Synthesis, crystal structure, and antinociceptive effects of some new riluzole derivatives

Article abstract of DOI:10.1007/s00044-018-2154-4

Nine N-alkylated derivatives of riluzole were synthesized in order to obtain new compounds with potential antinociceptive activity. Riluzole was firstly transformed into (6-trifluoromethoxy-benzothiazol-2-yl)-hydrazine, then it was chlorinated by SOCl₂ to obtain 2-chloro-6-trifluoromethoxy-benzothiazole. This intermediate product was treated with nine alkylamines to give N-alkylated derivatives of riluzole respectively. The structures of compounds were confirmed by means of elemental analysis, IR, ¹H NMR, and ¹³C NMR. The synthetic route was optimized and four novel crystals were obtained by recrystallization. This study investigated the antinociceptive activity of some N-alkylated derivatives of riluzole by hot plate test in mice. The relationship between antinociceptive activity and the doses of 4b, 4c, 4h, 4g, and riluzole had been studied. Compared with the control group (0 mg/kg), the effects of compounds 4b and 4h showed a significant increase (13.78 ± 2.89 s, 12.89 ± 2.94 s, respectively). Compound 4c showed extreme significant increase (18.07 ± 3.08 s) in the time mice spent on the hot plate. The compounds 4b, 4c, and 4h had increased the latency time compared to the blank solvent group. They have potential application in developing new drug candidates with antinociceptive activity.

Full text of DOI:10.1007/s00044-018-2154-4

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2154-4
ORIGINAL RESEARCH
Synthesis, crystal structure, and antinociceptive effects of some new
riluzole derivatives
1
Xiang-Long Wu1 Liu Liu1 You-Jia Li1 Jie Luo1 Dong-Wei Gai1 Ting-Li Lu1 Qi-Bing Mei
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Received: 28 September 2017 / Accepted: 10 February 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
Nine N-alkylated derivatives of riluzole were synthesized in order to obtain new compounds with potential antinociceptive
activity. Riluzole was firstly transformed into (6-trifluoromethoxy-benzothiazol-2-yl)-hydrazine, then it was chlorinated by
SOCl2 to obtain 2-chloro-6-trifluoromethoxy-benzothiazole. This intermediate product was treated with nine alkylamines to
give N-alkylated derivatives of riluzole respectively. The structures of compounds were confirmed by means of elemental
1
analysis, IR, H NMR, and 13C NMR. The synthetic route was optimized and four novel crystals were obtained by
recrystallization. This study investigated the antinociceptive activity of some N-alkylated derivatives of riluzole by hot plate
test in mice. The relationship between antinociceptive activity and the doses of 4b, 4c, 4h, 4g, and riluzole had been studied.
Compared with the control group (0 mg/kg), the effects of compounds 4b and 4h showed a significant increase (13.78
2.89 s, 12.89 2.94 s, respectively). Compound 4c showed extreme significant increase (18.07 3.08 s) in the time mice
spent on the hot plate. The compounds 4b, 4c, and 4h had increased the latency time compared to the blank solvent group.
They have potential application in developing new drug candidates with antinociceptive activity.
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Keywords Riluzole Benzothiazole derivatives Antinociceptive Synthesis Crystal structure
Introduction
Parkinson’s disease, Huntington’s disease and cerebral and
retinal ischemia (Jehle et al. 2000).
Riluzole [2-amino-6-(trifluoromethoxy) benzothiazole], is a
derivative of benzothiazole, a bicyclic ring compound,
which comprises a benzene ring, fused to a five-membered
ring containing one nitrogen and one sulfur atom (Cheah
et al. 2010). Riluzole, a novel anti-glutamate agent with
neuroprotective properties in animal models of neurode-
generative disease, has been approved by the U.S. Food and
Drug Administration for amyotrophic lateral sclerosis
(Cheung et al. 2006). It is effective in animal models of
2-(N-alkylamino) benzothiazoles exhibit a wide range of
pharmacological and physiological activities, and they are
very important structural units in many biologically active
compounds. Benzothiazole derivatives were first identified
as potential muscle relaxants in clinical medicine during the
1950s (Domino et al. 1952). Research into benzothiazole
derivatives subsequently subsided, although interest was
reignited with identification of the anti-convulsant properties
of riluzole during the 1980s (Mizoule et al. 1985). Ben-
zothiazole derivatives have demonstrated anti-tumor, anti-
microbial, anti-helmintic, anti-leishmanial, anti-inflamma-
tory, and psychotropic properties (Rana et al. 2007).
Riluzole can delay the onset of thermal and tactile
hypersensitivity and partially reverse established pain
behavior. It also can reduce microglial staining in the spinal
cord (Chew et al. 2014). Riluzole has undergone clinical
trials for spinal cord injury, and the promising results
obtained here suggest that its use may provide a new
treatment option for avulsion injuries, a very specific type of
spinal cord injury (Wilson and Fehlings 2013). Early
administration of riluzole is sufficient to relieve nerve root-
mediated pain by preventing development of neuronal
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00044-018-2154-4) contains supplementary
material, which is available to authorized users.
* Ting-Li Lu
lutinglixinxin@nwpu.edu.cn
* Qi-Bing Mei
gahpyudx@163.com
1
Key Laboratory for Space Bioscience and Biotechnology, School
of Life Sciences, Northwestern Polytechnical University, 710072
Xi’an, China

Medicinal Chemistry Research
dysfunction in the spinal cord and the nerve root (Nicholson
et al. 2014). Riluzole improved functional recovery and
preserved tissue in a rat model of mild gradual cervical
spinal cord compression. It can inhibit the induction and
maintenance of neuropathic pain. Riluzole represents a
novel therapeutic approach to attenuate neuropathic pain
and protect against neurological loss in cervical spondylotic
myelopathy (Moon et al. 2014).
Riluzole and other benzothiazoles protect against tran-
scriptional impact and adverse molecular networks following
neuronal stress (Liu et al. 2011). Suitability of riluzole for
symptom relief, as well as potential for prevention of neu-
rodegenerative consequences associating visceral hypersen-
sitivity do provide a new class of specific therapeutic agent
for treating irritable bowel syndrome (Mishra et al. 2014). 2-
Equiox-55 FTIR spectrometer. 1H and13C spectra were
recorded on a BRUKER AVANCE instrument. Elemental
analyses for C, H, and N were obtained using a Vario EL-III
analyzer. The X-ray diffraction data were collected on a
Bruker SMART AREX II CCD diffractometer equipped
with a graphite monochromated Mo Kα radiation (λ =
0.071073 nm) source by using the ω-2θ scan technique at
room temperature. The structure was solved by direct
methods with SHELXS-97, and refined used SHELXL-97.
Crystallographic data for the structures reported here have
been deposited with CCDC (Deposition No. CCDC-
1039246 (3), CCDC-1453892 (4e), CCDC-1045917 (4f),
and CCDC-1446747(4i)). These data can be obtained free
of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.
html or from CCDC, 12 Union Road, Cambridge CB2 1EZ,
UK, E-mail: deposit@ccdc.cam.ac.uk.
Amino-6-trifluoromethylthio-benzothiazole (SKA-19),
a
thioanalog of riluzole, as a potent, novel anticonvulsant, had
been reported. It exerts broad spectrum anticonvulsant and
analgesic effects (Coleman et al. 2015). Given the function of
riluzole and benzothiazole derivatives, we designed and
synthesized nine N-alkylated derivatives of riluzole.
Synthesis of (6-trifluoromethoxy-benzothiazol-2-yl)-
hydrazine (2)
6-Trifluoromethoxy-2-amino-benzothiazole (23.42 g, 0.10 mol),
hydrazinium sulfate (NH2NH2• H2SO4, 19.52 g, 0.15 mol)
and hydrazine hydrate (80% aqueous solution, 80 mL) were
added to ethylene glycol (350 mL). The mixture was heated
and stirred under nitrogen at 130 °C for 4 h. TLC (CH2Cl2/
MeOH = 20:1) was used to confirm the completion of the
reaction. After cooling to room temperature, the mixture
was poured into the ice-water. A lot of gray solid was
precipitated. The precipitate was filtered and washed three
times with water. The solid was dried in vacuum to constant
weight. Compound 2 (18.7 g, Yield 75%) was obtained.
Gray schistose; mp 205–206 °C; IR (KBr) υmax 3362, 3205,
The direct N-alkylation of 2-amino-benzothiazole is one
of the easiest methods for the preparation of 2-(N-alkyla-
mino) benzothiazole. However, such reactions with alkyl
halides afford 3-alkyl-2-imino-benzothiazolines other than
2-(N-alkylamino) benzothiazole because the endocyclic
nitrogen is more basic than the exocyclic one (Jimonet et al.
1999). Several transition metal-catalyzed methods have
been developed for the synthesis of 2-(N-alkylamino) ben-
zothiazoles, including oxidative C–H funcationalization of
benzothioureas (Joyce and Batey 2009), cyclization of
ortho- halobenzothioureas (Saha et al. 2009), and oxidative
decarbonylative coupling of benzothiazoles with for-
mamides. However, these methods usually suffer from the
multistep synthesis of the starting materials, use of expen-
sive metal catalysts and low functional group tolerance.
In this paper, we will report a revised approach to N-
alkylated derivatives of riluzole according to the reference
(Mignani et al. 1992). Nine N-alkylated derivatives of
riluzole were obtained. To our knowledge, 4b, 4d, 4e, 4f,
4g, 4h, and 4i were new compounds. To investigate the
antinociceptive activity of N-alkylated derivatives of rilu-
zole, the hot plate test was performed, which was a selective
test that uses heat as a nociceptive stimulus to identify
analgesic substances with a central effect.
3124, 2963, 2880, 2361, 1660, 1564, 1464, 1261, 1123 cm
−1
;
1H NMR (DMSO-d6, 500 MHz): δ = 9.22 (1H, s,
–NH–), 7.79 (1H, s, Ar–H), 7.35 (1H, d, J = 8.7 Hz, Ar–H),
7.17 (1H, d, J = 8.7 Hz, Ar–H), 5.12 (2H, s, –NH2); ESI-
MS m/z 248.39[M–H]-; C8H6F3N3OS (calcd. 249.21) Anal.
Calcd. for C8H6F3N3OS: C, 38.56; H, 2.43; N, 16.86.
Found: C, 38.44; H, 2.34; N, 16.75.
Synthesis of 2-chloro-6-trifluoromethoxy
benzothiazole (3)
Compound 2 (12.45 g, 0.05 mol) was slowly added in
portions to a solution of SOCl2 (120 mL) which was pre-
heated at 65 °C. The solution was stirred for 5 h at 60 °C.
Thionyl chloride was evaporated, then the residue was
dissolved in CH2Cl2 and solvent was evaporated again. This
process was repeated at least three times in order to remove
all SOCl2. The residue was dissolved in CH2Cl2 and washed
three times with water, dried over anhydrous Na2SO4. The
solvent was removed under reduced pressure. The crude
product was purified by column chromatography on silica
Materials and methods
Experimental general information
Melting points were measured on a X-4 micro melting point
apparatus. IR spectra in cm−1 were recorded on a Brucker

Medicinal Chemistry Research
gel eluted with petroleum ether (60–90 °C) to give compound
3 (10.71 g, Yield 85%) as a white solid. mp 38–39 °C;
IR (KBr) υmax 3099, 3078, 2361, 1480, 1452, 1262, 1164,
S–C–N), 150.98 (C, Ar–O), 143.54 (C, Ar–N), 130.84 (C,
Ar–S), 121.65(C, q, J = 237.5 Hz, –CF3), 119.68 (C, Ar-4),
118.87 (C, Ar-5), 114.06 (C, Ar-7), 47.44 (C, NH–C–C),
22.79 (C, –C–C),11.34(C, –CH3); Anal. Calcd for
C11H11F3N2OS: C, 47.82; H, 4.01; N, 10.14. Found: C,
47.69; H, 4.20; N, 10.03.
1016, 865 cm−1 1H NMR (CDCl3, 500 MHz): δ = 7.96
;
(1H, d, J = 8.9 Hz, Ar–H), 7.66 (1H, s, Ar–H), 7.37 (1H, d,
J = 8.9 Hz, Ar–H); ESI-MS: m/z 251.91 [M–H]-;
C8H3ClF3NOS
(calcd.252.96);
Anal.
Calcd
for
C8H3ClF3NOS: C, 37.88; H, 1.19; N, 5.52. Found: C,
37.95; H, 1.13; N, 5.59.
2-(N-n-butylamine)-6-trifluoromethoxy-benzothiazole (4c)
White solid; mp 76–78 °C; IR (KBr) υmax 3211, 2923, 2859,
1
General procedure for the preparation of N-
2361, 1626, 1566, 1462, 1264, 1166, 815 cm−1; H NMR
alkylated derivatives of riluzole (4a–4i)
(CDCl3, 500 MHz): δ = 7.47 (1H, d, J = 8.8 Hz, Ar–H),
7.45 (1 H, s, Ar–H), 7.15 (1H, d, J = 8.7 Hz, Ar–H), 5.74
(1H, s, –NH–), 3.41 (2H, t, J = 7.1 Hz, NHCH2), 1.71–1.65
(2H, m, NHCH2CH2), 1.48–1.41 (2H, m, –CH2CH3), 0.96
(3H, t, J = 7.4 Hz, CH3); Anal. Calcd for C12H13F3N2OS: C,
49.65; H, 4.51; N, 9.65. Found: C, 49.54; H, 4.46; N, 9.69.
2-Chloro-6-trifluoromethoxy-benzothiazole (1.27 g, 5 mmol)
was dissolved in 10 mL ethylamine aqueous solution. The
resultant mixture was stirred at room temperature overnight.
The solvent was removed under reduced pressure. The
residue was dissolved in CH2Cl2 and washed three times
with water, dried over anhydrous Na2SO4, filtered and the
solvent completely removed. The crude material was pur-
ified by column chromatography over silica used dichlor-
omethane as eluent to give compound 4a as a white
amorphous powder (1.11 g, 84.7%). Compounds 4b–4i
were also prepared by the same procedure from compound
3 with n-butylamine, diaethylamin, cyclohexylamine, pyr-
rolidine, piperidine, morpholine, and 4-methylpiperidine,
respectively. Spectroscopic data of N-alkylated derivatives
of riluzole were given as follow.
2-(N-diaethylamine)-6-trifluoromethoxy-benzothiazole (4d)
White solid; mp 38–39 °C; IR (KBr) υmax 3090, 2981, 2938,
2857, 2361, 1607, 1550, 1460, 1359, 1294, 1215, 1151 cm
−1
;
1H NMR (Acetone-d6, 500 MHz): δ = 7.72 (1H, s,
Ar–H), 7.46 (1H, d, J = 8.8 Hz, Ar–H), 7.21 (1H, d, J =
8.8 Hz, Ar–H), 3.62 (4H, q, J = 7.1 Hz, 2 × –CH2–), 1.27
(6H, t, J = 7.1 Hz, 2 × –CH3); 13C NMR (CDCl3,
100 MHz): δ = 167.81 (C, S–C–N), 152.15 (C, Ar–O),
142.94 (C, Ar–N), 131.27 (C, Ar–S), 121.94(C, q, J =
246 Hz, –CF3), 119.50 (C, Ar-4), 118.64 (C, Ar-5), 113.76
(C, Ar-7), 45.52 (C, N–C–C), 12.80 (C, –CH3); Anal. Calcd
for C12H13F3N2OS: C, 49.65; H, 4.51; N, 9.65. Found: C,
49.54; H, 4.62; N, 9.77.
2-(N-ethylamine)-6-trifluoromethoxy-benzothiazole (4a)
White solid; mp 133–134 °C; IR (KBr) υmax 3210, 2987,
2922, 2361, 1622, 1584, 1461, 1250, 1214, 1152, 807 cm
−1
;
1H NMR (CDCl3, 500 MHz): δ = 7.48 (1H, d, J =
2-(N-cyclohexylamine)-6-trifluoromethoxy-benzothiazole (4e)
White solid; mp 96–97 °C; IR (KBr) υmax 3427, 3231, 2933,
8.8 Hz, Ar–H), 7.46 (1H, s, Ar–H), 7.16 (1H, d, J = 8.8 Hz,
Ar–H), 5.69 (1H, s, –NH–), 3.47 (2H, q, J = 7.2 Hz,
–CH2–), 1.34 (3H, t, J = 7.2 Hz, –CH3); 13C NMR(CDCl3,
125 MHz): δ = 167.95 (C, S–C–N), 150.94 (C, Ar–O),
143.57 (C, Ar–N), 130.86 (C, Ar–S), 121.65(C, q, J =
237.5 Hz, –CF3), 119.68 (C, Ar-4), 118.92 (C, Ar-5),
114.07 (C, Ar-7), 40.43 (C, N–C–C), 14.83(C, –CH3);
Anal. Calcd for C10H9F3N2OS: C, 45.80; H, 3.46; N, 10.68.
Found: C, 45.93; H, 3.40; N, 10.76.
1
2858, 1616, 1548, 1456, 1249, 1220, 1162 cm−1; H NMR
(CDCl3, 500 MHz): δ = 7.46 (1H, d, J = 8.8 Hz, Ar–H),
7.44 (1H, s, Ar–H), 7.15 (1H, d, J = 8.7 Hz, Ar–H), 5.53 (s,
1H, –NH–), 3.56 (1H, t, J = 9.6 Hz, –CH–), 2.14–2.10(2H,
m, –CH2–), 1.80–1.76 (2H, m, –CH2–), 1.52–1.14 (6H, m,
3 × –CH2–); 13C NMR(CDCl3, 100 MHz): δ = 167.12 (C,
S–C–N), 151.26 (C, Ar–O), 143.45 (C, Ar–N), 130.98 (C,
Ar–S), 121.94(C, q, J = 230 Hz, –CF3), 119.62 (C, Ar-4),
118.85 (C, Ar-5), 113.98 (C, Ar-7), 54.64 (C, N–C–C),
33.20 (C, –CH2–), 25.42 (C, –CH2–), 24.68 (C, –CH2–);
Anal. Calcd for C14H15F3N2OS: C, 53.15; H, 4.78; N, 8.86.
Found: C, 53.33; H, 4.68; N, 8.67.
2-(N-propylamine)-6-trifluoromethoxy-benzothiazole (4b)
White solid; mp 88–89 °C; IR (KBr) υmax 3150, 2978, 2861,
2361, 1614, 1556, 1463, 1281, 1214, 1162, 808 cm−1; H
1
NMR (CDCl3, 500 MHz): δ = 7.47 (1H, d, J = 8.8 Hz,
Ar–H), 7.45 (1H, s, Ar–H), 7.16 (1H, d, J = 8.8 Hz, Ar–H),
5.75 (1H, s, –NH–), 3.39 (2H, t, J = 7.1 Hz, NH–CH2),
1.75–1.69 (2H, m, –CH2CH2CH3), 1.02 (3H, t, J = 7.4 Hz,
–CH3); 13C NMR (CDCl3, 125 MHz): δ = 168.23 (C,
2-(N-pyrrolidine)-6-trifluoromethoxy-benzothiazole (4f)
White solid; mp 129–130 °C; IR (KBr) υmax 3418, 2969,
2870, 2361, 1614, 1554, 1452, 1365, 1243, 1214, 1184 cm−1;

Medicinal Chemistry Research
1H NMR (CDCl3, 500 MHz): δ = 7.53 (1H, d, J =
8.8 Hz, Ar–H), 7.46 (1H, s, Ar–H), 7.15 (1H, d, J = 8.8 Hz,
Ar–H), 3.58 (4H, t, J = 6.3 Hz, CH2–N–CH2), 2.11–2.06
(4H, m, –CH2CH2–); 13C NMR (CDCl3, 100 MHz): δ =
165.79 (C, S–C–N), 152.15 (C, Ar–O), 142.91 C, Ar–N),
131.35 (C, Ar–S), 121.94(C, q, J = 235 Hz, –CF3), 119.49
(C, Ar-4), 118.74 (C, Ar-5), 113.89 (C, Ar-7), 49.52 (C, N–
C–C), 25.61 (C, –CH2–); Anal. Calcd for C12H11F3N2OS:
C, 49.99; H, 3.85; N, 9.72. Found: C, 49.87; H, 3.75; N,
9.61.
131.36 (C, Ar–S), 121.93 (C, q, J = 231 Hz, –CF3), 119.60
(C, Ar-4), 118.94 (C, Ar-5), 113.83 (C, Ar-7), 49.07 (C, N–
C–C), 33.43 (C, –CH2–), 30.79 (C, –CH–), 21.71 (C,
–CH3); Anal. Calcd for C14H15F3N2OS: C, 53.15; H, 4.78;
N, 8.86. Found: C, 53.76; H, 4.60; N, 8.70.
Animals
Six-week-old male Kunming mice (18–22 g, specific
pathogen-free grade, SPF) were obtained from the Academy
of Experimental Animal Center of The Fourth Military
Medical University (Xi’an, China). Mice were maintained
under normal conditions: 21–26 °C temperature, 60–70%
relative humidity, 12:12 h dark/light cycle, and free access
to laboratory standard diet and water. All mice were quar-
antined and adapted for 7 days after arriving. All animal
experiments were conducted under institutional guidelines
and approved by the Ethical Committee for Animal Care
and Use of the Northwestern Polytechnical University.
2-(N-piperidine)-6-trifluoromethoxy-benzothiazole (4g)
White solid; mp 74–75 °C; IR (KBr) υmax 3423, 2940, 2859,
2361, 1608, 1549, 1461, 1246, 1158 cm−1 1H NMR
;
(CDCl3, 500 MHz): δ = 7.49 (1H, d, J = 8.8 Hz, Ar–H),
7.45 (1H, s, Ar–H), 7.14 (1H, d, J = 8.8 Hz, Ar–H),
3.61–3.60 (4H, m, –CH2–N–CH2–), 1.72–1.71 (6H, m,
–CH2CH2CH2–); 13C NMR (CDCl3, 125 MHz): δ = 169.27
(C, S–C–N), 151.78 (C, Ar–O), 143.17 (C, Ar–N), 131.32
(C, Ar–S), 121.69(C, q, J = 266.5 Hz, –CF3), 119.56 (C,
Ar-4), 118.92 (C, Ar-5), 113.80 (C, Ar-7), 49.68 (C, N–
C–C), 25.27 (C, –CH2–), 24.17 (C, –CH2–); Anal. Calcd for
C13H13F3N2OS: C, 51.65; H, 4.33; N, 9.27. Found: C,
51.46; H, 4.42; N, 9.16.
Assessment of antinociceptive activity
Antinociceptive activity of the test compounds against
noxious thermal stimulation was measured by hot-plate test
in mice as described in references (Kaplancikli et al. 2012;
Turan-Zitouni et al. 2015; Queiroz et al. 2015). Mice were
placed into the hot plate apparatus in a quiet environment
and a hot stimulus (55 0.5 °C) used to determine their
initial pain threshold. The time (in seconds) that elapsed
between placing the mouse on the hot plate and the mani-
festation of signs of acute discomfort, such as licking of
the hind paw or jumping in an attempt to escape from the
heat, was taken as the reaction time or latency. The mice
pain threshold greater than 60 s or less than 5 s were
eliminated.
2-(N-morpholine)-6-trifluoromethoxy-benzothiazole (4h)
White solid; mp 103–105 °C; IR (KBr) υmax 3447, 2974,
2907, 2866, 1608, 1549, 1459, 1380, 1339, 1267, 1112,
1
1026 cm−1; H NMR (CDCl3, 500 MHz): δ = 7.52 (1H, d,
J = 8.8 Hz, Ar–H), 7.49 (1H, s, Ar–H), 7.17 (1H, d, J =
8.8 Hz, Ar–H), 3.84–3.82 (4H, m, –CH2–O–CH2–),
3.63–3.61 (4H, m, –CH2–N–CH2–); 13C NMR (CDCl3,
125 MHz): δ = 169.45 (C, S–C–N), 151.22 (C, Ar–O),
143.64 (C, Ar–N), 131.19 (C, Ar–S), 121.65(C, q, J =
223.8 Hz, –CF3), 119.86 (C, Ar-4), 119.57 (C, Ar-5),
113.98 (C, Ar-7), 66.18 (C, N–C–C), 48.51 (C, –CH2–);
Anal. Calcd for C12H11F3N2O2S: C, 47.37; H, 3.64; N, 9.21.
Found: C, 47.49; H, 3.54; N, 9.30.
Statistical analysis
Data were analyzed using SPSS 13.0 version. The results
were expressed as the mean S.E.M. The data were sub-
jected to one-way analysis of variance (ANOVA) followed
by Tukey–Kramer post-hoc analysis. The level of p < 0.05
was used as the criterion of statistical significance. P < 0.01
was considered extreme significant.
2-(N-4-methyl-1-piperidinyl)-6-trifluoromethoxy-
benzothiazole (4i)
White solid; mp 77–78 °C; IR (KBr) υmax 3419, 2940, 2805,
1
2727, 1614, 1470, 1358, 1271, 1184, 772 cm−1; H NMR
(CDCl3, 400 MHz): δ = 7.47 (1H, d, J = 8.7 Hz, Ar–H),
7.44 (1H, s, Ar–H), 7.13 (1H, d, J = 8.2 Hz, Ar–H), 4.09
(2H, d, J = 12.7 Hz, N–CH2–), 3.12 (2H, t, J = 12.7 Hz,
–CH2–N), 1.77 (2H, d, J = 12.7 Hz, –CH2–), 1.68–1.63
(1H, m, –CH–), 1.36–1.26 (2H, m, –CH2–), 1.00 (3H, d,
J = 6.5 Hz, –CH3); 13C NMR (CDCl3, 100 MHz): δ =
169.20 (C, S–C–N), 151.80 (C, Ar–O), 143.18 (C, Ar–N),
Results and discussion
Synthesis and structural confirmation
These N-alkylated derivatives of riluzole were synthesized
according to the synthetic procedure shown in Scheme 1.
Although the synthesis route of compound 3 had been

Medicinal Chemistry Research
F3CO
F3CO
S
F3CO
SOCl2
S
S
F3CO
NH2NH2 H2O
S
NHNH2
NH2
Cl
R
N
H
R
N
NH2NH2 H2SO4
N
N
3
4a-4i
2
1
4a: R=
4c: R=
NHCH2CH2CH2CH3 4d: R=
NHCH2CH3
4b: R=
NHCH2CH2CH3
N
4f: R=
4i: R=
N
4h: R=
N
O
N
4g: R=
N
4e: R=
N
H
Scheme 1 Synthesis of new N-alkylated derivatives of riluzole
Table 1 Preparation of novel N-alkylated derivatives of riluzole
reported, we optimized the synthetic condition and pro-
cessing method. For example, in the process of preparing
compound 2, the precipitate was vacuum filtered and tri-
turated in a mixture of water-diethyl ether according to the
references (Saha et al. 2009; Mignani et al. 1992). We
found that the yield was very low by this method because
precipitate was not complete. Therefore, our method was
that the reaction mixture was poured into ice water. In the
process of preparing compound 3, the residue SOCl2 was
destroyed by water. However, it was not a good method.
The SOCl2 should be evaporated, then the residue was
dissolved in CH2Cl2. The CH2Cl2 which contains residual
SOCl2 was evaporated again. It can be used repeatedly.
In the second step reaction, tail gas absorption equipment
was needed. As we can see from Scheme 1, compound 2-
chloro-6-trifluoromethoxy benzothiazole (3) was an
important intermediate. The chlorine atom in compound 3
had high reactivity, so it was very easy to be substituted by
the alkylamine. The reagent, reaction time, temperature,
yield and melting point were summarized in Table 1 for
these N-alkylated derivatives of riluzole.
As can be seen from Table 1, all of the compounds were
prepared with high yield (above 75%). The best yield of
these compounds could reach to 85.6%. The reaction tem-
perature was 45 °C or room temperature. The reaction
conditions were very mild and efficient. In the third step
reaction, the alkylamine was not only a substrate but also a
solvent. After the completion of the reaction, the alkylamine
could be recycled under reduced pressure. Therefore, the
solvent was saved owing to its re-use and the environmental
pollution was reduced.
R–H
Temp. Time 4(Yield/%) m.p.(°C)
1
2
3
4
5
6
7
8
9
Ethylamine
45 °C
45 °C
45 °C
45 °C
45 °C
rt
12 h
4 h
4a (75.4)
4b (79.7)
4c (84.8)
4d (75.9)
4e (80.3)
4f (85.6)
4g (81.4)
4h (83.3)
4i (85.1)
133–134
88–89
Propylamine
n-Butylamine
Diaethylamin
Cyclohexylamine
Pyrrolidine
4 h
76–78
12 h
12 h
3 h
38–39
96–97
129–130
74–75
Piperidine
rt
3 h
Morpholine
rt
3 h
103–105
77–78
4-Methylpiperidine rt
3 h
were listed in Table 2, Bond lengths and angles were
listed in Table s1–s4 (See Supplementary data). Crystals of
2-chloro-6-trifluoromethoxy benzothiazole (3) were obtained
by recrystallization from petroleum ether (bp. 60–90 °C). The
crystals of compound 3 were monoclinic, space group P2
(1)/n with four molecules per unit cell. In the crystal
structure of compound 3, the N(1)–C(1) in 0.1268(3) nm
was a double bond. However, the N(1)–C(2) in 0.1377(3)
nm was a single bond. The bond length of C(1)–Cl(1) was
0.1691(3) nm. The bond length of S(1)–C(1) (0.1722 nm)
was longer than S(1)–C(1) (0.1714 nm). It indicated that the
S(1) atom lies within the thiazole ring plane was close to
benzene ring. The bond lengths of C–F in the –CF3 were
different and the values of them were 0.1287, 0.1296, and
0.1303 nm, respectively. The thiazole ring and benzene ring
were coplanar.
Crystals
of
compound
2-(N-pyrrolidine)-6-tri-
Crystal structures
fluoromethoxy-benzothiazole (4f) were obtained by
recrystallization from ethanol. The compound 4f crystallizes
in the triclinic P-1 space group with two molecules per unit
cell. The N(1)–C(8) in 0.1299(4) nm was a double bond.
The N(1)–C(5) in 0.1378(4) nm was a single bond. The
bond length of N(2)–C(8) (0.1328 nm) was shorter than N
(2)–C(9) and N(2)–C(12) (0.1457 nm, 0.1453 nm,
Single crystals of compounds 3, 4e, 4f, and 4i were
obtained by recrystallization and their structures were fur-
ther confirmed by X-ray diffraction determination. The
perspectives were shown in Fig. 1. The crystal data,
details concerning data collection and structure refinement

Medicinal Chemistry Research
The packing drawing of compounds 3, 4e, 4f, and 4i
were given in the supplementary data (Figs. s1–s4). In the
crystal packing, as shown in Fig. s3 in supplementary data,
weak intermolecular hydrogen bonds are observed (showed
as dashed lines). In the crystal packing, as shown in Figs. s3
and s4, adjacent molecules were crossed stacked through
strong offset π···π aromatic stacking interactions. The
crystal structures have potential applications in identifying
the binding site for allosteric modulators of the receptor,
and should be useful in the area of medical or pharmaceu-
tical applications.
Compound 3
Hot plate test
This study investigated the antinociceptive activity of some
N-alkylated derivatives of riluzole by hot plate test in mice.
Figure 2 showed the antinociceptive effects of 4b, 4c, 4g,
and 4h at different doses on the time mice spent on the hot
plate. Compound 4b, at the doses tested (6 mg/kg: 17.06
4.72 s; 12 mg/kg: 17.60 4.01 s), showed extreme sig-
nificant increase in the time mice spent on the hot plate
when compared with the control group (7.96 2.13 s) (Fig.
2 compound 4b). At the doses tested (18 mg/kg: 13.78
2.89 s; 36 mg/kg: 13.61 2.43 s), compound 4b showed
significant increase in the reaction time. Compound 4c, at
all the doses tested, showed extreme significant increase in
the reaction time when compared with the control group
(Fig. 2 compound 4c).
Compound 4e
Compound 4f
Compound 4h, at the doses tested (6 mg/kg: 12.90
2.58 s; 12 mg/kg: 14.89 2.32 s), showed extreme sig-
nificant increase in the reaction time compared with the
control group 9.06 2.02 s). Compound 4h, at the dose
18 mg/kg i.g had a significant increase in the reaction time
(12.89 2.94 s) compared with the control group at dose
0 mg/kg (9.06 2.02 s). Compound 4g, only at the dose
12 mg/kg i.g had a significant increase in the reaction time
(13.37 4.24 s) compared with the control group at dose
0 mg/kg (9.07 2.65 s).
Compound 4i
Fig. 1 ORTEP drawings of compounds 3, 4e, 4f, and 4i
Animals treated with riluzole (compound 1) showed
increased latency time on the hot plate. From the Fig. 3,
riluzole had a significant effect on antinociceptive activity.
At a dose of 18 mg/kg, it had showed extreme significant
increase in latency time (15.40 2.83 s). When the intra-
gastric administration dose arrived at 24 mg/kg, the average
of latency time (14.13 2.99 s) did not increase with the
addition of the doses.
According to our previous study, we chose the same dose
(18 mg/kg) to compare antinociceptive activity (See
Fig. 4). The blank solvent group (BL) and compound 1
(riluzole) were control group and positive control group,
respectively. Compared to the control group (0 mg/kg, BL),
the effect of compounds 4b and 4h showed a significant
increase (13.78 2.89 s, 12.89 2.94 s, respectively).
respectively), which were belonged to the pyrrolidine ring.
The length of C(3)–C(2) and C(5)–C(4) in benzene ring
were 0.1732 and 0.1383 nm, respectively.
Crystals of compounds 4e and 4i were obtained by
recrystallization from ethanol. The compound 4e crystallize
in the monoclinic P21/n space group with four molecules
per unit cell and the compound 4i in the triclinic P-1
space groups. They had same properties in bond length. In
addition, the cyclohexane ring of compound 4e and the
piperidine ring of compound 4i were typical chair con-
formation. The piperidine ring was in the same plane with
benzothiazole ring. However, there was a dihedral angle
between the mean plane of benzothiazole and cyclohexane
ring.

Medicinal Chemistry Research
Table 2 Crystal data and structure refinement for 3, 4e, 4f, and 4i
Compound 3
Compound 4e
Compound 4f
Compound 4i
CCDC deposit no.
Empirical formula
Formula weight
Temperature (K)
Wavelength (nm)
Crystal system
Space group
a (nm)
1039246
C8H3ClF3NOS
252.96
1453892
C14H15F3N2OS
316.34
1045917
C12H11F3N2OS
288.29
1446747
C14H15F3N2OS
316.34
296(2)
296(2)
296(2)
296(2)
0.071073
Monoclinic
P2(1)/n
1.1797(10)
1.1152(10)
0.7379(7)
90
0.071073
Monoclinic
P2(1)/n
1.3401(5)
1.0138(4)
1.3401(5)
90
0.071073
Triclinic
P-1
0.071073
Triclinic
P-1
0.71573(16)
0.80484(14)
1.1949(3)
91.291(4)
99.683(4)
111.850(3)
0.627
0.6893(2)
0.8216(2)
1.4252(4)
88.664(5)
79.693(4)
72.664(5)
0.7577(4)
2
b (nm)
c (nm)
α (°)
β (°)
99.856
97.19
γ (°)
Volume (nm3)
90
90
0.9565
0.18062(4)
4
Z
4
2
Dcalc (g/cm3)
Abs.coefficient (mm−1
F(000)
1.761
1.325
1.516
1.382
)
0.632
0.218
0.285
0.245
504
752
294
326
Crystal size (mm3)
0.37 × 0.28 × 0.20
0.37 × 0.31 × 0.24
0.37 × 0.29 ×
0.130
0.35 × 0.30 × 0.24
θ limit (°)
1.75 to 25.10
2.03 to 25.10
1.74 to 25.10
1.45 to 25.10
Ranges/indices h. k. l.
−9/14, −13/13,
−8/7
−15/15, −12/11,
−15/13
−5/8, −9/9, −14/
13
−8/7, −9/9, −16/
11
Reflections collected/
unique/Rint
4644 / 1706
/0.0268
6197 / 2559/ 0.0472
3107 / 2206/
0.0170
3809 / 2680/
0.0206
Completeness to θ =
25.10°
100.0 %
80.1 %
98.7 %
99.1 %
Data/restraints/
parameters
1706 / 0 / 136
2680 / 0 / 191
2206 / 0 / 172
2680 / 0 / 191
Goodness of fit on F2
1.081
1.018
1.108
1.026
R1, wR2
0.0446, 0.0953
311 and −271
0.0694, 0.1847
314 and −179
0.0617, 0.1591
307 and −306
0.0735, 0.2161
517 and −336
Largest diff. peak and
hole (e•nm−3
)
Compounds 4c, 4f, and 4i showed extreme significant
increase (18.07 3.08 s, 17.75 3.23 s, and 15.36 2.01 s,
respectively) in the reaction time when compared to the
control group. However, at the dose of 18 mg/kg, no sig-
nificant differences were observed for the riluzole and
compounds 4c, 4f, 4i.
To compare the antinociceptive activity with riluzole, the
difference values were calculated by subtracting the riluzole
latency from the latency of 4a–4i (See Fig. 5). The positive
value indicated the analgesic effect was better than riluzole,
but the negative value indicated it was worse than riluzole.
As we can see, compounds 4c and 4f showed extreme
significant analgesic effect compared to the riluzole. The 2-
substituent groups of them were n-butylamine and
pyrrolidine, respectively. They all have four carbon atoms
in substituent group. The analgesic effect of compound 4i
was the same as riluzole. However, the antinociceptive
activities of other compounds were worse than riluzole.
Several lines of evidence had shown that nociception in
the hot plate test results from the accumulation of intra-
cellular Ca2+ concentration that, in turn, initiates a number
of second / third messenger- mediated intracellular cascades
(Sommer and Seeburg 1992). Riluzole can stabilize Na+
channels and inhibit glutamate release. Riluzole can also
inhibit Ca2+-dependent release of glutamate from neurons,
as well as interact with other cellular targets (Gegelashvili
and Bjerrum 2014). Therefore, our hot plate test results
suggested that the possible mechanisms of these title

Medicinal Chemistry Research
Fig. 2 The relationship between latency and intragastric administration
concentration of compound 4b, 4c, 4g, and 4h. ** p < 0.01 compared
with the 0 mg/kg group, *p < 0.05 compared with the 0 mg/kg group.
Each bar is the mean S.E.M. of individual determinations in 8 mice
of each group
compounds may be linked to Na+ channels, Ca2+ con-
centration or glutamate release.
Conclusion
In conclusion, we have reported a practical synthetic route
towards N-alkylated derivatives of riluzole. New single
crystals of compounds 3, 4e, 4f, and 4i were obtained by
recrystallization and their structures data were very useful in
the area of medical or pharmaceutical applications. To our
knowledge, with exception of compounds 2, 3, 4a, and 4c,
others have not been reported by references. The results
showed that 4c, 4f, and 4i had increased the latency time
compared to the blank solvent group. They have potential
application in developing new drug candidates with anti-
nociceptive activity.
Fig. 3 The relationship between latency and intragastric administration
concentration of compound 1 (riluzole). **p < 0.01 compared with the
0 mg/kg group, * p < 0.05 compared with the 0 mg/kg group. Each bar
is the mean S.E.M. of individual determinations in 8 mice of each
group

Medicinal Chemistry Research
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Acknowledgements The work was supported by the National Natural
Science Foundation of China (No. 21202130), the Fundamental
Research Funds for the Central Universities (No. 3102014JCQ15005),
the Natural Science Foundation Research Project of Shaanxi Province
(No. 2015JM2067), the Seed Foundation of Innovation and Creation
for Graduate Students in Northwestern Polytechnical University (No.
Z2017239), the national innovation experiment program for university
students (No.201610699258)
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.

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