Optimization of Novel Antagonists to the Neurokinin-3 Receptor for the Treatment of Sex-Hormone Disorders (Part II)

Abstract

Further lead optimization on N-acyl-triazolopiperazine antagonists to the neurokinin-3 receptor (NK₃R) based on the concurrent improvement in bioactivity and ligand lipophilic efficiency (LLE) is reported. Overall, compound 3 (LLE > 6) emerged as the most efficacious in castrated rat and monkey to lower plasma LH, and it displayed the best off-target safety profile that led to its clinical candidate nomination for the treatment of sex-hormone disorders.

The neurokinin-3 receptor (NK₃R) is a class A GPCR with neurokinin B (NKB) as its endogenous agonist. We present here the sequel on the lead optimization of N-acyl-triazolopiperazine NK₃R antagonists (1 and 2, Figure 1).¹

Figure 1.

Lead progression: iv POC (1), oral POC (2), and clinical candidate (3). (The “magic methyl” groups are shown in red.)

NK₃R antagonists were speculated as therapeutically relevant for CNS dysfunctions, e.g., schizophrenia, predicated on the “hyperdopaminergic hypothesis”, which repeatedly met with clinical failures in over a decade of efforts.² Meanwhile, emerging biology has unambiguously established the role of NK₃R/NKB signaling in reproductive neuroendocrinology. Importantly, recent studies have revealed NK₃R as a key regulatory component of the hypothalamic–pituitary–gonadal (HPG) axis wherein its tonic activation positively regulates the gonadotropin-releasing hormone (GnRH) pulse frequency.³ In turn, the GnRH pulse frequency is known to differentially control the circulating levels of luteinizing hormone (LH) versus follicle-stimulating hormone (FSH). Thus, high frequency pulses stimulate LH release, whereas low frequency pulses favor FSH induction.⁴ These gonadotropins ultimately act on the ovary and testis to promote production of gametes and sex-hormone release. In 2011, it was reported that patients with a loss of function mutation in NK₃R display a phenotype of normosomic congenital hypogonadotropic hypogonadism, low plasma LH, and attendant low LH/FSH ratios that could be restored through exogenous administration of GnRH.⁵ We have demonstrated elsewhere⁶ that the foregoing NK₃R antagonists slow the LH pulse and decrease circulating LH levels without affecting FSH, consistent with the literature reports.⁴ As such, these antagonists are subtle modulators of gonadotropin secretion unlike GnRH ligands that abrogate both LH and FSH with the consequent decline in plasma estrogen to castration levels thereby triggering menopausal-like adverse events such as bone mineral density loss and incidences of hot flashes.⁷ Hence, NK₃R antagonists offer a potentially safer therapeutic approach due to a decreased rather than abrogated GnRH pulse frequency. Collectively, these findings offer a strong rationale for repositioning NK₃R antagonists to address sex-hormones disorders such as polycystic ovary syndrome (PCOS) and uterine fibroids (UF), among others.⁸

The synthetic approach to the analogues herein was previously described.¹ With minor modifications, Scheme 1 was used for the GMP scale-up of 3 in overall 42% yield (2.7 kg) with 99.3% purity and >99.9% enantiomeric excess.

Scheme 1.

Reagents and conditions: (a) Et₃OBF₄, Na₂CO₃, CH₂Cl₂, 45 min, 68%; (b) MeOH, 70 °C, 8 h, 80%; (c) TFA, 2 h, >99% conversion; (d) 4-fluorobenzoyl chloride, NaHCO₃, 15 min, 97%; (e) recrystallization (EtOH/H₂O), 97% (DMB = 2,4-dimethoxybenzyl).

The in vitro bioactivity structure–activity relationship (SAR) was established through radioligand binding (pK_i) and aequorin functional assays (pIC₅₀) data from recombinant human NK₃R in CHO cells. The lead optimization strategy¹ of combined improvement in both bioactivity and ligand lipophilic efficiency (LLE = pK_i – logD_7.4)⁹ was maintained. An initial emphasis was placed on LLE as a predictive marker of improved safety profiles.¹⁰ Other efficiency metrics such as LE¹¹ and Fsp^3 12 (Table 1) were also tracked, though not as a primary focus. The previously discovered “magic methyl” groups in Rings B and D (Figure 1),¹ so-called due to their significant impact in improving potency and LLE, remained crucial and rendered feasible improvements in Fsp³ and LE as well (see below).

Table 1. Human NK₃R In Vitro Bioactivity, LogD_7.4, Ligand Efficiency Metrics,¹⁰⁻¹² and Off-Target Safety SAR.

^aN = 3, %RSD ≤ 5.

^bCYP 3A4, 2D6, 2C9, 2C19, and 1A2, respectively (N = 2, <10% variability).

^cN = 3, coefficient of variation < 6%.

To recall, replacing 2-methylthiazole (2) at Ring D with 3-methyl-1,2,4-thiadiazole (8) was reported earlier to markedly improve bioactivity and LLE (Table 1).¹ Moreover, 1,2,4-thiadiazole is regarded as a means of circumventing bioactivation liabilities potentially relevant to the thiazole ring.¹³ These considerations overall prompted us to retain the 1,2,4-thiadiazole, but to modify the 4-(thiophen-2-yl)phenyl in 8 to the 4-fluorophenyl Ring A (i.e., 3) present in the earlier lead structures.¹ Progression from 8 to 3 helped reduce lipophilicity (ΔlogD_7.4 = −1.5), which not surprisingly right-shifted bioactivity by nearly one log. However, in evaluating 3 against 8, the improved LLE and absence of the thiophene ring (a potential structural safety alert)¹⁴ was considered of greater importance due to the reduced toxicological risk. In addition, 3 was nearly equipotent to the oral POC lead 2, while >1-log superior in LLE. Narrowing our focus on the thiadiazole Ring D and related variants, we observed the following descending trend in bioactivity (Table 1): 1,2,4-thiadiazole (3) > 1,2,4-oxadiazole (10) ≫ 1,3,4-thiadiazole (9). As with the Ring A cases, increased lipophilicity in Ring D helped improve bioactivity albeit offset by a loss in LLE, e.g., 11 vs 10 (Table 1). The impact of Rings B and D magic methyl groups on SAR trends was quite pronounced, as expected based on previous results,¹ since the corresponding des-Me analogues of 3, whether at Ring B or D (12 and 13, respectively), were decidedly inferior in both bioactivity and LLE (Table 1). Gem dimethyl Ring B substitution substantially eroded the bioactivity (14 vs 3) in keeping with the unfavorable impact of (S)-Me at this Ring B position (data not shown).¹ Once again, improved bioactivity followed increased lipophilicity whether at Ring A (15) or at Ring D (16) positions, but this gain was negated by a deteriorated LLE against 3. Conversely, replacing 4-fluorophenyl with phenyl at Ring A, i.e., 17, helped diminish lipophilicity, but it also deteriorated bioactivity. Interestingly, a hydroxyethyl substitution at Ring B (18) afforded an alternative means of reducing lipophilicity (ΔlogD_7.4 = −0.5 vs 3) with minimal impact on bioactivity, thus resulting in a 0.4-log superior LLE vs 3. Despite this, 18 proved inferior to 3 due to P_gp efflux that in turn markedly diminished its brain exposure level (Table 2 and discussion further below).

Table 2. Permeability, Plasma and Brain Fraction Unbound, Brain Exposure, and PK Data^a.

	Caco-2 Papp (nm/sec)
Cpd	AB	BA	ER	species	plasma fu	brain fu	Cplasma,u (nM)	Cbrain,u (nM)	(B/P)u	iv ClT (min/mL/kg)	iv Vss (L/kg)	iv T1/2 (min)	%F
2b	339	224	0.7	rat	0.055	0.028	54.4	7.79	0.14	7.4	1.4	126	119
2c	−	−	−	monkey	0.043	−	17.9	−	−	16.5	1.52	210	12
3b	487	465	1.0	rat	0.639	0.525	1507	343	0.23	1.5	0.60	279	62.5
3c	−	−	−	monkey	0.532	−	5040	−	−	3.17	1.15	324	107
8d	360	221	0.6	rat	0.065	0.031	58.6	39.3	0.67	7.3	2.8	267	−
12d	477	528	1.1	rat	0.674	0.436	1767	212	0.12	1.94	0.65	239	126
15d	467	345	0.7	rat	0.408	−	−	−	−	2.2	0.91	294	73.6
16d	419	368	0.9	rat	0.291	0.109	348.0	85.4	0.25	1.07	2.25	1479	98.2
17d	437	519	1.2	rat	0.592	0.604	406.7	68.6	0.17	13.7	0.68	35	55
18d	91	345	3.8	rat	0.662	0.995	897.4	45.6	0.05	5.07	1.59	215	46.6

^aPK doses: iv, 1 mg/kg (rat), 10 mg/kg (monkey); oral, 3 mg/kg (rat), 5 mg/kg (monkey). Brain exposure dose: 1 mg/kg. Mean values for N = 3–4 rats, or 4 monkeys, per group. All rat data at 60 min: (B/P)_u = C_brain,u/C_plasma, u with C_brain,u = C_{brain, total} × bf_u and C_plasma,u = C_plasma,total × f_u. Monkey C_plasma,u data at 90 min (oral).

^bPK formulation: HPβCD.

^cPK formulation: iv HPβCD; oral 0.5% MC/water.

^dPK formulation: 1% DMSO, HPβCD in 0.9% NaCl.

The hERG SAR herein (Table 1) was governed by the interplay between lipophilicity and the hydrogen-bond acceptor (HBA) count in the heteroaryl Ring D, as previously reported.¹ For instance, in progressing from 8 to 3 (ΔlogD_7.4 = 1.5), the hERG IC₅₀ was improved by over 12-fold. However, the poor hERG IC₅₀ = 1.6 μM in 11 (logD_7.4 = 1.7) was in keeping with the increased HBA count in the Ring D oxadiazole (N + N + O) in stark contrast to the thiadiazole (N + N) Ring D variations (3 and 15–18), all of which displayed a superior hERG, IC₅₀ ≥ 39 μM (logD_7.4 = 1.3–2.4). Interestingly, the Ring B hydroxyl group in 18 did not adversely impact hERG (IC₅₀ = 50 μM) suggesting that the HBA effect on hERG SAR is primarily a Ring D related effect. Finally, the Ring B magic methyl also reduced hERG efficacy, i.e., 3 (IC₅₀ > 100 μM) vs 12 (IC₅₀ = 50 μM). Compound 3 was the best overall in the hERG and CYP safety profile evaluation.

Based on the free drug hypothesis, the unbound fraction rather than total drug is relevant for PKPD analysis.¹⁵ The NK₃R is mainly expressed on KNDy neurons in the ARC region of the hypothalamus³ that is part of the circumventricular organs lacking blood–brain barrier and are therefore exposed to blood solutes.¹⁶ As such, both the unbound plasma (f_u) and the unbound brain levels (bf_u) must be considered here (Table 2). While lipophilicity alone does not correlate well to albumin binding, this trend is often apparent in a congeneric series.¹⁷ Hence, a compound with balanced lipophilicity such as 3 (logD_7.4 = 1.5) displayed high f_u and bf_u levels (>50%) in contrast to the more lipophilic congeners, e.g., 16 (Table 2). It is noteworthy that despite an increase in unbound plasma concentration, the systemic clearance levels (CL_T) remained low (e.g., 3, Table 2). The comparatively lower CL_T in para substituted phenyl Ring A (3, 15) against the unsubstituted congener 17 is likely due to the metabolic blocking effect. All analogues except 18 displayed high Caco-2 permeability with no evidence of appreciable P_gp efflux (ER = 0.6–1.2), consistent with the high oral availability (%F) and brain-to-plasma ratios observed. The so-called P_gp rule-of-4 suggests that increasing the number of HBA atoms to (N + O) ≥ 8 tends to confer an increasing likelihood of P_gp efflux.¹⁸ This is in keeping with the P_gp efflux in 18 (ER = 3.8) given its HBA atom count (N + O = 8). As with 2,¹ a complete oral absorption (%F > 100) was also observed in rat with compound 12 and in monkey with 3. This phenomenon is well-known and various underlying causes have been reported.¹⁹ No drug accumulation was observed in 5-day once-daily oral dosing studies in rats (3 and 12) or monkeys (3), despite administration of elevated doses (e.g., up to 1 g/kg in rats with 12), in step with the relatively short half-life values and the previous related observations with 2.¹ Moreover, no adverse hepatotoxicity (AST, ALT, and bilirubin levels normal) was detected in these subchronic studies. Furthermore, 3 displayed the highest bf_u = 0.525 and brain unbound concentration (C_brain,u = 343 nM) herein (Table 2). In contrast, 18 although nearly completely unbound in the brain displayed a comparatively low C_brain,u = 45.6 nM consistent with its elevated P_gp efflux ratio.

The key PKPD parameters for interpreting the LH inhibition data are the unbound plasma and brain levels normalized with respect to the bioactivity, i.e., C_plasma,u/K_i and C_brain u/K_i (Table 3).¹ The plasma and brain levels were determined at the T_max for the minimum effective dose (MED). As noted before for 2 and 8,¹ a statistically significant effect was attained at C_plasma,u/K_i ≥ 7.6 and C_brain,u/K_i > 1 in rat oral LH inhibition studies. This was also the case here, i.e., for analogues 3, 12, and 16–18, with MED values ranging from 3 mg/kg (3) to 30 mg/kg (12 and 17). For example, in rats, 3 was 20-fold more efficacious in vivo against the initial POC lead 2 despite being 3-fold right-shifted in K_i. This ameliorated efficacy is reflected in their respective MED-normalized plasma and brain PKPD parameters (Table 3, the last two columns). Otherwise stated, the >1-log LLE superiority of 3 vs 2 underscores the greater unbound exposure levels and consequently the greater in vivo efficacy of 3. Likewise, the monkey LH data (Figure 2) mirrored these trends with 3 4-fold more efficacious (MED levels) although nearly equipotent to 2 in monkey K_i values (Table 3) in keeping with the significantly better MED-normalized plasma PKPD parameter for 3 vs 2.

Table 3. PKPD Analysis of the Oral LH Inhibition Studies^a.

Cpd	species	Ki (nM)	LLE	MED (mg/kg)	Tmax (min)	Cplasma,u/Ki	Cbrain,u/Ki	(Cplasma,u/Ki)/MED	(Cbrain,u/Ki)/MED
2	rat	76	4.0	60	150	16.4	2.32	0.273	0.039
2	monkey	20	4.6	20	60	13.3	−	0.665	−
3	rat	219	5.2	3	150	22.0	5.03	7.33	1.68
3	monkey	25	6.1	5	90	192	−	38.4	−
8	rat	22	4.6	10	150	15.0	12.4	1.5	1.24
12	rat	2033	4.5	30	150	22.5	2.77	0.75	0.09
16	rat	85	4.7	10	150	23.8	5.87	2.38	0.59
17	rat	573	4.9	30	45	47.3	7.71	1.58	0.26
18	rat	244	5.6	10	45	38.9	1.82	3.89	0.18

^aPlasma concentrations coincident with LH measurements. MED determined by a significant decrease (p < 0.05) in LH vs baseline with a lower nonsignificant dose established in all cases.

Figure 2.

Oral LH inhibition with 3 (0.5% MC/water) in castrated cynomolgus monkey (2-way ANOVA and Dunnet’s comparison to the vehicle; ***p < 0.001, **p < 0.01).

In summary, 3 proved a superior lead candidate based on bioactivity, LLE, LE, and Fsp³ (Table 1) criteria. Apart from its excellent hERG and CYP safety profile, 3 was highly efficacious in LH inhibition, showed >2.5-log selectivity against NK₁R and NK₂R subtypes, proved >300-fold selective against related HPG axis receptors¹ (KOR, GnRH, GnIH-R, GPR54), and was highly selective in the broad CEREP off-target screen (<25% inhib at 10 μM). Finally, 3 showed no effect either in Langendorff cardiac safety in rabbits (up to 30 μM) or in AMES genotoxicity test (up to 100 μM). Compound 3 (ESN364) is currently in phase 2 clinical trials for the treatment of PCOS and UF.

Glossary

ABBREVIATIONS

ARC

arcuate nucleus

AST

aspartate transaminase

ALT

alanine aminotransferase

bf_u

brain fraction unbound

(B/P)_u

unbound brain-to-plasma

CYP

cytochrome P-450

FSH

follicle-stimulating hormone

Fsp³

fraction sp³ carbon content

f_u

plasma fraction unbound

GnRH

gonadotropin-releasing-hormone

HBA

hydrogen-bond acceptor

hERG

human ether-à-go-go related gene

HPβCD

9% hydroxypropyl-β-cyclodextrin

HPG

hypothalamic–pituitary–gonadal

KNDy

kisspeptin-neurokinin B-dynorphin A neuron

LH

luteinizing hormone

LE

ligand efficiency

LLE

ligand lipophilicity efficiency

MC

methyl cellulose

MED

minimum effective dose

NKB

neurokinin B

NK₃R

neurokinin-3 receptor

P_gp

P-glycoprotein

PKPD

pharmacokinetic–pharmacodynamic

POC

proof-of-concept

T_1/2

elimination half-life

V_ss

steady-state volume of distribution

Biography

Hamid Hoveyda obtained his Ph.D. from the University of British Columbia (Vancouver, Canada) followed by postdoctoral stints at Harvard University (NSERC fellow) and University of Alberta (Edmonton, Canada). He began his industrial career at the Affymax Research Institute (CA, USA) working on the applications of diversity-oriented synthesis in drug discovery. Later, at Tranzyme Pharma (Canada), he led the discovery of two ghrelin agonists that advanced into clinical development for the treatment of GI disorders. Since 2007, he has led medicinal chemistry efforts on various GPCR targets at Euroscreen (Belgium). His scientific contributions have been captured in over fifty publications and patents.

Supporting Information Available

Experimental details for the synthesis and characterization of compounds, X-ray structure of 3 (accession code: CCDC 1052911), and pharmacology and profiling assays. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmedchemlett.5b00117.

This work was supported by the Ministry of Sustainable Development and Public Works, Walloon Region, Belgium.

The authors declare no competing financial interest.