Bicyclic [3.3.0]-Octahydrocyclopenta[c]pyrrolo Antagonists of Retinol Binding Protein 4: Potential Treatment of Atrophic Age-Related Macular Degeneration and Stargardt Disease

Abstract

Antagonists of retinol-binding protein 4 (RBP4) impede ocular uptake of serum all-trans retinol (1) and have been shown to reduce cytotoxic bisretinoid formation in the retinal pigment epithelium (RPE), which is associated with the pathogenesis of both dry age-related macular degeneration (AMD) and Stargardt disease. Thus, these agents show promise as a potential pharmacotherapy by which to stem further neurodegeneration and concomitant vision loss associated with geographic atrophy of the macula. We previously disclosed the discovery of a novel series of nonretinoid RBP4 antagonists, represented by bicyclic [3.3.0]-octahydrocyclopenta[c]pyrrolo analogue 4. We describe herein the utilization of a pyrimidine-4-carboxylic acid fragment as a suitable isostere for the anthranilic acid appendage of 4, which led to the discovery of standout antagonist 33. Analogue 33 possesses exquisite in vitro RBP4 binding affinity and favorable drug-like characteristics and was found to reduce circulating plasma RBP4 levels in vivo in a robust manner (>90%).

Figure 1

Retinol (1), fenretinide (2), A1120 (3), and 2-((3aR,5r,6aS)-5-(2-(trifluoromethyl)phenyl)octahydrocyclopenta[c]pyrrole-2-carboxamido)benzoic acid (4).

Figure 2

Medicinal chemistry work plan for pyrimidine-4-carboxylic acid containing antagonists of RBP4.

Figure 3

(A) Overlay of minimized bound conformations of RBP4 antagonist 10 (orange) and 3 (green) within 3fmz. (B) Expanded view of the anthranilic acid and 6-methylpyrimidine-4-carboxylic acid fragments of 3 and analogue 10. Both moieties are shown engaging in H-bond interactions with Arg121, Gln98, and Tyr90. H-bond interactions to 10 are indicated with blue lines. Residues making contact with ligands are labeled and illustrated in stick format. Molecular surface is colored by hydrophobicity (green) and hydrophilicity (purple) using contact preferences (MOE, Chemical Computing Group, Inc., Montreal, CA).

Figure 4

(A) Overlay of minimized bound conformations of RBP4 antagonist 33 (magenta) and 3 (orange) within 3fmz. (B) Expanded view of the anthranilic acid and 6-methylpyrimidine-4-carboxylic acid fragments of 3 (green) and analogue 33 (magenta). Both moieties are shown engaging in H-bond interactions with Arg121, Gln98, and Tyr90. H-bond interactions to 33 are indicated with blue lines. Residues making contact with ligands are labeled and illustrated in stick format. The molecular surface is colored by hydrophobicity (green) and hydrophilicity (purple) using contact preferences (MOE, Chemical Computing Group, Inc., Montreal, CA).

Figure 5

(A) Overlay of minimized bound conformations of RBP4 nicotinic acid antagonist 54 (yellow) and 3 (A1120, green) within 3fmz. (B) Expanded view of the anthranilic acid and nicotinic acid fragments of 3 (green) and analogue 54 (yellow). Both moieties are shown engaging in H-bond interactions with Arg121, Gln98, and Tyr90. H-bond interactions to 54 are indicated with blue lines. Residues making contact with ligands are labeled and illustrated in stick format. The molecular surface is colored by hydrophobicity (green) and hydrophilicity (purple) using contact preferences (MOE, Chemical Computing Group, Inc., Montreal, CA).

Figure 6

(A) Plasma exposure levels of analogue 33 in Sprague–Dawley rats following a single (Day 1) and 7-day (Day 7) QD oral dose administration at 18.2 µmol/kg (5 mg/kg). Data represented as the mean (SD). Each dosing group consisted of three drug naïve adult male Sprague–Dawley rats; the test article vehicle was 2% Tween 80 in 0.9% saline. The difference in plasma compound concentration between Day 1 and Day 7 samples was statistically significant at all time points studied (P < 0.05; two-tailed unpaired Student’s t test). (B) Plasma concentration of analogue 33 at predose time points during the 7-day QD oral dose administration in three rats from Group 3. Data represented as the mean (SD). The predose compound concentration was statistically significantly increased at Day 7 in comparison to the Day 2 predose level (P < 0.05; two-tailed unpaired Student’s t test). (C) Pharmacokinetic parameters for 33 in Sprague–Dawley rats after a single dose (Day 1) and seven QD oral doses (Day 7) at 18.2 µmol/kg. Data are represented as the mean with standard deviation shown in parentheses.

Figure 7

(A) Plasma RBP4 levels (µg/mL) in male Sprague–Dawley rats following single (Day 1, Group 1) and 7-day (Day 7, Group 3) 18.2 µmol/kg (5 mg/kg) QD dose administration of analogue 33. Data represented as the mean (SD). (B) Plasma RBP4 levels (µg/mL) at predose time-points during the 7-day 5 mg/kg QD oral dose administration of analogue 33 (Group 3) or test article vehicle (Group 2) in Sprague–Dawley rats. Data represented as the mean (SD).

Scheme 1^a

^aReagents and conditions: (a) (i) n-BuLi, THF, −78 °C, 40 min; (ii) 1-benzylpiperidin-4-one, THF, −78 °C; (b) SOCl₂, 0 °C, 2 h; (c) (i) HCO₂NH₄, 10% Pd/C, CH₃OH, reflux; (ii) 4.0 M HCl solution in 1,4-dioxane, CH₃CN, rt, 2 h; (d) methyl 2-chloro-6-methylpyrimidine-4-carboxylate, i-Pr₂NEt Et₃N, DMF, 60 °C, 16 h; (e) (i) LiOH·H₂O, H₂O, THF, rt, 16 h; (ii) 2 N aq HCl.

Scheme 2^a

^aReagents and conditions: (a) tert-butyl piperazine-1-carboxylate, Pd(OAc)₂, BINAP, NaOt-Bu toluene, 110 °C, 16 h; (b) 2.0 M HCl solution in Et₂O, CH₂Cl₂, rt, 3 h; (c) methyl 2-chloro-6-methylpyrimidine-4-carboxylate, i-Pr₂NEt, DMF, 60 °C, 16 h; (d) (i) LiOH·H₂O, H₂O, THF, rt, 16 h; (ii) 2 N aq HCl.

Scheme 3^a

^aReagents and conditions: (a) (3aR,6aS)-tert-butyl hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate, Pd(OAc)₂, BINAP, NaOt-Bu, toluene, 110 °C, 16 h; (b) 2.0 M HCl solution in Et₂O, CH₂Cl₂, rt, 3 h; (c) methyl 2-chloro-6-methylpyrimidine-4-carboxylate, i-Pr₂NEt, DMF, 60 °C, 16 h; (d) (i) LiOH·H₂O, H₂O, THF, rt, 16 h; (ii) 2 N aq HCl.

Scheme 4^a

^aReagents and conditions: (a) (i) LiAlH₄ (1.0 M solution in THF), THF, 70 °C, 16 h; (ii) Boc₂O, CH₂Cl₂, rt, 16 h; (b) (i) NaIO₄, RuO₂·H₂O, CH₃CN, CCl₄, H₂O, rt, 24 h; (c) Ac₂O, NaOAc, 120 °C, 3 h; (d) (i) LiHMDS (1.0 M solution in THF), THF, −78 °C, 30 min; (ii), PhN(SO₂CF₃)₂, THF, −78 °C to rt, 3 h; (e) (2-(trifluoromethyl)phenyl)boronic acid, Pd(PPh₃)₄, 2 M Na₂CO₃, DME, 80 °C, 6 h; (f) H₂ (40 psi), 10% Pd/C, CH₃OH, rt, 16 h; (g) 2.0 M HCl in Et₂O, CH₂Cl₂, 0 °C to rt, 24 h; (h) 2-chloro-6-substitutedpyrimidine-4-carboxylic acid methyl ester, Et₃N, DMF, 60 °C, 16 h; (i) (i) 2 N aq NaOH, THF, CH₃OH, rt, 16 h; (ii) 2 N HCl.

Scheme 5^a

^aReagents and conditions: (a) TFA, CH₂Cl₂, rt, 3 h; (b) H₂ (50 psi), 10% Pd/C, CH₃OH, rt, 6 h; (c) (i) 2-chloro-6-methylpyrimidine-4-carboxylic acid, Et₃N, DMF, 60 °C, 16 h; (ii) 2 N aq NaOH, THF, CH₃OH, rt, 16 h; (iii) 2 N aq HCl.

Scheme 6^a

^aReagents and conditions: (a) (i) 2,4-dichloro-5,6-dimethylpyrimidine, Pd(OAc)₂, Cs₂CO₃, JohnPhos, toluene, reflux, 16 h; (ii) Mo(CO)₆, Pd(OAc)₂, BINAP, CH₃OH, CH₃CN, 80 °C, 16 h; (iii) LiOH·H₂O, THF, CH₃OH, H₂O, rt, 16 h; (b) (i) methyl 2-chloro-5-methylpyrimidine-4-carboxylate, Et₃N, DMF, 60 °C, 16 h; (ii) 2 N aq NaOH, THF, CH₃OH, rt, 16 h; (c) (i) methyl 2-chloro-4-methylpyrimidine-5-carboxylate or methyl 2-chloropyrimidine-5-carboxylate, i-Pr₂NEt, 70 °C (microwave irradiation), 1.5 h or Et₃N, DMF, 70 °C, 16 h; (ii) LiOH·H₂O, THF, H₂O, rt, 16 h or 2 N aq NaOH, THF, CH₃OH, rt, 16 h; (iii) 2 N aq HCl.

Scheme 7^a

^aReagents and conditions: (a) (i) 2-chloro-6-methylpyrimidine-4-carbonitrile, i-Pr₂NEt, DMF, 60 °C, 16 h; (b) LiOH·H₂O, THF, H₂O, 70 °C, 18 h; (c) NaN₃, NH₄Cl, DMF, 130 °C (microwave irradiation), 1 h.

Scheme 8^a

^aReagents and conditions: (a) (i) methyl 6-chloro-4-methylpicolinate, Pd(OAc)₂, XantPhos, Cs₂CO₃, toluene, 110 °C, 16 h; (ii) 2 N aq NaOH, THF, CH₃OH, rt, 16 h; (iii) 2 N aq HCl; (b) (i) methyl 2-chloro-6-methylisonicotinate, Pd(OAc)₂, XantPhos, Cs₂CO₃, toluene, 110 °C, 16 h; (ii) 2 N aq NaOH, THF, CH₃OH, rt, 16 h; (iii) 2 N aq HCl; (c) (i) methyl 6-chloro-3-methylpicolinate, Pd(OAc)₂, JohnPhos, Cs₂CO₃, toluene, reflux, 14 h; (ii) LiOH·H₂O, THF, CH₃OH, H₂O, rt, 72 h; (iii) 2 N aq HCl; (d) (i) methyl 5-bromonicotinate, Pd₂(dba)₃, XantPhos, Cs₂CO₃, 1,4-dioxane, reflux, 16 h; (ii) LiOH·H₂O, THF, CH₃OH, H₂O, rt, 4 h; (iii) 2 N aq HCl; (e) methyl 3-bromobenzoate, Pd(OAc)₂, BINAP, NaOt-Bu, toluene, 110 °C, 16 h; (ii) LiOH·H₂O, THF, CH₃OH, H₂O, rt, 16 h; (iii) 2 N aq HCl; (f) (i) methyl 2-chloro-6-methylnicotinate, Pd₂(dba)₃, XantPhos, Cs₂CO₃, toluene, 100 °C, 16 h; (ii) LiOH·H₂O, THF, CH₃OH, H₂O, 50 °C, 30 min; (iii) 2 N aq HCl; (g) (i) methyl 2-chloronicotinate, Pd(OAc)₂, XantPhos, Cs₂CO₃, 1,4-dioxane, 110 °C, 16 h; (ii) LiOH·H₂O, THF, CH₃OH, H₂O, rt, 16 h; (iii) 2 N aq HCl.

Scheme 9^a

^aReagents and conditions: (a) (i) methyl 2-chloropyrazine-6-carboxylate or methyl 3-chloropyridazine-5-carboxylate or methyl 2-chloro-5-methylthiazole-4-carboxylate, Et₃N or i-Pr₂NEt, DMF, 60 °C, 16 h or i-Pr₂NEt, DMSO, 110 °C, 24 h; (ii) LiOH·H₂O, THF, CH₃OH, H₂O, rt, 30 min-16 h; (iii) 2 N aq HCl.

Scheme 10^a

^aReagents and conditions: (a) (i) substituted phenyl or pyridyl boronic acid, Pd(PPh₃)₄, 2.0 M Na₂CO₃, DME, 80 °C, 6 h; (b) H₂ (40 psi), 10% Pd/C, CH₃OH, rt, 16 h; (c) 2.0 M HCl in Et₂O, CH₂Cl₂, 0 °C to rt, 24 h; (d) (i) methyl 2-chloro-6-methylpyrimidine-4-carboxylate, Et₃N, DMF, 60 °C, 16 h; (ii) 2 N aq NaOH, THF, CH₃OH, rt, 16 h; (iii) 2 N aq HCl.