Development of MBRI-001, a deuterium-substituted plinabulin derivative as a potent anti-cancer agent

Article abstract of DOI:10.1016/j.bmcl.2017.01.096

Plinabulin, a drug targeting microtubule of cancer cells, has been currently tried in its phase III clinical study. However, low efficacy caused by poor pharmacokinetic (PK) properties has been considered to be the main obstacle to approved by the Food and Drug Administration. Herein, we introduced a deuterium atom as an isostere in its structure to become a new compound named (MBRI-001, No. 9 in a series of deuterium-substituted compounds). The structure of MBRI-001 was characterized by HRMS, NMR, IR and a single crystal analysis. MBRI-001 exhibited better pharmacokinetic characteristics than that of plinabulin. Additionally, its antitumor activity is in a low nanomolar level for a variety of cancer cell lines and high activity against human NCI-H460 xenograted in mice intravenous administration. Importantly, continuous administration of MBRI-001 exhibited lower toxicity compared to docetaxel. We thus suggest that MBRI-001 could be developed as a promising anti-cancer agent in near future.

Full text of DOI:10.1016/j.bmcl.2017.01.096

Accepted Manuscript
Development of MBRI-001, a Deuterium-substituted Plinabulin Derivative as
a Potent Anti-cancer Agent
Zhongpeng Ding, Hejuan Cheng, Shixiao Wang, Yingwei Hou, Jianchun Zhao,
Huashi Guan, Wenbao Li
PII:
S0960-894X(17)30120-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.01.096
BMCL 24672
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
12 November 2016
19 January 2017
31 January 2017
Please cite this article as: Ding, Z., Cheng, H., Wang, S., Hou, Y., Zhao, J., Guan, H., Li, W., Development of
MBRI-001, a Deuterium-substituted Plinabulin Derivative as a Potent Anti-cancer Agent, Bioorganic & Medicinal
Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.01.096
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Development of MBRI-001, a Deuterium-substituted Plinabulin Derivative as a
Potent Anti-cancer Agent
Zhongpeng Ding a, Hejuan Cheng a, Shixiao Wang b, Yingwei Hou a,b, Jianchun Zhao
a,b, Huashi Guan a,b, and Wenbao Li a,b,
*
a Key Laboratory of Marine Drugs of Ministry of Education of China, School of
Medicine and Pharmacy, Ocean University of China, Qingdao 266003, Peoples’
Republic of China
b Marine Biomedical Research Institute of Qingdao, Qingdao 266071, Peoples’
Republic of China
Corresponding author Professor Wenbao Li. Tel /fax: +86 532 85906861.
E-mail address: wbli92128@ouc.edu.cn.
Abstract: Plinabulin, a drug targeting microtubule of cancer cells, has been
currently tried in its phase III clinical study. However, low efficacy caused by
poor pharmacokinetic (PK) properties has been considered to be the main
obstacle to approved by the Food and Drug Administration. Herein, we
introduced a deuterium atom as an isostere in its structure to become a new
compound named (MBRI-001, No. 9 in a series of deuterium-substituted
compounds). The structure of MBRI-001 was characterized by HRMS, NMR, IR
and a single crystal analysis. MBRI-001 exhibited better pharmacokinetic
characteristics than that of plinabulin. Additionally, its antitumor activity is in a
low nanomolar level for a variety of cancer cell lines and high activity against
human NCI-H460 xenograted in mice intravenous administration. Importantly,
continuous administration of MBRI-001 exhibited lower toxicity compared to
docetaxel. We thus suggest that MBRI-001 could be developed as a promising

anti-cancer agent in near future.
Key words: Plinabulin; Deuterium substituted; Microtubule; Pharmacokinetic
properties; Docetaxel
Microtubules, composing of α/β-tubulin heterodimers, are a component of the
cytoskeleton essential function in maintenance shape, motility, and transmission of
cell signaling and mitosis.1-3 Microtubules also play an important role in eukaryotic
cells as a target for anticancer drugs.2,4 Anti-microtubule agents are currently
classified into two groups: microtubule-stabilizing agents which bind to the tubulin to
stable microtubule, and microtubule-destabilizing agents which locate in the tubulin
dimers to destabilize the microtubules.5,6 Anti-microtubule agents, namely the
microtubule stabilizing taxanes such as paclitaxel and docetaxel, and the microtubule
depolymerizing vinca alkaloids such as vinblastine, vincristine and vinorelbine, have
been used as first-line drugs for cancer therapy.7-11 However, colchicine, a
microtubule destabilizing agent, has not been approved for its extreme toxicity, such
as neurological and bone marrow toxicity.6,12 Further, cancer cells development of
resistance to colchicine might also the main reason for limited use of the drug.6
Development of new drug with high efficacy and lower toxicity of microtubule
inhibitors have still needed in the great market prospects.
Plinabulin is developed from the marine natural “phenylahistin” namely as (-)-1,
possessing a dehydropiperazine Structure (Figure 1).13,14 The bind of Plinabulin was
found at the interfacial region between α- and β- tubulins near the colchicine binding
site, but not inside the colchicine binding cavity.15-17 It has multi-faceted activities
reported as anti-cancer activity through the anti-angiogenesis, interruption of tumor
blood flow and induction of cancer apoptosis via the c-Jun N-terminal kinase (JNK)
pathway.18 To date, Plinabulin has been under phase III clinical trial for treatment of
lung cancers.10,19 In this study, we developed some Plinabulin derivatives, a series of
deuterium-substituted plinabulin derivatives, demonstrating potent anti-microtubule,
lower toxicity and better pharmacokinetic properties evaluated in vitro and in vivo

assays.
Figure 1. Structures of natural colchicine-site binder (−)- 1, plinabulin and colchicine.
Deuterium atom is the stable isotope of hydrogen atom. The volume of deuterium
atom is similar to that of hydrogen atom with nearly identical physiochemical
properties, but the carbon-deuterium bond is more stable than the carbon-hydrogen
bond. We thus suggest that it is the best nonradioactive bioisosteric replacement.20,21
In addition, a common feature is breaking the carbon-hydrogen in the process of drug
metabolism. Thus, if hydrogen atom of drug molecules is replaced by deuterium, it
might obtain the beneficial because of improving the metabolism such as
less-frequent dosing, lower toxicity.22-24 Many advantages have been reported with
deuterium replacing hydrogen as drugs, such as reducing gene toxicity of
D1-halothane, and increasing half-life of D6-nifedipine.25 Currently, a series of
deuterated drug candidates, such as SD809 as a vesicular amine transporter 2
(VMAT2) inhibitor and CTP-347 as a selective serotonin reuptake inhibitor, have been
developed in clinical trial. SD809, is a deuterated version of tetrabenazine, can
attenuate CYP2D6 metabolism to increase metabolite half-lives in patients with
Huntington disease. And CTP-347, is a deuterium substituted of paroxetine, can
decrease inactivation of CYP2D6 and reduce drug-drug interactions for improving the
metabolism profiles.26-28

We report the synthesis of a deuterated plinabulin derivative (compound 9) and
the evaluation of its pharmacokinetic properties assayed by in vitro and in vivo assays,
including human NCI-H460 lung carcinoma xenograted in mice.
The deuterium-plinabulin derivative was synthesized via a sequence of eight
linear steps (Scheme 1). The optimized synthesis route has been utilized to the
preparation of more than 30 g of the compound 9. First, to obtain oxazole, ethyl
isocyanoacetate and pivalic anhydride was condensed in the presence of 1,
8-diazabicyclo[5.4.0]undec-7-ene (DBU) for 48 h at room temperature. After
purification using silica gel column chromatography, the oxazole ester was converted
into the imidazole ester by the solvolysis reaction in formamide at 175oC. Then,
LiAlH4 was used to reduce imidazole ester to alcohol, and MnO2 was used for the
oxidation to prepare aldehyde. The imidazole aldehyde was further converted to the
deuterium-imidazole aldehyde by reduction reaction with using NaBD4 at -20oC and
again oxidation with using MnO2. Total yield to get deuterium-imidazolealdehyde was
24% by six steps.
Scheme 1. Synthesis of deuterium-plinabulin derivative compound 9.
Reagents, conditions and yield: (a) DBU, THF, rt, 48 h, 99%; (b) formamide, 175 °C, 36 h, 45%;
(c) LiAlH4, THF, 0 oC to rt, 6 h, 94%; (d) MnO2, acetone, rt, 48 h, 80%; (e) NaBD4, EtOH, 5 h,
-20 oC, 85% (f) MnO2, acetone, rt, 48 h, 84%; (g) 1,4-diacetylpiperazine-2,5-dione, Cs2CO3, DMF,
rt, 20 h, 54%; (h) benzaldehyde, Cs2CO3, DMF, 50 °C, 24 h, 89%.
Then a tandem aldol condensation with two different kinds of aldehydes onto the
diacetyl-2,5-piperazinedione ring was observed in the presence of Cs2CO3 in N,
N-dimethylformamide (DMF). Namely, diacetyl-2,5-piperazinedione was reacted
with the deuterium-imidazole aldehyde for 20 h at room temperature. The low

reactivity of the deuterium-imidazolealdehyde probably caused by the high steric
hindrance with a bulky tert-butyl group at the 5-position.13 The second condensation
reaction was completed between compound 8 and benzaldehyde at 50 oC in DMF
under an N2 atmosphere. The degassed condition was utilized to avoid probable
oxidation of the activated methylene in the core of diketopiperazine (DKP) ring in the
presence of Cs2CO3.13,29 During last condensation reaction, the dark condition had to
be used to avoid probable yield E configuration of deuterium plinabulin. The total
yield of synthesis of compound 9 was 11% in the eight steps. The chemical structures
of compound 9 was characterized by high resolution mass spectrometer (HRMS),
infrared radiation (IR), nuclear magnetic resonance (NMR) spectroscopies (see the
Supporting Information) and a single crystal analysis (Figure 2).
From analysis of the single crystal of the compound 9 (CCDC: 1509116), there is
a hydrogen bonding between NH of the DKP-amide core and imine of the imidazole
rings to form a pseudo tricyclic structure (Figure 2). A dihedral angle of -29o was
found in the left side phenyl ring and the core of the DKP structure, which indicated
that it is not a coplanar (Figure 2). As shown in Figure 2, a Z configuration was
observed for the double bond of C13-C2, which can be transformed into E
configuration in ultraviolet light.
Figure 2. ORTEP drawing of compound 9

The pharmacokinetic properties of compound 9 and plinabulin have been
evaluated in the plasma and in the liver microsomal of rat. The results showed that
both compound 9 and plinabulin were stable in the plasma from 0-100 mins. Both
compound 9 and plinabulin exhibited similar stability in rat liver microsomes. The
amide bond of the DKP structures were not hydrolyzed by the esterase in rat. (See the
Supporting Information)
Figure 3: Plasma concentration-curve of compound 9 and plinabulin (5 mg·kg-1) after single
intravenous injection to Wistar rats(n=5). Each time point is the mean± SD from five individual
mice.
Table 1: Pharmacokinetic parameters of compound 9 and plinabulin after single intravenous
injection at 5 mg·kg-1
Parameter(unit)
ke (h-1)
Plinabulin
0.68
Compound 9
0.50
t1/2 (h)
1.13
1.47
Tmax (h)
0.02
0.02
Cmax (ng·mL-1)
AUClast (h·ng·mL-1)
AUCINF_obs(h·ng·mL-1)
5037.50
720.76
723.08
7276.00
1519.32
1520.52

Vz_obs (L·kg-1)
CL_obs (L·h-1·kg-1)
MRTlast (h)
11.39
7.26
0.50
7.72
3.53
1.68
ke = elimination rate constant; t1/2 = halflife; Tmax = time to maximal plasma concentration; Cmax
=
maximal concentration; AUClast = area under the plasma concentration–time curve from 0 h to last
quantifiable concentration; AUCINF_obs = area under the plasma concentration–time curve from 0 h
to infinity; Vz_obs = apparent volume of distribution; CL_obs = clearance; MRT = mean residence
time.
The pharmacokinetic behaviors were further evaluated in the vivo assay. No oral
absorption was observed for both compound 9 and plinabulin in the Wistar rats. After
single intravenous injection with dosage 5 mg·kg-1, the concentration-curves of
plinabulin and compound 9 were shown in Figure 3. The pharmacokinetic parameters
were further calculated and summarized in Table 1. Compound 9 was showed to
display a better PK property compared with plinabulin at the dosage of 5.0 mg/kg for
a signal administration. Compound 9 had longer T1/2 at 1.47 h compared with
plinabulin at 1.13 h, and exhibited over 2-fold increase in the area under the plasma
concentration-time curve AUC0–∞ at 1520.52 h·ng·mL-1 compared with plinabulin at
723.08 h·ng·mL-1. The peak concentration (Cmax) of compound 9 at 7276 ng·mL-1 was
closed to 1.5-fold higher than that of plinabulin at 5037 ng·mL-1. Compound 9
exhibited an obviously decrease in the clearance (CL) at 3.53 L·h-1·kg-1 compared
with plinabulin at 7.26 L·h-1·kg-1, and also exhibited a longer total body mean
residence time (MRT) at 1.68 h compared with plinabulin at 0.50 h. These results
indicated that compound 9 exhibited better pharmacokinetic parameters than
plinabulin in rats.
Cytotoxic effect was determined in the cell lines of NCI-H460 (human lung
carcinoma), NCI-H446 (small cell lung cancer) and Jurkat (human T
lymphocyte carcinoma). As shown in Table 2，compound 9 and plinabulin have the
similar effect of cytotoxic activities against cancer cell growth.

Table 2. Cytotoxicity of compound 9 and plinabulin against human cancer cell lines.
a
Compound
Cytotoxicity IC50 (nM)
NCI-H460
33.9 ± 0.5
33.2 ± 0.5
NCI-H446
Jurkat
Plinabulin
44.2 ± 1.1
3.3 ± 1.3
4.3 ± 1.6
22.0 ± 2.4
9
a Values represent mean ± SD from at least three independent dose response curves.
Figure 4: Antitumor activity in vivo. A. The volumes of NCI-H460 xenografted in nude mice after
administrated with compound 9 by intravenous injection with Q3D7. The data were presented as
the mean ± SD. B. The images of excised tumors of xenograft.
Figure 5 : The body weights of nude mice after administrated with compound 9 by intravenous
injection with Q3D7. Data were presented as mean ± SD.
The anticancer activity of the compound 9 (MBRI-001) was further evaluated
against NCI-H460 xenograft models by intravenous injection with a dose at 3 mg/kg,
6 mg/kg, or 12 mg/kg every two days for consecutive 21 days. As a
microtubule-stabilizing agent with first-line therapy for lung cancers,5,30 docetaxel
was used as a positive control at a dose of 10 mg/kg. At the end of administration

period, the mean of final tumor volumes of vehicle, docetaxel and compound 9 at 3
mg/kg, 6 mg/kg, or 12 mg/kg were 2065.87, 1163.16, 1523.29, 1372.27 and 991.78
mm3 (Figure 4A), respectively. Average excised tumor weights of the corresponding
groups were 1.25 g, 0.75 g, 0.90 g, 0.69 g and 0.66 g, respectively. The response
calculated inhibitory rates were 40.0%, 28.8%, 44.8% and 48.0%, respectively
(Figure 4B). Overall, antitumor activity of compound 9 showed doses dependent in
the concentrations range of 3 to 12 mg/mL, which was agreement with measured
concentration of compound 9 with different doses at anatomic tumors. The tissue
concentration of compound 9 was 316.45 ng/g, 809.11 ng/g, 2493.72 ng/g at doses of
3 mg/kg, 6 mg/kg, 12 mg/kg, respectively. Compared with docetaxel at 10 mg/kg,
compound 9 at 6 mg/kg and 12 mg/kg caused a considerable suppression of the tumor
growth (Figure 4B). Significant decrease of body weights were not observed in all
groups of compound 9 treated mice (Figure 5). However, docetaxel caused a decrease
of body weights by 18.3% (Figure 5).
In summary, a deuterium-substituted plinabulin, compound 9 (MBRI-001) was
synthesized as an anti-microtubule agent. In comparison with plinabulin, its
pharmacokinetic properties were improved with better AUC. The
deuterium-substituted plinabulin compound 9 exhibited a considerable efficacy
against cancer growth and lower toxicity in xenografted mice compared with
docetaxel. Thus, Compound 9 (MBRI-001) has been suggested to be promising agent
in future clinical therapy.
Acknowledgment
This work was supported by “Zhufeng Scholar Program” of Ocean University of
China (841412016), “Major Projects of Independent Innovation” of Qingdao
(15-4-13-zdzx-hy) and “Outstanding Talents Plan” of Qingdao (15-10-3-15-(34)-zch)
to Dr. Wenbao Li.
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Graphical Abstract

1) MBRI-001 is a deuterium-substituted plinabulin derivative.
2) MBRI-001 exhibited better pharmacokinetic characteristics than plinabulin.
3) MBRI-001 showed high activity against human NCI-H460 xenograted in mice.
4) MBRI-001 and plinabulin have the similar cytotoxicity against cancer cell growth.
5) MBRI-001 exhibited lower toxicity compared to docetaxel as control.