Prodrug study of plinabulin using a click strategy focused on the effects of a replaceable water-solubilizing moiety

Article abstract of DOI:10.1248/cpb.c12-00216

Plinabulin (1) is a potent anti-microtubule agent, however, its low water solubility has to be improved for the advantage in pharmacokinetics and chemotherapy. In this report, the replaceable water-solubilizing moiety of the water-soluble prodrug of plina

Full text of DOI:10.1248/cpb.c12-00216

July 2012
Regular Article
Chem. Pharm. Bull. 60(7) 877–881 (2012)
877
Prodrug Study of Plinabulin Using a Click Strategy Focused on the
Effects of a Replaceable Water-Solubilizing Moiety
Fumika Yakushiji, Hironari Tanaka, Kyohei Muguruma, Takahiro Iwahashi, Yuri Yamazaki, and
Yoshio Hayashi*
School of Pharmacy, Tokyo University of Pharmacy and Life Sciences; 1432–1 Horinouchi, Hachioji, Tokyo 192–
0392, Japan. Received March 1, 2012; accepted April 9, 2012
Plinabulin (1) is a potent anti-microtubule agent, however, its low water solubility has to be improved
for the advantage in pharmacokinetics and chemotherapy. In this report, the replaceable water-solubilizing
moiety of the water-soluble prodrug of plinabulin (1) was investigated. The properties of the water-soluble
prodrugs of plinabulin (1), in which the water-solubilizing part was replaced with a new functionality, were
evaluated. The newly introduced water-solubilizing moiety provided interesting effects on the water solubil-
ity and half-life of the prodrugs.
Key words water-soluble prodrug; anti-microtubule agent; click chemistry; plinabulin; monolactim
Prodrug research is one of the most important fields of reaction, was shown to be cellularly non-toxic.11) These results
medicinal chemistry. In particular, improvements in water sol- suggested that it was worthwhile to pursue further optimiza-
ubility have been studied extensively.1) Paclitaxel, for example, tion of this prodrug system.
‘
’
exhibited potent anti-microtubule activity2) as intravenous (i.v.)
In this paper, we investigated the replaceable water-solubi-
injection. Due to its low water solubility (0.25µg/mL),3) it was lizing moiety to evaluate its influence on prodrug water solu-
initially formulated with the detergent cremophor EL, which bility and half-life. As a water-solubilizing moiety, we focused
induced hypersensitivity.4) Therefore, a variety of water-sol- on two types of polar groups. One was L-carnitine (3),25) which
uble paclitaxel prodrugs have been developed to improve the is an γ-amino acid derivative involved in lipid metabolism in
5–9)
’
patient s burden.
the human body. The unprotected polar functional group of
Previously, we reported the design and synthesis of a the azide 426) is a β-hydroxycarboxylic acid moiety, that is
water-soluble prodrug of plinabulin (1)10) as a monolactim similar to the serine-type structure used in prodrug 2, with
serine-type derivative 211) using click chemistry12–14) (Fig. 1). a higher number of carbon atoms. These slight changes were
Plinabulin (NPI-2358) (1) (cytotoxic activity: IC50=15nM in expected to provide interesting effects on the water solubility
HT-29 cells) is a derivative of S-(−)-phenylahistin15,16) and and half-life of the prodrug. And the other was β-D-galactose
phase II clinical trials have been undertaken worldwide17–19) (5), which is a monosaccharide configured as a biological
by way of i.v. injection as a promising vascular disrupting component. The four hydroxyl groups of 1-azide-1-deoxy-β-
agent (VDA).20–24) Plinabulin (1) showed low water solubility D-galactopyranoside (6) were predicted to be effective for the
(<0.1µg/mL), which forced the co-injection of a solubilizing water solubility like glycosides in natural resources (Fig. 2).
agent, such as paclitaxel. To improve the pharmacokinetics
and chemotherapeutic index, the water-soluble prodrug 2 was
developed. The water solubility of α-amino acid type deriva-
Results and Discussion
Synthesis of Water-Soluble Prodrugs First, we synthe-
tive 2 was determined to be 6.38mg/mL, and the in vitro half- sized the carnitine derivative 4 (Chart 1). (R)-Ethyl 4-chloro-
life (t1/2) in the presence of porcine liver esterase was found 3-hydroxybutanoate (7) was reacted with sodium azide to
to be 59.9min. These values make the compound appropriate obtain 8 in 73% yield. The ethyl ester part of 8 was then
for i.v. injection without inclusion of a solubilizing agent. Fur- hydrolyzed under basic conditions. The obtained spectral data
thermore, the water-solubilizing triazole auxiliary, which was was consistent with the structure of 4, which was used in a
cleaved from the mother skeleton via an esterase hydrolysis subsequent reaction without further purification.
Fig. 1. Structures of Plinabulin (1) and Monolactim Serine-Type Prodrug 2
The authors declare no conflict of interest.
*To whom correspondence should be addressed. e-mail: yhayashi@toyaku.ac.jp
© 2012 The Pharmaceutical Society of Japan

878
Vol. 60, No. 7
°
Reagents and conditions: a) NaN3, DMF, 100 C, 3h, 73%; b) KOH (1.3eq),
EtOH/H2O, rt, 15h, 99%.
Chart 1
Fig. 2. Structures of L-Carnitine (3), Carnitine Derivative 4, β-D-
Galactose (5) and β-D-Galactose Derivative 6
t
°
°
Reagents and conditions: a) 9, Cs2CO3, DMF, 50 C, microwave, 15min, 54%; b) 4 or 6, sodium ascorbate, CuSO4·5H2O, H2O/ BuOH/DMF (1:1:1), 50 C, microwave,
10min, then HPLC purification, 11: 79%, 13: 79%; c) ion exchange resin DIAION® WK11, eluent H2O/CH3CN (1:1); d) esterase solution from porcine liver (suspension
°
in 3.2M (NH4)2SO4 solution, pH 8.0) in 10mM PBS (pH 7.4), 37 C, then DMSO.
Chart 2
The resultant carnitine derivative 4 and commercially avail- be explained by a difference in the relative hydrophobicity
able 6 were then combined with the mother skeleton, respec- between the carnitine-type and serine-type water-solubilizing
tively (Chart 2). The diketopiperazine ring of plinabulin (1) moieties. Compound 12 provided a larger surface area for hy-
was skeletally transformed to the monolactim by O-alkylation dration, which incurred a high loss of entropy in water at the
of alkynoate 9 under the basic condition with microwave hydration layer, and the water solubility of 12 was adversely
irradiation.11) The obtained alkyne 10 was subsequently con- affected.27) This investigation revealed that a slight increase in
nected with 4 or 6 by cupper-catalyzed alkyne azide coupling the hydrophobic properties greatly influenced the water solu-
(CuAAC).14) These reactions proceeded without side reactions, bility of the prodrug. On the other hand, the glycoside-type
and the triazole derivatives 11 and 13 were obtained in 79% derivative 13 dissolved in water with the solubility of 7.5µg/
yield, respectively, after HPLC purification. Furthermore, the mL. Contrary to our expectations, the water solubility of 13
carboxylic acid moiety of 11 was converted to the correspond- indicated insignificant value. In addition, 13 was found to be
ing sodium salt 12 using an ion exchange resin. Regardless marginally stable.28) These aspects of the glycoside-type deriv-
of the azide-substrate, our synthetic strategy for the water- ative 13 were derived from the water-solubilizing moiety. The
soluble prodrug worked effectively and reproducibly.
number of hydroxyl groups as glycosides induced the modest
Water Solubility The water solubility of 11, 12 and 13 improvement of water solubility and the easy hydrolysis of the
was examined. Compound 11 dissolved in H2O only a little, ester part. However, 12 and 13 showed more improved water
however the solubility was not enough to be analyzed by solubility than 1, suggesting that the click chemistry functions
HPLC. In contrast, the sodium salt 12 was soluble in water as an effective strategy for the water-soluble prodrug develop-
with the solubility of 0.85mg/mL, which was over eighty five ment.
hundred times greater than that of plinabulin (1). This result
In Vitro Hydrolysis with Esterase The stable and rela-
indicated that the conversion to the sodium salt enhanced the tively high water-soluble prodrug 12 was dissolved in 10mM
efficacy for the water solubility. However, the observed mild phosphate buffered saline (PBS, pH 7.4) and hydrolyzed with
°
water solubility of 12 in comparison to 2 (6.38mg/mL) could the porcine liver esterase (PLE) at 37 C to reproduce parent

July 2012
879
Fig. 3. HPLC Charts of Esterase Hydrolysis of Prodrug 12 in 10mM
°
PBS (pH 7.4, 37 C)
drug 1. It was reported that the generated formaldehyde was
rapidly metabolized and the produced carbon dioxide was ex-
creted via expiration, as in the pivoxil series.29–32) The hydro-
lysates were analyzed by HPLC, as shown in Fig. 3. This hy-
drolysis reaction proceeded without any by-product formation.
The water-soluble auxiliary 14 was detected in the DMSO
peak area, as verified by comparison with HPLC analysis of
14, which was synthesized separately as a standard.
Fig. 4. Time Course of Hydrolysis of Prodrug 12 and Release of Parent
Drug 1 in 10mM PBS
The percentage was determined by HPLC. Data show the average in three ex-
periments.
Figure 4 shows that 12 decreased over the course of the hy-
drolysis reaction, as measured by HPLC analysis. The less sol-
uble plinabulin (1) was detected, accompanied by observation
of small amounts of its precipitate on the vessel. The half-life of the part also appeared to influence the half-life, although
(t1/2) of 12 was 22.4min, only one-third that of 2. This result the scissile ester bond was away from the soluble part. These
indicated that the water-solubilizing part certainly influenced observations warrant further investigation through optimiza-
the esterase hydrolysis reaction. In both of the prodrugs 2 and tion of the water-solubilizing moiety toward higher water solu-
12, the water-solubilizing moiety was distant from the reactive bility and a proper half-life profile. Our original strategy using
ester, which was expected to have little effect. However, the click chemistry facilitated this optimization, and a variety of
difference in t1/2 indicated that the distinct water-solubilizing water-soluble prodrugs of plinabulin (1) are currently under
moieties significantly influenced the esterase reaction. The consideration.
bulkiness of the polar functional group may contribute to
the hydrolysis rate. The water-solubilizing moiety of 12 was
sterically less hindered than that of 2, which shortened the
Experimental
General Procedures 1H-NMR were measured in CD3OD
half-life. The stereochemistries of the auxiliaries of 2 and 12 solution, and referenced to TMS (0.00ppm) using Varian Mer-
were opposite, which was also capable of having affected the cury-300 NMR (300MHz), Bruker DPX-400 NMR (400MHz),
hydrolysis rate.33–36) The presence of an asymmetric center at Bruker DPX-500 NMR (500MHz) spectrophotometers, unless
the water-solubilizing moiety is generally avoided to reduce otherwise noted. 13C-NMR were measured in CD3OD solution,
structural complexity, however, the stereochemistry of the and referenced to CD3OD (49.0ppm) using Bruker DPX-400
substrate did affect the enzyme reaction,37–40) suggesting that NMR (400MHz) and Bruker DPX-500 NMR (500MHz) spec-
this feature may be significant. In our case, the asymmetric trophotometers. Chemical shifts are reported in ppm (from
center in the water-solubilizing moiety, which was derived TMS). When peak multiplicities are reported, the following
from a natural amino acid, may have adjusted the prodrug abbreviations are used: s, singlet; d, doublet; t, triplet; q, quar-
half-life while improving the water solubility. Additionally, the tet; m, multiplet; br, broadened. Mass spectra were obtained
fact that a simple change in the carbon chain length in pro- on Waters MICRO MASS LCT-premier. Optical rotations
drugs 2 and 12 intriguingly affected their half-life is maybe were determined on JASCO DIP-730. For the preparative pur-
related to the hydrolysis mechanism of the esterase.
pose, HITACHI L-6250 Intelligent Pump were used. Column
PLE, a serine hydrase, reacts with substrates by forming an chromatography was performed on silica gel 60N (spherical,
acyl-enzyme intermediate at the active site.37) Therefore, slight neutral) (40–50µm or 63–210µm), thin layer chromatography
structural differences in the carbon chain and the stereochem- (TLC) was performed on precoated plates (0.25mm, silica gel
istry at the water-solubilizing moiety, which corresponds to Merk Kieselgel 60F245), and compounds were visualized with
“
”
the acid side in the esterase recognition, might be more UV light and phosphomolybdic acid stain. All reactions were
strictly recognized than the parent drug part (the alcohol side). performed in oven-dried glassware under positive pressure of
argon, unless otherwise noted. Reaction mixtures were stirred
magnetically. Solvents were freshly distilled prior to use or
Conclusion
We investigated the water-soluble prodrug of plinabulin purchased from KANTO Kagaku: N,N-dimethylformamide
(1) using a click chemistry strategy focused on the water- (DMF) was purchased from KANTO Kagaku (DMF, Dehy-
solubilizing moiety. L-Carnitine-type derivatives 11, 12 and a drated): methanol (MeOH), ethanol (EtOH) and tert-butanol
galactose-type derivative 13 were designed and synthesized. (tBuOH) were purchased from KANTO Kagaku (MeOH,
t
Among the derivatives, the sodium salt 12 showed relatively EtOH or BuOH, Dehydrated). Esterase was purchased from
high water solubility and proper stability. In comparison with Sigma Chemical Co. (Esterase solution, from porcine liver,
2, we found that increasing the carbon chain length of the suspension in 3.2M (NH4)2SO4 solution, pH 8.0).
water-solubilizing moiety by a single carbon atom affected the
(R)-4-(4-(4-((((3Z,6Z )-6-Benzylidene-3-((5-(tert-
water solubility. In addition, the bulkiness and stereochemistry butyl)-1H-imidazol-4-yl)methylene)-5-oxo-3,4,5,6-tetra-

880
Vol. 60, No. 7
hydropyrazin-2-yl)oxy)methoxy)-4-oxobutyl)-1H-1,2,3-tri- OBDTM 19×150mm (5mm, 12nm), gradient: milli-Q water
azol-1-yl)-3-hydroxybutanoic Acid (11) To a solution of (TFA 0.1%):CH3CN (TFA 0.1%)=30:70 to milli-Q water
alkyne 10 (10mg) in DMF/tBuOH (1:1) (0.60mL) were added (TFA 0.1%):CH3CN (TFA 0.1%)=40:60 over 40min, Flow
a solution of (R)-4-azido-3-hydroxybutanoic acid (4) (2.8mg) rate: 9.00mL/min, UV: 365, 230nm) to give the triazole 13
in H2O (0.30mL), CuSO4·5H2O (5mol%) and sodium ascor- (11mg, 79%). IR (KBr) νmax: 3131, 1749, 1659, 1623, 1586,
1
bate (15mol%) at room temperature. The mixture was stirred 1353, 1204, 1135cm−1; H-NMR (300MHz, CD3OD) δ: 8.27 (s,
°
for 10min at 50 C under microwave irradiation. Then the 1H), 8.09 (s, 1H), 8.07 (s, 1H), 7.88 (s, 1H), 7.40–7.37 (m, 3H),
mixture was cooled to rt and solvent was removed in vacuo. 7.27 (s, 1H), 6.58 (s, 1H), 6.16 (s, 2H), 5.48 (d, J=9.2Hz, 1H),
The residue was diluted with AcOEt and water and extracted 4.10 (t, J=9.4Hz, 1H), 3.97 (d, J=3.2Hz, 1H), 3.81–3.65 (m,
with AcOEt. The extracts were washed with brine, dried over 4H), 2.73 (t, J=7.6Hz, 2H), 2.50 (t, J=7.3Hz, 2H), 2.01–1.96
Na2SO4, filtered and concentrated. The residual mixture was (m, 2H), 1.41 (s, 9H); 13C-NMR (100MHz, CD3OD) δ: 173.62,
purified through reverse-phase HPLC (SunFireTMPrepC18 161.65, 153.26, 148.18, 141.94, 136.04, 134.91, 132.95, 132.48,
OBDTM 19×150mm (5mm, 12nm), gradient: milli-Q water 130.74, 130.56, 129.56, 122.15, 101.07, 90.17, 83.36, 79.92,
(TFA 0.1%):CH3CN (TFA 0.1%)=35:65 to milli-Q water 75.33, 71.41, 70.39, 62.47, 34.04, 33.09, 30.50, 25.46; HR-MS
(TFA 0.1%):CH3CN (TFA 0.1%)=75:25 over 40min, Flow (ESI) m/z Calcd for C32H40N7O9 [M+H]+ 666.2888, Found
rate: 9.00mL/min, UV: 365, 230nm) to give the triazole 11 666.2886.
(10mg, 79%). IR (KBr) νmax: 3180, 3050, 2972, 2925, 1663,
(R)-4-(4-(Carboxymethyl)-1H-1,2,3-triazol-1-yl)-3-hy-
1205, 1134cm−1; H-NMR (500MHz, CD3OD) δ: 8.08 (s, 1H), droxybutanoic Acid (14) 14 was synthesized from 5-hexy-
8.07 (s, 1H), 7.80 (s, 1H), 7.61 (s, 1H), 7.39–7.30 (m, 3H), 7.23 noic acid in 3 steps.12) 1H-NMR (300MHz, CD3OD) δ: 7.81 (s,
(s, 1H), 6.71 (s, 1H), 6.15 (s, 2H), 4.45–4.30 (m, 3H), 2.71 (t, 1H), 4.57–4.49 (m, 1H), 4.44–4.36 (m, 2H), 2.76 (t, J=7.5Hz,
J=7.6Hz, 2H), 2.51–2.47 (m, 3H), 2.39 (dd, J=16, 7.3Hz, 1H), 2H), 2.57–2.43 (m, 2H), 2.35 (t, J=7.3Hz, 2H), 1.96 (quint,
1.99 (quint, J=7.4Hz, 2H), 1.42 (s, 9H); 13C-NMR (125MHz, J=7.5Hz, 2H); HR-MS (ESI) m/z Calcd for C10H16N3O5
CD3OD) δ: 174.26, 173.69, 161.47, 154.03, 147.82, 142.28, [M+H]+ 258.1090, Found 258.1096.
1
136.36, 135.05, 132.98, 132.77, 131.22, 130.15, 129.46, 129.19,
Water Solubility and Stability of Prodrug Derivatives.
124.46, 122.93, 104.18, 83.25, 68.06, 56.29, 40.11, 34.07, Water Solubility Prodrug derivatives were saturated in
33.14, 31.00, 25.57, 25.40; high resolution (HR)-MS (electro- distilled water and shaken vigorously. The saturated solu-
spray ionization (ESI)) m/z: Calcd for C30H36N7O7 [M+H]+ tions were sonicated for 15min at 25 C and filtered through
°
606.2676, Found 606.2704.
the centrifugal filter (0.45nm filter unit, Ultrafree®-MC, Mil-
Sodium (R)-4-(4-(4-((((3Z,6Z)-6-Benzylidene-3-((5-(tert- lipore). The filtrate was injected into HPLC to analyze the
butyl)-1H-imidazol-4-yl)methylene)-5-oxo-3,4,5,6-tetra- water solubility.
hydropyrazin-2-yl)oxy)methoxy)-4-oxobutyl)-1H-1,2,3-tri-
Hydrolysis of 12 in the Presence of Esterase 12 (500µg)
azol-1-yl)-3-hydroxybutanoate (12) The triazole 11 was was dissolved in a 10mM PBS (500µL, pH 7.4) solution and
eluated with a solvent of H2O/CH3CN (1:1) through an ion the solution was separated to three eppendorf tubes by each
exchange resin at room temperature. The eluent was filtrated 50, 50 and 300µL. The two 50µL solutions were used as the
with H2O/CH3CN (1:1) and concentrated. The residue was negative control without the enzyme, one was incubated at rt
°
lyophilized to give the title compound 12 as a yellow solid. and the other was incubated at 37 C. To a solution of 10mM
IR (KBr) νmax: 3141, 3075, 2954, 1647, 1579, 1440, 1396, 1219, PBS (300µL, pH 7.4) was added a suspension of the porcine
1
1126cm−1; H-NMR (500MHz, CD3OD) δ: 8.07 (s, 1H), 8.06 liver esterase (6.0µL) (PLE; carboxylic–ester hydrolase; EC
(s, 1H), 7.65 (s, 1H), 7.63 (s, 1H), 7.39–7.30 (m, 3H), 7.21 (s, 3.1.1.1; E-2884), which is a model esterase having a good cor-
1H), 6.75 (s, 1H), 6.14 (s, 2H), 4.44 (dd, J=15, 2.8Hz, 1H), relation with plasma esterase in the stability assay and was
4.31–4.22 (m, 2H), 2.71 (t, J=7.5Hz, 2H), 2.48 (t, J=7.3Hz, obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.)
2H), 2.36 (dd, J=15, 5.0Hz, 1H), 2.27 (dd, J=15, 7.2Hz, 1H), as a suspension in a 3.2M (NH4)2SO4 solution (pH 8.0) con-
1.99 (quint, J=4.5Hz, 2H), 1.41 (s, 9H); 13C-NMR (125MHz, taining 40 units of enzyme per mL. Then the solution was
CD3OD) δ: 178.83, 173.72, 161.41, 154.24, 147.77, 142.38, separated to six eppendorf tubes by 50µL, followed by incu-
°
136.45, 135.09, 133.12, 132.70, 130.03, 129.42, 128.75, 124.28, bation at 37 C. DMSO (150µL) was added to each tubes and
°
121.83, 105.07, 83.24, 69.22, 56.56, 42.47, 34.10, 33.15, 31.15, the mixtures were sonicated for 15min at 25 C and filtered
25.63, 25.46; HR-MS (ESI) m/z Calcd for C30H35N7O7Na through the centrifugal filter (0.45nm filter unit, Ultrafree®-
[M+H]+ 628.2496, Found 628.2479.
MC, Millipore). The filtrate was injected into HPLC to ana-
(((3Z,6Z)-6-Benzylidene-3-((5-(tert-butyl)-1H-imida- lyze the conversion to the parent drug.
zol-4-yl)methylene)-5-oxo-3,4,5,6-tetrahydropyrazin-2-yl)-
oxy)methyl
4-(1-((2R,3R,4S,5R,6R)-3,4,5-Trihydroxy-6-
Acknowledgments This work was supported by Grants
(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-1H-1,2,3- from MEXT (Ministry of Education, Culture, Sports, Sci-
triazol-4-yl)butanoate (13) To a solution of alkyne 10 ence and Technology) of Japan, including a Grant-in-Aid for
(10mg) in DMF/tBuOH/H2O (1:1:1) (0.40mL) were add- Scientific Research (B) (No. 20390036), a Grant-in-Aid for
ed 1-azide-1-deoxy-β-D-galactopyranoside (6) (8.9mg), Research Activity Start-up (No. 21890262), and a Grant-in-Aid
CuSO4·5H2O (5mol%) and sodium ascorbate (15mol%) at for Young Scientists (B) (No. 21790118).
°
room temperature. The mixture was stirred for 10min at 50 C
under microwave irradiation. Then the mixture was cooled to
rt and solvent was removed in vacuo. The residue was filtered
through silica gel pad and concentrated. The residual mixture
was purified through reverse-phase HPLC (SunFireTMPrepC18
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