Benzothiophene piperazine and piperidine urea inhibitors of fatty acid amide hydrolase (FAAH)

Abstract

The synthesis and structure–activity relationships (SAR) of a series of benzothiophene piperazine and piperidine urea FAAH inhibitors is described. These compounds inhibit FAAH by covalently modifying the enzyme’s active site serine nucleophile. Activity-based protein profiling (ABPP) revealed that these urea inhibitors were completely selective for FAAH relative to other mammalian serine hydrolases. Several compounds showed in vivo activity in a rat complete Freund’s adjuvant (CFA) model of inflammatory pain.

Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme that degrades the fatty acid amide family of signaling lipids, including the endocannabinoidanandamide.¹ Genetic^1g or pharmacological^1f inactivation of FAAH leads to elevated endogenous levels of fatty acid amides and a range of behavioral effects including analgesic and anti-inflammatory phenotypes in rodents. Importantly, these behavioral phenotypes occur in the absence of alterations in motility, weight gain, or body temperature that are typically observed with direct cannabinoid receptor 1 (CB1) agonists, indicating that FAAH may represent an attractive therapeutic target for the treatment of inflammatory pain and related conditions.² Several classes of FAAH inhibitors have been reported including electrophilic ketones (e.g., OL-135³), carbamates (e.g., URB597⁴ and SA-47⁵) and, more recently, piperidine/piperazine ureas (e.g., PF-750⁶ and Takeda-25/JNJ-1661010⁷) (Fig. 1). We recently reported that PF-750 inhibits FAAH by covalently modifying the enzyme’s active site serine nucleophile and is completely selective for FAAH relative to other mammalian serinehydrolases.^6a In this Letter, we describe the synthesis and structure–activity relationships (SAR) of a series of benzothiophene piperazine and piperidine urea FAAH inhibitors.

Figure 1.

Structures of FAAH inhibitors.

A series of piperazine phenyl ureas, exemplified by 1, was discovered by high throughput screening of the Pfizer chemical file against human FAAH (Fig. 2). We prepared piperazine phenyl urea intermediate 2 and performed a series of reductive aminations with various aromatic aldehydes (Scheme 1, Eqs. 1 and 2). This allowed us to easily vary the aldehyde component and resulted in the identification of several bicyclic cores including PF-622^6a and a series of benzothiophenes represented by 4h, which is the subject of this manuscript (Scheme 1, Eq. 2).

Figure 2.

Structures of HTS lead (1) and PF-622.

Scheme 1.

Synthesis of piperazine ureas 4 and 6: (a) PhNCO, CH₂Cl₂, 92%; (b) TFA, CH₂Cl₂, 80–99% (c) NaBH(OAc)₃, DCE, 50–95%; (d) N-Boc-piperazine, NaBH(OAc)₃, CH₂Cl₂; then TFA, CH₂Cl₂, 64%; (e) RNCO (R = alkyl, Ph, 3-pyr; ie. 4d–g, 4h, 6a), CH₂Cl₂, 50–90%; (f) RNHCO₂Ph (R = heterocycle, ie. 4j–1, 6), DMSO, 60 °C, 50–80%.

We then explored the benzothiophene lead 4h in more detail. Therefore, the benzothiophene piperazine intermediate 5 was prepared according to Equation 3 and reacted with various commercially available alkyl and aryl isocyanates or phenyl carbamates of heterocyclic amines⁸ to give the corresponding piperazine ureas 4 and 6 (Scheme 1).

Scheme 2 and Scheme 3 outline the synthesis of representative piperidine urea FAAH inhibitors utilized in the current studies. Lithiation of benzothiophene 7 followed by reaction with Weinreb amide 8 provided ketone 9 in good yield.⁹ Ketone 9 was reduced with sodium borohydride to give alcohol 10 which underwent elimination when treated with p-toluene sulfonic acid (TsOH). The resulting double bond was hydrogenated to give piperidine 11 which was treated with isocyanate 12 as described previously to give the desired urea 13.⁸

Scheme 2.

Synthesis of piperidine urea 13: (a) n-BuLi, THF, −78 °C; then add 8, 70%, (b) NaBH₄, EtOH, quant; (c) TsOH, toluene, quant; (d) H₂(g), 20% Pd/C, MeOH, 87%; (e) 12, CH₂Cl₂, 60%.

Scheme 3.

Synthesis of linker analogs 15–18: (a) TFA, CH₂Cl₂, 99%; (b) 12, CH₂Cl₂, 85%; (c) Ph₃PCH₃Br, n-BuLi, THF, −20 °C to rt, 97%; d) H₂(g), Pd/C, MeOH, 76%; (e) chiral chromatography (CHIRALCEL^® OD); (f) NaBH₄, EtOH, quant.

The linker analogs 15–18 were synthesized according to Scheme 3. Deprotection of Boc-piperidine 14 with TFA followed by reaction with isocyanate 12 afforded urea 15 with a ketone linker. Wittig olefination of ketone 15 provided compound 16 with a methylene substituted linker. Hydrogenation of the double bond gave 17 with a methyl substituted linker as the racemate and the enantiomers were separated by chiral chromatography. Furthermore, ketone 15 was reduced to give racemic 18 with a hydroxy substituted linker.

As previously reported, these piperidine/piperazine ureas inhibit FAAH by covalent carbamylation of the catalytic Ser241 nucleophile.^6a,6b Therefore, the potency of these benzothiophene piperidine/piperazine urea inhibitors were determined by the second order rate constants k_inact/K_i values using an enzyme-coupled FAAH assay as described previously.^6b,10 Unlike IC₅₀ values, k_inact/K_i values do not change with various preincubation times and have been described as the best measure of potency for irreversible inhibitors.¹¹

Table 1 shows the SAR of the right hand portion of the urea. The alkyl ureas (4a–g) had very low potency for FAAH, which is similar to what has been observed for N-alkylurea analogs of URB597.^4a In contrast, we found that aryl ureas such as phenyl urea 4h were potent FAAH inhibitors. Next, we investigated a series of heteroaromatic ureas (4i–l, 6a) in part to avoid the generation of aniline upon covalent binding to FAAH. The 3-aminopyridyl group (6a) was preferred over the 2-aminopyridyl (4i) and 3-aminopyridazinyl (4j) groups in the benzothiophene series. The SAR of the heterocyclic group was highly sensitive. For example, isoxazole 4k retained potency, while the potency was lost with methylthiazole 4l, which is consistent with observations made by Keith et al. in a related thiadiazolopiperazinyl urea series.^7b Boger and co-workers also observed that subtle changes in the central heterocycle of α-ketoheterocycle FAAH inhibitors greatly influence inhibitor activity.^3c Also it is interesting to note that isoxazole 4k exhibited equal potency for hFAAH and rFAAH, while phenyl urea 4h and pyridazine urea 4j showed higher potency for hFAAH than rFAAH.

Table 1.

Human and rat FAAH potency (k_inact/K_i values)¹⁵ for compounds 4a–l, 6a showing SAR of the right hand side of the urea

	R	hkinact/Ki (M−1 s−1)	rkinact/Ki (M−1 s−1)
4a	Butyl	44	13
4b	CH2CH2OH	42	<10
4c	CH2C(O)NHMe	<10	<10
4d	iPr	70	<10
4e	Cyclohexyl	<10	<10
4f	tBu	<10	<10
4g	Bn	<10	<10
4h	Ph	1918	762
4i	2-pyr	1002	635
6a	3-pyr	2401	1083
4j		924	191
4k		1559	1433
4l		<10	<10

Next, we investigated the effect of substituents at the 6-position of the 3-amino pyridine (Table 2). The compounds with 6-methoxy (6g), 6-amino (6i) and 6-NHAc (6n) substituents are roughly equipotent to 6a, while the 6-Cl (6d) and 6-NHMe (6j) groups result in compounds that are about twofold less potent and the potency is further diminished by the 6-NHEt (6l) substituent (fourfold less potent than 6a). Furthermore, 6-CF₃ (6c) and 6-NEt₂ (6m) substituents are detrimental to activity. Taken together with the results in Table 1, it is clear that the nature of the aromatic amine portion of the urea is critical for activity, but the potency does not appear to correlate with the leaving group ability. There appears to be a steric limitation at the 6-position as hFAAH potency generally decreases with the larger substituents (i.e., h k_inact/K_i for NH₂ > NHMe > NMe₂ > NEt₂). Interestingly, several 6-amino compounds (6j–m) actually show a preference for rFAAH over hFAAH.

Table 2.

Human and rat FAAH potency (k_inact/K_i values)¹⁵ for compounds 6a–6n showing SAR of substituted pyridyl ureas

	R	hkinact/Ki (M−1 s−1)	rkinact/Ki (M−1 s−1)
6a	H	2401	1083
6b	Me	1481	390
6c	CF3	69	<10
6d	CI	1238	242
6e	Br	645	157
6f	Ph	631	1215
6g	OMe	2931	1060
6h	O-(3-Pyr)	605	101
6i	NH2	1871	1067
6j	NHMe	1108	1513
6k	NMe2	892	1811
61	NHEt	597	1891
6m	NEt2	90	227
6n	NHAc	2011	1719

Subsequent SAR studies focused on optimization of the benzothiophene substitution while maintaining the optimal 3-aminopyridyl urea (Table 3). It was found that incorporating an alkyl group at the 3-position of the benzothiophene (R¹) led to improved potency. For example, compound 6a was 3–4-fold more potent than the unsubstituted benzothiophene 19. In most cases for hFAAH, the piperidine analogs were slightly more potent than the corresponding piperazine analogs (i.e., 20 > 6a), which is most likely a result of more favorable Van der Waals interactions of the piperidine with the acyl chain binding channel of FAAH.^1b,6b Substitution was tolerated at the 4- and 5-position of the benzothiophene (24–36), but no significant improvement in potency was realized. Conversely, moving the substituent (R⁴) to the 6-position of the benzothiophene impaired potency (37–39), which is predicted to introduce steric clashes with the FAAH protein (Fig. 5).

Table 3.

Human and rat FAAH potency (k_inact/K_i values)¹⁵ for compounds 13, 19–39 showing SAR of substituted benzothiophenes

	R1	R2	R3	R4	R5	X	hkinact/Ki (M−1 s−1)	rkinact/Ki (M−1 s−1)
19	H	H	H	H	H	N	695	174
6a	Me	H	H	H	H	N	2401	1083
20	Me	H	H	H	H	C	5129	2613
21	Et	H	H	H	H	N	1402	774
22	Et	H	H	H	H	C	1668	1013
6b	Me	H	H	H	Me	N	1481	390
23	Cl	H	H	H	Me	N	900	133
24	Me	F	H	H	H	N	3321	1203
25	Me	F	H	H	H	C	4147	1547
26	Me	F	H	H	Me	N	1980	758
27	Me	H	F	H	H	N	3134	779
13	Me	H	F	H	H	C	4474	729
28	Me	H	Me	H	H	N	2635	557
29	Me	H	Me	H	H	C	3024	482
30	Me	H	Cl	H	H	N	2822	1133
31	Me	H	CF3	H	H	N	1389	219
32	Me	H	OMe	H	H	N	1381	500
33	Et	H	F	H	H	N	1808	617
34	Et	H	F	H	H	C	2292	1141
35	Me	H	F	H	Me	N	1871	260
36	Me	H	Me	H	Me	N	1169	183
37	Me	H	H	OMe	H	N	73	113
38	Me	H	H	OEt	H	N	14	<10
39	Cl	H	H	Me	Me	N	73	14

Figure 5.

Model of compound 38 covalently attached to Ser241 of h/rFAAH using coordinates from the PF-750-h/rFAAH crystal structure.^6b Overlay with PF-750 shown in blue.

As shown in Table 4, the potency is maintained when a ketone or methylene is incorporated within the linker (15 and 16, Scheme 3) whereas the reduced compounds 17 and 18 with methyl and hydroxyl substituents lose much of their activity. The sp2 hybridization of the ketone and methylene linker compounds 15 and 16 imposes a conformational constraint that appears to help position the benzothiophene in a more favorable binding orientation. The SAR in this linker region is sensitive as further evidenced by the >10-fold difference in potency for the enantiomers of 17.

Table 4.

Human and rat FAAH potency (k_inact/K_i values)¹⁵ for compounds 15–18 showing SAR of the piperidine linker

	hkinact/Ki (M−1 s−1)	rkinact/Ki (M−1 s−1)
15	3601	1226
16	5441	991
17-rac	114	48
17-E1	34	<10
17-E2	617	110
18-rac	200	79

We have proposed that the urea may be activated upon binding in the active site of FAAH by undergoing a conformational change that diminishes conjugation of the nitrogen lone pair with the carbonyl, which would activate the urea toward nucleophile attack as shown in Figure 3.^6a It appears that a cyclic secondary amine such as piperazine or piperidine is critical for activation of the urea as ureas prepared from primary amines are ineffective.^4a

Figure 3.

Proposed mechanism of covalent inhibition of FAAH by piperidine ureas. The catalytic triad Ser-Ser-Lys is shown.

Compound 13 (Fig. 4) and compound 38 (Fig. 5) were modeled,¹² using Sybyl 8.0, into the ‘humanized’ rat FAAH (h/rFAAH) crystal structure (2VYA).^6b Compound 13 fit within the binding site in almost identical fashion to that of PF-750 with only very slight movement of the surrounding residue atoms, in essence, occupying virtually identical space. The green surface depicting the binding site volume and the space-filled representations were generated from the compound 13-h/rFAAH model and are virtually identical to those generated from the PF-750-h/rFAAH crystal structure. In contrast, when the model of compound 38-h/rFAAH was minimized, there was a significant shift in the position of the side chain of Met436. As shown in Figure 5, the ethoxy portion of compound 38 extends beyond the green surface generated from the compound 13 model illustrating the increased size of the ligand and the subsequent displacement (i.e., push) of the Met436 side chain. This steric interaction with Met436 could explain the decreased potency of compound 38.

Figure 4.

Model of compound 13 covalently attached to Ser241 of h/rFAAH using coordinates from the PF-750-h/rFAAH crystal structure.^6b Overlay with PF-750 shown in blue.

To assess the selectivity of the benzothiophene piperidine/piperazine urea series, compounds 6b, 6g (PF-465), and 13 (PF-946) were profiled at 100 µM by competitiveactivity-based protein profiling (ABPP)¹³ in a number of different proteomes derived from mouse and human sources (Fig. 6). Competitive ABPP provides a global view of the proteome-wide selectivity of enzyme inhibitors as described previously.^13b,13c In the case of FAAH inhibitors, we used a reporter-tagged fluorophosphonate (FP) ABPP probe,^13c which serves as a general activity-based profiling tool for the serine hydrolase super family.^13d,13e Serine hydrolases that show significant reductions in FP probe labeling intensity in the presence of inhibitor are scored as targets of the compound. The selectivity of compounds 6b, 6g, and 13 was compared to that of URB597, which was profiled at 10 and 100 µM. Gel images of soluble proteomes of mouse liver, mouse kidney, human liver, and human heart and the membrane proteome of human brain are shown in Figure 6. Consistent with previous reports,^6a,14 multiple off-targets were observed in peripheral tissues for URB597 even at 10 µM, particularly amongst FP-labeled proteins migrating between 55 and 65 kDa. It is noteworthy that URB597, which has been shown to be highly selective for serine hydrolases in mouse brain proteomes, inhibits at least one additional hydrolase migrating at 55–60 kDa in the human brain membrane proteome. In contrast, no off-targets were observed for compounds 6b, 6g, and 13 even when tested at 100 µM (Fig. 6). Under the assay conditions, compounds 6b, 6g, 13, and URB597 completely inhibited FAAH from both mouse and human tissues.

Figure 6.

Selectivity profiling of FAAH inhibitors. Gel images of proteomes labeled with FP-rhodamine in the absence or presence of FAAH inhibitors (Lane 1, buffer; lane 2, 6b at 100 µM; lane 3, 6g at 100 µM; lane 4, 13 at 100 µM; lane 5, URB597 at 100 µM; lane 6, URB597 at 10 µM). Inhibited bands are shown with arrows. (A) soluble mouse liver proteome, (B) soluble mouse kidney proteome, (C) soluble human liver proteome, (D) soluble human heart proteome, and (E) human brain membrane proteome. Fluorescent gel signals shown in grayscale.

Next we selected compound 6g (PF-465) to assess its in vivo efficacy in a rat model of inflammatory pain. Subcutaneous injection of complete Freund’s adjuvant (CFA) into the plantar surface of the hind paw produced a significant decrease in mechanical paw weight threshold (PWT) at 5 days post injection (Fig. 7). Compound 6g dosed at 3, 10 and 30 mg/kg (ip) caused a dose-dependent inhibition of mechanical allodynia with a minimum effective dose (MED) of 10 mg/kg. At doses of 10 and 30 mg/kg, compound 6g inhibited pain responses to an equivalent degree as the nonsteroidal anti-inflammatory drug naproxen (10 mg/kg, ip).

Figure 7.

Anti-hyperalgesic effects of compound 6g (3–30 mg/kg, ip) in the CFA model of inflammatory pain. Compound 6g produces a dose-dependent reduction of mechanical allodynia (hyperalgesic) in rats (black bars). The effect of the non-steroidal anti-inflammatory drug naproxen (10 mg/kg, ip hatched bar) is shown for comparison. Anti-hyperalgesic responses were determined at 4 h following drug treatment and were significantly different between compound 6g- and vehicle-treated groups (p < 0.05). n = 8 rats/group.

In conclusion, we have described a series of benzothiophene piperazine/piperidine urea FAAH inhibitors that covalently modify the enzyme’s active site serinenucleophile. Activity-based protein profiling revealed that these urea inhibitors are highly selective for FAAH relative to other mammalian serine hydrolases. Furthermore, in vivo activity was demonstrated in a rat CFA model of inflammatory pain. Additional SAR development of this class of piperazine/piperidine urea FAAH inhibitors will be reported in due course.