Identifying Structural Features on 1,1-Diphenyl-hexahydro-oxazolo[3,4-a]pyrazin-3-ones Critical For Neuropeptide S Antagonist Activity

Abstract

A series of 7-substituted 1,1-diphenyl-hexahydro-oxazolo[3,4-a]pyrazin-3-ones were synthesized and tested for Neuropeptide S (NPS) antagonist activity. A concise synthetic route was developed, which features a DMAP catalyzed carbamate formation. 4-Fluorobenzyl urea (1c) and benzyl urea (1d) were identified as the most potent antagonists among the compounds examined. Structure-activity relationships (SARs) demonstrate that a 7-position urea functionality is required for potent antagonist activity and alkylation of the urea nitrogen (1e) or replacement with carbon or oxygen (5a) results in reduced potency. In addition, compounds with alpha-methyl substitution (1b) or elongated alkyl chains (1h and 1j) had reduced potency, indicating a limited tolerance for 7-position substituents.

Graphical abstract

Description: Structural features important for Neuropeptide S antagonist activity have been identified though the synthesis and testing of a series of 7-substituted 1,1-diphenyl-hexahydro-oxazolo[3,4-a]pyrazin-3-ones.

The coupling of putative transmitters with orphan receptors has led to the identification of several interesting ligand-receptor pairings that are now beginning to show novel pharmacology.¹ In particular, the neuropeptide S receptor (NPSR) system deorphanized by Sato and co-workers² has recently been shown to modulate a variety of physiological states such as sleep,³ feeding,^4,5 anxiety,^3,6,7 drug abuse,⁸ and inflammation.⁹

The NPS receptor is activated by an endogenous 20 amino acid peptide (Figure 1) resulting in elevated intracellular calcium via its cognate proteins G_q and G_s.³ Amino acids important for NPS agonist activity have been identified through Ala scanning mutagenesis. In particular, Phe 2, Arg 3, Asn 4, and Val 6 are critical for agonist activity whereas residues 5–13 are hypothesized to form an α-helical recognition sequence.¹⁰ NMR and circular dichroism studies on NPS have indicated a significant degree of flexibility among the amino acids critical for receptor activation, thus making identification of the bioactive NPS conformer difficult using currently available data.^10,11

Figure 1. Neuropeptide S and previously described NPS antagonists.

Several single nucleotide-polymorphisms (SNP) of the NPS receptor have been shown to exist. In particular, NPSR Asn107Ile and NPSR C-alt have been fully characterized for agonist sensitivity.¹² Radioligand binding of [¹²⁵I]Tyr¹⁰-NPS is unaltered among receptor variants, however, a 5–10-fold enhancement in functional sensitivity using calcium flux is observed for the Ile¹⁰⁷ variant.¹² Importantly, the SNP (Asn¹⁰⁷ to Ile¹⁰⁷) in the NPS receptor has been implicated as a risk factor for asthma and has been shown to modulate the functional properties of NPS.⁹

The unique pharmacological properties of NPS were first described by Xu and colleagues who determined that NPS was involved in both arousal and anxiety.³ Administration of NPS (i.c.v.) increased locomotor activity in both habituated and naïve mice. NPS-treated mice also demonstrated anxiolytic-like behaviors in the elevated plus maze, light dark box, and marble burying paradigm. In more recent studies, Rizzi and co-workers confirmed the arousal and anxiolytic promoting properties of NPS using stress-induced hypothermia, which is a behavioral model insensitive to alterations in locomotor activity.⁶

Recognizing that small molecule probes would greatly aid the pharmacological characterization of NPS receptors, our laboratory undertook a program to identify small molecule antagonists. The development of small molecule antagonists based on a computationally generated structure of the endogenous peptide was not considered reliable due to the significant conformational flexibility inherent in the peptide.¹¹ Therefore, our laboratory chose to employ a previously patented scaffold as a lead structure.¹³ Although no functional or receptor binding data was disclosed in the patent, structural features important for functional activity appeared to center around 7-position derivatives of 1,1-diphenyl-hexahydro-oxazolo[3,4-a]pyrazin-3-ones. Therefore, our laboratory synthesized and evaluated a diverse series of 7-substituted analogs (Tables 1 and 2). During the course of preparing this manuscript, two small molecules described in the Takeda patent were also synthesized and tested by Okamura et al.¹⁴ These compounds (SHA-66, SHA-68) were shown to be potent and selective antagonists of both the Asn¹⁰⁷ and Ile¹⁰⁷ variants of the NPS receptor (Figure 1).

Table 1. NPS Antagonist Activity for Urea Derivatives.

Compounda	R	NPS Ile107 Ke	NPS Asn107 Ke	Asn/Ile
1b		214 ± 42	588 ± 150	2.8
1cb		13.7 ± 2.8	52 ± 2	3.8
1dc		9.9 ± 1.6	47 ± 15	4.8
1e		317 ± 5	229 ± 75	0.7
1f		321 ± 120	326 ± 140	1.0
1g		567 ± 190	690 ± 390	1.2
1h		309 ± 52	417 ± 71	1.3
1i		537 ± 190	437 ± 150	0.8
1j		726 ± 107	1260 ± 250	1.7
1k		> 4 mM	> 4 mM	--
1l		109 ± 23	490 ± 173	4.5

^aAll target compounds were fully characterized using NMR and Mass Spectrometry.

^bSHA-68.

^cSHA-66.

Table 2. NPS Antagonist Activity for Alternate 7-Substitution.

Compound	R	NPS Ile107 Ke	NPS Asn107 Ke	Asn/Ile
5a		354 ± 51	353 ± 22	1.0
1m		339 ± 76	460 ± 130	1.4
1n		244 ± 42	267 ± 41	1.1
1o		911 ± 130	4640 ± 1400	5.1
1p		1110 ± 30	1100 ± 300	1.0
1q	−CH3	> 4 mM	> 4 mM	--
1r		3760 ± 470	2560 ± 70	0.7
1s		280 ± 41	354 ± 96	1.3

^aAll target compounds were fully characterized using NMR and Mass Spectrometry.

Target compounds (1b–1s), designed to identify structural features important for NPS antagonist activity, were prepared using the methods described in Schemes 1 and 2. The key intermediate 1,1-diphenyl-hexahydro-oxazolo[3,4-a]pyrazin-3-one (1a) was synthesized following a concise route as depicted in Scheme 1. Treatment of commercially available dibenzyl ester 2 with excess phenyllithium at -78 °C afforded (1,4-dib enzyl-piperazin-2-yl)-diphenyl-methanol in moderate yield. Subsequent debenzylation using palladium on barium sulfate under a hydrogen atmosphere with two equivalents of hydrochloric acid at 60 °C afforded 3 in nearly quantitative yield. Reprotection of the resulting amino groups as carbamates seemed ideal, since the desired cyclic carbamate 5 could be readily prepared upon subsequent heating of 4 with base. Based on this hypothesis, carboxybenzyl (Cbz), and 2-(trimethylsilyl)ethoxycarbonyl (Teoc) groups were investigated. Compounds 4a and 4b were obtained as planned and cyclization with sodium hydride in dimethyl formamide proceeded smoothly to provide mono-protected derivatives 5a and 5b. Unfortunately, removal of either protecting group proved problematic. Under normal deprotection conditions both cyclic carbamates 5a and 5b either collapsed to give 3 or decomposed. In an effort to circumvent these issues an alternate protecting group was explored. Knölker and colleagues reported that 1,2-aminoalcohols, when treated with di-tert-butyl dicarbonate (Boc₂O) and dimethylaminopyridine (DMAP), readily cyclize to provide oxazolidin-2-ones.¹⁵ Using this rationale, diamine 3 was treated with Boc₂O, triethylamine, and DMAP in tetrahydrofuran to afford compound 5c in excellent yield. Key intermediate 1a was then obtained in quantitative yield by treatment of 5c with trifluoroacetic acid (TFA) in dichloromethane. This approach now allows for the preparation of key intermediate 1a from diamine 3 in two high yielding steps.

Scheme 1. Synthesis of key intermediate 1a.

Reagents and Conditions. Synthesis of amine 1a: (a) PhLi, THF, -78 °C; (b) H₂, 40 psi, Pd/BaSO₄, EtOH, 2eq, HCl, 60 °C ; (c) 4a, CbzCl, K₂CO₃, H₂O/THF, 4b, Teoc-Succinimide, Et₃N, CH₃CN; (d) NaH, DMF; (e) Boc₂O, DMAP, Et₃N, THF; (f) TFA,CH₂Cl₂.

Scheme 2. Synthesis of target compounds 1b–1s.

Reagents and Conditions. Synthesis of target compounds 1b–s: (a) Appropriate isocyanate or isothiocyanate, CH₂Cl₂; (b) Mel, NaHMDS, THF; (c) Appropriate acid, BOP, Et₃H, THF; (d) TFA, CH₂Cl₂; (e) Appropriate aldehyde, NaB(OAc)₃H, dichloroethane; (f) N-(2-aminoethyl)piperidine, triphosgene, Et₃N, CH₂Cl₂.

The four-step procedure described in Scheme 1 provides key intermediate 1a in good yield. Although the yield of step a in Scheme 1 occurs in moderate yield (≈ 50%), steps b,e and f are accomplished in almost quantitative yield. Compared to the procedure adopted by both Takeda Pharmaceuticals¹³ and Okamura et al.,¹⁴ orthogonal protection of the two amino groups was not needed in our approach. In addition, the DMAP mediated cyclization of 3 to 5c makes this route an attractive alternative.

Target ureas 1b–l and thioureas 1r and 1s were prepared by reacting 1a with various commercially available isocyanates or isothiocyanates (Scheme 2). For 1l, where the isocyanate was not available, reaction of 1a with triphosgene and N-(2-aminoethyl)piperidine proceeded well to give 1l in moderate yield. Alternatively, 1a was coupled to a number of acids with benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP) in tetrahydrofuran to provide amides 1m–o. The N-alkyl amines 1p and 1q were prepared by reductive amination of 1a with 4-(4-fluorophenyl)-butanal, which was obtained through oxidation of 4-(4-fluorophenyl)butanol¹⁶ with pyridinium chlorochromate in dichloromethane, or formaldehyde using sodium triacetoxyborohydride in dichloroethane.¹⁷ Target compounds were fully characterized by NMR and mass spectroscopy.

Considering the potential pharmacological significance of small molecule NPS receptor antagonists, we have defined key structural features of 7-substituted 1,1-diphenyl-hexahydro-oxazolo[3,4-a]pyrazin-3-ones required for antagonist activity. In keeping with earlier reports,^12,14 NPS was more potent at the Ile¹⁰⁷ variant (EC₅₀ of 2.5 nM) than at the Asn¹⁰⁷ variant (EC₅₀ 16.7 nM) using a calcium flux functional assay.¹⁸ The number of spare receptors in an expression system can affect potency, however, taken together, these data support there being a functional difference between the two variants. Compound 1c and a closely related 1,2,3,6-tetrahydro-pyridine analog of 1l were resolved into their individual enantiomers in the Takeda Patent although no indication of biological activity was provided (Table 1).¹³ Okamura and colleagues recently reported K_b's of 27.9 nM (Ile¹⁰⁷) and 16.9 nM (Asn¹⁰⁷) for racemic 1c and 32.6 nM (Ile¹⁰⁷) and 21.7 nM (Asn¹⁰⁷) for the desfluoro analog 1d, respectively.¹⁴ In our hands, racemic 1c (K_e = 13.7 and 51 nM) and 1d (K_e = 9.9 and 47 nM) were the most potent NPS antagonists among the compounds described herein at both the Ile¹⁰⁷ and Asn¹⁰⁷ variants, respectively. In contrast to Okamura, we found these compounds to be slightly more selective for the Ile¹⁰⁷ variant. Methylation of urea 1c (1b, K_e = 214 and 588 nM) reduced potency by 16-fold at Ile¹⁰⁷ and 12-fold at Asn¹⁰⁷. Similarly, replacement of the urea nitrogen with carbon (1m, K_e = 339 and 460 nM) or oxygen (5a, K_e = 354 and 353 nM) significantly reduced potency at both receptor variants. This suggests a hydrogen on the 7-position urea is critical for antagonist activity. In order to determine if a hydrogen bond donor placed in close proximity to the urea hydrogen could serve as a suitable surrogate, phenylalanine derivative 1o was prepared. A 100-fold reduction in potency was observed for 1o (K_e = 911 nM and 4640 nM). Because the urea functionality appeared critical for NPS antagonist activity, we began to evaluate alternate substitution of the 7-position urea. Addition of an alpha methyl group (1e, K_e = 317 and 229 nM) reduced potency by 32- and 48-fold compared to 1d. Lengthening the alkyl spacer between the terminal phenyl ring to two carbons (1f, K_e = 321 nM) reduced potency by 25-fold compared to 1c. Further elongation of the alkyl spacer to three (1h, K_e = 309 nM) and four carbons (1i, K_e = 537 nM) significantly reduced potency compared to the parent 1d. These results suggest that the hypothetical receptor pocket has specific size requirements. In order to assess if hydrophilic or hydrophobic character of the urea substituent was essential for antagonist potency, carboxylic acid 1k and piperidine 1l were prepared. Intermediate ester 1j (K_e = 726 nM) and target acid 1k (K_e > 4 μM) were both inactive as antagonists. Interestingly, 2-piperidinoethyl analog 1l had intermediate potency at the Ile¹⁰⁷ receptor variant (K_e = 109 nM) indicating bulky amine substituents are tolerated, albeit at slightly reduced potency. Converting urea 1l to thiourea 1s moderately reduced potency (K_e = 280 nM) whereas replacement of the piperidine with a more water soluble morpholino group (1r, K_e = 3760 nM) dramatically reduced potency. Finally, evaluation of 4-(4-fluorophenyl)butyl analog 1p (K_e = 1100) and methyl analog 1q (K_e > 4 μM) were inactive as antagonists. In general, it appears that tolerance to 7-substitution is limited.

This study demonstrates that antagonists were generally more potent at the Ile¹⁰⁷ variant (1–5 fold) with limited exceptions (Tables 1 and 2). This suggests that future development of antagonists selective for one receptor variant may be possible. In addition, none of the compounds presented in this study possessed measurable intrinsic activity at 10 μM in the NPS Ile¹⁰⁷ cell line.

In conclusion, we have provided a novel synthetic route to 7-substituted 1,1-diphenyl-hexahydro-oxazolo[3,4-a]pyrazin-3-ones and begun to identify the key structural features required for NPS antagonist activity. In particular, we have demonstrated the importance of the urea functionality possessing a free hydrogen and that ethylpiperidine (1l) can serve as a substitute for the benzyl group of 1d. The combination of these results provides a basis for the design and testing of novel scaffolds with enhanced potency and drug-like properties for the NPS receptor.