A novel class of H₃ antagonists derived from the natural product guided synthesis of unnatural analogs of the marine bromopyrrole alkaloid dispyrin

Abstract

This Letter describes the natural product guided synthesis of unnatural analogs of the marine bromopyrrole alkaloid dispyrin, and the resulting SAR of H₃ antagonism. Multiple rounds of iterative parallel synthesis improved human H₃ IC₅₀ ~33-fold, and afforded a new class of H₃ antagonists based on the novel bromotyramine core of dispyrin.

The neurotransmitter histamine exerts its action through four distinct Class A GPCRs (H₁–H₄).^1–7 The histamine H₃ receptor, a Gi/o-coupled receptor in the CNS, is a pre-synaptic auto- and heteroreceptor that not only controls the release of histamine, but also other neurotransmitters (acetylcholine, noradrenaline, dopamine, GABA and serotonin).^1–7 Preclinically, H₃ antagonists/inverse agonists have demonstrated efficacy in a number of CNS pathologies including schizophrenia, epilepsy, depression, pain, decreasing food intake, drug abuse and addiction, sleep disorders/narcolepsy and cognitive enhancement.^1–7 Early reference H₃ antagonists contained imidazole moieties, such as thioperimide 1 and Perceptin (GT-2331) 2 (Fig. 1). Effort from multiple companies then focused on non-imidazole H₃ antagonists and include compounds such as UCL 1972 3, ABT-239 4, JNJ’s 5, Novo Nordisk’s 6, Eli Lilly’s 7 and GSK189254 8 to exemplify a few (Fig. 1). This intense effort from the pharmaceutical industry led to the evolution of a refined H₃ antagonist pharmacophore model 9.^1–8

Figure 1.

Imidiazole and non-imidazole H₃ antagonists 1–8 leading to a refined H₃ pharmacophore model 9.

We recently completed the first total synthesis of dispyrin 10,⁹ a bromopyrrole alkaloid with a novel bromotyramine core, isolated by Crews in 2007¹⁰ from the marine sponge Agelas dispar (Fig. 2). Upon recognition that dispyrin 10 possessed the basic features of the refined H₃ pharmacophore model 9, we evaluated our synthetic dispyrin against the human H₃ receptor. Gratifyingly, dispyrin was found to have modest activity as an H₃ antagonist (IC₅₀ = 2.35 μM, K_i = 1.04 μM).⁹ Based on these data, we initiated a natural product guided synthesis effort, employing iterative parallel synthesis¹¹ for molecular editing, aimed at improving H₃ inhibition and binding; moreover, we wanted to validate the marine natural product dispyrin 10 as a viable lead molecule due to the novel scaffold providing intellectual property in extremely crowded chemical space.

Figure 2.

Dispyrin 10, a novel bromopyrrole alkaloid from Agelas dispar with a bromotyramine core unprecedented in marine natural products.

The first generation 25-member library was based on a 5 × 5 two-dimensional design wherein the core was held constant and the amide R¹ and aminoalkyl moieties R² varied (Scheme 1). The library synthesis began with a simple DIC amide coupling employing commercially available 3-bromo-4-methoxyphenylethylamine 11 with one of five heterocyclic carboxylic acids R¹. These five scaffolds were then treated with BBr₃ to remove the methyl ether liberating the free phenols 13. Each of the five phenols 13 was then alkylated with one of five aminoalkyl chlorides to install R² under microwave-assisted conditions to afford unnatural dispyrin analogs 14 (Table 1).

Scheme 1.

First generation library synthesis. Reagents and conditions: (a) R¹COOH, DIC, HOBt, DIEA, DCM, rt, 12 h (69–99%); (b) BBr₃, DCM, −78 °C–rt, 1.5 h (50–95%); (c) 1.3 equiv ClR², CsCO₃, KI, DMF, mw, 160 °C, 20 min (72–93%). All analogs purified to >98% by mass-directed preparative HPLC.¹²

Table 1.

Structures and activities of dispyrin analogs 14

Compd	R1	R2	H3Kia (μm)	H3IC50a (μm)
14a			1.04	2.35
14b			1.61	3.72
14c			0.19	0.43
14d			12.9	29.1
14e			1.33	2.94
14f			1.36	2.92
14g			1.55	3.43
14h			0.15	0.38
14i			26.4	59.2
14j			1.52	3.63
14k			1.76	3.99
14l			2.13	4.54
14m			0.13	0.31
14n			17.6	39.6
14o			1.65	3.62
14p			1.61	3.61
14q			0.67	1.52
14r			0.08	0.18
14s			13.5	30.4
14t			1.25	3.73
14u			2.29	4.81
14v			2.72	5.93
14w			0.17	0.39
14x			32.5	73.2
14y			2.11	4.56

^aAverage of three independent determinations with human H₃.

This first generation library was highly informative. In general, all R¹s and R²s afforded modestly potent (K_is and IC₅₀s in the low micromolar range) H₃ antagonists. Potent H₃ antagonists (K_is < 200 nM, IC₅₀s < 430 nM) resulted for all of the heterocyclic amides R¹ in combination with the ethyl pyrrolidinyl R² (14c, 14h, 14m, 14r and 14w). In contrast, the ethyl morpholino congeners (14d, 14i, 14n, 14s and 14x) were uniformly weak (K_is > 12 μM, IC₅₀s > 29 μM). The most potent H₃ antagonist from the first generation library was 14r (R¹ = 4-bromo-thiophene, R² = ethyl pyrrolidine) with a K_i of 80 nM and an IC₅₀ of 180 nM—a 13-fold improvement over the parent natural product dispyrin 10 (IC₅₀ = 2.35 μM, K_i = 1.04 μM). Based on these data, the next library maintained R¹ = 4-bromo-thiophene and surveyed functionalized pyrrolidines at R² (Scheme 2).

Scheme 2.

Second generation library synthesis. Reagents and conditions: (a) BrCH₂CH(OMe)₂, Cs₂CO₃²⁻, KI, DMF, reflux (70%); (b) TosOH, mw, 160 °C, 10 min (60%); (c) functionalized pyrrolidine, MP-(OAc)₃H, DCM, rt (40–85%). All analogs purified to >98% by mass-directed preparative HPLC.¹²

Following Scheme 1, a large quantity of 15 was prepared. Then, the phenol was alkylated with 2-bromo-1,1-dimethoxy ethane to provide 16, which was then converted to the corresponding aldehyde 17 by treatment with tosylic acid. Finally, reductive amination employing a functionalized pyrrolidine and MP-B(OAc)₃H provided analogs 18. As shown in Table 2, analogs 18 were weaker H₃ antagonists than 14r, and there was no evidence of enantioselective inhibition (18a vs 18b). In agreement with the H₃ pharmacophore model, incorporation of β-fluorine atoms such as in 18c and 18d, which lowers the pK_a on the pyrrolidine nitrogen from 11 to 9, afforded diminished H₃ inhibition.¹³

Table 2.

Structures and activities of dispyrin analogs 18

Compd	R1	H3Kia (μm)	H3IC50a (μm)
18a	(S)-2-Me	0.31	0.71
18b	(S)-2-Me	0.33	0.75
18c	(S)-3-F	1.10	2.11
18d	(S)-3-F	1.15	2.52

^aAverage of three independent determinations.

We then prepared two singleton compounds following the synthetic route depicted in Scheme 1 with the appropriate substitutions, wherein the bromine in 14r was replaced with a chlorine 19 and a truncated version 20 (Fig. 3). A twofold diminution in potency was noted for 19, relative to 14r, and the truncated benzyl version lost over 13-fold compared to 14r; however, this highlighted that the heavy bromine atom was not required for H₃ inhibition.

Figure 3.

Chlorinated version and chlorinated truncated version of 14r.

The final library iteration was directed at surveying a wider range of alternative amides (heterocycles and functionalized aromatic moieties) while holding the preferred ethyl pyrrolidine ether and bromotyramine core constant. The synthesis began with 11 and conversion to the phthalimide congener 21. Standard BBr₃ deprotection provided 22 which was alkylated with chloroethyl pyrrolidine to deliver 23. Hydrolysis of the phthalimide with hydrazine, followed by amide coupling with a diverse collection of aryl and heteroaryl carboxylic acids generated library 24 (Scheme 3).

Scheme 3.

Third generation library synthesis. Reagents and conditions: (a) 1,2-dicarboxybenzene, DIC, HOBt, DIEA, DCM, rt, 12 h (99%); (b) BBr₃, DCM, −78 °C–rt, 1.5 h (95%); (c) 1.3 equiv Chloroethyl pyrrolidine, CsCO₃, KI, DMF, mw, 160 °C, 20 min (93%); (d) (i) N₂H₄, mw, 160 °C; (ii) SCX; (e) R¹COOH, DIC, HOBt, DIEA, DCM, rt, 12 h (64–98%). All analogs purified to >98% by mass-directed preparative HPLC.¹²

This third generation library was uniformly active, providing H₃ antagonists in the sub-micromolar range. Six-member heterocycles, such as pyridine 24a, were active, as were aryl amides with halogens (Cl and Br) or trifluoromethyl groups in the 3-position (24f–h). Five-member heterocycles (24b–e) proved optimal, with a 5-oxazole 24b (K_i = 32 nM, IC₅₀ = 83 nM) and 2-thiazole 24d (K_i = 32 nM, IC₅₀ = 72 nM) affording the most potent H₃ antagonists of the unnatural dispyrin analogs. For example, 24d improved the H₃ K_i and IC₅₀ ~33-fold over the natural product dispyrin, and required only three iterations of molecular editing and 40 analogs. Moreover, as dispyrin represented a novel chemotype, we were able to obtain composition of matter patents for the dispyrin analogs as H₃ antagonists within an incredibly crowded intellectual property landscape.¹⁴ This effort highlights the value of employing natural products as leads for therapeutically relevant targets (Table 3).

Table 3.

Structures and activities of dispyrin analogs 24

Compd	R1	H3Kia (μm)	H3IC50a (μm)
24a		0.27	0.56
24b		0.03	0.08
24c		0.43	0.97
24d		0.03	0.07
24e		0.12	0.26
24f		0.25	0.55
24g		0.21	0.45
24h		0.44	0.98

^aAverage of three independent determinations.

In summary, a natural products guided synthesis effort in molecular editing, employing iterative parallel synthesis, quickly optimized the weak H₃ antagonism of the marine natural product dispyrin 10 over 30-fold to afford unnatural analog 24d with low nanomolar potency and binding. By employing a novel natural product scaffold for lead optimization, we were able to establish an intellectual property position in an incredibly crowded intellectual property landscape. Although the role of natural products drug discovery efforts within the pharmaceutical industry is being significantly reduced, despite overwhelming success, the biological activity of dispyrin and its analogs argue further that natural products are viable drug leads and offer patenting advantages.