Discovery of a potent M₅ antagonist with improved clearance profile. Part 1: Piperidine amide-based antagonists

Abstract

The lack of potent and selective tool compounds with pharmaceutically favorable properties limits the in vivo understanding of muscarinic acetylcholine receptor subtype 5 (M₅) biology. Previously, we presented a highly potent and selective M₅ antagonist VU6019650 with a suboptimal clearance profile as our second-generation tool compound. Herein, we disclose our ongoing efforts to generate next-generation M₅ antagonists with improved clearance profiles. A mix and match approach between VU6019650 (lead) and VU0500325 (HTS hit) generated a piperidine amide-based novel M₅ antagonist series. Several analogs within this series, including 29f, provided good on-target potency with improved clearance profiles, though room for improvement remains.

The M₅ receptor has recently emerged as a potential drug target for the treatment of psychiatric issues such as substance abuse disorder, depression, and anxiety.^1-10 Although the field now knows much more about M₅ than ever before,^8,11-13 the lack of robust and pharmaceutically favorable tool compounds (agonists, PAMs, NAMs, and/or antagonists) limits the understanding of M₅ biology. A robust M₅ in vivo tool compound remains an unmet need for the field.

Fig. 1 shows previously disclosed M₅ antagonists 1–3.^14-16 Early tool compounds (especially 1 and 3) have good potencies and subtype selectivities, though suboptimal DMPK profile (especially in vivo clearance) restricts their use as advanced in vivo tools. Previously, we exploited a ‘mix and match’ approach between two HTS hits to discover the highly potent and selective 3 (VU6019650), which maintained a suboptimal clearance profile. We sought to further improve 3 by a similar approach (Fig. 2).

Fig. 1.

Reported M₅ antagonist tool compounds.^14-16

Fig. 2.

Next-generation M₅ antagonists design: ^a Calcium mobilization assay with hM₅-CHO cells was performed in the presence of an EC₈₀ fixed concentration of acetylcholine. Each experiment was performed in triplicate.

We hypothesized that replacing the known metabolic soft-spot thioether linkage of 3 would improve the clearance profile. While searching for a suitable mix and match counterpart, structurally related compound 4, identified in the same HTS hit list, drew our attention (Fig. 2). Despite lacking the thioether linkage, 4 still showed acceptable potency (hM₅ IC₅₀ = 2,803 nM) and exquisite subtype selectivity (hM₁ IC₅₀ > 10,000 nM and hM_2-4 IC₅₀ = inactive). To test our hypothesis, we designed and synthesized 5 and 6 following the synthetic route illustrated in Scheme 1.

Scheme 1.

Synthesis of piperidine amides: (a) DIPEA, DCM, 0 °C to rt, 1–8 h 89–96 %; (b) aq NaOH solution, 1,4-dioxane, rt, 2–8 h, 53–95 %; (c) aniline HATU, DIPEA, DMF, rt, 0.5–3 h, 20–89 %.

The synthesis of piperidine amide-containing analogs was short and straightforward (Scheme 1). Suitably substituted sulfonyl chlorides 7 were reacted with suitably substituted piperidines 8 to form a library of sulfonamides 9 under basic conditions. Base promoted ester hydrolysis of 9, followed by HATU coupling with commercially available anilines, provided desired products 11 in good yields. Alternatively, suitably substituted carboxylic acids 12 were coupled with commercially available anilines using HATU to provide intermediates 13 (Scheme 2). Boc deprotection from 13 under suitable acidic conditions generated piperidine salts 14. Piperidine salts 14 were then reacted with suitably substituted commercially available sulfonyl chlorides 7 to provide final products 11 in moderate to good yields. We think that sulfonyl chloride reagent conditions and poor compound solubility (in some cases) contributed to this wide range of isolated yields of final products.

Scheme 2.

Alternative synthesis of piperidine amides: (a) HATU, DIPEA, DMF, rt, 0.5–3 h, 44–99 %; (b) TFA or HCl, DCM, 1–6 h, 61–99 %; (c) 7, DIPEA, DCM, 0 °C to rt, 1–8 h, 25–83 %.

Sulfonyl chloride 19 was synthesized in 3 steps (Scheme 3). 16 was reacted with allyl bromide 15 under the basic conditions to provide intermediate 17. A radical cyclization reaction followed by sulfonyl chloride formation reaction with thionyl chloride provided desired sulfonyl chloride 19, which was pure enough to use without purification.

Scheme 3.

Synthesis of sulfonyl chloride 19: (a) allyl bromide (15), K₂CO₃, acetone, 60 °C, overnight, 96 %; (b) benzene, tributyltin hydride, 2,2′ -azobis(2-methylpropionitrile), 80 °C, overnight, 95 %; (c) i) SO₃.DMF under N₂, 85 °C, overnight, ii) SOCl₂, rt to 75 °C over the course of 1 h, assumed quantitative yield and used for the next step without purification.

Interestingly, while compound 6 lost all activity (hM₅ IC₅₀ = inactive), 5 maintained reasonable potency (hM₅ IC₅₀ = 111 nM) compared to 3 (hM₅ IC₅₀ = 36 nM). Although 5 still had a suboptimal in vitro clearance profile, we were encouraged by its potency and subtype selectivity and decided to explore the follow-up SAR starting from 5.

To improve clearance profiles of compound 5, we first tried to block potential sites of metabolism on 5 by introducing a methyl group in various positions (Table 1), hypothesizing that masking (or blocking) a site of metabolism would affect metabolic rates. Interestingly, a ‘monomethyl walk’ within 5 was generally well tolerated with exceptions of 2 positions: 1) the amide N—H (21, hM₅ IC₅₀ > 10,000 nM) and 2) the 2 position of the 2,3-dihydrobenzofuran (27, hM₅ IC₅₀ > 10,000 nM). Although potencies were maintained below 500 nM in many cases, clearance profiles remained suboptimal (Predicted CL_hep > 16 (h) and > 60 (r) mL/min/kg) in all cases (Table 1). Therefore, we decided to make changes on both sides (eastern and western) of the molecule.

Table 1.

Methyl-walk to mask a potential metabolic soft spot.

Cmpd	Structure	Synthetic Route (Scheme)	hM5 IC50 (nM)a	hM5 ACh Min (%)a	CLint (mL/min/kg) Predicted CLhep (mL/min/kg)
20		1	594c	19c	74 (h), 426 (r) 16 (h), 60 (r)
21		From 2b	>10,000	51	–
(rac)-22		2	419c	3c	199 (h), 550 (r) 19 (h), 62 (r)
(rac)-23		2	1,530c	3c	79 (h), 506 (r) 17 (h), 61 (r)
(rac)-24		2	134c	2c	264 (h), 572 (r) 19 (h), 62 (r)
(rac)-25		2	94	3	114 (h), 520 (r) 18 (h), 62 (r)
(rac)-26		2 and 3	109	3	263 (h), 1021 (r) 19 (h), 66 (r)
(rac)-27		2	>10,000	16	–

– means not tested.

^aCalcium mobilization assay with hM₅-CHO cells was performed in the presence of an EC₈₀ fixed concentration of acetylcholine. Each experiment was performed in triplicate.

^bMethylation condition: 5, MeI, NaH, THF, 0 °C to rt, 81% yield.

^cIC₅₀ value represents the average from two independent experiments. Each experiment was performed in triplicate.

Initial exploration of the eastern heteroaromatic ring SAR is depicted in Table 2. Despite retaining the overall size and shape of 5, many analogs completely lost their activity (28a-e, hM₅ IC₅₀ = inactive). Comparing compound 5 with the analogs from Table 2, we concluded the eastern heteroaromatic ring requires basic character for potency. Indeed, a moderately basic 1,2,3-benzothiadiazole 28g was equipotent with 5 (28g, hM₅ IC₅₀ = 143 nM). Moreover, quinoline 28f showed weak activity (28f, hM₅ IC₅₀ = 1,802 nM) despite altering the 3-D molecular shape and the location of heteroatom. Notably, 6 was inactive (Fig. 2) despite containing a basic N-methyl imidazole eastern ring, indicating the importance of the location of the basic nitrogen atom. Unfortunately, 28f and 28g still showed high in vitro clearance profiles (Table 2). Thus, we concluded a benzothiazole eastern heteroaromatic ring as near-optimal, and shifted our effort to the western heteroaromatic ring.

Table 2.

SAR exploration of eastern heteroaromatic ring.

Cmpd	R =	Synthetic Route (Scheme)	hM5 IC50 (nM)a	hM5 ACh Min (%)a	CLint (mL/ min/kg) Predicted CLhep (mL/ min/kg)
28a		1	inactive	inactive	–
28b		1	inactive	inactive	–
28c		1	inactive	inactive	–
28d		1	inactive	inactive	–
28e		1	inactive	inactive	–
28f		1	1,802	3	99(h), 740(r) 17(h), 64(r)
28g		1	143b	10b	108(h), 804 (r) 18(h), 64(r)

^aCalcium mobilization assay with hM₅-CHO cells was performed in the presence of an EC₈₀ fixed concentration of acetylcholine. Each experiment was performed in triplicate.

^bIC₅₀ value represents the average from two independent experiments. Each experiment was performed in triplicate.

Unlike the eastern ring SAR, exploration of the western ring SAR provided improved clearance profiles (Table 3). Replacing the 2,3-dihydrobenzofuran with the more polar benzothiazole (29a) decreased cLogP from 3.28 (5) to 2.99 (29a), resulting in a moderate improvement in the in vitro clearance profile (predicted CL_int = 13 (h) and 38 (r) mL/min/kg). The larger quinoline 29b (cLogP = 2.97) lost about 9-fold potency (hM₅ IC₅₀ = 959 nM), indicating that the pocket around the western ring cannot accommodate increased size. Therefore, we downsized the western ring (29c-f). We first tested 2-chloropyridine 29c (cLogP = 2.37), which retains the basic nitrogen and mimics the lone pair electrons of a chlorine atom. 29c maintained on-target potency (hM₅ IC₅₀ = 168 nM). Due to potentially problematic 2-chloropyridine electrophilicity in 29c, we replaced the chlorine atom with a methyl group (29d), which fortunately improved potency and clearance (hM₅ IC₅₀ = 105 nM; Predicted CL_hep = 4 (h) and 27 (r) mL/min/kg). To reduce molecular weight (MW), desmethyl analog 29e was synthesized and tested as well. Although 29e was slightly more potent (hM₅ IC₅₀ = 59 nM) than 29d (hM₅ IC₅₀ = 105 nM), the in vitro clearance profile of 29e was slightly worse (Predicted CL_hep = 10 (h) and 52 (r) mL/min/kg), potentially because pyridine is more susceptible to metabolism.

Table 3.

SAR exploration of western heteroaromatic ring.

Cmpd	het =	Synthetic Route (Scheme)	hM5 IC50 (nM)a	hM5 ACh Min (%)a	CLint (mL/min/ kg) Predicted CLhep (mL/ min/kg)
29a		2	266	2	36(h), 81(r) 13(h), 38(r)
29b		2	959	2	20(h), 212(r) 10(h), 53(r)
29c		2	168b	3b	21(h), 62(r) 11(h), 33(r)
29d		2	105	3	5(h), 43(r) 4(h), 27(r)
29e		2	59b	2b	19(h), 207(r) 10(h), 52(r)
29f		2	304	2	5(h), 45(r) 4(h), 28(r)

^aCalcium mobilization assay with hM₅-CHO cells was performed in the presence of an EC₈₀ fixed concentration of acetylcholine. Each experiment was performed in triplicate.

^bIC₅₀ value represents the average from two independent experiments. Each experiment was performed in triplicate.

Lastly, we synthesized compound 29f with a nitrogen atom is buried between two methyl groups. Despite 29f being about 3-fold less potent than 5 (29f, hM₅ IC₅₀ = 304 nM), the improved in vitro clearance profile (Predicted CL_hep = 4 (h) and 28 (r) mL/min/kg) motivated further exploration of substituted 5-membered heterocycles, while incorporating the beneficial 3-fluoropiperidine core from compound 3 (Fig. 2).¹⁷ Compounds bearing 5-membered western heterocycles along with a 3-fluoropiperidine core, such as (rac)-30b, showed exquisite potency (hM₅ IC₅₀ = 10 nM) and low predicted clearance (predicted CL_hep = 4 (h), 29 (r) mL/min/kg) (Table 4). Among those analogs, 29f, (rac)-30a-c were selected for follow-up studies.

Table 4.

Further optimization with fluorinated piperidine core.

Cmpd	R =	Synthetic Route (Scheme)	IC50 (nM)a	ACh Min (%)a	CLint (mL/min/kg) Predicted CLhep (mL/min/kg)
(rac)-30a)-30a		2	309b	3b	7(h), 260(r) 5(h), 55(r)
(rac)-30b		2	10	2	6(h), 49(r) 4(h), 29(r)
(rac)-30c		2	13	2	6(h), 81(r) 5(h), 37(r)

^aCalcium mobilization assay with hM₅-CHO cells was performed in the presence of an EC₈₀ fixed concentration of acetylcholine. IC₅₀ value represents the average from two independent experiments. Each experiment was performed in triplicate.

^bIC₅₀ value represents the average from three independent experiments. Each experiment was performed in triplicate.

Table 5 presents the detailed profiles of 29f and (rac)-30a-c. All four compounds showed good potencies (hM₅ IC₅₀ = 10–309 nM), without a significant species disconnect between human M₅ and rat M₅. However, only 29f and (rac)-30a maintained acceptable human M₁ subtype selectivity. 29f was advanced into an in vivo PK study and showed a good in vitro/in vivo correlation. However, there was still room for improvement. In general, this series was susceptible to P-gp efflux (ER = 110; both 29f and (rac)-30a) leading to low brain exposures (K_p = 0.02, 0.04; K_p.uu = 0.16, 0.38; 29f and (rac)-30a respectively). Additional optimizations toward a brain permeable tool, such as masking a hydrogen bond donor, are currently ongoing.

Table 5.

Subtype selectivity and DMPK profile data of selected compounds.

Property	29f VU6027634	(rac)-30a VU6029053	(rac)-30b VU6035460	(rac)-30c VU6035461
hM5 (nM)	304a	309c	10b	13b
[ACh Min (%)]	[2]	[3]	[2]	[2]
rM5 (nM)	1330c	572	–	–
[ACh Min (%)]	[4]	[3]c
M1 (nM)	>10,000d	>10,000d	110d	300d
[ACh Min (%)]	[50, 35]	[51]	[3]	[3]
(h or r)	(h, r)	(h)	(r)	(r)
hM4 (nM)	inactive	inactive	–	–
[ACh Min (%)]a
MW	419.5	437.5	457.9	440.0
cLogP	1.76	2.05	1.97	2.23
tPSA	94.4	97.2	97.2	92.3
Fu, plasma (rat)	0.03	0.02	0.005	0.007
Fu, plasma (human)	0.14	0.26	0.02	0.02
Fu, brain (rat)	0.20	0.17	0.05	0.06
Kp	0.02	0.04	0.005	0.007
Kp,uu	0.16	0.38	0.05	0.06
CLint (mL/min/kg)	5 (h), 45 (r)	7 (h), 260 (r)	6 (h), 49 (r)	6 (h), 81 (r)
Predicted CLhep (mL/min/kg)	4 (h), 28 (r)	5 (h), 55 (r)	4 (h), 29 (r)	5 (h), 37 (r)
Rat in vivo PK profile
CLp (mL/min/kg)	11.1	–	–	–
Vss (L/kg)	0.61	–	–	–
Elim. t1/2 (min)	86	–	–	–
MDCK-MDR1	110	110	–	–
P-gp ER
MDCK-MDR1	0.374	0.431
Papp (10−6 cm/s)

- means not tested.

^aCalcium mobilization assay with hM₅-CHO cells performed in the presence of an EC₈₀ fixed concentration of acetylcholine. Each experiment performed in triplicate.

^bIC₅₀ value represent average from two independent experiments. Each experiment performed in triplicate.

^cIC₅₀ value represent average from three independent experiments. Each experiment performed in triplicate.

^dCalcium mobilization assay with hM₁-CHO cells performed in the presence of an EC₈₀ fixed concentration of acetylcholine. Each experiment performed in triplicate.

In summary, we report a novel M₅ antagonist series containing a piperidine amide moiety. This novel M₅ antagonist series contains an amide linkage instead of a metabolically labile, flexible thioether linkage, and optimized 29f exhibits good potency and an improved in vitro and in vivo clearance profiles in some cases. However, low brain exposure due to P-gp efflux liability remains unresolved. Additional optimizations and follow-up in vivo studies are currently in progress and will be reported in due course.

Data availability

The data that has been used is confidential.