Synthesis of sunitinib-metastin conjugate, a novel esterase-sensitive prodrug system based on lactonization reaction

Article abstract of DOI:10.3987/COM-14-13042

We describe a strategy for preparing sunitinib-metastin conjugate, a prodrug composed of the anticancer agent sunitinib for renal cell carcinoma and the carrier protein metastin, which are conjugated to each other by a linker. We designed a modified L-homoserine linker, which is composed of an acyl group that acts as the masking group for hydrolysis with an esterase, as well as a carbon chain of appropriate length between sunitinib and metastin. The sunitinib-metastin conjugate was converted into a hydrolyte by hydrolysis of the acyl group with an esterase, and sunitinib was released by intramolecular lactonization. Sunitinib-metastin conjugate, an esterase-sensitive amide prodrug that has a modified L-homoserine linker that participates in the intramolecular lactonization, was synthesized.

Full text of DOI:10.3987/COM-14-13042

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HETEROCYCLES, Vol. 89, No. 8, 2014
HETEROCYCLES, Vol. 89, No. 8, 2014, pp. 1860 - 1876. © 2014 The Japan Institute of Heterocyclic Chemistry
Received, 12th June, 2014, Accepted, 4th July, 2014, Published online, 11th July, 2014
DOI: 10.3987/COM-14-13042
SYNTHESIS OF SUNITINIBMETASTIN CONJUGATE, A NOVEL
ESTERASE-SENSITIVE PRODRUG SYSTEM BASED ON
LACTONIZATION REACTION
a
b
a
a
Yuki Takahashi, Sunao Shoji, Takuya Morishige, Aya Katsumata,
a
a
a
Fumihiro Tsurifune, Mitsuhiro Tsutsumi, Yoshiharu Honda, Tomoyo
a
a
c
b
Hasuda, Yukio Hitotsuyanagi, Toshiro Terachi, Toyoaki Uchida, and
a,
Koichi Takeya *
a
School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1
b
Horinouchi, Hachioji, Tokyo 192-0392, Japan, Department of Urology, Tokai
University Hachioji Hospital, 1838 Ishikawa-machi, Hachioji, Tokyo 192-0032,
c
Japan, Department of Urology, Tokai University School of Medicine, 143
Shimokasuya, Isehara, Kanagawa 259-1193, Japan; E-mail: takeyak@toyaku.ac.jp
Abstract – We describe a strategy for preparing sunitinibmetastin conjugate, a
prodrug composed of the anticancer agent sunitinib for renal cell carcinoma and
the carrier protein metastin, which are conjugated to each other by a linker. We
designed a modified L-homoserine linker, which is composed of an acyl group that
acts as the masking group for hydrolysis with an esterase, as well as a carbon chain
of appropriate length between sunitinib and metastin. The sunitinibmetastin
conjugate was converted into a hydrolyte by hydrolysis of the acyl group with an
esterase, and sunitinib was released by intramolecular lactonization.
Sunitinibmetastin conjugate, an esterase-sensitive amide prodrug that has a
modified L-homoserine linker that participates in the intramolecular lactonization,
was synthesized.
INTRODUCTION
1
,2
The KISS-1 gene is located on human chromosome lq32. KISS-1 encodes a C-terminus peptide with 54
amino acid residues called metastin (kisspeptin), which is the ligand of the G-protein-coupled metastin

HETEROCYCLES, Vol. 89, No. 8, 2014
1861
3
receptor. Both metastin and the metastin receptor inhibit tumor invasion or migration through focal
adhesion kinase, paxillin, MAP kinase or Rho A, and have been implicated in melanoma, thyroid cancer,
esophageal squamous cell carcinoma, hepatocellular carcinoma, pancreatic carcinoma, breast cancer,
ovarian cancer, renal cell carcinoma (RCC), upper tract urothelial carcinoma, bladder cancer, and prostate
4
-6
cancer. Metastin and the metastin receptor are expressed in the hypothalamus, brainstem, spinal cord,
pituitary, ovary, prostate, and placenta in normal human tissue, and play a pivotal role in the control of
7
the hypothalamic-pituitary-gonadal axis via regulation of gonadotropin-releasing hormone secretion.
Moreover, it is reported that the metastin receptor is overexpressed in human RCC, and that metastin and
8
the metastin receptor are probable targets for suppressing RCC metastasis.
Drug delivery systems (DDSs), which are engineered technologies for the targeted delivery and
controlled release of a therapeutic agent, have been the focus of intensive studies. The delivery of an
anticancer agent directly and locally to an affected part of the body is expected to yield high curative
effects without producing any side effects. Generally, anticancer agents show low selectivity for tumor
sites, the development of anticancer agents having a protein or a monoclonal antibody that shows affinity
for tumor sites, and the control of their pharmacokinetics in vivo have been carried out. As the interaction
between a ligand and a receptor has high specificity, and the development of a prodrug that uses metastin
as the carrier protein would be a valuable contribution to the treatment of RCC. Although sunitinib is the
active drug for RCC treatment as tyrosine kinase inhibitor, there are many systemic side effects. As far as
we know, there is no report of a prodrug that uses metastin as the carrier protein and sunitinib. In order to
reduce the risk of systemic side effects of sunitinib, we decided to synthesize sunitinibmetastin
conjugate with metastin as the carrier protein. To this end, we embarked on the synthesis of the
sunitinibmetastin conjugate and examined its potential for use as a prodrug for RCC treatment.
The design concept of the linker between sunitinibandmetastin was considered as follows. Sunitinib
should be released upon lactonization of the hydrolyte produced by the esterase hydrolysis of the
9
sunitinibmetastin conjugate (Scheme 1). Therefore, the linker should have an acyl group as the masking
group, which should be removed by esterase hydrolysis in vivo, and should form a-lactone by
intramolecular lactonization. Moreover, if the linker has a short carbon chain between sunitinib and
metastin, the proximity of metastin to sunitinib would inhibit the binding of metastin to the metastin
receptor. Accordingly, we decided to conjugate metastin to the linker terminus with an extended carbon
chain. Finally, we designed a modified L-homoserine linker, which is composed of an acyl group that acts
as the masking group for hydrolysis with an esterase, as well as a carbon chain of appropriate length
between sunitinib and metastin.

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HETEROCYCLES, Vol. 89, No. 8, 2014
masking
group
sunitinib
O
O
sunitinib
O
OH
esterase
O
O
HN
CONH
HN
O
CONH
11
11
metastin
metastin
O
O
prodrug
hydrolyte
O
O
lactonization
sunitinib
＋
HN
O
CONH
11
metastin
O
Scheme 1. Sunitinib releasing model through lactonization of the L-homoserine linker
We initially investigated the binding position of sunitinib to the modified L-homoserine linker and the
optimization of the acyl group as the masking group. Because the structure of sunitinib made it difficult to
bind to the linker, we decided to connect the linker to the nitrogen atom of the oxyindole ring of sunitinib.
Furthermore, the linker terminus, which was connected to metastin, had to be a carboxylic acid. Therefore,
the linker was synthesized by using an acetyl or an isobutyryl group as the acyl group, and then connected
to sunitinib. Compounds having an acetyl or an isobutyryl group as the masking group were evaluated for
the release of sunitinib by enzyme hydrolysis under physiological conditions in vitro. An aliquot of the
reaction mixture was subjected to HPLC to monitor the reaction. The synthesis of the amide prodrug,
sunitinibmetastin conjugate, which has a modified L-homoserine linker, is described in detail below.
RESULTS AND DISCUSSION
The linker having an acetyl group or an isobutyryl group as the masking group was synthesized by the
following procedure (Scheme 2). Compounds 6 and 7 were synthesized from commercially available
Boc-L-homoserine. Boc-L-homoserine was converted into benzyl ester 1, which was then treated with
acetic anhydride in pyridine to provide acetate 2. The benzyl group of 2 was removed with hydrogen gas
and 10% Pd/C, and the resultant carboxylic acid was treated with pentafluorophenol in the presence of
2 2
N,N'-dicyclohexylcarbodiimide (DCC) in CH Cl to give activated ester 4. Treatment of 4 with sunitinib
in N,N-dimethylformamide (DMF) afforded compound 6. Compound 7, which possesses an isobutyryl

HETEROCYCLES, Vol. 89, No. 8, 2014
1863
group as the masking group, was synthesized in a similar way from compound 1 via compound 3 using
isobutyryl chloride instead of acetic anhydride.
OH
OH
OR
i) NaOH, EtOH, 24 h
Ac2O or isobutyryl chloride
pyridine, 12 h or 9 h
ii) benzyl bromide, DMF, 46 h
Y. 86% (2 steps)
BocHN
CO H
BocHN
2: R = Ac Y. 84%
: R = isobutyryl Y. 81%
CO Bn
2
BocHN
CO Bn
2
2
Boc-L-homoserine
1
3
O
N
OR
H
N
i) H , 10% Pd/C
MeOH, 30 min
2
N
sunitinib
F
H
ii) pentafluorophenol, DCC
CH Cl , 23 h
DMF, 5 days
O
BocHN
CO Pfp
2
N
2
2
4
5
: R = Ac Y. 76%
: R = isobutyryl Y. 48%
OR
O
NHBoc
6
: R = Ac Y. 25%
7
: R = isobutyryl Y. 45%
Scheme 2
Compounds 6 and 7 were evaluated to determine whether sunitinib is released by the intramolecular
lactonization of the hydrolyte obtained, by hydrolysis with porcine liver esterase (PLE) (Scheme 3).

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HETEROCYCLES, Vol. 89, No. 8, 2014
First, compound 6 was converted into the malic acid salt, and this was dissolved in phosphate-buffered
saline (PBS, pH 7.4) and left to stand at 36.6 °C with or without PLE. The reaction was monitored by
HPLC equipped with a photodiode array detector to measure the absorption spectrum of sunitinib at the
1
0
maximum absorption wavelength of 431 nm (Figures 1 and 2).

HETEROCYCLES, Vol. 89, No. 8, 2014
1865
The t
R
values for sunitinib, compound 6, and compound 7 were 8, 20, and 16 min, respectively. It was
observed that compound 6 having an acetyl group released sunitinib via the hydrolyte in the absence of

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HETEROCYCLES, Vol. 89, No. 8, 2014
PLE at 3 h after the start of the reaction (Figure 1A). In the case of compound 7 having an isobutyryl
group, sunitinib was released slightly in the absence of PLE at 8 h after the start of the reaction, and was
released in the presence of PLE at 3 h after the start of the reaction (Figure 2). The results indicated that
the isobutyryl group was a more suitable masking group than the acetyl group.

HETEROCYCLES, Vol. 89, No. 8, 2014
1867
Sunitinibmetastin conjugate 14 was synthesized by the following procedure (Scheme 4).
1
0-Undecenoic acid was treated with tert-butanol, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC·HCl), and 4-(N,N-dimethylamino)pyridine (DMAP) in THF to provide compound 9.
The Boc group of compound 3 was removed by treating 3 with CHCl /trifluoroacetic acid (TFA) (2.5:1),
and the product was reacted with allyl chloroformate in 1,4-dioxane/saturated aqueous NaHCO (3:1) to
3
3
afford compound 8. Compound 8 was reacted with compound 9 in the presence of 0.2 molar equiv of
nd
Grubbs 2 generation catalyst in CH
2
Cl
2
to yield compound 10. The benzyl group of compound 10 was
removed by treating 10 under an atmosphere of hydrogen with 10% Pd/C, and the product was then
treated with pentafluorophenol and DCC in CH Cl to provide compound 11. Compound 11 was mixed
2
2
with sunitinib in DMF at room temperature for two days to obtain compound 12. The t-butyl group of
compound 12 was removed by treating 12 with CHCl /TFA (4:1), and the product was then treated with
pentafluorophenol, EDC·HCl, and DMAP in CH Cl to provide compound 13. Compound 13 was mixed
3
2
2
with metastin in DMF at room temperature for five days to furnish sunitinibmetastin conjugate 14.
The properties of conjugate 14 were determined by ESI-TOF-MS analysis. Figure 3 shows the
ESI-TOF-MS spectrum of conjugate 14.
Figure 3. ESI-TOF-MS spectrum of compound 14

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HETEROCYCLES, Vol. 89, No. 8, 2014
The theoretical average m/z values for the individual charge states are given in the table. The
ESI-TOF-MS spectrum of conjugate 14 showed multiply charged ions at m/z 1031.2981, 1178.1360, and
1
374.0596. When the conjugate has three sunitinib-linker molecules per unit molecule of metastin, the
8
+
7+
6+
values for m/z 1031.2981, 1178.1360, and 1374.0596 were [M+8H] , [M+7H] , and [M+6H] ,
respectively. The results suggest that conjugate 14 is composed of about three sunitinib-linker molecules
per unit molecule of metastin.
EXPERIMENTAL
General Experimental Procedures
Thin layer chromatography (TLC) was performed on Merck precoated silica gel 60 F254 plates. Optical
rotations were measured on a JASCO P-1030 digital polarimeter. IR spectra were recorded on a JASCO
1
FT/IR 620 spectrophotometer, and NMR spectra, on a Bruker AV-600 spectrometer (600 MHz for H
1
3
1
NMR, 150 MHz for C NMR). H chemical shifts in CDCl were referenced to residual CHCl (7.26
3
3
1
3
3
ppm), and C chemical shifts, to CDCl (77.03 ppm). Mass spectra were recorded on a Waters
Micromass LCT spectrometer. Analytical HPLC was carried out on a PU-980 pump unit (JASCO)
equipped with a multiwavelength detector MD-910, and a NUCLEODUR 3CN column (3 m, 4.6 × 150
mm, Chemco Inc.). Preparative HPLC was carried out on a PU-986 pump unit (JASCO) equipped with a
UV-970 detector (254 nm), and a CAPCELL PAK TYPE UG80 column (5 m, 20 × 250 mm, Shiseido
Co., Ltd.).
(
S)-Benzyl 2-((tert-butoxycarbonyl)amino)-4-hydroxybutanoate (1): Boc-L-homoserine (300 mg, 1.3
mmol) was dissolved in EtOH (2.0 mL) to prepare a solution, to which 2.0 mol/L aqueous NaOH (690 L,

HETEROCYCLES, Vol. 89, No. 8, 2014
1869
1
.4 mmol) was added. The mixture was stirred at room temperature for 24 h. The solvent was removed in
vacuo, and the residue was dissolved in DMF (400 L) together with benzyl bromide (327 L, 2.7 mmol).
The mixture was stirred at room temperature for 46 h. The solvent was removed in vacuo and the residue
was dissolved in CHCl
whole was extracted with CHCl
mL), dried over Na SO , and filtered, and the solvent was removed in vacuo. The residue was subjected
to column chromatography (silica gel, 2:1 hexane/EtOAc) to afford 1 (345 mg, 86%) as a colorless
3
(10 mL). Saturated aqueous NaHCO
3
(10 mL) was added to the solution, and the
3
(3 × 10 mL). The combined CHCl
3
extracts were washed with brine (10
2
4
2
5
-1 1
amorphous gum: [] -38.5 (c 0.21, MeOH); IR (film)  3356, 1777, 1685, 1532, 1163 cm . H NMR
D
(600 MHz, CDCl
3
) 7.397.36 (4H, m), 7.327.29 (1H, m), 5.10 (1H, br s), 4.71 (2H, s), 4.45 (1H, t, J =
8
1
.8 Hz), 4.36 (1H, br s), 4.25 (1H, ddd, J = 11.3, 9.4, 5.8 Hz), 2.672.62 (1H, m), 2.112.06 (1H, m),
1
3
.47 (9H, s); C NMR (150 MHz, CDCl ) 175.3 (s), 155.5 (s), 140.9 (s), 128.6 × 2 (d), 127.7 (d), 127.0
3
×
2 (d), 80.7 (s), 65.8 (t), 65.4 (t), 50.2 (d), 30.7 (t), 28.3 × 3 (q); HR-ESIMS m/z calcd for C H NO
16 24 5
+
[
M+H] 310.1654, found 310.1635.
(
S)-Benzyl 4-acetoxy-2-((tert-butoxycarbonyl)amino)butanoate (2): A solution of 1 (23.8 mg, 0.077
mmol) in pyridine (2.0 mL) was treated with acetic anhydride (1.0 mL). The mixture was stirred at room
temperature for 12 h. The solvent was removed in vacuo and the residue was dissolved in CHCl (10 mL).
Saturated aqueous NaHCO (10 mL) was added to the solution, and the whole was extracted with CHCl
3 × 10 mL). The combined CHCl
3
3
3
(
3
extracts were washed with brine (10 mL), dried over Na
2
SO
4
, and
filtered, and the solvent was removed in vacuo. The residue was subjected to column chromatography
1
(
silica gel, 3:1 hexane/EtOAc) to afford 2 (22.7 mg, 84%) as a colorless amorphous gum: H NMR (600
MHz, CDCl )  7.427.33 (5H, m), 5.19 (2H, s), 5.15 (1H, d, J = 11.8 Hz), 4.484.44 (1H, m), 4.19 (1H,
3
dq, J = 12.3, 6.8 Hz), 4.12 (1H, dq, J = 12.3, 6.8 Hz), 2.232.17 (1H, m), 2.092.02 (1H, m), 2.01 (3H, s),
1
3
1
.45 (9H, s); C NMR (150 MHz, CDCl )  172.1 (s), 170.8 (s), 155.3 (s), 135.3 (s), 128.7 × 2 (d), 128.5
3
(
d), 128.3 × 2 (d), 80.1 (s), 67.3 (t), 60.4 (t), 51.0 (d), 31.3 (t), 28.3 × 3 (q), 20.8 (q); HR-ESIMS m/z
+
calcd for C18
H
26NO
6
[M+H] 352.1760, found 352.1763.
(
S)-Benzyl 2-((tert-butoxycarbonyl)amino)-4-(isobutyryloxy)butanoate (3): To a solution of 1 (413
mg, 1.34 mmol) in pyridine (400 L) was added isobutyryl chloride (141 L, 1.34 mmol), and the
mixture was stirred at room temperature for 9 h. The solvent was removed in vacuo and the residue was
dissolved in CHCl (10 mL). Saturated aqueous NaHCO (20 mL) was added to the solution, and the
3
3
whole was extracted with CHCl
3
3
(3 × 10 mL). The combined CHCl extracts were washed with brine (20
mL), dried over Na
2 4
SO , and filtered, and the solvent was removed in vacuo. The residue was subjected
to column chromatography (silica gel, 3:1 hexane/EtOAc) to afford 3 (413 mg, 81%) as a colorless
2
5
-1 1
amorphous gum: [] -26.6 (c 0.26, MeOH); IR (film)  3369, 2975, 1715, 1516, 1366, 1159 cm . H
D

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HETEROCYCLES, Vol. 89, No. 8, 2014
NMR (600 MHz, CDCl
3
)  7.407.35 (5H, m), 5.19 (2H, s), 5.165.13 (1H, m), 4.48 (1H, q, J = 6.2 Hz),
4
2
.194.14 (1H, m), 4.11 (1H, dd, J = 12.4, 5.3 Hz), 2.51 (1H, quint, J = 6.8 Hz), 2.222.17 (1H, m),
1
3
.061.99 (1H, m), 1.45 (9H, s), 1.16 (6H, d, J = 6.8 Hz); C NMR (150 MHz, CDCl )  176.9 (s), 172.1
3
(
(
s), 155.2 (s), 135.2 (s), 128.6 × 2 (d), 128.5 (d), 128.3 × 2 (d), 80.1 (s), 67.3 (t), 60.4 (t), 51.1 (d), 33.9
+
d), 31.2 (t), 28.3 × 3 (q), 18.9 × 2 (q); HR-ESIMS m/z calcd for C20
80.2074.
S)-Pentafluorophenyl 4-acetoxy-2-((tert-butoxycarbonyl)amino)butanoate (4): 10% Pd/C (44.0 mg)
H30NO
6
[M+H] 380.2073, found
3
(
was added to a solution of 2 (44.0 mg, 0.13 mmol) in MeOH (2.0 mL), and the mixture was stirred at
room temperature under an atmosphere of hydrogen for 30 min. The catalyst was filtered off, and the
filtrate was concentrated to dryness. The residue was dissolved in CH
pentafluorophenol (116 mg, 0.63 mmol) and DCC (36.4 mg, 0.18 mmol). The mixture was stirred at room
temperature for 23 h. Saturated aqueous NaHCO (30 mL) was added, and the whole was extracted with
4
CHCl (3 × 10 mL). The combined CHCl extracts were washed with brine (30 mL), dried over Na SO ,
2 2
Cl (500 L) together with
3
3
3
2
and filtered, and the solvent was removed in vacuo. The residue was subjected to column chromatography
1
(
silica gel, 4:1 hexane/EtOAc) to afford 4 (42.3 mg, 76%) as a colorless amorphous gum: H NMR (600
MHz, CDCl )  5.19 (1H, d, J = 7.3 Hz), 4.784.77 (1H, m), 4.314.26 (1H, m), 4.21 (1H, dq, J = 12.3,
3
1
3
7

6
4
3
.3 Hz), 2.412.33 (1H, m), 2.242.16 (1H, m), 2.01 (3H, s), 1.46 (9H, s); C NMR (150 MHz, CDCl )
170.8 (s), 168.7 (s), 155.1 (s), 141.8 (d), 140.6 (d), 140.2 (d), 138.8 (d), 137.1 (d), 124.7 (d), 80.8 (s),
+
0.0 (t), 50.9 (d), 30.9 (t), 28.2 × 3 (q), 20.8 (q); HR-ESIMS m/z calcd for C H F NO Na [M+Na]
1
7
18
5
6
50.0952, found 450.0942.
(
S)-Pentafluorophenyl 2-((tert-butoxycarbonyl)amino)-4-(isobutyryloxy)butanoate (5): 10% Pd/C
102 mg) was added to a solution of 3 (151 mg, 0.40 mmol) in MeOH (6.1 mL), and the mixture was
(
stirred at room temperature under an atmosphere of hydrogen for 30 min. The catalyst was filtered off,
and the filtrate was concentrated to dryness. The residue was dissolved in CH Cl (1.5 mL) together with
2
2
pentafluorophenol (355 mg, 1.93 mmol) and DCC (172 mg, 0.83 mmol). The mixture was stirred at room
temperature for 20 h. Saturated aqueous NaHCO (30 mL) was added, and the whole was extracted with
3
CHCl
and filtered, and the solvent was removed in vacuo. The residue was subjected to column chromatography
silica gel, 4:1 hexane/EtOAc) and then HPLC (60:40 MeCN/H O) to afford 5 (87.7 mg, 48%) as a
3 3 2 4
(3 × 10 mL). The combined CHCl extracts were washed with brine (30 mL), dried over Na SO ,
(
2
2
5
colorless amorphous gum: [] -20.3 (c 0.55, CHCl ); IR (film)  3359, 2978, 1794, 1718, 1521, 1157
D
3
-1 1
cm . H NMR (600 MHz, CDCl
3
)  5.21 (1H, br d, J = 8.0 Hz), 4.77 (1H, dd, J = 11.5, 6.4 Hz), 4.28 (1H,
quint, J = 5.6 Hz), 4.224.19 (1H, m), 2.56 (1H, sept, J = 7.0 Hz), 2.382.36 (1H, m), 2.202.17 (1H, m),
1
3
1
3
.45 (9H, s), 1.176 (3H, d, J = 7.0 Hz), 1.175 (3H, d, J = 7.0 Hz); C NMR (150 MHz, CDCl ) 176.9

HETEROCYCLES, Vol. 89, No. 8, 2014
1871
(
s), 168.6 (s), 155.0 (s), 141.9 (s), 141.8 (s), 140.2 (s), 138.8 (s), 137.1 × 2 (s), 80.8 (s), 60.1 (t), 51.1 (d),
+
22 5 6
33.9 (d), 30.9 (t), 28.2 × 3 (q), 18.89 (q), 18.86 (q); HR-ESIMS m/z calcd for C19H F NO Na [M+Na]
4
78.1265, found 478.1257.
(
S,Z)-3-((tert-Butoxycarbonyl)amino)-4-(3-((4-((2-(diethylamino)ethyl)carbamoyl)-3,5-dimethyl-1H-
pyrrol-2-yl)methylene)-5-fluoro-2-oxoindolin-1-yl)-4-oxobutyl acetate (6): Free sunitinib was
obtained by the following operations. A solution of sunitinib malate in CHCl (30 mL) was treated with
aqueous NaOH (1.0 mol/L, 30 mL), washed with brine (30 mL), dried over Na SO , and filtered, and the
3
2
4
solvent was removed in vacuo. Compound 4 (10.2 mg, 0.024 mmol) and sunitinib (5.4 mg, 0.014 mmol)
were dissolved in DMF (200 μL), and the mixture was stirred at room temperature for five days. The
solvent was removed in vacuo, and the residue was subjected to column chromatography (silica gel, 7:1
1
CHCl
3
/MeOH) to afford 6 (2.2 mg, 25%) as an orange amorphous solid: H NMR (600 MHz, CDCl
3
) 
1
2.74 (1H, br s), 8.18 (1H, dd, J = 8.9, 4.7 Hz), 7.40 (1H, s), 7.19 (1H, dd, J = 8.4, 2.6 Hz), 6.92 (1H, td,
J = 8.9, 2.6 Hz), 6.65 (1H, br s), 5.92 (1H, br s), 5.57 (1H, br d, J = 7.6 Hz), 4.28 (2H, dq, J = 26.0, 5.9
Hz), 3.543.49 (2H, m), 2.73 (2H, br s), 2.632.59 (4H, m), 2.53 (3H, s), 2.42 (3H, s), 2.362.31 (1H, m),
1
3
2

1
3
.091.97 (1H, m), 1.94 (3H, s), 1.56 (9H, s), 1.08 (6H, t, J = 7.0 Hz); C NMR (150 MHz, CDCl )
173.3 (s), 170.8 × 2 (s), 168.2 (s), 160.1 (s), 159.6 (s), 155.5 (s), 140.1 (s), 133.8 (d), 128.3 (s), 126.5 (s),
25.2 (s), 120.8 (s), 117.8 (d), 113.2 (d), 111.1 (s), 103.9 (d), 80.2 (s), 60.9 (t), 52.6 (d), 51.4 (t), 46.6 × 2
(
[
(
t), 36.7 (t), 32.0 (t), 28.4 × 3 (q), 20.9 (q), 14.4 (q), 11.5 × 3 (q); HR-ESIMS m/z calcd for C H FN O
33 45 5 7
+
M+H] 642.3303, found 642.3317.
S,Z)-3-((tert-Butoxycarbonyl)amino)-4-(3-((4-((2-(diethylamino)ethyl)carbamoyl)-3,5-dimethyl-1H-
pyrrol-2-yl)methylene)-5-fluoro-2-oxoindolin-1-yl)-4-oxobutyl isobutyrate (7): Compound 5 (14.6 mg,
.032 mmol) and sunitinib (7.9 mg, 0.020 mmol) were dissolved in DMF (300 μL), and the mixture was
stirred at room temperature for five days. The solvent was removed in vacuo, and the residue was
subjected to column chromatography (silica gel, 7:1 CHCl /MeOH) to afford 7 (6.0 mg, 45%) as an
0
3
2
5
-1 1
orange amorphous solid: [] +42.8 (c 0.23, CHCl
3
); IR (film)  2974, 1698, 1570, 1521, 1474 cm . H
)  12.74 (1H, s), 7.93 (1H, br t, J = 5.7 Hz), 7.31 (1H, s), 7.15 (1H, dd, J = 8.7,
.5 Hz), 6.83 (1H, td, J = 8.5, 2.5 Hz), 6.79 (1H, dd, J = 8.5, 4.4 Hz), 5.955.85 (1H, m), 5.33 (1H, br d, J
D
NMR (600 MHz, CDCl
3
2
=
6.8 Hz), 4.174.15 (1H, m), 4.12 (2H, t, J = 6.7 Hz), 3.82 (2H, t, J = 5.7 Hz), 3.24 (1H, t, J = 5.7 Hz),
3
.12 (4H, q, J = 7.3 Hz), 2.55 (3H, s), 2.512.49 (1H, m), 2.49 (3H, s), 2.172.11 (1H, m), 1.941.91 (1H,
m), 1.41 (9H, s), 1.36 (3H, t, J = 7.3 Hz), 1.35 (3H, t, J = 7.3 Hz), 1.13 (3H, d, J = 6.8 Hz), 1.12 (3H, d, J
13
=
3
6.8 Hz); C NMR (150 MHz, CDCl ) 177.1 (s), 176.5 (s), 166.5 (s), 159.9 (s), 158.3 (s), 155.6 (s),
1
38.9 (s), 133.3 (s), 131.0 (s), 128.8 (s), 124.2 (d), 120.1 (s), 114.3 (s), 112.8 (s), 112.7 (d), 109.9 (d),
05.1 (d), 79.2 (s), 61.4 (t), 52.7 (t), 52.4 (d), 48.0 × 2 (d), 35.8 (t), 35.6 (t), 33.9 (d), 28.4 × 3 (q), 19.9 ×
1

1872
HETEROCYCLES, Vol. 89, No. 8, 2014
+
2
6
5 7
(q), 14.2 (q), 11.3 (q), 8.6 × 2 (q); HR-ESIMS m/z calcd for C35H49FN O [M+H] 670.3616, found
70.3611.
(
S)-Benzyl 2-(((allyloxy)carbonyl)amino)-4-(isobutyryloxy)butanoate (8): A solution of 3 (284 mg,
.75 mmol) in CHCl /TFA (2.5:1, 3.5 mL) was stirred at room temperature for 30 min. The solvent was
0
3
removed in vacuo, and the residue was dissolved in 1,4-dioxane (2.1 mL) together with allyl
chloroformate (159 L, 1.50 mmol). The mixture was stirred at room temperature for 5 min, and saturated
3
NaHCO solution (0.70 mL) was added. Then, the mixture was stirred at room temperature for 10 h.
Aqueous HCl (1 M, 20 mL) was added, and the whole was extracted with CHCl (3 × 10 mL). The
3
combined CHCl
3 2 4
extracts were washed with brine (10 mL), dried over Na SO , and filtered, and the
solvent was removed in vacuo. The residue was subjected to column chromatography (silica gel, 5:1:1
2
5
CHCl /hexane/EtOAc) to afford 8 (216 mg, 80%) as a colorless oil: [] +0.4 (c 0.24, CHCl ); IR (film)
3
D
3
-
1 1

3350, 2974, 1730, 1528, 1157 cm . H NMR (600 MHz, CDCl
J = 5.5 Hz), 5.89 (1H, d, J = 7.6 Hz), 5.30 (1H, d, J = 17.3 Hz), 5.21 (1H, d, J = 8.7 Hz), 5.18 (2H, d, J =
.2 Hz), 4.56 (2H, d, J = 5.5 Hz), 4.53 (1H, dd, J = 12.8, 7.6 Hz), 4.17 (1H, dd, J = 12.8, 7.0 Hz), 4.09
1H, dd, J = 11.7, 7.0 Hz), 2.48 (1H, sept, J = 7.0 Hz), 2.22 (1H, sept, J = 7.0 Hz), 2.06 (1H, sept, J = 7.0
3
)  7.387.32 (5H, m), 5.89 (1H, octet,
4
(
13
Hz), 1.136 (3H, d, J = 7.0 Hz), 1.134 (3H, d, J = 7.0 Hz); C NMR (150 MHz, CDCl
3
)  176.8 (s), 171.7
(
(
s), 155.7 (s), 135.1 (s), 132.5 (d), 128.6 × 2 (d), 128.5 (d), 128.2 × 2 (d), 117.9 (t), 67.4 (t), 65.9 (t), 60.2
+
6
t), 51.5 (d), 33.8 (d), 31.2 (d), 18.9 × 2 (q); HR-ESIMS m/z calcd for C19H25NO Na [M+Na] 386.1580,
found 386.1576.
tert-Butyl undec-10-enoate (9): 10-Undecenoic acid (500 mg, 2.7 mmol) was dissolved in THF (0.5 mL)
together with tert-butanol (1.1 mL, 11.6 mmol) and DMAP (33.2 mg, 0.27 mmol), and EDC·HCl (522 mg,
2
.7 mmol) was added at 0 °C. The mixture was stirred at this temperature for 1 h, and then at room
temperature for 20 h. Saturated aqueous NaHCO (10 mL) was added, and the mixture was extracted with
CHCl (3 × 10 mL). The combined CHCl extracts were washed with brine (10 mL), dried over Na SO ,
3
3
3
2
4
and filtered, and the solvent was removed in vacuo. The residue was subjected to column chromatography
2
5
(
silica gel, 20:1 hexane/EtOAc) to afford 9 (355 mg, 55%) as a colorless oil: [] -3.6 (c 0.18, CHCl
3
);
D
-1 1
IR (film)  3077, 2978, 2927, 2855, 1732, 1640 cm . H NMR (600 MHz, CDCl )  5.80(1H, ddt, J =
3
1
7.1, 10.2, 6.7 Hz), 4.98 (1H, dd, J = 17.1, 3.7 Hz), 4.91 (1H, d quint, J = 10.2, 1.2 Hz), 2.19 (2H, t, J =
.5 Hz), 2.03 (2H, qt, J = 6.7, 1.5 Hz), 1.56 (2H, quint, J = 7.5 Hz), 1.44 (9H, s), 1.36 (2H, quint, J = 7.5
7
1
3
Hz), 1.28 (8H, br s); C NMR (150 MHz, CDCl
3
)  173.3 (s), 139.1 (d), 114.1 (t), 79.9 (s), 35.6 (t), 33.8
t), 29.3 × 2 (t), 29.2 (t), 29.0 (t), 28.9 (t), 28.1 × 3 (q), 25.1 (t); HR-ESIMS m/z calcd for C H O
2
(
[
15
29
+
M+H] 241.2168, found 241.2168.

HETEROCYCLES, Vol. 89, No. 8, 2014
1873
(
S,E)-tert-Butyl
1
2-(((1-(benzyloxy)-4-(isobutyryloxy)-1-oxobutan-2-yl)carbamoyl)oxy)dodec-10-enoate (10):
2 2
Compounds 8 (122 mg, 0.33 mmol) and 9 (161 mg, 0.67 mmol) were dissolved in CH Cl (434 μL)
nd
together with Grubbs 2 generation catalyst (28.4 mg, 0.033 mmol), and the mixture was stirred at 55 °C
under an atmosphere of argon for 45 h. The solvent was removed in vacuo, and the residue was dissolved
®
in CH
2
Cl
2
. Chromatorex NH silica gel (500 mg) was added to the solution, and the mixture was stirred
at room temperature for 1 h. Insoluble material was filtered off, and the filtrate was concentrated to
dryness. The residue was subjected to HPLC (85:15 MeOH/H O) to provide 10 (54.9 mg, 29%) as a
2
2
5
-1 1
colorless oil: [] +2.3 (c 0.29, CHCl
3
); IR (film)  3354, 2974, 2929, 2857, 1730, 1154 cm . H NMR
)  7.387.31 (5H, m), 5.74 (1H, quint, J = 7.0 Hz), 5.53 (1H, quint, J = 7.0 Hz), 5.37
1H, d, J = 7.9 Hz), 5.17 (2H, dd, J = 12.5, 4.5 Hz), 4.534.51 (1H, m), 4.49 (2H, d, J = 7.0 Hz), 4.16 (1H,
D
(
600 MHz, CDCl
3
(
dq, J = 11.7, 6.9 Hz), 4.08 (1H, dq, J = 12.1, 6.9 Hz), 2.47 (1H, sept, J = 6.8 Hz), 2.232.21 (1H, m), 2.18
2H, t, J = 7.6 Hz), 2.0682.006 (1H, m), 2.02 (2H, q, J = 6.9 Hz), 1.56 (2H, quint, J = 7.0 Hz), 1.43 (9H,
(
13
s), 1.361.34 (2H, m), 1.27 (8H, br s), 1.129 (3H, d, J = 7.0 Hz), 1.128 (3H, d, J = 7.0 Hz); C NMR
150 MHz, CDCl ) 176.8 (s), 173.3 (s), 171.8 (s), 155.8 (s), 136.4 (d), 135.1 (s), 128.6 × 2 (d), 128.5 (d),
(
3
1
2
28.2 × 2 (d), 123.9 (d), 79.8 (s), 67.3 (t), 66.1 (t), 60.2 (t), 51.4 (d), 35.6 (t), 33.8 (d), 32.2 (t), 31.2 (t),
9.25 (t), 29.20 (t), 29.08 (t), 29.04 (t), 28.1 × 3 (q), 25.1 (t), 18.88 (q), 18.79 (q); HR-ESIMS m/z calcd
+
for C32
H49NO
8
Na [M+Na] 598.3356, found 598.3350.
(
S)-tert-Butyl
1
1
2-(((4-(isobutyryloxy)-1-oxo-1-(pentafluorophenoxy)butan-2-yl)carbamoyl)oxy)dodecanoate (11):
0% Pd/C (5.0 mg) was added to a solution of 10 (36.5 mg, 0.063 mmol) in CH Cl /EtOAc (2:1, 3.0 mL),
2
2
and the mixture was stirred at room temperature under an atmosphere of hydrogen for 30 min. The
catalyst was filtered off, and the filtrate was concentrated to dryness. The residue was dissolved in
CH
mmol). The mixture was stirred at room temperature for 4 h. Saturated aqueous NaHCO
added, and the mixture was extracted with CHCl (3 × 10 mL). The combined CHCl
2 2
Cl (200 L) together with pentafluorophenol (35.0 mg, 0.19 mmol) and DCC (13.1 mg, 0.063
3
(10 mL) was
3
3
extracts were
washed with brine (10 mL), dried over Na SO , and filtered, and the solvent was removed in vacuo. The
2
4
residue was subjected to column chromatography (silica gel, 6:1 hexane/EtOAc) to afford 11 (33.2 mg,
2
5
8
1
4
7
1%) as a colorless oil: [] -19.7 (c 0.33, MeOH); IR (film)  3342, 2975, 2931, 2857, 1730, 1523,
D
-1 1
155, 999 cm . H NMR (600 MHz, CDCl )  5.42 (1H, d, J = 8.0 Hz), 4.83 (1H, td, J = 8.0, 5.2 Hz),
3
.30 (1H, quint, J = 5.0 Hz), 4.20 (1H, dq, J = 12.8, 5.0 Hz), 4.08 (2H, t, J = 6.2 Hz), 2.55 (1H, sept, J =
.0 Hz), 2.422.37 (1H, m), 2.242.22 (1H, m), 2.19 (2H, t, J = 7.6 Hz), 1.621.60 (2H, m), 1.571.53
13
(
2H, m), 1.43 (9H, s), 1.321.25 (12H, m), 1.17 (3H, d, J = 7.0 Hz), 1.16 (3H, d, J = 7.0 Hz); C NMR

1874
HETEROCYCLES, Vol. 89, No. 8, 2014
(150 MHz, CDCl
3
) 176.9 (s), 173.5 (s), 168.5 (s), 156.1 (s), 141.83 (s), 141.78 (s), 140.1 (s), 137.12 (s),
1
2
37.06 × 2 (s), 80.1 (s), 65.9 (t), 60.1 (t), 51.2 (d), 35.6 (t), 33.9 (d), 31.0 (t), 29.5 × 2 (t), 29.4 (t), 29.3 (t),
9.2 (t), 29.1 (t), 28.9 (t), 28.1 × 3 (q), 25.8 (t), 25.1 (t), 18.9 × 2 (q); HR-ESIMS m/z calcd for
+
C
31
H
44
F
5
NO
8
Na [M+Na] 676.2885, found 676.2906.
(
S,Z)-tert-Butyl
1
2
1
2-(((1-(3-((4-((2-(diethylamino)ethyl)carbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-
-oxoindolin-1-yl)-4-(isobutyryloxy)-1-oxobutan-2-yl)carbamoyl)oxy)dodecanoate (12): Compound
1 (16.9 mg, 0.026 mmol) and sunitinib (6.9 mg, 0.017 mmol) were dissolved in DMF (200 μL), and the
mixture was stirred at room temperature for two days. The solvent was removed in vacuo, and the residue
was subjected to column chromatography (silica gel, 20:1 CHCl /MeOH) to afford 12 (13.7 mg, 61%) as
+36.0 (c 0.38, MeOH); IR (film)  3411, 2975, 2928, 2855, 1728,
3
2
5
an orange amorphous solid: []
D
-1 1
1
154 cm . H NMR (600 MHz, CDCl
3
)  12.68 (1H, s), 8.16 (1H, q, J = 4.5 Hz), 8.148.11 (1H, m),
7.38 (1H, s), 7.20 (1H, dd, J = 8.1, 2.0 Hz), 6.90 (1H, td, J = 8.7, 2.0 Hz), 5.955.91 (1H, m), 5.74 (1H, d,
J = 8.7 Hz), 4.27 (2H, t, J = 6.4 Hz), 4.06 (2H, t, J = 6.8 Hz), 3.713.69 (2H, m), 3.073.04 (2H, m), 2.94
4H, quint, J = 6.6 Hz), 2.60 (3H, s), 2.51 (3H, s), 2.492.43 (1H, m), 2.392.34 (1H, m), 2.19 (2H, t, J =
.7 Hz), 2.112.06 (1H, m), 1.561.52 (2H, m), 1.43 (9H, s), 1.291.24 (16H, m), 1.22 (6H, m), 1.12 (3H,
(
7
1
3
d, J = 7.0 Hz), 1.11 (3H, d, J = 7.0 Hz); C NMR (150 MHz, CDCl ) 176.8 (s), 173.4 (s), 169.9 (s),
3
1
1
4
2
68.2 (s), 165.8 (s), 159.7 (s), 156.4 (s), 140.5 (s), 133.4 (s), 132.0 (s), 128.3 (s), 126.4 (s), 125.3 (d),
20.7 (s), 117.8 (d), 113.0 (d), 111.1 (s), 104.1 (d), 79.9 (s), 65.5 (t), 60.8 (t), 53.2 (d), 51.5 (t), 47.1 (t),
7.0 (t), 35.9 (t), 35.6 (t), 33.9 (d), 31.8 (t), 29.53 (t), 29.46 (t), 29.33 (t), 29.31 (t), 29.11 (t), 29.06 (t),
9.0 (t), 28.1 × 3 (q), 25.9 (t), 25.1 (t), 18.94 (q), 18.91 (q), 14.4 (q), 11.3 (q), 9.5 (q), 9.4 (q); HR-ESIMS
+
m/z calcd for C47
H71FN O
5 9
[M+H] 868.5236, found 868.5256.
(
S,Z)-Pentafluorophenyl
1
2
2-(((1-(3-((4-((2-(diethylamino)ethyl)carbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoro-
-oxoindolin-1-yl)-4-(isobutyryloxy)-1-oxobutan-2-yl)carbamoyl)oxy)dodecanoate (13): A solution
of 12 (74.0 mg, 0.085 mmol) in CHCl
3
/TFA (4:1, 2.0 mL) was stirred at room temperature for 3 h, and
the solvent was removed in vacuo. The residue was dissolved in CH Cl (200 L) together with
2
2
pentafluorophenol (49.2 mg, 0.27 mmol) and DMAP (0.14 mg, 0.0011 mmol), and EDC·HCl (37.5 mg,
.20 mmol) was added at 0 °C. The mixture was stirred at this temperature for 1 h, and then at room
temperature for 21 h. Saturated aqueous NaHCO (10 mL) was added, and the mixture was extracted with
CHCl (3 × 10 mL). The combined CHCl extracts were washed with brine (10 mL), dried over Na SO
and filtered, and the solvent was removed in vacuo. The residue was subjected to column chromatography
0
3
3
3
2
4
,
2
5
(
3
silica gel, 20:1 CHCl /MeOH) to afford 13 (60 mg, 72%) as a yellow amorphous solid: [] +43.8 (c
D

HETEROCYCLES, Vol. 89, No. 8, 2014
1875
-
1 1
0
1
6
.23, CHCl
3
); IR (film)  3364, 2927, 2855, 2360, 1791, 1698, 1520 cm . H NMR (600 MHz, CDCl ) 
3
1.62 (1H, br s), 8.16 (1H, q, J = 4.5 Hz), 8.148.12 (1H, m), 7.39 (1H, s), 7.17 (1H, dd, J = 8.3, 2.6 Hz),
.91 (1H, td, J = 8.7, 2.6 Hz), 5.955.91 (1H, m), 5.72 (1H, d, J = 8.3 Hz), 4.27 (2H, t, J = 5.8 Hz), 4.07
(
(
2H, t, J = 6.4 Hz), 3.89 (2H, br t, J = 4.5 Hz), 3.313.29 (2H, m), 3.203.16 (4H, m), 2.67 (3H, s), 2.65
2H, t, J = 7.5 Hz), 2.60 (3H, s), 2.462.41 (1H m), 2.372.31 (1H m), 2.092.06 (1H, m), 1.76 (2H,
quint, J = 7.5 Hz), 1.6391.580 (2H, m), 1.444 (3H, t, J = 7.2 Hz), 1.441 (3H, t, J = 7.2 Hz), 1.331.32
1
3
(
2H, m), 1.321.24 (12H, m), 1.13 (3H, d, J = 7.2 Hz), 1.09 (3H, d, J = 7.2 Hz); C NMR (150 MHz,
CDCl ) 176.9 (s), 173.0 (s), 169.8 (s), 168.2 (s), 166.0 (s), 159.7 (s), 156.3 (s), 142.0 × 2 (s), 141.0 (s),
40.3 (s), 138.7 × 2 (s), 137.7 (s), 134.1 (s), 132.0 (s), 128.3 (s), 126.5 (s), 125.4 (d), 119.6 (s), 117.9 (d),
3
1
1
3
13.1 (d), 111.3 (s), 104.1 (d), 65.5 (t), 60.8 (t), 53.6 (t), 53.1 (d), 48.86 (t), 48.83 (t), 35.5 (t), 33.9 (d),
3.4 (t), 31.8 (t), 29.5 (t), 29.4 (t), 29.28 (t), 29.25 (t), 29.1 (t), 29.0 (t), 28.9 (t), 25.8 (t), 24.8 (t), 18.92
+
(
q), 18.90 (q), 14.7 (q), 11.7 (q), 8.7 × 2 (q); HR-ESIMS m/z calcd for C49
H
61
F
6
N
5
O
9
[M+H] 978.4452,
found 978.4474.
Sunitinibmetastin conjugate (14): Compound 13 (3.7 mg, 0.0038 mmol) and metastin (1.1 mg,
.00018 mmol) were dissolved in DMF (600 μL), and the mixture was stirred at room temperature for
five days. The mixture was passed through a PD-10 desalting column (GE Healthcare Life Sciences) with
O as eluent to give a fraction containing the conjugate. The solution was lyophilized by using
0
H
2
8
+
FDU-2200 (EYELA) to provide conjugate 14 (0.96 mg, 64%); ESI-TOF-MS m/z 1031.2981 [M+8H] ,
7
+
6+
m/z 1374.0596 [M+7H] , m/z 1778.1360 [M+6H] .
Enzymatic Hydrolysis of Compounds 6 and 7
An equivalent molar amount of malic acid was added to compound 6 or 7. The mixture was dissolved in
methanol, and the solution was concentrated to dryness. The residue (each 290 g) was dissolved in PBS
(
450 L, pH 7.4) and the solution was left to stand at 36.6 °C with or without PLE (100 g, 17 units/mg,
SIGMA). The reaction was monitored by injecting a 10 L aliquot of the reaction mixture into an
analytical HPLC equipped with DP-L910W software, using the following conditions: mobile phase,
5
R
5:45 MeCN20 mM sodium acetate buffer; flow rate, 1 mL/min; = 431 nm. The t values for sunitinib,
compound 6, and compound 7 were 8, 20, and 16 min, respectively.
ACKNOWLEDGEMENT
We are grateful to Dr. Tetsuya Ohtaki of Takeda Chemical Industries Ltd., for the gift of metastin.
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1876
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C. D. Johnson and S. Lane, J. Org. Chem., 1988, 53, 5130.
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0. B. Blanchet, C. Saboureau, A. S. Benichou, B. Billemont, F. Taieb, S. Ropert, A. Dauphin, F.
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