Design and synthesis of novel <i>α</i>-aminoamides derivatives as Nav1.7 inhibitors for antinociception

Design and synthesis of novel α-aminoamides derivatives as Nav1.7 inhibitors for antinociception

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Dengqi Xue^a^,^b^,¹, Yani Liu^c^,¹^,^*, Yilin Zheng^a, Heling Niu^c, Liying Dong^c, Xiangshuo Ouyang^a, Siyu Song^a, Denggao Zhang^a, Qianwei Ge^a, Kewei Wang^c, Liming Shao^a^,^b^,^*

Chinese Chemical Letters | 2022, 33(3) : 1643 - 1646

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Chinese Chemical Letters | 2022, 33(3): 1643-1646

• Communications •

Design and synthesis of novel α-aminoamides derivatives as Nav1.7 inhibitors for antinociception

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Dengqi Xue^a^,^b^,¹, Yani Liu^c^,¹^,^*, Yilin Zheng^a, Heling Niu^c, Liying Dong^c, Xiangshuo Ouyang^a, Siyu Song^a, Denggao Zhang^a, Qianwei Ge^a, Kewei Wang^c, Liming Shao^a^,^b^,^*

Affiliations

^aSchool of Pharmacy, Fudan University, Shanghai 201203, China

^bState Key Laboratory of Medical Neurobiology, Fudan University, Shanghai 200032, China

^cDepartment of Pharmacology, School of Pharmacy, Qingdao University Medical College, Qingdao 266071, China

Published: 2022-03-15 doi: 10.1016/j.cclet.2021.08.026

Outline

Abstract

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Three novel series of α-aminoamides derivatives were designed and synthesized based on ralfinamide, and their Nav1.7 inhibitory activities were evaluated using manual patch clamp electrophysiology. Active compounds inhibited Nav1.7 with half maximal inhibitory concentration (IC₅₀) values ranging from 2.9 µmol/L to 21.4 µmol/L. Among them, the most potent compound 19h exhibited about 12-fold potency better than ralfinamide. The investigation of their structure-activity relationship gives a strategy to improve the Nav1.7 inhibition of ralfinamide analogues. Compound 19h was efficacious in antinociception in the mouse spared nerve injury (SNI) model of neuropathic pain without causing sedation in the open field test.

Key words

α-Aminoamides / Sodium channel / Nav1.7 inhibitor / Chronic pain / Analgesia

Cite this Article

Dengqi Xue, Yani Liu, Yilin Zheng, Heling Niu, Liying Dong, Xiangshuo Ouyang, Siyu Song, Denggao Zhang, Qianwei Ge, Kewei Wang, Liming Shao. Design and synthesis of novel α-aminoamides derivatives as Nav1.7 inhibitors for antinociception[J]. Chinese Chemical Letters, 2022 , 33 (3) : 1643 -1646 . DOI: 10.1016/j.cclet.2021.08.026

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Chronic pain has serious and negative impact on quality of life [1-3. Currently available analgesics are limited with either greater side-effects or low levels of analgesic efficacy, thus presenting an unmet medical need [4]. Voltage-gated sodium channel Nav1.7 encoded by SCN9A gene plays a key role in transmission of pain signaling [5, 6]. The loss-of-function mutations in Nav1.7 lead to congenital insensitivity to pain (CIP) [7, 8]. Conversely, the gain-of-function mutations in Nav1.7 underlie inherited erythromelalgia, paroxysmal extreme pain disorder and idiopathic small fiber neuropathies [5, 9]. These lines of evidence indicate that Nav1.7 is a promising target for pain therapy. Ralfinamide (Fig. 1), a Nav1.7 inhibitor, was developed by Newron Pharmaceuticals, it also shows inhibition toward to Cav2.2 and N-methyl-d-aspartate (NMDA) receptor [10-14]. Ralfinamide was under phase III clinical trial for low back pain before discontinued [15]. Nav1.7 is a promising target for pain therapy, so optimizing the structure of ralfinamide may lead to a compound with higher Nav1.7 inhibition and better in vivo efficacy in antinociception.

In this study, three series of novel α-aminoamides derivatives were designed and synthesized for investigation of their structure–activity relationship (SAR). Started with investigation of the influence of the length of carbon linkage between the α-amino group and the aryl group (Fig. 1, I), then, we modified the α-aminoamide groups (Fig. 1, II) before various substituents were introduced into the benzyl group for investigation of the SAR (Fig. 1, III). Seventeen novel α-aminoamides derivatives were obtained and their inhibitions of human Nav1.7 currents were tested in whole-cell patch clamp recording assay. A lead compound was further evaluated for its in vitro metabolic stability and in vivo antinociception.

The syntheses of α-aminoamides derivatives were depicted in Scheme 1. Detailed procedures and compound characterizations can be found in Supporting information. 4-Hydroxyphenethyl alcohol as a starting material reacted with 2-fluorobenzyl bromide in acetone to give compound 2 and then it was oxidized by the Dess-Martin periodinane to yield the compound 3 [16, 17]. Compound 3 reacted further with L-alaninamide hydrochloride in the presence of sodium cyanoborohydride to yield the target compound 4. The compound 6 was achieved by the reaction of methyl 3-(4-hydroxyphenyl) propionate with 2-fluorobenzyl bromide in acetonitrile, subsequently through the hydrolysis of carboxylic ester to produce the compound 7. Compound 7 was reduced with lithium aluminum hydride to the alcohol 8, which was oxidized by Dess-Martin periodinane to yield the compound 9. The target compound 10 was obtained by the reaction of compound 9 with L-alaninamide hydrochloride. 4-(4-Hydroxyphenyl)butanoic acid was refluxed with 2-fluorobenzyl bromide in acetone to afford the compound 12. Compound 12 was reduced with lithium aluminum hydride and then it was oxidized by the Dess-Martin periodinane to yield the compound 14. The compound 14 reacted further with L-alaninamide hydrochloride to give the target compound 15. The target compound 16 was achieved by the reaction of compound 10 with formaldehyde. The compound 9 reacted with appropriate α-aminoamides hydrochloride to yield the target compounds 17a–d. The compound 18 was obtained by removing the ortho-fluoro-benzyl group from compound 10, which reacted further with a series of substituted benzyl alcohol to yield the target compounds 19a–i.

All synthesized compounds (4, 10, 15, 16, 17a–d and 19a–i) were tested for their inhibition of human Nav1.7 channel currents using whole-cell patch clamp recordings technique (Supporting information) [18, 19]. Ralfinamide was selected as reference compound. As summarized in Table 1 and Fig. 2, all compounds exhibited a dose-dependent inhibition of Nav1.7 currents with half maximal inhibitory concentration (IC₅₀) values ranging from 2.9 µmol/L to 21.4 µmol/L. Among them, the compound 19h (IC₅₀ = 2.9 µmol/L) was the most potent with about 12-fold more potency than ralfinamide (IC₅₀ = 35.2 µmol/L). The compounds 4, 10 and 15 showed better potency than ralfinamide, indicating that increasing the carbon chain length between the α-amino and the aryl group was beneficial for the bioactivity. Among the three compounds, the compound 10 (IC₅₀ = 5.0 µmol/L) with ideal three carbon atoms of the chain length showed the most potent inhibition of Nav1.7 current. The compound 16 demonstrated weaker inhibition than the compound 10, indicating that the methyl substitution on the α-amino group was not beneficial for the bioactivity. Removing the methyl group from α-position of aminoamides (compound 17a) or adding methyl group to α-position of aminoamides (compound 17b) reduced inhibitory activity, it also showed a steric effect of the substituents at α-position of aminoamides with a potency order of CH₃ (compound 10) > CH₂CH₃ (compound 17c) > CH(CH₃)₂ (compound 17d). Introduction of a methyl group to the orthro-fluoro benzyl position (compounds 19a and 19b) led to a decrease potency with a little chirality effect. 2-Cl (compound 19c), 2-OCH₃ (compound 19d) and 2-CH₃ (compound 19e) substitution on the phenyl ring led to a decreased efficiency. Alternatively, extra fluorine in either the para- or the orthro-position (compounds 19f and 19g) showed no improvement on Nav1.7 inhibition. Whereas substituents such as 1, 2-methylenedioxy (compound 19h) showed the best potency with IC₅₀ = 2.9 µmol/L, which is better than a bulkier substituted group such as 1, 2-ethylenedioxy (compound 19i).

It is important to study the metabolic stability of compound, because the most of the drugs are metabolized by cytochrome P450 (CYP450) in the liver [20-23]. The metabolic stability of the compound 19h was tested in human and mouse liver microsomes (Table 2 and Supporting information), and 19h appeared less stable than ralfinamide likely due to its higher clearance.

The spared nerve injury (SNI) model of neuropathic pain is characterized with a significant and continuous increase in mechanical sensitivity of mice [24]. Detailed procedures can be found in supporting information. The withdrawal threshold of the ipsilateral paw after surgery (0.03 ± 0.002 g) was significantly lower than the sham operation group (1.03 ± 0.07 g) on the sixth day after operation, indicating a successful establishment of mouse SNI model (Fig. 3). After intraperitoneal administrations of compound 19h (5, 10 and 20 mg/kg) for 10 days, the average paw withdrawal threshold was dose-dependently increased to 0.44 ± 0.05 g, 0.75 ± 0.03 g and 1.06 ± 0.07 g, respectively, as compared with the value of 0.04 ± 0.001 g for vehicle control or 0.60 ± 0.03 g for ralfinamide at 10 mg/kg. In addition, the paw withdrawal threshold for the group with compound 19h at 20 mg/kg (1.06 ± 0.07 g) was comparable to the sham operation group (1.17 ± 0.05 g) and morphine (1.25 ± 0.07 g) at 5 mg/kg.

Locomotor activity is commonly used for evaluation of psychostimulative or sedative effects. We next tested the effect of compound 19h on locomotion by assessing the total travel distance and average speed in the open field test (Supporting information). As shown in Fig. 4, mice treated with 20 mg/kg compound 19h (i.p.) had no significant differences in the total travel distance and the average speed, as compared with the vehicle control group. This result suggests that the compound 19h induced-antinociception was less likely resulted from the sedation effect.

With ralfinamide as a starting point, we synthesized a variety of α-aminoamides derivatives that were evaluated against Nav1.7 channel using whole-cell patch clamp recordings. Among the tested compounds, the compound 19h was the most potent with about 12-fold more potency than ralfinamide on inhibition of Nav1.7 channel. Our study provides valuable insights into the structure–activity relationship of ralfinamide analogues towards Nav1.7 channel. It gives a strategy to improve the Nav1.7 inhibition of ralfinamide analogues. The compound 19h was more efficacious in antinociception in the mouse model of neuropathic pain induced by spared nerve injury compared with ralfinamide, although 19h shows a slightly shorter half-life and faster clearance liver microsomes. The open field test result suggests that the compound 19h had no alteration on locomotion in mice.

Declaration of competing interest

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

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Financial supports by National Natural Science Foundation of China (Nos. 82003565 and 81973162) and the Science and Technology Commission of Shanghai Municipality (No. 20S11902300, China).

Supplementary materials

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Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2021.08.026

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Year 2022 volume 33 Issue 3

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Article Info

doi: 10.1016/j.cclet.2021.08.026

Receive Date：2021-07-04
Online Date：2025-12-12
Published：2022-03-15

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History

Received：2021-07-04
Revised：2021-07-19
Accepted：2021-08-06

Affiliations

^aSchool of Pharmacy, Fudan University, Shanghai 201203, China

^bState Key Laboratory of Medical Neurobiology, Fudan University, Shanghai 200032, China

^cDepartment of Pharmacology, School of Pharmacy, Qingdao University Medical College, Qingdao 266071, China

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表12种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Fig. 1 Chemical structures of ralfinamide and its designed compounds (I, II and III).

Scheme 1 Synthesis of α-aminoamides derivatives. Reaction conditions: (a) 2-Fluorobenzyl bromide, K₂CO₃, acetone, reflux, 63%–96%. (b) Dess-Martin periodinane, CH₂Cl₂, r.t., 43%–88%. (c) L-Alaninamide hydrochloride, NaBH₃CN, MeOH, AcOH, r.t. to 60 ℃, 43%–69%. (d) 2-Fluorobenzyl bromide, K₂CO₃, acetonitrile, 90 ℃, 70%. (e) NaOH, MeOH, THF, 60 ℃, 93%. (f) LiAlH₄, THF, 80 ℃, 60%. (g) 37% Formaldehyde (aq), NaBH₃CN, MeOH, AcOH, r.t., 88%. (h) Appropriate α-aminoamides hydrochloride, NaBH₃CN, MeOH, AcOH, r.t., 44%–71%. (i) Hydrogen, 5% palladium on activated carbon, EtOH, 45 ℃, 90%. (j) Substituted benzyl alcohol derivatives, PPh₃, di-tert-butyl azodicarboxylate (DBAD), THF, 90 ℃, 18%–57%.

Fig. 2 The dose-dependent inhibition of Nav1.7 currents by α-aminoamides derivatives in whole-cell patch clamp recordings of HEK293 cells stably expressed Nav1.7 channel. (a) Top panel, voltage protocol used in compound inhibition experiments. To activate Nav1.7 from a holding voltage of −40 mV, a 20 msec prepulse to −150 mV is applied to recover the current from inactivation followed by a 0 mV pulse to open the channel with 1 s intervals. Bottom panel, representative current traces elicited by 0 mV before and after applications of compound 19h. The dashed line represents zero current level. (b) Dose-response curve of compound 19h was fitted using logistic function with IC₅₀ of 2.9 ± 0.8 μmol/L, n = 7–8. (c) and (d) Curves were fitted using logistic function for concentration-dependent inhibition of Nav1.7 by α-aminoamides derivatives, n = 4–8. All data were expressed as the mean ± SEM.

Fig. 3 The dose-dependent attenuation of nociception by compound 19h in SNIinduced mechanical allodynia in mice. Top panel, a schematic time-course of paw mechanical withdrawal threshold (PMWT) measured by von Frey hair stimulation in mouse model of SNI. Bottom panel, a dose-dependent antinociception by 19h(5, 10 and 20 mg/kg, i.p.), morphine (5 mg/kg) and ralfinamide (10 mg/kg) in SNI model. Two-way ANOVA followed by Bonferroni post hoc tests revealed a significant decrease of PMWT between group sham and group vehicle, ^***P < 0.001; a significant increase of PMWT in 19h (5, 10 and 20 mg/kg, i.p.) injected groups, compared with group vehicle, ^###P < 0.001; and also a significant difference between group 19h (10 mg/kg) and group ralfinamide (10 mg/kg), ^$P < 0.05. Data were expressed as the mean ± SEM and ^***P < 0.001 (vs. Sham), n = 5–9.

Fig. 4 Compound 19h lack of altering locomotion. (a) Representative traces of mouse travels in the open field test after injections of saline, compound 19h (20 mg/kg, i.p.) and urethane (10%, i.p.). (b) The total distance traveled in the open field for 5 min after injections of saline, compound 19h (20 mg/kg, i.p.) and urethane (10%, i.p.). (c) The mean travel speed in open field for 5 min after injection of Saline, compound 19h (20 mg/kg, i.p.) and urethane (10%, i.p.). One-way ANOVA followed by Tukey test, n = 6–8. All data were expressed as the mean ± SEM.

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