Water-soluble prodrug of antimicrotubule agent plinabulin: Effective strategy with click chemistry

Article abstract of DOI:10.1002/chem.201102293

Click for improved solubility: A water-soluble prodrug of plinabulin was designed and synthesized efficiently by using click chemistry in three steps (see scheme). The product was highly water-soluble, and the parent compound could be regenerated by ester

Full text of DOI:10.1002/chem.201102293

COMMUNICATION
DOI: 10.1002/chem.201102293
Water-Soluble Prodrug of Antimicrotubule Agent Plinabulin:
Effective Strategy with Click Chemistry
Fumika Yakushiji, Hironari Tanaka, Kyohei Muguruma, Takahiro Iwahashi,
Yuri Yamazaki, and Yoshio Hayashi*[a]
The diketopiperazine (DKP)-containing natural product
phenylahistin (1) was isolated from Aspergillus ustus in 1997
and found to have potential anti-microtubule activity (cyto-
toxicity: IC50 =394 nm in HT-29 cells).[1] The total synthesis
of 1 was achieved by our group,[2a] Couladouros and co-
workers,[2b,c] as well as Joulliꢀ and co-workers.[2d] The dedi-
cated structure-activity relationship studies of 1 led us to the
discovery of plinabulin[3] (2; NPI-2358, cytotoxicity: IC50 =
15 nm in HT-29 cells) and phase II clinical trials with intra-
venous (i.v.) injection were undertaken in the US[4] as a
promising vascular disrupting agent (VDA).[5]
ated with reconstructing 2, and at the same time, maintain
the attractive activity of the compound.
To produce the desired candidates efficiently by using a
short-step synthesis, complicated transformations should be
avoided and an aqueous substituent had to be introduced di-
rectly without the use of protective polar functional groups.
As a result of repeated examination, we planned the effec-
tive strategy by using esterase hydrolysis and click chemis-
try.[7,8] The Huisgen reaction, in particular, was adequate for
the purpose of high chemoselectivity, high yield, and mild
conditions with unprotected substituents.[9] Our strategy is
shown in Scheme 1.
Although 2 exhibited potential antitumor activity, its solu-
bility was not compatible with the requirements of a drug
(<0.1 mgmLÀ1). Compound 2 has been injected with a solu-
bilizing agent, but long-term administration may have the
possibility of unexpected safety issues or problems in the
quality of life of the patient. Hence, we attempted to im-
prove the aqueous solubility of 2 to increase its pharmaceut-
ical value. Here, we report a novel monolactim prodrug
form of plinabulin.
Two methods are generally used to improve the solubility
of a drug in water. First, the mother skeleton may be trans-
formed into a soluble structure, and second, aqueous sub-
stituents may be attached to the original skeleton.[6] In our
case, the latter was selected to prevent complications associ-
Scheme 1. The strategy for preparing the water-soluble prodrug of plina-
bulin (2) using click chemistry.
[a] Dr. F. Yakushiji, H. Tanaka, K. Muguruma, T. Iwahashi, Y. Yamazaki,
Prof. Dr. Y. Hayashi
The C12 amide part of 2 was O-alkylated[10] with alky-
noate 3, then the alkyne part of 4 was reacted with the
aqueous substituent 5 by using the Huisgen reaction. The
ester moiety of 6 was hydrolyzed by esterase to produce the
parent compound 2. By this process, formaldehyde was gen-
erated, but prompt metabolic activity produced carbon diox-
ide that could be exhaled, as reported.[11]
School of Pharmacy
Tokyo University of Pharmacy and Life Sciences
1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan)
Fax : (+81)42-676-4475
E-mail: yhayashi@toyaku.ac.jp
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201102293.
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Table 1. Synthesis of the alkyne-containing monolactim 8.
Entry
7
Base
Solvent
T
[8C]
t
Yield
[%]
[equiv]
[h]
1
2
3
4
5
6
7
5.0
2.5
1.2
2.0
2.0
2.0
2.0
DBU[a]
tBuOK
Cs2CO3
Cs2CO3
Cs2CO3
Cs2CO3
Cs2CO3
DMF
THF
50
RT
RT
8
8
8
0.25
0.25
0.25
0.25
18
15
29
54
44
51
52
DMF
DMF
DMF
DMSO
DMSO
50[b]
50[b]
50[b]
50[b]
[c]
[c]
[a] Tetrabutylammonium hydrogen sulfate was added. [b] Microwave ir-
radiation. [c] Tetrabutylammonium iodide was added.
Scheme 2. Synthesis of the water-soluble prodrug candidate 11. Reagents
and conditions: a) Compound 9, sodium ascorbate, CuSO4·5H2O, H2O/
tBuOH/DMF (1:1:1), 508C, microwave, 10 min then HPLC purification,
67%; b) ion exchange resin DIAION WK11, eluent H2O/CH3CN (1:1),
96%.
According to Scheme 1, plinabulin (2) was O-alkylated
with the linker 7 under basic conditions (Table 1). The alky-
noate 7 was synthesized from the readily commercially
available 5-hexynoic acid and chloromethyl chlorosulfate.[12]
Under the conditions, in the presence of 1,8-diazabicyclo-
b-hydroxy carboxylic acid can contribute to the water solu-
bility of a compound and ease of handling.
A
Compound 8 was combined with 9 by Huisgen reaction
with CuSO4·5H2O and sodium ascorbate in H2O/tBuOH/
DMF under microwave irradiation. This reaction proceeded
smoothly within 10 min and 10 was obtained as a single
isomer (Scheme 2). An aqueous substituent was introduced
easily, but contrary to expectations, 10 was not so water-
soluble because of an intramolecular hydrogen bond be-
tween the carboxylic acid and the triazole moiety, which was
was recovered, even though the phase transfer catalyst was
co-present (Table 1, entry 1). tBuOK gave the desired mono-
lactim 8 in only 15%, because the product tended to under-
go decomposition (Table 1, entry 2). When other bases
(NaH, lithium diisopropylamide (LDA), and lithium hexam-
ethyldisilazide (LHMDS)) were examined, O or N-alkyla-
tion did not proceed significantly and the starting material
was recovered. Fortunately, Cs2CO3 provided the desired
compound 8 in a moderate yield (Table 1, entry 3–7). In par-
ticular, microwave irradiation provided the monolactim 8 in
54% yield (Table 1, entry 4) and the starting material was
also recovered. The phase transfer catalyst and solvent were
examined, but much higher yield was not obtained (Table 1,
entry 5–7). The effects of the reaction temperature were
also measured; temperatures exceeding 508C led to decom-
position of the product. In addition, iodine or bromide alky-
noates were tested, however, the yield of 8 was not im-
proved. The tautomerization of the C12 amide and the vul-
nerable ester moiety made this O-alkylation challenging, but
8 was obtained preferentially. The structure of 8 was deter-
mined by X-ray crystallography.[13]
1
apparent in the H and 13C NMR spectra. Therefore, the car-
boxylic acid of 10 was exchanged to form the sodium salt 11
in the presence of a methacrylic acid ion exchange resin,
DIAION WK11. After filtration and lyophilization, a yellow
crystalline powder was obtained, and the spectra indicated
the presence of the sodium salt 11. Compound 11 showed a
water solubility of 6.38 mgmLÀ1, which is 64000 times great-
er than that of 2.
Next, the utility of 11 as a prodrug was examined. Ac-
cording to our plan, synthesized 11 was solved to the phos-
phate buffered saline (PBS) solution (pH 7.4) and hydro-
lyzed with esterase (porcine liver) at 378C to reproduce 2
(Scheme 3).
With the alkyne-containing lactim 8 in hand, we turned
our attention to the synthesis of the water-soluble prodrug
derivatives. The serine derivative 9 (Scheme 2) was prepared
to introduce an aqueous substituent, which was obtainable
through a diazotransfer reaction in three steps as reported
by Tirrell.[14] Amino acid derivatives are commonly used to
produce water-soluble prodrugs[15] because of their high sol-
ubility and low toxicity. In particular, a serine residue with a
Scheme 3. Hydrolysis of 11 with esterase. Reagents and conditions: a) Es-
terase solution from porcine liver (suspension in 3.2m (NH4)2SO4 solu-
tion, pH 8.0), PBS solution (10 mm, pH 7.4), 378C then DMSO.
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Water-Soluble Prodrug of Anti-Microtubule Agent Plinabulin
COMMUNICATION
Table 2. Cytotoxic activity toward human colon HT-29 cells.
The results of the hydrolysis, as monitored by HPLC, are
shown in Figure 1 and Figure 2. This reaction proceeded
without any side reactions and only the parent drug 2 was
produced (Figure 1). The water-soluble auxiliary 12 was de-
Entry
Compound
IC50
[nm][a]
1
2
3
4
11
12
101.4Æ5.7
>2mm
13.5Æ2.2
16.6Æ0.9
plinabulin (2)
colchicine
[a] Concentration of compound that produced 50% inhibition of cell pro-
liferation.
from cells to medium, or the inner part of the cells directly.
On the other hand, the water-soluble auxiliary 12 proved to
be non-toxic even when the concentration was raised to
2 mm (Table 2, entry 2). From these results, this water-solu-
ble prodrug system would be safe and efficacious in humans.
In summary, we have designed and synthesized a water-
soluble prodrug of plinabulin (2) using click chemistry,
which produced a unique monolactim skeleton with high
water solubility, and regeneration of the parent compound 2
upon esterase hydrolysis. The safe and stable prodrug 11
was transformed in three steps from the parent compound.
In addition, alkyne 8 can be functionalized with various
aqueous azide substituents. This provided significant advan-
tages to the novel prodrug system, including facile optimiza-
tion of the water-soluble substituent. Higher-order function-
al assessment and investigation of water-solubilizing group
are now in progress.
Figure 1. HPLC charts of esterase hydrolysis of prodrug 11 in PBS
(pH 7.4, 378C).
Experimental Section
General procedure for click chemistry: A solution of azide (28.8 mg,
0.22 mmol) in H2O (0.40 mL), CuSO4·5H2O (2.7 mg) and sodium ascor-
bate (65 mg), were added to a solution of alkyne (50 mg, 0.11 mmol) in
DMF (0.40 mL) and tBuOH (0.40 mL), and the mixture was stirred for
10 min at 508C under microwave irradiation. Then, the mixture was
cooled to RT and the solvent was removed in vacuo. The residue was di-
luted with AcOEt and water, and extracted with AcOEt. The extract was
washed with brine, dried over Na2SO4, filtered and concentrated. The re-
sidual oil was purified through reverse phase high performance liquid
chromatography (SunFire PrepC18 OBD 19ꢂ150 mm (5 mm, 12 nm),
gradient: milli-Q water (TFA 0.1%)/CH3CN (TFA 0.1%)=35:65 to
milli-Q water (TFA 0.1%)/CH3CN (TFA 0.1%)=75:25 over 40 min,
Flow rate: 9.00 mLminÀ1, UV: 365 nm and 230 nm) to give the desired
product as a yellow solid.
&
*
Figure 2. Time course of hydrolysis of prodrug 11 ( ) and release of 2 ( )
in PBS (10 mm). The percentage was determined by HPLC. The data
points are calculated from the average (Æstandard error) of three assays.
tected in the peak of the DMSO area, which was ascer-
tained by distinct HPLC analysis.[16] In addition, esterase hy-
drolysis was confirmed to occur at the ester moiety, because
the absence of esterase in PBS gave only the starting materi-
al. The concentration of 11 decreased concurrently with the
production of 2 (Figure 2). The half-life (t1/2) of 11 was
59.9 min, which is appropriate for water-soluble prodrug sys-
tems delivered by i.v. injection without a solubilizing agent.
Although hydrolysis was complete, as shown in Figure 2, the
detection of 2 reached a maximum at 80% because insolu-
ble 2 precipitated onto the vessel. The high solubility and
appropriate t1/2 indicates that the designed and synthesized
11 shows potential as a water-soluble prodrug of plinabulin.
In addition, the sodium salt 11 was stable over one month at
48C without decomposition.
Acknowledgements
This work was supported by grants from MEXT (Ministry of Education,
Culture, Sports, Science and Technology), Japan, including the Grant-in-
Aid for Scientific Research (B) (to Y.H.; No. 20390036), Grant-in-Aid
for Research Activity Start-up (to F.Y.; No. 21890262) and Grant-in-Aid
for Young Scientists (B) (to Y.Y.; No. 21790118).
The cytotoxic activities of 11 and 12 were verified in vitro
with human colon HT-29 cell lines (Table 2).[17] Compound
11 exhibited one-tenth of the IC50 of 2 under normal condi-
tions (Table 2, entry 1).[18] This result may be explained by
the fact that the monolactim prodrug 11 reacted with small
amounts of esterases produced by cells, which was exuded
Keywords: antitumor agents · click chemistry · monolactim ·
peptides · prodrugs
Chem. Eur. J. 2011, 17, 12587 – 12590
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
12589

Y. Hayashi et al.
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contained the a,b-unsaturated diketopiperazine ring, which assisted
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mentary crystallographic data for phosphorous oxymethyl deriva-
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Received: July 26, 2011
Published online: September 29, 2011
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