4392 J. Med. Chem.

2006, 49, 4392-4408

Semisynthetic Maytansine Analogues for the Targeted Treatment of Cancer

Wayne C. Widdison, Sharon D. Wilhelm, Emily E. Cavanagh, Kathleen R. Whiteman, Barbara A. Leece, Yelena Kovtun,
Victor S. Goldmacher, Hongsheng Xie, Rita M. Steeves, Robert J. Lutz, Robert Zhao, Lintao Wang, Walter A. Blättler, and
Ravi V. J. Chari*
ImmunoGen, Inc., 128 Sidney Street, Cambridge, Massachusetts 02139

ReceiVed March 20, 2006

Maytansine, a highly cytotoxic natural product, failed as an anticancer agent in human clinical trials because
of unacceptable systemic toxicity. The potent cell killing ability of maytansine can be used in a targeted
delivery approach for the selective destruction of cancer cells. A series of new maytansinoids, bearing a
disulfide or thiol substituent were synthesized. The chain length of the ester side chain and the degree of
steric hindrance on the carbon atom bearing the thiol substituent were varied. Several of these maytansinoids
were found to be even more potent in vitro than maytansine. The targeted delivery of these maytansinoids,
using monoclonal antibodies, resulted in a high, specific killing of the targeted cells in vitro and remarkable
antitumor activity in vivo.

Introduction tissue and can extend the in vivo half-life of the drug (typically,
minutes to a few hours for the unconjugated drug) to as long
Maytansine (1) was originally isolated from the bark of the
as two weeks, thereby accumulating a proportionally larger
African shrub Maytenus oVatus by Kupchan and colleagues1
amount at the malignant tissue than at the healthy tissues.8 Thus,
and is the first compound of a class of benzoansamacrolide
this method of delivery should effectively contribute to a wider
antibiotics named maytansinoids.2 Maytansinoids are antimitotic
therapeutic window for the maytansinoids and make them
agents that bind to tubulin and inhibit microtubule assembly.3
effective anticancer agents. Preclinical evaluations of the first
They bind to the same site on tubulin as the Vinca alkaloids
set of antibody-maytansinoid conjugates have demonstrated that
do, and similar inhibitory constants were measured for both drug
this targeted delivery approach greatly improves the tumor
classes.4 However, many maytansinoids are 100 to 1000-times
specificity and the antitumor activity of the maytansinoid.9,10
more cytotoxic than vincristine and vinblastine toward cancer
On the basis of this promising preclinical data, the first
cell lines in vitro.5 Maytansine was extensively tested in
preclinical and clinical settings.6 Several phase I and phase II antibody-maytansinoid conjugate has undergone a phase I
clinical trials that were performed with patients suffering from clinical evaluation in patients with colorectal and pancreatic
diverse types of cancers established the toxicity profile in cancers.11,12
humans, but failed to demonstrate therapeutic benefits at Delivery of the maytansinoid by a monoclonal antibody
tolerable doses. Further development stopped, and maytansine requires that the maytansinoid is stably linked to the antibody.
became one of the many cytotoxic agents that were discontinued However, once the antibody has bound to the antigen on the
for the lack of a sufficiently large therapeutic window. cancer cells and the antibody-antigen complex is internalized
Although, maytansine was not effective in human clinical into the cell, the cytotoxic agent needs to be released inside the
trials, the unusually high cytotoxic activity of the maytansinoids cell to enable it to efficiently arrive at the target and inactivate
makes them attractive candidates for tissue-specific delivery. it. If one considers that the cytoplasm of cells contains
Tumor-reactive monoclonal antibodies are large proteins that millimolar concentrations of the thiol-containing tripeptide,
bind with high affinity to receptors that are selectively expressed glutathione, whereas the concentration of glutathione in human
on the surface of cancer cells. Although these antibodies display blood is about 1000-fold lower (generally in the micromolar
high specificity for the tumor, a majority of them are not potent range), then a disulfide bond appears to have ideal chemical
enough to be therapeutically useful. Imaging studies with characteristics.13 It is thermodynamically very stable (bond
radiolabeled antibodies have demonstrated the localization of energy of about 65 kcal/mol) but kinetically labile in the
antibodies at the tumor site in cancer patients; however, the presence of sulfhydryl groups. The strength of the disulfide bond
concentration of antibodies achieved at the tumor was quite low.7 can be manipulated to achieve maximal stability during circula-
The in vitro cytotoxic inhibitory concentrations (IC50 values) tion in the blood stream, while allowing for efficient cleavage
of maytansinoids are even lower than the antibody concentra- inside the target cell. This is achieved by the introduction of
tions achieved at the tumor, making them suitable candidates methyl substituents on the carbon atoms geminal to the disulfide
for targeted delivery using antibodies. The selective delivery link, conferring varying degrees of steric hindrance. We,
to tumor tissue has the effect of changing the in vivo distribution therefore, set out to synthesize maytansinoid analogues that
and pharmacokinetics of the cytotoxic agent. Humanized contained sulfhydryl groups and showed equal or enhanced
monoclonal antibodies typically have a circulation half-life of cytotoxic activity relative to that of the parent compound,
about two weeks in humans and are largely found in the vascular maytansine. Some of these maytansinoids were linked to a
and extravascular plasma space. Thus, linking a small drug monoclonal antibody, and the effect of disulfide bond strength
molecule to the antibody will prevent penetration into healthy correlated with antitumor activity.
Structure-activity studies with maytansinoids had identified
* Corresponding author. Tel: (617) 995-2500. Fax: (617) 995-2510. the C3 N-acyl-N-methyl-L-alanyl ester side chain, the C4-C5
E-mail: ravi.chari@immunogen.com. epoxide moiety, the C9 carbinol function, and the position of
10.1021/jm060319f CCC: $33.50 © 2006 American Chemical Society
Published on Web 06/10/2006
Semisynthetic Maytansine Analogues Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4393

tocins that are maytansinoids with C3 alkanoic acid esters.16,17

Because ansamitocins lack the C3 N-acyl-N-methyl-L-alanyl side
chain, they have diminished cytotoxicity; however, they con-
stitute a ready starting material for the preparation of maytansi-
noid analogues with novel C3 side chains.

Results and Discussion

Chemistry. The chemistry followed the scheme originally
Figure 1. Structures of maytansine and disulfide-containing maytansi- designed by Kupchan at al.,5,15 who first reported the chemical
noid. conversion of maytansine to the C3 hydroxy compound,
maytansinol, and its subsequent conversion to different C3
the conjugated C11 and C13 double bonds as critical elements esters. Esterification of maytansinol with alkanoic acids gave
for activity.14 This left the phenyl ring and the N′-acyl group as the bacterial ansamitocins. Kawai et al.,18 have described the
chemically modifiable entities. Although the N′-acyl group is esterification of maytansinol with a variety of N-acyl- N-methyl-
required for biological activity, studies by Kupchan et al.15 have L-alanyl compounds to produce highly active C3 ester analogues
shown that the nature of the acyl group can be varied without of maytansine.
a significant loss in activity. Thus, we initially replaced the The synthetic route for the preparation of disulfide-containing
N-acetyl group in maytansine (1) with a 3-methyldithiopropionyl maytansinoid esters is shown in Schemes 1 and 2. Maytansinol
group, thus producing disulfide-containing maytansinoid 2b (4) was synthesized from ansamitocins (3), which in turn were
(DM1SMe), (Figure 1). On the basis of the promising in vitro obtained from the fermentation of the microorganism Actino-
potency data of this compound, we then synthesized a variety synnema pretiosum. Ansamitocins were isolated as a mixture
of disulfide and thiol-containing maytansinoids bearing different of C3 esters of maytansinol, including propanoate, butyrate,
N-acyl-N-methyl-L-alanyl ester moieties at the C3 position. A isobutyrate, and pentanoate esters. The predominant species
representative set of these maytansinoids were then conjugated (∼80%) was the C3 isobutyrate ansamitocin P-3. Because the
to monoclonal antibodies to provide conjugates with varying C3 ester group in maytansinoids is susceptible to elimination
disulfide bond strengths. The synthesis of these new maytansi- under mild basic conditions (pH >9) to give the R,β-unsaturated
noids and the results from their biological testing in free form maytansinoid maysine,19 ester hydrolysis was achieved through
or as conjugates of monoclonal antibodies is reported herein. a reductive cleavage process. The previously described method15
Biosynthetically, maytansinoids are complex polyketides. for the reductive cleavage of the C3 ester used lithium aluminum
Consequently, soon after the initial isolation from plants, a hydride and gave low yields of maytansinol with several side
bacterial actinomycetes strain Actinosynnema pretiosum was products. Several other reducing agents, including DIBAL,
isolated, which produces ansamycin antibiotics called ansami- NaBH4, Red-Al, or Red-Al + 1 eq methanol, gave poor yields

Scheme 1. Synthesis of Maytansinola

a (a) LiAlH(OMe)3, THF, -30 to -40 °C; (b) pH 12, 30 min (c) pH 6-7, (d) pH 2, 2 h or pH 12, 30 min.
4394 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 Widdison et al.

Scheme 2. Synthesis of Disulfide-Containing Maytansinoidsa

a Reaction conditions: (a) DCC/ZnCl2/CH2Cl2, rt.

and multiple side products. Attempted enzymatic cleavage of reacting with methyl methanethiolsulfonate or aryl disulfides,
the ester using a wide panel of commercially available esterases respectively (Scheme 3). The dithio-carboxylic acids were
and lipases was also unsuccessful, with no detectable hydrolysis. converted into their N-hydroxysuccinimide esters, followed by
However, the ester group in the ansamitocins was efficiently reaction with N-methyl-L-alanine to form the desired C3 N-acyl-
cleaved using the mild reducing agent lithium trimethoxyalu- N-methyl-L-alanine side chains for reaction with maytansinol
minum hydride, under controlled temperature (-30 to -40 °C), (4). The two enantiomeric 4-(methydithio)pentanoic acids
to give maytansinol in good yields. Higher reaction temperatures (compounds 13 and 18) were prepared from the corresponding
led to epoxide opening, whereas colder reaction temperatures optically pure 1,3-dihdroxybutanes as shown in Schemes 4 and
resulted in a high proportion of unreacted starting material. 5. The conversion of the 1,3-dihdroxybutanes to the ditosylates,
Maytansinol was obtained after quenching the reaction mixture followed by the displacement of the primary tosyl group by
at -40 °C with water, which resulted in a pH of about 12, and cyanide, and the substitution of the secondary tosyl group with
holding at this pH for 30 min, followed by subsequent a xanthate moiety gave compounds 12 and 17. Base hydrolysis,
neutralization with formic acid. Interestingly, when the reaction followed by disulfide exchange with methyl methanethiolsul-
mixture was neutralized with acid to a pH between 6 and 7, the fonate gave compounds 13 and 18, which were then converted
very stable acetal intermediate 4c (presumably formed via 4a, to their respective N-hydroxysuccinimide esters 14 and 19.
4b) was obtained. This intermediate could be isolated and stored. Acylation of N-methyl-L-alanine with the N-hydroxysuccinimde
It was converted to maytansinol (4) either by acidification and ester of S- and R-4-(methyldithio)pentanoic acids gave the two
holding at pH 2 for 2 h or by adding a base and holding at diastereomeric acyl N-methyl-L-alanyl side chains 5g and 5h
basic pH (∼11) for 30 min (Scheme 1). for condensation with maytansinol. To synthesize a maytansi-
Mercaptocarboxylic acids of varying linear chain lengths were noid ester bearing a sterically hindered thiol substituent, the
converted into methyldithio or aryldithio carboxylic acids by N-acyl-N-methyl-L-alanine compound 5i, bearing two methyl
Semisynthetic Maytansine Analogues Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4395

Scheme 3. Synthesis of N-Acyl-N-methyl-L-alaninesa at the R-carbon of the N-methyl-L-alanine moiety takes place
during the reaction. In addition, unreacted maytansinol (typically
10-20%) was also recovered. The addition of another aliquot
of the carboxylic acid, DCC, and ZnCl2 did not result in any
further reaction. Several other coupling agents, solvents, and
reaction conditions were tried but generally gave poorer yields.
Of the Lewis acids investigated, zinc chloride gave the best
yields. The substitution of zinc chloride with acyl transfer
catalysts such as (dimethylamino)pyridine or hydroxybenzo-
triazole gave little or no product. The diastereomeric maytansi-
noid esters were readily separated by normal phase HPLC, using
a cyano-bonded silica column. On this column, the two
diastereomers had retention times that were about 10 min apart,
enabling a facile separation. In some cases, the diastereomers
could also be separated by preparative TLC on silica gel. In
a Reaction conditions: (a) CH SSO CH , (PhSSPh for 8d); (b) N-
one case, the maytansinoid ester could not be isolated in a
3 2 3
hydroxysuccinimide, EDC; (c) N-methyl-L-alanine, triethylamine.
reasonable yield. In this case, the maytansinoid DM2SMe (2e)
was found to be unstable under the reaction conditions and
Scheme 4. Synthesis of underwent rapid decomposition, presumably because of facile
N-Methyl-N-[(4-(S)-methyldithio)-1-oxopentyl]-S-alaninea β-elimination of the methydithio substituent to give the substi-
tuted alkene, which is the thermodynamically favored product.
Because maytansinoids are sensitive to bases, nucleophiles,
and strong reducing agents, the reduction of disulfide-containing
maytansinoids to the thiol compounds had to be achieved using
very mild and disulfide-specific reagents. The reduction of the
disulfide-containing maytansinoids (2b,c,e,f,i) proceeded smoothly
upon treatment, in neutral buffered aqueous solution, with a
small excess (2-3 equiv) of dithiothreitol (DTT), a reducing
a Reaction conditions: (a) TsCl. Py; (b) NaCN; (c) KSSCOEt; (d) NaOH;
agent commonly used by biochemists for the scission of
(e) CH3SSO2CH3; (f) N-hydroxysuccinimide, EDC; (g) N-methyl-L-alanine,
triethylamine.
disulfide bonds in proteins (Scheme 7). Initial attempts to obtain
pure thiol-containing drugs by reverse phase HPLC using a
substituents on the carbon bearing the disulfide bond, was water-acetonitrile mobile phase resulted in a considerable loss
synthesized from isobutylene sulfide using the steps shown in of product because of the rapid oxidation of the thiols to dimeric
Scheme 6. Ring opening of episulfide 20 with lithiated aceto- maytansinoids that had low solubility in aqueous solutions. The
nitrile gave nitrile 21. The hydrolysis of the nitrile, followed thiol-containing maytansinoids could be efficiently purified
by disulfide exchange with methylmethanethiolsulfonate gave using nonaqueous solvents on a normal phase cyano-bonded
carboxylic acid 23. The condensation of 23 with N-hydroxy- HPLC column to provide mercapto-L-aminoacyl-maytansinoids
succinimide in the presence of EDC gave hydroxysuccinimide 25a (DM1), 25b (DM3), and 25c (DM4) (Scheme 7). Similarly,
ester 24, followed by the reaction with N-methyl-L-alanine to the reduction of the disulfide-containing-D-aminoacyl maytansi-
give 5i. noids (6b,c,f,i) gave the thiol-containing maytansinoids 26a-
Disulfide-containing maytansinoid esters (2a-i), in which c. The water soluble reducing agent tris-(2-carboxyethyl)-
both the chain length and degree of branching in the acyl group phosphine (TCEP) could also be used instead of DTT with
were varied, were prepared by condensing maytansinol (4) with similar results. Once purified, using nonaqueous solvents, the
the N-acyl-N-methyl-L-alanyl derivatives described above in the thiol-containing drugs were stable upon extended storage in dry
presence of the coupling reagent 1,3-dicyclohexylcarbodiimide form. For a subsequent reaction with antibodies, the thiol-
(DCC) along with catalytic amounts of zinc chloride (Scheme containing maytansinoids had to be reconstituted in a water-
2). In all cases, two diastereomeric products (2a-i and 6a-i) miscible solvent that would keep the drug in solution in the
containing the L- and D-aminoacyl side chain, respectively, were predominantly aqueous milieu (<5% organic solvent) and also
obtained, despite the fact that enantiomerically pure acylamino compatible with proteins. Dimethylacetamide (DMAa) was
acids were used in the reaction. This indicates that epimerization found to be the ideal solvent, once it was re-purified and freed

Scheme 5. Synthesis of N-Methyl-N-[(4-(R)-methyldithio)-1-oxopentyl]-S-alaninea

a Reaction conditions: (a) TsCl. Py; (b) NaCN; (c) KSSCOEt; (d) NaOH; (e) CH SSO CH ; (f) N-hydroxysuccinimide, EDC; (g) N-methyl-L-alanine,
3 2 3
triethylamine.
4396 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 Widdison et al.

Scheme 6. Synthesis of Scheme 8. Synthesis of Maytansinoid Dimersa

N-Methyl-N-[4-methyl-(4-methyldithio)-1-oxopentyl)-S-alaninea

a Reaction conditions: (a) CH CN, nBu-Li; (b) NaOH; (c) CH SSO CH ;

3 3 2 3
(d) N-hydroxysuccinimide, EDC; (e) N-methyl-L-alanine.

Scheme 7. Synthesis of Thiol-Containing Maytansinoidsa

Scheme 9. Preparation of Antibody-Maytansinoid Conjugatesa

a Reaction conditions: (a) dithiothreitol/RT.

of metal ions. This was achieved by the careful glass-distillation

of high-grade commercial DMA.
Maytansinoids 2j (DM1-TPA) and 2k (DM4-TBA) were succinimidyl 4-(2-pyridyldithio)butanoate (SPDB, 28) to intro-
sometimes obtained as byproducts of the conjugation reaction duce an active disulfide-containing group. The treatment with
of thiol-containing maytansinoids with antibodies. To determine these bifunctional cross-linking agents resulted in the formation
the potency of these compounds, they were independently of stable amide bonds with the -amino group of lysine residues
synthesized from the corresponding thiol-containing maytansi- to generate a modified antibody bearing, on an average, four to
noids 25a and 25c, respectively, by a disulfide exchange with five linked pyridyldithio groups per antibody molecule. Modi-
the appropriate pyridyldithioalkanoic acids. The disulfide dimers fication with SPP results in the introduction of a sterically
25d and 25e of DM1 and DM4, respectively, were also hindered center on the carbon atom bearing the disulfide moiety,
sometimes obtained as byproducts of the conjugation reaction. whereas the treatment with SPDB results in the introduction of
These two dimers were synthesized either by disulfide exchange a nonhindered disulfide. In the second step, the treatment with
or by metal-ion-catalyzed oxidation of the parent thiol-contain- a thiol-containing maytansinoid led to disulfide exchange with
ing maytansinoids 25a and 25c, respectively (Scheme 8). the pyridyldithio group to give antibody conjugates containing,
Antibody Conjugates. The antibody used in this study was on the average, three to four maytansinoid drugs linked via the
huC242, a humanized version of the murine monoclonal newly formed disulfide bonds. The strength of the disulfide bond
antibody C24220 that binds to the CanAg antigen that is highly was varied by using maytansinoids 25b and 25c that bear
expressed on the surface of several human solid tumors, sterically hindered thiols or by utilizing the SPP cross-linker,
including colon, pancreatic, gastric, and some lung tumors. This which would introduce a methyl substituent on the antibody
antibody displayed limited cross-reactivity with human normal side of the disulfide link. For example, the huC242-DM1
tissues. The conjugation of the maytansinoid to the monoclonal conjugate cantuzumab mertansine contains one methyl substitu-
antibodies was accomplished in two steps (Scheme 9). First, ent on the antibody side of the disulfide link. The huC242-
the monoclonal antibody was modified either with N-succin- DM3 conjugate bears one methyl substituent on the drug side
imidyl 4-(2-pyridyldithio)pentanoate (SPP, 27) or with N- of the disulfide link, whereas the huC242-DM4 conjugate bears
a Abbreviations: DMA, dimethylacetamide; SPP, N-succinimidyl 4-(2-
two methyl substituents on the drug side of the disulfide link.
pyridyldithio)pentanoate; SPDB, N-succinimidyl 4-(2-pyridyldithio)bu- A structural representation of these conjugates is shown in
tanoate. Figure 2. The conjugates were monomeric, soluble in neutral
Semisynthetic Maytansine Analogues Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4397

Table 1. Comparison of the in Vitro Potency of Maytansine (1) and

Disulfide-Containing Maytansinoid, DM1SMe (2b) after 72 h of
Exposure to the Drugs
IC50 (nM)
cell line maytansine (1) maytansinoid 2b
KB 0.034 ( 0.005 0.029 ( 0.003
Sk-Br-3 0.030 ( 0.005 0.014 ( 0.006
A-431 0.040 <0.030
A-549 0.160 ( 0.040 0.120
SW-620a >0.100 0.040 ( 0.004
Ovcar-3a 0.100 ( 0.020 0.050 ( 0.010
HCT-15 0.750 0.130
MCF-7 0.044 ( 0.002 <0.03
A-375 0.037 0.030 ( 0.015
a 24 h exposure.

Table 2. Cytotoxicity of Various Maytansinoids in Vitro toward Tumor

Cell Lines (72 h of Exposure)
IC50 (nM)
compd KB cells SK-Br-3 cells
4 >3 1.2
1 0.034 0.030
2a 0.190 nd
2b 0.029 0.014
2c 0.100 nd
2d 0.0085 0.038
2f 0.011 0.0042
2g 0.040 0.032
2h 0.042 0.027
2i 0.0011 0.0032
2j 1.80 3.40
2k 23.0 7.10
6b 3.0 6.0
6d 2.0 nd
6f 1.20 0.22
6g >3.0 >3.0
6h 2.40 2.20
Figure 2. Structural representation of maytansinoid conjugates with 6i >0.30 >0.30
the huC242 antibody. The differences in the linker region are shadowed. 25a 1.10 1.10
25b 0.077 0.031
buffered aqueous solution, and stable for up to two years upon 25c 0.290 0.120
25d 0.140 0.100
storage at 4 °C. 25e 0.0016 0.0012
Biological Studies. Free Drugs. The disulfide-containing 26a >3.0 >3.0
maytansinoid drugs and thiol-containing maytansinoid drugs 26c >1.0 3.3
were evaluated for their ability to suppress the proliferation of
human tumor cell lines in vitro. KB (human epidermoid in the C3 side chain of maytansine with a methyldithiopropanoyl
carcinoma) and SK-BR-3 (human breast tumor) cells were group not only preserved the cytotoxic activity of maytansine
exposed to the compounds for 24 or 72 h, and the surviving but, in some cases, even enhanced its potency toward cancer
fractions of cells were measured using a clonogenic plating cell lines.
efficiency assay. The IC50 values were then calculated from these We then examined the effect of the chain length of the acyl
data. The L-aminoacyl maytansinoid 2b (DM1SMe), bearing a group on potency (Table 2). Maytansinoid 2d, bearing the
methyldithio-propanoyl substituent in the C3 ester side chain, methyldithiobutanoyl side chain (one methylene longer than 2b),
was found to be highly potent with IC50 values of 0.029 and displayed similar potency to that of 2b. In contrast, the
0.014 nM toward KB and SK-BR-3 cells, respectively (Table methyldithioacetyl maytansinoid 2a (one methylene unit shorter
1). This methyldithio-maytansinoid displayed cytotoxicity simi- than 2b) was about 10-fold less cytotoxic than 2b, suggesting
lar to that of the parent drug maytansine toward KB cells and that a minimum of two methylene groups are required in the
increased potency compared to that of maytansine toward SK- N-acyl side chain to achieve high potency. The phenyldithio
BR-3 cells. The potency of 2b was then compared to that of analogue 2c was also about 10-fold less potent than the
maytansine on a wider panel of human tumor cell lines (see corresponding methydithio-maytansinoid 2b. Maytansinoids
Table 1). Compound 2b was potent toward SW-620 (colon bearing a branched L-aminoacyl side chain, incorporating one
adenocarcinoma) with an IC50 value of 0.040 nM, about 3-fold methyl substituent (DM3SMe, 2f,g,h) or two methyl substituents
more potent than maytansine. Both 2b and maytansine were (DM4SMe, 2i) on the carbon atom bearing the methydithio
less potent toward HCT-15 (colon carcinoma) cells with IC50 substituent, were highly potent, with IC50 values ranging from
values of 0.13 and 0.75 nM, respectively. The lower potency 0.0042 to 0.011 nM for 2f and 0.011 to 0.0032 nM for 2i.
of these drugs toward HCT-15 cells line can be explained by Maytansinoid 2f bears a new chiral center in the acyl side chain.
the reported expression of the multidrug resistance gene (mdr1/ Maytansinoids 2g and 2h bearing the R and S configurations in
P-glycoprotein) in this cell line.21 Compound 2b was about twice the acyl side chains, respectively, were found to display
as cytotoxic (IC50 ) 0.05 nM) as maytansine against the ovarian potencies similar to that of 2f, which bears the racemic acyl
cancer line OVCAR3. Thus, the replacement of the acetyl group side chain. Although the stereochemistry in the acyl side chain
4398 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 Widdison et al.

was not important for activity, the stereochemistry of the

N-methylalanyl moiety had a dramatic influence on potency.
Thus, the maytansinoids 6b,d,f,g,h,i bearing the unnatural
N-methyl-D-alanyl moiety were about 100-fold less cytotoxic
than their corresponding L-epimers toward both cell lines.
In all cases, the mercapto-containing maytansinoids were less
potent than their corresponding methydisulfide analogues. Thus,
maytansinoid 25a (DM1), bearing the mercaptopropanoyl side
chain, had an IC50 value of 1.1 nM toward KB and SK-BR-3
cells, rendering it about 30- and 100-fold less potent than 2b
toward these cell lines. This decrease in potency may be
attributed to the instability of thiol-containing maytansinoids
such as 25a under cell culture conditions wherein the oxidation
of 25a to its disulfide dimer 25d was observed. However, dimer
25d was independently synthesized and found to be only about
3- to 10-fold less potent than 2b, suggesting that 25a might be
inactivated additionally by some other mechanism. For example,
the thiol group could react with a cell culture component such
as cystine to provide a charged maytansinoid carboxylate, which
is incapable of internalization into the cell, resulting in
diminished potency. Maytansinoids 25b,c bearing sterically
hindered thiols were 3- to 12-fold more potent than 25a, which
is consistent with the predicted lower reactivity and conse-
quently, the greater stability of these compounds in the cell
culture milieu. The dimer (25e) of DM4 was the most potent
maytansinoid tested, with IC50 values of 0.0012 and 0.0016 nM
toward the two cell lines tested. Maytansinoids 2j and 2k bear
a terminal carboxyl group that renders them at least 100-fold Figure 3. Top panel: in vitro potency of unconjugated drug DM4SMe
(2i) toward COLO 205 and A-375 cells. Bottom panel: in vitro potency
less potent than the parent compounds 2b and 2i, suggesting of huC242-DM4 conjugate toward target COLO 205 cells and no-
that the charged carboxylate residue might interfere with the target A-375 cells.
internalization into cells, resulting in diminished potency.
Antibody-Drug Conjugates. Cytotoxicity in Vitro. The
conjugation of the huC242 antibody, via disulfide bonds, with
thiol-containing maytansinoids DM1 (25a), DM3 (25b), and
DM4 (25c) gave the conjugates labeled huC242-DM1, huC242-
DM3, and huC242-DM4, respectively (Figure 2). In these
conjugates, the antibody modifying agent and the maytansinoids
were chosen with the goal of producing conjugates of varying
disulfide bond stability. All three conjugates displayed high
potency in vitro toward the antigen-expressing COLO 205 cell
line, with IC50 values (in protein concentration) of 1.1 × 10-11,
1.3 × 10-11, and 3.8 × 10-11 M for huC242-DM4, huC242-
DM3, and huC242-DM1, respectively. Thus, the disulfide bond
strength does not appear to play an important role in determining
the magnitude of in vitro potency toward target cells. Impor-
tantly, all three conjugates were about 200-fold less cytotoxic Figure 4. Comparison of the antitumor activity of huC242-maytansi-
toward antigen-negative A-375 cells, demonstrating the specific- noid conjugates in a human colon tumor xenograft model established
subcutaneously with COLO 205 cells.
ity of the cytotoxic effect. Figure 3 shows an example of how
an antibody can confer target specificity to an otherwise relative efficacy of the three conjugates, we selected a treatment
nonspecifically cytotoxic drug. Both the COLO 205 and A-375 regimen that used a low dose of the conjugate (∼20% of the
cell lines were killed equally well by the nonconjugated maximum tolerated dose) that would result in a delay in tumor
maytansinoid 2i (DM4SMe). In contrast, the conjugated may- growth but would not be curative. The results are shown in
tansinoid huC242-DM4 kills the target COLO 205 cells at a Figure 4. The tumors in the control group of mice grew to a
1000-fold lower concentration than the nontarget A-375 cell, size of nearly 900 mm3 in 24 days. The treatment with huC242-
establishing that the linkage to antibodies confers tumor DM1 (linked maytansinoid dose of 75 µg/kg, qd × 5) resulted
specificity to the otherwise indiscriminately toxic maytansinoid in a tumor growth delay of 20 days, whereas the treatment with
drugs. huC242-DM3, at an equivalent dose, resulted in a greater
Antitumor Activity in Vivo. The antitumor activity of therapeutic effect and caused complete tumor regressions lasting
the three huC242-maytansinoid conjugates, huC242-DM1, 45 days. Treatment at an equivalent dose of the huC242-DM4
huC242-DM3, and huC242-DM4, were compared in human conjugate was even more efficacious, resulting in cures of all
colon tumor xenograft models established with COLO 205 cells. of the treated animals. Interestingly, the antitumor activity of
We have previously shown10 that C242-DM1 is curative in huC242-DM3 is superior to that of huC242-DM1, although
this model and that the free DM1 and the unconjugated C242 both conjugates would have been predicted to display similar
antibody show little or no antitumor effect. To compare the stability in vivo. The huC242-DM3 conjugate bears one methyl
Semisynthetic Maytansine Analogues Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4399

group on the carbon atom bearing the disulfide bond on the shifts are reported in δ values relative to that of an internal
drug side of the conjugate, whereas huC242-DM1 bears one tetramethylsilane (Me4Si) standard. UV spectra were recorded on
methyl substituent at the carbon atom on the antibody side of a Beckman DU-640 spectrophotometer. The optical rotations were
the disulfide link. However, cleavage of the disulfide bond in determined using a Rudolph AUTOPOL IV polarimeter. Mass
huC242-DM3 releases the thiol-containing maytansinoid DM3, spectra were obtained on a Bruker Daltonics spectrometer or a
Waters QTOF-API-US spectrometer. Elemental analyses were
which is about 10-fold more potent than DM1, the active performed by Atlantic Microlabs, Atlanta, GA. High performance
component released from huC242-DM1, which could explain liquid chromatography (HPLC) was performed on a Beckman or
the greater efficacy observed with the DM3 conjugate. Varian instrument (analytical or preparative scale) equipped with
The greater antitumor activity of huC242-DM4 compared a diode array detector.
to that of huC242-DM1 can be attributed to at least two Synthesis of Maytansinol (4). To a 200 mL flask was added a
factors: (a) the greater stability of the disulfide bond between 1 M solution of lithium aluminum hydride (45 mmol, 45 mL) in
the antibody and the drug in huC242-DM4, which results in a THF. The solution was placed under an argon atmosphere and
longer circulation time of the intact conjugate and thus cooled to -40 °C. A mixture of anhydrous methanol (5.4 mL, 135
potentially greater accumulation at the tumor site, and (b) the mmol) in THF (25 mL) was added dropwise. The temperature was
greater stability of the released DM4 drug, which bears a thiol maintained between -30 and -40 °C. After the addition was
complete, the reaction mixture was stirred at this temperature for
substituent at a tertiary carbon center. Studies to determine the
an additional period of 10 min to give lithium trimethoxyaluminum
parameters contributing to the different in vivo activities of the hydride (LiAlH(OMe)3). In a separate 250 mL flask, ansamitocin
conjugates are ongoing. (3.2 g, 5.0 mmol) was dissolved in anhydrous THF (30 mL), and
the solution was placed under an argon atmosphere and cooled in
Conclusions a dry ice-acetone bath to -40 °C. The solution of LiAlH(OMe)3
To harness the potent cytotoxic activity of maytansinoids for was transferred via a cannula over 10 min into the chilled solution
the treatment of cancer, we prepared analogues of maytansine of ansamitocins. The reaction temperature was maintained between
-30 and -35 °C under argon and stirred for 2 h. Deionized water
that can be covalently linked to monoclonal antitumor antibodies
(22 mL) was added dropwise while the reaction mixture was
for targeted treatment. Such analogues not only have to maintain maintained at -30 and -35 °C. After 30 min, 88% formic acid
the activity of maytansine but also have to be able to readily (1.8 mL) and ethyl acetate (50 mL) were added. The mixture was
form a stable chemical bond with a protein in aqueous solution. allowed to warm up to room temperature. The precipitate was
For manufacturing reasons, bond formation must be efficient removed by vacuum filtration, and the filtrate was concentrated
and occur in high yield. Sulfhydryl groups or their respective under vacuum. The residue was dissolved in a minimum volume
thiolate anions react readily in aqueous solutions with maleimido of dichloromethane and loaded onto a silica gel column packed in
moieties in a Michael-type addition reaction to form thioethers dichloromethane. The column was eluted with dichloromethane/
and with disulfide groups in a disulfide exchange reaction to methanol (95:5, v/v). The fractions containing the desired product
form new disulfide bonds. We, therefore, prepared and tested were pooled and evaporated under reduced pressure to give 2.3 g
of maytansinol (81% yield) as a white solid: mp 205-207 °C; 1H
maytansinoid compounds with reactive sulfhydryl groups, which
NMR (CDCl3) δ 0.82 (3H, s), 1.28 (3H, d, J ) 6 Hz), 1.52-1.54
during synthesis and for some biological testing were protected (1H, m), 1.67 (3H, s), 1.80 (br s s), 2.07-2.29 (4H, m), 2.56 (1H,
in the form of simple disulfide compounds. d, J ) 11 Hz), 2.95 (br s), 3.10 (1H, d, J ) 12 Hz), 3.19 (3H, s),
Thus, we have synthesized a series of new maytansinoids 3.41 (3H, s), 3.44-3.51 (3H, m), 3.97 (3H, s), 4.34 (1H, t, J ) 11
bearing a disulfide or thiol substituent. The series was chosen Hz), 5.50 (1H, dd, J ) 9 and 15 Hz), 6.13 (1H, d, J ) 11 Hz),
so as to evaluate the influence of the chain length of the ester 6.37 (1H, s), 6.42 (1H, dd, J ) 11 and 15 Hz), 6.79 (1H, d, J )
side chain and the degree of steric hindrance on the carbon atom 1.5 Hz), 7.02 (1H, d, J ) 1.5 Hz); HRMS calcd for C28H33ClN2O8
bearing the thiol substituent on in vitro cytotoxicity. Several of (M + H)+ m/z ) 565.2317; found, 565.2329. HPLC analysis was
these maytansinoids were found to be even more potent than performed on a Kromasil analytical C-8 column (length: 100 mm,
the parent drug maytansine in killing human tumor cells in vitro. i.d.: 4.6 mm), at a flow rate of 1 mL/min, eluting with a gradient
of water and acetonitrile (30% to 45% CH3CN over 30 min). Under
Antibody conjugates bearing varying degrees of steric hindrance these conditions, maytansinol eluted as a sharp peak with a retention
around the disulfide link between the maytansinoid and antibody time of 11.15 min.
were shown to be highly active in selectively killing target cells Acetal 4c. A solution of ansamitocins (3.0 g, 4.72 mmol) in THF
to which the antibody was capable of binding. Nontarget cells (15 mL) was cooled to -35 °C, and a solution of 0.67 M LiAl-
were not affected even at a 100-fold higher concentration, (OMe)3H (56 mL, 37.7 mmol) in THF was added dropwise by
demonstrating that the high potency of the maytansinoids could syringe using a syringe pump. The temperature of the reaction was
be directed toward the specific killing of target cells. Remarkable maintained between -30 and -40 °C throughout the addition. After
antitumor efficacy was observed in a xenograft model. These addition was complete, the reaction was stirred for 2 h at between
studies, in part, have led to the development of huC242-DM4 -34 and -37 °C. A solution of 88% formic acid (1.85 mL, 2.16
for evaluation as an antitumor agent in human clinical trials in g, 41.5 mmol) in deionized water (23 mL) was added dropwise to
patients expressing the antigen for C242. the flask at a rate that did not produce excessive frothing, followed
by the addition of ethyl acetate (60 mL). The cooling bath was
removed, and the mixture was allowed to warm up to room
Experimental Section
temperature. The pH of the mixture was checked with pH paper
All reagents were obtained from the Aldrich Chemical Co., unless and found to be approximately 6. Precipitated aluminum-based
otherwise stated. Maytansine (NCI 158358/26/0) was obtained from byproducts were removed by vacuum filtration, and the solvent
the National Cancer Institute. Ansamitocin P-3 was produced by was removed from the filtrate by rotary evaporation under vacuum.
fermentation of an Actinosynnema pretiosum strain derived from Butyl acetate (10 mL) was added to the residue, and the solvent
several rounds of mutagenesis of the parent ATCC 31565 strain. was then evaporated to remove the residual water. The residue was
Human tumor cell lines used in biological assays were obtained purified by silica chromatography using dichloromethane/methanol
from the American Type Culture Collection (ATCC). Melting points 95:5 (v:v), giving a later eluting band (maytansinol) and an early
were determined on an electrothermal melting point apparatus. eluting band. The maytansinol band was collected, and the solvent
Proton magnetic resonance (1H NMR) spectra were obtained on a was removed by rotary evaporation to give 1.55 g of maytansinol
Bruker Avance spectrometer operating at 400 MHz. The chemical (58%). The solvent was removed from the earlier eluting band,
4400 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 Widdison et al.

and the material was dissolved in a minimum volume of acetonitrile mixture was diluted with ethyl acetate, washed successively with
and then purified by preparative reverse-phase HPLC. The solvent cold 1.0 M NaH2PO4 at pH 7.5, water, and 1.0 M aqueous NaH2-
was removed by rotary evaporation to give 440 mg (15% yield) of PO4 at pH 4.0. The organic layer was separated and dried over
4c, R ) -CH(CH3)2; 1H NMR (CDCl3) δ 0.78 (s, 3H), 0.97 (d, MgSO4, filtered, and then evaporated to give 2.63 g of crude R-3-
3H, J ) 6.9), 1.04 (d, 3H, J ) 6.7), 1.23 (m, 1H), 1.28 (d, 3H, O-p-toluenesulfonyl-pentanenitrile. MS: m/z 275.80 (M + Na)+.
J ) 6.4), 1.54 (m, 1H), 1.66 (s, 3H), 1.72 (m, 2H), 2.03 (dd, 1H, The product was used directly without further purification. To the
J ) 14, 3.6 Hz), 2.3 (d, 1H, J ) 14), 2.49 (dd, 1H, J ) 11.7, 14), solution of crude of R-3-O-p-toluenesulfonyl-pentanenitrile (2.63
2.92 (d, 1H, J ) 9.5 Hz), 3.14 (s, 3H), 3.12 (m, 1H), 3.37, (s, 3H), g) in ethanol (15 mL) was added potassium O-ethylxanthate (4.55
3.52 (m, 3H), 3.65 (m, 1H), 3.75 (m, 1H), 3.97 (s, 1H), 4.31 (m, g) in ethanol (50 mL). After stirring overnight under argon, the
2H), 5.52 (dd, 1H, J ) 16, 8.7 Hz), 6.13(d, 1H, J ) 11 Hz), 6.34 mixture was concentrated, diluted with ethyl acetate, and filtered
(s, 1H), 6.45 (dd, 1H, J ) 16, 11 Hz), 6.80 (d, 1H, J ) 1.5 Hz), through a short silica column. The eluant was concentrated and
6.92 (d, 1H, J ) 1.5 Hz); MS m/z 619.3 (M +H)+. purified by chromatography on silica gel, eluting with 1:4 (v/v)
3-(Methyldithio)propanoic Acid (8b). To a stirred solution of EtOAc/hexane, to give 1.54 g (63%, 2 steps) of the title compound
3-mercaptopropanoic acid (7b) (5.00 g, 0.047 mol) in water (150 (Rf ) 0.40 (1:4 EtOAc/hexane)) as a colorless oil. 1H NMR (CDCl3)
mL) cooled in an ice bath was added methyl methanethiolsulfonate δ 4.67 (dd, 2H, J ) 7.1, 14.2 Hz), 3.86 (ddd, 1H, J ) 7.0, 14.0,
(6.54 g, 0.052 mol) in absolute ethanol (75 mL). The reaction 21.9 Hz), 2.50 (t, 2H J ) 7.3 + 7.6 Hz), 2.06 (m, 2H), 1.44 (m,
mixture was stirred overnight at room temperature. The mixture 6H); 13C NMR 213.04, 119.16, 70.28, 44.57, 32.10, 20.20, 15.21,
was then diluted with saturated aqueous NaCl (400 mL) and 13.93; MS: m/z 226.51 (M + Na) +, 242.51 (M + K) +.
extracted with ether (3 × 150 mL), and the combined ether extracts S-(+)-4-(Methyldithio)pentanoic Acid (13). To a solution of
were then washed with saturated NaCl. The solution was dried over S-4-O-Ethylxanthic-pentanenitrile (12, 1.95 g, 9.61 mmol) in a
Na2SO4, concentrated, and then distilled to afford a colorless liquid mixture of ethanol (10 mL) and water (150 mL) was added NaOH
(6.47 g, 90%); bp (1 mm) ) 105 °C; 1H NMR (CDCl3) δ 2.32 (5 g). The reaction mixture was refluxed overnight under argon.
(3H, s), 2.80 (4H, m), 11.20 (1H, s). The mixture was cooled to room temperature and diluted with water
4-(Methyldithio)butanoic Acid (8c). To a stirred solution of (150 mL) and extracted with 1:1 EtOAc/hexane (2 × 100 mL).
bis-(3-carboxypropyl)disulfide (1.00 g, 4.20 mmol) in methanol (20 The aqueous layer was acidified with H3PO4 to pH 2.5-3.0 and
mL) was added a solution of dithiothreitol (0.647 g, 4.20 mmol) in extracted with EtOAc (6 × 75 mL). The organic layers were
H2O (20 mL). A solution of 10 M NaOH (0.842 mL, 8.42 mmol) combined, dried over MgSO4, filtered, and evaporated to dryness
was then added and the mixture was allowed to stir overnight at to give the crude S-4-mercaptopentanoic acid. This crude product
room temperature to effect complete reduction. Methyl methane- was used directly in the next step without further purification. To
thiolsulfonate (1.17 g, 9.24 mmol) was added, and the reaction a solution of crude S-4-mercaptopentanoic acid (1.2 g) in a mixture
mixture was stirred for another 3 h. The mixture was then diluted of ethanol (50 mL) and 0.5 M NaH2PO3 buffer (75 mL) at pH 7.0
with saturated aqueous NaCl (150 mL), acidified (aqueous HCl), was added dropwise methyl methanethiolsulfonate (1.47 g, 11.65
and extracted with ethyl ether (3 × 100 mL). The combined organic mmol) in dry THF (5 mL) over 45 min at 0 °C. After stirring under
layers were washed with saturated NaCl, dried (Na2SO4), concen- argon at 0 °C for 30 min followed by stirring at room temperature
trated, and chromatographed on silica gel, eluting with dichlo- for 2 h, the mixture was concentrated and extracted with dichlo-
romethane/ethyl acetate to give 0.867 g (56%) of a colorless liquid; romethane (2 × 50 mL). The aqueous layer was acidified with H3-
1H NMR (CDCl ) δ 2.12 (2H, m), 2.40 (3H, s), 2.74 (2H, m), 11.18
3 PO4 to pH 2.5-3.0 and extracted with EtOAc (4 × 100 mL). The
(1H, s). organic layers were combined, dried over MgSO4, filtered, and
3-(Phenyldithio)propanoic Acid (8d). To a stirred solution of evaporated. The residue was purified by chromatography over silica
diphenyl disulfide (3.0 g, 13.8 mmol) in a mixture of ether (10 gel, eluting with 1:100:400 HOAc/EtOAc/hexane to give 1.43 g
mL) and methanol (20 mL) under a nitrogen atmosphere at room (83%) of the desired product (Rf ) 0.32, 1:100:400 HOAc/EtOAc/
temperature was added a solution of 3-mercaptopropanoic acid (7b) hexane) as a white solid; mp 290-293 °C (dec); 1H NMR (CDCl3)
(0.49 g, 4.6 mmol) in ether (5 mL), followed by a solution of 10 δ 2.91 (ddd, 1H, J ) 6.8, 13.7, 20.5 Hz), 2.53 (t, 2H, J ) 7.7 +
M NaOH (0.46 mL, 4.6 mmol). The reaction mixture was stirred 7.4 Hz), 2.42 (s, 3H), 1.94 (m, 2H), 1.36 (d, 3H, J ) 6.8 Hz); 13C
at room temperature for 20 h and then stripped of the solvents under NMR 179.18, 45.35, 31.58, 30.73, 24.70, 21.05; MS: (m/z) 202.92
reduced pressure. The product was purified by column chroma- (M + Na)+; [R]25D ) +41.35 (c ) 2, CH3OH).
tography on silica gel, eluting with ethyl acetate/hexane. The S-1,3-Di-O-(para-toluenesulfonyl)butane (16). A solution of S-
product was obtained as a white solid (0.56 g, 56.6%); mp 57-59 (-)-1,3-butanediol (15, 2.00 g, 22.22 mmol) in a mixture of dry
°C; 1H NMR (CDCl3, TMS) δ 2.6-3.0 (4H, m), 7.12-7.60 (5H, pyridine (40 mL) and dry toluene (60 mL) was treated with
m), 10.6 (1H, s). p-toluenesulfonyl chloride (12.70 g, 66.84 mmol) under argon at 0
R-1,3-Di-O-(para-toluenesulfonyl)butane (11). A solution of °C. After stirring at 0 °C for 5 min followed by stirring at room
R-(-)-1,3-butanediol (10, 2.00 g, 22.22 mmol) in a mixture of dry temperature for 2 h, the mixture was evaporated under vacuum.
pyridine (40 mL) and dry toluene (60 mL) was treated with The residue was redissolved in ethyl acetate and washed with 0.1
p-toluenesulfonyl chloride (12.70 g, 66.84 mmol) under argon at 0 M aqueous NaHCO3 and saturated NaCl. The organic layer was
°C. After stirring at 0 °C for 5 min followed by stirring at room separated, dried over MgSO4, filtered, and evaporated. The residue
temperature for 2 h, the mixture was evaporated under vacuum, was purified by chromatography over silica gel, eluting with 1:2
redissolved in ethyl acetate, and washed with 0.1 M aqueous ethyl acetate/hexane to give 6.25 g (71%) of the title compound
NaHCO3, followed by saturated aqueous NaCl. The organic layer (Rf ) 0.40 (1:1 EtOAc/hexane)). Recrystallization from ether/
was dried over MgSO4 and filtered, and the solvent was evaporated. hexane afforded a white solid; mp 56-58 °C; 1H NMR (CDCl3) δ
Purification by chromatography on silica gel, eluting with 1:2 7.76 (dd, 4H, J ) 1.0, 8.0 Hz), 7.35 (dt, 4H, J ) 0.4, 8.0 +8.0
(v/v) ethyl acetate/hexane gave 6.51 g (74%) of the title compound Hz), 4.70 (m, 1H), 4.03 (m, 1H), 3.94 (m, 1H), 2.46 (s, 6H), 1.92
(Rf ) 0.40, 1:1 EtOAc/hexane) as a white solid; mp 56-58 °C; 1H (m, 2H), 1.26 (d, 3H, J ) 6.3 Hz); 13C NMR 145.17, 133.00,
NMR (CDCl3) δ 7.76 (dd, 4H, J ) 1.0, 8.0 Hz), 7.35 (dt, 4H, J ) 130.11, 128.12, 127.91, 76.28, 66.21, 36.08, 21.86, 21.06; MS: m/z
0.4, 8.0 +8.0 Hz), 4.70 (m, 1H), 4.03 (m, 1H), 3.94 (m, 1H), 2.46 420.99 (M + Na)+.
(s, 6H), 1.92 (m, 2H), 1.26 (d, 3H, J ) 6.3 Hz); 13C NMR 145.17, R-4-O-(Ethylxanthic)pentanenitrile (17). A solution of S-1,3-
133.00, 130.11, 128.12, 127.91, 76.28, 66.21, 36.08, 21.86, 21.06; di-O-(p-toluenesulfonyl)butane (16, 6.25 g, 15.70 mmol) in 60 dry
MS: m/z 420.99 (M + Na)+. DMSO (50 mL) was treated with NaCN (0.85 g). The reaction
S-4-(O-Ethylxanthic)pentanenitrile (12). A solution of R-1,3- mixture was stirred under argon for 18 h at room temperature. The
di-O-p-toluenesulfonyl-butane (11, 4.80 g, 12.06 mmol) in dry reaction mixture was then diluted with ethyl acetate, washed
DMSO (50 mL) was treated with NaCN (0.65 g, 13.26 mmol). sequentially with cold 1.0 M NaH2PO4 buffer at pH 7.5, water,
After stirring at room temperature under argon for 18 h, the reaction and 1.0 M NaH2PO4 buffer at pH 4.0. The organic layer was dried
Semisynthetic Maytansine Analogues Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4401

over MgSO4, filtered, and evaporated to give 3.62 g of crude S-3- concentrated HCl and then extracted with ethyl acetate (3 × 75
O-(p-toluenesulfonyl)pentanenitrile. The product was used directly mL). The organic layers were dried over anhydrous Na2SO4, and
without further purification. To a solution of crude S-3-O-p- the solvent was removed by rotary evaporation under vacuum to
toluenesulfonyl-pentanenitrile (3.62 g) in ethanol (50 mL) was give 10 g of product 22 (39% yield). The material was used without
added potassium O-ethylxanthate (5.72 g) in ethanol (100 mL). further purification.1H NMR (CDCl3) δ 1.38 (6H, s), 1.87-1.93
After stirring overnight under argon, the mixture was concentrated, (2H, m), 2.08 (1H, s), 2.51-2.57 (2H, m).
diluted with ethyl acetate, and filtered through a short column of 4-Methyl-4-(methyldithio)pentanoic Acid (23). A solution of
silica gel. The eluant was concentrated, and the residue was purified 4-mercapto-4-methylpentanoic acid (22) (6.0 mL, 40 mmol) was
by chromatography over silica gel, eluting with 1:4 EtOAc/hexane dissolved in deionized water (50 mL) in a 250 mL flask. The
to give 2.0 g (62%, 2 steps) of the title compound (Rf ) 0.40 (1:4 solution was magnetically stirred as sodium carbonate (6.4 g, 60
EtOAc/hexane)) as a colorless liquid. 1H NMR (CDCl3) δ 4.67 mmol) was added to the acid at a rate that would not cause excessive
(dd, 2H, J ) 7.1, 14.2 Hz), 3.86 (ddd, 1H, J ) 7.0, 14.0, 21.9 Hz), frothing. The flask was equipped with a 100 mL addition funnel,
2.50 (t, 2H J ) 7.3 + 7.6 Hz), 2.06 (m, 2H), 1.44 (m, 6H); 13C which was charged with a solution of methyl methanethiolsulfonate
NMR 213.04, 119.16, 70.28, 44.57, 32.10, 20.20, 15.21, 13.93; (7.5 g, 60 mmol) dissolved in absolute ethanol (30 mL). The flask
MS: m/z 226.51 (M + Na) +, 242.51 (M + K) +. was cooled in an ice/water bath, and the system was maintained
R-(-)-4-(Methyldithio)pentanoic Acid (18). A solution of R-4- under an argon atmosphere. The methyl methanethiolsulfonate
O-(ethylxanthic)pentanenitrile (17, 2.0 g, 9.85 mmol) in a mixture solution was added dropwise to the flask as rapidly as possible but
of ethanol (10 mL) and water (200 mL) was treated with NaOH without causing excessive frothing. The cooling bath was removed,
(6.0 g). The reaction mixture was refluxed overnight under argon. and the reaction mixture was allowed to stir at room temperature
The mixture was diluted with water (150 mL) and extracted with for an additional 3 h. The solvent was removed by rotary
1:1 EtOAc/hexane (2 × 100 mL). The aqueous layer was acidified evaporation under vacuum, until approximately 20 mL remained.
with H3PO4 to pH 2.5-3.0 and extracted with EtOAc (6 × 75 mL). Then, saturated sodium bicarbonate (10 mL) and deionized water
The organic layers were combined, dried over MgSO4, filtered, and (30 mL) were added. The mixture was washed with ethyl acetate
evaporated to dryness to give crude R-4-mercaptopentanoic acid. (3 × 25 mL), and the aqueous layer was separated and adjusted to
This crude product was used directly in the next step without further approximately pH 2 with 5 M HCl and then extracted with ethyl
purification. To a solution of 1.60 g of the crude R-4-mercapto- acetate (2 × 120 mL). The organic layers were combined and
pentanoic acid in a mixture of ethanol (50 mL) and 0.5 M NaH2- washed with a solution composed of saturated NaCl (16 mL) and
PO4 buffer at pH 7.0 (75 mL) was added dropwise a solution of 1 M HCl (4 mL). The organic layer was then dried over 14 g of
methyl methanethiolsulfonate (1.96 g, 15.53 mmol) in dry THF (7 anhydrous sodium sulfate and filtered, and the solvent was removed
mL) over 45 min at 0 °C. The reaction mixture was stirred under by rotary evaporation under vacuum to give 5.4 g of product 23
argon at 0 °C for 30 min and then at room temperature for 2 h. (70% yield) as a white solid. 1H NMR (CDCl3) δ 1.54 (6H, s),
The mixture was concentrated and extracted with dichloromethane 2.15-2.21 (2H, m), 2.64 (3H, s), 2.69-2.72 (2H, m); MS: m/z
(2 × 50 mL). The aqueous layer was acidified with H3PO4 to pH 217.1 (M + Na) +.
2.5-3.0 and extracted with EtOAc (4 × 100 mL). The organic
layers were combined, dried over MgSO4, filtered, and evaporated. General Method for the Synthesis of N-Hydroxysuccinimide
The residue was purified by chromatography over silica gel, eluting Esters 9a-d, 14, 19, and 24. (Methyldithio)-alkanoic acid (15
with HOAc/EtOAc/hexane (1:100:400) to give 1.65 g (93%) of mmol) was dissolved in dichloromethane (20 mL) and stirred
the title compound (Rf ) 0.32 (HOAc/EtOAc/hexane, 1:100:400). magnetically as N-hydroxysuccinimide (2.65 g, 23 mmol) was
Recrystallization from ether/hexanes afforded a white solid; mp added followed by 1-[3-(dimethylamino)propyl]-3-ethylcarbodiim-
157-160 °C; 1H NMR (CDCl3) δ 2.91 (ddd, 1H, J ) 6.8, 13.7, ide hydrochloride (EDC, 4.4 g, 23 mmol). The mixture was stirred
20.4 Hz), 2.53 (t, 2H, J ) 7.7 + 7.4 Hz), 2.42 (s, 3H), 1.96 (m, under an argon atmosphere for 2 h. The reaction mixture was poured
2H), 1.36 (d, 3H, J ) 6.8 Hz); 13C NMR 179.46, 45.67, 31.91, into a 125 mL separatory funnel, 40 mL of ethyl acetate was added,
31.07, 25.02, 21.36; MS: 202.9 (M + Na)+, 203.9; [R]25D ) -39.16 and the solution was washed with 50 mM potassium phosphate
(c ) 2, CH3OH). buffer at pH 6.0 (2 × 20 mL) and saturated sodium chloride (20
4-Mercapto-4-methylpentanoic Acid (22). Compound 21 was mL). The organic layer was dried over anhydrous Na2SO4, and the
prepared by the method previously described.22 A 500 mL flask solvent was removed by rotary evaporation under vacuum to give
was equipped with a stir bar and a 150 mL addition funnel. The the desired product, which was used without further purification.
system was placed under an argon atmosphere, and anhydrous THF General Method for the Synthesis of N-Acyl-N-methyl-L-
(150 mL) and n-BuLi (2.5 M, 75 mL, 18.7 mmol) in hexanes were alanines (5a-i). N-Methyl-L-alanine (18.0 mmol) was dissolved
added via a cannula, and the solution was cooled in a -78 °C dry in a mixture of dimethoxyethane (25 mL) and deionized water (25
ice/acetone bath. Acetonitrile (9.4 mL, 18 mmol) was added mL) in a 125 mL flask equipped with a magnetic stir bar.
dropwise via a syringe over approximately 5 min. The reaction Triethylamine (6.9 g, 36 mmol) was added, and the solution was
mixture was stirred for 30 min, while a white precipitate of lithiated vigorously stirred as the N-hydroxysuccinimidyl ester (9a-d, 14,
acetonitrile was formed. Isobutylene sulfide (15.0 g, 17 mmol) was 19, 24) (18 mmol) dissolved in 40 mL of the same solvent mixture
dissolved in anhydrous THF (100 mL) and added dropwise over was added dropwise over approximately 5 min. After 2 h, the
approximately 30 min via the addition funnel. The cooling bath reaction mixture was concentrated to approximately 40 mL by rotary
was removed, and the reaction was allowed to stir at room evaporation under vacuum. Deionized water (10 mL) and 1 M HCl
temperature for 3 h. The flask was cooled in an ice/water bath as were added to give a pH of approximately 2. The mixture was
0.5 M HCl (38 mL) was added dropwise. The THF layer was poured into a separatory funnel and extracted with ethyl acetate (2
retained, and the aqueous layer was washed twice with 75 mL of × 50 mL). The organic layers were combined and washed with
ethyl acetate. The THF and ethyl acetate layers were combined, saturated sodium chloride solution (10 mL). The organic layer was
dried over approximately 20 g of anhydrous sodium sulfate, and dried over 8.0 g of anhydrous Na2SO4, and the solvent was removed
transferred to a 250 mL flask. The solvent was removed by rotary by rotary evaporation under vacuum. The residue was taken up in
evaporation under reduced pressure to give crude 21. Ethanol (30 a minimum volume of ethyl acetate and purified by chromatography
mL) was added, and the contents were stirred. A solution of NaOH on silica (silica: 40 micron flash grade, silica bed: 24 × 3.0 cm,
(8 g) in water (30 mL) was slowly added. The flask was equipped mobile phase: hexanes/ethyl acetate/acetic acid, 50:48:2). The
with a reflux condenser and placed under an argon atmosphere. fractions containing the desired product were combined, and the
The reaction was refluxed overnight and then cooled to room solvent was removed under vacuum. Residual acetic acid was
temperature. Water (60 mL) was added, and the mixture was removed by dissolving the residue in a minimum volume of ethyl
extracted twice with 25 mL portions of a 2:1 mixture of ethyl acetate acetate and precipitating the product by the rapid but dropwise
and hexane. The aqueous layer was acidified to pH 2 with addition of hexane with stirring. Hexane was added until the product
4402 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 Widdison et al.

was no longer detected in the supernatant by TLC analysis. The N2′-Deacetyl-N2′-[2-(methyldithio)-1-oxoethyl]maytansine (D-
precipitate was vacuum-dried for 4 h to give the desired products M0SMe, 2a). Maytansinol (4) was reacted with N-methyl-N-[(2-
5a-i. methyldithio)-1-oxoethyl]-L-alanine (5a) as described above. The
N-Methyl-N-[(2-methyldithio)-1-oxoethyl]-L-alanine (5a). Pale desired maytansinoid 2a, and the corresponding D-aminoacyl isomer
yellow solid (19% yield); 1H NMR (CDCl3) δ 1.458 (3H, d, J ) 6a were isolated by purification using preparative TLC, eluting with
7.6), 2.446 (3H, s), 3.069 (3H, s), 3.666 (2H, s), 5.185 (1H, m); 5% methanol in ethyl acetate to give a white solid. 6a 1H NMR δ
MS: m/z 246.0 (M + Na)+. 0.796 (3H, s), 1.252 (1H, m), 1.306 (3H, d, J ) 6.4 Hz), 1.322
N-Methyl-N-[(3-methyldithio)-1-oxopropyl]-L-alanine (5b). (3H, d, J ) 6.8 Hz), 1.344 (3H, d, J ) 10.8 Hz), 1.414 (2H, m),
White solid (60% yield); mp 103-104 °C; 1H NMR (CDCl3) δ 1.553 (1H, d, J ) 14.4 Hz), 1.640 (3H, s), 2.182 (1H, dd, J )
1.449 (3H, d, J ) 7.6), 2.422 (3H, s), 2.821 (2H, m), 2.989 (2H,m), 12.4, 2.8 Hz), 2.416 (3H, s), 2.624 (1H, dd, J ) 12, 2.4 Hz), 2.914
3.017 (3H, s), 5.194 (1H, m). Anal. (C8H15NO3S2) C, H, N. MS: (3H, s), 3.022 (1H, d, J ) 9.6 Hz), 3.099 (1H, d, J ) 12.4 Hz),
m/z 259.9 (M + Na)+. 3.210 (3H, s), 3.357 (3H, s), 3.499 (1H, d, J ) 9.2 Hz), 3.571 (1H,
N-Methyl-N-[(3-phenyldithio)-1-oxopropyl]-L-alanine (5c). d, J ) 14 Hz), 3.980 (3H, s), 4.266 (1H, dt, J ) 12, 1.6 Hz), 4.787
White solid (60% yield); mp 96-97 °C; 1H NMR (CDCl3) δ 1.42 (1H, dd, J ) 9.2, 2.8 Hz), 5.379 (1H, m), 5.674 (1H, dd, J ) 9.2,
(2H, d), 2.7-3.0 (7H, m), 5.24 (1H, q), 7.24-7.60 (5H, m). 6 Hz), 6.287 (1H, s), 6.422 (1H, dd, J ) 11.2, 4 Hz), 6.678 (1H,
N-Methyl-N-[(4-methyldithio-1-oxobutyl]-L-alanine (5d). White d, J ) 1.6 Hz), 6.691 (1H, d, J ) 8.8 Hz) and 6.822 (1H, d, J )
crystals (44% yield); mp 92-93 °C; 1H NMR (CDCl3) δ 1.439 1.6 Hz). HRMS calcd for C35H48ClN3O10S2Na (M + Na)+,
(3H, d, J ) 7.2), 2.089 (2H, t, J ) 7.2), 2.405 (3H, s), 2.517 (2H, 792.2367; found, 792.2350.
t, J ) 7.2), 2.779 (2H, m), 2.989 (3H, s), 5.123 (1H, m); MS: m/z 6a. 1H NMR δ 0.861 (3H, s), 1.273 (1H, m), 1.275 (3H, d, J )
290.0 (M + K)+. 6.4 Hz), 1.428-1.478 (2H, m), 1.519 (3H, d, J ) 7.6 Hz), 1.689
N-Methyl-N-[(4-(R,S)-methyldithio-1-oxopentyl)] -L-alanine (3H, s), 1.765 (1H, d, J ) 13.6 Hz), 2.214 (1H, dd, J ) 11.2, 3.2
(5f). White solid (60% yield). 1H NMR δ 1.35 (3H, d, J ) 7), 1.41 Hz), 2.487 (3H, s), 2.667 (1H, dd, J ) 12, 2.4 Hz), 2.856 (1H, d,
(3H, d, J ) 7), 1.94-2.03 (2H, m), 2.43(3H, s), 2.50-2.55 (2H, J ) 9.6 Hz), 3.114 (3H, s), 3.168 (3H, s), 3.203 (1H, d, J ) 13.2
m), 2.83-2.93 (1H, m), 2.98 (3H, s), 5.14 (1H, q, J ) 7). MS: Hz), 3.340 (3H, s), 3.458 (1H, d, J ) 9.2 Hz), 3.508 (1H, d, J )
m/z 288.1 (M + Na)+. 12.8 Hz), 3.636 (2H, J ) 6 Hz), 3.995 (3H, s), 4.293 (1H, t), 4.888
N-Methyl-N-[(4-(S)-methyldithio)-1-oxopentyl]-S-alanine (5g). (1H, dd, J ) 8.8, 3.2 Hz), 5.126 (1H, m), 5.869 (1H, dd, J ) 9.6,
White solid (62% yield); 1H NMR δ 1.36 (3H, d, J ) 7), 1.42 6 Hz), 6.230 (1H, d, J ) 10.8 Hz), 6.331 (1H, s), 6.423 (1H, dd,
(3H, d, J ) 7), 1.93-1.98 (2H, m), 2.40 (3H, s), 2.50-2.53 (2H, J ) 11.2, 4 Hz), 6.801 (1H, d, J ) 1.2 Hz) and 6.852 (1H, d, J )
m), 2.90-2.95 (1H, m), 2.99 (3H, s), 5.14 (1H, q, J ) 7), MS: 1.6 Hz). HRMS calcd for C35H48ClN3O10S2Na (M + Na)+,
m/z 288.1 (M + Na)+ 792.2367; found, 792.2350.
N-Methyl-N-[(4-(R)-methyldithio)-1-oxopentyl]-S-alanine (5h). N2′-Deacetyl-N2′-(3-methyldithio-1-oxopropyl)-maytansine
White solid (60% yield); 1H NMR δ 1.35 (3H, d, J ) 7), 1.42 (DM1SMe, 2b) and Its D-Alanyl Isomer D-DM1SMe (6b).
(3H, d, J ) 7), 1.93-2.00 (2H, m), 2.43 (3H, s), 2.50-2.55 (2H, Maytansinol (4) was reacted with N-methyl-N-[(2-methyldithio)-
m), 2.88-2.95 (1H, m), 3.00 (3H, s), 5.14 (1H, q, J ) 7), MS: 1-oxopropyl]-L-alanine (5b) as described above. Purification by
m/z 288.1 (M + Na)+. column chromatography over silica gel, eluting with a mixture of
N-Methyl-N-[4-methyl-(4-methyldithio)-1-oxopentyl)-L-ala- dichloromethane/ethyl acetate/methanol to give a mixture of
nine (5i). White solid (51% yield) mp 77-79 °C; 1H NMR (CDCl3) DM1SMe (2b) and the D-aminoacyl isomer D-DM1SMe (6b). The
δ 1.32 (6H, s), 1.42 (3H, d, J ) 7 Hz), 1.90-97 (2H, m), 2.40 two diastereomers were separated by HPLC on a Kromasil cyano
(3H, s), 2.42-2.49 (2H, m), 2.9 (3H, s), 5.15 (1H, q, J ) 7 Hz). preparative HPLC column (250 mm × 50 mm, 10 micron particle
MS: m/z 302.0. (M + Na)+. size). The column was equilibrated in a mixture of hexanes/2-
General Procedure for the Esterification of Maytansinol to propanol/ethyl acetate (17:2:6, v/v/v) at a flow rate of 150 mL/
Give Maytansinoids 2a-i and 6a-i. In a typical experiment, min. Under these conditions, the desired L-DM1SMe (2b) had a
maytansinol (4, 25 mg, 0.044 mmol) and carboxylic acids 5a-i retention time of 42 min, whereas the D-DM1SMe isomer (6b)
(0.264 mmol) were dissolved in dichloromethane (3 mL) and stirred elutes at 56 min. Optically pure products were obtained by this
under an argon atmosphere. A solution of dicyclohexylcarbodiimide method (ee >99.9%). HPLC analysis using a Vydac analytical C-18
(DCC, 57.2 mg, 0.277 mmol) in dichloromethane (0.7 mL) was column (length: 100 mm, i.d.: 4.6 mm, particle size: 3 microns)
added. After 1 min, a solution of 1 M ZnCl2 in diethyl ether (0.055 at a flow rate of 1 mL/min, eluting with a gradient of water and
mL, 0.055 mmol) was added. The mixture was stirred at room acetonitrile (0-3 min, 35% CH3CN; 3-13 min, 35-65% CH3-
temperature for 2 h, then ethyl acetate (5 mL) was added, and the CN). Under these conditions L-DM1SMe (2b) eluted with a
mixture was vacuum filtered through course filter paper. The filtrate retention time of 10.76 min.
was washed with a solution of saturated aqueous sodium bicarbonate DM1SMe (2b). White solid (32% yield); 1H NMR (CDCl3) δ
(2 mL) followed by a saturated sodium chloride solution (1 mL). 0.84 (3H, s), 1.11-1.23 (1H, m), 1.31 (3H, d, J ) 6 Hz), 1.35
The organic layer was separated, dried over anhydrous sodium (3H, d, J ) 7 Hz), 1.46-1.52 (1H, m), 1.68 (3H, s), 1.97 (1H, d,
sulfate, and filtered, and the solvent was removed under reduced J ) 9 Hz), 2.24 (1H, dd, J ) 12, 15 Hz), 2.30 (3H, s), 2.65 (1H,
pressure. The residue was purified by silica chromatography using dd, J ) 12, 15 Hz), 2.73-2.86 (2H, m), 2.90 (3H, s), 2.92-3.03
a mixture of dichloromethane and methanol to remove the unreacted (2H, m), 3.08 (1H, d, J ) 9 Hz), 3.14 (1H, d, J ) 12 Hz), 3.28
maytansinol. The fractions containing the desired product were (3H, s), 3.39 (3H, s), 3.54 (1H, d, J ) 9 Hz), 3.72 (1H, d, J ) 13
combined, and the solvent was removed under vacuum to give a Hz), 4.02 (3H, s), 4.31 (1H, t, J ) 11 Hz), 4.82 (1H, dd, J ) 3, 12
mixture of diastereomeric maytansinoids bearing the N-methyl-N- Hz), 5.45 (1H, q, J ) 7 Hz), 5.69 (1H, dd, J ) 9, 15 Hz), 6.25
acyl-L- and D-alanine eaters. The residue was taken up in a (1H, s), 6.47 (1H, dd, J ) 11, 15 Hz), 6.67 (1H, d, J ) 1.5 Hz),
minimum volume of ethyl acetate and purified by one of three 6.77 (1H, d, J ) 11 Hz), 6.85 (1H, d, J ) 1.5 Hz). HRMS calcd
ways: (1) preparative TLC on silica, (2) HPLC on an analytical for C36H51ClN3O10S2 (M + H)+, 784.2704; found, 784.2693;
Kromasil cyano-bonded silica column (4.6 mm × 250 mm), using extinction coefficient 280 nm (ethanol) ) 5545 M-1 cm-1, 252 nm )
an isocratic elution at a flow rate of 1 mL/min, with hexane/ethyl 25 700 M-1 cm-1.
acetate/2-propanol (68:24:8, v/v/v), or (3) HPLC using a preparative D-DM1SMe (6b). White solid (30% yield); mp 170-171 °C;
(50 cm × 250 cm, 10 micron) Kromasil or Diazem cyano-bonded 1H NMR (CDCl ) δ 0.85 (3H, s), 1.11-1.20 (1H, m 1.22-1.29
3
silica column using as mobile phase a 68:8:24 mixture of hexane, (3H, m), 1.49 (3H, d, J ) 7.5 Hz), 1.69 (3H, s), 2.20 (1H, dd, J )
2-propanol, and ethyl acetate. The flow rate was 118 mL/min. Under 3, 14 Hz), 2.40 (3H, s), 2.65 (1H, dd, J ) 12, 14 Hz), 2.79-2.84
either HPLC condition, typically, the desired product L-aminoacyl (3H, m), 2.92-2.96 (2H, m), 3.04 (3H, s), 3.15 (3H, s), 3.33 (3H,
maytansinoid (2a-i) eluted about 8-10 min before the correspond- s), 3.43 (1H, d, J ) 9 Hz), 3.50 (1H, d, J ) 12 Hz), 3.98 (3H, s),
ing D-aminoacyl isomer (6a-i). 4.28 (1H, m), 4.90 (1H, dd, J ) 3, 12 Hz), 5.17 (1H, q, J ) 7 Hz),
Semisynthetic Maytansine Analogues Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4403

5.88 (1H, dd, J ) 9 Hz), 6.22-6.26 (2H, m), 6.41 (1H, dd, J ) 6.820 (1H, d, J ) 2 Hz). HRMS calcd for C37H52ClN3O10S2Na
11, 15 Hz), 6.78 (1H, d, J ) 1.6 Hz), 6.84 (1H, d, J ) 1.6 Hz). (M + Na)+, 820.2680; found, 820.2667.
HRMS calcd for C36H51ClN3O10S2 (M + Na)+, 806.2524; found, 6d. 1H NMR (CDCl3) δ 0.874 (3H, s), 1.193-1.429 (3H, m),
806.2518. 1.290 (3H, d, J ) 6.4 Hz), 1.314 (3H, d, J ) 6.8 Hz), 1.696 (3H,
N2′-Deacetyl-N2′-[2-(phenydithio)-1-oxopropyl]maytansine s), 1.777 (1H, d, J ) 13.6 Hz), 2.058 (2H, m), 2.209 (1H, dd, J )
(DMISPh, 2c) and Its D-Alanyl Isomer D-DM1SPh (6c). May- 11.2, 3.2 Hz), 2.420 (3H, s), 2.529 (2H, m), 2.662 (1H, dd, J )
tansinol (4) was reacted with N-methyl-N-[(2-phenyldithio)-1-oxo- 12, 3.6 Hz), 2.727-2.801 (2H, m), 2.826 (1H, d, J ) 9.6 Hz),
proyl]-L-alanine (5c) as described above. The product was purified 3.044 (3H, s), 3.167 (3H, s), 3.204 (1H, d, J ) 11.2 Hz), 3.342
by preparative TLC of the residue on silica gel, and eluting twice (3H, s), 3.439 (1H, d, J ) 9.2 Hz), 3.514 (1H, d, J ) 7.6 Hz),
with 5% MeOH/CHCL3 gave two strongly UV absorbing bands 3.999 (3H, s), 4.351 (1H, t), 4.933 (1H, dd, J ) 8.8, 3.2 Hz), 5.082
with Rf ) 0.5 and 0.6. The two products were isolated by extraction (1H, m), 5.844 (1H, dd, J ) 9.6, 5.6 Hz), 6.231 (1H, d, J ) 8.8
with ethyl acetate. The higher Rf band was found to be the Hz), 6.242 (1H, s), 6.434 (1H, dd, J ) 11.2, 4.4 Hz), 6.799 (1H,
D-aminoacyl ester 6c of maytansinol, and the lower band was the d, J ) 2 Hz) and 6.851 (1H, d, J ) 2 Hz). HRMS calcd for C37H52-
L-aminoacyl ester 2c. The products were separated and further ClN3O10S2Na (M + Na)+, 820.2680; found, 820.2658.
purified by HPLC on a Waters Radialpak C-18 column, eluting at N2′-Deacetyl-N2′-[4-(R,S)-(methyldithio)-1-oxopentyl]may-
a flow rate of 1.5 mL/min with a linear gradient of acetonitrile/ tansine (DM3-SMe, 2f). Maytansinol (4) was reacted with N-meth-
water (70-90% acetonitrile/10 min). Under these conditions, both yl-N-[(4-(R,S)-methyldithio-1-oxopentyl)]-S-alanine (5f) as de-
isomers had an identical retention time of 6.0 min. scribed above. The reaction mixture was purified by silica
DM1SPh (2c). 1H NMR (CDCl3) δ 0.82 (3H, s), 1.11-1.25 (1H, chromatography using a mixture of dichloromethane and methanol
m), 1.33 (3H, d, J ) 3 Hz), 1.61 (3H, s), 1.63 (3H, d, J ) 14 Hz), to remove unreacted maytansinol. The fractions containing the
2.19 (1H, dd, J ) 3, 15 Hz), 2.61 (1H, dd, J ) 12, 15 Hz), 2.78 desired product were combined, and the solvent was removed under
(3H, s), 2.68-3.03 (2H, m), 3.07 (1H, d, J ) 9 Hz), 3.20 (3H, s), vacuum to give a mixture of the diastereomers 2f and 6f. The
3.38 (3H, s), 3.53 (1H, d, J ) 9 Hz), 3.63 (1H, d, J ) 13 Hz), 3.68 residue was purified on a 50 cm × 250 cm, 10 micron Diazem CN
(3H, s), 4.01 (3H, s), 4.30 (1H, t, J ) 11 Hz), 4.79 (1H, dd, J ) column using as mobile phase a 68:8:24 mixture of hexane,
3, 8 Hz), 5.43 (1H, q, J ) 7 Hz), 5.68 (1H, dd, J ) 9, 15 Hz), 6.23 2-propanol, and ethyl acetate. The flow rate was 118 mL/min. The
(1H, s), 6.45 (1H, dd, J ) 12, 15 Hz), 6.60 (1H, d, J ) 1.5 Hz), desired product 2f eluted with a retention time of 11 min, and the
6.75 (1H, d, J ) 12 Hz), 6.77 (1H, d, J ) 1.5 Hz), 7.22-7.40 (5H, undesired diastereomer 6f had a retention time of 19 min.
m). HRMS cacld for C41H52ClN3O10S2Na (M +Na)+, 868.2680; 2f. White solid (36% yield). 1H NMR (CDCl3) δ 0.80 (3H, s),
found, 868.2670. 1.19-1.23(1H,m), 1.28-1.36 (9H, m), 1.42-1.46(1H, m), 1.53-
D-DM1SPh (6c). 1H NMR (CDCl3) δ 0.89 (3H, s), 1.31 (3H, d, 1.63 (2H, m), 1.64 (3H, s), 1.80-1.89 (1H, m), 1.90-2.09 (1H,
J ) 6.4 Hz), 1.48-1.53 (m, 1H), 1.51 (3H, d), 1.72 (3H, s), 2.10 m), 2.18 (1H, dd, J ) 3 and 14 Hz), 2.32 (3H, s), 2.33-2.42 (1H,
(1 H, d, J ) 6 Hz), 2.15 (1H, dd, J ) 3, 15 Hz), 2.69 (1H, dd, m), 2.49-2.62 (2H, m), 2.88 (3H, s), 3.04 (1H, d, J ) 9 Hz), 3.11
J ) 12, 15 Hz), 2.81-2.89 (2H, m), 2.96 (3H, s), 3.02 (1H, d, J ) (1H,d,J ) 11 Hz), 3.23 (3H,s), 3.35 (3H,s), 3.49 (1H, d, J ) 9
7 Hz), 3.20 (3H, s), 3.36 (3H, s), 3.47 (1H, d, J ) 9 Hz), 3.55 (1H, Hz), 3.63 (1H, d, J ) 12 Hz), 3.98 (3H, s), 4.27 (1H, t, J ) 10
d, J ) 13 Hz), 3.68 (3H, s), 4.04 (3H, s), 4.31 (1H, t, J ) 11 Hz), Hz), 4.79 (1H, dd, J ) 3 and 12 Hz), 5.41 (1H, q, J ) 7 Hz), 5.66
4.93 (1H, dd, J ) 3, 12 Hz), 5.20 (1H, q, J ) 7 Hz), 5.90 (1H, dd, (1H, dd J ) 9 and 15 Hz), 6.21 (1H, s), 6.42 (1H, dd, J ) 11 and
J ) 9, 15 Hz), 6.25 (1H, s), 6.27 (1H, d, J ) 12 Hz), 6.45 (1H, dd, 15 Hz), 6.65 (1H, d, J ) 1.5 Hz), 6.73 (1H, d, J ) 11 Hz), 6.81
J ) 12, 15 Hz), 6.83 (1H, d, J ) 1.5 Hz, 6.89 (1H, d, J ) 1.5 Hz), (1H, d, J ) 1.5 Hz). HRMS calcd for C38H54ClN3O10S2Na (M +
7.27-7.49 (5H, m). HRMS cacld for C41H52ClN3O10S2Na (M Na)+, 834.2837; found, 834.2819.
+Na)+, 868.2680; found, 868.2672. 6f. White solid (30% yield) 1H NMR δ 0.869 (3H, s), 1.200-
N2′-Deacetyl-N2′-[3-(methyldithio)-1-oxobutyl]maytansine (D- 1.315 (2H, m), 1.283 (3H, d, J ) 6.8 Hz), 1.373 (3H, dd, J ) 5.6,
M2′SMe, 2d). Maytansinol (4) was reacted with N-methyl-N-3- 1.2 Hz), 1.498 (3H, d, J ) 7.6 Hz), 1.600 (1H, d, J ) 14.4 Hz),
(methyldithio)-1-oxobutyl]-L-alanine (5d) as described above. The 1.691 (3H, s), 1.772 (1H, m), 1.929 (2H, m), 2.203 (1H, dd, J )
reaction mixture was purified by preparative TLC on silica gel, 11.6, 3.2 Hz), 2.421 (3H, s), 2.518 (2H, m), 2.654 (1H, t), 2.814
eluting twice with 7% MeOH in CHCl3. Two new UV absorbing (1H, dd, J ) 8, 1.6 Hz), 2.902 (1H, m), 3.038 (3H, d, J ) 3.2 Hz),
bands (Rf ) 0.65, 0.75) were obtained. The products were isolated 3.164 (3H, s), 3.200 (1H, d, J ) 13.2 Hz), 3.339 (3H, s), 3.436
by extraction with ethyl acetate. The higher Rf band was determined (1H, d, J ) 8 Hz), 3.507 (1H, d, J ) 12.8 Hz), 3.996 (3H, s),
to be the D-aminoacyl ester 6d (31%), whereas the lower Rf band 4.309 (1H, t), 4.936 (1H, dd, J ) 4.8, 4 Hz), 5.093 (1H, m), 5.851
was the desired L-aminoacyl ester 2d (44%). Both isomers were (1H, m), 6.233 (1H, d, J ) 11.2 Hz), 6.269 (1H, s), 6.429 (1H, dd,
further purified by HPLC using a Waters Radialpak C-18 column, J ) 10.8, 4.4 Hz), 6.792 (1H, s) and 6.848 (1H, s). HRMS calcd
eluting at a flow rate of 2 mL/min with a linear gradient of for C38H54ClN3O10S2Na (M + Na)+, 834.2837; found, 834.2830.
acetonitrile/water (50-80% acetonitrile/10 min). Under these N2′-Deacetyl-N2′-[4-(S)-(methyldithio)-1-oxopentyl]maytan-
conditions, the D-aminoacyl ester had a retention time of 7.4 min, sine (2g). Maytansinol (4) was coupled with N-methyl-N-[(4-(S)-
whereas the L-aminoacyl isomer had a retention time of 7.6 min. methyldithio)-1-oxopentyl]-S-alanine (5g) using DCC and zinc
The diastereomeric purity of 2d and 6d were determined by HPLC chloride in dichloromethane as described above for the synthesis
analysis using a Kromasil cyano column (250 mm × 4.6 mm, 10 of 2f. A mixture of 2 diastereomers bearing the N-methyl-L-alanyl
micron particle size) at a flow rate of 1.50 mL/min, eluting with moiety (2g, S,S) and the N-methyl-D-alanyl moiety (6g, R,S) were
an isocratic mixture of ethyl acetate/hexane/2-propanol (24:66:10/ obtained. The diastereomers were separated by HPLC on a Kromasil
v/v). Under these conditions, 2d eluted with a retention time of cyano column (4.6 mm × 250 mm), using an isocratic elution at a
11.96 min, and its D-alanyl 6d isomer eluted with a retention time flow arte of 1 mL/min with hexane/ethyl acetate/2-propanol (68:
of 17.30 min. 24:8, v/v/v). Under these conditions, the isomer 2g (S,S) eluted at
2d. 1H NMR (CDCl3) δ 0.803 (3H, s), 1.265 (2H, m), 1.280 24.5 min. The peak for the other isomer 6g (R,S) was well separated
(3H, d, J ) 2.4 Hz), 1.309 (3H, d, J ) 2.4 Hz), 1.638 (1H, d, J ) and eluted at 34.6 min.
13.6 Hz), 1.643 (3H, s), 1.760 (1H, m), 2.051 (2H, m), 2.179 (1H, 2g. 1H NMR (CDCl3) δ 0.798 (3H, s), 1.197-1.267 (2H, m),
dd, J ) 11.2, 2.8 Hz), 2.318 (3H, s), 2.365-2.536 (2H, m), 2.611 1.292 (3H, d, J ) 7.2 Hz), 1.283 (3H, d, J ) 2 Hz), 1.300 (3H, d,
(1H, dd, J ) 12.4, 2 Hz), 2.719 (2H, m), 2.852 (3H, s), 3.033 (1H, J ) 1.2 Hz), 1.462 (1H, m), 1.536 (1H, d, J ) 8 Hz), 1.640 (3H,
d, J ) 9.6 Hz), 3.110 (1H, d, J ) 12.4 Hz), 3.219 (3H, s), 3.355 s), 1.834-2.020 (2H, m), 2.176 (1H, dd, J ) 11.6, 2.8 Hz), 2.326
(3H, s), 3.498 (1H, d, J ) 10.8 Hz), 3.657 (1H, d, J ) 12.4 Hz), (3H, s), 2.373-2.522 (2H, m), 2.603 (1H, dd, J ) 12.4, 2 Hz),
3.983 (3H, s), 4.284 (1H, t), 4.872 (1H, dd, J ) 8.8, 2.8 Hz), 5.660 2.847 (3H, s), 2.871-2.903 (1H, m), 3.028 (1H, d, J ) 9.6 Hz),
(1H, dd, J ) 8.8, 5.6 Hz), 6.197 (1H, s), 6.430 (1H, dd, J ) 11.2, 3.108 (1H, d, J ) 12.4 Hz), 3.223 (3H, s), 3.351 (3H, s), 3.499
4 Hz), 6.648 (1H, d, J ) 2 Hz), 6.740 (1H, d, J ) 11.6 Hz) and (1H, d, J ) 9.2 Hz), 3.639 (1H, d, J ) 12.8 Hz), 3.983 (3H, s),
4404 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 Widdison et al.

4.279 (1H, t), 4.627 (1H, dd, J ) 8.8, 3.2 Hz), 5.388 (1H, m), (1H,d,J ) 11 Hz), 3.23 (3H,s), 3.35 (3H,s), 3.49 (1H, d, J ) 9
5.658 (1H, dd, J ) 8.8, 6.4 Hz), 6.207 (1H, s), 6.426 (1H, dd, J ) Hz), 3.63 (1H, d, J ) 12 Hz), 3.98 (3H, s), 4.27 (1H, t, J ) 10
11.2, 4.4 Hz), 6.639 (1H, d, J ) 1.6 Hz), 6.737 (1H, d, J ) 11.2 Hz), 4.79 (1H, dd, J ) 3 and 12 Hz), 5.41 (1H, q, J ) 7 Hz), 5.66
Hz) and 6.818 (1H, d, J ) 2 Hz). MS: m/z 834.2 (M + Na)+. (1H, dd J ) 9 and 15 Hz), 6.21 (1H, s), 6.42 (1H, dd, J ) 11 and
6g. 1H NMR (CDCl3) δ 0.867 (3H, s), 1.251-1.383 (3H, m), 15 Hz), 6.65 (1H, d, J ) 1.5 Hz), 6.73 (1H, d, J ) 11 Hz), 6.81
1.284 (3H, d, J ) 6 Hz), 1.374 (3H, d, J ) 6.8 Hz), 1.498 (3H, d, (1H, d, J ) 1.5 Hz). HRMS (M + H)+ calcd for C39H57ClN3O10S2.
J ) 7.6 Hz), 1.691 (3H, s), 1.763 (1H, d, J ) 14 Hz), 1.932 (2H, (M + H)+, 826.3174; found, 826.3150; extinction coefficient
m), 2.206 (1H, dd, J ) 11.2, 3.2 Hz), 2.421 (3H, s), 2.518 (2H, (ethanol) 280 nm ) 5140 M-1 cm-1, 252 nm ) 24 700 M-1 cm-1.
m), 2.653 (1H, dd, J ) 12.4, 2.4 Hz), 2.817 (1H, d, J ) 9.6 Hz), 6i (D-DM4SMe). White solid; mp 165-167 °C; 1H NMR
2.911 (1H, m), 3.034 (3H, s), 3.165 (3H, s), 3.200 (1H, d, J ) (CDCl3) δ 0.869 (3H, s), 1.265 (3H, d, J ) 7.2 Hz), 1.327 (6H, s),
13.2 Hz), 3.339 (3H, s), 3.438 (1H, d, J ) 9.2 Hz), 3.508 (1H, d, 1.495 (3H, d, J ) 7.6 Hz), 1.590 (2H, m), 1.690 (3H, s), 1.785
J ) 12.8 Hz), 3.996 (3H, s), 4.311 (1H, t), 4.929 (1H, dd, J ) 8.8, (1H, m), 1.915 (2H, m), 2.201 (1H, dd, J ) 17.6 and 11.6 Hz),
3.2 Hz), 5.095 (1H, m), 5.856 (1H, dd, J ) 9.2, 6 Hz), 6.236 (1H, 2.422 (3H, s), 2.452 (2H, m), 2.646 (1H, d, J ) 26.8 and 2.4 Hz),
d, J ) 13.2 Hz), 6.253 (1H, s), 6.428 (1H, dd, J ) 11.2, 4 Hz), 2.800 (1H, d, J ) 9.6 Hz), 3.041 (3H, s) 3.165 (3H, s), 3.195 (1H,
6.794 (1H, d, J ) 1.6 Hz) and 6.849 (1H, d, J ) 1.2 Hz). MS: m/z d, J ) 13.2 Hz), 3.340 (3H, s), 3.431 (1H, d, J ) 9.2 Hz), 3.508
834.2 (M + Na)+. (1H, d, J ) 12.8 Hz), 3.995 (3H, s), 4.313 (1H, t), 4.960 (1H, dd,
N2′-Deacetyl-N2′-(4-(R)-methyldithio-1-oxopentyl)maytan- J ) 15.2 and 8.8 Hz), 5.117 (1H, q, J ) 7.6 Hz), 5.838 (1H, dd,
sine (2h). Maytansinol (4) was coupled with N-methyl-N-[(4-(R)- J ) 24.4 and 6 Hz), 6.233 (1H, d, J ) 10 Hz), 6.246 (1H, s),
methyldithio)-1-oxopentyl]-S-alanine (5h) using DCC and zinc 6.428 (1H, dd, J ) 26 and 4 Hz), 6.785 (1H, d, J ) 1.6) and 6.847
chloride in dichloromethane as described above. A mixture of 2 (1H, d, J ) 1.6 Hz). HRMS calcd C39H56ClN3O10S2Na (M + Na)+,
diastereomers bearing the N-methyl-S-alanyl moiety (2h, S,R) and 848.2993; found, 848.2975.
the N-methyl-R-alanyl moiety (6h, R,R) were obtained. The N2′-Deacetyl-N2′-(3-mercapto-1-oxopropyl)-maytansine (DM1,
diastereomers were separated by HPLC on a Kromasil cyano 25a). A solution of N2′-deacetyl-N2′-(3-methyldithio-1-oxopropyl)-
column (4.6 mm × 250 mm), using an isocratic elution at a flow maytansine (2b) (1.95 g, 2.5 mmol) in a mixture of ethyl acetate
rate of 1 mL/min, with hexane/ethyl acetate/2-propanol (68:24:8, (140 mL) and methanol (210 mL) was stirred at room temperature
v/v/v). Under these conditions, isomer 2h (S.R) eluted at 23.9 min. under an argon atmosphere and treated with a solution of dithio-
The peak for the other isomer 6h (R,R) was well separated and threitol (0.95 g, 6.2 mmol) in 0.05 M potassium phosphate buffer
eluted at 33.7 min. (140 mL) at pH 7.5 containing 2 mM ethylenediaminetetraacetic
2h. 1H NMR (CDCl3) δ 0.796 (3H, s), 1.230 (2H, m), 1.290 acid (EDTA). The progress of the reaction was monitored by HPLC
(3H, d, J ) 5.6 Hz), 1.297 (3H, d, J ) 1.2 Hz), 1.305 (3H, d, J ) and was complete in three hours. The reaction mixture was treated
6.4 Hz), 1.447 (1H, m), 1.575 (1H, d, J ) 13.6 Hz), 1.637 (3H, s), with a solution of 0.2 M potassium phosphate buffer (250 mL) at
1.815-2.002 (2H, m), 2.170 (1H, dd, J ) 11.2, 3.2 Hz), 2.303 pH 6.0 containing 2 mM EDTA and then extracted with ethyl
(3H, s), 2.357-2.547 (2H, m), 2.606 (1H, dd, J ) 12, 2.4 Hz), acetate (3 × 600 mL). The organic layers were combined, washed
2.846 (3H, s), 3.027 (1H, d, J ) 9.6 Hz), 3.108 (1H, d, J ) 12.8 with brine (100 mL), and then dried over sodium sulfate. Evapora-
Hz), 3.215 (3H, s), 3.350 (3H, s), 3.494 (1H, d, J ) 9.6 Hz), 3.648 tion of the solvent gave a residue of crude thiol-containing
(1H, d, J ) 12.8 Hz), 3.978 (3H, s), 4.273 (1H, t), 4.775 (1H, dd, maytansinoid 25a. The crude residue was purified by HPLC using
J ) 9.2, 2.8 Hz), 5.384 (1H, m), 5.665 (1H, m), 6.227 (1H, s), a preparative Diazem cyano HPLC column (250 mm × 50 mm,
6.634 (1H, d, J ) 1.6 Hz), 6.734 (1H, d, J ) 11.2 Hz) and 6.816 10 micron particle size) that was equilibrated in a mixture of
(1H, d, J ) 1.6 Hz). MS: m/z 834.2 (M + Na)+. hexanes/2-propanol/ethyl acetate (78.0:5.5:16.5, v/v/v) and run at
6h. 1H NMR (CDCl3) δ 0.867 (3H, s), 1.280 (3H, d, J ) 6.4 a flow rate of 150 mL/min. The desired product 25a eluted as a
Hz), 1.359 (2H, m), 1.384 (3H, d, J ) 6.4 Hz), 1.446 (1H, m), peak centered at 16 min. The fractions containing the product were
1.495 (3H, d, J ) 7.2 Hz), 1.689 (3H, s), 1.780 (1H, d, J ) 13.4 evaporated to give 25a as a white solid (76% yield); mp 190-192
Hz), 1.886-1.964 (2H, m), 2.200 (1H, dd, J ) 11.6, 2.8 Hz), 2.419 °C (dec); 1H NMR (CDCl3) δ 0.84 (3H, s), 1.33 (3H, d, J ) 5 Hz),
(3H, s), 2.470-2.564 (2H, m), 2.651 (1H, dd, J ) 12, 2.4 Hz), 1.35 (3H, d, J ) 5 Hz), 1.60 (3H, s), 1.68 (3H, s), 2.22 (1H, dd,
2.808 (1H, d, J ) 9.6 Hz), 2.893 (1H, m), 3.040 (3H, s), 3.161 J ) 3 and 14 Hz, 2.60-2.82 (2H, m), 2.88 (3H, s), 3.08-3.20
(3H, s), 3.196 (1H, d, J ) 13.2 Hz), 3.338 (3H, s), 3.432 (1H, d, (2H, m), 3.25 (3H, s), 3.39 (3H, s), 3.55 (1H, d, J ) 9 Hz), 3.71
J ) 9.2 Hz), 3.504 (1H, d, J ) 12.8 Hz), 3.993 (3H, s), 4.303 (1H, (1H, d, J ) 12 Hz), 4.02 (3H, s), 4.32 (1H, t, J ) 10 Hz), 4.81
t), 4.938 (1H, dd, J ) 8.8, 3.2 Hz), 5.091 (1H, m), 5.842 (1H, m), (1H, dd, J ) 3 and 12 Hz), 5.45 (1H, q, J ) 7 Hz), 5.67 (1H, dd
6.234 (1H, d, J ) 12.8 Hz), 6.250 (1H, s), 6.428 (1H, dd, J ) J ) 9 and 15 Hz), 6.25 (1H, s), 6.47 (1H, dd, J ) 11 and 15 Hz),
10.8, 4.4 Hz), 6.788 (1H, d, J ) 1.6 Hz) and 6.847 (1H, d, J ) 1.6 6.70 (1H, d, J ) 1.5 Hz), 6.75 (1H, d, J ) 11 Hz), 6.86 (1H, d,
Hz). MS: m/z 834.2 (M + Na)+. J ) 1.5 Hz). HRMS calcd for C35H49ClN3O10S (M + H)+ 738.2827;
N2′-Deacetyl-N2′-(4-methyl-4-(methyldithio)-1-oxopentyl)may- found, 738.2820; [R]25D ) -113.2, (c ) 0.306, CHCl3); extinction
tansine (DM4-SMe, 2i) and Its D-Alanyl Isomer D-DM4SMe. coefficient (methanol): 280 nm ) 5422 M-1 cm-1, 252 nm ) 25 800
Maytansinol (4) was reacted with N-methyl-N-[4-methyl-4-(meth- M-1 cm-1.
yldithio)-1-oxopentyl]-L-alanine (5i), as described above. The HPLC Analysis. HPLC analysis was performed using a Waters
reaction mixture was purified by silica chromatography using a symmetry shield analytical C-8 column (length: 150 mm, i.d.: 3.9
mixture of dichloromethane and methanol to remove the unreacted mm, particle size: 5 microns) operating at 40 °C and at a flow
maytansinol. The fractions containing the desired product were rate of 1 mL/min, eluting with a linear gradient of water (containing
combined, and the solvent was removed under vacuum to give a 0.05% TFA) and acetonitrile (containing 0.05% TFA) from 35%
mixture of diastereomers 2i and 6i. The residue was taken up in a to 45% acetonitrile over 30 min. Under these conditions, DM1 (25a)
minimum volume of ethyl acetate and purified on a 50 cm × 250 eluted with a retention time of 12.13 min. The diastereomeric purity
cm, 10 micron Diazem CN column using as mobile phase a mixture of 25a was determined by HPLC analysis using a Diazem cyano
of hexane, 2-propanol, and ethyl acetate at a ratio of 68:8:24. The column (length: 250 mm, i.d.: 4.6 mm, particle size: 5 microns)
flow rate was 118 mL/min. Under these conditions, the desired at a flow rate of 1 mL/min, eluting with a isocratic mixture of ethyl
product 2i eluted with a retention time of 11 min, and the undesired acetate/hexane/2-propanol (24:68:8 v/v respectively). Under these
diastereomer 6i had a retention time of 19 min. conditions, 25a eluted with a retention time of 13.70 min. There
2i (DM4SMe). White solid (36% yield); 1H NMR (CDCl3) δ was no detectable signal for the D-isomer 26a, which elutes at 17.3
0.80 (3H, s), 1.28-1.36 (13H, m), 1.42-1.46(2H, m), 1.53-1.63 min.
(2H, m), 1.64 (3H, s), 1.75-1.85 (1H, m), 1.90-2.10 (1H, m), D-DM1 (26a). A solution of D-DM1SMe (6b) (20 mg, 0.027
2.18 (1H, dd, J ) 3 and 14 Hz), 2.31 (3H,s), 2.40-2.49 (1H, m), mmol) in a mixture of ethyl acetate (0.5 mL) and methanol (1.0
2.50-2.65 (1H, m), 2.85 (3H, s), 3.04 (1H, d, J ) 9 Hz), 3.11 mL) was stirred at room temperature under an argon atmosphere
Semisynthetic Maytansine Analogues Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4405

and treated with a solution of dithiothreitol (6.1 mg, 0.039 mmol) hexane, 2-propanol, and ethyl acetate at a ratio of 70:8:22. The
in 0.05 M potassium phosphate buffer (0.5 mL) at pH 7.5 containing flow rate was 22 mL/min. The desired product eluted with a
2 mM ethylenediamine-tetraacetic acid (EDTA). The progress of retention time of 10 min. The fractions containing product were
the reaction was monitored by HPLC and was complete in 2 h. combined, and the solvent was removed under vacuum to give 11
The reaction mixture was then extracted with ethyl acetate (3 × 1 mg of 25c (97% yield) as a white solid; mp 185-187 °C (dec); 1H
mL). The organic layers were combined and dried over sodium NMR (CDCl3) δ 0.80 (3H, s), 1.19-1.23(1H,m), 1.28-1.36 (12H,
sulfate. Evaporation of the solvent gave a residue of crude thiol- m), 1.42-1.46(2H, m), 1.53-1.63 (2H, m), 1.64 (3H, s), 1.75-
containing maytansinoid 26a. The crude residue was purified by 1.85 (1H, m), 1.90-2.10 (1H, m), 2.18 (1H, dd, J ) 3 and 14 Hz),
HPLC using a semipreparative Diazem cyano HPLC column (250 2.40-2.49 (1H, m), 2.50-2.65 (2H, m), 2.88 (3H, s), 3.04 (1H, d,
mm × 10 mm, 10 micron particle size) that was equilibrated in a J ) 9 Hz), 3.11 (1H,d,J ) 11 Hz), 3.23 (3H,s), 3.35 (3H,s), 3.49
mixture of hexanes/2-propanol/ethyl acetate (65:10:25, v/v/v) and (1H, d, J ) 9 Hz), 3.63 (1H, d, J ) 12 Hz), 3.98 (3H, s), 4.27 (1H,
run at an initial flow rate of 2 mL/min. After 2 min, the flow rate t, J ) 10 Hz), 4.79 (1H, dd, J ) 3 and 12 Hz), 5.41 (1H, q, J )
was increased to 4.5 mL/min for the remainder of the run. The 7 Hz), 5.66 (1H, dd J ) 9 and 15 Hz), 6.21 (1H, s), 6.42 (1H, dd,
desired product eluted as a peak centered at 15 min. The fractions J ) 11 and 15 Hz), 6.65 (1H, d, J ) 1.5 Hz), 6.73 (1H, d, J ) 11
containing the product were evaporated to give 26a as a white solid; Hz), 6.81 (1H, d, J ) 1.5 Hz). HRMS calcd for C38H54ClN3O10-
mp 187-192 °C (dec); 1H NMR (CDCl3) δ 0.85 (3H, s), 1.26- SNa (M + Na)+, 802.3101; found, 802.3116. [R]25D ) -106.7,
1.29 (4H, m), 1.50 (3H, d, J ) 7.6 Hz), 1.69 (3H, s), 2.21 (1H, dd, (c ) 0.057, CHCl3), extinction coefficient (ethanol): 280 nm ) 5180
J ) 3 and 14 Hz), 2.65 (1H, dd, J ) 12 Hz and 14 Hz), 2.79-2.84 M-1 cm-1, 252 nm ) 26 200 M-1 cm-1. HPLC analysis: purity was
(3H, m), 2.92-2.96 (6H, m), 3.03 (3H, s), 3.16 (3H, s), 3.34 (3H, determined by HPLC analysis using a Vydac analytical C-18
s), 3.45 (1H, d, J ) 9 Hz), 3.51 (1H, d, J ) 12 Hz), 4.00 (3H, s), column (250 mm × 4.6 mm, 3 micron particle size) at a flow rate
4.04 91H, m), 4.29 (1H, m), 4.88 (1H, dd, J ) 3 and 12 Hz), 5.20 of 1.00 mL/min in a gradient of water and acetonitrile, eluting with
(1H, q, J ) 7 Hz), 5.90 (1H, dd, J ) 9 and 15 Hz), 6.23-6.27 37-58% acetonitrile over 25 min. Under these conditions L-DM4
(2H, m), 6.43 (1H, dd, J ) 11 and 15 Hz), 6.80 (1H, d, J ) 1.6 (25c) eluted with a retention time of 11.8 min. The purity of L-DM4
Hz), 6.85 (1H, d, J ) 1.6 Hz). HRMS calcd for C35H48ClN3O10- was 98.8%. The diastereomeric purity of 25c was determined by
SNa m/z: 760.2647 (M + H)+; found, 760.2637; [R]25D ) -135.0, HPLC analysis using a Diazem cyano column (50 mm × 4.6 mm,
(c ) 0.294, CHCl3). HPLC analysis: The diastereomeric purity of 5 micron particle size) at a flow rate of 1.00 mL/min, eluting with
26a was determined as described above for 26a. The isomer 26a a isocratic mixture of ethyl acetate/hexane/2-propanol (20:75:5
eluted with a retention time of 17.25 min. There was no detectable v/v/v). Under these conditions, 25c eluted with a retention time of
signal for the L-isomer, which elutes at 13.7 min, 4.52 min. There was no detectable signal for the D-alanyl isomer,
N2′-Deacetyl-N2′-(4-mercapto-1-oxopentyl)maytansine (DM3, which elutes at 5.42 min, indicating that 25c was a diastereomeri-
25b). DM3-SMe (2f, 12 mg, 0.015 mmol) was dissolved in 1.0 cally pure compound.
mL of a 1:1 mixture of ethyl acetate and methanol. A solution of
D-DM4 (26c). A solution of D-DM4SMe (6i) (40.0 mg, 0.048
dithiothreitol (18 mg, 0.117 mmol) in 0.50 mL of 50 mM phosphate
mmol) in a mixture of ethyl acetate (0.2 mL) and methanol (0.5
buffer at pH 7.5 was then added. The reaction solution was
mL) was stirred at room temperature under an argon atmosphere
magnetically stirred under an argon atmosphere for 3 h, then 1 mL
and treated with a 0.50 mL solution containing dithiothreitol (59.7
of 200 mM phosphate buffer at pH 6.0 was added, and the mixture
mg, 0.387 mmol) in 0.050 M potassium phosphate buffer (0.5 mL)
was extracted three times with 2 mL portions of ethyl acetate. The
at pH 7.5 and 2 mM ethylenediamine-tetraacetic acid (EDTA). The
organic layers were combined and washed with 1 mL of saturated
progress of the reaction was monitored by HPLC and was complete
sodium chloride solution and then dried over 1 g of anhydrous
after stirring overnight. The reaction mixture was treated 0.05 M
sodium sulfate. The solvent was removed under vacuum, and the
residue was taken up in a minimum of ethyl acetate and purified potassium phosphate buffer (1.0 mL) at pH 6.0 containing 2 mM
on a 50 cm × 250 cm, 10 micron Diazem CN column using as EDTA, and the product was extracted with ethyl acetate (3 × 1
mobile phase a 70:8:22 mixture of hexane, 2-propanol, and ethyl mL). The organic layers were combined and dried over sodium
acetate. The flow rate was 22 mL/min. The desired product eluted sulfate. Evaporation of the solvent gave a residue of crude thiol-
with a retention time of 10 min. The fractions containing the pure containing maytansinoid 26c. The crude residue was purified
product were combined, and the solvent was removed under vacuum by HPLC using a preparative Diazem cyano HPLC column
to give 11 mg of product 25b (97% yield). 1H NMR (CDCl3) δ (250 mm × 10 mm, 10 micron particle size) that was equilibrated
0.80 (3H, s), 1.19-1.23(1H,m), 1.28-1.36 (9H, m), 1.42-1.46- in a mixture of hexanes/2-propanol/ethyl acetate (65:10:25, v/v/v)
(1H, m), 1.53-1.63 (2H, m), 1.64 (3H, s), 1.80-1.89 (1H, m), and run at an initial flow rate of 118 mL/min. The desired product
1.90-2.09 (1H, m), 2.18 (1H, dd, J ) 3 and 14 Hz), 2.33-2.42 26c eluted as a peak centered at 12.0 min. The fractions containing
(1H, m), 2.49- -2.62 (2H, m), 2.88 (3H, s), 3.04 (1H, d, J ) 9 Hz), the product were evaporated to give 26c as a white solid.
1H NMR (CDCl ) δ 0.866 (3H, s), 1.282 (3H, d, J ) 6.4 Hz),
3.11 (1H,d,J ) 11 Hz), 3.23 (3H,s), 3.35 (3H,s), 3.49 (1H, d, J ) 3
9 Hz), 3.63 (1H, d, J ) 12 Hz), 3.98 (3H, s), 4.27 (1H, t, J ) 10 1.409 (6H, s), 1.500 (3H, d, J ) 6.4 Hz), 1.625 (2H, m), 1.689
Hz), 4.79 (1H, dd, J ) 3 and 12 Hz), 5.41 (1H, q, J ) 7 Hz), 5.66 (3H, s), 1.766 (1H, m), 1.875 (2H, m), 2.205 (1H, dd, J ) 17.6
(1H, dd J ) 9 and 15 Hz), 6.21 (1H, s), 6.42 (1H, dd, J ) 11 and and 11.2 Hz), 2.578 (2H, m), 2.646 (1H, dd, J ) 26.8 and 2.8 Hz),
15 Hz), 6.65 (1H, d, J ) 1.5 Hz), 6.73 (1H, d, J ) 11 Hz), 6.81 2.811 (1H, d, J ) 9.6 Hz), 3.168 (3H, s), 3.196 (1H, d, J ) 12.4
(1H, d, J ) 1.5 Hz). HRMS calcd for C37H52ClN3O10SNa (M + Hz), 3.341 (3H, s), 3.431 (1H, d, J ) 9.2 Hz), 3.511 (1H, d, J )
Na)+, 788.2960; found, 788.2957. 13.2 Hz), 3.995 (3H, s), 4.300 (1H, t), 4.946 (1H, dd, J ) 15.2 and
N2′-Deacetyl-N2′-(4-mercapto-4-methyl-1-oxopentyl)maytan- 8.8 Hz), 5.156 (1H, q, J ) 7.6 Hz), 5.836 (1H, dd, J ) 24.4 and
sine (DM4, 25c). DM4SMe (2i) (12 mg, 0.015 mmol) was dissolved 6 Hz), 6.243 (1H, d, J ) 10.4 Hz), 6.246 (1H, s),6.426 (1H, dd,
in 1.0 mL of 1:1 ethyl acetate/methanol. A solution of dithiothreitol J ) 26.4 and 4 Hz), 6.790 (1H, d, J ) 1.6 Hz) and 6.848 (1H, d,
(18 mg, 0.117 mmol) in 0.50 mL of 50 mM phosphate buffer at J ) 1.6 Hz). HRMS calcd for C38H54ClN3O10SNa (M + Na)+,
pH 7.5 was then added. The solution was magnetically stirred under 802.3101; found, 802.3099; [R]25D ) -90.4, (c ) 0.052, CHCl3).
an argon atmosphere for 3 h, then 1 mL of 200 mM phosphate HPLC analysis: purity was determined by HPLC analysis using a
buffer at pH 6.0 was added, and the mixture was extracted three Vydac analytical C-18 column (250 mm × 4.6 mm, 3 micron
times with 2 mL portions of ethyl acetate. The organic layers were particle size) at a flow rate of 1.00 mL/min with a gradient of water
combined and washed with 1 mL of saturated sodium chloride and acetonitrile, eluting with 20-50% acetonitrile over 30 min.
solution and then dried over 1 g of anhydrous sodium sulfate. The Under these conditions, D-DM4 (26c) eluted with a retention time
solvent was removed under vacuum, and the residue was taken up of 17.85 min. The purity of 26c was 97.8%. The diastereomeric
in a minimum of ethyl acetate and purified on a 50 cm × 250 cm, purity of 26c was determined as described above for DM4 (25c).
10 micron Diazem CN column using as mobile phase a mixture of The isomer eluted with a retention time of 5.42 min. There was no
4406 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 Widdison et al.

detectable signal for the 25c isomer, which elutes at 4.52 min, under reduced pressure. The resulting aqueous layer was extracted
indicating that 26c was a diastereomerically pure compound. with ethyl acetate (3 × 5 mL), and the combined organic layer
Synthesis of N2′-Deacetyl-N2′-[3-(3-carboxy-1-methyl-propyl- was washed with saturated sodium chloride solution (1 × 5 mL)
dithio)-1-oxopropyl]-maytansine (DM1-TPA, 2j). A solution of and then dried over sodium sulfate and filtered. The solvent was
4-(2-pyridyldithio)pentanoic acid (24 mg, 0.10 mmol) and DM1 evaporated under reduced pressure, and the residue was purified
(25a, 30 mg, 0.041 mmol) in glass distilled methanol (5 mL) was by HPLC using a Metachem Inertsil ODS column, eluting with a
vigorously stirred, and 3 mL of an aqueous buffer (200 mM KH2- gradient of water and acetonitrile to provide dimer 25d as a white
PO4, 2 mM EDTA at pH 7.6) was added dropwise. The reaction solid (13.7 mg, 52% yield). 1H NMR (CDCl3) δ 0.75 (3H, s), 1.23-
was strirred overnight, and the product was purified by HPLC using 1.26 (1H, m), 1.28 (3H, d, J ) 6 Hz), 1.30 (3H, d, J ) 7 Hz),
a Vydac C-18 column (10 × 250 mm) operating at 30 °C and a 1.42-1.52 (1H, m), 1.66 (3H, s), 2.15 (1H, m), 2.59 (1H, dd, J )
flow rate of 4.75 mL/min, eluting with a linear gradient of 6 and 14 Hz), 2.71-2.82 (2H, m), 2.84 (3H, s), 3.00 (1H, d, J )
acetonitrile (15% to 85% over 30 min) in 40 mM ammonium acetate 9 Hz), 3.09 (1H, d, J ) 12 Hz), 3.18 (3H, s), 3.35 (3H, s), 3.48
buffer at pH 7.2. DM1-TPA (2j) eluted with a retention time of 12 (1H, d, J ) 9 Hz), 3.61 (1H, d, J ) 13 Hz), 3.97 (3H, s), 4.26 (1H,
min. The product was collected as the ammonium salt was taken t, J ) 11 Hz), 4.76 (1H, dd, J ) 3 and 12 Hz), 5.33 (1H, q, J )
up in ethyl acetate (15 mL). The solution was washed with 1 M 7 Hz), 5.61 (1H, dd, J ) 9 and 15 Hz), 6.28 (1H, s), 6.41 (1H, dd,
HCl (4 mL) followed by saturated sodium chloride (3 mL). The J ) 11 and 15 Hz), 6.61 (1H, d, J ) 1.6 Hz), 6.65 (1H, d, J ) 11
organic layer was dried over anhydrous sodium sulfate, and the Hz), 6.81 (1H, d, J ) 1.5 Hz). HRMS: calcd for C70H94Cl2N6O20-
solvent was removed under vacuum to give 15 mg (37% yield) of S2Na (M + Na)+, 1495.5239, found 1495.5242; extinction coef-
product 2j. 1H NMR (400 MHz, CDCl3) δ 0.81 (3H, s), 1.21- ficients (methanol): 280 nm ) 11 195 M-1 cm-1, 252 nm ) 68 035
1.37 (11H, m), 1.44-1.48 (1H, m), 1.61 (1H, m), 1.65 (3H, s), M-1 cm-1. HPLC analysis was performed using a Vydac analytical
1.77-1.93 (3H, m), 2.20 (1H, d, J ) 12 Hz), 2.39-2.49 (2H, m), C-18 column (length: 100 mm, i.d.: 4.6 mm, particle size: 3
2.60 (1H, d, J ) 12 Hz), 2.70 (1H, t, J ) 9 Hz), 2.78-3.00 (7H, microns) at a flow rate of 1 mL/min, eluting with a gradient of
m), 3.12 (1H, d, J ) 14 Hz), 3.22 (3H, s), 3.36 (3H, s), 3.48 (1H, water and acetonitrile as follows: 0 min 40% CH3CN, 5 min 40%
d, J ) 9 Hz), 3.64 (1H, d, J ) 12 Hz), 3.98 (3H, s), 4.31 (1H, t, CH3CN, 5-30 min 40-60% CH3CN. Under these conditions, DM1
J ) 10 Hz), 4.82 (1H, d, J ) 12 Hz), 5.15-5.20 (1H, m), 5.63 dimer eluted with a retention time of 13.69 min.
(1H, dd J ) 9 and 15 Hz), 6.39-6.45 (2H, m), 6.59 (1H, t, J ) 12 Synthesis of N2′-Deacetyl-N2′-(4-mercapto-4-methyl-1-oxopen-
Hz), 6.67 (1H, d, J ) 9 Hz), 6.82 (1H, s). HRMS calcd for C40H56- tyl)-maytansine Dimer (DM4 Dimer, 25e). A solution of 25c
ClN3O12S2Na (M + Na)+, 892.2891; found, 892.2955. HPLC (100.0 mg, 0.128 mmol) in a 1:1 (v/v) mixture of dichloromethane
analysis was performed using a Vydac analytical C-18 column and ethyl acetate (4 mL) was treated with a solution of CuCl2 (85.76
(length: 100 mm, i.d.: 4.6 mm, particle size: 3 microns) at a flow mg, 0.640 mmol) in 0.50 M HCl (3 mL). The reaction mixture
rate of 1 mL/min, eluting with a gradient of water and acetonitrile was stirred at room temperature for 2 h. The product was extracted
as follows: 0 min: 5% CH3CN, 5 min: 5% CH3CN, 5-65 min: with ethyl acetate (5 mL), and the aqueous phase was separated.
5-95% CH3CN. Under these conditions DM1-TPA eluted with a An additional extraction of the aqueous phase with ethyl acetate
retention time of 23.75 min. (3 mL) was combined with the original mixture and washed with
Synthesis of N2′-Deacetyl-N2′-[4-methyl-4-(3-carboxy-propyl- brine (2 mL) and dried over anhydrous Na2SO4 and filtered. The
dithio)-1-oxopentyl]-maytansine (DM4-TBA, 2k). A solution of solvent was evaporated under reduced pressure to give the L-DM4-
4-(2-pyridyldithio)butanoic acid (41.1 mg, 0.179 mmol) and DM4 dimer 25e (11.7 mg, 12% yield). 1H NMR (CDCl3) δ 0.796 (6H,
(25c, 70.0 mg, 0.09 mmol) in glass distilled methanol (7 mL) was s), 1.136 (6H, d, J ) 4 Hz), 1.227 (2H, m), 1.298 (12H, s), 1.299
vigorously stirred, and 5 mL of an aqueous buffer (50 mM KH2- (6H, d, J ) 12 Hz), 1.463 (2H, m), 1.633 (6H, s), 1.670-1.842
PO4, 2 mM EDTA at pH 7.5) was added dropwise. The reaction (4H, m), 2.171 (2H, dd, J ) 17.2 and 11.2 Hz), 2.262-2.456 (4H,
was monitored by HPLC and was complete after 3 h. The solvent m), 2.596 (2H, dd, J ) 26.4 and 2 Hz), 2.842 (6H, s), 3.028 (2H,
was evaporated, and the residue was purified by column chroma- d, J ) 9.6 Hz), 3.105 (2H, d, J ) 12.4 Hz), 3.209 (6H, s), 3.353
tography over silica gel, eluting with a mixture of dichloromethane/ (6H, s), 3.489 (2H, d, J ) 8.8 Hz), 3.634 (2H, d, J ) 12.8 Hz),
methanol (97:3, v/v containing 0.1% glacial acetic acid) to give 45 3.966 (6H, s), 4.277 (2H, t), 4.766 (2H, dd, J ) 14.8 and 9.2 Hz),
mg (57% yield) of the purified product DM4-TBA (2k). 1H NMR 5.366 (2H, q, J ) 6.8 Hz), 5.644 (2H, dd, J ) 24.4 and 6.4 Hz),
(CDCl3) 0.800 (3H, s), 1.239 (6H, s), 1.302 (3H, d, J ) 2 Hz), 6.346 (2H, s), 6.417 (2H, dd, J ) 26.4 and 4 Hz), 6.623 (2H, d,
1.303 (3H, d, J ) 15.6 Hz), 1.414-1.513 (1H, m), 1.601 (2H, m), J ) 1.6 Hz), 6.708 (2H, d, J ) 11.2 Hz) and 6.816 (2H, d, J ) 1.6
1.642 (3H, s), 1.813-1.991 (2H, m), 1.932 (2H, t), 2.188 (1H, dd, Hz). HRMS calcd for C76H106Cl2N6O20S2Na (M + Na)+, 1579.6178;
J ) 17.2 and 11.2 Hz), 2.346-2.530 (2H, m), 2.415 (2H, t), 2.636 m/z: found, 1579.6180. HPLC analysis was performed using a
(1H, dd, J ) 18 and 12 Hz), 2.667 (2H, t), 2.875 (3H, s), 3.001 Vydac analytical C-18 column (length: 100 mm, i.d.: 4.6 mm,
(1H, d, J ) 9.6 Hz), 3.121 (1H, d, J ) 12.4 Hz), 3.219 (3H, s), particle size: 3 microns) at a flow rate of 1 mL/min, eluting with
3.356 (3H, s), 3.492 (1H, d, J ) 8.8 Hz), 3.641 (1H, d, J ) 12.8 a gradient of water and acetonitrile (0 min: 5% CH3CN, 5 min:
Hz), 3.981 (3H, s), 4.293 (1H, t), 4.791 (1H, dd, J ) 15.2 and 8.8 5% CH3CN, 5-65 min: 5-95% CH3CN. HPLC analysis was
Hz), 5.324 (1H, q, J ) 6.8 Hz), 5.667 (1H, dd, J ) 24.4 and 6.4 performed using a Vydac analytical C-18 column (length: 100 mm,
Hz), 6.399 (1H, s), 6.421 (1H, dd, J ) 26.4 and 4 Hz), 6.500 (1H, i.d.: 4.6 mm, particle size: 3 microns) at a flow rate of 1 mL/min,
d, J ) 1.6 Hz), 6.674 (1H, d, J ) 11.2 Hz) and 6.826 (1H, d, J ) eluting with a gradient of water and acetonitrile (37-58% CH3CN
1.6 Hz). HRMS calcd for C42H60ClN3O12S2Na (M + Na)+, over 25 min). Under these conditions, the DM4 dimer eluted with
920.3186; found, 920.3192. HPLC analysis was performed using a retention time of 22.48 min.
a Vydac analytical C-18 column (length: 100 mm, i.d.: 4.6 mm, N-Succinimidyl 4-(2-pyridyldithio)pentanoate (SPP, 27). A
particle size: 3 microns) at a flow rate of 1 mL/min, eluting with solution of 2.2′-dithiodipyridine (300 g, 1.36 mol) in a mixture of
a linear gradient of 40 mM ammonium acetate buffer at pH 7.2 ethanol (1 L) and glacial acetic acid (42 mL) was placed in a flask
and acetonitrile as follows: 0 min: 5% CH3CN, 5 min: 5% CH3- and stirred under an argon atmosphere. A solution of 4-mercapto-
CN, 5-35 min: 5-35% CH3CN, Under these conditions DM4- pentanoic acid (101.8 g, 0.76 mol) in ethyl acetate (400 mL) was
TBA eluted with a retention time of 18.40 min. added dropwise over 15 min, and the reaction was stirred for an
Synthesis of N2′-Deacetyl-N2′-(3-mercapto-1-oxopropyl)-may- additional 2 h. The solvent was removed by rotary evaporation,
tansine dimer (DM1 dimer, 25d). A solution of 25a (26.4 mg, and the residue was purified by column chromatography over silica
0.035 mmol) in a 1:1 (v/v) mixture of ethanol and 0.05 M potassium gel, eluting with a mixture of hexanes/ethyl acetate/acetic acid (4:
phosphate buffer at pH 7.5 (5 mL) was treated with a solution of 1:0.1) to give 4-(2-pyridyldithio)pentanoic acid (40 g, 21.4% yield).
2,2′-dithiodipyridine (3.9 mg, 0.017 mmol) in ethanol (1 mL). The 1H NMR in CDCl δ 1.34 (3H, d, J ) 7.0 Hz), 1.72-1.95 (2H,
3
reaction mixture was stirred at room temperature for 2 h. Most of m), 2.52-2.63 (2H, m), 2.91-3.02 (1H, m), 7.08-7.13 (1H, m),
the ethanol in the reaction mixture was removed by evaporation 7.62-7.77 (2H, m), 8.46-8.49 (1H, m), 11.5 (1H, br s).
Semisynthetic Maytansine Analogues Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4407

A solution of 4-(2-pyridyldithio)pentanoic acid (30 g, 123 mmol) 280 nm ) 217 560 M-1 cm-1. The number of pyridylthio groups
in dichloromethane (525 mL) was treated with N-hydroxysuccin- introduced per antibody molecule was determined using a spec-
imide (14.3 g, 124 mmol) and 1-[3-(dimethylamino)propyl]-3- trophotometric assay. An aliquot of the modified antibody was
ethylcarbodiimide (EDC, 31.8 g, 165 mmol). The contents were treated with an excess (>20 equiv) of dithiothreitol and the release
stirred at room temperature for 2 h after which ethyl acetate (750 of pyridine-2-thione determined using the known extinction coef-
mL) was added. The solution was washed with 0.5% aqueous acetic ficients of 343 nm ) 8080 M-1 cm-1 and 280 nm ) 5100 M-1 cm-1
acid (3 × 300 mL) and then with saturated aqueous NaCl (150 for pyridine-2-thione.
mL). The organic layer was separated and dried over anhydrous
The modified antibody was diluted to 2.5 mg/mL in aqueous
sodium sulfate and filtered, and the solvent was evaporated. The
buffer (50 mM potassium phosphate, 50 mM sodium chloride, 2
residue was purified by column chromatography over silica gel,
mM ethylenediaminetetraacetic acid disodium salt) at pH 6.5. The
eluting with hexanes/ethyl acetate (1:1). The fractions containing
the pure product were combined, and the solvent was removed by SPP-modified antibody was treated with 1.7 equiv of DM1 (25a),
rotary evaporation. The resulting oil (31 g) was taken up in a whereas the SPDB-modified antibody was treated with a 1.7 equiv
minimum volume of warm ethanol and stirred as ethyl ether (350 of either DM3 (25b) or DM4 (25c) dissolved in glass-distilled DMA
mL) was added, followed by hexanes (100 mL). The resulting (final concentration of DMA was 3% v/v). The reaction mixture
precipitate was collected by vacuum filtration and dried in a vacuum was incubated for 18 h at room temperature. The reaction mixture
oven at 30 °C for 12 h, to give SPP (27) as a white solid (18.7 g, was purified by passage through a Sephadex G25 gel filtration
45% yield); mp 48-9 °C; 1H NMR (CDCl3) δ 1.36(3H, d, J ) 7.0 column to remove the unconjugated drug and other low molecular
Hz), 1.97-2.8 (2H, m), 2.73-2.86 (2H, m), 2.84 (4H, br s), 3.02- weight species. The concentration of the conjugate was determined
3.11 (1H, m), 7.08-7.13 (1H, m), 7.62-7.77 (2H, m), 8.46-8.49 spectrophotometrically using the known extinction coefficients for
(1H, m); Anal. (C14H16N2O4S2) C, H, N, S. the antibody (280 nm ) 217 560 M-1 cm-1 and 252 nm ) 80 062
N-Succinimidyl 4-(2-pyridyldithio)butanoate (SPDB, 28). A M-1 cm-1) and the drugs. The resulting conjugate was monomeric
solution of 2,2′-dithiopyridine (3.8 g, 17.3 mmol) in a mixture of and contained, on the average, 3.2-3.5 maytansinoid molecules
acetic acid (0.5 mL) and ethyl alcohol (20 mL) was stirred under linked per antibody molecule.
an argon atmosphere. A solution of 4-mercaptobutyric acid (7c, Cytotoxicity Assays in Vitro. The cell lines used in cytotoxicity
1.39 g, 11.6 mmol) in ethyl acetate (10 mL) was added dropwise assays were (1) KB (ATCC CCl-17), a cell line of human epithelial
over approximately 2 min. The contents were stirred for 2 h, and origin, (2) SK-BR-3 (ATCC HTB-30), a cell line established from
the solvent was evaporated under reduced pressure. The residue a human breast adenocarcinoma, (3) COLO 205 (ATCC CCL-222),
was taken up in a minimum volume of ethyl acetate and purified a human colon tumor cell line, and (4) A-375 (ATCC CRL 1619),
by column chromatography on silica gel, eluting with a mixture of a human melanoma cell line. The cell lines were grown in
hexanes/ethyl acetate/acetic acid (65:33:2). The fractions containing Dulbecco’s modified Eagles Medium (DMEM, Biowhittaker,
the pure product were combined, and the solvent was removed under Walkersville, MD) with L-glutamine supplemented with 10% fetal
vacuum to give 1.5 g (6.55 mmol, 56% yield) of 4-(2-pyridyldithio)-
bovine serum (Hyclone, Logan, UT) and 50 µg/mL of gentamycin
butanoic acid. 1H NMR (CDCl3) δ 2.0-2.1(2H, m), 2.505(1H, t,
sulfate (Life Technologies, Rockville, MD). The cells were
J ) 7 Hz), 2.508 (1H, t, J ) 7), 2.74 (1H, t, J ) 7 Hz), 2.86 (1H,
maintained at 36-37.5 °C in a humidified atmosphere that
t, J ) Hz), 7.10-7.13 (1H, m), 7.67 (1H, dt, J ) 1, J ) 5), 7.73
(1H, d, J ) 8 Hz), 8.49 (1H, dd, J ) 0.7, J ) 5 Hz), 11.81 (1H, contained 6% CO2.
br s). The cytotoxicity study was performed using a clonogenic assay
A flask containing a stir bar was charged with 4-(2-pyridyldithio)- as previously described.23,24 The test cell lines were plated into
butanoic acid (1.1 g, 4.8 mmol) in dichloromethane (20 mL). 6-well culture dishes at a constant number of 1000 cells per well.
N-Hydroxysuccinimide (0.64 g, 5.6 mmol) and 1-[3-(dimethylami- The cells were incubated with varying concentrations (0 to 3 nM)
no)propyl]-3-ethylcarbodiimide (EDC, 1.1 g, 5.6 mmol) were then of the various maytansinoids (free or conjugated to antibodies) for
added. The contents were stirred at room temperature for 2 h after 72 h. The medium was then aspirated from the plates and replaced
which ethyl acetate (35 mL) was added. The reaction mixture was with fresh medium. The cultures were allowed to grow and form
washed with 0.5 M HCl (3 × 10 mL) and then with saturated colonies for a total of 7-10 days after plating. The cultures were
aqueous NaCl (1 × 10 mL). The organic layer was separated and then fixed and stained with 0.2% crystal violet in 10% formalin/
dried over anhydrous sodium sulfate and filtered. The solvent was PBS, and the colonies were counted. Plating efficiency of nontreated
evaporated, and the residue was purified by column chromatography cells (medium alone) was determined by dividing the number of
on silica gel, eluting with a mixture of hexanes/ethyl acetate/acetic colonies counted by the number of cells plated. The surviving
acid (71:28.8:0.2). The fractions containing the pure product were fraction of cells exposed to the drugs was determined by dividing
combined, and the solvent was removed under vacuum. The the number of colonies in wells that were exposed to the drug by
resulting oil was taken up in a minimum volume of warm reagent- the number of colonies in the control wells.
grade ethanol and stirred while ethyl ether (30 mL) was added,
Antitumor Activity in Vivo. The in vivo efficacy of conjugates
followed by hexanes (9.0 mL). The resulting precipitate was
of maytansinoids DM1 (25a), DM3 (25b), and DM4 (25c) with
collected by filtration and dried in a vacuum oven at 30 °C for 12
the huC242 antibody was evaluated in a human colon tumor
h to give 0.50 g of SPDB (28) as a white solid (∼31% yield); mp
47-48 °C; 1H NMR in CDCl3 δ 2.16 (2H, tt, J ) 7, J ) 15 Hz), zenograft model established with COLO 205 cells. Five-week-old
2.795 (2H, t, J ) 7 Hz), 2.83(4H, br s), 2.91(1H, t, J ) 7 Hz), female SCID mice (20 animals) were inoculated subcutaneously
7.09 (1H, m), 7.65-7.67 (2H, m), 8.48 (1H, d, J ) 5 Hz); Anal. in the right flank with COLO 205 human colon carcinoma cells
(C13H14N2O4S2) C, H, N, S. (1.5 × 106 cells/mouse) in 0.1 mL of serum-free medium. The
Preparation of Antibody Conjugates with Maytansinoids 25a, tumors were grown for 11 days to an average size of 100 mm3.
25b, and 25c. A solution of huC242 antibody (8 mg/mL) in aqueous The animals were then randomly divided into four groups (5 animals
buffer (50 mM potassium phosphate, 50 mM sodium chloride, 2 per group). The first group of mice served as the control group
mM ethylenediaminetetraacetic acid disodium salt) at pH 6.5 was and were treated with the phosphate-buffered saline vehicle. The
incubated for 2 h with a 7- to 10-fold molar excess of either remaining three groups were treated with either huC242-DM1
N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP, 27, or N-suc- (DM1 dose of 75 µg/kg, qd × 5), huC242-DM3 (DM3 dose of
cinimidyl-4-(2-pyridyldithio)butanoate (SPDB, 28). The reaction 75 µg/kg, qd × 5), or huC242-DM4 conjugate (DM4 dose of 75
mixture was purified by passage through a Sephadex G25 gel µg/kg, qd × 5), administered intravenously. Tumor sizes were
filtration column to remove low molecular weight material. The measured twice weekly, and the tumor volumes were calculated
concentration of the antibody was determined spectrophoto- using the formula tumor volume ) 1/2(length × width × height).
metrically using the known extinction coefficients for the antibody The weight of the animals was also measured twice per week.
4408 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 Widdison et al.

Supporting Information Available: Analytical data (elemental (12) Helft, P. R.; Schilsky, R. L.; Hoke, F. J.; Williams, D.; Kindler, H.
analysis and HRMS and HPLC purity data) for target compounds. L.; Sprague, E.; DeWitte, M.; Martino, H. K.; Erickson, J.; Pandite,
This material is available free of charge via the Internet at http:// L.; Russo, M.; Lambert, J. M.; Howard, M.; Ratain, M. J. A phase
I study of cantuzumab mertansine administered as a single intravenous
pubs.acs.org. infusion once weekly in patients with advanced solid tumors. Clin.
Cancer Res. 2004, 10, 4363-4468.
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