Chem Biol Drug Des 2016; 88: 38–42 Research Article

Design, Synthesis, and Biological Evaluation of Novel CNS 7056 Derivatives as Sedatives in Rats and Rabbits Yan Liu1, Xiangqing Xu2, Jianyong Xie1, Huan Ma1, Tao Wang2, Guisen Zhang3,* and Qingeng Li1,* 1

Division of Medicinal Chemistry, School of Pharmacy, Chongqing Medical University, Chongqing 400010, China 2 R&D Department, Jiangsu Nhwa Pharmaceutical Corporation Ltd., Jiangsu 221009, China 3 School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China *Corresponding author: Guisen Zhang, [email protected]; Qingeng Li, [email protected] A new water-soluble benzodiazepine derivative, CNS 7056 (named as remimazolam), has been undergoing many reactions in recent years to provide an intravenous agent with a predictable fast-onset, short duration of action, and rapid recovery profile. Based on the structure of CNS 7056 with proven activity, seven new CNS 7056 derivatives were designed, and their sedative activities upon mouse, rats, and rabbits were examined. Sedative activities of EL-001~007 were screened. The results indicated that the shorter the side chain at C3 position is, the higher the sedative activity is. EL-001 was chosen as the optimal compound for studies of ED50, LD50, latency to LRR and the duration of LRR, and its anesthetic activity was compared with that of CNS 7056 in rats and rabbits. Studies showed that EL-001 is a potent sedative in rodent and lagomorpha, with a short duration of action. Compared with CNS 7056, EL-001 has a shorter period of induction despite a slightly longer sedative duration and recovery time. Key words: CNS 7056 derivatives, rats and rabbits, sedation

inhibitory neurotransmitter, GABA (5), which has sedative, hypnotic, anxiolytic, anticonvulsant and muscle relaxant properties. Midazolam, the first water-soluble short-acting central nervous system (CNS) depressant of the benzodiazepine class, is used widely for sedation and amnesia before medical procedures (6). However, prolonged recovery from sedation induced by midazolam is due to the production of active metabolites (7) and a reliance on the liver enzyme cytochrome P450 3A4 for metabolism (8). A new water-soluble benzodiazepine derivative, CNS 7056 (named as remimazolam), has been undergoing many reaction in recent years to provide an intravenous agent with a predictable fast-onset, short duration of action, and rapid recovery profile (9). The sedation properties of CNS 7056 were compared with those of midazolam and propofol in sheep. CNS 7056 had advantages over midazolam, such as more rapid offset and greater depth of sedation, and is better over propofol, due to more rapid onset and less dose dependency of depth of sedation (10). CNS 7056 is a methyl ester, and its hydrolysis product CNS 7054 has low activity at the GABAA receptor (5). Ester hydrolysis of CNS 7056 to CNS 7054 is an important mechanism (Scheme 1), which limits the duration and intensity of sedative effect in vivo (11). Some studies concluded that in sheep, CNS 7056 showed high metabolic clearance, small distribution volumes, and rapid onset and offset of sedative effect. Its predictable effects are over a range of doses (7). Initial clinical data suggest that rapid recovery after sedation with CNS 7056 has been seen in men as well (12). Based on the structure of CNS 7056 with proven activity, seven new CNS 7056 derivatives were designed, and their bioactivities upon mouse, rats, and rabbits were investigated.

Received 20 September 2015, revised 5 January 2016 and accepted for publication 21 January 2016

Methods and Materials Midazolam is a short-acting central nervous system (CNS) depressant of the benzodiazepine class developed by Hoffmann-La Roche in the 1970s (1). The drug is used for the treatment of acute seizures. It causes moderate to severe insomnia, and it will induce sedation and amnesia before medical procedures (2). Benzodiazepines are effective on the c-aminobutyric acid type A (GABAA) receptor (3,4) and enhance the action of the 38

General Benzenesulfonate of CNS 7056, which used as positive control, was purchased from Jiangsu Nhwa pharmaceutical Co., Ltd (Xuzhou, Jiangsu, China). Sodium chloride injection (LN. 1208068201) was purchased from Cisen Pharmaceutical Co., Ltd (Chongqing, China). All other reagents were purchased from Shanghai Chemical Reagent Company (Shanghai, China). Column chromatography (CC): silica gel 60 (100–200 mesh). Thin-layer ª 2016 John Wiley & Sons A/S. doi: 10.1111/cbdd.12731

Novel CNS 7056 Derivatives as Sedatives

Scheme 1: Ester hydrolysis of CNS 7056 to CNS 7054 in vivo.

chromatography (TLC): silica gel 60 F254 plates (250 mm; Qingdao Ocean Chemical Company, Qingdao, China). M.p.: capillary tube; uncorrected. IR spectra: Shimadzu FTIR-8400S spectrophotometer; in cm 1. 1H NMR spectra: Bruker ACF-400Q apparatus at 400 MHz, in DMSOd6 unless otherwise indicated; d in ppm relative to Me4Si, J in Hz. 13C NMR spectra: Bruker ACF-400Q apparatus at 100 MHz, in DMSO-d6 CDCl3 unless otherwise indicated; d in ppm relative to Me4Si, J in Hz. Mass spectrometry (MS):Waters UPLC/MS/MS: ACQUITY UPLC/TQD, in m/z; elemental analyses: CHN-O-Rapid instrument; %purity of the target compounds (>97%) were determined by HPLC analysis (UV detector, wavelength: 254 nm).

Animals and treatments Sedative activities screened in vivo upon mice Healthy male BALB/c mice weighting 16 ~ 20 g and healthy New Zealand White rabbit weighting 1.5 ~2.5 kg were purchased from laboratory animal center of Nanjing Qinglongshan and Pizhou, respectively. Mice were kept at 24  1 °C and 55  10% humidity, with 12 h of light (artificial illumination; 08:00–20:00) for 1 week. The test compound was administered intravenously to animals through tail veins, while those animals were placed in plastic restrainers. When onset of loss of righting reflex was recorded, the animals were immediately removed from the restrainers. The loss of ability to right itself from supine positions was defined as LRR. Once an animal was able to right itself for the first time, it was placed on its back once again. If they recovered itself consecutively two more times back to normal, the animals actions were labeled as recovered from LRR. Each compound, including positive controls, was prepared in a 0.9% wt/vol saline solution. A dose of 9.0 mg/kg b.w. was administered by rapid bolus via the lateral tail vein to six mice.

Measurement of sedative ED50 and LD50 in rats and rabbits Healthy male Sprague Dawley rats weighting 180 ~ 320 g and healthy New Zealand White rabbits weighting 1.5 ~2.5 kg were purchased from laboratory animals center of Chem Biol Drug Des 2016; 88: 38–42

Chongqing Medical University. Animals were kept at 24  1 °C and 55  10% humidity, with 12 h of light (artificial illumination; 08:00–20:00) for 1 week. Sedative ED50 and LD50 were tested upon each animal by sequential procedure. Rats or rabbits, placed in plastic restrainers, were injected with EL-001 or CNS 7056 (saline solution) by intravenous route (lateral tail vein in 10 seconds for rats and 30 seconds for rabbits). The values of ED50 and LD50 were determined by the dose of loss of righting reflex or death.

Measurement of latency to LRR and the duration of LRR in rats and rabbits Animals used were the same as mentioned above. Each animal, placed in plastic restrainer, was injected with EL-001 or positive control (saline solution) by the intravenous route (lateral tail vein for rats and lateral ear vein for rabbits; doses adjusted to two times of ED50). The latency to LRR and the duration of LRR were recorded. The righting reflex test was performed as soon as the animals appeared sedated, at approximately 20- to 30-seconds intervals. Once the righting reflex was absent, LRR was measured by testing for the return of the righting reflex approximately every 20–30 seconds thereafter. Eight rats or rabbits were studied by groups, and the drugs were administered intravenously by rapid bolus.

Intravenous guttae test in rabbits Rabbits used were the same as mentioned above. Each rabbit, placed in plastic restrainer, was injected with either EL-001 or positive control (saline solution) by the intravenous route (lateral ear vein; doses adjusted to three times of ED50) to induce anesthesia. Thereafter, anesthetic condition was maintained by minipump with the dose of 22.5 mg/kg/h for 30 min. The behavior in anesthesia and elimination half-life were recorded.

Results and Discussion Chemistry The synthetic route employed to prepare the novel CNS 7056 derivatives is illustrated in Scheme 2. Hydrolysis of 39

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Scheme 2: Synthesis of CNS 7056 derivatives. Reagents and conditions: (a) NaHCO3, aqueous NaOH; ethyl acetate/methanol, r.t.; (b) EDCI/DMAP, substituted alcohols, R = ethyl, isopropyl, propyl, isobutyl, cyclohexyl, isopentyl and neopentyl; (c) tosylic acid; ethyl acetate, recrystallize.

CNS 7056 was carried out in the presence of NaHCO3 and NaOH with ethyl acetate as solution to give the corresponding CNS 7054. The ester derivatives IIa~g were afforded by treatment in IIa~g and various alcohol with EDCI, DMAP as catalyst. The tosylates of the target compounds were prepared by crystalizing them in ethyl acetate with tosylic acid to obtain EL-001~007. The details of chemical synthesis are presented in supporting information.

Sedative activities screen Intravenous administration of each CNS 7056 derivative (EL-001 ~ 007, 9.0 mg/kg) brought about various behaviors and latency/duration time in mice (Table 1). Group treated with EL-001 showed a significantly increased sedation and duration of LRR time compared to others, which indicated that the shorter the side chain at C3 position is, the higher the sedative activity is. EL-001 was chosen as the optimal compound for further studies.

ED50 and LD50 of EL-001 and CNS 7056 in rats and rabbits ED50 and LD50 value of both EL-001 and CNS 7056 are shown in Table 2. The therapeutic index was also determined via therapeutic ratio. Compared to CNS 7056, the ED50 and LD50 values of EL-001 were lower, and the TI 40

was almost close. All the results demonstrated that EL001 was an effective and safe derivative for the following assays.

Latency to LRR and the duration of LRR in rats and rabbits Intravenous administration of both CNS 7056 and EL-001 (dose is two times of ED50, according to above result; n = 8) brought about observed and immediate loss of the righting reflex both in rats and in rabbits (Table 3). When tested during LRR, rats receiving CNS 7056 exhibited an appropriate sedation while the hindlimb retained hypokinesia. However, rats receiving EL-001 manifested faster onset of LRR. A similar behavior was also observed in tests upon rabbits. With the treatment of an equivalent dose of single bolus, the animal receiving EL-001 obtained a significantly shorter period of latency to LRR than the one receiving CNS 7056 (in the rats group latency to LRR decreased to 38% with p = 0.06, while in the rabbits group that decreased to 42% with p < 0.05). Besides, the duration of LRR in animals who receiving EL-001 is slightly longer than in those animals who receiving CNS 7056 [the duration of LRR in rats and rabbits groups was prolonged to 10% and 29%, respectively (p > 0.05)]. It indicated that rats treated with equivalent dose of EL-001 have shorter time of latency to LRR when compared to the rats treated with equivalent dose of CNS 7056. Chem Biol Drug Des 2016; 88: 38–42

Novel CNS 7056 Derivatives as Sedatives Table 1: Structures and sedative activities of target compounds EL-001~007 in micea

Treatment (mg/kg)

R

EL-001 EL-002 EL-003 EL-004 EL-005 EL-006 EL-007 CNS 7056 Blank

Ethyl Isopropyl Propyl Isobutyl Cyclohexyl Isopentyl Neopentyl

a

Number of animals with LRR/total animals

Latency to LRR (seconds)

7/8 7/8 1/8 0/8 0/8 0/8 0/8 8/8 0/8

65 78 17 – – – – 87 –

LRR duration (seconds) 240  80 170  160 57 – – – – 242.2  68 –

The values were presented as the mean  standard deviation of mean.

Table 2: Determination of the ED50 and LD50 values for EL-001 (n = 10 or 20, for rabbits and rats, respectively) CNS 7056

EL-001

Species

ED50, mg/kg

LD50, mg/kg

TI

ED50, mg/kg

LD50, mg/kg

TI

Rats Rabbits

5.0 0.5

61.4 >40.0

12.3 >87.0

4.5 0.5

42.4 Not test

9.4 Not test

Table 3: Determination of latency to LRR and the duration of LRR for EL-001 in rats and rabbits (n = 8)a EL-001

CNS 7056 Species

Dose, mg/kg

Latency to LRR, min

Duration of LRR, min

Dose, mg/kg

Latency to LRR, min

Duration of LRR, min

Rats Rabbits

10.0 1.0

0.9  0.8 4.3  2.4

4.1  1.1 10.2  2.7

10.0 1.0

0.34  0.2 1.8  0.8

5.2  1.5 13.2  3.5

a

The values were presented as the mean  standard deviation of mean.

Table 4: Evaluation of anesthetic activity for EL-001 in rabbits (n = 8)a

Inductive dose, mg/kg Maintenance dose, mg/kg Recovery time, min

CNS 7056

EL-001

1.5 22.5 33.91  10.28

1.5 22.5 45.20  12.59

The values were presented as the mean  standard deviation of mean.

a

Anesthetic activity evaluation in rabbits With an equivalent dose of intravenous guttae, the rabbits receiving EL-001 would recover in approximately 45.2 min after cessation of infusion, while the rabbits receiving CNS 7056 could recover in 33.9 min (Table 4). During intravenous guttae with both CNS 7056 and EL-001, the Chem Biol Drug Des 2016; 88: 38–42

rabbits stayed steadily with the loss of eyelash reflex, no salivation, lacrimation or snore observed. The rabbits receiving EL-001 showed longer time of palinesthesia than others. It can be concluded that the recovery time with EL-001 treatment extended 30% of that with CNS 7056 (p > 0.05, no significant difference).

Conclusions In conclusion, the present studies showed that EL-001 is a potent sedative in rodent and lagomorpha, with a short duration of action. Compared with CNS 7056, EL-001 has a shorter period of induction despite a slightly longer sedative duration and recovery time. Further studies in exploring its mechanisms of metabolism in vivo and safety are in progress. 41

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8. Yuan R., Flockhart D.A., Balian J.D. (1999) Pharmacokinetic and pharmacodynamic consequences of metabolismbased drug interactions with alprazolam, midazolam, and triazolam. J Clin Pharmacol;39:1109–1125. 9. Kilpatrick G.J., McIntyre M.S., Cox R.F., Stafford J.A., Pacofsky G.J., Lovell G.G., Wiard R.P., Feldman P.L., Collins H., Waszczak B.L., Tilbrook G.S. (2007) CNS 7056: a novel ultra-short-acting Benzodiazepine. Anesthesiology;107:60–66. 10. Upton R.N., Martinez A., Grant C. (2009) Comparison of the sedative properties of CNS 7056, midazolam, and propofol in sheep. Br J Anaesth;103:848–857. 11. Upton R.N., Somogyi A., Martinez A., Colvill J., Grant C. (2010) Pharmacokinetics and pharmacodynamics of the short-acting sedative CNS 7056 in sheep. Br J Anaesth;105:798–809. 12. Sneyd J.R., Rigby-Jones A. (2010) New drugs and technologies, intravenous anaesthesia is on the move (again). Br J Anaesth;105:246–254.

Supporting Information Additional Supporting Information may be found in the the supporting information tab for this article Data S1. Supporting Information.

Chem Biol Drug Des 2016; 88: 38–42

Design, Synthesis, and Biological Evaluation of Novel CNS 7056 Derivatives as Sedatives in Rats and Rabbits.

A new water-soluble benzodiazepine derivative, CNS 7056 (named as remimazolam), has been undergoing many reactions in recent years to provide an intra...
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