High-affinity and selective dopamine D₃ receptor full agonists

Abstract

We have designed, synthesized and evaluated a series of new compounds with the goal to identify potent and selective D₃ ligands. The two most potent and selective new D₃ ligands are compounds 38 and 52, which bind to the D₃ receptors with a K_i value of <1 nM and display a selectivity of 450–494 times over the D₂ receptors and >10,000 times over the D₁ receptors. Both 38 and 52 are full agonists with high potency at the D₃ receptor in a D₃ functional assay.

Dopaminergic neurotransmission is mediated by five dopamine receptors (D₁–D₅), which can be grouped into the D₁-like (D₁ and D₅) and D₂-like (D₂, D₃, and D₄) receptor subtypes. The dopamine 3 (D₃) subtype receptor has been identified as an important target for agents currently used clinically for the treatment of schizophrenia, Parkinson’s disease, depression, and other neurological diseases^1–4 and a number of studies have provided strong evidence that potent and selective D₃ ligands may have therapeutic potential as novel therapies for the treatment of drug abuse.^5–8 Hence, considerable effort has been devoted to the discovery and development of potent and selective D₃ ligands.^7–32

A number of known potent and selective D₃ ligands, including antagonists, partial agonists and full agonists, are shown in Figure 1. Evaluated in in vitro binding assays either using cloned human dopamine receptors or rat brain,^7,8 these ligands were shown to bind to the D₃ receptor with high affinities and selectivities of 100–500 fold over the D₂ receptor and >1,000 over the D₁-like receptors. SB-277011A (1) is a potent and selective D₃ antagonist⁹ which has been extensively used in animals to investigate the role of the D₃ receptor in drug abuse.⁸ A shortcoming of SB-277011A is that relatively high doses are needed to produce an in vivo effect, suggesting only moderate CNS penetration. Subsequent optimization efforts led to the identification of SB-414796 (2), which is a potent and selective D₃ antagonist with excellent bioavailability in the rat, and good CNS penetration.¹⁶ However, SB-414796 was not advanced into clinical development because it inhibits p450 and has potential cardiotoxicity due to its strong binding to the hERG potassium channel.^8,32 Very recently, compound 3 was designed as a highly potent and selective D₃ antagonist.³² This compound has excellent oral bioavailability and good CNS penetration, shows weak inhibition on all the P450 isoforms and at the hERG channel, thus defusing the major issues associated with compound 2. BP 897 (4) was initially described as a potent D₃ partial agonist with a modest selectivity of < 100 over D₂,⁶ but subsequent studies have shown that it may behave as a D₃ antagonist.^33,34 NGB 2904 (5) is a potent and selective D₃ antagonist¹⁰ and has been extensively evaluated in vivo.⁸ A large number of new analogues, including compounds 6,¹⁸ 7³⁰ and 8,³⁰ have been designed and synthesized in an effort to improve D₃ selectivity and solubility. Among them, compound 8 is the most potent and selective D₃ antagonist and displays a selectivity of >400-fold over D₂ in in vitro binding assays.³⁰ Significantly, compound 8 is also more soluble than compound 5.³⁰

Figure 1.

Chemical structures of representative known D3 ligands.

Pramipexole (9) is a highly potent D₃ full agonist but with only modest selectivity for the D₃ receptor over the D₂ receptor.^25,28 Pramipexole has been approved for the treatment of Parkinson’s disease and restless legs syndrome and has excellent safety and pharmacological properties in humans. Hence, Pramipexole serves as an excellent leading compound for the design of potent and selective D3 ligands. In an effort to improve its selectivity for D₃ over D₂, we have previously designed and synthesized a series of compounds based upon the core structure of pramipexole.²⁵ Our study led to the identification of several potent D₃ ligands such as compounds 10 and 11, which are much more selective for D₃ over D₂ ²⁵ than pramipexole. Unfortunately, notwithstanding their high binding affinity to the D₃ receptor (K_i < 1nM) and excellent aqueous solubility, compounds 10 and 11 were found to be active at the D₃ receptor only at 32 mg/kg in the yawning assay in the rat, indicating that these compounds have poor bioavailability in the brain.²⁵

In the present study, we have performed additional chemical modifications based upon the chemical structures of compounds 10 and 11 with the goal of identifying new D₃ ligands with high affinity and excellent selectivity.

Since the D₃ radioligand [³H]PD128907 used previously in our D₃ binding assays recently became unavailable, it became necessary to switch to another radioligand [³H]-R(+)-7-hydroxy-N,N-di-n-propyl-2-aminotetralin ([³H]-R(+)-7-OH-DPAT) for our D₃ receptor binding assay. Accordingly, we re-evaluated compounds 10 and 11 in both D₃ and D₂ binding assays using this new D₃ radioligand. Compounds 10 and 11 were found to bind to the D₃ receptor with K_i values of 1.0 ± 0.04 nM (Table 1) and 1.1 ± 0.3 nM (Table 2), respectively, as compared to 0.41 ± 0.031 nM and 0.40 ± 0.057 nM, respectively, in our previous D₃ assay²⁵ using [³H]PD128907. Compounds 10 and 11 were found to bind to the D₂ receptor with K_i values of 239 ± 60 nM (Table 1) and 185±61 nM (Table 2), respectively, as compared to 330 ± 69 nM and 307±38 nM, respectively, as we previously reported.²⁵ The selectivity of compounds 10 and 11 for D₃ over D₂ are 239- and 168-times, respectively, as compared to 800- and 763-times, respectively, in our previous report.²⁵ This is due primarily to the 2.5-fold increase in K_i values in the D₃ receptor binding assay.

Table 1.

Binding Affinities at the D₁-like, D₂-like, and D₃ Receptors in Binding Assays Using Rat Brain.

	Ki±SEM (nM)				Selectivity
ligand	R	D3 ([3H]-R(+)-7-OH-DPAT)	D2-like ([3H]Spiperone)	D1-like ([3H]SCH23390)	D2-like/D3	D1-like/D3
9		0.78	3.1±0.3	>100,000	4.0	>100,000
10	H	1.0 ± 0.04	239 ± 60	NT	239	NT
15	4-F	1.0 ± 0.2	66 ± 10	4,674±466	66	71
16	3-F	9.9 ± 2.4	80 ± 2	6,606±556	8	666
17	2-F	2.9 ± 0.3	92 ± 32	10,022±1692	32	3,513
18	4-Cl	2.5 ± 0.7	27 ± 4	4,032±394	11	1,596
19	3-Cl	1.2 ± 0.3	52 ± 7	5,766±508	43	4,644
20	2-Cl	1.2 ± 0.2	30 ± 5	5,436±389	26	4,646
21	4-OMe	6.7 ± 1.5	94 ± 9	8,026±776	14	1,196
22	3-OMe	9.3 ± 1.7	122 ± 23	8,890±928	13	953
23	2-OMe	8.1 ± 0.8	102 ± 10	7,916±712	13	983

Table 2.

Binding Affinities at the D₁-like, D₂-like, and D₃ Receptors in Binding Assays Using Rat Brain.

	Ki±SEM (nM)				Selectivity
ligand	R	D3 ([3H]-R(+)-7-OH-DPAT)	D2-like ([3H]Spiperone)	D1-like ([3H]SCH23390)	D2-like/D3	D1-like/D3
11		1.1±0.3	185±61	NT	168	NT
24		1.6±0.24	88±4.8	11,305±864	55	6,054
25		1.6±0.25	72±5.7	44,047±2,317	45	27,106
26		5.9±1.4	64±16	26,450±2,782	11	4,503
27		2.0±0.55	45±3.3	46,877±5,199	23	23,755
28		1.9±0.03	23±2.6	37,163±2,354	12	19,909
29		3.0±0.69	43±5.9	59,660±5,488	14	19,877
30		1.1±0.26	32±2.3	24,667±1,894	29	21,790
31		3.0±0.29	52±0.43	14,407±1,184	18	4,867
32		2.4±0.51	56±5.2	14,033±1,691	23	5,959
33		1.3±0.12	442±41	34,613±3,625	345	27,041
34		2.1±0.15	328±53	17,748±921	155	8,381
35		2.0±0.19	470±53	10,912±756	231	5,366
36		3.5±0.57	633±100	11,410±765	180	3,245
37		1.5±0.23	491±70	5,000±495	339	3,448
38		0.73±0.028	359±58	9,937±888	494	13,678

We first performed modifications on 10 by installation of an F, Cl or CH₃O group in the para-, meta- or ortho-positions in the phenyl ring to yield compounds 15–23 (Table 1). Compounds 15–23 were tested for their binding affinities to the dopamine D₁, D₂ and D₃ receptors and the results are summarized in Table 1. Our binding data showed that substitution of the phenyl ring in compound 10 with F, Cl or OCH₃ improves neither the potency to D₃ nor the selectivity for D₃ over D₂.

We next synthesized compounds 24–26 to investigate the effect of replacing a carbon atom in the naphthyl group with a nitrogen atom at each of three different positions in compound 11 (Table 2). These three compounds bind to D₃ with K_i values of 1.6, 1.6 and 5.9 nM, respectively, and are somewhat less potent than 11. They are also less selective than 11 for D₃ over D₂.

A series of compounds (27–32) in which a phenyl group connected to different 2–3 nitrogen-containing hetero-cyclic systems replaces the naphthyl group in 11 (Table 2) was synthesized. These compounds bind to D₃ with K_i values of 1.1 to 3.0 nM and have a modest selectivity of 12–29 times for D₃ over D₂.

We synthesized compound 33, in which the naphthyl group is replaced by a 4-(5-methyl-1,2,4-oxadiazol-3-yl)phenyl group, which had been used in the design of compound 2 (Table 2). Compound 33, binds to D₃ with the same high affinity (K_i = 1.3 nM) as 11, but its selectivity over the D₂ receptor is improved to 345-fold due to its decreased binding affinity to D₂. Encouraged by this promising binding and selectivity data, we further modified 33 with different 1,2,4-oxadiazol-3-yl-phenyl groups, to obtain compounds 34–38 (Table 2). Replacement of the methyl group in 33 with ethyl (34), isopropyl (35) or cyclopropyl (36) decreased the binding affinity to D₃ and also the selectivity for D₃ over D₂ by factors of 2–3 as compared to 33. Compounds 37 and 38, in which the 4-(5-methyl-1,2,4-oxadiazol-3-yl)phenyl group in 33, has been replaced by either 3-(5-ethyl-1,2,4-oxadiazol-3-yl)phenyl or 3-(5-methyl-1,2,4-oxadiazol-3-yl)phenyl, bind to D₃ with K_i values of 1.5 nM and 0.73 nM, respectively and their selectivity for D₃ over D₂ is 339 and 494, respectively.

Compounds 39–53, which all contain a sulfonamide group in place of the amide group in the linker region (Table 3), were made. Compound 39, in which a sulfonamide group was used to replace the amide group in 11, binds to D₃ with a similar affinity (1.5 nM) as 11, but its selectivity over D₂ is decreased due to its increased binding affinity to D₂. Compound 40, in which the naphthyl group is connected to the sulfonamide group via its alpha position instead of the beta position in 39, binds to D₃ with a K_i value of 1.5 nM and has a selectivity of 21-times for D₃ over D₂. Compound 41, in which the naphthyl group in 39 is replaced by a phenyl group, binds to the D₃ receptor with a K_i of 0.79 nM, and due to its decreased binding affinity to D₂, its selectivity over D₂ is improved to 72-fold, 8 times more selective than 39. Compound 42, in which the naphthyl group in 39 is replaced by a biphenyl group, binds to D₃ with a K_i value of 41 nM, much weaker than compound 39.

Table 3.

Binding Affinities at the D₁-like, D₂-like, and D₃ Receptors in Binding Assays Using Rat Brain.

	Ki±SEM (nM)				Selectivity
ligand	R	D3 ([3H]-R(+)-7-OH-DPAT)	D2-like ([3H]Spiperone)	D1-like ([3H]SCH23390)	D2-like/D3	D1-like/D3
39		1.5 ± 0.2	14 ± 2	3,689 ± 366	9	2,427
40		1.5 ± 0.4	31 ± 3	4,667 ± 476	21	3,164
41		0.79 ± 0.08	57 ± 8	23,370 ± 1,331	72	29,686
42		41 ± 3	75 ± 10	1,800 ± 168	2	44
43		0.72 ± 0.11	136 ± 17	10,359 ± 805	190	14,467
44		0.78 ± 0.11	38 ± 3	21,330 ± 1,006	49	27,487
45		1.1 ± 0.1	127 ± 15	5,552 ± 231	117	5,094
46		0.95 ± 0.01	71 ± 5	8,287 ± 257	75	8,754
47		0.68 ± 0.06	66 ± 11	9,484 ± 630	98	14,015
48		0.89 ± 0.04	62 ± 8	8,399 ± 672	70	9,472
49		4.5 ± 0.1	133 ± 16	8,147 ± 836	30	1,810
50		5.0 ± 0.4	101 ± 12	3,149 ± 263	20	626
51		0.63 ± 0.04	28 ± 3	8,191 ± 937	44	12,933
52		0.38 ± 0.05	170 ± 19	17,443 ± 1,727	451	46,310
53		0.93 ± 0.10	213 ± 11	10,716 ± 623	229	11,523

Compound 41 has a high binding affinity to D₃ but a modest selectivity for D₃ over D₂. We next modified the phenyl ring of 41 using different substituents, which yielded compounds 43–50. Compound 43, which has a meta-F substituent, has a high binding affinity to D₃ (K_i = 0.72 nM) and displays 190-fold selectivity over D₂. Compound 44, which has a methoxyl group at the para position of the phenyl ring, binds to D₃ with a high affinity (K_i = 0.78 nM), but its selectivity over D₂ is decreased to 49-fold due to its improved binding affinity to D₂. Compounds 45–47 with a Cl substituent at three different positions in the phenyl ring have high binding affinities (K_i = 0.68–1.1 nM) to D₃ and 100-fold selectivity over D₂. Compound 48 with a meta methyl substituent binds to D₃ with a high affinity (0.89 nM) and has a selectivity of 70-fold over D₂. However, an isopropyl (49) or a tert-butyl (50) substituent at the para position of the phenyl ring decreases the binding affinity to the D₃ receptor by a factor of 6 as compared to 41. These two compounds also have a modest selectivity of 20–30 times over D₂.

Both 46 and 47 have a high affinity to D₃ but a modest selectivity for D₃ over D₂. We replaced one carbon atom with a nitrogen atom in the phenyl ring in 46 and 47 to investigate the effect on binding and selectivity. Compound 51, in which a nitrogen atom was inserted into the meta position of the phenyl ring in 46, binds to D₃ with the same affinity as 46 but is slightly less selective than 46 over D₂. Compound 52, in which a nitrogen atom was inserted into the ortho position with respect to the Cl substituent in the phenyl ring in 47, binds to D₃ with a K_i value of 0.38 nM and has a selectivity of 451 times over D₂. Compound 53, in which a nitrogen atom was inserted into the para position with respect to the Cl substituent in the phenyl ring in 47, binds to D₃ with a K_i value of 0.93 nM and has a selectivity of 229 times over D₂.

Hence, our chemical modifications of compound 11 have identified a number of new ligands with high affinities to D₃ and an excellent selectivity for D₃ over D₂. Of these, 38 (UM-206) and 52 (UM-226) bind to D₃ with K_i values of 0.73 nM and 0.38 nM, respectively, and have a selectivity of 494 and 451 times, respectively, over D₂. These two compounds also display a selectivity of >10,000 times for D₃ over the D₁-like receptors.

We evaluated 38 and 52 in the D₃ functional mitogenesis assay using CHO cells transfected with the human D₃ receptor. The functional data showed that both 38 and 52 function as full agonists with EC₅₀ values of 4.3 ± 1.2 nM and 1.5 ± 0.5 nM, respectively.

The synthesis of compounds 15–23 is shown in Scheme 1. Pramipexole 9 was allowed to react with commercially available tert-butyl trans-4-(2-oxoethyl)cyclohexylcarbamate to give key intermediate 13 which was treated with TFA to afford the amine 14. Compound 14, when reacted with different cinnamoyl chlorides, gave compounds 15–23. The synthesis of compounds 24–38 is straightforward and is shown in Scheme 2. Compound 14 was condensed with the appropriate carboxylic acid to give the designed compounds, 24–38. The synthesis of compounds 39–53 is described in Scheme 3. Compound 14 was reacted with appropriate sulfonyl chloride in the presence of diisopropylethylamine (DIPEA) to give compounds 39–53.

Scheme 1.

Synthesis of compounds 15–23. Conditions and reagents: (i) tert-butyl trans-4-(2-oxoethyl)cyclohexyl carbamate, NaBH(OAc)₃, MOAc, DCM, 75%; (ii) TFA, DCM, RT, 12 h, 90%; (iii) respective cinnamoyl chloride, DIPEA, DCM, RT, 2 h, 50–76%.

Scheme 2.

Synthesis of compounds 24–38. Conditions and reagents: (i) RCO₂H, EDCI, HOBt, DIPEA, DCM, 55–78%.

Scheme 3.

Synthesis of compounds 39–53. Conditions and reagents: (i) RSO₂CI, DIPEA, DCM, RT, 2 h, 60–75%.

In summary, through chemical modifications of our previously reported two D₃ ligands, compound 10 and 11, we have identified a number of new D₃ ligands with high binding affinities to the D₃ receptor and an excellent selectivity over the D₂ and D₁ receptors based upon our in vitro binding data. Compounds 38 and 52 are the two most potent and selective D₃ ligands among these new analogues. Both compounds bind to D₃ with a K_i value of <1 nM and display a selectivity of 450–494 times over D₂ and >10,000 selectivity over D₁. Both 38 and 52 are full agonists with a high potency at the D₃ receptor in a D₃ functional assay. Further in vivo characterizations of these compounds are in progress and will be reported in due course.