Synthesis and characterization of a cobalamin-colchicine conjugate as a novel tumor-targeted cytotoxin

Article abstract of DOI:10.1021/jo049953w

Colchicine was derivatized at C7 with p-alkoxyacetophenone and conjugated to cobalamin (vitamin B₁₂) through an acid-labile hydrazone linker. The cobalamin moiety leads to preferential uptake of the cobalamin-colchicine prodrug by cancer cells,

Full text of DOI:10.1021/jo049953w

VOLUME 69, NUMBER 26
DECEMBER 24, 2004
© Copyright 2004 by the American Chemical Society
Synthesis and Characterization of a Cobalamin-Colchicine
Conjugate as a Novel Tumor-Targeted Cytotoxin
Joshua D. Bagnato,† Alanna L. Eilers,‡ Robert A. Horton,† and Charles B. Grissom*,†
Department of Chemistry, University of Utah, 315 S. 1400 E., Salt Lake City, Utah 84112-0850, and
MantiCore Pharmaceuticals, Inc., 2401 Foothill Dr., Salt Lake City, Utah 84109-1405
grissomc@chemistry.utah.edu
Received January 7, 2004
Colchicine was derivatized at C7 with p-alkoxyacetophenone and conjugated to cobalamin (vitamin
B12) through an acid-labile hydrazone linker. The cobalamin moiety leads to preferential uptake of
the cobalamin-colchicine prodrug by cancer cells, whereupon the hydrazone linker undergoes
hydrolysis in the lysosome to unmask colchicine, which acts as a potent cytotoxin by stabilizing
microtubules and causing cell death. The bioconjugate is stable in cell culture media and at neutral
pH but undergoes hydrolysis with a half-life of 138 min at pH 4.5. The colchicine-cobalamin
bioconjugate exhibits nanomolar LC50 values against breast, brain, and melanoma cancer cell lines
in culture. Attachment of colchicine to cobalamin is expected to increase the therapeutic index of
the drug by limiting the side effects caused by the current nonselective administration of tubulin-
targeted chemotherapeutic drugs.
A major challenge in cancer therapy is to selectively
target cytotoxic agents to tumor cells. In an effort to
decrease the undesirable side effects of small molecule
anticancer agents, many targeting approaches have been
examined. One of the most promising methods involves
the covalent attachment of a potent cytotoxic warhead
to a macromolecular carrier. Many carriers have been
tested with varying success, including soluble polymers,1-4
nanoparticles, proteins,5 micelles, liposomes, folate, and
antibodies.6-8
Vitamin B12 (hereafter referred to as cobalamin to
describe the naturally occurring 5′-deoxyadenosylcobal-
amin, methylcobalamin, hydroxocobalamin, cyanocobal-
amin, or a synthetic derivative that includes the corrin
ring and is recognized by B12 transport proteins and
enzymes) shows promise as a drug delivery system to
target the delivery of cytotoxic drugs to cancer cells.9
Rapidly dividing cells require abnormally high quantities
of cobalamin for the synthesis of thymidine to support
DNA replication prior to cell division.10 Cobalamin can
be covalently modified to allow the attachment of a
variety of drugs without disrupting the corrin ring
pharmacophore that is recognized by B12 transport
proteins.9 Several such cobalamin bioconjugates have
been prepared in our laboratory and tested in vitro and
in vivo.11-15
Colchicine has been used since the 16th century for a
broad range of diseases, including the treatment of gout
† University of Utah.
‡ MantiCore Pharmaceuticals, Inc.
(9) Hogenkamp, H., P. C.; Collins, D. A.; Grissom, C. B.; West, F.
G. In Chemistry and Biochemistry of B12; Banerjee, R., Ed.; John Wiley
& Sons, Inc.: New York, 1999; p 921.
(1) Luo, Y.; Prestwich, G. D. Bioconjugate Chem. 1999, 10, 755-
63.
(2) Duncan, R.; Kopeckova, P.; Strohalm, J.; Hume, I. C.; Lloyd, J.
B.; Kopecek, J. Br. J. Cancer 1988, 57, 147-56.
(3) Putnam, D.; Kopecek, J. Adv. Polym. Sci. 1995, 122, 59-123.
(4) Omelyanenko, V.; Kopeckova, P.; Gentry, C.; Shiah, J. G.;
Kopecek, J. J. Drug Target 1996, 3, 357-73.
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enault, F.; Cazabat, A.; Schumacher, P.; Unger, C.; Falken, U. J.
Pharm. Sci. 1998, 87, 338-46.
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8, 498-502.
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F. G.; Grissom, C. B. Enzym. Mech. 1999, 27, 150-54.
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B.; West, F. G.; Aziza, M. A.; El-Ahmady, O. Abstracts of Papers, 214th
National Meeting of the American Chemical Society, Las Vegas, NV,
September 7-11, 1997; American Chemical Society: Washington, DC,
1996; MEDI-123.
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G. R.; Greenfield, R. S.; Vyas, D. M. Bioconjugate Chem. 1991, 2, 133-
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R.; Bell, D. H.; Berkenkamp, S.; Hillenkamp, F. Anal. Chem. 1997,
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D.; Holcomb, R.; Hallett, W.; Tsou, H. R.; Upeslacis, J.; Shochat, D.;
Mountain, A.; Flowers, D. A.; Bernstein, I. Bioconjugate Chem. 2002,
13, 47-58.
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B.; West, F. G.; Aziza, M. A.; El-Ahmady, O. Abstracts of Papers, 213th
National Meeting of the American Chemical Society, San Francisco,
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1997; MEDI-123.
(15) Pinson, P. R. Ph.D. Thesis, University of Utah, 2002.
10.1021/jo049953w CCC: $27.50 © 2004 American Chemical Society
Published on Web 06/03/2004
J. Org. Chem. 2004, 69, 8987-8996
8987

Bagnato et al.
SCHEME 1. Synthesis of Colchicine Analog 2a
and acute gouty arthritis.16 Colchicine is cytotoxic and
has a mechanism of action similar to that of the taxanes
by acting as a spindle poison. Colchicine prevents tubulin
polymerization and blocks cell growth at metaphase in
mitosis. As a result of the success of the taxanes in cancer
therapy, colchicine research has experienced renewed
interest because of its inherent water solubility and
similar mechanism of action, as well as its commercial
availability and low cost. Colchicine has not been useful
for the treatment of cancer to date because of its over-
bearing systemic toxicity that produces unacceptable side
effects when administered intravenously.17-22 By using
cobalamin to preferentially deliver colchicine to cancer
cells, these side effects may be dramatically decreased.
Our previous work has focused on the delivery of the
alkylating agent chlorambucil and the topoisomerase
poison doxorubicin.11-14 These DNA-targeted drugs must
ultimately reach the nucleus to exert their cytotoxic
effect. To further probe the versatility of cobalamin as a
cancer drug delivery agent, we chose to deliver colchicine
as the cytotoxic warhead because it has a cytosolic target
that does not require transport across the nuclear
membrane. A cytosolic target should be more readily
delivered to its site of action as the drug does not have
to both escape the lysosome and accumulate in the
nucleus to achieve cell death. In addition, colchicine is a
cytotoxin significantly more potent than those previously
conjugated to cobalamin. The LC50 values range from low
micromolar to nanomolar in all cancer cell lines tested.
A major limitation to cobalamin delivery is that only a
finite amount of cobalamin can be transported into a cell
before all of the cellular receptors have been saturated.
The potent toxin colchicine may result in greater efficacy
because less of the drug is required to initiate cell death.
An acid-labile hydrazone has been used as the key
linkage between cobalamin and colchicine. The use of an
acid-labile hydrazone for the release of a drug at low pH
has been discussed widely in the literature.5-8,15,23-28
Gemtuzumab ozogamicin (Mylotarg) was the first anti-
body-cytotoxin bioconjugate that was approved by the
FDA for the treatment of acute myeloid leukemia. Gem-
tuzumab ozogamicin contains a calicheamicin warhead
that is linked to a monoclonal antibody via an acid-labile
hydrazone bond.8 A similar linker using 4-acetylphenoxy-
acetic acid was used as the electron-rich ketone in our
work. Because a hydrazone is stable at neutral pH, this
provides a stable bond until the bioconjugate undergoes
endocytosis and localization in the low pH environment
of the lysosome. Once the bioconjugate is inside of the
lysosome, the hydrazone undergoes hydrolysis to release
the active drug.
a (a) Boc2O, DMAP, TEA in CH3CN; (b) 2 M NaOCH3 in CH3OH;
(c) TFA (85% yield, 3 steps); (d) APAA, BOP, NMM (84% yield).
is a very low energy bond that can be cleaved by exposure
to visible light. Cancer cells were incubated with these
cobalamin bioconjugates and then exposed to light to
unmask the cytotoxin inside of the cell. Evidence in our
lab suggests that once the bioconjugate is taken into a
cell, the endosome becomes a lysosome and the intrave-
sicular pH drops. As an alternate method to light-
triggered release of the drug, we have used a pH-sensitive
linker that releases the drug in the acidic intralysosomal
environment.
Several analogues of colchicine have been prepared in
the past.23,29-31 Upon reviewing the literature it became
evident that modifications of the B ring at the C7 acetate
appeared to have the least effect on the overall toxicity
of the resulting analogue. Modifications of the A ring and
C ring tend to decrease toxicity of the analogue dramati-
cally, and therefore modification at the C7 position of
colchicine was selected for this study.
Results and Discussion
The C7 acetamido group of colchicine can be hydrolyzed
under forcing acidic conditions;32 however, this also
results in isomerization of the C ring, and requires a
tedious purification process. To avoid this isomerization,
a three-step process based on a modification of a pub-
lished procedure33 was utilized to obtain the nucleophilic
deacetylcolchicine (Scheme 1). The amide is first acylated
(23) Baker, M. A.; Gray, B. D.; Ohlsson-Wilhelm, B. M.; Carpenter,
D. C.; Muirhead, K. A. J. Controlled Release 1996, 40, 89-100.
(24) Hinman, L. M.; Hamann, P. R.; Wallace, R.; Menendez, A. T.;
Durr, F. E.; Upeslacis, J. Cancer Res. 1993, 53, 3336-3342.
(25) King, H. D.; Dubowchik, G. M.; Mastalerz, H.; Willner, D.;
Hofstead, S. J.; Firestone, R. A.; Lasch, S. J.; Trail, P. A. J. Med. Chem.
2002, 45, 4336-43.
(26) Kratz, F.; Beyer, U.; Schutte, M. T. Crit. Rev. Ther. Drug Carrier
Syst. 1999, 16, 245-88.
(27) Poole, B.; Ohkuma, S. J. Cell. Biol. 1981, 90, 665-9.
(28) Trail, P. A.; Willner, D.; Lasch, S. J.; Henderson, A. J.;
Greenfield, R. S.; King, D.; Zoeckler, M. E.; Braslawsky, G. R. Cancer
Res. 1992, 52, 5693-700.
(29) Gelmi, M. L.; Mottadelli, S.; Pocar, D.; Riva, A.; Bombardelli,
E.; De Vincenzo, R.; Scambia, G. J. Med. Chem. 1999, 42, 5272-6.
(30) Medrano, F. J.; Andreu, J. M.; Gorbunoff, M. J.; Timasheff, S.
N. Biochemistry 1989, 28, 5589-99.
(31) Plourde, R.; Phillips, A. T.; Wu, C. H.; Hays, R. M.; Chowdhury,
J.; Chowdhury, N.; Wu, G. Y. Bioconjugate Chem. 1996, 7, 131-7.
(32) Raffauf, R. F.; Farren, A. L.; Ullyot, G. E. J. Am. Chem. Soc.
1953, 75, 752-58.
(33) Lebeau, L.; Ducray, P.; Mioskowski, C. Synth. Commun. 1997,
27, 293-6.
In previous experiments a cytotoxin was covalently
bound to the â-face of cobalamin by direct attachment to
the central cobalt atom core.15 The resulting C-Co bond
(16) Levy, M.; Spino, M.; Read, S. Pharmacotherapy 1991, 11, 196-
211.
(17) Kubler, P. A. Med. J. Aust. 2000, 172, 498-9.
(18) Simons, R. J.; Kingma, D. W. Am. J. Med. 1989, 86, 356-7.
(19) Iacobuzio-Donahue, C. A.; Lee, E. L.; Abraham, S. C.; Yardley,
J. H.; Wu, T. T. Am. J. Surg. Pathol. 2001, 25, 1067-73.
(20) Luciani, I. J. Emerg. Nurs. 1989, 15, 80-2.
(21) Stanley, M. W.; Taurog, J. D.; Snover, D. C. Clin. Exp.
Rheumatol. 1984, 2, 167-71.
(22) Stapczynski, J. S.; Rothstein, R. J.; Gaye, W. A.; Niemann, J.
T. Ann. Emerg. Med. 1981, 10, 364-9.
8988 J. Org. Chem., Vol. 69, No. 26, 2004

Cobalamin-Colchicine Conjugate as Cytotoxin
SCHEME 2. Synthesis of Cobalamin Hydrazide 5a
a (a) tert-Butyl-carbazate in CH2Cl2 (61% yield); (b) Zn, NH4Cl in H2O; (c) hydrazide 3 in CH3OH; (d) TFA (95% yield, 3 steps).
SCHEME 3. Synthesis of Cobalamin Bioconjugate
6a
dride, followed by activation of the resulting carboxylic
acid with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
(EDCI) and N-hydroxysuccinimide (NHS) to generate 7.
Compound 7 was added to the 5′-modified cobalamin 8,
as prepared previously,34 to generate stabile bioconjugate
9.
NMR Characterization of 6 in DMSO-d6. The
1
structure of 6 (Figure 1) was determined using H, 13C,
DEPT, DQ-COSY, NOESY, gHSQC, and gHMBC mul-
tidimensional NMR experiments in DMSO-d6.
Evidence of two isomers is observed in the NMR
spectrum. The two compounds result from isomerization
about the C-N bond commonly seen in other acylhydra-
zones.35 By careful analysis of the data, the structure of
the two major isomers was assigned (Table 1). Because
no HMBC correlations to the terminal side chain amide
carbons are observed, the amide side chain protons were
assigned by reference to a previous structure.34
a (a) Colchicine analog 2 in CH3OH (40% yield).
To distinguish the two isoforms from one another, a
prime designation (′) is used to denote the assignment of
the second set of resonances. The majority of the side
chain methylene carbons on cobalamin had two distinct
HSQC correlations, so each proton was denoted as either
a or b. In the case of the colchicine portion, if chirality
was present, each proton was denoted as either R or â
corresponding to the relative position of the proton as
drawn in Figure 1.
A number of NMR assignments of cobalamin have been
published previously.34,36-40 This discussion will focus on
the assignment of the â ligand of the bioconjugate. The
NMR resonances from carbon atoms that are R and â to
cobalt show severe line broadening because of the electric
quadrapole moment of cobalt.40 Knowing this, there are
two carbons that have a rather broad carbon resonance
with Boc-anhydride using DMAP to form an N-acetyl-
carbamate, followed by selective methanolysis of the
acetate and subsequent deprotection with TFA to yield
the desired compound 1. With the primary amine in
hand, acylation with acetylphenoxyacetic acid (APAA)
and Castro’s reagent (BOP) as activating agent proceeded
smoothly to yield compound 2 in 84% yield.
The efficient synthesis of a hydrazide moiety on cobal-
amin may be useful as a general starting material for
the attachment of a number of chemotherapeutic agents.
To that end, 4-chlorobutyric acid chloride was reacted
with tert-butyl carbazate to give protected hydrazide 4
(Scheme 2). The primary alkyl chloride was combined
with hydroxocobalamin under reducing conditions to
yield a protected version of the desired hydrazide. Depro-
tection occurs smoothly, and this synthetic approach is
scaleable to the production of multigram quantities to
give compound 5 in high yields with no purification.
Preparation of the colchicine-cobalamin bioconjugate
occurs rapidly in CH3OH at 25 °C by mixing colchicine
analogue 2 with the prepared cobalamin hydrazide 5 to
yield bioconjugate 6 (Scheme 3).
(34) Horton, R. A.; Bagnato, J. D.; Grissom, C. B. J. Org. Chem.
2003, 68, 7108-11.
(35) Palla, G.; Predieri, G.; Domiano, P.; Vignali, C.; Turner, W.
Tetrahedron 1986, 42, 3649-54.
(36) Brown, K. L.; Brooks, H. B.; Gupta, B. D.; Victor, M.; Marques,
H. M.; Scooby, D. C.; Goux, W. J.; Timkovich, R. Inorg. Chem. 1991,
30, 3430-3438.
(37) Anton, D. L.; Hogenkamp, H. P. C.; Walker, T. E.; Matwiyoff,
N. A. J. Am. Chem. Soc. 1980, 102, 2215-2219.
(38) DiFeo, T. J.; Schiksnis, R. A.; Kohli, R. K.; Opella, S. J.; Nath,
A. Magn. Reson. Chem. 1989, 27, 127-129.
(39) Bax, A.; Summers, M. F. J. Am. Chem. Soc. 1986, 108, 2093-
4.
As a control, a light- and pH-stabile colchicine-
cobalamin bioconjugate was prepared as depicted in
Scheme 4. Compound 9 does not have a labile hydrazone
and was expected not to be cytotoxic if colchicine must
be released from cobalamin to induce cell death. N-
deacetylcolchicine 1 was acylated with glutaric anhy-
(40) Vitamin B12; Dolphin, D., Ed.; John Wiley and Sons: New York,
1982; Vol. 1.
J. Org. Chem, Vol. 69, No. 26, 2004 8989

Bagnato et al.
SCHEME 4. Synthesis of Stable Bioconjugate 9a
a (a) Glutaric anhydride in DMSO; (b) EDCI, NHS in DMSO (34% yield, 3 steps).
FIGURE 1. Numbering scheme of cobalamin bioconjugate 6.
at 25.72 and 26.45 ppm. The HSQC correlations to these
carbons are shifted downfield due to deshielding. The
carbon at 25.72 ppm assigned to the R carbon (relative
to cobalt, T1) has HSQC correlations to protons at 0.46
and 1.32 ppm, and the carbon assigned to the â position
(T2) at 26.45 has protons associated at 0.39 and -0.12
ppm. These values are supported by their similarity to
the published values for 13C enriched ethylcobalamin, in
which the R carbon is reported to be at 24.4 ppm and
the associated protons are at 0.566 and 0.847 ppm. The
â carbon is unidentified because of quadrapolar broaden-
ing, but the protons are assigned at -0.42 and 0.04
ppm.40 The protons on the γ carbon (T3a and T3b) are
then assigned on the basis of COSY correlations to these
8990 J. Org. Chem., Vol. 69, No. 26, 2004

Cobalamin-Colchicine Conjugate as Cytotoxin
TABLE 1. NMR Assignments for Bioconjugate 6 in DMSO-d6
carbon
freq ppm
DEPT
assign
gHSQC
gHMBCa
D11, D8
DQ-COSY
NOESY
177.95
175.38
175.31
174.00
173.81
173.42
172.89
172.79
171.43
171.33
170.64
168.29
167.15
167.02
163.62
163.57
162.65
158.58
158.36
152.96
150.43
150.29
146.39
142.42
140.74
138.57
135.09
134.49
134.49
134.17
131.23
131.10
130.98
130.80
130.57
129.59
127.65
127.15
125.36
118.33
118.33
114.32
112.18
112.18
110.47
107.78
104.60
104.39
103.78
103.66
93.82
D9
C16
C4
C53, C54, C55
C35
C50b
C32
C9
C43
C11
C57/C61
C27
C38
T4
C46, C47
T4NH′
A1
A2, D7NH
A2′, D7NH
C35, C36, C37
D8, D11, D12, D20
C53
A1′
C6
D10
C14
A3
A2, A5/A7
A2′, A5′/A7′
D4, D19
A3′
D3
D1
D4, D17
D16
A9
D7, D8, D11
A5/A7, A10, T4NH
R1
CH
B2
7.24
D2
D4, D18
B9
B2, B7
B9′
B2, B7
CH
CH
D12
D11
D14
A6
7.03
7.11
D8, D11
D11
D12
D12
D20
D4, D5R, D6â
A4/A8, A10
A4/A8, A10
B11
A6′
B5
B6
B10
CH
D8
7.22
D7NH
B8
B2
CH
CH
A5/A7
A5′/A7′
D13
B4
7.65
7.51
A4/A8
A4′/A8′
A4/A8, A10
A10′
D5R, D5â, D4, D8, D12
CH
CH
CH
CH
6.43
6.49
6.93
7.02
C20, C30b, C31a, C31b
C30b, C31a, C31b, C41a
A2/A2′, A5′/A7′, D17
A2/A2′, A5/A7
B4′
A4′/A8′
A4/A8
D15
B7
A5′/A7′
A5/A7
D8, D12
B11
CH
CH
7.21
6.78
R1, R3, R4, B11
D5â, D19
D4
D5R, D5â
C35
C5
C5′
C35
C15
C15′
C10
C10′
C1
C53
C53
CH
CH
6.12
6.12
C8, C37a, C46, C47
C8′, C37a, C46, C47
93.45
85.06
C20
Pr3
84.94
CH
R1
6.15
3.95
4.50
4.06
4.14
4.06
4.58
4.58
3.53
3.52
3.78
R2
B7, B11, C56b, N59, R2, R3, R4, R5
B7, C48a, N59, Pr2, R1, R3
R1, R2, R4, R5
82.03
CH
R4
R3, R5
R2, R4
C18
74.13
CH
R3
74.13
CH
C19
Pr2
R2
C18, C26a, C26b, C54, N63a, T1a, T2a
N59, Pr3, R4, R5
70.41
CH
Pr1a, Pr3
R1, R3, R7
69.74
CH
C56b, R1, R3, R5
66.70
CH2
CH2
CH2
CH3
CH3
A2
A4/A8, D7NH
66.59
A2′
A4′/A8′, D7NH
61.60
R5
R4, R8
B11, C53, Pr2, R1, R2, R3
D18, D20
60.80
D17
D18
C17
D20
D19
C3
60.67
D17
57.81
C54
56.04
CH3
CH3
CH
CH
CH
CH
CH
CH
3.87
3.83
4.23
4.35
3.76
3.66
3.16
3.08
A4′/A8′, C26b, D11
D4
55.84
54.85
C25
C25
C10, C36
C10,C36
C46, C47
C46′, C47
C30b
C30b
C25, C26a, C26b, C35
C25, C26′, C30′, C35′
C10, C35, C37a, C41a, C41b
C10
54.55
C3′
53.63
C8
C41a, C41b
C41a,C41b
C48a
53.63
C8′
52.24
C13
C13′
C53
52.24
C48a
C46′, C47, C48a, C48b, C53
J. Org. Chem, Vol. 69, No. 26, 2004 8991

Bagnato et al.
Table 1 (Continued)
carbon
assign
freq ppm
DEPT
CH
gHSQC
4.41
gHMBCa
DQ-COSY
NOESY
51.24
49.11
48.93
46.34
46.23
45.63
45.24
D7
C7
D6R, D5â, D8, D12
C36
D6R
D6â, D7NH
C7′
C12
C12′
C2
C36
C10, C46
C10, C46′
C25
CH2
CH2
Pr1
2.83a
3.44b
2.05a
2.22b
2.16
1.71a
2.41b
2.74
2.01R
2.26â
1.85a
2.24b
1.73a
2.20b
2.11a
2.47b
1.77
Pr3
N59, Pr1b, Pr2
N59, Pr1a
N59, Pr1b, Pr2, Pr3
N59, Pr1a, Pr3
C18, C19, C25, C30a, N29a, N29b, N34a
Pr3
42.57
C26
C25
C25
C3, C19
42.33
41.95
CH2
CH2
C26′
C37
C25′
B4, B4′, C3, C3′, C31, N34b
C8, C10, C37b, T1b
C37a, C41a
C19, C20, C25, C26b, C56a, C56b, N63b
D5â, D7, D7NH
D5R, D7, D7NH
T3b, T4NH
C36
C37b
C37a
C19, C60b
D7
C36
38.62
35.46
CH
CH2
C18
D6
C54, C55a
D5R, D5â, D7
D5R, D5â, D7
D5R
33.54
32.47
32.20
CH2
CH2
CH2
T3
T2b
T2a
T3a
C31
C60
N34
N34
C30a, C31b
C30a, C31a
B4, B4′
C25, C26′
C18
C56a, T1a
31.78
31.56
CH2
CH2
C49
C42
C48a, C48b
C41a
C41a
C47
2.31a
2.34b
1.01
C41b
C41b
31.34
31.34
29.88
CH3
CH3
CH2
C46
C46′
C56
C47
C47
C10, C47
0.97
C10, C13′, C47, T1a
1.96a
2.46b
2.61R
2.23â
1.18a
1.23b
1.83a
1.96b
1.07a
1.77b
0.39a
-0.12b
1.75
C56b
C56a
D5â, D6â
D5R
C18, C54, C56b, C60, N59, N63a, N63b
C18, C54, R1, R2
29.14
28.96
26.73
26.45
26.45
25.72
25.72
CH2
CH2
CH2
CH2
CH
D5
D4, D7
D4, D7
C54
D6R
D4
C55
C48
C41
T2
C54
C13, C13′
C13′, C47, N52b
C13′, C47, N52a, R4
B4′, C8, C8′, C37b, C41b
C41a, C42
C8, C8′
C8, C8′, C42a, C42b
T1a, T2b, T3a
T1a, T1b, T2a,T3a
C3, C3′
C19
C19
CH2
CH2
C30
T1
C26a
1.81
C31a, C31b
T1b, T2a, T2b
T1a, T2a, T2b
C3, C3′
0.46a
1.32b
0.45
C19, C46, C46′, C60, T1b
T1a, C37a
21.49
21.27
20.76
20.67
20.12
19.90
19.79
18.99
18.81
17.78
17.61
16.60
15.79
15.57
15.46
13.84
13.20
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C20
C20′
C47
C47′
Pr3
B10
B11
C36
C36′
C25
C25′
C54
C35
C35′
C53
A10′
A10
B4′, C18, C25′
0.45
1.38
C46
C46′
C10, C13, C46, C48a, C48b, C49
C10, C13′, C46′, C48a, C48b, C49
N59, Pr1a, Pr1b, Pr2
1.38
1.06
Pr2
2.27
2.19
B7
B7, R1, R5
N40a, N40b
1.68
1.68
1.28
C3, C18, C20, C26a, C31a, N29a, N29b
C3′, C18, C20, C26a, C31a, N29a, N29b
C19, C19, C53, C56a, C56b, N63b
C3, C3′, C8, N40a, N40b
C3, C3′, C8, N40a, N40b
C13, C13′, C54, N52a, R5
A5′/A7′, T4NH′
1.23
1.25
2.41
2.41
2.25
2.08
2.06
A5/A7, T4NH
a This is the proton correlation. b Italics indicate an assignment that is made by reference to previous structures because of a lack of
HMBC correlations.
â protons, and the corresponding γ carbon (T3) is as-
signed from HSQC correlations. The hydrazide proton is
determined on the basis of a NOESY correlation observed
between T3a and a proton at 9.94 ppm (assigned T4NH′)
that is consistent with a hydrazide proton. The two
isomers of the hydrazone are shown in Figure 2 with the
corresponding correlations depicted to clarify the assign-
ments made. The T4NH′ proton also has a weak HMBC
correlation to T4, further confirming this assignment.
This hydrazide proton at T4NH′ also has a NOESY
correlation to the A10′ methyl that serves as a bridge to
correlate the alkyl chain (T1-T4) to the acetophenone
portion of 2.
As expected from two isomers, there are two distinct
A10 carbons (13.84 and 13.20 ppm) and corresponding δ
1H of 2.08 and 2.06 ppm. A very strong NOESY correla-
8992 J. Org. Chem., Vol. 69, No. 26, 2004

Cobalamin-Colchicine Conjugate as Cytotoxin
TABLE 2. Calculated Half-Life of Bioconjugate 6 in
Various Buffers
buffer
pH 4.5 NaOAc
half-life
138 min
13 h
pH 5.5 succinic acid buffer
pH 7.0 (H3N)2HPO4 buffer
MEM buffer (10% FBS)
9.4 d
>7 da
>7 da
Dulbecco-MEM buffer (10% FBS)
a Monitoring stopped after 10 days as a result of bacterial
growth.
HMBC correlation from the D19 proton to a quaternary
carbon at 152.96 is then used to assign D3. Two strong
HMBC correlations from the D4 proton to two quaternary
carbons at 150.29 and 140.74 ppm are used to identify
D13 and D2. An HMBC correlation from one of the
methoxy singlets at 3.78 ppm to the same carbon at
140.74 ppm confirmed this carbon as D2, and by default
the carbon at 150.29 ppm is D13. An HMBC correlation
to D13 from a proton at 7.03 ppm, which has an HSQC
correlation to a carbon at 134.46 ppm is then assigned
D12. This D12 proton also has an HMBC correlation to
a carbon at 163.57 ppm that is assigned D10. An HMBC
correlation from 3.87 ppm to this carbon is used to
identify the proton at D20, and HSQC identifies the D20
carbon at 56.04 ppm. The only remaining methoxy proton
at 3.52 ppm is then assigned to D17 by default. This
proton shows an HMBC correlation to the resonance at
150.43 ppm that is assigned D10.
The rest of the core ring structure and side chains of
cobalamin was assigned as reported previously.34 One
interesting observation of note was that there are several
NOESY correlations from T1a to unique sites on the
corrin ring. NOESY correlations between T1a to C60b
as well as T1a to C19 and even T1a to C46 suggest that
the Co-C bond does not freely rotate on axis and the
upper ligand has constrained movement placing the T1a
C-H bond pointing toward the southern portion of the
molecule between the e and f side chains. There may be
additional NOESY correlations to T1b as well as T2a and
T2b to sites on the corrin ring, but they are indistin-
guishable because of the large number of protons in the
region between 1 and 2 ppm.
Serum Stability Studies. Bioconjugate 6 was tested
for stability in a variety of buffers, and the results were
fitted to first-order decay kinetics (data not shown) to
calculate the half-lives (Table 2). As expected, the hy-
drazone undergoes clean hydrolysis with a short half-
life at pH 4.5 of under 2.5 h. The half-life increases to 13
h at pH 5.5 and to over 9 days at pH 7.0. The bioconjugate
was further tested for stability in the same cell media
used in the cell viability assays in the presence of 10%
heat-inactivated fetal bovine serum (FBS), and biocon-
jugate 6 had a half-life of >7 days in both media.
Cell Viability Results. The data and results that
follow are divided into two groups in which either the
CellTiter-Glo Luminescent Cell Viability Assay (Luc.
Assay) or the CellTiter 96 Non-Radioactive Cell Prolif-
eration Assay (MTT Assay) was used to quantify cell
viability. The cell viability versus concentration was
plotted on a log scale, and the LC50 values were estimated
from the graph and tabulated in Table 3.
FIGURE 2. Two isomers of the hydrazone of bioconjugate 6
and 2D NMR correlations of each isomer. The proton reso-
nances are listed for key protons.
tion between a proton at 10.04 ppm (assigned T4NH) and
2.06 ppm indicates that this is the E isomer. The Z isomer
has a much weaker NOESY correlation between T4NH′
(9.95 ppm) and A10′ (2.08 ppm). Interestingly, the E
isomer shows no NOESY correlation between the T4NH
and the T3a proton, but there is a weak HMBC correla-
tion to A9. The remainder of the acetophenone proton
(A3-A10) is assigned via HMBC and COSY correlations.
Carbon A2 has two resonances assigned to carbons
66.70 ppm (A2) and 66.59 ppm (A2′), yet HMBC correla-
tions for each carbon have protons at 4.78 ppm. NOESY
correlations between A2/A2′, A4/A8, and A4′/A8′ as well
as a NOESY correlation to a proton at 8.95 (assigned
D7NH) aid in the assignment of this portion of the
molecule. An HMBC correlation between A2/A2′ and A3
as well as to A1 confirms these assignments. The D7NH
proton is then correlated to the D7 proton via a NOESY
interaction. Using a combination of COSY and NOESY
correlations, the relative stereochemistry of the prochiral
D6 and D5 protons are assigned on the basis of the
Karplus relationship, and the corresponding carbons are
assigned from the HSQC correlations. The stereochem-
istry of these protons is assigned on the basis of a NOESY
correlation between the D4 proton (6.78 ppm) and the
2.61 ppm resonance (assigned D5R). As a result of the
geometry of the ring system, only the R-proton is suf-
ficiently close to show a NOESY correlation based on
computer modeling. The rest of the colchicine portion of
the molecule is assigned using HMBC and HSQC cor-
relations. To distinguish D1-D3, which are in nearly
equivalent environments, first a NOESY correlation
between D4 and a large singlet peak at 3.83 ppm in the
1H spectra (assigned D19) is identified. Because there are
four large singlet absorption peaks in the 1H spectra that
correspond to the four methoxy groups of colchicine, these
are used to distinguish the corresponding quaternary
carbons, as they each show one HMBC correlation. An
The dose response curve for 2 and 6 using either cell
viability assay was nearly identical to that of colchicine
J. Org. Chem, Vol. 69, No. 26, 2004 8993

Bagnato et al.
TABLE 3. LC50 Values for Cancer Cell Lines Treated
with Colchicine, Colchicine Analogues 2, and Two
Colchicine-Cobalamin Bioconjugates 6 and 9
cytotoxicity of 6, which was not seen in the stable
cobalamin bioconjugate 9.
Luc. assay
MTT assay
Experimental Section
SK-BR-3b SK-N-MC B16-F1 SK-BR-3 SK-N-MC B16-F1
Materials. All chemicals were purchased from commercial
suppliers and used without further purification. Mass spec-
trometric analysis of the analogues was carried out using an
electrospray mass spectrometer in positive ion mode.
Standard NMR Conditions. All NMR samples were
dissolved in 250-300 µL of DMSO to a final concentration of
∼30 mmol in a 5-mm Shigemi NMR tube, and all NMR spectra
were collected at 500 MHz unless otherwise noted. 13C NMR
data were collected using a 5-mm switchable broadband probe,
and all 1H and 2-D NMR data sets were collected using a 5-mm
indirect pulse field gradient probe.
colchi-
16
31
65
6
19
67
cinea
2
6
9
64
99
20 000
30
43
76 000
11
60
17
23
61 000
80
302
66 000
90 000 18 000
a Average of 3 independent cell viability experiments b SK-BR-
3 is a human breast adenocarcinoma cell line; SK-N-MC is a
human neuroblastoma cell line; and B16-F1 is a mouse melanoma
cell line.
Standard HPLC Conditions. Analytical runs were per-
formed using a C18 15 µm, 100 Å, 3.9 mm × 300 mm column
with detection at 254 nm. Preparatory runs were carried out
using a C18 15 µm, 100 Å, 25.0 × 200 mm column, monitored
at 254 nm. The buffers used in all chromatography runs were
as follows: Buffer A, 0.05 M phosphoric acid, pH ) 3.0 (or pH
) 7.0 for compound 6 only) with NH4OH. Buffer B, 9:1
acetonitrile/ddH2O.
Analytical HPLC Method. A flow rate of 2.0 mL/min was
used; 0-2 min, with isocratic elution of 95:5 A:B; 2-10 min,
linear gradient to 70:30 A:B; at 10-15 min, linear gradient to
0:100 A:B; isocratic elution of 0:100 A:B; and 18-27 min, linear
gradient to 95:5 A:B.
Semipreparative HPLC Method. A flow rate of 40 mL/
min was used; 0-2.7 min, isocratic elution of 95:5 A:B; 2.7-
13.7 min, linear gradient to 70:30 A:B; 13.7-20.5 min, linear
gradient to 5:95 A:B; 20.5-21.2 min, linear gradient to 0:100
A:B; 21.2-24.7 min, isocratic elution of 0:100 A:B; and at 24.7-
37 min, linear gradient to 95:5 A:B.
General Desalting Procedure. All cobalamins were de-
salted using a C18 column by first rinsing the column with
two column volumes of methanol followed by three column
volumes of ddH2O. The cobalamin analogue was applied to the
column and rinsed with three column volumes of ddH2O
followed by elution with CH3OH. The CH3OH was removed
via rotary evaporation, and the solid was dried over P2O5 at
50-100 mTorr.
Cell Culture. All cell lines were obtained from ATCC and
incubated at 37° C with 5% CO2. SK-N-MC cells were
maintained in Minimum Essential Medium (MEM) with
Earle’s salts, with L-glutamine. SK-BR-3 and B16-F1 cells were
maintained in Dulbecco’s Modified Eagle Medium (D-MEM)
with GlutaMAX, with high glucose, with pyridoxine hydro-
chloride, without sodium pyruvate. All media were supple-
mented with 10% heat-inactivated defined fetal bovine serum,
penicillin-streptomycin to a final concentration of 10 units
penicillin and 10 µg of streptomycin per mL and an additional
2 mM L-glutamine. For the viability assays, cells were plated
in 96-well plates in 100 µL of growth media. Initial cell density
varied from 1000 to 20000 cells/well depending on cell type
and assay.
Determination of Bioconjugate Cytotoxicity. The effect
of colchicine and the cobalamin-colchicine bioconjugates on
cell viability was determined using two commercially available
assays. The CellTiter-Glo luminescent cell viability assay (Luc.
Assay) quantifies the ATP present in cells, which correlates
with metabolically active cells. The CellTiter 96 non-radioac-
tive cell proliferation assay (MTT Assay) is based on the
reduction of a blue tetrazolium salt into a purple formazan
product by living cells. These two assays measure different
cellular activities and thus allow for independent confirmation
of the effects on viability obtained in each assay.
in SK-N-MC neuroblastoma cells. For all agents, con-
centrations of 0.001 and 0.01 µM drug had no cytotoxic
effect, whereas concentrations of 0.1 µM and higher
resulted in a substantial decrease in SK-N-MC cell
viability. In SK-BR-3 human breast adenocarcinoma
cells, concentrations above 0.1 µM produced a significant
decrease in viability. For this cell line, colchicine appears
to be more toxic than 2 and 6, showing a 4- and 6-fold
decrease in toxicity, respectively, for the Luc. Assay. The
MTT assay had a similar trend, but bioconjugate 6
showed a 10-fold decrease in toxicity relative to colchi-
cine. In the final cell line tested, B16-F1 cells, analogue
2 had a slight decrease in toxicity, but 6 had a 5-fold
decrease in toxicity. For all cell lines tested, a dramatic
decrease in toxicity was observed for the stable biocon-
jugate 9, in most cases greater than a three log decrease,
thereby confirming the expectation that the drug does
need to be released from cobalamin for efficacy.
Conclusions
Colchicine was functionalized at the C7 position with
a p-alkoxyacetophenone linker to produce analogue 2.
Hydroxycobalamin was functionalized with a small hy-
drazide tether, and the two analogues were combined to
form the hydrazone-containing cobalamin bioconjugate
6. The bioconjugate showed excellent stability in cell
media and in neutral pH buffer, while undergoing rapid
and clean hydrolysis at slightly acidic pH as anticipated.
The acid-labile hydrazone is expected to be hydrolyzed
within lysosomes inside the cells and thereby release
active drug. In vitro data show that colchicine analogue
2 exhibits only a minimal loss in toxicity compared to
authentic colchicine, and the colchicine-cobalamin bio-
conjugate 6 shows a 10-fold maximum decrease in toxicity
in the three cancer cell lines tested. The inherent
advantage of this bioconjugate is to target cancer cells
over healthy cells to decrease the side effects of current
nonselective chemotherapeutic drugs. The cytotoxicity of
this bioconjugate in vitro is comparable to current
chemotherapeutic drugs such as paclitaxel and docetaxel,
yet the bioconjugate is highly water-soluble and signifi-
cantly less expensive (less than $50 per gram) than either
paclitaxel or docetaxel. The stable control bioconjugate
9 shows a dramatic 3-log decrease in cytotoxicity, indicat-
ing that the release of colchicine from cobalamin is
necessary to cause cell death. This decrease in toxicity
also suggests that the acid-labile hydrazone undergoes
hydrolysis within the cell, as indicated by the nM
Stock solutions of colchicine and compounds 2, 6, and 9 were
prepared in 5% DMSO, stored at -20 °C, and protected from
light. Each compound was tested at six concentrations covering
10-fold dilutions from 100 µM to 10 nM, except 6 for which
8994 J. Org. Chem., Vol. 69, No. 26, 2004

Cobalamin-Colchicine Conjugate as Cytotoxin
100 µM was not tested. Ten-fold stock solutions were prepared
and 10 µL was added to each of three wells containing cells.
The compounds were added 24 h after the cells were plated.
Untreated control cells received 10 µL of 5% DMSO. Three
wells containing media alone were included to determine
background absorbance. The plates were incubated at 37 °C
with 5% CO2. The incubation times for the different cell lines
were 48 h for SK-N-MC cells, 96 h for SK-BR-3, and either 72
or 96 h for B16-F1 cells.
At the end of incubation, cell viability was quantified as
described. The CellTiter-Glo luminescent cell viability assay
was carried out according to the manufacturers protocol.
Luminescence was recorded over an integration time of 0.1 s.
The CellTiter 96 non-radioactive cell proliferation assay
protocol was used with the following changes: Incubation with
the dye solution was reduced to 1 h at 37 °C. Following
addition of the solubilization/stop solution, the plate was
incubated at 37° C for 30 min. The contents of the wells were
mixed, and the plate was returned to 37° C for 1 h to ensure
complete solubilization of the formazan crystals. Absorbance
of the solutions at 570 and 650 nm was measured, with 650
nm used as the reference wavelength.
Calculations and Data Representation. Data from both
assays were processed in the same manner. The average value,
either luminescence or absorbance, of the three wells contain-
ing media alone was subtracted from the raw data values to
give corrected values. Measured absorbance values were to
correct for the background absorbance imparted by the red
color of high concentrations of 9 by subtraction of the back-
ground value for cells treated with 100 µM 9 or the lumines-
cence of media + 100 µM 9, instead of just medium alone. The
three corrected values for wells dosed with the same concen-
tration of compound were averaged and the standard deviation
was calculated. These data were plotted as average absorbance
(av abs ( SD) or average luminescence (av lum ( SD) versus
concentration of compound (data not shown).
Percent cell viability was also calculated by considering the
values from untreated cells as representing 100% viability. The
corrected absorbance or luminescence for each value was
divided by the average corrected absorbance or luminescence
of untreated cells and multiplied by 100 to give the percent
cell viability. Average percent cell viability of the triplicate
wells and the standard deviation were calculated. These data
were plotted as average percent cell viability (av % viability
( SD) versus concentration of compound (data in Supporting
Information).
Synthesis. N-Deacetylcolchicine (1). Colchicine (515.8
mg, 1.29 mmol) was dissolved in 5 mL of acetonitrile. To this
were added 4-(dimethylamino)pyridine (157.5 mg, 1.29 mmol),
triethylamine (350 µL, 2.52 mmol), and di-tert-butyl dicarbon-
ate (678 mg, 3.11 mmol). The reaction flask was attached to a
reflux condenser and placed into an oil bath at 100 °C. The
reaction was monitored over time via HPLC (product tR ) 16.7
min). After 1 h the reaction stalled at 70% conversion, so
additional di-tert-butyl dicarbonate (610 mg, 2.79 mmol) was
added. The liquid chromatogram showed only one new peak
at 16.7 min, with colchicine having a retention time of 14.0
min. The reaction was complete in 3 h. The reaction was
quenched by the addition of 35 mL of CHCl3 and was washed
with 3 × 50 mL of saturated aqueous citric acid. The combined
aqueous layers were back extracted with 30 mL of CHCl3, and
the organic layers combined. The organic layer was washed
with 30 mL of saturated brine and then concentrated to a
reddish brown solid. The crude compound was >95% pure via
HPLC, and the only impurity was unreacted colchicine. The
crude sample was carried on through the next step.
was dried over Na2SO4, filtered, and concentrated to a crude
solid (∼90% purity via HPLC). The only other peak evident
by HPLC was unreacted colchicine, and the crude sample was
carried through the next reaction.
The crude N,N′-Boc-deacetylcolchicine was dissolved in 1
mL of neat trifluoroacetic acid (TFA) and stirred for 5 min.
The TFA was removed via rotary evaporation to yield a crude
sample of the desired product (>87% pure via HPLC, tR ) 12.5
min). Again, the only detected impurity was unreacted colchi-
cine. The product was purified at this point by extraction of
N-deacetylcolchicine into saturated aqueous citric acid, allow-
ing for the isolation of the unreacted colchicine. Pure N-
deacetylcolchicine was isolated by adjusting the aqueous layer
pH to 10 with 1 M NaOH and extracting with several portions
of CH2Cl2, resulting in a product with >95% purity (542 mg,
85% yield). ES+ calcd for C20H24NO5 (M + H+) 358.17, found
358.2 and for C20H23NO5Na (M + Na+) 380.17, found 380.2.
2-(4-Acetylphenoxy)-N-acetamidocolchicine (2). N-
Deacetylcolchicine 1 (504.6 mg, 1.1 mmol) was dissolved in 20
mL of CHCl3. To this was added 4-methylmorpholine (400 L,
3.6 mmol) followed by addition of acetylphenoxyacetic acid (242
mg, 1.2 mmol) and BOP (580 mg, 3.0 mol). The reaction was
complete after 90 min (product tR ) 15.4 min). The reaction
was diluted with 10 mL of CHCl3 and washed with 3 × 35 mL
of saturated citric acid. The combined aqueous layers were
back extracted with 2 × 15 mL of CHCl3. The combined organic
layers were washed with 2 × 40 mL of saturated NaHCO3 and
30 mL of saturated brine. The crude sample was evaporated
to a brown oil via rotary evaporation and dried to solid 2 with
P2O5 under high vacuum (540 mg, 84% yield): mp 115 °C dec;
1H NMR (500 MHz, DMSO) δ 8.90 (d, J ) 7.6 Hz, 1H), 7.92
(d, J ) 8.6 Hz, 1H), 7.23 (s, 1H), 7.12 (d, J ) 10.7 Hz, 1H),
7.00-7.06 (m, 3H), 6.78(s, 1H), 4.68 (s, 2H), 4.36-4.44 (m, 1H),
3.87 (s, 3H), 3.83 (s, 3H), 3.78 (s, 3H), 3.52 (s, 3H), 2.60 (dd, J
) 5.8, 13.2 Hz, 1 H), 2.51 (s, 3H), 2.21-2.30 (m, 1H), 1.95-
2.10 (m, 2H); 13C NMR (125 MHz, DMSO) δ 197.25, 177.91,
166.66, 163.56, 161.53, 152.95, 150.42, 150.15, 140.75, 135.02,
134.45, 134.12, 130.54, 130.36, 130.30, 125.34, 114.41, 112.11,
107.75, 66.58, 60.78, 60.65, 56.00, 55.82, 55.24, 35.45, 29.14,
26.35; ES+ calcd for C30H32NO8 (M + H+) 534.20, found 534.2
and for C30H31NO8Na (M + Na+) 556.20, found 556.2.
N-(4-Chloro-butanoyl)-hydrazinecarboxylic Acid tert-
Butyl Ester (3). In a 50 mL flask, tert-butyl-carbazate (2.5 g,
18.9 mmol) was dissolved in 20 mL of dry CH2Cl2, and
4-methylmorpholine was added (2.1 mL, 22.5 mmol). To the
stirring solution at 0 °C was added 4-chlorobutylacid chloride
(2.1 mL, 11.8 mmol) dropwise. The reaction was checked after
10 min via TLC (1:1 EtOAc/hexanes and visualized with
ninhydrin stain). The reaction was worked up upon completion
of the reaction (Rf ) 0.29). The product was extracted with 40
mL of CH2Cl2 and was washed with 3 × 50 mL of 50% citric
acid solution. The organic layer was dried over MgSO4 and
filtered, and the solvent was removed under vacuum. The
crude oil was crystallized from hot EtOAc and hexanes yielding
1
small needle crystals (2.68 g, 61% yield): mp 65 °C; H NMR
(300 MHz, CDCl3) δ 7.65 (br s, 1 H), 6.65 (br s, 1H), 3.62 (t, J
) 6.0 Hz, 2H), 2.42 (t, J ) 7.0 Hz, 2H), 2.15 (p, J ) 6.6 Hz,
2H), 1.44 (s, 9H); 13C NMR (75 MHz, CDCl3) δ 171.6, 155.6,
82.0, 44.1, 30.6, 28.1, 27.7.
Cobalamin Hydrazide (5). Hydroxocobalamin (990 mg,
0.71 mmol) was placed into a 25-mL round-bottom flask and
dissolved in 8 mL of ddH2O. To this was added NH4Cl (414
mg, 7.7 mmol). In a separate 50-mL round-bottom flask excess
zinc dust was added (467 mg, 7.1 mmol). In a 10-mL round-
bottom flask, 3 (344 mg, 1.5 mmol) was dissolved in 1 mL of
MeOH. The three flasks were purged in series with nitrogen
for 30 min. The dissolved hydroxocobalamin solution was
transferred via cannula to the flask with zinc dust and allowed
to react for 20 min to form reactive Co(I). The solution of 3
was then transferred via cannula to the Co(I) reaction and
monitored via HPLC. Hydroxocobalamin has a retention time
The crude N-Boc-colchicine was dissolved in 1 mL of CH3-
OH. To this was added 2 M NaOCH3 in CH3OH (130 µL). The
solution was stirred for 30 min at room temperature and
monitored via HPLC (product tR ) 16.1 min). After 30 min
the solution was transferred to 40 mL of brine, and the product
was extracted with 3 × 30 mL of diethyl ether. The product
J. Org. Chem, Vol. 69, No. 26, 2004 8995

Bagnato et al.
of 8.3 min, and the product has a retention time of 12.9 min.
After 30 min the reaction was complete and was worked up.
The excess zinc was filtered off using a Buchner funnel, and
then the reaction was desalted on a 1 g C18 column as above
to obtain a red solid 4 at >98% purity by HPLC. The Boc group
was removed via treatment with 1 mL of neat TFA for 10 min.
The reaction mixture was added to 100 mL of 2:1 Et2O/CH2-
Cl2 and a red precipitate was filtered and triturated with 2 ×
10 mL CH2Cl2 and 3 × 10 mL Et2O. The red solid 5 was
checked via HPLC, and one peak was evident at 10.8 min at
>97% purity (1.05 g, 95% yield). ES+ calcd for C66H98-
CoN15O15P (M + H+) 1430.64, found 1430.6 and for C68H97-
CoN15O15PNa (M + Na+)1452.64, found 1452.7.
CNCbl-5′-[(6-Amino)-hexylcarbamate] (8). Cobalamin 8
was prepared as reported previously.34 In brief, CNCbl was
dissolved in DMSO at 30 °C followed by addition of carbon-
ylditriazole (CDT). After 15 min the reaction was quenched
by addition to Et2O/EtOAc and filtration to removed excess
CDT. The compound is redissolved in DMSO, and excess 1,6-
diaminohexane was added. The progress of the reaction was
monitored via HPLC (tR ) 12.7 min) and was completed in 15
min. ES+ calcd for C70H103CoN16O15P (M + H+) 1497.68, found
1497.5 and for C70H102CoN16O15PNa (M+Na+) 1519.68, found
1519.6.
Cobalamin Bioconjugate (9). Cobalamin 8 (54 mg, 36.0
mol) was dissolved in 1.0 mL of DMSO. To this was added the
crude compound 7 (50 mg, 88.0 mol). The reaction was
monitored via HPLC, and a new peak was evident after 20
min (tR ) 13.7 min). The reaction was complete in 1 h and
was transferred to 60 mL of 2:1 Et2O.CH2Cl2. The resulting
red precipitate was filtered using a medium frit and washed
with 3 × 5 mL of Et2O. The sample was purified via prepara-
tory HPLC, and pure fractions were desalted as above to yield
Cobalamin Bioconjugate (6). Cobalamin hydrazide 5 (176
mg, 0.11 mmol) was dissolved in 2 mL of MeOH. To this was
added colchicine analogue 2 (60 mg, 0.11 mmol). The solution
was stirred for 12 h at room temperature and monitored via
HPLC (product tR ) 14.1 min). The sample was then trans-
ferred to 60 mL of a 2:1 mixture of Et2O/CH2Cl2, resulting in
the precipitation of the product. The crude sample was filtered
using a medium glass fritted filter and triturated with 3 × 15
mL of Et2O, yielding a sample of bioconjugate 6 at >75%
purity. The sample was purified via preparatory HPLC, and
pure fractions were desalted as above to yield pure bioconju-
pure 9 (23 mg, 34% isolated yield). ES+ calcd for C95H130
CoN17O22P (M + H+) 1950.9, found 1950.7 and for C95H129
CoN17O22PNa (M + Na+) 1972.9, found 1972.6.
-
-
Stability Studies. Bioconjugate 6 (2.0 mg, 1.0 mol) was
dissolved in 100 µL of DMSO followed by dilution with 5.0 mL
of the corresponding buffer (50 mM). Each sample was
monitored over time for a least three half-lives via HPLC. All
data were fitted to a first-order rate equation, and the
calculated half-lives are tabulated in Table 2.
gate 6 (88.8 mg, 40% isolated yield). ES+ calcd for C96H127
CoN16O22P (M + H+) 1945.83, found 1945.2 and for C96H126
CoN16O22PNa (M + Na+) 1967.82, found 1967.2.
-
-
N,N′-Deacetyl-colchicine-hemiglutaramide-NHS (7).
N-Deacetylcolchicine (105 mg, 0.22 mmol) was dissolved in 500
µL DMSO. To this was added excess 4-methyl morpholine
followed by glutaric anhydride (26 mg, 0.23 mmol). The
reaction was complete in 15 min (as monitored by HPLC,
product tR ) 14.1 min). To the stirring reaction was added 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide (69 mg, 0.36 mmol)
and N-hydroxysuccinimide (29 mg, 0.25 mmol), and the
reaction was complete in 24 h (product tR ) 14.8 min). The
reaction mixture was diluted with 30 mL of CHCl3 and washed
with 3 × 30 mL of saturated aqueous NaHCO3 followed by 30
mL of saturated brine. The solvent was removed via rotary
evaporation to yield crude product that was used without
further purification (100 mg).
Acknowledgment. We would like to thank the
National Institute of Health for funding (Grant CA73003)
to C.B.G. and F. G. West.
Supporting Information Available: Cell viabilty versus
concentration plots in three cell lines, as well as NMR spectra
of compounds 2, 3, 5, and 6 and mass spectra of compounds 5
and 6. This material is available free of charge via the Internet
at http://pubs.acs.org.
JO049953W
8996 J. Org. Chem., Vol. 69, No. 26, 2004