Design, synthesis and neuroprotective effects of Fenazinel derivatives

Article abstract of DOI:10.1016/j.cclet.2017.02.003

In search of novel neuroprotective agents with higher potency and lower hERG liability, a series of novel Fenazinel derivatives were designed and synthesized, among which compounds 8m–o containing amide moiety exhibited good neuroprotective effects in vitro and in vivo. Especially, the representative compound 8o showed lower activity in a patch clamp hERG K⁺ ion channel screen and could be considered as a lead compound for further development. These findings provided an alternative approach to the development of drugs more potent than Fenazinel for the intervention of ischemic stroke.

Full text of DOI:10.1016/j.cclet.2017.02.003

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Title: Design, synthesis and neuroprotective effects of
Fenazinel derivatives
Authors: Qing-Wei Zhang, Ling Jiang, Guan Wang, Jian-Qi Li
PII:
S1001-8417(17)30051-7
DOI:
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http://dx.doi.org/doi:10.1016/j.cclet.2017.02.003
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Please cite this article as: Qing-Wei Zhang, Ling Jiang, Guan Wang, Jian-Qi Li, Design,
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Original article
Design, synthesis and neuroprotective effects of Fenazinel derivatives
Qing-Wei Zhanga,b,* , Ling Jianga,b,c, Guan Wanga,b, Jian-Qi Lia,b,*
a Novel Technology Center of Pharmaceutical Chemistry, Shanghai Institute of Pharmaceutical Industry, Shanghai 201203, China
b Shanghai Engineering Research Center of Pharmaceutical Process, Shanghai 201203, China
c School of Engineering, China Pharmaceutical University, Nanjing 211198, China
＊ Corresponding author at: Novel Technology Center of Pharmaceutical Chemistry, Shanghai Institute of Pharmaceutical Industry, Shanghai 201203, China.
E-mail address: sipiqingwei@163.com (Q.-W. Zhang), lijianqb@126.com (J.-Q. Li).

Graphical Abstract
Design, synthesis and neuroprotective effects of Fenazinel derivatives
＊
＊
Qing-Wei Zhanga,b, , Ling Jianga,b,c, Guan Wanga,b, Jian-Qi Lia,b,
a Novel Technology Center of Pharmaceutical Chemistry, Shanghai Institute of Pharmaceutical Industry, Shanghai 201203, China
b Shanghai Engineering Research Center of Pharmaceutical Process, Shanghai 201203, China
c School of Engineering, China Pharmaceutical University, Nanjing 211198, China
In search of novel neuroprotective agents with higher potency and lower hERG liability, a series of novel Fenazinel derivatives were designed
and synthesized.
ARTICLE INFO
ABSTRACT
In search of novel neuroprotective agents with higher potency and lower hERG liability, a
series of novel Fenazinel derivatives were designed and synthesized, among which compounds
8m~o containing amide moiety exhibited good neuroprotective effects in vitro and in vivo.
Especially, the representative compound 8o showed lower activity in a patch clamp hERG K+
ion channel screen and could be considered as a lead compound for further development.
These findings provided an alternative approach to the development of drugs more potent than
Fenazinel for the intervention of ischemic stroke.
Article history:
Received 24 November 2016
Received in revised form 23 December 2016
Accepted 16 January 2017
Available online
Keywords:
Fenazinel
Synthesis
hERG
Neuroprotective
Ischemic stroke
1. Introduction
Stroke is a major cause of morbidity and mortality, with a high case fatality worldwide. In China, stroke is the second-leading cause
for mortality in all diseases[1, 2]. Particularly, ischemic strokes account for 60%~80% of these strokes[3, 4]. For nearly two decades,
only tissue plasminogen activator (tPA) [5, 6] is an FDA-approved drug treatment, and it is used in less than 5% of stroke patients
because it is associated with an increased risk of intracranial hemorrhage (ICH)[7-9]. Edaravone (Fig. 1)[10-12], also known as MCI-
186, is a recently developed neuroprotective drug that has been successfully used for treating acute stroke caused by cerebral
thrombosis and embolism, while its potency is limited unless administered with other pharmacological agents[13-15]. Hence, there is
an urgent need for other effective neuroprotective agents to treat ischemic strokes. In previous work, we designed and synthesized
several dicarbonylalkyl piperazine derivatives to explore their neuroprotective properties [16,17]. Especially the compound
2a(Fenazinel, Fig. 1) [18-22] and compound 7o [23]exhibited good neuroprotection in vitro PC12 cells and in vivo rat focal cerebral
ischemic animal model, and Fenazinel was developed into clinical trials as a novel neuroprotective agent.
However, side effects with use of Fenazinel were found in phase I clinical trials: two cases had reactions that activity of serum
creatine phosphokinase (CPK) increased, and one case suffered potentially atrial premature.
The overall ancillary pharmacology profile of Fenazinel was evaluated to establish if there is any significant off-target activity or
other metabolic liabilities associated with the compound. Subsequent studies found that Fenazinel had weak activity in the hERG patch
clamp K+ channel binding assay with a IC50=8.64 μmol/L, while the hERG IC50 for its main metabolite M1 was 0.43 μmol/L in the
same assay, which predicts a potential liability for M1 to cause drug-induced QT prolongation or other drug-related cardiac toxicity.

With an increased awareness by regulatory agencies on the liabilities associated with drug-induced QT prolongation, we felt that it is
necessary to minimize the hERG activity for compounds within this chemical series [24]. We detail below our efforts to identify a
novel compound with the same promising biological profile as Fenazinel but with reduced hERG liability.
2. Results and discussion
Based on the previous study, the metabolizing of the carbonyl moiety into hydroxyl on Fenazinel was believed to play a very
important role in drug-related cardiac toxicity.
To further discover potential neuroprotectant with less hERG activity, we designed and synthesized a novel series of piperazine
derivatives bearing benzenesulfonic acyloxy or the benzoheterocycle-one groups replacing the acetophenone moiety of Fenazinel.
Furthermore, we introduced substituent groups at the β-site of carbonyl moiety and replaced carbonyl group with amide-containing
group to find new chemical entities with better neuroprotective activity and weaker hERG liability (Fig. 2). Herein, a total of 15 target
compounds were designed and synthesized.
In order to study the potential neuroprotective activities of the title compounds, a preliminary screening was performed
investigating neuroprotection on impairment induced by oxygen–glucose deprivation (OGD) in PC12 cells, as evaluated by MTT assay.
The results are showed in Table 1.
Unexpectedly, compounds 8a~j showed potent cytotoxicity against PC12 cells for all three test concentrations (0.1, 1.0, 10
μmol/L), indicating that the neuroprotective activities disappeared after introduction of the benzenesulfonic acyloxy or the
benzoheterocycle--one groups connecting bridge into structures. It was surprising that the potency and toxicity were highly sensitive to
structural variations, though the mechanism still need to be further explored.
Meanwhile, the target compounds 8k~o showed moderate to good neuroprotective effect at three levels of concentrations against
OGD-induced neurotoxicity in PC12 cells, with a dose-dependent survival rate from 63.5% to 95.7%.
Among the derivatives, compounds 8k and 8l with β-substituent groups in carbonyl moiety displayed similar survival rate as
Fenazinel, revealing that the β-substituted group seems to be little difference for the neuroprotective inhibitory activity. Also, it was
found that the amide moiety addition of the compounds 8m~o caused concentration-dependent neuroprotective effects in vitro PC12
cells with the maximal effect observed at 10 μmol/L (cell protection: 92.9%, 95.7% and 94.8%, respectively), even stronger than that
of their precursor compound Fenazinel (90.6% viable rate at 10 μmol/L), clearly indicating that the introduction of amide moiety could
significantly increase their neuroprotective activity.
Based upon the finding that the amide moiety derivatives are the most potent compounds among the different position analogues,
compound 8m~o was further evaluated in hERG binding assay and in hypoxia tolerance model in mice (Table 2). Compounds 8m~o
were inactive in our hERG binding assay with a IC50 > 30 μmol/L, which does not predict a liability for the compounds to cause drug-
induced QT prolongation. Hypoxia tolerance assay in vivo showed that compounds 8m~o could prolong the survival time of mice
under hypoxic condition at dose of 6 mg/kg and 20 mg/kg than the control group, and were comparable with with Fenazinel group.
Especially, the analog 8o exhibited a highly potent neuroprotective activity in vivo of prolonging the survival time of mice compared to
Fenazinel at 6mg/kg (4565 s vs 2829 s), therefore, it can be considered as a new lead compound for further development in specific
tests for a potential neuroprotective agent.
3. Conclusion
In conclusion, a novel class of Fenazinel derivatives were synthesized and evaluated on their neuroprotective activity based on our
previous studies. The result obtained indicated that the analog 8m~o with amide moiety exhibited neuroprotective activity in OGD test.
Particularly, compound 8o, which was inactive in hERG binding assay and showed prolonged life time of mice in hypoxia tolerance
model, may be a promising candidate for further intensive study. Further investigation on in vitro/in vivo assay of compound 8o is in
progress and will be reported in due course.
4. Experimental
The synthesis of the compounds 8a~o was shown in Scheme 1. Step “ a” was commenced with 2-chloroacetyl chloride with
benzylamine under ice-water bath for 6 h to give 5. Intermediate 7 was obtained by alkylation of 5 with piperazine hydrochloride in
EtOH via refluxing in 81% yield. 7 and HCHO were dissolved in CH3CH2OH and the solution was stirred at room temperature for 20
min. To the solution benzenesulfonic acid or benzoheterocycle-one was added. The resulting mixture was stirred at room temperature
for 15 h, followed by acidification with HCl/EA to give the targeted compounds 8a~j in 21%~42% yield. The reaction of 7 with
various commercially available aryl carbamic chloride, followed by acidification with HCl/EA, afforded compounds 8k~o in
40%~55% yield.
The structures of new compounds 8a~o are illustrated in Table 1. All the compounds were evaluated on their neuroprotective
activity via in vitro (oxygen–glucose deprivation test in PC12 cells). Several potential analogs were further evaluated in vivo assays
(hypoxia tolerance model in mice ) and hERG patch clamp K+ channel binding assay .
The detailed experimental procedures, characterization data and 1H NMR and MS spectra of target compounds, in vitro and in vivo
test methods are available in Supporting information.
Acknowledgment

This work was financially supported by Science and Technology Commission of Shanghai Municipality (No.
14YF1412800,14431901600) and by the National Natural Science Foundation of China (No. 81273373).
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Fig. 1. Edaravone, Fenazinel, 7o and the main metabolite of Fenazinel (M1)

Fig. 2. Design novel derivatives based on Fenazinel

Scheme 1. Synthetic route of the designed compounds. Reagents and conditions: (a) 2-chloroacetyl chloride, TEA, CH3CN, 0-15 ℃, 6 h; (b) EtOH, reflux, 1.5 h;
(c) benzenesulfonic acid or benzoheterocycle-one, HCHO, CH3CH2OH, r.t., 15 h; HCl/EA, r.t., 1 h; (d) 2-chloro-N-phenylpropanamide or 2-chloro-1-
cyclopropyl-2-(2-fluorophenyl)ethanone,CH3COCH3, K2CO3, KI, 40 ℃, 8h; HCl/EA,
chloride,CH3COCH3, K2CO3, KI, 40 ℃, 8 h; HCl/EA, r.t., 1 h.
r.t., 1 h; (e) corresponding aromatic-containing carbamic

Table 1. In vitro neuroprotective activity of the targeted compounds of 8a~o.
OGD testa
Survival rate (%)
Compd.
Ar, Y
0.1 μmol/L
1 μmol/L
10 μmol/L
8a
69.8±0.39
65.3±6.9
69.4±4.1
53.7±7.3
8b
54.3±0.87
51.0±2.9
8c
69.1±4.9
53.6±4.4
42.4±9.1
8d
72.3±11.7
64.7±13.0
48.0±10.3
8e
65.6±1.6
58.6±8.2
44.2±2.2
8f
70.5±4.9
74.1±11.0
69.5±4.3
73.6±1.6
69.4±16.3
62.6±2.9
77.6±1.6
70.4±9.0
44.5±2.3
8g
8h
8i
64.8±5.0
53.9±2.8
51.9±2.7
8j
8k
8l
67.7±9.1
69.1±1.5
69.3±0.3
55.5±10.3
77.2±6.2
77.1±3.3
49.9±3.4
78.6±3.4
84.8±4.1
8m
8n
63.5±8.0
75.3±2.6
79.8±8.2
81.3±8.5
92.9±8.9
95.7±0.28
8o
69.3±0.3
66.6±4.6
87.1±3.3
94.8±4.1
90.6±3.2
Fenazinel
80.0±14.9
a Oxygen–glucose deprivation (OGD) test as described in Ref [23].

Table 2. In vitro and in vivo data for selected compounds
Hypoxia tolerance assayb
Survival time (s)
6 mg/kg
2829.1±129.8
2484.7±390.4
3130.0±239.4
——
a
hERG IC50
(μmol/L)
Compd.
20 mg/kg
3025.2±199.9
3474.2±567.0
4565.0±260.7
2761.1±409.6
8m
8n
8o
Fenazinel
>30
>30
>30
8.64
——
2355.6±356.7
Control（2% DMSO)
a
hERG Patch clamp screen as described in Ref [25]. IC50 values represent the concentration to inhibit 50% of hERG current (IKr). Numbers represent IC50
generated from 3-point concentration response relationships in duplicate.
b Hypoxia tolerance assay in mice as described in Ref [23].
values