Novel cryptophycin antitumor agents: Synthesis and cytotoxicity of fragment 'B' analogues

Article abstract of DOI:10.1021/jm980706s

A general synthetic approach to novel cryptophycin analogues 6 is described. N-Hydroxysuccinimide active ester 15, a key common intermediate, was converted to β-epoxide 6 in three steps, via initial coupling with unprotected amino acid 9, followed by deprotection/macrolactamization of acyclic precursor 16, and final oxidation of styrene 7 to install the C7-C8 β-epoxide. Cryptophycin styrenes 7 and β-epoxides 6, bearing diverse side chains in fragment 'B', were evaluated for cytotoxic activity, β-Epoxides 6, in general, were significantly more potent than the corresponding α-epoxides 17 and styrenes 7. A benzyl side chain was required for potent activity, with β-epoxide 6u, possessing a 3-Cl,4-(dimethylamino)benzyl moiety, as the most potent cytotoxic agent prepared, with an IC₅₀ = 54 pM, only 2-fold-less than that of Cryptophycin-52 (3).

Full text of DOI:10.1021/jm980706s

2588
J . Med. Chem. 1999, 42, 2588-2603
Novel Cr yp top h ycin An titu m or Agen ts: Syn th esis a n d Cytotoxicity of F r a gm en t
“B” An a logu es
Vinod F. Patel,*,† Sherri L. Andis,§ J oseph H. Kennedy,‡ J ames E. Ray,† and Richard M. Schultz§
Lilly Research Laboratories, A Division of Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285
Received December 15, 1998
A general synthetic approach to novel cryptophycin analogues 6 is described. N-Hydroxysuc-
cinimide active ester 15, a key common intermediate, was converted to â-epoxide 6 in three
steps, via initial coupling with unprotected amino acid 9, followed by deprotection/macrolac-
tamization of acyclic precursor 16, and final oxidation of styrene 7 to install the C7-C8
â-epoxide. Cryptophycin styrenes 7 and â-epoxides 6, bearing diverse side chains in fragment
“B”, were evaluated for cytotoxic activity. â-Epoxides 6, in general, were significantly more
potent than the corresponding R-epoxides 17 and styrenes 7. A benzyl side chain was required
for potent activity, with â-epoxide 6u , possessing a 3-Cl,4-(dimethylamino)benzyl moiety, as
the most potent cytotoxic agent prepared, with an IC50 ) 54 pM, only 2-fold less than that of
Cryptophycin-52 (3).
Ch a r t 1
In tr od u ction
The first cryptophycin, Cryptophycin A (Chart 1, 1),
was isolated from terrestrial Nostoc sp. ATCC 53789 by
Schwartz1 at Merck and found to be a potent antifungal
agent. Subsequently, in 1994, Moore and colleagues2
discovered the potent and selective antitumor properties
of the cryptophycin class of agents which revived
interest in these novel depsipeptides. In addition to 1,
designated Cryptophycin A by Moore, several other
cytotoxic analogues were isolated from blue-green algae
(cyanobacteria) belonging to Nostocaceae.1,3 Subse-
quently, studies were conducted to determine structure,
chemical stability, and antitumor activity of these new
analogues and provided a structure-activity relation-
ship (SAR).3 Cryptophycin A (1) displayed highly potent
in vitro cytotoxicity in several tumor cell lines (IC50
)
9-20 pM); however, analogues lacking the epoxide in
unit A (Chart 1),4 the chlorine or the O-methyl group
in unit B, and the intact macrolide ring showed dimin-
ished cytotoxicity which was consistent with marginal
to negative activities in vivo. Information on the mo-
lecular target of 1 was first provided by Smith5 who
revealed that it disrupted microtubule structure. In
further studies the binding site of 1 was identified to
be the same as or overlapping with that of the Vinca
alkaloids.6 Cellular studies, however, comparing clini-
cally used antimicrotubule agents, Paclitaxel, Vinblas-
tine, and Colchicine, with 1 demonstrated that the
cryptophycins were poor substrates for the drug efflux
pump P-glycoprotein, a novel and potentially important
property.5
(IC50 ) 5 pg/mL). In vivo, however, 2 showed nil to
marginal antitumor activity which was not surprising
based on the reported SAR for cryptophycin 1.8
A synthetic analogue, Cryptophycin-52 (3) (Chart 1),
was designed to enhance the hydrolytic stability of the
C-D ester linkage in 1.3 In addition to meeting this
criterion, the introduction of a methyl group at C2 in
unit C of 1 also eliminated an asymmetric center.
Cryptophycin-52 (3) is an exceptionally potent, broad-
spectrum antitumor agent,9,10 which retains activity
against cell lines resistant to clinically used oncolyt-
ics.10,11 It displayed impressive tumor growth inhibition
of “wild-type”12 and, more importantly, “resistant”13,14
human tumor xenografts.13 Recently, detailed investiga-
tions were performed to elucidate the effects of 3 on
microtubule organization and mass. Interestingly, in
addition to mitotic inhibition similar to that observed
with other antimitotic agents, it exhibited some unique
Also in search of bioactive substances, Kobayashi and
Kitagawa7 isolated a related depsipeptide, Arenastatin
A (Chart 1, 2), from an Okinawan marine sponge, also
exhibiting potent in vitro cytotoxicity against KB cells
* To whom correspondence should be addressed at: Kinetix Phar-
maceuticals Inc., 200 Boston Ave, Suite 4700, Medford, MA 02155.
E-mail: patel@kinetixpharm.com.
† Discovery Chemistry Research Division.
‡ Chemical Process Research and Development Division.
§ Cancer Research Division.
10.1021/jm980706s CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/23/1999

Novel Cryptophycin Antitumor Agents
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2589
Sch em e 1. Retrosynthetic Analysis
characteristics including the formation of microtubule
bundles in cells with reduced microtubule polymer mass
and the induction of asters particularly in prophase
cells.14 Encouraging preclinical data, including its po-
tency and unique mechanistic profile, has supported the
advancement of 3 (LY355703) to clinical trials for
treatment of human cancers.
The formidable challenges associated with the syn-
thesis of 16-membered cyclic depsipeptide 1 bearing
multiple stereogenic centers were elegantly solved first
by Tius and Moore,15 who employed a disconnection at
the amide and ester linkages to give units A-D.
Synthesis and assembly of each unit, followed by mac-
rolactamization to form the B-C amide as the final
bond, led to the cryptophycins. Subsequently, several
formal16-18 and total19-24 syntheses of members in this
class of agents have appeared in the literature. In a
collaborative effort,25 a program at Lilly was initiated
to expand the initial SAR3 by preparing analogues with
structural variations in fragments A,26 B, C,27 and D.28
In this report, we describe the synthesis and cytotoxic
activity of novel fragment “B” analogues 6 (Scheme 1).
odology, developed in our laboratory,30 that would allow
acylation of unprotected amino acid 9 with intermediate
8 to first form the A-B amide linkage. Subsequent
deprotection of the amine and macrolactamization,
under conditions previously described by Tius,15 should
lead to cryptophycin 7.
Methyl ester 1015,31 was hydrolyzed to carboxylic acid
1115 which, without extensive purification, was con-
verted to N-hydroxysuccinimide ester 12 in excellent
overall yield (Scheme 2). Fluoride-mediated desilylation
of 12, under acidic conditions,15 cleanly furnished alco-
hol 13 as white crystals. We next turned our attention
to the nontrivial esterification of fragment A alcohol 13
with Boc-protected amino acid 14.24a,31 Preactivation of
acid 14 with EDC in the presence of catalytic DMAP
followed by addition of alcohol 13 at a final reaction
concentration of 0.2 M provided key intermediate 15.
Under these specific conditions the coupling was repro-
ducible and scalable on multigram quantities.32
In previous work,29,30 we discovered that N-hydroxy-
succinimide esters, in the presence of N,O-bis(trimeth-
ylsilyl)acetamide (BSA), exclusively acylate aliphatic
amines over other nucleophiles such as alcohols and
carboxylic acids. Complete chemoselectivity is observed
presumably because in situ silylation of oxygen provides
protection, while silylation of amines serves to increase
nucleophilicity.33 Typically, couplings were performed
by reacting intermediate 15 with 1.5 equiv of D-amino
acid 9 in the presence of excess BSA and at elevated
temperatures to afford acyclic precursor 16 (Scheme 3).
A particularly noteworthy aspect of this reaction was
the ease with which product 16 was isolated in high
purity and yields (61-93%, see Table 1) by a simple
aqueous acid wash to remove excess amine and byprod-
ucts. The commercial availability of amino acids 9,
except 9r ,s, was an advantage of this methodology and
allowed very rapid access to precursor 16. 3-Cl,4-OH-
D-phenylalanine (9r ) was obtained in one step by
demethylation of known amino acid 9q15 using condi-
tions described by Nakamoto34 (Scheme 4). The re-
maining aniline 9s was prepared from commercially
available p-amino-D-phenylalanine (9k ) (Scheme 5).
Esterification of 9k gave methyl ester 18 which was
Ch em istr y
Our general approach to the synthesis of cryptophycin
fragment “B” analogues 6 is described by the retrosyn-
thetic analysis outlined in Scheme 1. Introduction of the
C7-C8 â-epoxide was planned as the final step in the
synthesis, a strategy commonly utilized for the prepara-
tion of cryptophycins,15-24,26-28 presumably to avoid
limitations imparted by the enhanced reactivity of the
epoxide of a styryl system. Oxidation of Cryptophycin
C (4) reveals that epoxidation occurs with excellent
regioselectivity to afford the desired C7-C8 epoxide;
however, the transformation is nonstereoselective to
provide a mixture of 1:2 R:â-isomers, which could be
separated to give â-isomer 1.15 To date, the efficient
incorporation of the â-epoxide functionality remains a
major challenge in the total synthesis of the cryptophy-
cins.29 Nevertheless, disconnection of styrene 7 at the
two amide linkages (A-B and B-C) leads to key
intermediate 8 and unnatural D-amino acid 9. To avoid
protecting groups, we elected to utilize a novel meth-

2590 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Patel et al.
Sch em e 2a
a
Reagents: (a) 1 N aq LiOH, acetone, quantitative; (b) EDC, N-hydroxysuccinimide, DMF, quantitative; (c) 48% aq HF, 0-25 °C,
CH3CN, 78%; (d) EDC, cat. DMAP, 14, 0.2 M CH2Cl2, 80%.
Sch em e 3a
a
Reagents: (a) general method A: D-amino acid, 9, N,O-bis(trimethylsilyl)acetamide, DMF, 55 °C, see Table 1; (b) general method B:
(i) neat trifluoroacetic acid, 25 °C, (ii) pentafluorophenyl diphenylphosphinate, DIEA, 25 °C, DMF, see Table 3; (c) general method C:
mCPBA (1.1 equiv), 0.2 M CH2Cl2, 25 °C; RP-HPLC separation, see Table 5.
converted to diacetate 1935 in good overall yield. Fully
protected amino acid 19 was chlorinated using 1.4 equiv
of N-chlorosuccinimide36 to deliver desired chloride 20,
with no evidence of dichlorination. Finally, a one-pot
global deprotection of 20, first using basic conditions to
hydrolyze the methyl ester and one of the acetate groups
followed by treatment with strong acid to remove the
remaining acetate, furnished 3-Cl,4-NH2-D-phenylala-
nine (9s). Overall the methodology for formation of
amide 16 (Scheme 3) was remarkably general as indi-

Novel Cryptophycin Antitumor Agents
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2591
Ta ble 1. Elemental Analyses, Optical Rotations, and Yields for Carboxylic Acids 16
2
compd
R
formula1
[R]D
+32
yield (%)
93
a
H
C
33H48N2O9‚0.5H2O
(c ) 0.50)
+21.7
b
c
d
e
f
CH(Me)2
C36H54N2O9‚0.3H2O
96
89
92
88
72
79
75
88
87
72
86
67
61
75
77
78
65
85
(c ) 1.01)
+16.9
CH(OH)Me*
C
C
35H52N2O10‚0.5H2O
36H52N2O9‚0.5H2O
(c ) 1.07)
+66
pyrrolidine
(c ) 0.67)
+25.3
CH2cyclohexyl
CH2CH2Ph
C40H60N2O9‚0.75H2O
C41H56N2O9
(c ) 1.03)
+20.8
(c ) 1.06)
+2.1
g
h
i
CH2OCH2Ph
C41H56N2O10‚0.6H2O
(c ) 0.97)
-20
(p-OH)Ph
C39H52N2O10‚1.0H2O
(c ) 0.65)
+9.5
CH2Ph
C40H54N2O9‚0.5H2O
C40H54N2O10‚0.5H2O
C40H55N3O9
(c ) 1.06)
+12
j
CH2(p-OH)Ph
CH2(p-NH2)Ph
CH2(p-I)Ph
(c ) 0.60)
+5.0
k
p
r
(c ) 0.82)
-4.1
C40H53IN2O9‚0.5H2O
C40H53N2O10‚1.0H2O
C40H54ClN3O9‚1.0H2O
C40H54N2O11‚0.5H2O
C44H56N2O9
(c ) 0.99)
+6.3
CH2(m-Cl,p-OH)Ph
CH2(m-Cl,p-NH2)Ph
CH2(m,p-diOH)Ph
CH2(R-naphthyl)
CH2(â-naphthyl)
CH2(3-pyridyl)
CH2(3-indolyl)
(c ) 0.96)
-18
s
(c ) 0.57)
+12
v
w
x
y
z
(c ) 0.84)
+49
(c ) 0.99)
-9.8
C44H56N2O9‚1.0H2O
C39H53N3O9‚1.0H2O
C424H55N3O9‚1.0H2O
(c ) 1.02)
+7.7
(c ) 0.52)
-4.8
(c ) 0.84)
1 Compounds were analyzed for C,H,N; the results agreed to within (0.4% of the theoretical values. 2 All optical rotations were performed
in methanol at 589 nm. *(S)-Configuration at C3, derived from D-threonine.
Sch em e 4a
anilines, 16k ,s; and pyridines, 16y (see Table 3).
Unfortunately, indolyl derivative 16z, for reasons un-
determined, failed to provide the desired cyclized prod-
uct under these conditions. Proton NMR data for cyclic
depsipeptide 7 indicated it to be a single diastereomer
for which the 2-H in unit B was shifted downfield
compared to acyclic precursor 16 (see representative
examples in Table 4) and in agreement with the assign-
ments for Cryptophycin C (4).15
a
Reagents: (a) 48% HBr, PhOH, reflux, 69%.
cated by the diverse amino acid side chains that were
tolerated in the coupling reaction, including alcohols 9c,
phenols 9h ,j,r ,v, anilines 9k ,s, and nitrogen hetero-
cycles 9y,z (see Table 1). Epimerization at C2 in unit
B, a possible problem, was not evident under these
conditions. Physical and chemical data for acid 16,
including proton NMR data (see representative ex-
amples in Table 2), was consistent with the structural
assignments.
Conversion of precursor 16 to cyclic depsipeptide 7
was accomplished employing the procedure developed
by Moore and Tius15 in the synthesis of Cryptophycin
C (4) (Chart 1). Accordingly, removal of the Boc group
with neat trifluoroacetic acid followed by macrolactam-
ization of the resulting amino acid under high-dilution
conditions and using pentafluorophenyl diphenylphos-
phinate, as an activating agent, gave styrene 7 (Scheme
3). To our delight, the two-step cyclization sequence
proved quite general, tolerating side chain hydroxyl
groups, 16c; phenols, 16h ,j,r ,v; benzyl ethers, 16g;
The final step in the sequence to target agent 6
involved an oxidation to introduce the â-epoxide func-
tionality.15-22,24,26-28 We elected to employ conditions
previously reported for the regioselective, but nonste-
reoselective, C7-C8 epoxidation of Cryptophycin C (4).15
Treatment of styrene 7 with m-chloroperbenzoic acid
(mCPBA) led to the expected mixture of R:â-isomers
which were separated by reverse-phase HPLC to afford
â-epoxide 6 (Scheme 3). Interestingly, regardless of the
side chain on fragment B, the R:â-epoxide ratio re-
mained at 1:2 (see Table 5). Assignment of the epoxide
isomers was based on the shift of 8-H in unit A which
resonates at ∼3.7 ppm for the â-isomer 6 (see repre-
sentative examples in Table 6) and at ∼3.6 ppm for the
corresponding R-isomer 17, both appearing as doublets
(J ) ∼1.6 Hz). It was found, however, that use of excess
oxidant led to significant levels of bis-epoxidation,
particularly in the case of sytrene 7d where the C2-
C3 olefin is more nucleophilic due to the presence of the

2592 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Patel et al.
Sch em e 5a
a
Reagents: (a) AcCl, MeOH, reflux, 100%; (b) Ac2O, pyridine, 25 °C, 81%; (c) NCS, CH3CN, reflux, 50%; (d) (i) 1 N aq NaOH, dioxane,
reflux, (ii) 5 N aq HCl, 95 °C, (iii) SCX cation exchange, 72%.
Ta ble 2. Proton NMR Data for Representative Carboxylic Acids 16*
16a : glycine
16b: valine
16f: homoPhe
16h : ([p-OH]Ph)glycine
16j: tyrosine
7.35-7.16 (m)
6.52-6.56 (m)
6.39 (d, J 16.0)
6.06 (dd, J 16.1/8.12)
6.01 (d, J 15.2)
4.81-4.75 (m)
2.55-2.35 (m)
2.55-2.35 (m)
1.00 (d)1
Unit A
PhH5
3-H
8-H
7-H
2-H
5-H
6-H
4-H2
6-Me
7.36-7.15 (m)
6.65-6.48 (m)
6.41 (d, J 15.8)
6.08 (dd, J 16.1/8.9)
6.01 (d, J 15.4)
4.79-4.77 (m)
2.57-2.47 (m)
2.47-2.37 (m)
1.10 (d, J 5.9)
7.36-7.10 (m)
7.39-7.07 (m)
6.61-6.51 (m)
6.41 (d, J 15.9)
6.08 (dd)1
6.07 (d, J , 15.7)
4.81-4.77 (m)
2.63-2.52 (m)
2.63-2.52 (m)
1.04 (d)1
7.35-7.09 (m)
6.57-6.52 (m)
6.40 (d, J 15.9)
6.07 (dd, J 16.2/8.3)
6.14 (d, J 15.7)
4.79-4.75 (m)
2.57-2.37 (m)
2.57-2.37 (m)
1.02 (d)1
6.59-6.49 (m)
6.41 (d, J 15.9)
6.08 (dd, J 16.4/8.5)
6.15 (d, J 16.5)
4.80-4.76 (m)
2.57-2.46 (m)
2.46-2.36 (m)
1.02 (d)1
Unit B
12.5 (br s)
CO2H 12.5 (br s)
12.6 (br s)
12.6 (br s)
12.6 (br s)
NH
2-H
R
8.22 (t, J 5.4)
8.05 (d, J 8.5)
4.19 (dd, J 8.2/6.1)
8.26 (t, J 7.7)
4.22-4.15 (m)
8.49 (d, J 7.5)
5.21 (d, J 7.2)
8.15 (d, J 7.9)
4.39-4.32 (m)
9.15 (s, OH)
6.96 (d, J 8.2, PhH2)
6.59 (d, J 8.4, PhH2)
2.90 (dd, J 13.7/4.7, 3-H)
2.71 (dd, J 13.8/4.7, 3′-H)
3.76 (d, J 5.64, 2-H2) 0.84 (d, J 6.7, 5H3/4-Me) 7.39-7.07 (m, PhH5) 9.45 (s, OH)
2.63-2.52 (m, 4-H2)
2.46-2.40 (m, 3-H2)
7.70 (d, J 8.2, PhH2)
7.14 (d, J 8.0, PhH2)
Unit C
6.69 (t, J 5.6)
3.18-3.12 (m)
3.08-3.05 (m)
1.32 (s)
NH
3-H
3-H′
Boc
6.69 (t, J 6.1)
3.24-3.08 (m)
3.08-2.97 (m)
1.32 (s)
6.69 (t, J 6.1)
3.18-3.12 (m)
3.04-2.97 (m)
1.32 (s)
∼6.7 (t)1
6.48 (t, J 7.8)
3.14 (dd, J 13.4/6.9)
3.00 (dd, J 13.4/6.5)
1.32 (s)
3.24-3.05 (m)
3.03-2.97 (m)
1.32 (s)
2-Me
2-Me
1.05 (s)
1.02 (s)
1.05 (s)
1.02 (s)
1.05 (s)
1.03 (s)
1.05 (s)
1.01 (s)
1.05 (s)
1.01 (s)
Unit D
2-H
3-H2
4-H
5-H3
4-Me
4.91-4.89 (m)
1.63-1.44 (m)
1.63-1.44 (m)
0.75 (d, J 6.7)2
0.73 (d, J 6.5)2
4.93 (app q, J 5.4)4
1.62-1.50 (m)
1.50-1.45 (m)
0.75 (d, J 6.4)2
0.73 (d, J 6.4)2
4.92 (app q, J 5.4)d
1.61-1.50 (m)
1.50-1.45 (m)
0.75 (d, J 6.5)2
0.73 (d, J 6.6)2
4.90-4.88 (m)
1.66-1.56 (m)
1.50-1.38 (m)
0.75 (d, J 6.4)2
0.73 (d, J 6.4)2
4.88 (app q, J 5.7)4
1.64-1.42 (m)
1.64-1.42 (m)
0.75 (d, J 7.0)2
0.72 (d, J 6.7)2
1
2
4
3
*In DMSO-d6 at 300 MHz. Partially buried. Overlapping. Buried. app, apparent.
tertiary amide. It was essential, therefore, to use only
a slight excess of oxidant which, in turn, required the
reactions to be conducted at high concentrations to
ensure complete conversions. Although dichloromethane
was the solvent of choice, in cases where solubility of
substrate was an issue (e.g., phenol 7j), a polar solvent,
such as tetrahydrofuran, was added without affecting
regio- and stereoselectivity or yield. In cases where the
substrate contained a free amino group (e.g., 7k ,s), it
was necessary to apply a protecting group prior to
epoxidation. Acid sensitivity of the epoxide functionality
and the presence of labile ester linkages were factors
considered in selecting Fmoc, a protecting group which
is readily removed under mild basic conditions.29 Treat-
ment of anilines 7k ,s with Fmoc chloride under stan-
dard conditions37 gave 7m ,t, which on epoxidation led
to the expected mixture of epoxides 21 and 22, respec-
tively (Scheme 6). Deprotection of 21 and 22 with
piperidine was rapid and clean to give the corresponding
aniline which was separated to furnish the desired
â-epoxides 6k ,s, respectively. Access to dimethylaniline
analogue 6u was gained through simple methylation of
the corresponding aniline 6s (Scheme 6). Although data
in Table 4 reflect low yields for the formation of
â-epoxide 6, partly attributable to the difficult separa-
tions of the R:â-mixtures, this method was generally
applicable for a broad range of substrates 7 and satis-
factory. Attempts to epoxidize 7v, however, led to
significant decomposition, possibly due to oxidation of
the 3,4-dihydroxyphenyl system to the corresponding
o-quinone. Although protection of the hydroxyls would
overcome this problem, it was not attempted. In the case

Novel Cryptophycin Antitumor Agents
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2593
Ta ble 3. Elemental Analyses, Optical Rotations, and Cyclization Yields for Styrenes 7
2
compd
R
formula1
28H38N2O6
[R]D
yield (%)
76
a
H
C
-40
(c ) 0.50)
+21
b
c
d
e
f
CH(Me)2
C31H44N2O6‚0.5H2O
61
51
74
56
62
57
59
55
80
58
22
42
62
52
56
65
71
(c ) 0.24)
+54
CH(OH)Me*
C
C
30H42N2O7‚0.5H2O
31H42N2O6‚0.75H2O
(c ) 0.24)
+41
pyrrolidine
(c ) 0.52)
+25
CH2cyclohexyl
CH2CH2Ph
C35H50N2O6
(c ) 0.85)
+43
C36H46N2O6
(c ) 0.90)
+29
g
h
i
CH2OCH2Ph
C36H46N2O7‚0.4H2O
(c ) 0.28)
+15
(p-OH)Ph
C34H42N2O7‚0.3H2O
(c ) 0.39)
+31
CH2Ph
C35H44N2O6‚0.2H2O
C35H44N2O7‚0.3H2O
C35H45N3O6‚0.25H2O
C35H43IN3O6
(c ) 0.97)
+30
j
CH2(p-OH)Ph
CH2(p-NH2)Ph
CH2(p-I)Ph
(c ) 0.53)
+33
k
p
r
(c ) 0.73, MeOH)
+25
(c ) 0.28)
+29
CH2(m-Cl,p-OH)Ph
CH2(m-Cl,p-NH2)Ph
CH2(m,p-diOH)Ph
CH2(R-naphthyl)
CH2(â-naphthyl)
CH2(3-pyridyl)
C35H43ClN2O7
(c ) 0.84)
+32
s
C35H44ClN3O6
(c ) 0.94)
+34
v
w
x
y
C35H44N2O8‚1.3H2O
C39H46N2O6‚0.3H2O
C39H46N2O6‚0.3H2O
C34H43N3O6‚0.5H2O
(c ) 0.59, MeOH)
+48
(c ) 0.86)
+15
(c ) 0.60)
+30
(c ) 0.67, MeOH)
1 Compounds were analyzed for C,H,N; the results agreed to within (0.4% of the theoretical values. 2 All optical rotations were performed
in chloroform, unless stated otherwise, at 589 nm. *(S)-Configuration at C3, derived from D-threonine.
of pyridyl 7y, treatment with 1 equiv of mCPBA acid
initially gave the N-oxide, which with further oxidant
led to the corresponding epoxide. However, the reaction
was not clean, and attempts to isolate the desired
â-isomer 4w were unsuccessful.
The synthetic approach described above proved re-
markably general for accessing a variety of structurally
complex cryptophycin analogues, styrene 7 and â-epox-
ide 6. Consequently, we were able to probe the SAR in
this region of the molecule for these two series.
These results are consistent with the Cryptophycin A
(1) series, where the chlorophenol analogue (Crypto-
phycin-17) was 2.4-5-fold less potent than the parent
(Cryptophycin-3).39 Removal of the chlorine in 7r ,s, to
give tyrosine derivative 7j and aniline 7k , respectively,
led to a further 3-fold loss in potency. Conversely, the
same alteration in the Cryptophycin A (1) series was
reported to produce a 6-9-fold increase in activity.39
This exemplifies the caution that must be taken when
comparing data derived from different tumor cell lines.
Interestingly, the cytotoxicity of unsubstituted aromatic
7i is comparable to that of disubstituted aromatic 7r ,s.
This result may be explained by considering cellular
transport, a critical factor for cellular activity. Under
passive conditions, it is reasonable to assume that the
more hydrophobic 7i diffuses across the cell membrane
more easily than polar derivatives 7r ,s. Further in-
creasing hydrophobicity in region B, e.g., naphthyl
derivatives 7w ,x, however, led to total loss of activity.
The bulky Fmoc moiety at C4 in the phenyl ring of 7m ,t
was not well-tolerated. Replacing the phenyl moiety in
7i with a cyclohexyl (7e), a group with similar hydro-
phobicity, abolishes activity suggesting that the phenyl
ring is probably important for interaction at the molec-
ular target. Substitution of the 4-hydroxyl group in 7j
with 4-iodide (7p ) did not change the potency implying
that H-bonding at this position is not required. In fact,
losses in activity were observed on introduction of polar
functionality, e.g., dihydroxy analogue 7v and pyridine
derivative 7y. The methylene linkage between the core
Str u ctu r e-Activity Rela tion sh ip s
Biological activity was measured in an in vitro cellular
cytotoxicity assay using a human leukemia CCRF-CEM
tumor cell line (see Experimental Section for the assay
protocol).38 Cryptophycin-52 (3), with an IC50 of 22 pM
(see Table 7), is one of the most potent cytotoxic agents
in this class of agents. The corresponding styrene,
Cryptophycin-51 (5) (Chart 1), was approximately 630-
fold less potent (IC50 ) 13.9 nM) than 3. It should be
noted that during the total synthesis of Cryptophycin
C (4), Tius and Moore15 also prepared the epimer at C2
in unit B. This analogue, which corresponds to incor-
poration of the natural L-amino acid in fragment B, was
14-24-fold less active than Cryptophycin C (4).
In general, styrene analogues 7 were 20- to >140-fold
less potent than Cryptophycin-51 (5). Chlorophenol 7r ,
a styrene analogue lacking a methyl group, resulted in
a 20-fold loss in cellular activity, while the correspond-
ing chloroaniline 7s displayed 36-fold less potency.

2594 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Ta ble 4. Proton NMR Data for Representative Styrenes 7*
Patel et al.
7a : glycine
7b: valine
7f: homoPhe
Unit A
7.36-7.18 (m)
7h : ([p-OH]Ph)glycineX
16j: tyrosineX
PhH5
3-H
8-H
7-H
2-H
5-H
7.36-7.20 (m)
7.36-7.16 (m)
7.36-7.15 (m)
6.79-6.69 (m)
6.54 (d, J 15.8)
7.42-7.26 (m)
6.67 (ddd, J 15.2/8.8/5.8) 6.81 (ddd, J 15.1/10.3/4.7) 6.79-6.70 (m)
6.73-6.65 (m)3
6.40 (d, J 15.8)
6.44 (d, J 15.9)
6.02 (dd, J 15.8/8.8)
5.92 (d, J 15.8)
5.07-5.00 (m)
6.43 (d, J 15.8)
6.03 (dd, J 15.8/8.8)
5.88 (d, J 15.7)
6.42 (d, J 15.8)
6.03 (dd, J 15.8/8.8) 5.98 (dd, J 15.8/8.8)
5.70 (d, J 15.6)
6.00 (dd, J 16.1/8.8)
5.82 (d, J 15.1)
5.02 (m)
2.59-2.52 (m)
2.33 (app q, J 11.2)
1.13 (d, J 6.8)
5.85 (d, J 14.8/1.0)
5.06-5.00 (m)
2.61-2.49 (m)
2.39-2.30 (m)
1.10 (d, J 6.8)
5.06 (ddd, J 8.3/6.8/1.72) 5.07-5.04 (m)
4-H/6-H 2.63-2.53 (m)
2.62-2.54 (m)
2.47-2.35 (m)
1.15 (d, J 6.9)
2.61-2.55 (m)
2.40-2.31 (m)
1.15 (d, J 6.9)
4′-H
6-Me
2.49-2.38 (m)
1.15 (d)1
Unit B
5.50 (d, J 7.60)
4.53-4.45 (m)
NH
2-H
R
6.07-5.98 (d)3
5.59 (d, J 8.2)
4.40 (dd, J 8.0/4.3)
7.65 (d, J 6.3)
5.28 (d, J 6.5)
not observed
4.57 (dd, J 9.6/3.8)
3.92 (dd, J 16.9/5.8)
4.05 (dd, J 16.9/7.0, 2-H′) 2.47-2.35 (m, 3-H)
7.36-7.18 (m, PhH5) 7.90 (dd, J 10/1.68, OH) 7.56 (d, J 7.6, OH)
1.00 (d, J 6.9, 5-H3)
0.90 (d, J 7.0, 3-Me)
2.73 (t, J 7.6, 4-H2) 7.10 (d, J 8.5, PhH2)
2.40-2.31 (m, 3-H) 6.71 (d, J 8.4, PhH2)
1.99-1.86 (m, 3-H′)
7.03 (d, J 8.4, PhH2)
6.72 (d, J 8.3, PhH2)
3.02 (dd, J 13.3/1.6, 3-H)
2.80 (dd, J 14.2/10.1, 3′-H)
Unit C
NH
3-H
3-H′
2-Me
2-Me
7.08-7.04 (m)
3.44 (dd, J 13.5/8.4)
3.18 (dd, J 13.4/4.3)
1.24 (s)
7.36-7.16 (m)
3.40 (dd, J 13.4/8.4)
3.17 (dd, J 13.5/3.8)
1.23 (s)
7.36-7.18 (m)
7.36-7.15 (m)
7.22-7.20 (m)
3.44 (dd, J 13.3/9.7)
3.14 (dd, J 14.4/3.6)
1.18 (s)
3.37 (dd, J 13.5/8.3) 3.51 (dd, J 13.3/10.1)
3.18 (dd, J 13.4/3.8) 3.00 (d, J 13.4)
1.23 (s)
1.18 (s)
1.19 (s)
1.16 (s)
1.18 (s)
1.18 (s)
1.17 (s)
Unit D
2-H
4.91 (dd, J 10.0/3.2)
4.91 (dd, J , 10.1/3.5)
1.74-1.48 (m)
1.40-1.33 (m)
0.76 (d, J 6.8)
4.91 (dd, J 9.9/3.4)
1.71-1.63 (m)
1.39-1.25 (m)
0.76 (d, J 6.7)
0.73 (d, J 6.6)
4.90 (dd, J 10/3.5)
1.66-1.54 (m)
1.32-1.25 (m)
0.69 (d, J 7.1)2
0.66 (d, J 7.1)2
4.99-4.86 (m)
1.65-1.55 (m)
1.34-1.24 (m)
0.71 (d, J 6.6)
0.69 (d, J 6.5)
3-H/4-H 1.76-1.62 (m)
3′-H
5-H3
4-Me
1.52-1.28 (m)
0.78 (d, J 6.4)
0.73 (d, J 6.4)
0.74 (d, J 6.7)
1
2
3
4
*In CDCl3 at 300 MHz. X In CDCl3/CD3OD (1:1) at 300 MHz. Partially buried. Overlapping. Buried. app, apparent.
nucleus and the phenyl group was shown to be impor-
tant for activity. A one-carbon increase in chain length
(e.g., 7i vs 7f) produced a 4-fold loss in activity, while a
one-carbon decrease (e.g., 7j vs 7h ) led to a >2.3-fold
loss. More drastic alterations in region B, including
removal of the side chain (i.e., 7a ), replacement with
aliphatic side chains (e.g., 7b,c), and introducing a
tertiary amide (e.g., 7d ) all produced inactive crypto-
phycin styrene analogues.
the Fmoc derivatives act as prodrugs which cleave in
vitro to provide the more potent 6k ,s, respectively. In
the epoxide series, insertion of a methylene linkage (i.e.,
6i to 6f) led to a dramatic 3200-fold decrease in activity.
Adding an oxygen atom in ethyl-bridged 6f to produce
ether-linked derivative 6g returned some activity, as
observed in the styrene series. Eliminating the linkage
between the aromatic moiety and the core nucleus (i.e.,
6j to 6h ) led to ∼70-fold loss in activity. It is noteworthy
that activity in the styrene series 7 was not necessarily
a good predictor for activity in the epoxide series 6. For
example, styrenes 7e,w ,x showed no activity, while
their counterpart epoxides 6e,w ,x, respectively, were all
moderately active. Cyclohexyl analogue 6e was the only
nonaromatic side chain analogue that showed any
reasonable potency. All others (6a -d ) were completely
inactive.
In the epoxide series, cryptophycins 6, bearing a
C7-C8 epoxide in the â-configuration, were 4-60-fold
more potent than the corresponding R-isomers 17 (Table
7) and generally more potent than the corresponding
styrenes 7. Similar trends were found in the SAR of
fragment “A” in the Cryptophycin A (1)3 and Crypto-
phycin-52 (3)26 series. Our data showed, for example,
that for derivatives i,j,k ,m ,r ,s,t,w ,x possessing a benzyl
side chain, epoxides 6 were 18-2000-fold more potent
than styrenes 7. In the â-epoxide family 6, desmethyl
agent 6r was 24-fold less potent than parent 3, which
correlated well with the 27-40-fold decrease observed
in the Cryptophycin A (1) series.3 Replacing the oxygen
in 6r for nitrogen in 6s maintained the potency at the
subnanomolar level. Since the methylated phenols were
more active, we prepared dimethylaniline 6u and,
indeed, realized a 10-fold increase in activity compared
to aniline 6s. With an IC50 of 54 pM, 6u represented
the most potent fragment “B” analogue, only 2-fold less
potent than parent 3. To our surprise, phenyl derivative
6i was only ∼3-fold less active than 6u , while mono-
substituted analogues 6j,k were significantly less cyto-
toxic. Unexpectedly, intermediates 21 and 22, bearing
a bulky C4 Fmoc group, were relatively active consider-
ing they were a mixture of isomers. It is conceivable that
Con clu sion s
Our synthesis of fragment “B” analogues 6 featured
a novel and efficient amide bond-forming reaction
between an N-hydroxysuccinimide ester of key inter-
mediate 15 and an unprotected amino acid 9. This
approach provided access to analogues bearing a diverse
range of side chains in fragment B. Biological evaluation
of these analogues in an in vitro cytotoxicity assay
revealed a structure-activity relationship. In general,
â-epoxides 6 were more potent than the corresponding
R-epoxides 17 and styrenes 7. Furthermore, a benzyl
side chain was required for potent activity with 3-chloro-
4-dimethylamino substitution in the phenyl ring, 6u ,
leading to an exquisitely, potent cytotoxic agent (IC50
) 54 pM). The results clearly demonstrate that cytotoxic
activity is sensitive to substitution in region “B”;

Novel Cryptophycin Antitumor Agents
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2595
Ta ble 5. Physical-Chemical Data and Yields for â-Epoxides 6
RP-HPLC
2
compd
R
formula1
[R]D
tR (min)3
yield (%)4
48
â/R-ratio5
a
H
C28H38N2O7‚0.75H2O
-22.7
10.1a
1.7
(c ) 0.275)
+23.6
b
c
d
e
f
CH(Me)2
C31H44N2O7‚0.5H2O
19.2a
8.80a
18.0a
29.0b
26.4b
26.3b
13.3a
27.7a
4.67a
22.5a
19.8b
19.4a
28.8b
28.3b
30
22
30
18
25
30
22
25
34b
23
35
32
27
18
1.9
1.6
2.2
1.9
2.0
1.7
2.0
2.0
2.2
2.1c
2.0
2.0a
1.8
1.9
(c ) 0.445)
+57.3
CH(OH)Me*
pyrrolidine
C
C
30H42N2O8‚1.0H2O
(c ) 0.345)
+50.0
31H42N2O7
(c ) 0.435)
+16
CH2cyclohexyl
CH2CH2Ph
C35H50N2O7‚0.3H2O
C36H46N2O7‚1.0H2O
HRMSa
(c ) 0.99)
+11.1
(c ) 1.14)
+19
g
h
i
CH2OCH2Ph
(p-OH)Ph
(c ) 0.38)
-22.0
C
34H42N2O8‚1.4H2O
(c ) 0.185, MeOH)
+31
CH2Ph
C35H44N2O6‚0.2H2O
C35H44N2O8‚0.5H2O
C35H45N3O7
(c ) 0.47)
+34.7
j
CH2(p-OH)Ph
CH2(p-NH2)Ph
CH2(m-Cl,p-OH)Ph
CH2(m-Cl,p-NH2)Ph
CH2(R-naphthyl)
CH2(â-naphthyl)
(c ) 0.215)
+31
k
r
(c ) 0.42)
+16
C35H43ClN2O8‚0.5H2O
C35H44ClN3O7
(c ) 0.97, MeOH)
+28
s
(c ) 0.88)
+32
w
x
C39H46N2O7‚0.25H2O
(c ) 0.91)
+13.3
b
C39H46N2O7
(c ) 1.02)
1
a
Compounds were analyzed for C,H,N; the results agreed to within (0.4% of the theoretical values. HRMS calcd for C36H47N2O8 m/z
b
requires 635.3332, found 635.3324 (∆ -0.8 mmu). Elemental analysis requires C, 71.54; found C, 72.10. HRMS calcd for C39H47N2O7
2
m/z requires 655.3383, found 655.3380 (∆ -0.3 mmu). All optical rotations were performed in chloroform, unless stated otherwise, at
3
a
589 nm. Epoxide (1 mg in 2 mL CH3CN/H2O (50/50)) was analyzed by RP-HPLC (λmax ) 220 nm, flow rate ) 1.5 mL/min). Nova-Pak
b
C18 (3.9 × 150 mm) column: isocratic elution 50/50 CH3CN/H2O. Kromasil C18 (4.6 × 250 mm) column: gradient elution 50/50-60/40
4
a
CH3CN/H2O (0-30 min). Isolated yield of â-epoxide after preparative RP-HPLC separation. 25% starting material was recovered.
5
a
Determined from the crude reaction mixture. Ratio of epoxides after Fmoc deprotection, see Scheme 5. *(S)-Configuration at C3, derived
from D-threonine.
elemental analyzer for C, H, and N and a Lilly/Brinkman
setup, employing a combustion titration method, for other
elements.
however, additional studies are required to understand
the effects of these agents at the molecular target. The
availability of these analogues will allow studies to
further probe the mechanism of action of this exciting
class of novel antitumor agents. In addition, work is
planned to further evaluate 6u , with special emphasis
on the effect of resistance factors on activity, solubility
properties, and in vivo efficacy.
Thin-layer chromatography (TLC) was performed on E.
Merck silica gel 60 F254 plates. Spots were detected using a
combination of UV and chemical detection [plates were dipped
in a ceric ammonium molybdate solution (150 g of ammonium
molybdate and 8 g of ceric(IV) sulfate in 1000 mL of 10% (v/v)
aqueous sulfuric acid) and then heated on a hot plate]. “Silica
gel flash column chromatography” was performed on E. Merck
silica gel 60 (230-400 mesh, 40-63 µm). Analytical HPLC
separations were performed on a LDC system with a CM 4000
multiple solvent delivery system, a spectromonitor 5000 pho-
todiode array detector set to record at 254 or 220 nm, a Hitachi
D-2500 chromato-integrator, and a Gilson FC204 fraction
collector. Analytical reverse-phase (RP) HPLC was performed
on a Kromasil C18 6-mm column (3.9 × 150 mm) using
acetonitrile:water at a flow rate of 1.0 mL/min. Preparative
RP-HPLC was performed on a 2.0-cm × 25-cm Kromasil C18
column using acetonitrile:water as the mobile phase and a
flow rate of 28 mL/min. All HPLC solvents were filtered
through a 0.22-mm membrane and then thoroughly degassed
under vacuum. All solvents and reagents were obtained
commercially and used as supplied unless otherwise stated.
In those cases where solvents and reagents were dried and
purified, literature procedures were followed. Where necessary,
reactions were performed under a flow of dry nitrogen in a
flame-dried flask.
Exp er im en ta l Section
Spectra were routinely obtained on a General Electric QE-
300 instrument operating at 300 MHz for 1H and a Bruker
AC-250 instrument operating at 62.9 MHz for 13C. Bruker
AMX-500 and Varian 400 spectrometers were used, where
1
stated, operating at 500 and 400 MHz, respectively, for H and
125 and 100 MHz, respectively, for 13C spectra. Spectral data,
in various solvents (specified), are reported as parts per million
(δ) values downfield from tetramethylsilane. Multiplicities of
resonances are described as apparent (app), broad (br), singlet
(s), doublet (d), triplet (t), quartet (q), or multiplet (m). 1H and
13C chemical shift assignments are based on comparison with
previously reported data4 and detailed analysis of two-
dimensional NMR spectra (HMQC, HMBC, COSY, ROESY)
for Cryptophycin-52 (3).29 IR spectra were run as KBr pellets
or CHCl3 solutions on a Nicolet 510P FT-IR spectrometer.
Optical rotations were measured on an Autopol III automatic
polarimeter or a Perkin-Elmer 341 instrument. UV spectra
were obtained on a Shimadzu 2101PC instrument. Mass
spectral data were obtained on either a VG-70SE (for FD), a
SCIEX API-100 (for ESI), or a VG-ZAB2SE (for FAB and
accurate mass measurements) spectrometer. Elemental analy-
ses were performed using a Control Equipment Corp. 440
(5S)-[[(1,1-Dim eth yleth yl)dim eth ylsilyl]oxy]-(6R)-m eth -
yl-8-p h en yl-(2E,7E)-octa d ien oic Acid , 11.15 To a stirred
solution of methyl ester 1031 (1.00 g, 2.673 mmol) in acetone
(44 mL) was added 1 N aqueous LiOH (26 mL) at room
temperature. The cloudy mixture was further diluted with

2596 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Ta ble 6. Proton NMR Data for Representative â-Epoxides 6*
Patel et al.
6a : glycine
6b: valine
6f: homoPhe
6h : ([p-OH]Ph)glycineX
16j: tyrosineX
Unit A
PhH3
PhH2
3-H
2-H
5-H
8-H
7-H
4-H
6-H
7.41-7.32 (m)
7.31-7.25 (m)
7.40-7.31 (m)
7.28-7.13 (m)
7.46-7.35 (m)
7.33-7.25 (m)
7.36-7.27 (m)
7.26-7.16 (m)
6.79-6.70 (m)
5.91 (dd, J 15.5/1.9)
5.23-5.18 (m)
3.75 (d, J 1.7)
2.96 (dd, J 7.4/2.0)
2.72-2.67 (m)
2.44-2.39 (m)
1.88-1.81 (m)
1.13 (d, J 6.9)
7.36-7.31 (m)
7.21-7.30 (m)
6.78-6.70 (m)
5.67 (d, J 15.6)
5.18 (dd, J 10/4.6)
3.66 (s)
2.90 (dd, J 1.9/1.6)
2.57-2.53 (m)
2.46-2.37 (m)
1.78-1.69 (m)
1.12 (d, J 7.0)
6.66 (ddd, J 15.1/8.4/6.4) 6.80 (ddd, J 15.1/10.4/4.6) 6.83-6.76 (m)
5.90 (d, J 15.6)
5.20-5.15 (m)
3.71 (d, J 1.6)
2.94 (dd, J 7.3/1.9)
2.58-2.51 (m)
2.58-2.51 (m)
1.87-1.81 (m)
1.16 (d, J 7.3)2
5.85 (d, J 15.0)
5.23 (dd, J 10.9/4.8)
3.71 (d, J 1.1)
5.75 (d, J 14.5)
5.29-5.23 (m)
3.75 (d, J 1.6)
2.99 (dd, J 7.3/1.7)
2.56-2.47 (m)
2.69-2.63 (m)
1.85-1.61 (m)
1.18 (d, J 6.8)
2.94 (dd, J 7.5/1.4)
2.69-2.36 (m)4
2.66-2.36 (m)
4-H′
6-Me
1.84-1.74 (m)
1.17 (d, J 5.9)2
Unit B
NH
2-H
R
5.98 (t, J 6.3)
4.01 (dd, J 16.8/6.7)
5.53 (d, J 8.0)
4.36 (dd, J 8.2/4.5)
5.60 (d, J 7.8)
4.67-4.51 (m)
not observed
5.27 (s)
7.33-7.21 (m, PhH5) OH not observed
5.55 (br d, J 9)
4.71 (app q, J 7.5)4
OH not observed
3.94 (dd, J 16.7/5.9, 2-H′) 2.52-2.36 (m, 3-H)4
0.99 (d, J 6.9, 4-H3)
2.77 (t, J 7.7, 4-H2)
2.42-2.35 (m, 3-H)
1.98-1.86 (m, 3-H′)
7.13 (d, J 8.5, PhH2)
6.74 (d, J 8.5, PhH2)
6.98 (d, J 8.3, PhH2)
6.71 (d, J 8.1, PhH2)
3.04-2.99 (m, 3-H2)
0.89 (d, J 7.3, 3-Me)
Unit C
NH
3-H
3-H′
2-Me
2-Me
7.08-7.04 (m)
3.45 (dd, J 13.5/8.4)
3.16 (dd, J 13.5/4.0)
1.24 (s)
7.23-7.13 (m)4
3.41 (dd, J 13.4/8.6)
4.14 (dd, J 13.5/3.2)
1.23 (s)
7.46-7.21 (m)4
3.41 (dd, J 13.4/8.4)
3.22 (dd, J 13.4/3.5)
1.28 (s)
not observed
3.53 (d, J 13.6)
3.46 (d, J 7.2)
1.20 (s)
7.36-7.21 (m)3
3.44 (dd, J 13.7/9.1)
3.08 (dd, J 14.2/4.9)
1.20 (s)
1.18 (s)
1.18 (s)
1.22 (s)
1.18 (s)
1.14 (s)
Unit D
2-H
4.90 (dd, J 10/3.2)
4.89 (dd, J , 9.9/3.1)
1.84-1.60 (m)
1.38-1.25 (m)
0.87 (d, J 6.4)2
0.84 (d, J 7.3)2
4.94 (dd, J 10.4/3.5)
1.85-1.61 (m)
1.40-1.29 (m)
0.89 (d, J 6.4)
4.93 (dd, J 10/3.2)
1.69-1.59 (m)
1.30-1.22 (m)
0.79 (d, J 6.2)2
0.78 (d, J 6.3)2
4.81 (dd, J 10.1/3.4)
1.70-1.65 (m)
1.31-1.24 (m)
0.82 (d, J 6.7)
3-H/4-H 1.78-1.66 (m)
3-H′
5-H3
4-Me
1.42-1.33 (m)
0.87 (d, J 6.3)
0.86 (d, J 6.3)
0.88 (d, J 6.3)
0.80 (d, J 6.4)
1
2
3
*In CDCl3 at 300 MHz. X In CDCl3/CD3OD (1:1) at 300 MHz. †In CDCl3 at 400 MHz. Partially buried. Overlapping. Buried.
4
app, apparent.
Sch em e 6a
a
Reagents: (a) Fmoc-Cl, NaHCO3, dioxane-H2O, 88% (7m ) and 65% (7t); (b) mCPBA, 0.2 M CH2Cl2, 25 °C, quantitative (21 and 22);
(c) piperidine, DMF, 25 °C, then RP-HPLC separation, 23% (6k ) and 32% (6s); (d) MeI, K2CO3, CH3CN, 70 °C, 47%.
acetone (20 mL), and the resulting yellow mixture stirred at
room temperature for 23.5 h. The reaction was diluted with
diethyl ether (400 mL), and the organics were washed with 1
N HCl (120 mL), brine (200 mL), and H2O (160 mL). The
organics were dried (MgSO4) and concentrated in vacuo to
leave a yellow oil which was purified by column chromatog-
raphy (gradient: 5% AcOH in 20-40% EtOAc/n-hexanes) to
give carboxylic acid 11 as a yellow oil (960 mg, 100%): [R]D
+87.6° (c ) 1.05, CHCl3); [lit.15 [R]D +87.0° (c ) 1.4, CHCl3)];
1H NMR (CDCl3) δ 7.38-7.19 (m, PhH5), 7.09 (ddd, J ) 15.2,

Novel Cryptophycin Antitumor Agents
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2597
Ta ble 7. In Vitro Cytotoxicity of Styrenes 7, R-Epoxides 17, and â-Epoxides 6 in a CCRF-CEM Tumor Cell Line
IC50 (nM)1
compd
R
styrene 7
R-epoxide 17
â-epoxide 6
CH2 (m-Cl,p-OMe)Ph
H
CH(Me)2
CH(OH)Me*
pyrrolidine
CH2cyclohexyl
CH2CH2Ph
CH2OCH2Ph
(p-OH)Ph
CH2Ph
CH2(p-OH)Ph
CH2(p-NH2)Ph
CH2(p-NHFmoc)Ph
CH2(p-I)Ph
CH2(m-Cl,p-OH)Ph
CH2(m-Cl,p-NH2)Ph
CH2(m-Cl,p-NHFmoc)Ph
CH2(m-Cl,p-NMe2)Ph
CH2(m,p-diOH)Ph
CH2(R-naphthyl)
CH2(â-naphthyl)
CH2(3-pyridyl)
C-51 (5): 13.85
>2000
C-52 (3):2 0.022
>1940
>1800
1060
1350
8.8
598
249
274
a
b
c
d
e
f
g
h
i
>1850
>1840
>1860
>1680
1660
856
>1620
975
>1690
412
0.183
4.108
10.11
j
734
k
m
p
r
s
t
u
v
w
x
y
1540
>1200
607
269
500
>1250
R:â ) 1:2 (21); 115
31
0.52
0.58
R:â ) 1:2 (22); 15.3
0.054
1301
>1570
>1570
997
327
866
35
48
1
2
See Experimental Section for assay protocol. C-52 (3) was run as a positive control in each assay: SD ) 0.0062 nM (n ) 6); coefficient
of variation ) SD/mean ) 28%. *(S)-Configuration at C3, derived from D-threonine.
7.6 and 7.9 Hz, 3-H), 6.38 (d, J ) 16 Hz, 8-H), 6.16 (dd, J ) 16
and 8 Hz, 7-H), 5.85 (d, J ) 15.8 Hz, 2-H), 3.81-3.75 (m, 5-H),
2.49-2.37 (m, 6-H, 4-CH2), 1.12 (d, J ) 6.7 Hz, 6-Me), 0.91 (s,
SiCMe3), 0.065 (s, SiMe), 0.068 (s, SiMe); 13C NMR (CDCl3) δ
171.5 (C), 149.1 (CH), 137.5 (CH), 131.7 (CH), 130.4 (CH),
128.4 (CH), 127.0 (CH), 126.0 (CH), 122.5 (CH), 74.8 (CH), 42.8
(CH), 37.5 (CH2), 25.8 (CH3), 18.0 (C), 16.0 (CH3), -4.5 (CH3),
-4.7 (CH3); IR (CHCl3) υmax 2957, 2930, 2858, 1697, 1258,
1098, 838 cm-1; UV (EtOH) λmax 251 (ꢀ ) 17428), 292 (ꢀ )1571)
nm; FDMS m/z 360.2 (M+, 100).
[R]D589 -57.8° (c ) 1.06, CHCl3); 1H NMR (CDCl3) δ 7.41-7.20
(m, PhH5, 3-H), 6.48 (d, J ) 16 Hz, 8-H), 6.15-6.07 (m, 7-H,
2-H), 3.71-3.65 (m, 5-H), 2.83 (br s, CH2CH2), 2.60-2.33 (m,
6-H,4-CH2),1.95 (brs, 5-OH), 1.14 (d, J ) 6.8 Hz, 6-Me); 13C
NMR (CDCl3) δ 169.2 (C), 161.0 (C), 152.4 (CH), 136.8 (C),
132.1 (CH), 130.6 (CH), 128.5 (CH), 127.4 (CH), 126.1 (CH),
117.4 (CH), 73.4 (CH), 43.5 (CH), 37.7 (CH2), 25.5 (CH2), 16.7
(CH3); IR (KBr) υmax 3457, 1804, 1773, 1735, 1724, 1209, 1099,
1067, 1049, 975, 744, 694 cm-1; UV (EtOH) λmax 250 (ꢀ )
20535) nm; FDMS m/z 343.2 (M+, 100). Anal. Calcd for C19H21
-
NO5: C, 66.46; H, 6.16; N, 4.08. Found: C, 66.49; H, 6.16;
N, 4.07.
1-[[(5S)-[[(1,1-Dim eth yleth yl)d im eth ylsilyl]oxy]-(6R)-
m et h yl-1-oxo-8-p h en yl-(2E,7E)-oct a d ien yl]oxy]-2,5-p yr -
r olid in ed ion e, 12. To a stirred solution of carboxylic acid 11
(720 mg, 2.0 mmol) in dry dimethylformamide (5.50 mL) were
added 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC)
(459 mg, 2.4 mmol) and N-hydroxysuccinimide (299 mg, 2.6
mmol) at room temperature. The mixture was stirred for 28 h
and diluted with EtOAc (100 mL), and the organics were
washed with 1 N aqueous HCl (2 × 50 mL) and H2O (75 mL),
dried (MgSO4), and concentrated in vacuo to leave an oil. Crude
product was purified by column chromatography (gradient:
5-30% EtOAc/n-hexanes) to give active ester 12 as a pale-
(2S )-[3-[[(1,1-Dim e t h yle t h oxy)ca r b on yl]a m in o]-2,2-
dim eth yl-1-oxopr opoxy]-4-m eth ylpen tan oic Acid, 14. Car-
boxylic acid 14 was prepared31 according to the literature
procedure:24a [R]D589 -29.9° (c ) 1.1, MeOH); 1H NMR (DMSO-
d6) δ unit C′ 6.61 (t, J ) 6.2 Hz, NH), 3.16 (dd, J ) 13.6 and
7.15 Hz, 3-H), 3.04 (dd, J ) 13.6 and 5.7 Hz, 3′H), 1.33 (br s,
CMe3), 1.04 (br s, 6H, Me2); unit D 13.08 (br s, CO2H), 4.78
(dd, J ) 8.78 and 3.72 Hz, 2-H), 1.72-1.60 (m, 3-H2), 1.59-
1.54 (m, 4-H), 0.88 (d, J ) 6.7 Hz, 4-Me), 0.85 (d, J ) 6.6 Hz,
5-H3); 13C NMR (DMSO-d6) δ 175.3 (C), 171.9 (C), 155.8 (C),
77.7 (C), 70.5 (CH), 47.9 (CH2), 43.2 (C), 39.2 (CH2), 28.1 (3 ×
CH3), 24.3 (CH), 22.8 (CH3), 22.4 (CH3), 22.2 (CH3), 21.5 (CH3);
IR (CHCl3) υmax 2980, 2964, 1711, 1512, 1368, 1270, 1254, 1151
cm-1; ESMS (PI) m/z 232 ([M - Boc + 1]+, 100); (NI) m/z 300
([M - 1]-, 100). Anal. Calcd for C16H29NO6: C, 57.99; H, 8.82;
N, 4.23. Found: C, 57.95; H, 8.93; N, 4.28.
(1S,3E)-5-[(2,5-Dioxo-1-p yr r olid in yl)oxy]-1-[(1R,2E)-1-
m et h yl-3-p h en yl-2-p r op en yl]-5-oxo-3-p en t en yl-(2S)-[3-
[[(1,1-d im et h ylet h oxy)ca r b on yl]a m in o]-2,2-d im et h yl-1-
oxop r op oxy]-4-m eth ylp en ta n oa te, 15. To a suspension of
carboxylic acid 14 (1.28 g, 3.87 mmol) in dry dichloromethane
(6 mL) were added EDC (742 mg, 3.87 mmol) and a catalytic
amount of 4-(dimethylamino)pyridine (DMAP) (73 mg, 0.60
mmol), and the mixture stirred at room temperature, under a
nitrogen atmosphere, for 0.5 h. A solution of alcohol 13 (1.02
g, 2.97 mmol) in dichloromethane (5.5 mL) was then added,
and the reaction mixture stirred for a further 0.3 h. The
reaction was diluted with dichloromethane (200 mL), and the
organics were washed consecutively with 1 N aqueous HCl (2
× 50 mL), saturated aqueous NaHCO3 (2 × 50 mL), and
H2O (50 mL). The organics were dried (MgSO4) and concen-
trated in vacuo to leave an oily residue, which was purified
by column chromatography (gradient: 10-30% EtOAc/n-
hexanes) to provide desired ester 15 as a yellow solid (1.68 g,
yellow oil (724 mg, 80%): [R]D +71.3° (c ) 1.01, CHCl3); 1H
589
NMR (CDCl3) δ 7.36-7.20 (m, PhH5, 3-H), 6.38 (d, J ) 16 Hz,
8-H), 6.14 (dd, J ) 16.1 and 8.0 Hz, 7-H). 6.03 (d, J ) 16 Hz,
2-H), 3.79 (q, J ) 4.3 Hz, 5-H), 2.94 (brs, CH2CH2), 2.58-2.42
(m, 6-H, 4-CH2), 1.10 (d, J ) 6.8 Hz, 6-Me), 0.90 (s, SiCMe3),
0.05 (s, SiMe2); 13C NMR (CDCl3) δ 169.2 (C), 161.1 (C), 152.9
(CH), 137.3 (C), 131.5 (CH), 130.6 (CH), 128.4 (CH), 127.1
(CH), 126.0 (CH), 117.2 (CH), 74.6 (CH), 42.9 (CH), 38.0 (CH2),
25.7 (CH3), 25.5 (CH2), 18.0 (C), 15.8 (CH3), -4.5(CH3), -4.6
(CH3); IR υmax (CHCl3) 2957, 2931,2858, 1772, 1741, 1648,
1364, 1254, 1092, 1069, 838 cm-1; UV (EtOH) λmax 249 (ꢀ )
11184), 292 (ꢀ ) 1185) nm; FDMS m/z 457 (M+, 100). Anal.
Calcd for C25H35NO5: C, 65.61; H, 7.71; N, 3.06. Found: C,
65.51; H, 7.56; N, 3.02.
1-[[(5S)-Hyd r oxy-(6R)-m eth yl-1-oxo-8-p h en yl-(2E,7E)-
octa d ien yl]oxy]-2,5-p yr r olid in ed ion e, 13. To a stirred
solution of silyl ether 12 (2.50 g, 5.47 mmol) in CH3CN (130
mL) was added 48% aqueous HF (15 mL) at 0 °C, and the
solution stirred at 0 °C for 0.75 h and at room temperature
for 4 h. The reaction was diluted with diethyl ether (300 mL),
and the organics were washed with H2O until the aqueous
extract was ∼pH 7. Organics were dried (MgSO4) and concen-
trated in vacuo to give a yellow residue which crystallized
from Et2O to give alcohol 13 as white crystals (1.46 g, 78%):

2598 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Patel et al.
79%): [R]D589 +39.5° (c ) 1.04, CHCl3); 1H NMR (CDCl3) δ unit
A 7.35-7.20 (m, PhH5, 3-H), 6.43 (d, J ) 15.8 Hz, 8-H), 6.12
(d, J ) 15.9 Hz,2-H), 5.99 (dd, J ) 8.5 and 15.8 Hz, 7-H), 5.06-
5.08 (m, 5-H), 2.85 (br s, CH2CH2), 2.68-2.61 (m, 6-H, 4-CH2),
1.13 (d, J ) 6.8 Hz, 6-Me); unit C 5.32 (br t, J ) 6.0 Hz NH),
3.28-3.25 (m, 3-CH2),1.43 (s, CMe3), 1.21 (s, 2-Me), 1.19 (s,
2-Me); unit D 4.95 (dd, J ) 9.8 and 3.8 Hz, 2-H), 1.73-1.64
(m, 3-H, 4-H), 1.59-1.49 (m, 3-H′), 0.85 (d, J ) 6.4 Hz, 5-Me),
0.82 (d, J ) 6.4, 4-Me); 13C NMR (CDCl3) δ 176.2 (C), 170.6
(C), 169.0 (C), 160.7 (C), 156.3 (C), 149.4 (CH), 136.7 (C), 131.7
(CH), 129.6 (CH), 128.5 (CH), 127.5 (CH), 126.1 (CH), 118.5
(CH), 78.9 (C), 75.8 (CH), 71.1 (CH), 69.3 (CH), 48.4 (CH2),
43.8 (C), 40.6 (CH), 39.5 (CH2), 35.0 (CH2), 28.3 (CH3), 25.5
(CH2), 25.4 (C), 24.7 (CH), 22.8 (CH3), 22.7 (CH3), 22.4
(CH3), 22.3 (CH3), 21.5 (CH3), 21.2 (CH3), 16.7 (CH3); IR
(KBr) υmax 3400, 2975, 1743,1367, 1206, 1126, 1145, 1068
cm-1; UV (EtOH) λmax 251 (ꢀ ) 16086), 284 (ꢀ ) 1953), 293 (ꢀ
) 1389) nm; FDMS m/z 657 (M+, 100). Anal. Calcd for
C35H48N2O10: C, 64.01; H, 7.37; N, 4.27. Found: C, 64.19; H,
7.27; N, 4.52.
acid 19 (2.78 g, 10 mmol) in dry acetonitrile (175 mL) was
added N-chlorosuccinimide (1.86 g, 14 mmol), and the mixture
was heated under reflux for 19.5 h. The solvent was removed
in vacuo and the residue diluted with EtOAc (200 mL) and
washed with H2O (2 × 50 mL). The organic phase was dried
(MgSO4) and concentrated in vacuo to leave an oily residue
which was purified by column chromatography (SiO2; gradient
elution: 30-100% EtOAc/n-hexanes) to provide chloride 20
589
as a white powder (1.56 g, 50%): [R]D
-27.4° (c ) 0.80,
1
MeOH); H NMR (DMSO-d6) δ 9.42 (s, NH), 8.31 (d, J ) 7.8
Hz, NH), 7.55 (d, J ) 8.2 Hz, PhH), 7.30 (d, J ) 1.3 Hz, PhH),
7.10 (dd, J ) 8.3 and 1.4 Hz, PhH), 4.44-4.37 (m, 2-H), 3.57
(s, OMe), 2.96 (dd, J ) 13.8 and 5.4 Hz, 3-H), 2.79 (dd, J )
13.9 and 9.5 Hz, 3-H′), 2.03 (s, CH3CdO), 1.75 (s, CH3CdO);
IR (KBr) υmax 3291 (br), 1754, 1745, 1663, 1653, 1555, 1528,
1404, 1377, 1302 cm-1; UV (EtOH) λmax 244 (ꢀ ) 10197) nm;
FDMS m/z 312 (M+, 100). Anal. Calcd for C14H17ClN2O4: C,
53.77; H, 5.48; N, 8.96; Cl, 11.34. Found: C, 54.01; H, 5.54;
N, 8.89; Cl, 11.57.
4-Am in o-3-ch lor o-D-p h en yla la n in e, 9s. To a stirred solu-
tion of protected amino acid 20 (1.00 g, 3.21 mmol) in dioxane
(20 mL) was added 1 N aqueous NaOH (12.8 mL, 12.8 mmol).
The mixture was heated under reflux for 24 h and the solvent
removed in vacuo to give a solid. Crude product was redis-
solved in 5 N aqueous hydrochloric acid (30 mL) and heated
at 95 °C for 18 h. The reaction solution was concentrated in
vacuo and the resulting solid residue triturated with methanol
(6 × 50 mL) to remove the majority of the inorganic salts. The
product was further purified using cation-exchange resin (SCX
column; 10 g for 400 mg of crude product) and eluting, under
gravity, first with CH3CN:H2O (1:1) until eluent was pH 7 to
remove the remaining inorganics and then with 2 M NH3-
MeOH:CH3CN (1:1) to provide amino acid 9s as an off-white
3-Ch lor o-D-tyr osin e, 9r . A suspension of O-methyl-D-
tyrosine (9q)15 (1.20 g, 5 mmol) in 48% aqueous hydrobromic
acid (4 mL) and phenol (1.6 g, 17 mmol) was heated under
reflux for 6 h. The reaction solution was concentrated to a dry
solid, diluted with a mixture of n-BuOAc/EtOAc (1:9, 50 mL),
and extracted with H2O (2 × 5 mL). The aqueous phase was
adjusted to pH 7 with concentrated NH4OH and the resulting
precipitate filtered, washed with H2O (10 mL), and dried under
vacuum to give chlorotyrosine 9r as a white solid (760 mg,
589
1
69%): [R]D -12.0° (c ) 3.05, MeOH); H NMR (DMSO-d6/
D2O) δ 7.16 (d, J ) 1.6 Hz, PhH), 6.97 (dd, J ) 8.11 and 1.7
Hz, PhH), 6.81 (d, J ) 8.3 Hz, PhH), 3.37 (dd, J ) 8.1 and 4.1
Hz, 2-H), 3.97 (dd, J ) 14.5 and 4.2 Hz, 3-H), 2.73 (dd, J )
14.4 and 8.2 Hz, 3-H′); 13C NMR (DMSO-d6) δ 170.4 (C), 152.4
(C), 130.8 (CH), 129.2 (CH), 126.5 (C), 119.6 (C), 116.8 (CH),
53.4 (CH), 34.7 (CH2); FDMS m/z 215 (M+, 100). Anal. Calcd
for C9H10NClO3: C, 50.13; H, 4.67; N; 6.50. Found: C, 50.40;
H, 4.57; N, 6.81.
589
1
solid (493 mg, 72%): [R]D +23.4° (c ) 0.26, H2O); H NMR
(CD3OD) δ 7.20 (d, J ) 8.1 and 2.0 Hz, PhH), 6.83 (d, J ) 8.2
Hz, PhH), 3.71 (dd, J ) 8.7 and 4.4 Hz, 2-H), 3.18 (dd, J )
14.7 and 4.3 Hz, 3-H), 2.90 (dd, J ) 14.6 and 8.7 Hz, 3-H′); IR
(KBr) υmax 3400 (br), 2900 (br), 1598, 1508, 1402, 1313 cm-1
;
UV (EtOH) λmax 243 (ꢀ ) 10754), 298 (ꢀ ) 2277) nm; FABMS
m/z 215 ([M + 1]+, 28). Anal. Calcd for C9N11ClN2O2‚0.1H2O:
C, 49.94; H, 5.22; N, 12.95; Cl, 16.38. Found: C, 49.93; H, 4.97;
N, 12.57; Cl, 16.40.
4-Am in o-D-p h en yla la n in e Meth yl Ester , Dih yd r och lo-
r id e, 18. Acetyl chloride (20 mL, 0.28 mmol) was added
dropwise to dry methanol (300 mL) at 0 °C under a dry
nitrogen atmosphere. The solution was stirred at room tem-
perature for 0.5 h and p-amino-D-phenylalanine (9k ) (10 g,
0.056 mol) added in one portion. The mixture was heated at
P r ep a r a tion of Ca r boxylic Acid 16 (Gen er a l Meth od
A). To a stirred solution of active ester 15 (1.0 mmol) in
dimethylformamide (10 mL) were added N,O-bis(trimethylsi-
lyl)acetamide (BSA) (3.0 mmol) and amino acid 9 (1.5 mmol).
The mixture was heated at 55-60 °C, under a nitrogen
atmosphere, until all active ester was consumed as judged by
TLC analysis (reaction time ) 10-30 h). The reaction solution
was poured into 1 N aqueous HCl (200 mL) and extracted with
EtOAc (3 × 50 mL). Combined organics were washed with 1
N aqueous HCl, brine, and H2O (100 mL each), dried (MgSO4),
and concentrated in vacuo to provide carboxylic acid 16 as a
solid. Further purification, where necessary, was performed
using column chromatography (SiO2; gradient elution: 2-5%
AcOH in CH2Cl2-15% MeOH:CH2Cl2). 1H NMR data were
consistent with the assigned structures (see Table 2 for
representative examples).
reflux for 3.5 h and concentrated in vacuo to provide methyl
589
ester 18 as a hygroscopic, white solid (15.0 g, 100%): [R]D
-
5.9° (c ) 2.02, H2O); 1H NMR (DMSO-d6) δ 7.30-7.24 (m,
PhH4), 4.19 (t, J ) 6.1, 2-H), 3.61 (s, OMe), 3.10-3.07 (m,
3-H2); IR (KBr) υmax 3414 (br), 2877 (br), 2589, 1744, 1509,
1245 cm-1; UV (EtOH) λmax 243 (ꢀ ) 10037), 292 (ꢀ ) 1347)
nm; ESMS (NI) m/z 265.1 ([M - 1]-, 48). Anal. Calcd for
C10H16N2Cl2O2‚0.5H2O: C, 43.49; H, 6.20; N, 10.14; Cl, 25.68.
Found: C, 43.85; H, 7.09; N, 9.86; Cl, 25.28.
N-Acetyl-4-(a cetyla m in o)-D-p h en yla la n in e Meth yl Es-
ter , 19. To a stirred solution of diamine 18 (14.5 g, 0.054 mmol)
in dry pyridine (256 mL) was added acetic anhydride (77 mL,
0.815 mol), and the mixture stirred at room temperature for
17.5 h. The resulting purple solution was concentrated in
vacuo, diluted with H2O (250 mL), neutralized to pH 7 with 5
N aqueous HCl, and extracted with a mixture of THF/EtOAc
(1:5; 5 × 200 mL). Combined, dried (MgSO4) organics were
concentrated in vacuo to furnish fully protected amino acid
19 as a yellow powder (12.2 g, 81%): [R]D589-40.1° (c ) 0.85,
N-[(1,1-Dim eth yleth oxy)ca r bon yl]-2,2-d im eth yl-â-a la -
n yl-(2S)-2-h yd r oxy-4-(m eth ylp en ta n oyl)-(2E,5S,6R,7E)-5-
h yd r oxy-6-m eth yl-8-p h en yl-2,7-octa d ien oylglycin e, 16a .
Active ester 15 was coupled with glycine (9a ), according to
general method A, to give 16a as a pale-yellow foam (93%):
FABMS m/z 617.5 ([M + 1]+, 25), 517.4 ([M - Boc + 1]+, 100).
1
MeOH); H NMR (DMSO-d6) δ 9.88 (s, NH), 8.28 (d, J ) 7.7
Hz, NH), 7.43 (d, J ) 8.4 Hz, PhH2), 7.07 (d, J ) 8.4 Hz, PhH2),
4.35 (app. q, J ) 2.8 Hz, 2-H), 3.54 (s, OMe), 2.89 (dd, J )
13.8 and 5.7 Hz, 3-H), 2.76 (dd, J ) 13.7 and 9.1 Hz, 3-H′),
1.98 (s, CH3CdO), 1.75 (s, CH3CdO); IR (KBr) υmax 3255 (br),
N-[(1,1-Dim eth yleth oxy)ca r bon yl]-2,2-d im eth yl-â-a la -
n yl-(2S)-2-h yd r oxy-4-(m eth ylp en ta n oyl)-(2E,5S,6R,7E)-5-
h ydr oxy-6-m eth yl-8-ph en yl-2,7-octadien oyl-D-valin e, 16b.
Active ester 15 was coupled with D-valine (9b), according to
general method A, to give 16b as a pale-yellow foam (96%):
ESMS (PI) m/z 659.5 ([M + 1]+, 48), 559.4 ([M - Boc + 1]+,
100).
N-[(1,1-Dim eth yleth oxy)ca r bon yl]-2,2-d im eth yl-â-a la -
n yl-(2S)-2-h yd r oxy-4-(m eth ylp en ta n oyl)-(2E,5S,6R,7E)-5-
h yd r oxy-6-m e t h yl-8-p h e n yl-2,7-oct a d ie n oyl-D -t h r e o-
3066, 2401, 2375, 1746, 1651, 1542, 1519, 1403, 1214 cm-1
;
UV (EtOH) λmax 247 (ꢀ ) 15859) nm; FDMS m/z 278 (M+, 100).
Anal. Calcd for C14H18N2O4: C, 60.42; H, 6.52; N, 10.07.
Found: C, 60.48; H, 6.48; N, 9.95.
N-Ace t yl-4-(a ce t yla m in o)-3-ch lor o-D-p h e n yla la n in e
Meth yl Ester , 20. To a stirred suspension of protected amino

Novel Cryptophycin Antitumor Agents
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2599
n in e, 16c. Active ester 15 was coupled with D-threonine (9c),
according to modified general method A using BSA (4 equiv),
to give 16c as a pale-yellow foam (89%): ESMS (PI) m/z 661.4
([M + 1]+, 33), 561.3 ([M - Boc + 1]+, 100).
h yd r oxy-6-m et h yl-8-p h en yl-2,7-oct a d ien oyl-3-ch lor o-D-
tyr osin e, 16r . Active ester 15 was coupled with 3-chloro-D-
tyrosine (9r ), according to modified general method A using
BSA (4 equiv), to give 16r as a pale-yellow solid (67%): ESMS
(PI) m/z 757.5 ([M + 1]+, 50), 657.4 ([M - Boc + 1]+, 100).
N-[(1,1-Dim eth yleth oxy)ca r bon yl]-2,2-d im eth yl-â-a la -
n yl-(2S)-2-h yd r oxy-4-(m eth ylp en ta n oyl)-(2E,5S,6R,7E)-5-
h yd r oxy-6-m et h yl-8-p h en yl-2,7-oct a d ien oyl-4-a m in o-3-
ch lor o-D-p h en yla la n in e, 16s. Active ester 15 was coupled
with 3-chloro-4-amino-D-phenylalanine (9s), according to modi-
fied general method A using BSA (6 equiv), to give 16s as a
golden solid (61%): ESMS (PI) m/z 756.6 ([M + 1]+, 41), 656.4
([M - Boc + 1]+, 100).
N-[(1,1-Dim eth yleth oxy)ca r bon yl]-2,2-d im eth yl-â-a la -
n yl-(2S)-2-h yd r oxy-4-(m eth ylp en ta n oyl)-(2E,5S,6R,7E)-5-
h yd r oxy-6-m eth yl-8-p h en yl-2,7-octa d ien oyl-3-h yd r oxy-D-
tyr osin e, 16v. Active ester 15 was coupled with 3,4-dihydroxy-
D-phenylalanine (D-DOPA) (9v), according to modified general
method A using BSA (5 equiv), to give 16v as a pale-yellow
solid (75%): ESMS (PI) m/z 739.6 ([M + 1]+, 46), 639.3 ([M -
Boc + 1]+, 100).
N-[(1,1-Dim eth yleth oxy)ca r bon yl]-2,2-d im eth yl-â-a la -
n yl-(2S)-2-h yd r oxy-4-(m eth ylp en ta n oyl)-(2E,5S,6R,7E)-5-
h yd r oxy-6-m et h yl-8-p h en yl-2,7-oct a d ien oyl-3-(1-n a p h -
th a len yl)-D-a la n in e, 16w . Active ester 15 was coupled with
3-(1-naphthyl)-D-alanine (9w ), according to general method A,
to give 16w as a pale-yellow solid (77%): ESMS (PI) m/z 757.6
([M + 1]+, 39), 657.4 ([M - Boc + 1]+, 100).
N-[(1,1-Dim eth yleth oxy)ca r bon yl]-2,2-d im eth yl-â-a la -
n yl-(2S)-2-h yd r oxy-4-(m eth ylp en ta n oyl)-(2E,5S,6R,7E)-5-
h yd r oxy-6-m et h yl-8-p h en yl-2,7-oct a d ien oyl-3-(2-n a p h -
th a len yl)-D-a la n in e, 16x. Active ester 15 was coupled with
3-(2-naphthyl)-D-alanine (9x), according to general method A,
to give 16x as a pale-yellow solid (78%): ESMS (PI) m/z 757.6
([M + 1]+, 60), 657.4 ([M - Boc + 1]+, 100).
N-[(1,1-Dim eth yleth oxy)ca r bon yl]-2,2-d im eth yl-â-a la -
n yl-(2S)-2-h yd r oxy-4-(m eth ylp en ta n oyl)-(2E,5S,6R,7E)-5-
h yd r oxy-6-m eth yl-8-p h en yl-2,7-octa d ien oyl-3-(3-p yr id i-
n yl)-D-a la n in e, 16y. Active ester 15 was coupled with 3-(3-
pyridyl)-D-alanine (9y), according to general method A, to give
16y as a pale-yellow solid (65%): ESMS (PI) m/z 708.5 ([M +
1]+, 100), 608.3 ([M - Boc + 1]+, 57).
N-[(1,1-Dim eth yleth oxy)ca r bon yl]-2,2-d im eth yl-â-a la -
n yl-(2S)-2-h yd r oxy-4-(m eth ylp en ta n oyl)-(2E,5S,6R,7E)-5-
h y d r o x y -6-m e t h y l-8-p h e n y l-2,7-o c t a d ie n o y l-D -t r y p -
top h a n , 16z. Active ester 15 was coupled with D-tryptophan
(9z), according to general method A, to give 16z as an off-white
solid (85%): ESMS (PI) m/z 746.4 ([M + 1]+, 60), 646.7 ([M -
Boc + 1]+, 100).
N-[(1,1-Dim eth yleth oxy)ca r bon yl]-2,2-d im eth yl-â-a la -
n yl-(2S)-2-h yd r oxy-4-(m eth ylp en ta n oyl)-(2E,5S,6R,7E)-5-
h ydr oxy-6-m eth yl-8-ph en yl-2,7-octadien oyl-D-pr olin e, 16d.
Active ester 15 was coupled with D-proline (9d ), according to
general method A, to give 16d as a yellow foam (92%): FABMS
m/z 657.5 ([M + 1]+, 60), 557.5 ([M - Boc + 1]+, 100).
N-[(1,1-Dim eth yleth oxy)ca r bon yl]-2,2-d im eth yl-â-a la -
n yl-(2S)-2-h yd r oxy-4-(m eth ylp en ta n oyl)-(2E,5S,6R,7E)-5-
h ydr oxy-6-m eth yl-8-ph en yl-2,7-octadien oyl-3-cycloh exyl-
D-a la n in e, 16e. Active ester 15 was coupled with cyclohexyl-
D-alanine (9e), according to general method A, to give 16e as
a pale-yellow foam (88%): ESMS (PI) m/z 713.7 ([M + 1]+,
65), 613.4 ([M - Boc + 1]+, 100).
1-(1,1-Dim et h ylet h yl)-(7S,10S,12E,16R)-4,4-d im et h yl-
10-[(1R,2E)-1-m et h yl-3-p h en yl-2-p r op en yl]-7-(2-m et h yl-
p r op yl)-5,8,14-t r ioxo-16-(2-p h en ylet h yl)-6,9-d ioxa -2,15-
diazah eptadec-12-en edioate, 16f. Active ester 15 was coupled
with D-homophenylalanine (9f), according to general method
A, to give 16f as a pale-yellow foam (72%): ESMS (PI) m/z
721.5 ([M + 1]+, 55), 621.6 ([M - Boc + 1]+, 100).
N-[(1,1-Dim eth yleth oxy)ca r bon yl]-2,2-d im eth yl-â-a la -
n yl-(2S)-2-h yd r oxy-4-(m eth ylp en ta n oyl)-(2E,5S,6R,7E)-5-
h yd r oxy-6-m et h yl-8-p h en yl-2,7-oct a d ien oyl-O-(p h en yl-
m eth yl)-D-ser in e, 16g. Active ester 15 was coupled with
O-benzyl-D-serine (9g), according to general method A, to give
16g as a pale-yellow foam (72%): ESMS (PI) m/z 737.5 ([M +
1]+, 70), 637.3 ([M - Boc + 1]+, 100).
(2R)-N-[(1,1-Dim et h ylet h oxy)ca r b on yl]-2,2-d im et h yl-
â-a la n yl-(2S)-2-h yd r oxy-4-(m eth ylp en ta n oyl)-(2E,5S,6R,-
7E)-5-h yd r oxy-6-m et h yl-8-p h en yl-2,7-oct a d ien oyl-2-(4-
h yd r oxyp h en yl)glycin e, 16h . Active ester 15 was coupled
with d-(4-hydroxyphenyl)glycine (9h ), according to modified
general method A using BSA (4 equiv), to give a yellow solid.
Crude product was dissolved in 10% AcOH/CH2Cl2, stirred for
1 h, and concentrated in vacuo to give a solid which was
purified by column chromatography (SiO2; gradient elution:
3% AcOH in CH2Cl2-15% MeOH:CH2Cl2) to give 16h as an
off-white foam (75%): ESMS (PI) m/z 709.5 ([M + 1]+, 50),
609.3 ([M - Boc + 1]+, 100).
N -[(1,1-Dim e t h yle t h oxy)ca r b on yl]-2,2-d im e t h yl-â-
alan yl-(2S)-2-h ydr oxy-4-(m eth ylpen tan oyl)-(2E,5S,6R,7E)-
5-h yd r oxy-6-m eth yl-8-p h en yl-2,7-octa d ien oyl-D-p h en yl-
a la n in e, 16i. Active ester 15 was coupled with D-phenylala-
nine (9i), according to general method A, to give 16i as a pale-
yellow foam (88%): ESMS (PI) m/z 707.5 ([M + 1]+, 50), 607.4
([M - Boc + 1]+, 100).
N-[(1,1-Dim eth yleth oxy)ca r bon yl]-2,2-d im eth yl-â-a la -
n yl-(2S)-2-h yd r oxy-4-(m eth ylp en ta n oyl)-(2E,5S,6R,7E)-5-
h ydr oxy-6-m eth yl-8-ph en yl-2,7-octadien oyl-D-tyr osin e, 16j.
Active ester 15 was coupled with D-tyrosine (9j), according to
modified general method A using BSA (4 equiv), to give 16j
as an off-white solid (87%): ESMS (PI) m/z 723.5 ([M + 1]+,
4), 623.5 ([M - Boc + 1]+, 100).
N-[(1,1-Dim eth yleth oxy)ca r bon yl]-2,2-d im eth yl-â-a la -
n yl-(2S)-2-h yd r oxy-4-(m eth ylp en ta n oyl)-(2E,5S,6R,7E)-5-
h yd r oxy-6-m et h yl-8-p h en yl-2,7-oct a d ien oyl-4-a m in o-D-
p h en yla la n in e, 16k . Active ester 15 was coupled with
p-amino-D-phenylalanine (9k ), according to modified general
method A using BSA (6 equiv), to give 16k as an orange foam
(72%): ESMS (PI) m/z 722.5 ([M + 1]+, 54), 622.6 ([M - Boc
+ 1]+, 100).
N -[(1,1-Dim e t h yle t h oxy)ca r b on yl]-2,2-d im e t h yl-â-
alan yl-(2S)-2-h ydr oxy-4-(m eth ylpen tan oyl)-(2E,5S,6R,7E)-
5-h yd r oxy-6-m et h yl-8-p h en yl-2,7-oct a d ien oyl-4-iod o-D-
p h en yla la n in e, 16p . Active ester 15 was coupled with p-iodo-
D-phenylalanine (9p ), according to general method A, to give
16p as a pale-yellow solid (86%): ESMS (PI) m/z 833.7 ([M +
1]+, 57), 733.4 ([M - Boc + 1]+, 100).
P r ep a r a tion of Styr en e 7 (Gen er a l Meth od B). A
solution of Boc amine 16 (1 mmol) in trifluoroacetic acid (10-
20 mL) was stirred at room temperature and under a nitrogen
atmosphere for 2-3 h. The solvent was removed in vacuo and
the crude residue azeotroped with toluene (3 × 50 mL) and
dried under high vacuum (5-24 h) to provide the correspond-
ing amine salt as a solid, which was used in the next step
without further purification. To a stirred solution of amine salt
(1 mmol) in dry dimethylformamide (200 mL, 5 mM) was
added N,N-diisopropylethylamine (DIPA) (3.0 mmol) followed
by pentafluorophenyl diphenylphosphinate (FDPP) (1.5 mmol).
The mixture was stirred under a nitrogen atmosphere for 15-
40 h. The solvents were removed in vacuo and the residue
purified by column chromatography (SiO2; gradient elution:
1
20-80% EtOAc/n-hexanes) to provide styrene 7 as a solid. H
NMR data were consistent with the assigned structures (see
Table 4 for representative examples).
Cyclo[2,2-d im eth yl-â-a la n yl-(2S)-2-h yd r oxy-4-(m eth yl-
p en ta n oyl)-(2E,5S,6R, 7E)-5-h yd r oxy-6-m eth yl-8-p h en yl-
2,7-octa d ien oylglycyl], 7a . Boc amine 16a was deprotected
and the resulting amino acid cyclized, according to general
method B, to provide styrene 7a as a white foam (76%): FDMS
m/z 498 (M+, 100).
N-[(1,1-Dim eth yleth oxy)ca r bon yl]-2,2-d im eth yl-â-a la -
n yl-(2S)-2-h yd r oxy-4-(m eth ylp en ta n oyl)-(2E,5S,6R,7E)-5-
Cyclo[2,2-d im eth yl-â-a la n yl-(2S)-2-h yd r oxy-4-(m eth yl-
p en ta n oyl)-(2E,5S,6R, 7E)-5-h yd r oxy-6-m eth yl-8-p h en yl-

2600 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Patel et al.
2,7-octa d ien oyl-D-va lyl], 7b. Boc amine 16b was deprotected
and the resulting amino acid cyclized, according to general
method B, to provide styrene 7b as an off-white solid (61%):
FDMS m/z 541 (M+, 100).
5.75 (d, J ) 15.3 Hz, 2-H), 5.07-5.02 (m), 2.57-2.50 (m, 4-H,
6-H), 2.43-2.31 (m, 4-H′), 1.14 (buried d, 6-Me); unit B 8.06-
7.99 (br s, NH), 7.80 (d, J ) 7.4 Hz, PhH2), 7.62 (d, J ) 7.4
Hz, PhH2), 7.43 (t, J ) 7.4 Hz, PhH2), 7.36-7.11 (m, PhH4),
7.08 (d, J ) 8.4 Hz, PhH2), 5.47 (d, J ) 7.7 Hz, NH), 4.77 (app.
q, J ) 6.0 Hz, Fmoc-CH), 4.51 (d, J ) 6.8 Hz, Fmoc-CH2), 4.31
(app. q, J ) 6.8 Hz, 2-H); unit C 7.36-7.11 (m), 3.39 (dd, J )
13.3 and 8.3 Hz, 3-H), 3.17-3.11 (m, 3-H′), 0.74 (overlapping
d, J ) 5.9 Hz, 2-Me), 0.72 (overlapping d, J ) 6.0 Hz, 2-Me);
unit D 4.84 (dd, J ) 9.9 and 2.5 Hz, 2-H), 1.72-1.52 (m, 3-H,
4-H), 1.37-1.28 (m, 3-H′), 1.23 (s, 5-H3), 1.16 (s, 4-Me); IR
(KBr) υmax 3450 (br), 2960, 1740, 1717, 1680, 1525, 1479, 1152
cm-1; UV (EtOH) λmax 227 (ꢀ ) 29075), 247 (ꢀ ) 41236); 288 (ꢀ
) 5785), 299 (ꢀ ) 5349) nm; ESMS (PI) m/z 860.7 (M+, 80).
Anal. Calcd for C50H54ClN3O8: C, 69.80; H, 6.33; N, 4.88; Cl,
4.12. Found: C, 70.04; H, 6.22; N, 4.78; Cl, 4.43.
Cyclo[2,2-d im eth yl-â-a la n yl-(2S)-2-h yd r oxy-4-(m eth yl-
p en ta n oyl)-(2E,5S,6R, 7E)-5-h yd r oxy-6-m eth yl-8-p h en yl-
2,7-octa d ien oyl-D-th r eon yl], 7c. Boc amine 16c was depro-
tected and the resulting amino acid cyclized, according to
general method B, to provide styrene 7c as a white powder
(51%): FDMS m/z 542 (M+, 100), 498 ([M - C2H4O]+, 65).
Cyclo[2,2-d im eth yl-â-a la n yl-(2S)-2-h yd r oxy-4-(m eth yl-
p en ta n oyl)-(2E,5S,6R, 7E)-5-h yd r oxy-6-m eth yl-8-p h en yl-
2,7-octa d ien oyl-D-p r olyl], 7d . Boc amine 16d was depro-
tected and the resulting amino acid cyclized, according to
general method B, to provide styrene 7d as an off-white solid
(61%): FDMS m/z 541 (M+, 100).
Cyclo[2,2-d im eth yl-â-a la n yl-(2S)-2-h yd r oxy-4-(m eth yl-
p en ta n oyl)-(2E,5S,6R, 7E)-5-h yd r oxy-6-m eth yl-8-p h en yl-
2,7-octa d ien oyl-4-iod o-D-p h en yla la n yl], 7p . Boc amine 16p
was deprotected and the resulting amino acid cyclized, accord-
ing to general method B, to provide styrene 7p as a yellow
solid (22%): FDMS m/z 715 (M+, 100).
Cyclo[3-cycloh exyl-D-alan yl-2,2-dim eth yl-â-alan yl-(2S)-
2-h ydr oxy-4-(m eth ylpen tan oyl)-(2E,5S,6R,7E)-5-h yd r oxy-
6-m eth yl-8-p h en yl-2,7-octa d ien oyl], 7e. Boc amine 16e was
deprotected and the resulting amino acid cyclized, according
to general method B, to provide styrene 7e as a white solid
(56%): FDMS m/z 595 (M+, 100).
Cyclo[2,2-d im eth yl-â-a la n yl-(2S)-2-h yd r oxy-4-(m eth yl-
p en ta n oyl)-(2E,5S,6R, 7E)-5-h yd r oxy-6-m eth yl-8-p h en yl-
2,7-octa d ien oyl-3-ch lor o-D-tyr osyl], 7r . Boc amine 16r was
deprotected and the resulting amino acid cyclized, according
to general method B, to provide styrene 7r as a white solid
(42%): FDMS m/z 638 (M+, 100).
Cyclo[2,2-d im eth yl-â-a la n yl-(2S)-2-h yd r oxy-4-(m eth yl-
p en ta n oyl)-(2E,5S,6R, 7E)-5-h yd r oxy-6-m eth yl-8-p h en yl-
2,7-octa d ien oyl-3-ch lor o-4-a m in o-D-p h en yla la n yl], 7s. Boc
amine 16s was deprotected and the resulting amino acid
cyclized, according to modified general method B using DIPA
(6 equiv) and FDPP (3 equiv), to provide styrene 7s as a yellow
solid (62%): ESMS (PI) m/z 638.3 ([M + 1]+, 100).
(3S,10R,13E,16S)-6,6-Dim eth yl-16-[(1R,2E)-1-m eth yl-3-
ph en yl-2-pr open yl]-3-(2-m eth ylpr opyl)-10-(2-ph en yleth yl)-
1,4-d ioxa -8,11-d ia za cycloh exa d ec-13-en e-2,5,9,12-t et r o-
n e, 7f. Boc amine 16f was deprotected and the resulting amino
acid cyclized, according to general method B, to provide styrene
7f as an off-white solid (62%): FDMS m/z 602.3 (M+, 100).
Cyclo[2,2-d im eth yl-â-a la n yl-(2S)-2-h yd r oxy-4-(m eth yl-
p en ta n oyl)-(2E,5S,6R, 7E)-5-h yd r oxy-6-m eth yl-8-p h en yl-
2,7-octa d ien oyl-O-(p h en ylm eth yl)-D-ser yl], 7g. Boc amine
16g was deprotected and the resulting amino acid cyclized,
according to general method B, to provide styrene 7g as an
off-white solid (57%): FDMS m/z 619 (M+, 100).
Cyclo[2,2-d im eth yl-â-a la n yl-(2S)-2-h yd r oxy-4-(m eth yl-
p en ta n oyl)-(2E,5S,6R, 7E)-5-h yd r oxy-6-m eth yl-8-p h en yl-
2,7-octa d ien oyl-(2R)-2-(4-h yd r oxyp h en yl)glycyl], 7h . Boc
amine 16h was deprotected and the resulting amino acid
cyclized, according to general method B, to provide styrene 7h
as a white solid (59%): FDMS m/z 590 (M+, 100).
Cyclo[2,2-d im eth yl-â-a la n yl-(2S)-2-h yd r oxy-4-(m eth yl-
p en ta n oyl)-(2E,5S,6R, 7E)-5-h yd r oxy-6-m eth yl-8-p h en yl-
2,7-octa d ien oyl-D-p h en yla la n yl], 7i. Boc amine 16i was
deprotected and the resulting amino acid cyclized, according
to general method B, to provide styrene 7i as a white solid
(55%): ESMS (PI) m/z 589.5 ([M + 1]+, 100).
Cyclo[2,2-d im eth yl-â-a la n yl-(2S)-2-h yd r oxy-4-(m eth yl-
p en ta n oyl)-(2E,5S,6R, 7E)-5-h yd r oxy-6-m eth yl-8-p h en yl-
2,7-octa d ien oyl-D-tyr osyl], 7j. Boc amine 16j was depro-
tected and the resulting amino acid cyclized, according to
general method B, to provide styrene 7j as a yellow solid
(80%): FDMS m/z 604 (M+, 100).
Cyclo[2,2-d im eth yl-â-a la n yl-(2S)-2-h yd r oxy-4-(m eth yl-
p en ta n oyl)-(2E,5S,6R, 7E)-5-h yd r oxy-6-m eth yl-8-p h en yl-
2,7-octa d ien oyl-4-a m in o-D-p h en yla la n yl], 7k . Boc amine
16k was deprotected and the resulting amino acid cyclized,
according to modified general method B using DIPA (6 equiv)
and FDPP (3 equiv), to provide styrene 7k as an orange foam
(58%): ESMS (PI) m/z 604.4 ([M + 1]+, 100).
Cyclo[2,2-d im eth yl-â-a la n yl-(2S)-2-h yd r oxy-4-(m eth yl-
p en ta n oyl)-(2E,5S,6R, 7E)-5-h yd r oxy-6-m eth yl-8-p h en yl-
2,7-octa d ien oyl-4-[[(9H-flu or en -9-ylm eth oxy)ca r bon yl]-
a m in o]-D-p h en yla la n yl], 7m . To a stirred solution of aniline
7k (205 mg, 0.340 mmol) in a mixture of dioxane-H2O (5:1,
20 mL) was added NaHCO3 (43 mg, 0.510 mmol) followed by
Fmoc-Cl (106 mg, 0.408 mmol) at room temperature. The
mixture was stirred for 31.5 h, diluted with EtOAc (100 mL),
and washed with H2O (100 mL). The dried (MgSO4) organic
phase was concentrated in vacuo and the resulting crude oil
purified by column chromatography (SiO2; gradient elution:
25-75% EtOAc/n-hexanes) to give aniline 7m as an off-white
solid (248 mg, 88%): [R]D589 +16.4° (c ) 0.37, MeOH); 1H NMR
(CDCl3) δ unit A 7.36-7.11 (m, PhH5), 6.82-6.72 (m, 3-H),
6.41 (d, J ) 15.8 Hz, 8-H), 6.01 (dd, J ) 15.9 and 8.8 Hz, 7-H),
Cyclo[2,2-d im eth yl-â-a la n yl-(2S)-2-h yd r oxy-4-(m eth yl-
p en ta n oyl)-(2E,5S,6R, 7E)-5-h yd r oxy-6-m eth yl-8-p h en yl-
2,7-oct a d ien oyl-3-ch lor o-4-[[(9H -flu or en -9-ylm et h oxy)-
ca r bon yl]a m in o]-D-p h en yla la n yl], 7t. To a stirred solution
of aniline 7s (305 mg, 0.470 mmol) in a mixture of dioxane-
H2O (5:1, 20 mL) was added NaHCO3 (60 mg, 0.0718 mmol)
followed by Fmoc-Cl (120 mg, 0.575 mmol). The mixture was
stirred at room temperature for 48 h, diluted with EtOAc (150
mL), and washed with H2O (100 mL). The dried (MgSO4)
organic phase was concentrated in vacuo and the resulting
crude oil purified by column chromatography (SiO2; gradient
elution: 50-70% EtOAc/n-hexanes) to provide desired aniline
589
7t as a yellow foam (284 mg, 69%): [R]D +29.2° (c ) 0.96,
CHCl3); 1H NMR (CDCl3) δ unit A 7.42-7.31 (m, PhH5), 6.86-
6.77 (m, 3-H), 6.45 (d, J ) 15.8 Hz, 8-H), 6.05 (dd, J ) 16 and
8.9 Hz, 7-H), 5.77 (d, J ) 15.3 Hz, 2-H), 5.12-5.06 (m), 2.62-
2.55 (m, 4-H, 6-H), 2.46-2.31 (m, 4-H′), 1.17 (d, J ) 6.9 Hz,
6-Me); unit B 7.83 (d, J ) 7.5 Hz, PhH2), 7.66 (d, J ) 7.3 Hz,
PhH2), 7.46 (t, J ) 7.4 Hz, PhH2), 7.42-7.31 (m, PhH4), 7.16
(d, J ) 8.2 Hz, PhH2), 5.62 (d, J ) 7.8 Hz, NH), 4.80 (app. q,
J ) 6.7 Hz, Fmoc-CH), 4.57 (d, J ) 6.7 Hz, Fmoc-CH2), 4.32
(app. q, J ) 6.7 Hz, 2-H); unit C 7.29-7.24 (m), 3.48 (dd, J )
13.6 and 9.0 Hz, 3-H), 3.21-3.08 (m, 3-H′), 0.78 (d, J ) 4.7
Hz, 2-Me), 0.76 (d, J ) 5.2 Hz, 2-Me); unit D 4.89 (dd, J ) 9.9
and 3.4 Hz, 2-H), 1.76-1.60 (m, 3-H, 4-H), 1.41-1.30 (m, 3-H′),
1.26 (s, 5-H3), 1.20 (s, 4-Me); IR (KBr) υmax 3430 (br), 3010,
2964, 1736, 1717, 1680, 1525, 1486, 1451, 1317, 1304, 1187,
1152 cm-1; UV (EtOH) λmax 246 (ꢀ ) 47652), 288 (ꢀ ) 5650);
300 (ꢀ ) 5419) nm; ESMS (PI) m/z 826.7 ([M + 1]+, 33). Anal.
Calcd for C50H55N3O8: C, 72.71; H, 6.71; N, 5.09. Found: C,
72.40; H, 6.53; N, 4.98.
Cyclo[2,2-d im eth yl-â-a la n yl-(2S)-2-h yd r oxy-4-(m eth yl-
p en ta n oyl)-(2E,5S,6R, 7E)-5-h yd r oxy-6-m eth yl-8-p h en yl-
2,7-octa d ien oyl-3-h yd r oxy-D-tyr osyl], 7v. Boc amine 16v
was deprotected and the resulting amino acid cyclized, accord-
ing to general method B, to provide styrene 7v as a white solid
(52%): ESMS (PI) m/z 621.6 ([M + 1]+, 100).
Cyclo[3-(1-n a p h th a len yl)-D-a la n yl-2,2-d im eth yl-â-a la -
n yl-(2S)-2-h yd r oxy-4-(m eth ylp en ta n oyl)-(2E,5S,6R,7E)-5-

Novel Cryptophycin Antitumor Agents
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2601
h ydr oxy-6-m eth yl-8-ph en yl-2,7-octadien oyl], 7w. Boc amine
16w was deprotected and the resulting amino acid cyclized,
according to general method B, to provide styrene 7w as a tan
foam (56%): FDMS m/z 638 (M+, 100).
Cyclo[3-(2-n a p h th a len yl)-D-a la n yl-2,2-d im eth yl-â-a la -
n yl-(2S)-2-h yd r oxy-4-(m eth ylp en ta n oyl)-(2E,5S,6R,7E)-5-
h ydr oxy-6-m eth yl-8-ph en yl-2,7-octadien oyl], 7x. Boc amine
16x was deprotected and the resulting amino acid cyclized,
according to general method B, to provide styrene 7x as a tan
foam (65%): FDMS m/z 638.2 (M+, 100).
5.22 (m, 5-H), 3.66 (s, 8-H), 2.99 (d, J ) 7.7 Hz, 7-H), 2.75-
2.64 (m, 4-H, 6-H), 1.84-1.70 (m, 4-H′), 1.11 (d, J ) 7 Hz,
6-Me); unit B 7.46-7.21 (m, PhH5), 5.62 (d, J ) 7.4 Hz, NH),
4.59-4.52 (m, 2-H), 2.79 (dd, J ) 7.9 and 5.8 Hz, 4-H2), 2.44-
2.36 (m, 3-H), 2.08-1.86 (m, 3-H′); unit C 3.47-3.40 (m, 3-H),
3.25 (dd, J ) 14 and 3.7 Hz, 3-H′), 1.30 (s, 2-Me), 1.25 (s, 2-Me);
unit D 5.04 (dd, J ) 9.7 and 2 Hz, 2-H), 1.84-1.70 (m, 3-H2,
4-H), 0.96 (d, J ) 6.6 Hz, 5-H3), 0.91 (d, J ) 6.6 Hz, 4-Me);
FDMS m/z 618.2 (M+, 100). Anal. Calcd for C36H46N2O7: C,
70.68; H, 8.47; N, 4.71. Found: C, 70.53; H, 8.16; N, 4.44.
Cyclo[2,2-d im eth yl-â-a la n yl-(2S)-2-h yd r oxy-4-(m eth yl-
p en ta n oyl)-(2E,5S,6S)-5-h yd r oxy-6-[(2R,3R)-3-p h en ylox-
ir a n yl]-2-h ep ten oyl-O-(p h en ylm eth yl)-D-ser yl], 6g, a n d
Cyclo[2,2-d im et h yl-â-a la n yl-(2S)-2-h yd r oxy-4-(m et h yl-
p en ta n oyl)-(2E,5S,6S)-5-h yd r oxy-6-[(2S,3S)-3-p h en ylox-
ir a n yl]-2-h ep ten oyl-O-(p h en ylm eth yl)-D-ser yl], 17g. Sty-
rene 7g was epoxidized, according to modified general method
C using dichloromethane:toluene (2:1), to provide (i) â-epoxide
6g as a white solid (30%): FDMS m/z 635.1 (M+, 100) and (ii)
R-epoxide 17g as a white solid (10%): tR ) 27.4 minb (refer to
Cyclo[3-(3-p yr id in yl)-D-a la n yl-2,2-d im et h yl-â-a la n yl-
(2S)-2-h yd r oxy-4-(m eth ylp en ta n oyl)-(2E,5S,6R,7E)-5-h y-
d r oxy-6-m eth yl-8-p h en yl-2,7-octa d ien oyl], 7y. Boc amine
16y was deprotected and the resulting amino acid cyclized,
according to modified general method B using DIPA (4 equiv)
and FDPP (2.0 equiv), to provide styrene 7y as a white powder
(71%): ESMS (PI) m/z 590.5 ([M + 1]+, 100).
P r ep a r a tion of â-Ep oxid e 6 (Gen er a l Meth od C). To a
solution of styrene 7 (1 mmol) in dichloromethane (5 mL, 0.2
M) was added purified m-chloroperbenzoic acid (mCPBA) (1.1
mmol), and the mixture was stirred, under a nitrogen atmo-
sphere, until all starting material 7 was consumed as judged
by TLC and HPLC analysis (24-48 h). The reaction was
diluted with dichloromethane (100 mL) and washed with 10%
aqueous sodium meta-bisulfite (Na2S2O5) (100 mL), H2O (100
mL), saturated aqueous NaHCO3 (100 mL), and H2O (100 mL).
The dried (Na2SO4) organic phase was concentrated in vacuo
to give a mixture of R:â-epoxides, which were separated by
RP-HPLC (Kromasil C18; gradient elution: CH3CN-H2O; flow
rate ) 28 mL/min; UV detection at λmax ) 220 nm) to provide
the desired â-epoxide 6 (in a few examples R-epoxide 17 was
also isolated). 1H NMR data for â-epoxide 6 was consistent
with assigned structures (see Table 6 for representative data).
Cyclo[2,2-d im eth yl-â-a la n yl-(2S)-2-h yd r oxy-4-(m eth yl-
p en ta n oyl)-(2E,5S,6S)-5-h yd r oxy-6-[(2R,3R)-3-p h en ylox-
ir a n yl]-2-h ep ten oylglycyl], 6a . Styrene 7a was epoxidized,
according to general method C, to provide 6a as a white solid
(48%): ESMS (PI) m/z 515.5 ([M + 1]+, 100).
Cyclo[2,2-d im eth yl-â-a la n yl-(2S)-2-h yd r oxy-4-(m eth yl-
p en ta n oyl)-(2E,5S,6S)-5-h yd r oxy-6-[(2R,3R)-3-p h en ylox-
ir a n yl]-2-h ep ten oyl-D-va lyl], 6b. Styrene 7b was epoxidized,
according to general method C, to provide 6b as a white solid
(30%): ESMS (PI) m/z 557.1 ([M + 1]+, 100).
Cyclo[2,2-d im eth yl-â-a la n yl-(2S)-2-h yd r oxy-4-(m eth yl-
p en ta n oyl)-(2E,5S,6S)-5-h yd r oxy-6-[(2R,3R)-3-p h en ylox-
ir a n yl]-2-h ep ten oyl-D-th r eon yl], 6c. Styrene 7c was epoxi-
dized, according to general method C, to provide 6c as a white
solid (22%): ESMS (PI) m/z 559.4 ([M + 1]+, 100), 541.3 ([M
- H2O + 1]+, 90).
Cyclo[2,2-d im eth yl-â-a la n yl-(2S)-2-h yd r oxy-4-(m eth yl-
p en ta n oyl)-(2E,5S,6S)-5-h yd r oxy-6-[(2R,3R)-3-p h en ylox-
ir a n yl]-2-h ep ten oyl-D-p r olyl], 6d . Styrene 7d was epoxi-
dized, according to general method C, to provide 6d as a white
solid (30%): ESMS (PI) m/z 555.3 ([M + 1]+, 100).
Cyclo[3-cycloh exyl-D-alan yl-2,2-dim eth yl-â-alan yl-(2S)-
2-h yd r oxy-4-(m eth ylp en ta n oyl)-(2E,5S,6S)-5-h yd r oxy-6-
[(2R,3R)-3-p h en yloxir a n yl]-2-h ep ten oyl], 6e. Styrene 7e
was epoxidized, according to modified general method C using
dichloromethane:toluene (2:1), to provide 6e as a white solid
(18%): FDMS m/z 610.1 (M+, 100).
(3S,10R,13E,16S)-6,6-Dim eth yl-3-(2-m eth ylp r op yl)-10-
(2-p h en ylet h yl)-16-[(1S)-1-[(2R,3R)-3-p h en yloxir a n yl]-
eth yl]-1,4-d ioxa -8,11-d ia za cycloh exa d ec-13-en e-2,5,9,12-
tetr on e, 6f, an d (3S,10R,13E,16S)-6,6-Dim eth yl-3-(2-m eth yl-
pr opyl)-10-(2-ph en yleth yl)-16-[(1S)-1-[(2S,3S)-3-ph en yloxi-
r a n yl]et h yl]-1,4-d ioxa -8,11-d ia za cycloh exa d ec-13-en e-
2,5,9,12-tetr on e, 17f. Styrene 7f was epoxidized, according
to modified general method C using dichloromethane:toluene
(2:1), to provide (i) â-epoxide 6f as a white solid (25%): FDMS
m/z 618.2 (M+, 100) and (ii) R-epoxide 17f as a white solid
(10%): tR ) 27.5 minb (refer to Table 5, footnote 3); [R]D589 +3.8°
(c ) 0.59, CHCl3); 1H NMR (CDCl3) δ unit A 7.46-7.21 (m,
Ph5), 6.88-6.78 (m, 3-H), 5.81 (d, J ) 15.4 Hz, 2-H), 5.28-
589
Table 5, footnote 3); [R]D +35.0°(c ) 0.06, CHCl3); HRMS
(FAB) for C36H47N2O8 m/z requires 635.3332, found 635.3336
(∆ +0.4 mmu).
Cyclo[2,2-d im eth yl-â-a la n yl-(2S)-2-h yd r oxy-4-(m eth yl-
p en ta n oyl)-(2E,5S,6S)-5-h yd r oxy-6-[(2R,3R)-3-p h en ylox-
ir a n yl]-2-h ep ten oyl-(2R)-2-(4-h yd r oxyp h en yl)glycyl], 6h .
Styrene 7h was epoxidized, according to general method C, to
provide 6h as a white solid (22%): ESMS (PI) m/z 607.3 ([M
+ 1]+, 100).
Cyclo[2,2-d im eth yl-â-a la n yl-(2S)-2-h yd r oxy-4-(m eth yl-
p en ta n oyl)-(2E,5S,6S)-5-h yd r oxy-6-[(2R,3R)-3-p h en ylox-
ir a n yl]-2-h ep ten oyl-D-p h en yla la n yl], 6i. Styrene 7i was
epoxidized, according to general method C, to provide 6i as a
white amorphous solid (25%): ESMS (PI) m/z 605.4 ([M + 1]+,
100).
Cyclo[2,2-d im eth yl-â-a la n yl-(2S)-2-h yd r oxy-4-(m eth yl-
p en ta n oyl)-(2E,5S,6S)-5-h yd r oxy-6-[(2R,3R)-3-p h en ylox-
ir a n yl]-2-h ep ten oyl-D-tyr osyl], 6j. Styrene 7j was epoxi-
dized, according to modified general method C using mCPBA
(1.5 equiv) added over 4 days, to provide 6j as a white solid
(34%): ESMS (PI) m/z 621.5 ([M + 1]+, 100).
Cyclo[2,2-d im eth yl-â-a la n yl-(2S)-2-h yd r oxy-4-(m eth yl-
p en ta n oyl)-(2E,5S,6S)-5-h yd r oxy-6-[(2R,3R)-3-p h en ylox-
ir a n yl]-2-h ep ten oyl-4-a m in o-D-p h en yla la n yl], 6k . Styrene
7m was epoxidized, according to general method C, and the
crude reaction product purified by column chromatography
(SiO2; gradient elution: 25-75% EtOAc/n-hexanes) to provide
epoxide 21 as a 1:2 mixture of R (tR ) 44.5 mina):â (tR ) 41.6
mina) (refer to Table 5, footnote 3) epoxides as an off-white
solid (100%): HRMS (FAB) for C50H56N3O9 m/z requires
842.3938, found 842.3954 (∆ +1.60 mmu). Anal. Calcd for
C
50H56N3O9: C, 71.72; H, 6.58; N, 4.99. Found: C, 71.52; H,
7.30; N, 4.62.
To a solution of Fmoc amine 21 (195 mg, 0.232 mmol) in
dry DMF (10 mL) was added piperidine (70 µL, 0.696 mmol),
and the mixture stirred at room temperature for 6 h. The
solvent was removed in vacuo to leave a solid residue which
was purified by column chromatography (SiO2; 20-100%
EtOAc/n-hexanes-5% MeOH/CH2Cl2), and the resulting R:â-
epoxides were separated by RP-HPLC to provide â-epoxide 6k
as a white glass (23%): ESMS (PI) m/z 620.7 [M + 1]+, 100).
Cyclo[2,2-d im eth yl-â-a la n yl-(2S)-2-h yd r oxy-4-(m eth yl-
p en ta n oyl)-(2E,5S,6S)-5-h yd r oxy-6-[(2R,3R)-3-p h en ylox-
ir a n yl]-2-h ep ten oyl-3-ch lor o-D-tyr osyl], 6r , a n d Cyclo-
[2,2-d im e t h yl-â-a la n yl-(2S )-2-h yd r oxy-4-(m e t h ylp e n -
tan oyl)-(2E,5S,6S)-5-h ydr oxy-6-[(2S,3S)-3-ph en yloxir an yl]-
2-h ep ten oyl-3-ch lor o-D-tyr osyl], 17r . Styrene 7r was ep-
oxidized, according to modified general method C using
dichloromethane:toluene (2:1), to provide (i) â-epoxide 6r as a
white solid (35%): FDMS m/z 654.3 (M+, 100) and (ii) R-ep-
oxide 17r as a white solid (19%): tR ) 21.0 minb (refer to Table
5, footnote 3); [R]D589 +10.7° (c ) 1.32, MeOH); FDMS m/z 654
(M+, 100); Anal. Calcd for C35H43N2O8‚0.5H2O: C, 63.29; H,
6.68; N, 4.22. Found: C, 63.30; H, 6.81; N, 4.24.

2602 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Patel et al.
Cyclo[2,2-d im eth yl-â-a la n yl-(2S)-2-h yd r oxy-4-(m eth yl-
p en ta n oyl)-(2E,5S,6S)-5-h yd r oxy-6-[(2R,3R)-3-p h en ylox-
ir an yl]-2-h epten oyl-4-am in o-3-ch lor o-D-ph en ylalan yl], 6s.
Styrene 7t was epoxidized, according to general method C, and
the crude reaction product purified by column chromatography
(SiO2; gradient elution: 20-75% EtOAc:n-hexanes) to provide
epoxide 22 as a 1:2 mixture of R:â-epoxides (determined by
1H NMR) as a white solid (100%): HRMS (FAB) for C50H54N3-
ClNaN3O9 m/z requires 898.3446, found 898.4438 (∆ -0.8
mmu). Anal. Calcd for C50H54ClN3O9‚2H2O: C, 65.81; H, 6.41;
N, 4.60. Found: C, 65.92; H, 5.73; N, 4.27.
To a solution of Fmoc amine 22 (195 mg, 0.223 mmol) in
dry DMF (4.5 mL) was added piperidine (66 µL, 0.669 mmol),
and the mixture stirred at room temperature for 3 h. The
solvent was removed in vacuo to leave a solid residue which
was purified by column chromatography (SiO2; 20-100%
EtOAc/n-hexanes) and the resulting R:â-epoxide mixture
separated by RP-HPLC to provide â-epoxide 6s as a white solid
(32%): ESMS (PI) m/z 654.4 [M + 1]+, 100).
dilutions were made in DMSO using a Biomek Automated
workstation (Beckman, Fullerton, CA). Micropipet tips were
changed with each dilution. We have previously shown that
cryptophycins need to be serially diluted in DMSO to reduce
drug adsorption onto plastic and glass surfaces. Log-phase
human CCRF-CEM leukemia cells were added to wells on 24-
well plates (Costar, Cambridge, MA) at 4.8 × 104 cells/2 mL
of assay medium/well. Assay medium consisted of RPMI-1640
medium supplemented with 10% dialyzed fetal bovine serum
and 25 mM HEPES buffer. The series of compound dilutions
in DMSO were added to duplicate wells at 10 µL/well. Two
wells on each individual plate received 10 µL of DMSO as
controls. The final concentration of DMSO was 0.5%. Plates
were incubated for 72 h at 37 °C in a humidified 5% CO2-in-
air atmosphere. After incubation, the leukemia cells growing
in suspension were quantitated using a ZBI Coulter counter,
and an IC50 was determined.
Ack n ow led gm en t. We thank the Physical Chem-
istry Department at Eli Lilly for their assistance. We
are also grateful to our colleagues in the Chemical
Process R&D Division for providing key intermediates
and co-workers in the Cancer Research Division for
valuable discussions. Finally, we acknowledge the col-
laboration with our colleagues at the University of
Hawaii and Wayne State University.
Cyclo[2,2-d im et h yl-â-a la n yl-(2S)-2-h yd r oxy-4-(m et h -
ylp en t a n oyl)-(2E,5S,6S)-5-h yd r oxy-6-[(2R,3R)-3-p h en -
yloxir a n yl]-2-h ep t en oyl-3-ch lor o-4-(d im et h yla m in o)-D-
p h en yla la n yl], 6u . To a stirred solution of epoxide 6s (37.5
mg, 57.4 umol) in acetonitrile (7 mL) were added crushed,
anhydrous K2CO3 (980 mg, 7.2 mmol) and MeI (2.1 g, 14.4
mmol), and the mixture was heated at 70 °C in a sealed vessel
for 6 h. The mixture was filtered and the filtrate concentrated
in vacuo to give a solid residue, which was further purified by
column chromatography (SiO2; gradient elution: 20-90%
EtOAc/n-hexanes) to give dimethylaniline 6u as a white
Refer en ces
589
1
(1) Schwartz, R. E.; Hirsch, C. F.; Sesin, D. F.; Flor, J . E.; Chartrain,
M.; Fromtling, R. E.; Harris, G. H.; Salvatore, M. J .; Liesch, J .
M.; Yundin, K. Pharmaceuticals from cultured algae. J . Ind.
Microbiol. 1990, 5, 113-124.
(2) Trimuturlu, G.; Ohtani, I.; Patterson, G. M. L.; Moore, R. E.;
Corbett, T. H.; Valeriote, F. A.; Demchik, L. Total structures of
cryptophycins, potent antitumor desipeptides from the blue-
green alga Nostoc sp. strain GSV 224. J . Am. Chem. Soc. 1994,
116, 4729-4737.
(3) Golakoti, T.; Ogino, J .; Heltzel, C. E.; Husebo, T. L.; J ensen, C.
M.; Larsen, L. K.; Patterson, G. M. L.; Moore, R. E.; Mooberry,
S. L.; Corbett, T. H.; Valeriote, F. A. Structure determination,
conformational analysis, chemical stability studies, and antitu-
mor evaluation of the cryptophycins. Isolation of 18 new
analogues from Nostoc sp. strain GSV 224. J . Am. Chem. Soc.
1995, 117, 12030-12049.
(4) The numbering system for subunits A-D, shown in Chart 1,
was adopted from ref 15 and used for the NMR assignments.
(5) Smith, C. D.; Zhang, X.; Mooberry, S. L.; Patterson, M. L.; Moore,
R. E. Cryptophycin: A new antimicrotubule agent active against
drug-resistant cells. Cancer Res. 1994, 54, 3779-3784.
(6) (a) Kersiek, K.; Mejillano, M.; Schwartz, R. E.; Georg, G. I.;
powder (18.2 mg, 47%): [R]D +27.6° (c ) 0.31, CHCl3); H
NMR (CDCl3, 400 MHz) δ unit A 7.35-7.30 (m, PhH3), 7.24-
7.22 (m, PhH2), 6.76 (ddd, J ) 15.2, 10.8 and 4.2 Hz, 3-H),
5.71 (d, J ) 15.9, 2-H), 5.19 (dd, J ) 10.4 and 3.4 Hz, 5-H),
3.66 (d, J ) 1.6 Hz, 8-H), 2.90 (dd, J ) 7.8 and 1.9 Hz, 7-H),
2.57 (br d, J ) 14.5 Hz, 4-H), 2.49-2.40 (m, 4-H′), 1.13 (buried
d, J ) 6.7 Hz); unit B 7.24-7.22 (m, PhH2), 7.19-7.17 (m,
PhH), 5.46 (d, J ) 7 Hz, NH), 4.73 (app. q, J ) 6.5 Hz, 2-H),
3.09-2.98 (m, 3-H2), 2.83 (br s, NMe2); unit C 7.03-7.00 (m,
NH), 3.47-3.38 (m, 3-H), 3.09-2.98 (m, 3-H′), 1.21 (s, 2-Me),
1.14 (s, 2-Me); unit D 4.80 (dd, J ) 10.2 and 3.2 Hz, 2-H), 1.79-
1.65 (m, 3-H, 4-H), 1.33-1.27 (m, 3-H′), 0.83 (d, J ) 6.4 Hz,
5-H3), 0.81 (d, J ) 6.4 Hz, 4-Me); ESMS (PI) m/z 682.4 (M+,
100). Anal. Calcd for C37H48ClN3O7: C, 65.14; H, 7.09; N, 6.16.
Found: C, 65.10; H, 7.20; N, 6.09.
Cyclo[3-(1-n a p h th a len yl)-D-a la n yl-2,2-d im eth yl-â-a la -
n yl-(2S)-2-h yd r oxy-4-(m eth ylp en ta n oyl)-(2E,5S,6S)-5-h y-
dr oxy-6-[(2R,3R)-3-ph en yloxir an yl]-2-h epten oyl], 6w, an d
Cyclo[3-(1-n a p h th a len yl)-D-a la n yl-2,2-d im eth yl-â-a la n yl-
(2S)-2-h ydr oxy-4-(m eth ylpen tan oyl)-(2E,5S,6S)-5-h ydr oxy-
6-[(2S,3S)-3-p h en yloxir a n yl]-2-h ep ten oyl], 17w . Styrene
7w was epoxidized, according to modified general method C
using dichloromethane:toluene (2:1), to provide (i) â-epoxide
6w as a white solid (27%): FDMS m/z 654.3 (M+, 100) and (ii)
R-epoxide 17w as a white solid (12%): tR ) 29.7 minb (refer to
Himes, R. Interaction of cryptophycin
1 with tubulin and
microtubulins. FEBS Lett. 1995, 377, 59-61. (b) Smith, C. D.;
Zhang, X. Mechanism of action of cryptophycin. Interaction with
the Vinca alkaloid domain of tubulin. J . Biol. Chem. 1996, 271,
6192-6198.
(7) Kobayashi, M.; Aoki, S.; Ohyabu, N.; Kurosu, M.; Wang, W.;
Kitagawa, I. Arenastatin A, a potent cytotoxic depsipeptide from
the Okinawan marine sponge Dysidea Arenaria. Tetrahedron
Lett. 1994, 35, 7969-7972.
(8) Moore, R. E.; Corbett, T. H.; Patterson, G. M. L.; Valeriote, F.
A. The search for new antitumor drugs from blue-green algae.
Curr. Pharm. Des. 1996, 2, 317-330.
589
Table 5, footnote 3); [R]D +23.1° (c ) 0.53, CHCl3); FDMS
m/z 654 (M+, 100); HRMS (FAB) for C39H47N2O7 m/z requires
655.3383, found 655.3392 (∆ +0.9 mmu).
Cyclo[3-(2-n a p h th a len yl)-D-a la n yl-2,2-d im eth yl-â-a la -
n yl-(2S)-2-h yd r oxy-4-(m eth ylp en ta n oyl)-(2E,5S,6S)-5-h y-
d r oxy-6-[(2R,3R)-3-p h en yloxir a n yl]-2-h ep ten oyl], 6x, a n d
Cyclo[3-(2-n a p h th a len yl)-D-a la n yl-2,2-d im eth yl-â-a la n yl-
(2S)-2-h ydr oxy-4-(m eth ylpen tan oyl)-(2E,5S,6S)-5-h ydr oxy-
6-[(2S,3S)-3-p h en yloxir a n yl]-2-h ep ten oyl], 17x. Styrene 7x
was epoxidized, according to modified general method C using
dichloromethane:toluene (2:1), to provide (i) â-epoxide 6x as
a white solid (18%): FDMS m/z 654.3 (M+, 100) and (ii)
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R-epoxide 17x as a yellow solid (9%): tR ) 29.2 minb (refer to
589
Table 5, footnote 3); [R]D
+8.6° (c ) 0.65, CHCl3); FDMS
m/z 654.2 (M+, 100).
In Vitr o Cytotoxicity Assa y.38 Dose-response curves
were generated to determine the concentration required for
50% inhibition of growth (IC50). Test compounds were dissolved
initially in DMSO at a concentration of 0.2 mg/mL. Serial 1:3

Novel Cryptophycin Antitumor Agents
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2603
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J M980706S