Structure-Activity Relationships of (+)-Naltrexone-Inspired Toll-like Receptor 4 (TLR4) Antagonists

Abstract

Activation of Toll-like receptors has been linked to neuropathic pain and opioid dependence. (+)-Naltrexone acts as a Toll-like receptor 4 (TLR4) antagonist and has been shown to reverse neuropathic pain in rat studies. We designed and synthesized compounds based on (+)-naltrexone and (+)-noroxymorphone and evaluated their TLR4 antagonist activities by their effects on inhibiting lipopolysaccharide (LPS) induced TLR4 downstream nitric oxide (NO) production in microglia BV-2 cells. Alteration of the N-substituent in (+)-noroxymorphone gave us a potent TLR4 antagonist. The most promising analog, (+)-N-phenethylnoroxymorphone ((4S,4aR,7aS,12bR)-4a,9-dihydroxy-3-phenethyl-2,3,4,4a,5,6-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-7(7aH)-one, 1j) showed ∼75 times better TLR-4 antagonist activity than (+)-naltrexone, and the ratio of its cell viability IC50, a measure of its toxicity, to TLR-4 antagonist activity (140 μM/1.4 μM) was among the best of the new analogs. This compound (1j) was active in vivo; it significantly increased and prolonged morphine analgesia.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1

Synthetic targets 2 – 7

Figure 2

(+)-Naltrexone 1a, (+)-β-naltrexol 2 and (+)-α-naltrexol 3 inhibit LPS induced NO over-production in a dose-dependent manner. BV-2 cells were treated with 200 ng/mL LPS and various concentrations of compounds for 24 h. NO in the supernatant was measured by the 2,3-diaminonaphthalene method., The NO in the LPS (200 ng/mL) control group was normalized as 100%.

Figure 3

(+)-Desoxynaltrexone 4 inhibits LPS induced NO over-production in a dose-dependent manner. (+)-Naltrexone 1a served as a control. BV-2 cells were treated with 200 ng/mL LPS and various concentrations of the test compounds for 24 h. NO in the supernatant was measured by the 2,3-diaminonaphthalene method., The NO in the LPS (200 ng/mL) control group was normalized to 100%.

Figure 4

(+)-Didesoxynaltrexone 5 inhibits LPS induced NO over-production in a dose-dependent manner. (+)-Naltrexone 1a served as the control compound for comparison. BV-2 cells were treated with 200 ng/mL of LPS and various concentrations of test compounds for 24 h. NO in the supernatant was measured by the 2,3-diaminonaphthalene method., The NO in the LPS (200 ng/mL) group was normalized as 100%.

Figure 5

(+)-N-Hexylnoroxymorphone 1d inhibits LPS induced NO over-production in a dose-dependent manner. (+)-Naltrexone 1a served as the control compound for comparison. BV-2 cells were treated with 200 ng/mL LPS and various concentrations of test compounds for 24 h. NO in the supernatant was measured by the 2,3-diaminonaphthalene method., The NO in the LPS (200 ng/mL) control group was normalized to 100%.

Figure 6

(+)-N-Phenethylnoroxymorphone 1j inhibits LPS induced NO over-production in a dose-dependent manner. (+)-Naltrexone 1a served as the control compound for comparison. BV-2 cells were treated with 200 ng/mL LPS and various concentrations of test compounds for 24 h. NO in the supernatant was measured by the 2,3-diaminonaphthalene method., The NO in the LPS (200 ng/mL) group was normalized to 100%.

Figure 7

(+)-Didesoxy-N-hexylnoroxymorphone 6 inhibits LPS induced NO over-production in a dose-dependent manner. (+)-Naltrexone 1a served as the control compound for comparison. BV-2 cells were treated with 200 ng/mL LPS and various concentrations of test compounds for 24 h. NO in the supernatant was measured by the 2,3-diaminonaphthalene method., The NO in the LPS (200 ng/mL) group was normalized to 100%.

Figure 8

Intrathecal coadministration of morphine (15 μg) with 1j (60 μg) produced a significant potentiation of morphine tailflick analgesia. n = 6/group *P < 0.05, **P < 0.01: relative to vehicle; #P < 0.05: relative to baseline.

Scheme 1

Synthesis of (+)-Naltrexone 1a from Commercially Available Sinomenine 8

Scheme 2

Stereoselective Reduction of (+)-Naltrexone 1a

Scheme 3

Synthesis of (+)-Desoxynaltrexone 4 from (+)-Naltrexone 1a

Scheme 4

Synthesis of (+)-Didesoxynaltrexone 5 from (+)-O-Methylnaltrexone 20

Scheme 5

N-Alkyl and N-Arylalkyl Substituents in (+)-Noroxymorphone 1b–1k

Scheme 6

Synthesis of (+)-Didesoxy-N-hexylnoroxymorphone 6 from (+)-N-Hexylnoroxymorphone 1d

Scheme 7

Synthesis of (+)-N-Phenethylnoroxycodone 35