Total Synthesis and Antitubulin Activity of C10 Analogues of Cryptophycin-24

Article abstract of DOI:10.1021/jm030278c

The unsubstituted, 3′-Cl, 4′-C1, and 3′,4′-diCl C10 analogues of cryptophycin-24 were prepared via total synthesis and tested in vitro for cytotoxicity against MCF-7 and multi-drug-resistant MCF-7/ADR breast cancer cell lines and in a tubulin assembly assay. The ED₅₀ values ranged from 7.2 to 15.8 μM in the tubulin assay and from 0.05 to 3.4 nM in the cell assays. The presence of a 3′-C1 and/or 4′-C1 substituent on the C10 phenyl ring increased cytotoxicity in the MCF-7 cell line compared to the unsubstituted phenyl ring. The most potent compound in this series possessed a 3′-C1 substituent on the C10 phenyl ring. The 3′-C1 analogue had ED₅₀ values of 50 and 580 pM in the MCF-7 and MCF-7/ADR cell lines, respectively. Its activity was very similar to the parent compound cryptophycin-24. Substitution of the 4′-MeO group in cryptophycin-24 with a 4′-C1 moiety did not significantly affect cytotoxicity against MCF-7 and MCF-7/ADR cells compared to the parent compound. These results demonstrated that the 4′-MeO group in cryptophycin-24 is not essential and can be replaced with 3′-C1 or 4′-C1 substituents.

Full text of DOI:10.1021/jm030278c

696
J . Med. Chem. 2004, 47, 696-702
Tota l Syn th esis a n d An titu bu lin Activity of C10 An a logu es of Cr yp top h ycin -24
Suzanne B. Buck,† J acquelyn K. Huff,‡ Richard H. Himes,‡ and Gunda I. Georg*,†
Department of Medicinal Chemistry and Department of Molecular Biosciences, University of Kansas, 1251 Wescoe Drive,
Lawrence, Kansas 66045-7582
Received J une 9, 2003
The unsubstituted, 3′-Cl, 4′-C1, and 3′,4′-diCl C10 analogues of cryptophycin-24 were prepared
via total synthesis and tested in vitro for cytotoxicity against MCF-7 and multi-drug-resistant
MCF-7/ADR breast cancer cell lines and in a tubulin assembly assay. The ED50 values ranged
from 7.2 to 15.8 µM in the tubulin assay and from 0.05 to 3.4 nM in the cell assays. The presence
of a 3′-C1 and/or 4′-C1 substituent on the C10 phenyl ring increased cytotoxicity in the MCF-7
cell line compared to the unsubstituted phenyl ring. The most potent compound in this series
possessed a 3′-C1 substituent on the C10 phenyl ring. The 3′-C1 analogue had ED50 values of
50 and 580 pM in the MCF-7 and MCF-7/ADR cell lines, respectively. Its activity was very
similar to the parent compound cryptophycin-24. Substitution of the 4′-MeO group in
cryptophycin-24 with a 4′-C1 moiety did not significantly affect cytotoxicity against MCF-7
and MCF-7/ADR cells compared to the parent compound. These results demonstrated that the
4′-MeO group in cryptophycin-24 is not essential and can be replaced with 3′-C1 or 4′-C1
substituents.
In tr od u ction
The cryptophycins1-4 are antimitotic, cyanobacterial
metabolites isolated from the blue-green algae Nostoc
sp. ATCC 537895 and Nostoc sp. GSV 224.6,7 When
cryptophycin-1 (1, Figure 1) was first isolated,5 Schwartz
and co-workers chose the name cryptophycin because
of the compound’s highly potent fungicidal activity
against filamentous fungi and yeast of the genus Cryp-
tococcus. Because of the narrow breadth of antifungal
activity of cryptophycin-1 (1) and its low therapeutic
index,8 the development of cryptophycin-1 as an anti-
F igu r e 1. Structures of the cryptophycins.
fungal compound was not pursued. Cryptophycin-1 was
The cryptophycins are microtubule-destabilizing agents
that elicit cytotoxic effects by binding noncovalently to
a site at, or overlapping, the Vinca site on tubulin and
at the rhizoxin/maytansine site.10,12,15-18 Cryptophycin-1
also induces the phosphorylation of Bcl-219 and activates
the apoptosis process.20,21 Similar to other known an-
timitotic agents,22 cryptophycin-1 slows microtubule
dynamics at low concentrations23,24 while at subnano-
molar concentrations it causes complete loss of micro-
tubules within cells.23 Through its action on microtu-
bules and possibly other yet to be discovered targets,
cryptophycin induces potent cytotoxicity by activating
the apoptotic pathway.20,21
later isolated by Moore and co-workers who screened
over 1000 extracts of blue-green algae in search of
compounds that exhibited antitumor activity.6 Crypto-
phycin-1, the most abundant cytotoxin of the crypto-
phycins,9 has potent activity in the pM range in the
multi-drug-resistant (MDR) cell lines SKVLB1 and
MCF-7/ADR,10 thereby surpassing vinblastine, pacli-
taxel, and colchicine in the ability to retain cytotoxic
activity in MDR cell lines.
A structurally simpler cryptophycin, cryptophycin-24
(arenastatin A, 2), was isolated from an Okinawan
marine sponge,11 Dysidea arenaria. Although crypto-
phycin-24 lacks the chlorine substituent on the aryl ring
of the C10 side chain and the methyl group at C6, it
was extremely potent against KB cells (ED50 ) 5 pM).11
Unfortunately, cryptophycin-24 has a half-life of ap-
proximately 10 min in mouse serum12 due to ester
hydrolysis. Therefore, cryptophycin-52 (3), the C6 gem-
dimethyl analogue of cryptophycin-1, was prepared to
decrease the rate of ester hydrolysis in vivo.13 This
synthetic analogue is the first cryptophycin to undergo
clinical trials for the treatment of cancer.14
One of the strong attributes of cryptophycin is that it
lacks cross resistance with paclitaxel, doxorubicin,25 and
other families of drugs such as DNA alkylators, topos-
iomerase II inhibitors, and antimetabolites26 that are
generally used in combination protocols for the treat-
ment of cancer. Cryptophycin-1 has also been found to
exhibit excellent antiproliferative activity in vivo against
five solid tumors of murine origin (i.e. colon adenocar-
cinomas 38 and 51, taxol-sensitive and taxol-resistant
mammary adenocarcinoma M16, and pancreatic ductal
adenocarcinoma) that were subcutaneously trans-
planted into mice.6,27 In vitro, cryptophycin-1 was shown
* To whom correspondence should be addressed. Tel: (785) 864-
4498. Fax: (785) 864-5836. e-mail: georg@ku.edu.
† Department of Medicinal Chemistry.
‡ Department of Molecular Biosciences.
10.1021/jm030278c CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/12/2003

C10 Analogues of Cryptophycin-24
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 3 697
to be highly toxic to the human nasophyrngeal carci-
noma and the human colorectal adenocarcinoma cell
lines.6
Structure-activity relationship (SAR) studies of the
C10 side chain of cryptophycin-1 demonstrated that
introduction of a second chloro substituent at the C5′
position of the C10 side chain decreased activity by a
factor of 120, while absence of the C3′-Cl decreased
activity by a factor of 10.27 A factor of 30 decrease in
activity was observed when the C4′-O-methyl group was
absent.27 These data suggested that the 3′-C1 and 4′-
MeO are required for optimal activity. More recently,
it was revealed that the cytotoxicity of cryptophycin can
be more dramatically affected by altering the C10 side
chain. Patel and co-workers prepared analogues of
cryptophycin-52 (3) that were tested in the human
leukemia CCRF-CEM tumor cell line.13 Substitutions
for the benzyl moiety and alternative substitution
patterns on the aromatic ring were made. Substitution
of the C10 aryl ring with a naphthyl ring, altering the
tether length between the macrocycle and the aryl ring,
removing the aromatic ring, or replacing it with a
cyclohexyl group, led to analogues with varying degrees
of reduced activity. Variations in the substitution pat-
tern of the benzyl ring of the cryptophycin-52 analogues
revealed that the 3′-Cl substituent provides an increased
in vitro activity, whereas compounds lacking the 3′-Cl
substituent were active only in the 4 to 120 nM range
with the exception of the analogue containing an un-
substituted benzyl ring at C10 which was active and
had an ED50 value of 183 pM. The compound with the
most activity was the 3′-C1, 4′-NMe2 analogue, which
had an IC50 value 2.5 times that of cryptophycin-52.
The SAR determined from isolated analogues of
cryptophycin-1 and synthesized cryptophycin analogues
of cryptophycin-52 suggested that any analogue syn-
thesized should retain the following on the C10 side
chain for optimal activity: (1) the R stereochemistry at
C10, (2) a benzyl moiety, and (3) a chloro substitution
at the 3′ (meta) position. We chose to conduct a study
of the C10 substitution pattern of the benzyl ring using
a series of chlorinated analogues of cryptophycin-24 that
carry no other substituents (such as a methoxy or amino
groups) to further define the importance of the 3′-CI
substituent and evaluate the importance of the 4′-MeO
substituent with regard to tubulin interaction and
cytotoxicity. To this end, cryptophycin-24 analogues 4-7
(Figure 2) were synthesized and tested in an in vitro
tubulin assembly assay and in the MCF-7 and the MCF-
7/ADR breast cancer cell lines.
F igu r e 2. Structures of novel C10 analogues.
Sch em e 1a
a
Reagent and conditions: (a) TFA; (b) DCC, HOBT, DIEA, acid,
66-99%, two steps; (c) Pd(OH)2, H2, 81-96%; (d) DIEA, DMAP,
2,4,6-trichlorobenzoyl chloride, 21, 77-96%; (e) TFA; (f) HBTU,
DIEA, 55-88%; (g) DMD, 59-74%.
palladium(II) hydroxide and hydrogen gas to reveal
acids 17-20. Acids 17-20 were activated using the
Yamaguchi reagent, 2,4,6-trichlorobenzoyl chloride, and
coupled to 21, to yield esters 22-25.28,29 The N-Boc
group and the tert-butyl ester were simultaneously
cleaved from intermediates 22-25 using TFA.31 The
macrocycles were subsequently closed using HBTU (O-
benzotriazol-l-yl-N,N,N′,N′-tetramethyluronium hexaflu-
orophosphate) as the coupling reagent,31 thus forming
26-29. Epoxidation of styrenes 26-29 with dimethyl-
dioxirane (DMD)32 provided mixtures of the R and â
epoxides 4-7. The â epoxides were separated from the
ac epoxides using HPLC and tested in vitro in a tubulin
assembly assay and a cytotoxicity assay.
Ch em istr y
Analogues 4-7 were synthesized from two key frag-
ments: the “northern half” 21 and the “southern halves”
17-20 (Scheme 1). Northern half 21 was obtained
through asymmetric synthesis,28,29 while the southern
halves28 were formed through the coupling of three
commercially available building blocks: (1) L-leucic acid,
(2) â-alanine, and (3) a D-phenylalanine moiety.
Synthesis of the C10 analogues 4-7 began with
benzyl-protected 8.30 The Boc group was cleaved using
TFA and the resulting amine was coupled to the N-Boc-
phenylalanine moieties 9-12 to provide 13-16 (Scheme
1).30 The benzyl esters of 13-16 were cleaved using

698 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 3
Buck et al.
Ta ble 1. Biological Results for in Vitro Tubulin Assembly
Assay and Cytotoxicity Studies of C10 Analogues 4-7
the 4′-Cl analogue 6 against MCF-7/ADR cells compared
to cryptophycin-24 is not substantial. The 6-fold and 14-
fold decrease in activity of the unsubstituted analogue
4 and the 3′,4′-diCl analogue 7, respectively, is more
significant.
The results demonstrate that the 4′-MeO is not
essential for cytotoxicity against MCF-7/ADR cells in
the cryptophycin-24 series of compounds. The 4′-MeO
moiety can be replaced by a 3′-C1 or a 4′-C1 group
without significant effects on the cytotoxicity against
MCF-7/ADR cells when compared to the parent com-
pound cryptophycin-24.
It is interesting to note that the decrease in activities
of analogues 4-7 in the MCF-7/ADR cell line compared
to the MCF-7 cell line was significantly more (5 to 40-
fold) when compared to cryptophycin-l and cryptophy-
cin-24 which only displayed 2-fold decreases.
The most potent cryptophycin-24 analogue prepared
was the 3′-C1 analogue 5, which was very similar to the
parent compound cryptophycin-24. Analogue 5 was
twice as active as cryptophycin-24 in the tubulin as-
sembly assay and in the cell cytotoxicity assay against
MCF-7 cells and about 2-fold less active against the
MCF-7/ADR cell line.
tubulin assay:
ED50 (µM)
MCF-7:
ED50 (nM)
MCF-7/ADR:
ED50 (nM)
compound
1
2
4
5
6
7
3.4 ( 0.80
15.8 ( 0.14
13.1 ( 1.0
7.2
12.5 ( 0.7
12.6 ( 6.7
0.009 ( 0.003
0.13 ( 0.06
0.34 ( 0.13
0.05 ( 0.03
0.12 ( 0.04
0.08 ( 0.03
0.018 ( 0.007
0.25 ( 0.20
1.57 ( 1.11
0.58 ( 0.21
0.66 ( 0.40
3.4 ( 1.6
Biologica l Testin g
Cryptophycin-24 (2) and its analogues were active in
the tubulin assembly assay33 with ED50 values in the
range of 7.2-15.8 µM (Table 1). The 3′-C1 analogue 5
was the most active derivative (ED50 ) 7.2 µM). The
4′-C1 analogue 6 (ED50 ) 12.5 µM), unsubstituted 4
(ED50 ) 13.1 µM), and 3′,4′-diCl derivative 7 (ED50
)
12.6 µM) were essentially as active as cryptophycin-24
(2, ED50 ) 15.8 µM). Cryptophycin-1 (ED50 ) 3.4 µM)
was about 4 times as active as cryptophycin-24. Al-
though the analogues 4-7 were all active in the MCF-7
breast cancer cell line with ED50 values ranging from
0.05 to 0.34 nM (Table 1), none were as cytotoxic as
cryptophycin-1 (ED50 ) 0.009 nM). The activity of
cryptophycin-24, ED50 ) 0.13 nM, was within the range
of the synthesized analogues. The most active com-
pounds were 3′-C1 analogue 5 (ED50 ) 0.05 nM) and
3′,4′-diCl analogue 7 (ED50 ) 0.08 nM), whereas 4′-Cl
derivative 6 (ED50 ) 0.12 nM) and unsubstituted 4
(ED50 ) 0.34 nM) were slightly less active. In the MCF-
7/ADR breast cancer cell line, no analogue was as
cytotoxic as cryptophycin-1 with an ED50 value of 0.018
nM. The most active analogues, 3′-C1 5 and 4′-C1 6 had
ED50 values of 0.58 and 0.66 nM, respectively. The two
other analogues, unsubstituted 4 and 3′,4′-diC1 7, had
activities about 40 to 80% less than 5 and 6.
Exp er im en ta l Section
In str u m en ts a n d An a lyses. Proton and carbon nuclear
magnetic resonance spectra were recorded on a DRX400 MHz
or a Bruker DRX500 MHz. All chemical shifts were recorded
in parts per million (ppm), and CDCl3 was used as the internal
standard. Mass spectra were obtained from a ZAB HS mass
spectrometer (VG Analytical Ltd., Manchester, U. K.) equipped
with a 11/250 data system. Fast atom bombardment mass
spectrometry (FABMS) experiments were performed with a
Xenon gun operated at 8 keV energy and 0.8 mA emission at
the MS laboratory at the University of Kansas. Fast atom
bombardment high-resolution mass spectra (FAB HRMS) were
recorded at 1:10 000 resolution using linear voltage scans
under data system control and collected data in a multichannel
analyzer mode (MCA). A Perkin-Elmer 1420 Ratio Recording
Infrared Spectrophotometer or an Avatar 320 FTIR was used
to record infrared spectra. All optical rotations were obtained
using a Perkin-Elmer model 214 Polarimeter or an Autopol
IV Automatic Polarimeter at room temperature. Melting points
were obtained using a Fisher-J ohns melting point apparatus
and are uncorrected.
Ma ter ia ls a n d Meth od s. Tetrahydrofuran and diethyl
ether were freshly distilled over sodium and benzophenone.
Methylene chloride was freshly distilled over CaH2. All silica
gel (230-400 mesh) used for column chromatography was
purchased from VWR Scientific Products. All moisture-sensi-
tive reactions were carried out using oven-dried glassware and
under a positive pressure of argon. Solvents and reagents that
are commercially available were used without further purifica-
tion unless otherwise noted.
The N-Boc-D-phenylalanines required for the C10 analogue
syntheses were obtained from the PepTech Corporation. Cryp-
tophycin-1 (1) was isolated from the Nostoc sp. ATCC 53789
algae.5,6 Cryptophycin-24 (2) was prepared as described in ref
28.
Tu bu lin Assem bly Assa y.33 Varying concentrations of the
cryptophycin-24 analogues were incubated (total volume of 100
µL) at 37 °C for 15 min with tubulin (1.5 mg/mL) in PEM buffer
(pH 6.9, consisting of 0.1 M PIPES, 1 mM EGTA, 1 mM
MgSO4) containing 0.5 mM GTP and 8% DMSO. Following
polymerization, centrifugation for 4 min (37 °C at 50 000 rpm)
sedimented the microtubules formed, leaving any unassembled
tubulin in the supernatant. The Bradford method34 was used
to quantitate the protein concentration (unpolymerized tubulin
concentration) in the supernatant from which the protein
Discu ssion
The results showed that the unsubstituted phenyl
analogue 4, 3′-Cl 5, 4′-Cl 6, and the 3′,4′-diCl 7 ana-
logues had similar activities in the tubulin assembly
assay and the MCF-7 cell line assay compared to
cryptophycin-24. It can therefore be concluded that the
4′-MeO group of cryptophycin-24 is not essential for
tubulin interaction and cytotoxicity against MCF-7 cells
and that the 4′-MeO can be replaced with 3′-Cl, 4′-Cl
or 3′,4′-diCl moieties. Overall, the 2 to 3-fold decrease
in activity for the analogues and cryptophycin-24 in
these two assays, in comparison to cryptophycin-1, is
not substantial.
In the MCF-7/ADR cell line, cryptophycin-1 displayed
a 2-fold decrease in activity compared to the non-multi-
drug-resistant cell line. The difference between the
activities of cryptophycin-24 and analogues 4-7 is more
pronounced, varying from a 5-fold to a 43-fold decrease
in activity in the MCF-7/ADR cell line when compared
to cryptophycin-1. This could be due to a change in the
interaction of these analogues with the P-glycoprotein
present in the multi-drug-resistant cell line.
As was seen for cryptophycin-1, cryptophycin-24
displayed a 2-fold decrease of cytotoxicity (ED50 ) 0.25
nM) in the MCF-7/ADR cells when compared to its
activity against MCF-7 cells (ED50 ) 0.13 nM). The 2-
to 3-fold decrease of activity of the 3′-C1 analogue 5 and

C10 Analogues of Cryptophycin-24
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 3 699
concentration in the pellet was calculated. The concentration
of the cryptophycin-24 analogue that reduced the amount of
microtubules by 50% was defined as the ED50 value.
3H, J ) 6.4 Hz); 13C NMR (100 MHz, CDCl3) δ 171.9, 171.6,
171.5, 155.6, 135.6, 135.4, 133.1, 131.3, 131.1 (2C), 129.1 (2C),
129.0 (2C), 128.7 (2C), 80.4, 71.6, 67.8, 55.9, 39.9, 38.6, 35.6,
34.6, 28.7 (3C), 25.0, 23.4, 21.9; IR (CDCl3) 3440, 2980, 2940,
1740, 1710, 1680, 1500 cm-1; HRMS (FAB, PEG300) m/e calcd
for C30H41N2O7Cl (M + H)+: 575.2524, found 575.2500; [R]D
-27.0 (c 0.512, CHCl3).
Cell Cytotoxicity Assa y.33 The cytotoxicity of the crypto-
phycin-24 analogues was determined using the MCF-7 and
MCF-7/ADR breast cancer cell lines. Approximately 2000 cells
per well were plated in 96-well culture dishes in Dulbecco’s
MEM/F12 containing 10% fetal calf serum. New medium,
containing varying concentrations of the cryptophycin-24
analogue, was substituted for the original medium at 24 h.
After incubation for 72 h, cell proliferation, using sulfo-
rhodamine B dye was measured.35 The ED50 value was defined
as the concentration of the cryptophycin-24 analogue that
reduced cell proliferation by 50%.
P r ep a r a tion of Ben zyl-P r otected Sou th er n Ha lves
13-16 (Sa m p le P r oced u r e). To a solution of Boc-protected
benzyl ester 8 (1.97 g, 5.02 mmol) in CH2Cl2 (17.5 mL) at 0 °C
was added TFA (3.9 mL, 50.2 mmol) dropwise. The ice bath
was removed, and the solution was stirred at room tempera-
ture for 3.5 h. Dry toluene (1.0 mL) was added, and the solution
was concentrated. Quantitative yield was assumed and the
crude salt was used directly in the next reaction. To a solution
of the benzyl ester salt (5.02 mmol) in THF (62 mL) were added
HOBT (0.68 g, 5.02 mmol), DIEA (2.00 mL, 11.5 mmol), and
N-Boc-D-phenylalanine (9, 2.00 g, 7.54 mmol). After cooling
the solution to 0 °C, DCC (1.14 g, 5.52 mmol) was added. The
solution was stirred at 0 °C for 30 min, then allowed to warm
to room temperature and stirred for 16 h. After removing the
urea byproduct by filtration, the solution was concentrated and
dissolved in EtOAc. Following extraction once each with a 5%
KHSO4 solution and a saturated NaHCO3 solution, the organic
layer was dried (MgSO4). The product was filtered, concen-
trated, and purified by flash column chromatography on silica
gel (10% acetone:hexanes).
Ben zyl (2S)-2-[3-[(2R)-2-ter t-Bu toxyca r bon yla m in o-3-
(3,4-d ich lor op h en yl)p r op ion yla m in o]p r op ion yloxy]-4-
m eth ylp en ta n oa te (16). The total yield of 16, a white foam,
was 1.00 g, 68%: 1H NMR (400 MHz, CDCl3) δ 7.44-7.32
(m, 7H), 7.04 (dd, 1H, J ) 1.6 Hz, 8.6 Hz), 6.84-6.82 (m, 1H),
5.25-5.15 (m, 4H), 4.38-4.36 (m, 1H), 3.65 (dd, 1H, J ) 6.0
Hz, 12.8 Hz), 3.55-3.47 (m, 1H), 3.09 (dd, 1H, J ) 6.2 Hz,
13.8 Hz), 2.95 (dd, 1H, J ) 6.6 Hz, 7.5 Hz), 2.58 (t, 2H, J )
5.8 Hz), 1.82-1.57 (m, 3H), 1.41 (s, 9H), 0.94 (d, 3H, J )
6.3 Hz), 0.92 (d, 3H, J ) 6.3 Hz); 13C NMR (100 MHz, CDCl3)
δ 171.8; 171.6, 171.2, 155.6, 137.7, 135.3, 132.6, 131.8, 131.2,
130.7, 129.3, 129.1 (2C), 129.0, 128.6 (2C), 80.4, 71.6, 67.9, 55.7,
39.8, 38.4, 35.6, 34.7, 28.7 (3C), 25.0, 23.4, 21.9; IR (CDCl3)
3420, 3320, 2960, 2940, 1740, 1720, 1680, 1510 cm-1
;
HRMS (FAB, PEG600) m/e calcd for C30H39N2O7Cl2 (M + H)+:
609.2134, found 6019.2109; [R]D -19.8 (c 0.466, CHCl3).
P r ep a r a tion of Acid s 17-20 (Sa m p le P r oced u r e). After
degassing a solution of the benzyl ester 13 (1.79 g, 3.13 mmol)
in EtOAc (45 mL) for 5 min with argon, Pd(OH)2 (0.22 g, 0.31
mmol) was added. The solution was stirred under an atmos-
phere of hydrogen gas using a hydrogen filled balloon. After 2
h, the solution was filtered and concentrated. The crude
product obtained was 95% pure as determined by 1H NMR and
was used without further purification.
(2S)-2-[3-[(2R)-2-ter t-Bu toxyca r bon yla m in o-3-(p h en yl)-
p r op ion yla m in o]p r op ion yloxy]-4-m eth ylp en ta n oic Acid
(17). The total yield of 17, a white foam, was 0.87 g, 81%: 1H
NMR (400 MHz, CDCl3) δ 7.27-7.17 (m, 5H), 6.86 (m, 1H),
5.26-5.20 (m, 1H), 5.10-5.00 (m, 1H), 4.39-4.37 (m, 1H),
3.52-3.50 (m, 2H), 3.23-3.08 (m, 1H), 3.00-2.99 (min, 1H),
2.57-2.55 (m, 2H), 1.82-1.71 (m, 3H), 1.40 (s, 9H), 0.98 (d,
3H, J ) 6.2 Hz), 0.95 (d, 3H, J ) 6.1 Hz); 13C NMR (125 MHz,
CDCl3) δ 171.7 (2C), 171.4, 155.8, 136.4, 129.3 (2C), 128.4 (2C),
126.8, 80.6, 71.3, 55.6, 39.4, 38.8, 35.2, 33.4, 28.2 (3C), 24.6,
23.0, 21.4; IR (CDCl3) 3500-2500, 3440, 3380, 2980, 2900,
1720, 1680 cm-1; HRMS (FAB, PEG300) m/e calcd for
Ben zyl (2S)-2-[3-[(2R)-2-ter t-Bu toxyca r bon yla m in o-3-
(p h en yl)p r op ion yla m in o]p r op ion yloxy]-4-m et h ylp en -
ta n oa te (13). The total yield of 13, a white foam, was 2.70 g.
99%: 1H NMR (400 MHz, CDCl3) δ 7.41-7.37 (m, 5H), 7.27-
7.19 (m, 5H), 6.63 (br d, 1H), 5.23-5.12 (m, 4H), 4.39-4.37
(m, 1H), 3.59-3.58 (m, 1H), 3.34 (dd, 1H, J ) 6.4 Hz, 12.6
Hz), 3.12-3.04 (m, 2H), 2.57-2.54 (m, 2H), 1.81-1.69 (m, 2H),
1.68-1.61 (m, 1H), 1.40 (s, 9H), 0.94 (d, 3H, J ) 6.3 Hz), 0.92
(d, 3H, J ) 6.4 Hz); 13C NMR (125 MHz, CDCl3) δ 171.4, 171.3,
170.8, 155.2, 136.6, 135.0, 129.4, 129.3 (2C), 128.6 (2C), 128.5
(2C), 128.4, 128.2 (2C), 79.8, 71.1, 67.2, 55.7, 39.4, 38.8, 35.0,
34.0, 28.2 (3C), 24.5, 22.9, 21.5; IR (CDCl3) 3450, 2980, 2960,
1740, 1710, 1680 cm-1; HRMS (FAB, PEG600) m/e calcd for
C
23H35N2O7 (M + H)+: 451.2444, found 451.2438; [R]D -20.0
(c 0.49, CHCl3).
(2S)-2-[3-[(2R)-2-ter t-Bu toxyca r bon yla m in o-3-(3-ch lo-
r op h en yl)p r op ion yla m in o]p r op ion yloxy]-4-m eth ylp en -
ta n oic Acid (18). The total yield of 18, a white foam was 1.28
g, 85%: 1H NMR (500 MHz, CDCl3) δ 7.25-7.19 (m, 3H), 7.07
(d, 1H, J ) 2.7 Hz), 6.93-6.90 (m, 1H), 5.31-5.28 (m, 1H),
5.13-5.10 (m, 1H), 4.40-4.38 (m, 1H), 3.58-3.56 (m, 2H),
3.15-3.10 (m, 1H), 2.95-2.92 (m, 1H), 2.62-2.60 (m, 2H),
1.84-1.82 (m, 2H), 1.74-1.73 (m, 1H), 1.41 (s, 9H), 0.99 (d,
3H, J ) 6.4 Hz), 0.97 (d, 3H, J ) 6.4 Hz); 13C NMR (125 MHz,
CDCl3) δ 173.1, 171.8, 171.3, 155.8, 138.6, 134.0, 129.6, 129.4,
127.5, 126.9, 80.6, 71.3, 55.2, 39.4, 38.3, 35.2, 33.9, 28.1 (3C),
24.6, 22.9, 21.4; IR (CDCl3) 3500-2500, 3420, 3340, 2980, 2940,
2880, 1720, 1680 cm-1; HRMS (FAB, PEG300, NBA) m/e calcd
for C23H34ClN2O7 (M + H)+: 485.2054, found 485.2051; [R]D
-20.8 (c 0.528, CHCl3).
C
30H41N2O7 (M + H)+: 541.2914, found 541.2924; [R]D -27.3
(c 0.498, CHCl3).
Ben zyl (2S)-2-[3-[(2R)-2-ter t-Bu toxyca r bon yla m in o-3-
(3-ch lor op h en yl)p r op ion yla m in o]p r op ion yloxy]-4-m eth -
ylp en ta n oa te (14). The total yield of 14, a white foam, was
2.09 g, 82%: 1H NMR (500 MHz, CDCl3) δ 7.43-7.36 (m, 5H),
7.29-7.21 (m, 3H), 7.10-7.08 (m, 1H), 6.75-6.73 (m, 1H),
5.25-5.16 (m, 4H), 4.38 (d, 1H, J ) 6.4 Hz), 3.64-3.63 (m,
1H), 3.54-3.49 (m, 1H), 3.11 (dd, 1H, J ) 6.3 Hz, 13.7 Hz),
3.01-3.00 (m, 1H), 2.58-2.57 (m, 2H), 1.81-1.71 (m, 2H),
1.69-1.64 (m, 1H), 1.42 (s, 9H), 0.95 (d, 3H, J ) 6.4 Hz), 0.93
(d, 3H, J ) 6.4 Hz); 13C NMR (125 MHz, CDCl3) δ 171.9, 171.5,
171.2, 155.6, 139.3, 135.4, 134.6, 130.2, 130.0, 129.1 (2C),
129.0, 128.7 (2C), 128.0, 127.4, 80.4, 71.6, 67.9, 55.9, 39.9, 39.0,
35.6, 34.7, 28.7 (3C), 25.0, 23.4, 21.9; IR (CDCl3) 3460, 2980,
2960, 1740, 1710, 1680 cm-1; HRMS (FAB, PEG600) m/e calcd
for C30H40N2O7Cl (M + H)+: 575.2524, found 575.2500; [R]D
-21.8 (c 0.476, CHCl3).
Ben zyl (2S)-2-[3-[(2R)-2-ter t-Bu toxyca r bon yla m in o-3-
(4-ch lor op h en yl)p r op ion yla m in o]p r op ion yloxy]-4-m eth -
ylp en ta n oa te (15). The total yield of 15, a white foam, was
1.70 g, 66%: 1H NMR (500 MHz, CDCl3) δ 7.46-7.33 (m, 5H),
7.29-7.23 (m, 2H), 7.16-7.12 (m, 2H), 6.78-6.76 (m, 1H),
5.23-5.14 (m, 4H), 4.39-4.37 (m, 1H), 3.62-3.60 (m, 1H),
3.50-3.46 (m, 1H), 3.08 (dd, 1H, J ) 6.2 Hz, 13.6 Hz), 3.00-
2.95 (m, 1H), 2.58-2.55 (m, 2H), 1.81-1.71 (m, 2H), 1.70-
1.61 (m, 1H), 1.41 (s, 9H), 0.94 (d, 3H, J ) 6.4 Hz), 0.92 (d,
(2S)-2-[3-[(2R)-2-ter t-Bu toxyca r bon yla m in o-3-(4-ch lo-
r op h en yl)p r op ion yla m in o]p r op ion yloxy]-4-m eth ylp en -
ta n oic Acid (19). The total yield of 19, a white foam, was
1.00 g, 68%: 1H NMR (400 MHz, CDCl3) δ 10.9-10.7 (br s,
1H), 7.22 (d, 2H J ) 7.6 Hz), 7.11 (d, 2H, J ) 7.6 Hz), 6.29-
6.27 (m, 1H), 5.55 (d, 1H, J ) 7.6 Hz), 5.12-5.08 (m, 1H),
4.40-4.38 (m, 1H), 3.52-3.50 (m, 2H), 3.03-3.00 (m, 1H),
2.91-2.90 (m, 1H), 2.54-2.52 (m, 2H), 1.90-1.80 (m, 3H), 1.38
(s, 9H), 0.97 (d, 3H, J ) 6.0 Hz), 0.95 (d, 3H, J ) 6.0 Hz); 13
C
NMR (100 MHz, CDCl3) δ 172.1, 171.4 (2C), 155.7, 135.0,
132.6, 130.7 (2C), 128.5 (2C), 80.6, 71.5, 53.7, 39.5, 37.9, 35.2,
33.9, 28.1 (3C), 24.6, 23.0, 21.4; IR (CDCl3) 3460, 3000, 2880,
1750, 1680 cm-1; HRMS (FAB, PEG300) m/e calcd for C23H34
-

700 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 3
Buck et al.
ClN2O7 (M + H)+: 485.2055, found 485.2035; [R]D -20.0 (c
pion yloxy-4-m eth ylpen tan oyloxy]-6-m eth yl-8-ph en ylocta-
(2E,7E)-d ien oa te (24). The total yield of 24, pale yellow oil,
was 0.19 g, 96%: 1H NMR (500 MHz, CDCl3) δ 7.36-7.18 (m,
9H), 7.16-7.14 (m, 1H), 7.00-6.82 (m, 1H), 6.43 (d, 1H, J )
15.9 Hz), 6.03 (dd, 1H, J ) 8.7 Hz, 15.9 Hz), 5.89 (d, 1H, J )
15.7 Hz), 5.63 (d, 1H, J ) 8.5 Hz), 5.07-5.05 (m, 1H), 4.98
(dd, 1H, J ) 3.6 Hz, 10.1 Hz), 4.46 (d, 1H, J ) 5.6 Hz), 3.62-
3.61 (m, 1H), 3.50-3.49 (m, 1H), 3.26 (dd, 1H, J ) 5.0 Hz,
13.7 Hz), 2.96 (dd, 1H, J ) 8.6 Hz, 13.2 Hz), 2.63-2.45 (m,
5H), 1.75-1.62 (m, 1H), 1.50 (s, 9H), 1.40 (s, 9H), 1.14 (d, 3H,
J ) 6.8 Hz), 0.96-0.89 (m, 2H), 0.86 (d, 3H, J ) 6.5 Hz), 0.80
(d, 3H, J ) 6.5 Hz); 13C NMR (100 MHz, CDCl3) δ 171.7, 171.3,
170.8, 166.3, 155.9, 142.3, 137.2, 136.4, 132.8, 132.2, 131.2
(2C), 130.4, 129.0 (2C), 128.8 (2C), 128.0, 126.6 (2C), 126.5,
80.9, 80.0, 76.8, 71.6, 56.0, 41.6, 40.0, 38.4, 35.7, 35.5, 34.8,
28.7 (3C), 28.6 (3C), 25.0, 23.3, 21.7, 17.5; IR (CDCl3) 3480,
3000, 2960, 1740, 1710, 1680 cm-1; HRMS (FAB, PEG600) m/e
calcd for C42H58N2O9Cl (M + H)+: 769.3831, found 769.3859;
[R]D -1.3 (c 2.2, CHCl3).
ter t-Bu tyl (5 S,6R)-5-[(2S)-2-[3-[(2R)-2-ter t-Bu toxyca r -
b on yla m in o-3-(3,4-d ich lor op h e n yl)p r op ion yla m in o]-
p r op ion yloxy-4-m et h ylp en t a n oyloxy].-6-m et h yl-8-p h e-
n ylocta -(2E,7E)-d ien oa te (25). The total yield of 25, a pale
yellow oil, was 0.15 g, 82%: 1H NMR (500 MHz, CDCl3) 7.37-
7.11 (m, 8H), 7.11-7.10 (m, 1H), 6.86-6.83 (m, 1H), 6.43 (d,
1H, J ) 15.9 Hz), 6.02 (dd, 1H, J ) 8.7 Hz, 15.9 Hz), 5.86 (d,
1H, J ) 15.7 Hz), 5.72 (d, 1H, J ) 7.9 Hz), 5.04-5.02 (m, 1H),
4.97 (dd, 1H, J ) 3.5 Hz, 10.1 Hz), 4.46 (d, 1H, J ) 4.8 Hz),
3.60-3.55 (m, 1H), 3.54-3.53 (m, 1H), 3.29-3.25 (m, 1H),
2.94-2.92 (m, 1H), 2.61-2.55 (m, 3H), 2.45-2.44 (m, 2H),
1.64-1.53 (m, 1H), 1.50 (s, 9H), 1.38 (s, 9H), 1.14 (d, 3H, J )
6.8 Hz), 0.92-0.86 (m, 2H), 0.85 (d, 3H, J ) 6.5 Hz), 0.78 (d,
3H, J ) 6.5 Hz); 13C NMR (125 MHz, CDCl3) δ 171.3 (2C),
170.9, 165.9, 155.4, 141.9, 137.8, 136.7, 131.8, 131.4, 130.5,
130.1, 129.9, 128.8, 128.5 (2C), 128.3, 127.5, 126.1 (2C), 126.0,
80.4, 79.6, 76.3, 71.2, 55.2, 41.2, 39.5, 37.7, 35.3, 35.0, 34.3,
28.2 (3C), 28.1 (3C), 24.5, 22.8, 21.2, 17.0; IR (CDCl3) 3440,
2980, 2960, 1740, 1710, 1680 cm-1; HRMS (FAB, PEG600) m/e
calcd for C42H57Cl2N2O9 (M + H)+: 803.3441, found 803.3460;
[R]D -4.84 (c 3.00, CHCl3).
0.574, CHCl3).
(2S )-2-[3-[(2R )-2-t er t -Bu t oxyca r b on yla m in o-3-(3,4-
d ich lor op h en yl)p r op ion yla m in o]p r op ion yloxy]-4-m eth -
ylp en ta n oic Acid (20). The total yield of 20, a white foam
was 0.13 g, 95%: 1H NMR (500 MHz, CDCl3) δ 7.34 (d, 1H, J
) 7.8 Hz), 7.02 (d, 2H, J ) 8.2 Hz), 5.96-5.88 (m, 1H), 5.33-
5.32 (m, 1H), 5.09-5.07 (m, 1H), 4.36-4.35 (m, 1H), 3.57-
3.53 (m, 2H), 3.11-3.10 (m, 1H), 3.00-2.90 (m, 1H), 2.67-
2.58 (m, 2H), 1.86-1.81 (m, 2H), 1.75-1.70 (m, 1H), 1.41 (s,
9H), 0.99 (d, 3H, J ) 6.4 Hz), 0.97 (d, 3H, J ) 6.4 Hz); 13C
NMR (125 MHz, CDCl3) δ 173.4, 171.8, 171.3, 155.7, 136.8,
132.1, 131.3, 130.8, 130.2, 128.8, 80.7, 71.4, 55.0, 39.4, 37.7,
35.3, 34.0, 28.1 (3C), 24.6, 22.9, 21.4; IR (CDCl3) 3440, 3380,
2980, 2940, 2900, 3500-2500, 1730, 1720, 1580 cm-1; HRMS
(FAB, PEG300) m/e calcd for C23H33N2O7Cl (M + H)+: 519.1665,
found 519.1658; [R]D -22.4 (c 0.438, CHCl3).
P r ep a r a tion of ter t-Bu tyl Ester s 22-25 (Sa m p le P r o-
ced u r e). After drying over MgSO4 for 30 min, a solution of
acid 17 (0.21 g, 0.46 mmol) in THF (0.46 mL) was removed
via syringe and cooled to 0 °C. To this were added DIEA (0.10
mL, 0.58 mmol), 2,4,6- trichlorobenzoyl chloride (0.08 mL, 0.51
mmol), and DMAP (0.002 g, 0.02 mmol). After 2 h, a solution
of alcohol 21 (0.07 g, 0.23 mmol) in THF (3.4 mL) was added
and stirred at room temperature for 2 h. The solution was
diluted with CH2Cl2 and rinsed with a saturated NaHCO3
solution. The aqueous layer was extracted three times with
CH2Cl2, and the combined organic layers were dried (MgSO4).
After filtering and concentrating, the crude product was
purified using column chromatography on silica gel (100%
hexanes to 70% EtOAc:hexanes).
ter t-Bu tyl (5S,6R)-5-[(2S)-2-[3-[(2R)-2-ter t-Bu toxyca r -
b on yla m in o-3-p h en ylp r op ion yla m in o]p r op ion yloxy-4-
m eth ylp en ta n oyloxy]-6-m eth yl-8-p h en ylocta -(2E,7E)-d i-
en oa te (22). The total yield of 22, a pale yellow oil, was 0.14
g, 81%: 1H NMR (400 MHz, CDCl3) δ 7.32-7.19 (m, 10H),
6.74-6.72 (m, 1H), 6.49-6.46 (m, 1H), 6.43 (d, 1H, J ) 15.8
Hz), 6.02 (dd, 1H, J ) 8.7 Hz, 15.8 Hz), 5.86 (d, 1H, J ) 15.6
Hz), 5.50 (d, 1H, J ) 6.9 Hz), 5.06-5.05 (m, 1H), 4.98 (dd, 1H,
J ) 3.7 Hz, 9.9 Hz), 4.36-4.35 (m, 1H), 3.58-3.56 (m, 1H),
3.48-3.39 (m, 1H), 3.25-3.22 (m, 1H), 3.05-3.00 (m, 1H),
2.64-2.49 (m, 5H), 1.74-1.61 (m, 1H), 1.50 (s, 9H), 1.37 (s,
9H), 1.13 (d, 3H, J ) 6.8 Hz), 0.94-0.88 (m, 2H), 0.86 (d, 3H,
J ) 6.4 Hz), 0.81 (d, 3H, J ) 6.4 Hz); 13C NMR (100 MHz,
CDCl3) δ 171.9, 171.8, 171.2, 166.2, 156.9, 142.2, 137.7, 137.2,
132.2, 130.4, 129.8 (2C), 129.0 (2C), 128.8 (2C), 127.9, 127.0,
126.8, 126.6 (2C), 80.6, 79.9, 76.8, 71.6, 56.2, 41.4, 40.0, 39.1,
35.6, 35.2, 34.7, 28.7 (3C), 28.6 (3C), 25.0, 23.3, 21.8, 14.6; IR
(CDCl3) 3480, 2980, 2960, 1740, 1710, 1680 cm-1; HRMS (FAB,
PEG600) m/e calcd for C42H59N2O9 (M + H)+: 735.4221, found
735.4220; [R]D +1.52 (c 2.76, CHCl3).
ter t-Bu tyl (5S,6R)-5-[(2S)-2-[3-[(2R)-2-ter t-Bu toxyca r -
b on yla m in o-3-(3-ch lor op h e n yl)p r op ion yla m in o]p r o-
pion yloxy-4-m eth ylpen tan oyloxy]-6-m eth yl-8- ph en ylocta-
(2E,7E)-d ien oa te (23). The total yield of 23, a pale yellow
oil, was 0.13 g, 77%: 1H NMR (500 MHz, CDCl3) δ 7.33-7.13
(m, 9H), 7.03-7.00 (m, 1H), 6.84-6.82 (m, IH), 6.42 (d, 1H, J
) 15.8 Hz), 6.02 (dd, 1H, J ) 8.7 Hz, 15.8 Hz), 5.85 (d, 1H, J
) 15.6 Hz), 5.65 (d, 1H, J ) 7.5 Hz), 5.09-5.07 (m, 1H), 4.98
(dd, 1H, J ) 3.6 Hz, 10.0 Hz), 4.45-4.44 (m, 1H), 3.59-3.57
(m, 1H), 3.52-3.50 (m, 1H), 3.26 (dd, 1H, J ) 4.7 Hz, 13.6
Hz), 2.96-2.92 (m, 1H), 2.63-2.55 (m, 3H), 2.50-2.45 (m, 2H),
1.70-1.62 (m, 1H), 1.50 (s, 9H), 1.37 (s, 9H), 1.14 (d, 3H, J )
6.8 Hz), 0.94-0.89 (m, 2H), 0.85 (d, 3H, J ) 6.5 Hz), 0.79 (d,
3H, J ) 6.5 Hz); 13C NMR (125 MHz, CDCl3) δ 171.3, 171.1,
170.8, 165.8, 155.4, 141.7, 139.5, 136.7, 133.9, 131.7, 130.0,
129.5, 129.3, 128.5 (2C), 127.5, 127.4, 126.9, 126.7, 126.1 (2C),
80.4, 79.5, 76.4, 71.2, 55.4, 41.1, 39.5, 38.3, 35.2, 34.9, 34.3,
28.2 (3C), 28.1 (3C), 24.5, 22.8, 21.2, 16.9; IR (CDCl3) 3420,
3320, 1730, 1700, 1660 cm_; HRMS (FAB, PEG600) m/e calcd
for C42H58N2O9Cl (M + H)+: 769.3831, found 769.3845; [R]D
+0.37 (c 2.70, CHCl3).
P r ep a r a tion of Styr en es 26-29 (Sa m p le P r oced u r e).
To a solution of Boc-protected tert-butyl ester 22 (0.08 g, 0.11
mmol) in CH2Cl2 (5.0 mL) was added TFA (0.38 mL, 4.4
mnmol) dropwise. After stirring at room temperature for 2 h,
dry toluene (0.2 mL) was added. The solution was concentrated
in vacuo and pumped on overnight. The crude product was
used directly without further purification in the next reaction.
To a solution of the TFA salt (0.11 mmol) in CH3CN (4.0 mL)
were added DIEA (0.57 mL, 0.33 mmol) and HBTU (0.05 g,
0.13 mmol). After stirring at room temperature for 3 h, the
solution was quenched with a solution of saturated NaHCO3.
The organic layer was extracted three times with CH2Cl2 and
dried (MgSO4). Following filtration and concentration, the
crude product was purified using column chromatography on
silica gel (10% acetone:CH2Cl2 containing 1% HOAc to 20%
acetone:CH2Cl2 containing 1% HOAc).
Dem eth oxyd esep oxycr yp top h ycin -24 (26). The total
yield of 26, a white solid, was 0.033 g, 55%: mp 242-243 °C;
1H NMR (400 MHz, CDCl3) δ 7.36-7.21 (m, 10H), 7.04-7.02
(m, 1H), 6.73 (ddd, 1H, J ) 4.9 Hz, 10.3 Hz, 15.2 Hz), 6.41 (d,
1H, J ) 15.9 Hz), 6.04 (dd, 1H, J ) 8.8 Hz, 15.9 Hz), 5.75 (dd,
1H, J ) 1.1 Hz, 15.2 Hz), 5.65 (d, 1H, J ) 8.3 Hz), 5.07 (ddd,
1H, J ) 2.0 Hz, 4.5 Hz, 6.5 Hz), 4.92 (dd, 1H, J ) 3.6 Hz, 9.9
Hz), 4.82 (dd, 1H, J ) 2.2 Hz, 7.9 Hz), 3.54-3.46 (m, 2H), 3.33
(dd, 1H, J ) 5.5 Hz, 14.4 Hz), 3.11 (dd, 1H, J ) 7.7 Hz, 14.4
Hz), 2.58-2.53 (m, 4H), 2.39-2.36, (m, 1H), 1.68-1.60 (m, 2H),
1.37-1.30 (m, 1H), 1.15 (d, 3H, J ) 6.9 Hz), 0.76 (d, 3H, J )
6.4 Hz), 0.73 (d, 3H, J ) 6.4 Hz); 13C NMR (125 MHz, CDCl3)
δ 173.3, 171.2, 171.1, 165.9, 142.0, 137.1, 137.0, 132.2, 130.6,
129.6 (2C), 129.1 (2C), 129.0 (2C), 128.0, 127.3, 126.6 (2C),
125.5, 76.8, 71.9, 54.4, 42.7, 40.1, 36.8, 36.5, 34.7, 32.9, 24.7,
23.0, 21.6, 17.7; IR (CH2Cl2) 3440, 3060, 3000, 1740, 1680,
1650, 1430, 1220 cm-1; HRMS (FAB, PEG600) m/e calcd for
ter t-Bu tyl (5S,6R)-5-[(2S)-2-[3-[(2R)-2-ter t-Bu toxyca r -
b on yla m in o-3-(4-ch lor op h e n yl)p r op ion yla m in o]p r o-
C
33H41N2O6 (M + H)+: 561.2965, found 561.2960; [R]D +31 (c

C10 Analogues of Cryptophycin-24
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 3 701
0.40, CHCl3). 10-(3′-Chlorobenzyl)demethoxybenzyldesepoxy-
(dd, 1H, J ) 2.1 Hz, 7.9 Hz), 3.70 (d, 1H, J ) 1.9 Hz), 3.58-
3.53 (m, 1H), 3.48-3.44 (m, 1H), 3.25 (dd, 1H, J ) 5.5 Hz,
14.5 Hz), 3.13-3.07 (m, 1H), 2.94 (dd, 1H, J ) 1.9 Hz, 7.5 Hz),
2.61-2.54 (m, 3H), 2.49-2.43 (m, 1H), 1.75-1.65 (m, 3H),
1.34-1.30 (m, 1H), 1.16 (d, 3H, J ) 6.7 Hz), 0.86 (d, 3H, J )
6.3 Hz), 0.85 (d, 3H, J ) 6.3 Hz); 13C NMR (125 MHz, CDCl3)
δ 172.7, 170.6, 170.5, 165.3, 141.0, 136.6, 136.5, 129.1 (2C),
128.7 (2C), 128.6 (2C), 128.5, 127.0, 125.5 (2C), 125.1, 75.8,
71.1, 63.0, 59.0, 53.9, 40.6, 39.4, 36.3, 36.0, 34.2, 32.4, 24.3,
22.7, 21.2, 13.4; IR (film on KBr) 3286, 2958, 2925, 2853, 1743,
cryptophycin-24 (27). The total yield of 27, a white solid, was
1
0.077 g, 88%: mp 213-215 °C; H NMR (400 MHz, CDCl3) δ
7.36-7.22 (m, 9H), 7.13-7.11 (m, 1H), 6.72 (ddd, 1H, J ) 4.8
Hz, 10.0 Hz, 15.2 Hz), 6.43 (d, 1H, J ) 15.8 Hz), 6.03 (dd, 1H,
J ) 8.8 Hz, 15.8 Hz), 5.78 (d, 1H, J ) 15.4 Hz), 5.67 (d, 1H, J
) 8.6 Hz), 5.08-5.07 (m, 1H), 4.91 (dd, 1H, J ) 3.5 Hz, 10.0
Hz), 4.83 (dd, 1H, J ) 7.9 Hz, 14.0 Hz), 3.58-3.55 (m, 1H),
3.49-3.45 (m, 1H), 3.24 (dd, 1H, J ) 5.6 Hz, 14.4 Hz), 3.05
(dd, 1H, J ) 7.6 Hz, 14.4 Hz), 2.61-2.52 (m, SH), 1.32-1.23
(m, 1H), 1.15 (d, 3H, J ) 6.8 Hz), 0.96-0.90 (m, 2H), 0.77 (d,
3H, J ) 6.4 Hz), 0.73 (d, 3H, J ) 6.4 Hz); 13C NMR (125 MHz,
CDCl3) δ 172.5, 170.7, 170.4, 165.6, 141.3, 138.9, 136.6, 134.3,
131.7, 130.1, 129.8, 129.3, 128.5 (2C), 128.4, 127.5, 127.3,
127.0, 126.1 (2C), 125.2, 71.5, 53.6, 42.2, 39.6, 36.3, 35.7, 34.5,
32.4, 24.3, 22.6, 21.1, 17.2; IR (CH2Cl2) 3420, 2980, 2950, 1750,
1680, 1630, 1510, 1390 cm-1; HRMS (FAB, PEG600) m/e calcd
for C33H40N2O6Cl (M + H)+: 595.2575, found 595.2559; [R]D
+19 (c 0.28, CHCl3).
1678, 1536, 1453, 1371, 1261, 1173, 1012, 751, 698 cm-1
;
HRMS (FAB, PEG600) m/e calcd for C33H41N2O7 (M + H)+:
577.2914, found 577.2903; [R]D +41 (c 0.07, CHCl3).
10-(3′-Ch lor ob en zyl)d em et h oxyb en zylcr yp t op h ycin -
24 (5). The total yield of a mixture of 5 and R-5 (â:R, 2:1), a
white solid, was 8.3 mg, 59%. 5, tR ) 25.5 min; mp 213-214
°C; 1H NMR (400 MHz, CDCl3) δ 7.40-7.22 (m, 8H), 7.13-
7.11 (m, 1H), 6.93 (br t, 1H, 5.3 Hz), 6.70 (ddd, 1H, J ) 5.0
Hz, 10.0 Hz, 15.1 Hz), 5.75 (d, 1H, J ) 15.6 Hz), 5.71 (d, 1H,
J ) 8.7 Hz), 5.21 (dd, 1H, J ) 5.0 Hz, 11.0 Hz), 4.90 (dd, 1H,
J ) 3.3 Hz, 10.0 Hz), 4.81 (dd, 1H, J ) 8.0 Hz, 14.0 Hz), 3.71
(d, 1H, J ) 1.5 Hz), 3.60-3.52 (m, IH), 3.50-3.44 (m, 1H),
3.24 (dd, 1H, J ) 5.6 Hz, 14.3 Hz), 3.05 (dd, 1H, J ) 8.1 Hz,
14.4 Hz), 2.95 (dd, 1H, J ) 1.8 Hz, 7.5 Hz), 2.59-2.53 (m, 3H),
2.51-2.41 (m, 1H), 1.73-1.66 (m, 2H), 1.35-1.31 (m, 2H), 1.17
(d, 3H, J ) 6.8 Hz), 0.86 (d, 3H, J ) 6.2 Hz), 0.85 (d, 3H, J )
6.1 Hz); 13C NMR (125 MHz, CDCl3) δ 172.5, 170.6, 170.3,
165.4, 141.0, 138.8, 136.6, 134.3, 129.8, 129.3, 128.7 (2C),
128.5, 127.2, 127.1, 125.5 (2C), 125.2, 75.9, 71.1, 63.0, 59.0,
53.6, 40.5, 39.6, 36.6, 35.6, 34.4, 32.4, 24.3, 22.8, 21.2, 13.4;
IR (film on KBr) 3404, 3277, 2959, 2927, 2854, 1745, 1731,
10-(4′-Ch lor oben zyl)d em et h oxyb en zyld esep oxycr yp -
top h ycin -24 (28). The total yield of 28, a white solid, was
1
0.051 g, 83%: mp 225-227 °C; H NMR (400 MHz, CDCl3) δ
7.36-7.17 (m, 9H), 6.90-6.88 (m, 1H), 6.72 (ddd, 1H, J ) 5.3
Hz, 10.0 Hz, 15.3 Hz), 6.45 (d, 1H, J ) 15.8 Hz), 6.04 (dd, 1H,
J ) 8.8, 15.8 Hz), 5.77 (d, 1H, J ) 15.4 Hz), 5.66 (d, 1H, J )
8.5 Hz), 5.05 (dd, 1H, J ) 6.6 Hz, 9.4 Hz), 4.92 (dd, 1H, J )
3.5 Hz, 9.4 Hz), 4.83 (dd, 1H, J ) 7.7 Hz, 14.3 Hz), 3.61-3.55
(m, 1H), 3.48-3.41 (m, 1H), 3.23 (dd, 1H, J ) 4.7 Hz, 14.4
Hz), 3.08 (dd, 1H, J ) 7.6 Hz, 14.4 Hz), 2.60-2.53 (m, 4H),
2.42-2.35 (m, IH), 1.71-1.68 (m, 2H), 1.39-1.32 (m, 1H), 1.15
(d, 3H, J ) 5.0 Hz), 0.78 (d, 3H, J ) 6.4 Hz), 0.74 (d, 3H, J )
6.4 Hz); 13C NMR (125 MHz, CDCl3) δ 172.5, 170.8, 170.4,
165.6, 141.4, 136.6, 135.2, 132.7, 131.8, 130.5 (2C), 130.0, 128.7
(2C), 128.5 (2C), 127.5, 126.1 (2C), 125.1, 71.5, 53.6, 42.2, 39.6,
36.3, 35.3, 34.4, 32.3, 24.3, 22.6, 21.1, 17.2; IR (CH2Cl2) 3000,
2960, 1750, 1720, 1650, 1380 cm-1; HRMS (FAB, PEG600) m/e
calcd for C33H40CIN2O6 (M + H)+: 595.2575, found 595.2570;
[R]D +33 (c 0.08, CHCl3).
1679, 1673, 1536, 1467, 1371, 1243, 1173, 1077, 751, 698 cm-1
;
HRMS (FAB, PEG600) m/e calcd for C33H40N2O7Cl (M + H)+:
611.2524, found 611.2529; [R]D +17 (c 0.36, CHCl3).
10-(4′-Ch lor ob en zyl)d em et h oxyb en zylcr yp t op h ycin -
24 (6). The total yield of a mixture of 6 and R-6 (â:R, 2:1), a
white solid, was 13.4 mg, 74%. 6, tR ) 24.1 min; mp 223-225
°C; 1H NMR (400 MHz, CDCl3) δ 7.41-7.35 (m, 5H), 7.28-
7.25 (m, 2H), 7.17-7.15 (m, 2H), 6.90 (br t, 1H, J ) 5.6 Hz),
6.70 (ddd, 1H, J ) 5.0 Hz, 10.0 Hz, 15.2 Hz), 5.73 (dd, 1H, J
) 1.0 Hz, 15.3 Hz), 5.64 (d, 1H, J ) 8.5 Hz), 5.21 (ddd, 1H, J
) 2.0 Hz, 5.0 Hz, 11.1 Hz), 4.90 (dd, 1H, J ) 3.4 Hz, 10.0 Hz),
4.80 (dd, 1H, J ) 2.0 Hz, 8.2 Hz), 3.71 (d, 1H, J ) 2.0 Hz),
3.54-3.45 (m, 2H), 3.22 (dd, 1H, J ) 5.9 Hz, 14.3 Hz), 3.07-
3.05 (m, 1H), 2.94 (dd, 1H, J ) 2.0 Hz, 7.6 Hz), 2.61-2.55 (m,
4H), 1.83-1.80 (m, 2H), 1.33-1.27 (m, 2H), 1.17 (d, 3H, J )
10-(3′,4′-Dich lor ob en zyl)d em et h oxyb en zyld esep oxy-
cr yp top h ycin -24 (29). The total yield of 29, a white solid,
was 0.049 g, 80%: mp 225-226 °C; 1H NMR (400 MHz, CDCl3)
δ 7.35-7.24 (m, 7H), 7.10 (dd, 1H, J ) 2.0 Hz, 8.2 Hz), 6.83-
6.81 (m, 1H), 6.70 (ddd, 1H, J ) 5.6 Hz, 9.7 Hz, 15.3 Hz), 6.44
(d, 1H, J ) 15.8 Hz), 6.03 (dd, 1H, J ) 8.8 Hz, 15.8 Hz), 5.80
(d, 1H, J ) 15.6 Hz), 5.72 (d, 1H, J ) 8.9 Hz), 5.03 (dd, 1H, J
) 6.6 Hz, 9.2 Hz), 4.91 (dd, 1H, J ) 3.5 Hz, 10.0 Hz), 4.83 (dd,
1H, J ) 7.4 Hz, 15.0 Hz), 3.65-3.64 (m, 1H), 3.40-3.35 (m,
1H), 3.23 (dd, 1H, J ) 6.0 Hz, 14.3 Hz), 3.04 (dd, 1H, J ) 7.5
Hz, 14.3 Hz), 2.62-2.53 (m, 4H), 2.44-2.38 (m, 1H), 1.67-
1.60 (m, 2H), 1.37-1.33 (m, 1H), 1.16 (d, 3H, J ) 6.9 Hz), 0.78
(d, 3H, J ) 6.4 Hz); 0.73 (d, 3H, J ) 6.4 Hz); 13C NMR (125
MHz, CDCl3) δ 172.3, 170.8, 170.3, 165.7, 141.3, 137.2, 136.6,
132.4, 131.8, 131.2, 130.9, 130.4, 130.0, 128.6 (2C), 128.5,
127.5, 126.1 (2C), 125.2, 71.4, 53.4, 42.2, 39.6, 36.3, 35.1, 34.6,
32.3, 24.3, 22.6, 21.1, 17.2; IR (CH2Cl2) 3420, 2980, 2950, 2900,
1740, 1680, 1630, 1510 cm-1; HRMS (FAB, PEG600) mle calcd
for C33H39Cl2N2O6 (M + H)+: 629.2185; [R]D +18.3 (c 0.231,
CHCl3).
P r ep a r a tion of Ep oxid es 4-7 (Sa m p le P r oced u r e). To
a solution of styrene 26 (32.0 mag, 0.057 mmol) in acetone (2.0
mL) was added a solution of dimethyldioxirane30 in acetone
(2.0 mL). The solution was stirred at room temperature for 5
h. After concentrating, the residue was purified using column
chromatography on silica gel (40% EtOAc:hexanes). The total
yield of a mixture of 1:2 R:â-epoxides that were separated by
HPLC (Vydac C18, internal diameter 8 mm, eluent (isocratic)
MeOH:H2O 65:35, flow 3 mL/min).
7.2 Hz), 0.86 (d, 3H, J ) 6.4 Hz), 0.85 (d, 3H, J ) 6.4 Hz); 13
C
NMR (125 MHz, CDCl3) δ 172.5, 170.7, 170.4, 165.4, 141.0,
136.6, 135.1, 130.7, 130.5 (2C), 128.7 (2C), 128.6 (2C), 128.5,
125.5 (2C), 125.2, 75.8, 71.1, 63.0, 58.9, 53.6, 40.6, 39.4, 36.6,
35.3, 34.4, 32.3, 24.4, 22.8, 21.2, 13.4; IR (film on KBr) 3278,
2958, 2928, 2872, 1744, 1677, 1536, 1493, 1371, 1242, 1173,
1072, 1014, 752, 698 cm-1; HRMS (FAB, PEG600) m/e calcd
for C33H40N2O7Cl (M + H)+: 611.2524, found 611.2517; [R]D
+35 (c 0.08, CHCl3).
10-(3′,4′-Dich lor oben zyl)d em eth oxyben zylcr yp top h y-
cin -24 (7). The total yield of a mixture of 7 and R-7 (â:R, 2:1),
a white solid, was 13.8 mg, 65%. 7, tR ) 32.3 min; mp 192-
194 °C; 1H NMR (400 MHz, CDCl3) δ 7.39-7.34 (m, 6H), 7.26
(d, 1H, J ) 1.4 Hz), 7.11 (dd, 1H, J ) 2.0 Hz, 10.0 Hz), 6.82
(br t, 1H, J ) 5.6 Hz), 6.70 (ddd, 1H, J ) 5.3 Hz, 9.8 Hz, 15.2
Hz), 5.75 (d, 1H, J ) 15.5 Hz), 5.71 (d, 1H, J ) 8.7 Hz), 5.20
(dd, 1H, J ) 5.0 Hz, 11.1 Hz), 4.90 (dd, 1H, J ) 3.4 Hz, 10.2
Hz), 4.82 (dd, 1H, J ) 7.8 Hz, 14.6 Hz), 3.71 (d, 1H, J ) 1.9
Hz), 3.68-3.62 (m, 1H), 3.42-3.35 (m, 1H), 3.23 (dd, 1H, J )
5.9 Hz, 14.5 Hz), 3.03 (dd, 1H, J ) 7.6 Hz, 14.4 Hz), 2.95 (dd,
1H, J ) 1.9 Hz, 7.5 Hz), 2.65-2.55 (m, 3H), 251-2.42 (m, 1H),
1.85-1.81 (m, 1H), 1.74-1.67 (m, 2H), 1.37-1.32 (m, 1H), 1.18
(d, 3H, J ) 6.9 Hz), 0.87 (d, 3H, J ) 6.4 Hz), 0.86 (d, 3H, J )
6.4 Hz); 13C NMR (125 MHz, CDCl3) δ 172.3, 170.7, 170.2,
165.5, 140.9, 137.1, 137.1, 136.6, 132.4, 131.2, 130.9, 130.4,
128.7 (2C), 128.5, 128.4, 125.5 (2C), 75.9, 71.1, 62.9, 58.9, 53.4,
40.5, 39.4, 36.6, 35.1, 34.6, 32.3, 24.4, 22.8, 21.2, 13.4; IR (film
on KBr) 3289, 2958, 2926, 2852, 1741, 1673, 1537, 1470, 1373,
Dem eth oxycr yp top h ycin -24 (4). The total yield of a
mixture of 4 and R-4 (â:R, 2:1), a white solid, was 21.9 mg,
1
66%. 4, tR ) 10.4 min; mp 234-235 °C; H NMR (400 MHz,
CDCl3) δ 7.40-7.21 (m, 10H), 7.00 (br t, 1H, J ) 5.1 Hz), 6.71
(ddd, 1H, J ) 4.9 Hz, 10.4 Hz, 15.2 Hz), 5.72 (dd, 1H, J ) 1.2
Hz, 15.2 Hz), 5.59 (d, 1H, J ) 8.1 Hz), 5.24 (ddd, 1H, J ) 2.0
Hz, 5.0 Hz, 11.0 Hz), 4.91 (dd, 1H, J ) 3.5 Hz, 10.0 Hz), 4.82

702 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 3
Buck et al.
1243, 1173, 1030, 751, 698 cm-1; HRMS (FAB, PEG600) m/e
calcd for C33H39N2O7Cl2 (M + H)+: 645.2134, found 645.2144;
[R]D +18 (c 0.20, CHCl3).
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Ack n ow led gm en t . The authors thank Dmitry
Yakovlev of the Biological Research Services Laboratory
for performing all epoxide separations via HPLC. S. B.
Buck thanks the Department of Defense for a predoc-
toral fellowship (DAMD17-98-1-8200), and the NIH for
a predoctoral fellowship (NIH-GM-07775). The authors
also thank the NCI for funding (CA70369).
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