d, J = 8.6 Hz, 1H), 6.91 (m, 2H), 7.00 (m, 2H), 7.15 (dd, J = 2.5, 9.1 Hz, 1H), 7.24 (d, J = 2.0 Hz, 1H), 7.36 (m, 1H), 7.58 (d, J = 7.1 Hz, 1H), 7.61 (dd, J = 1.8, 7.8 Hz, 1H), 8.43 (d, J = 9.3 Hz, 1H), 8.88 (bs, 2H), 9.42 (s, 1H), 12.93 (s, 1H). ¹³C NMR (125 MHz, DMSO-d₆) δ 20.6, 55.8, 110.9, 111.3, 112.5, 119.5, 119.8, 121.1, 126.2, 127.2, 127.3, 127.8, 129.1, 129.3, 134.7, 135.1, 139.2, 144.5, 153.5, 156.2, 158.4, 162.2. LCMS: (A) retention time 1.65 min, m/z 436 [M + H]⁺.

5-(5-Methyl-2-nitrophenoxy)benzo[d]thiazol-2-amine (18).

The title compound was prepared as described for the preparation of 8a except 1-fluoro-2-nitrobenzene 6c was used in place of 1-fluoro-4-methyl-2-nitrobenzene 6a and 2-aminobenzo[d]thiazol-5-ol 17 in place of 4-hydroxybenzonitrile 4a. Crude material was purified using a Biotage Isolera One flash purification system with a silica gel cartridge (DCM/EtOAc, 0.5% Et₃N), Yellow powder (84% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 2.30 (s, 3H), 6.85 (s, 1H), 6.95 (dd, J = 2.8, 8.8 Hz, 1H), 7.09 (d, J = 8.8 Hz, 1H), 7.34 (d, J = 8.6 Hz, 1H), 7.49 (m, 3H), 7.94 (d, J = 8.3 Hz, 1H).

N-(2-(2-Aminobenzo[d]thiazol-5-yloxy)-4-methylphenyl)-2-methoxybenzenesulfonamide (19).

To a solution of compound 18 (228 mg, 0.840 mmol) in pyridine (10 mL) was added 2-methoxybenzenesulfonyl chloride (347 mg, 1.68 mmol) at rt. The reaction was stirred at rt for 10 min, then heated at 65 °C overnight. Pyridine was removed in vacuo and the crude material was partitioned between water and EtOAc. The organic layer was washed with brine, dried over magnesium sulfate and concentrated in vacuo. The residue was purified using a Biotage Isolera One flash purification system with a silica gel cartridge (CHCl₃/MeOH) to give 19 as orange solid (97% yield), mp 198 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 2.20 (s, 3H), 3.67 (s, 3H), 6.58 (d, J = 8.3 Hz, 1H), 6.65 (dd, J = 2.5, 8.6 Hz, 1H), 6.85 (dd, J = 2.0, 8.3 Hz, 1H), 6.96–7.01 (m, 3H), 7.20 (d, J = 2.0 Hz, 1H), 7.28 (d, J = 8.8 Hz, 1H), 7.47 (m, 1H), 7.64 (dd, J = 1.8, 7.8 Hz, 1H), 8.43 (bs, 2H), 9.07 (s, 1H). ¹³C NMR (125 MHz, DMSO-d₆) δ 20.4, 55.9, 111.6, 112.7, 116.8, 117.3, 118.1, 119.7, 125.7, 126.9, 127.2, 127.5, 129.4, 132.6, 134.7, 147.6, 150.8, 151.8, 156.5, 158.1, 167.2. LCMS: (A) retention time 1.78 min, m/z 442 [M + H]⁺.

In Vitro MrgV1 receptor assay.

HEK293 cells stably transfected with human MrgprX1 were plated in 96 well plates at 25,000 cell/well and incubated 2 days before imaging. On the day of imaging cells were incubated in 100 μL HBSS with 2 μM Fluo 4AM and 1% Trypan Red for 50 minutes at 37°C. The cells were then equilibrated for 10 minutes at room temperature before imaging. Test compounds were dissolved in HBSS and diluted in a serial dilution. Test compounds, BAM 8–22 (positive control) or HBSS (negative control) were added (50 μL into 100 μL) and cells were imaged on the FLIPR for 2 minutes. Data was exported as maximum – minimum fluorescent signal.

DRG sensory neuronal cultures.

DRGs from 3–4-week-old MrgprX1 mice were collected in cold DH10 medium (DMEM/F-12 with 10% fetal bovine serum and 1% penicillin/streptomycin, Gibco) and treated with enzyme solution (5 mg/mL dispase and 1 mg/mL collagenase Type I in HBSS without Ca²⁺ and Mg²⁺, Gibco) at 37°C. After trituration and centrifugation, cells were re-suspended in DH10 with nerve growth factor (50 ng/mL, Upstate Biotechnology, Lake Placid, NY) and glial cell line-derived neurotrophic factor (25 ng/mL, R&D Systems), plated on glass coverslips coated with poly-D-lysine (100 μg/mL, Biomedical Technologies) and laminin (10 μg/mL, Invitrogen), cultured at 37°C, and used after 20–40 hours.²⁴

Whole-cell recordings of cultured DRG neurons.

Whole cell currents of cultured DRG neurons from MrgprX1 mice with MrgprA3-GFP marker were recorded with an Axon 700B amplifier and pCLAMP 9.2 software (Molecular Devices, Sunnyvale, CA, USA). Extracellular solution contained (in mM) 130 N-methyl-D-glucamine chloride (NMDG-Cl), 5 BaCl₂, 1 MgCl₂, 10 HEPES, and 10 glucose, with pH of 7.4 adjusted with 1 M NMDG. Osmolality was adjusted to 310 mOsm/kg with sucrose. Electrodes were pulled (Model pp-830; Narishige, East Meadow, NY, USA) from borosilicate glass (World Precision Instruments, Sarasota, FL, USA) with resistances of 2–4 MΩ. Pipette solution contained (in mM) 140 tetraethylammonium chloride, 10 EGTA, 1 MgCl₂, 10 HEPES, 0.5 GTP, and 3 ATP, with pH of 7.4 and osmolality of approximately 300 mOsm/kg. The voltage protocol was modified from a previously published method.³ Briefly, cells were held at −80 mV and evoked to −40 mV for 20 milliseconds (ms) to activate low-voltage Ca²⁺ channels, and then held to −60 mV for 20 ms and evoked to −10 mV for 40 ms to activate HVA Ca²⁺ channels. Leak currents were subtracted with P/4 protocol. Liquid junction potentials and whole cell capacitances were offset, and series resistances were compensated by 70%. All experiments were performed at room temperature (21–23°C).

Opioid Receptor Binding Affinity.

As previously reported in detail,²⁵ the binding affinities of compounds 11g and 16 to δ-, μ- and κ-opioid receptor sites were measured using [³H]DADLE, [³H]DAMGO, or [³H]U69,593 as ligands, respectively.

In Vivo Pharmacokinetic Studies in Mice.

All pharmacokinetic studies in mice were conducted according to protocols approved by the Animal Care and Use Committee at Johns Hopkins University. Male CD 1 mice between 25 and 30 g were obtained from Harlan, Inc., and maintained on a 12-hour light-dark cycle with ad libitum access to food and water. Compound 16 was administered to mice (n=3) as a single intravenous dose of 10 mg/kg. The mice were sacrificed at predetermined time points post drug administration. For collection of plasma and spinal cord tissue, animals were euthanized with CO₂, and blood samples were collected in heparinized microtubes by cardiac puncture. Spinal cords were dissected and immediately flash frozen (−80 °C). Plasma was obtained by centrifugation of blood at 3,000 g for 15 minutes, and stored at −80 °C until LC/MS analysis.

Prior to extraction, frozen samples were thawed on ice. The calibration curves were developed using plasma and spinal cord from naïve animals as a matrix. Plasma samples (50 μL), were processed using a single liquid extraction method by addition of 300 μL of acetonitrile as with internal standard (losartan [5 μM]), followed by vortexing for 30 s and then centrifugation at 10000 rcf for 10 min. For spinal cord tissue, homogenized samples were vortexed and centrifuged as above. A 10 μL aliquot of supernatant was diluted with 40 μL water containing 0.5 μM losartan as internal standard. Extracts were centrifuged at 10000 rcf at 4 °C for 10 minutes. Supernatants were transferred to 250 μL polypropylene autosampler vials sealed with a Teflon cap. A volume of 3 μL was injected onto the ultra-performance liquid chromatography (UPLC) instrument for quantitative analysis by LC/MS/MS. Calibration curves over the 0.01–10 nmol/mL plasma and spinal cord were constructed from the peak area ratio of the analyte to the internal standard using linear regression with a weighting factor of 1/(nominal concentration). Correlation coefficient of greater than 0.99 was obtained in all analytical runs for all analytes.

ABBREVIATIONS USED

MRGPRX1

mas-related G-protein coupled receptor member X1

DRG

dorsal root ganglion

HTS

high throughput screening

HVA

high-voltage activated