Synthesis and cytotoxicity studies of new cryptophycin analogues

Article abstract of DOI:10.1002/ardp.200900067

Two analogues of cryptophycin were synthesized and biologically evaluated for their in-vitro cytotoxicities against several solid tumors and leukemia cell lines. The results revealed that both analogues exhibited a broad range of cytotoxic activity with observed IC₅₀ values in the μM-range, and compound 4 was more effective than compound 3 in most assays studied.

Full text of DOI:10.1002/ardp.200900067

Arch. Pharm. Chem. Life Sci. 2009, 342, 577–583
W. L. Liu et al.
577
Full Paper
Synthesis and Cytotoxicity Studies of New Cryptophycin
Analogues
Wen Lu Liu1, Jian Cun Zhang2, Fa Qin Jiang1, and Lei Fu1
1 School of Pharmacy, Shanghai Jiaotong University, Shanghai, P.R. China
2
Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, P.R. China
Two analogues of cryptophycin were synthesized and biologically evaluated for their in-vitro
cytotoxicities against several solid tumors and leukemia cell lines. The results revealed that both
analogues exhibited a broad range of cytotoxic activity with observed IC50 values in the lM-
range, and compound 4 was more effective than compound 3 in most assays studied.
Keywords: Anticancer / Cryptophycin / Cytotoxicity / Synthesis / Taxol /
Received: March 30, 2009; accepted: June 14, 2009
DOI 10.1002/ardp.200900067
Introduction
The cryptophycins are a family of macrocyclic depsipepti-
des isolated from terrestrial blue-green algae (Nostoc sp),
which are potent tubulin-binding antimitotic agents
with excellent activities against multi-drug resistant
(MDR) cancer cell lines and mammary-derived tumors [1,
2]. Cryptophycin-1 (1, Fig. 1) is the key cytotoxin in Nostoc
sp and displays remarkable cytotoxic activity [3, 4]. More
importantly, compared to vinblastine, colchicine, and
paclitaxel, the cryptophycin-1 presents reduced suscepti-
bility to P-glycoprotein-mediated multiple drug resist-
ance [5].
By 2005, twenty eight cryptophycin analogues had
been isolated, while hundreds of synthetic analogues
had been synthesized for the structure-activity relation-
ship (SAR) studies in search for more potent and drugable
compounds [1, 6]. Some synthetic cryptophycin ana-
logues have demonstrated superior activities and dru-
gable properties to their natural compounds. The closely
related synthetic analogue cryptophycin-52 (2, LY355703,
Fig. 1) was identified by Eli Lilly as the pharmacologically
Figure 1. Structures of cryptophycin-1 (1), –52 (2), and the ole-
fin analogues 3 and 4.
most promising first-generation clinical candidate, and
is currently in clinical studies as an antitumor agent [8–
11].
However, the low bio-availability in in-vivo cytotoxic
assays of cryptophycin-1 caused by its poor stability and
low solubility [12] led some research teams to initiate an
attempt to improve it through structure modifications.
In this work, we intend to introduce a lactam on C4
and polar groups (for instants: sulfur element of Met, OH
of Ser, lactam of Asp) on C6 of cryptophycin-1 in order to
alter its solubility. Two lactam analogues of cryptophy-
cin-1, 3 and 4 (Fig. 1) were synthesized for the first time,
and their in-vitro cytotoxicities were evaluated by the
MTT assay.
Correspondence: Lei Fu and Fa Qin Jiang, School of Pharmacy, Shang-
hai Jiaotong University, Shanghai 200240, P.R. China.
E-mail: leifu@sjtu.edu.cn and jfq2008@sjtu.edu.cn
Fax: +86 21 3420-4787
Results and discussion
Abbreviations: 2,3-Dichloro-5,6-dicyanobenzoquinone (DDQ); 4-(Dime-
thylamino)-pyridin (DMAP); 1-Ethyl-3-(3-dimethylaminopropyl)carbodi-
imide hydrochloride (EDC N HCl); Hydroxybenzotriazole (HOBT)
The retrosynthetic analysis of compounds 3 and 4 sug-
gested that they can be derived from two main building
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W. L. Liu et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 577–583
protection of the secondary alcohol in 9 with tert-butyldi-
methylsilyl chloride and triethylamine afforded the silyl
ether 10 in 92% yield. Deprotection of the p-methoxyben-
zyl ether with (2,3-dichloro-5,6-dicyanobenzoquinone)
DDQ followed by Swern Oxidation of the resulting alco-
hol furnished the aldehyde 11 in 54% yield over two
steps. Wittig–Horner olefination of 11 provided the a,b-
unsaturated tert-butyl ester 12 in 79% yield. Heck cou-
pling on 12 with iodobenzene in the presence of palla-
dium acetate and sodium bicarbonate furnished the
required ester 13 in 60% yield. Removal of the silyl ether
with tetrabutylammonium fluoride led to the formation
of the desired octadienoate ester 5 in 71% yield.
In the second synthon, tripeptides 6a and 6b, were syn-
thesized starting from the D-tyrosine derivative (R)-2-(tert-
butoxycarbonylamino)-3-(3-chloro-4-methoxyphenyl) pro-
panoic acid 14 (Scheme 3). Compound 14 was prepared
from D-tyrosine, through chlorination by SOCl2, Boc pro-
tection of the NH2 group by (Boc)2O, methylation by
Me2SO4, and deprotection of the methyl ester. The use of
diazomethane afforded the diazoketone derivative 15 in
85% yield, followed by addition of trifluoroacetic acid sil-
ver salt; the homo-amino acid derivative (R)-3-(tert-butoxy-
carbonylamino)-4-(3-chloro-4-methoxyphenyl) butanoic
acid 16 was afforded in 60% yield. Activated by 1-ethyl-3-
Scheme 1. The retrosynthetic analysis of the olefin analogues 3
and 4.
(3-dimethylaminopropyl)carbodiimide
hydrochloride
blocks, the octadienoate ester 5 and the tripeptide 6
(Scheme 1), respectively.
(EDC N HCl) and hydroxybenzotriazole (HOBT), 16 was
coupled with (S)-methyl 2-aminopropanoate and (S)-
methyl 2-amino-4-(methylthio)butanoate, respectively,
subsequently treated with NaOH, this furnished the
dipeptide derivatives 17a (67%) and 17b in 64% yield over
two steps. In a same procedure, 17a and 17b were coupled
with (S)-methyl 2-amino-4-methylpentanoate to obtain
the tripeptide derivatives 6a (59%) and 6b (52%) over two
steps.
Based on the above retrosynthetic analysis, 5 was pre-
pared using the crotylboration approach [13, 14]. Com-
pound 7 was prepared from 4-methyloxidyl-benanhy-
dride in several steps (Scheme 2). The key step in Scheme
2 utilized the crotylboration with crotyl diisopinocam-
pheylborane 8 (prepared from (+)-B-methoxydiisopina-
campheyl-borane) to generate the desired stereochemis-
try at both chiral centers of 9 in 55% yield (91% ee). Silyl
Scheme 2. Synthesis of the octadienoate ester 5.
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Arch. Pharm. Chem. Life Sci. 2009, 342, 577–583
New Cryptophycin Analogues
579
Scheme 3. Synthesis of the cryptophycin analogues 3 and 4.
Table 1. Cytotoxicity of compounds 3 and 4 against fifteen cell lines.
Comp.
Cytotoxicity (IC50, lM)
Solid tumor cells
Leukemia cells
Normal cells
293T
>100 >100 43.53 >100 5.23 68.71
T47D H460 Hela DV145 LoVo HepG2 HCT116 A549 SGC-7901 L1210 K562 RAW CHO VEC
Taxol 1.39
2.42 15.68 66.87 >100 >100 >100
21.47 29.62 69.84 94.81 43.10 52.66
26.64 24.73 53.73 >100 36.25 64.96
>100
77.84 38.09
54.30 19.06
5.36
3
4
3.50
3.78
51.44 34.32 38.78 >100 57.99 >100
37.23 18.34 34.96 >100 34.55 >100
Each experiment was performed independently three times.
The tripeptide derivatives 6a and 6b were activated by study the possible cytotoxicity in normal cells, the
DCC and a catalytic amount of 4-(dimethylamino)-pyri- growth inhibition of compounds 3 and 4 in 293T (human
din (DMAP), addition of the octadienoate ester 5 to the kidney epithelium cell), CHO (china hamster ovary cell),
mixed anhydride afforded the intermediate 18a (52%) and VEC (human blood vessel bast cell) were evaluated.
and 18b (50%), respectively (Scheme 3). Simultaneous The IC50 values were measured and are listed in Table 1.
deprotection of the tert-butyl ester and the N-Boc with tri-
As shown in Table 1, compounds 3 and 4 had demon-
fluoroacetic acid in CH2Cl2 produced the cyclized precur- strated effective growth inhibitory activities in almost all
sor and by EDC N HCl and HOBT activation provided the of the cell lines studied, broader than that of Taxol. In the
desired compounds 3 (45%) and 4 (42%), respectively.
leukemia cell lines studied, Taxol presented poor or no
A preliminary in-vitro evaluation of the cytotoxic activ- activity, whereas both compounds 3 and 4 were active.
ity of 3 and 4 was performed by MTT assays. The nine solid Similarly, in the solid tumor cell lines of HepG2, HCT116,
cancer cell lines (T47D: human breast cancer cell, NCI- and A549 both were active, but Taxol was a poor inhibi-
H460: human lung cancer cell, Hela: human cervical can- tor.
cer, HepG2: human liver cancer, LoVo and HCT116:
In DV145, similar IC50 were observed for both Taxol
human colon cancer, A549: human lung cancer, DV145: and the compounds synthesized. Taxol presented slightly
human prostate cancer, SGC-7901: human stomach can- better growth inhibitory activities in T47D and SGC-7901
cer) and three leukemia cell lines (K562: multi-drug cell lines, and much better ones in H460. The inhibitory
resistant human leukemia cell, L1210: mouse acute activities in Hela were comparable to both Taxol and the
lymph leukemia cell, RAW: mouse leukemic monocyte compounds synthesized. In LoVo, neither Taxol nor our
macrophage cell) were employed in the present study. To compounds were active.
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The IC50 values of compounds 3 and 4 in normal cell (3S,4R)-3-[(tert-Butyldimethylsilyl)oxy]-1-(4-
lines are all larger than that of Taxol, and most of them methoxybenzyloxy)-4-methyl-5-hexene 10
Alcohol 9 (0.15 g, 0.60 mmol) was dissolved in CH2Cl2 (6 mL) and
are above 100 lM except in VEC. This result implies that
cooled to –788C. 2, 6-Lutidine (0.14 mL, 1.20 mmol) was added,
compounds 3 and 4 are generally safer than Taxol.
followed by TBSOTf (0.17 mL, 0.72 mmol) dropwise via a syringe.
In summary, compounds 3 and 4 exhibited a broader
After 10 min, the reaction was quenched with saturated aqueous
cytotoxicity than Taxol both in solid tumors and leuke-
mia cell lines. In most cell lines tested, compound 4 was
slightly more active than compound 3, and the thio-sub-
stitution on C6 of compound 4 was believed to produce a
positive effect on the cytotoxic activity. Though more evi-
dence is needed, this encouraging observation has led us
to design potentially more potent compounds with
different polar group at position C6 of compound 4.
NaHCO3. The aqueous layer was extracted with CH2Cl2. The com-
bined organic extracts were dried (MgSO4), filtered, concen-
trated, and purified by flash column chromatography (hexane/
EtOAc, 9:1) to provide a colorless oil (0.20 g, 92%).
1H-NMR (CDCl3) d: 7.14–7.06 (d, J = 8.6 Hz, 2H, CH), 6.81–6.69
(d, J = 8.6 Hz, 2H, CH), 5.80–5.71 (m, 1H, CH), 5.00–4.92 (br d, J =
5.0 Hz, 2H, CH2), 4.44 (s, 2H, CH2), 3.80 (s, 3H, OCH3), 3.77–3.73
(m, 1H,CH), 3.48–3.45 (dt, J = 2.1, 7.0 Hz, 2H, CH2), 2.29–2.26 (m,
1H, CH), 1.70–1.63 (m, 2H, CH2), 1.00–0.98 (d, J = 6.9 Hz, 3H,
CH3), 0.88 (s, 9H, t-Bu-H), 0.08 (s, 3H, SiCH3), 0.04 (s, 3H, SiCH3).
We thank the National Natural Science Foundation of China
(20472050) for the financial support. The biological testing was
conducted by the Professor Yongxiang WANG's research group
at the Shanghai Jiaotong University.
(3S)-3-[(tert-Butyldimethylsilyl)oxy]-4-methyl-5-hexenal
11
p-Methoxybenzyl ether 10 (1.60 g, 4.39 mmol) was dissolved in
CH2Cl2 (20 mL). Water (1.30 mL) and solid DDQ (0.99 g, 4.39
mmol) were added. The mixture was stirred for 1 h, then diluted
with CH2Cl2 (200 mL), washed with saturated aqueous NaHCO3
(40 mL) and brine, dried (MgSO4), concentrated, and purified by
flash column chromatography (hexane/EtOAc, 4:1) to provide a
mixture of the desired primary alcohol and p-methoxybenzalde-
hyde which was used without further purification in the next
step.
The authors have declared no conflict of interest.
A mixture of CH2Cl2 (25 mL) and oxalyl chloride (2.2 mL, 20
mmol) was cooled to –50 to –608C as DMSO (3.4 mL, 40 mmol)
was added. The mixture was stirred for 2 min and the primary
alcohol (10 mmol in 10 mL CH2Cl2) was added; 15 min later, trie-
thylamine (7.0 mL, 50 mmol) was added. The mixture was stirred
for 5 min and then warmed to room temperature. H2O (50 mL)
was added and the aqueous layer was extracted with CH2Cl2 (50
mL). The organic layers were combined, washed with brine (100
mL), dried (MgSO4), concentrated, and purified by flash column
chromatography (hexane/EtOAc, 10:1) to provide product 11 as
an oil (0.57g, 54% over two steps).
Experimental
Chemistry
Melting points were recorded on a B-540 melting point appara-
tus (Bꢀchi Labortechnik, Flawil, Switzerland) and are uncor-
1
rected. The H-NMR spectra were recorded on a Bruker Avance
DMX-400 NMR-spectrometer (400 MHz, 298C; Bruker Bioscience,
Billerica, MA, USA). Chemical shifts are given in ppm relative to
tetramethylsilane (TMS) as internal standard (0 ppm). Mass spec-
tra were recorded on an Esquire-LC-00075 mass spectrometer
(Bruker). All reagents were purchased from commercial suppli-
ers and were purified when necessary.
1H-NMR (CDCl3) d: 9.78–9.72 (t, J = 2.2 Hz, 1H, CHO), 5.79–5.70
(dd, J = 7.0, 17.1 Hz, 1H, CH), 5.06–5.04 (m, 1H, CH2), 5.05-4.99
(m, 1H, CH2), 4.20–4.17 (m, 1H, CH), 2.50–2.43 (dt, J = 2.0, 6.2 Hz,
2H, CH2), 2.39–2.33 (m, 1H, CH), 1.03–1.01 (d, J = 6.9 Hz, 3H,
CH3), 0.87 (s, 9H, t-Bu-H), 0.08 (s, 3H, SiCH3), 0.04 (s, 3H, SiCH3).
(3S,4R)-1-(4-Methoxybenzyloxy)-4-methyl-5-hexen-3-ol
9
tert-Butyl(5S,6R,2E)-5-[(tert-butyldimethylsilyl)oxy]-6-
methyl-2,7-octadienoate 12
Aldehyde 7 (0.62 g, 3.0 mmol) was dissolved in dry ether (4 mL)
and cooled to –788C in N2 atmosphere. 8 (3.5 mmol, preparation
method as described [13]) was dissolved in dry ether (2 mL) drop-
wise via syringe at –788C in a period of 10 min. The mixture was
stirred at -788C for 2 h and heated to room temperature. NaOH (2
N, 3 mL) and H2O2 (55%, 6 mL) were added dropwise and the mix-
ture was stirred for another 2 h. The reaction was quenched
with saturated aqueous NaHCO3. The aqueous layer was
extracted with CH2Cl2. The combined organic extracts were
dried (MgSO4), filtered, concentrated, and purified by flash col-
umn chromatography (hexane/EtOAc, 8:1) to provide a colorless
tert-Butyldiethylphosphonoacetate (1.16 mL, 4.95 mmol), DBU
(0.44 mL, 2.97 mmol), and LiCl (0.15 g, 3.47 mmol) were stirred
vigorously at room temperature for 30 min in anhydrous CH3CN
(12 mL). The solution was added dropwise to a solution of anhy-
drous aldehyde 11 (0.60 g, 2.48 mmol) in CH3CN (6 mL). After 2 h,
saturated aqueous NH4Cl solution was added. The organic layers
were further washed with brine. The aqueous layers were
extracted with EtOAc, dried (MgSO4), concentrated, and purified
(hexane/EtOAc, 10:1) to get the product as a colorless oil (0.66 g,
79%).
1
oil (0.41 g, 55%). H-NMR (CDCl3) d: 7.28–7.26 (d, J = 8.7 Hz, 2H,
CH), 6.91–6.88 (d, J = 8.7 Hz, 2H, CH), 5.87–5.78 (m, 1H, CH),
5.08–5.06 (br d, J = 5.0 Hz, 2H, CH2), 4.47 (s, 2H, CH2), 3.82 (s, 3H,
OCH3), 3.74–3.61 (m, 3H, CH2, CH), 2.82–2.81 (d, J = 3.0 Hz, 1H,
CH), 1.76–1.69 (m, 2H, CH2), 1.07–1.05 (d, J = 6.9 Hz, 3H, CH3).
1H-NMR (CDCl3) d: 6.82–6.75 (dt, J = 8.0, 16.2 Hz, 1H, CH), 5.80–
5.71 (m, 1H, CH), 5.74–5.70 (br d, J = 16.1 Hz, 1H, CH), 5.03 (br s,
1H, CH2), 5.01–4.99 (br d, J = 9.1 Hz, 1H, CH2), 3.67–3.63 (m, 1H,
CH), 2.29–2.23 (m, 3H, CH2, CH), 1.46 (s, 9H, t-Bu-H), 1.00–0.99
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Arch. Pharm. Chem. Life Sci. 2009, 342, 577–583
New Cryptophycin Analogues
581
(d, J = 6.8 Hz, 3H, CH3), 0.88 (s, 9H, t-Bu-H), 0.03 (s, 3H, SiCH3), 0.03
(s, 3H, SiCH3).
H2O, dried over MgSO4, and co-evaporation with toulene
afforded a solid acid 16 (128 mg, 60%).
1H-NMR (CDCl3) d: 7.25 (s, 1H, CH), 7.01–7.12 (d, J = 8.3 Hz, 1H,
CH), 6.87–6.94 (d, J = 8.1 Hz, 1H, CH), 3.95–4.08 (m, 1H, CH), 3.93
(s, 3H, OCH3), 2.77–2.85 (m, 2H, CH2), 2.45–2.54 (m, 2H, CH2),
1.45 (s, 9H, Boc-H).
tert-Butyl-(5S,6R,2E,7E)-5-[(tert-butyldimethylsilyl)-oxy]-
6-methyl-8-phenyl-2,7-octadienoate 13
Olefin 12 (75 mg, 0.22 mmol) was dissolved in anhydrous CH3CN
(2 mL) under argon atmosphere. Then, iodobenzene (49 mg, 0.24
mmol), Pd(OAc)2 (2.50 mg, 0.01 mmol), and TEA (0.31 mL, 2.2
mmol) was added. The tube was sealed and heated at 80 to 858C
for 24 h. The mixture was concentrated and subjected directly to
column chromatography (hexane/EtOAc, 9:1) to provide product
13 as an oil (55 mg, 60%).
(S)-2-((R)-3-(tert-Butoxycarbonylamino)-4-(3-chloro-4-
methoxyphenyl)butanamido)propanoic acid 17a
The solid acid 16 (110 mg, 0.32 mmol) was dissolved in CH2Cl2 (6
mL). EDC N HCl (92 mg, 0.48 mmol), and HOBT (65 mg, 0.32
mmol) was added and the mixture was stirred for 30 min at
room temperature. Then, (S)-methyl 2-aminopropanoate (50 mg,
0.48 mmol) and Et3N (132 lL, 0.96 mmol) were added and the
mixture was stirred overnight at room temperature in N2 atmos-
phere. The mixture was diluted by CH2Cl2 (6 mL), washed by 0.1
N HCl and brine, dried with Na2SO4 and purified by column chro-
matography (Hex/EtOAc, 1:1). The methyl-ester of 17a was
obtained as a pale yellow solid.
tert-Butyl-(5S,6R,2E,7E)-5-hydroxy-6-methyl-8-phenyl-
2,7-octadienoate 5
The ester 13 (25 mg, 0.06 mmol) was dissolved in THF (500 lL).
TBAF solution (1 M in THF, 66 lL, 0.07 mmol) was added drop-
wise. After 2 h, saturated aqueous Na2CO3 solution was added.
The aqueous layers were extracted with EtOAc and the organic
layers were further washed with brine, concentrated, and sub-
jected to a short plug of silica (hexane/EtOAc, 4:1), providing a
pale yellow oil (13 mg, 71%).
The methyl-ester of 17a was dissolved in MeOH (6 mL) and 2 N
NaOH (650 lL, 0.65 mmol) was added dropwise. The solution was
stirred for 2 h at room temperature and the excess MeOH was
distilled in vacuum. H2O (50 mL) was added to obtain a solution
of the sodium salt of 17a. In an ice bath, 1 N HCl was added drop-
wise until pH 2–3 and a white suspension was formed. The mix-
ture was extracted with EtOAC (20 mL) twice. EtOAC layer was
combined, dried (Na2SO4) and distilled in vacuo to yield a pale yel-
low solid 17a (90 mg, 67%).
[a]2d0 = + 668 (c = 0.80, CHCl3); 1H-NMR (CDCl3) d: 7.37–7.36 (d, J =
7.3 Hz, 2H, CH), 7.32–7.28 (dt, J = 7.0 Hz, 2H, CH), 7.25–7.20 (t, J =
7.2 Hz, 1H, CH), 6.95–6.88 (dt, J = 7.5, 15.6 Hz, 1H, CH), 6.48–6.44
(d, J = 16.0 Hz, 1H, CH), 6.17–6.11 (dd, J = 8.6, 16.4 Hz, 1H, CH),
5.86–5.82 (br d, J = 15.6 Hz, 1H, CH), 3.67–3.62 (m, 1H, CH),
2.48–2.38 (m, 1H, CH), 2.36–2.28 (m, 1H, CH), 1.86 (br s, 1H, CH),
1.47 (s, 9H, t-Bu-H), 1.15 (s, 3H, CH3); 13C-NMR (100 MHz, CDCl3) d:
165.7, 144.0, 137.0, 131.8, 130.9, 128.5 (2C), 127.4, 126.1 (2C),
125.4, 80.2, 73.8, 43.2, 37.1, 28.1 (3C), 16.8; MS (ESI) m/z: 325 [M +
Na]. HRMS (FAB, NBA) calcd. for C19H27O3: [M + H] 303.1960;
found: 303.1980.
1H-NMR (CDCl3) d: 7.05–7.21 (m, 2H, CH), 6.81–6.89 (m, 1H,
CH), 4.68 (s, 1H, CH), 4.07 (s, 1H, CH), 3.85 (s, 3H, OCH3), 2.19–
2.78 (m, 4H, CH2 ), 1.52 (s, 3H, CH3), 1.41 (s, 9H, Boc-H).
(S)-2-((R)-3-(tert-Butoxycarbonylamino)-4-(3-chloro-4-
methoxyphenyl)butanamido)-4-(methylthio)butanoic acid
17b
(R)-tert-Butyl 1-(3-chloro-4-methoxyphenyl)-4-diazo-3-
oxobutan-2-ylcarbamate 15
The solid acid 16 was treated with (S)-methyl 2-amino-4-(methyl-
thio)butanoate according to the same process as 17a to yield 17b
as a pale yellow wax (100 mg, 64%).
To a solution of (R)-2-(tert-butoxycarbonylamino)-3-(3-chloro-4-
methoxyphenyl) propanoic acid 14 (329 mg, 1 mmol) in CH2Cl2
(8 mL) at 08C, triethylamine (0.17 mL, 1.2 mmol) and ethyl
chloroformate (0.11 mL, 1.1 mmol) was added. The mixture was
stirred at 08C for 10 min. CH2N2 ether solution (4 mL, 1.2 mmol)
was then added dropwise. The mixture slowly warmed up to
room temperature and was then stirred for 1 h. Partition of the
mixture between EtOAc and H2O, successive washing with 0.1 N
HCl and saturated NaHCO3, drying over MgSO4, followed by con-
centration gave the crude product which was further purified
by column chromatography (Hex/EtOAc, 2:1) to yield the diazo
compound 15 (220 mg, 62%). 1H-NMR (CDCl3) d: 7.24 (s, 1H, CH),
7.01–7.14 (d, J = 8.2 Hz, 1H, CH), 6.84–6.92 (d, J = 8.3 Hz, 1H, CH),
5.32 (s, 1H, CH), 4.41 (br s, 1H, CH), 3.92 (s, 3H, OCH3), 2.83–3.05
(m, 2H, CH2), 1.46 (s, 9H, Boc-H).
1H-NMR (CDCl3) d: 7.27–7.12 (m, 2H, CH), 6.84–6.72 (m, 1H,
CH), 4.65 (s, 1H, CH), 3.95 (s, 1H, CH), 3.77 (s, 3H, OCH3), 2.24–
2.79 (m, 8H, CH2), 1.99 (s, 3H, SCH3), 1.40 (s, 9H, Boc-H).
(6R,10S,13S)-6-(3-Chloro-4-methoxybenzyl)-13-isobutyl-
2,2,10-trimethyl-4,8,11-trioxo-3-oxa-5,9,12-
triazatetradecan-14-oic acid 6a
The peptide 17a was treated with (S)-methyl 2-amino-4-methyl-
pentanoate to yield a pale yellow solid 6a (68 mg, 59%).
1H-NMR (CDCl3) d: 7.39–7.45 (m, 2H, NH), 7.01–7.19 (m, 2H,
CH), 6.77–6.83 (m, 1H, CH), 4.46–4.61 (m, 2H, CH), 4.10–4.17
(m, 1H, CH), 3.82 (s, 3H, OCH3), 2.39–2.75 (m, 4H, CH2), 2.02 (m,
3H, CH2, CH), 1.62 (s, 3H, CH3), 1.40 (s, 9H, Boc-H), 0.89 (s, 6H,
CH3).
(R)-3-(tert-Butoxycarbonylamino)-4-(3-chloro-4-
methoxyphenyl)butanoic acid 16
The solid diazo compound 15 (220 mg, 0.63 mmol) was dissolved
in THF (6 mL) and H2O (0.3 mL). CF3CO2Ag (100 mg, 0.45 mmol)
was added and the mixture was stirred at room temperature
overnight. Brine was then added and the mixture was stirred for
2 h. The mixture was then filtered through celite and the solid
residue was washed with EtOAc. The filtrate was washed with
(6R,10S,13S)-6-(3-Chloro-4-methoxybenzyl)-13-isobutyl-
2,2-dimethyl-10-(2-(methylthio)ethyl)-4,8,11-trioxo-3-oxa-
5,9,12-triazatetradecan-14-oic acid 6b
A white solid 6b (60 mg, 52%) was obtained from the peptide 17b
according to the same process as for 6a.
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

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W. L. Liu et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 577–583
1H-NMR (CDCl3) d: 7.41(m, 2H, NH), 7.10–7.23 (m, 2H, CH),
6.71–6.83 (m, 1H, CH), 4.42–4.69 (m, 2H, CH), 4.12–4.21 (m, 1H,
CH), 3.94 (s, 3H, OCH3), 2.39–2.75 (m, 8H, CH2), 2.08 (m, 3H, CH2,
CH), 1.65 (s, 3H, CH3), 1.45 (s, 9H, Boc-H), 0.86 (s, 6H, CH3).
1H-NMR (CDCl3) d: 7.16–7.27 (m, 6H, CH), 6.57–6.74 (m, 2H,
CH), 6.26–6.37 (dd, J = 6.1, 12.0 Hz, 1H, CH), 6.18–6.24 (d, J = 12.5
Hz, 1H, CH), 6.08–6.16 (d, J = 12.1 Hz, 1H, CH), 5.66–5.73 (dd, J
=6.4, 12.2 Hz, 1H, CH), 4.70–4.85 (m, 1H, CH), 4.33–4.49 (m, 1H,
CH), 4.05–4.18 (m, 1H, CH), 3.92–4.15 (m, 1H, CH), 3.65 (s, 3H,
OCH3), 2.21–2.66 (m, 6H, CH2), 1.74–1.96 (m, 2H, CH2), 1.61–
1.84 (m, 1H, CH), 1.47 (s, 3H, CH3), 0.89 (s, 6H, CH3); 13C-NMR (100
MHz, CDCl3) d: 171.3, 169.5, 165.7, 164.0, 151.8, 147.5, 137.0,
131.8, 130.9, 128.5 (3C), 127.4, 126.1 (3C), 125.4, 122.7, 114.8,
78.2, 58.3, 52.8 (2C), 46.1, 42.3, 41.8 (2C), 40.6, 39.1, 23.1 (2C),
22.2, 17.7; MS (ESI) m/z: 660 [M + Na]. HRMS (FAB, NBA) calcd. for
C35H44ClN3O6: [M + H] 638.2930; found: 638.2910.
(6R,10S,13S)-((1E,3R,4S,6E)-8-tert-Butoxy-3-methyl-8-
oxo-1-phenylocta-1,6-dien-4-yl)6-(3-chloro-4-
methoxybenzyl)-13-isobutyl-2,2-dimethyl-10-(2-
(methylthio)ethyl)-4,8,11-trioxo-3-oxa-5,9,12-
triazatetradecan-14-oate 18a
6a (70 mg, 0.13 mmol) was dissolved in CH2Cl2 (5 mL). DCC (41
mg, 0.20 mmol) and DMAP (catalyst) was added and stirred for
30 min at room temperature. Then, the octadienoate ester 5 (40
mg, 0.14 mmol) was added and stirred overnight in N2 atmos-
phere. The mixture was diluted by CH2Cl2 (10 mL), washed by 0.1
N HCl and brine, dried with Na2SO4, and purified by silica gel col-
umn (Hex/EtOAc, 2:1) to yield 8a (53 mg, 52%) as a pale yellow
solid.
(3S,6S,10R,16S,E)-10-(3-Chloro-4-methoxybenzyl)-3-
isobutyl-6-(2-(methylthio)ethyl)-16-((R,E)-4-phenylbut-3-
en-2-yl)-1-oxa-4,7,11-triazacyclohexadec-13-ene-
2,5,8,12-tetraone 4
The compound 4 from compound 18b was obtained as a white
solid (17 mg, 42%) according to the same process of for 3.
1H-NMR (CDCl3) d: 7.29–7.44 (m, 6H, CH), 6.64–6.81 (m, 2H,
CH), 6.32–6.54 (dd, J = 6.3, 12.5 Hz, 1H, CH), 6.05–6.38 (d, J = 12.1
Hz, 1H, CH), 6.01–6.21 (d, J = 12.3 Hz, 1H, CH), 5.61–5.98 (dd, J =
6.2, 12.0 Hz, 1H, CH), 4.62–4.84 (m, 1H, CH), 4.16–4.41 (m, 1H,
CH), 3.89–4.15 (m, 1H, CH), 3.80–4.02 (m, 1H, CH), 3.71 (s, 3H,
OCH3), 2.25–2.79 (m, 10H, CH2), 2.01–2.29 (m, 3H, CH2 CH), 1.69
(s, 3H, SCH3), 0.91 (s, 6H, CH3); 13C-NMR (100 MHz, CDCl3) d: 173.5,
171.5, 167.4, 164.9, 153.2, 146.5, 134.6, 131.3, 129.9, 127.5 (3C),
126.9, 125.5 (3C), 124.2, 123.7, 115.8, 80.2, 60.5, 54.8 (2C), 47.7,
44.2, 42.8 (2C), 41.5, 40.1, 31.5, 29.8, 22.3 (2C), 21.8, 17.3; MS (ESI)
m/z: 720 [M + Na]. HRMS (FAB, NBA) calcd. for C37H48ClN3O6S: [M +
H] 698.3420; found: 698.3370.
1H-NMR (CDCl3) d: 7.23–7.40 (m, 6H, CH), 6.66–6.88 (m, 2H,
CH), 6.35–6.44 (dd, J = 6.1, 12.0 Hz, 1H, CH), 6.24–6.31 (d, J = 12.3
Hz, 1H, CH), 5.96–6.04 (d, J = 12.0 Hz, 1H, CH), 5.83–5.92 (dd, J =
6.4, 12.6 Hz, 1H, CH), 4.65–4.74 (m, 1H, CH), 4.37–4.43 (m, 1H,
CH), 4.17–4.22 (m, 1H, CH), 4.02–4.11 (m, 1H, CH), 3.75 (s, 3H,
OCH3), 2.28–2.68 (m, 6H, CH2), 1.82–1.85 (m, 2H, CH2), 1.83–
1.87 (m, 1H, CH), 1.48 (s, 3H, CH3), 1.45 (s, 9H, t-Bu-H), 1.40 (s, 9H,
Boc-H), 0.89 (s, 6H, CH3).
(6R,10S,13S)-((1E,3R,4S,6E)-8-tert-Butoxy-3-methyl-8-
oxo-1-phenylocta-1,6-dien-4-yl)6-(3-chloro-4-
methoxybenzyl)-13-isobutyl-2,2-dimethyl-10-(2-
(methylthio)ethyl)-4,8,11-trioxo-3-oxa-5,9,12-
triazatetradecan-14-oate 18b
A white solid 18b (56 mg, 50%) was obtained from the peptide 6b
according to the same process as for 18a.
Cell culture and cytotoxicity assay
All cancer cell lines were cultured in RPMI-1640 medium with
heat-inactivated 10% fetal bovine serum at 378C. Taxol was used
as the positive control. All tested compounds were dissolved in
DMSO at the concentrations 1.0 mg/100 lL and then diluted to
the appropriate concentrations. Cells were seeded in 96-well
microtiter plates (56105 cells/well). After 24-hour incubation in
appropriate medium, cells were treated with various concentra-
tions (50, 10, 1.0, 0.1, 0.01, 0.001 lg/mL) of test compounds, and
incubated for another 48 h. Afterwards, 10 lL of stock 3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT,
Sigma) solution was added to each well (final: 0.25 mg/mL) for
another 4 h of incubation. After 4 h, DMSO (100 lL) was added
and the optical density (OD) was read at 570 nm. The IC50 values
were calculated using the PrismPad computer program (Graph-
Pad Software, Inc. San Diego, CA, USA). All experiments were per-
formed in triplicate and the IC50 values were derived from the
mean OD values.
1H-NMR (CDCl3) d: 7.21–7.38 (m, 6H, CH), 6.56–6.77 (m, 2H,
CH), 6.38–6.45 (dd, J = 6.2, 12.1 Hz, 1H, CH), 6.17–6.25 (d, J = 12.4
Hz, 1H, CH), 5.89–5.95 (d, J = 12.0 Hz, 1H, CH), 5.72–5.83 (dd, J =
6.1, 12.3 Hz, 1H, CH), 4.63–4.91 (m, 1H, CH), 4.22–4.32 (m, 1H,
CH), 4.10–4.16 (m, 1H, CH), 3.96–4.05 (m, 1H, CH), 3.68 (s, 3H,
OCH3), 2.24–2.72 (m, 10H, CH2), 1.87–1.94 (m, 3H, CH2, CH), 1.73
(s, 3H, SCH3), 1.43(s, 9H, Bu-H), 1.40(s, 9H, Boc-H), 0.88(s, 6H, CH3).
(3S,6S,10R,16S,E)-10-(3-Chloro-4-methoxybenzyl)-3-
isobutyl-6-methyl-16-((R,E)-4-phenylbut-3-en-2-yl)-1-
oxa-4,7,11-triazacyclohexadec-13-ene-2,5,8,12-tetraone
3
18a (50 mg, 0.06 mmol) was dissolved in 20% trifluroacetic acid/
CH2Cl2 (1 mL). The mixture was stirred at room temperature
overnight and then diluted with toluene (2 mL). Evaporation
and co-evaporation with 6% HCl/EtOH gave a brownish solid.
The solid was dissolved in CH2Cl2 (2 mL) and EDCI (18 mg, 0.09
mmol), HOBT (13 mg, 0.09 mmol), and triethylamine (26 lL, 0.19
mmol) were added successively to the mixture. After stirring
overnight at room temperature, the mixture was washed with
0.1 N HCl, H2O, and saturated NaHCO3. The organic layer was
dried (Na2SO4), concentrated in vacuo and purified by flash chro-
matography (Hex/EtOAC, 1:3) to get 3 as a pale yellow solid (18
mg, 45%).
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