4714 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21
Brady et al.
gradient elution 95% f 70% A/70 min at a flow rate of 80 mL/
min. The lyophilizate from pooled homogeneous fractions was
passed through BioRad AG4 × 4 ion-exchange resin (acetate)
to give the title compound as the HOAc salt: retention time
(system C) 23.4 min, 99.8% pure; FAB-MS m/z 866.5 (calcd
[M + H]+ 866.7); amino acid compositional analysis (theory/
found) Pro 1.08/1.01 mmol/gm.
of Biorad AG4 × 4 ion-exchange resin (acetate cycle), followed
by freeze-drying to give 300 mg (38% isolated yield) of the title
compound as a lyophilized powder: anal. HPLC (system A)
26.7 min, 98.8% (<1% D-Ser isomer, 26.0 min); ES/FT-MS m/z
1637.0 (calcd [M + H]+ 1636.8); amino acid compositional
analysis (theory/found), Ser 4.0/2.54 (uncorr.), Glu 1.0/1.01
(Gln f Glu), Chg 1.0/1.09, Hyp 1.0/0.88, Pro 1.0/1.03.
Fractions containing at least 90% of the title major compo-
nent were combined and concentrated in vacuo, then repassed
through preparative HPLC as above. Pooled fractions contain-
ing <2% D-Ser isomer, after concentration and ion exchange
as above, yielded an additional 100 mg (13% yield) of the title
compound.
In a scaled-up preparation of conjugate 5, modifications were
implemented to reduce the extent of racemization at the
C-terminal serine of peptide 7b. Thus, when 4.95 g (6.28 mmol)
of peptide carboxylic acid 7b and 4.50 (3.73 mmol) of 4-O-(Pro)-
dAc-VIN 2c were coupled in 300 mL of DMF using 1.72 g (10.5
mmol) of the additive ODHBt, using NMM as base and 1.88 g
(9.8 mmol) of EDC, the extent of racemization, determined by
monitoring by HPLC as above, was reduced to <10%, as
determined by analytical HPLC (system A) of the crude
product prior to preparative HPLC.
4-O-(Ac-H yp -Se r -Se r -Ch g-D-Gln -D-Se r -Se r -P r o)-d es-
a cetyl Vin bla stin e 6. Samples of 125 mg (0.16 mmol) of
peptide carboxylic acid 7c and 130 mg (0.13 mmol) of 4-O-
(Pro)-dAc-VIN 2c, prepared as above, were dissolved in 10 mL
of DMF under N2. Then 44 mg (0.27 mmol) of ODHBT was
added, and the pH was adjusted to 6.5-7 (moistened 5-10
range pH paper) with NMM, followed by cooling to 0 °C and
addition of 49 mg (0.25 mmol) of EDC. Stirring was continued
at 0-5 °C until completion of the coupling as monitored by
analytical HPLC (system A), approximately 4 h. Analysis
showed the major component at 25.9 min. After 24 h the
reaction was worked up by concentrating to a small volume
in vacuo and dissolving in 50 mL of 5% HOAc, followed by
preparative HPLC on a Waters C18 Delta-Pak column 15µM
300A (A ) 0.1% TFA/H2O; B ) 0.1% TFA/CH3CN), gradient
elution 85 f 65% A/90 min. Homogeneous fractions were
pooled and concentrated to a volume of ∼50 mL and passed
through approximately 30 mL of Biorad AG4 × 4 ion-exchange
resin (acetate cycle), followed by freeze-drying to give 77 mg
(33% isolated yield) of the title compound as a lyophilized
powder: anal.HPLC (system A) 24.6 min, 98.9%; ES/FT-MS
m/z 1636.8 (calcd [M + H]+ 1636.8); amino acid compositional
analysis (theory/found), Ser 4.0/3.59 (corr.), Glu 1.0/0.94
(Gln f Glu), Chg 1.0/1.06, Hyp 1.0/1.05, Pro 1.0/0.96.
4-O-(Ac-Hyp -Ser -Ser -Ch g-Gln -Ser -P r o)-d es-a cetyl Vin -
bla stin e 3c. A sample of 30.5 mg (0.025 mmol) of 4-O-(prolyl)
des-acetyl vinblastine TFA salt 2c was dissolved in 6 mL of
DMF under N2, along with 40.3 mg (0.06 mmol) of peptide 7a ,
and the solution was cooled to 0 °C. Then 13.5 mg (0.10 mmol)
of HOAt was added, followed by 2,4,6-collidine to ensure a pH
of g6.5 (3 drops). After dissolution was complete, 13.2 mg (0.07
mmol) of EDC was added, followed by stirring of the solution
at 0-5 °C until completion of the coupling as monitored by
analytical HPLC (system A), approximately 8 h. After 20 h
the reaction was worked up by addition of 1 mL of H2O and,
after stirring 1 h, concentrated to a small volume in vacuo.
Upon dissolving in ca. 40 mL of 5% HOAc, the sample was
subjected to preparative HPLC on a Waters C18 Delta-Pak
column 15mM 300A (A ) 0.1% TFA/H2O; B ) 0.1% TFA/CH3-
CN), gradient elution 85 f 65% A/70 min at a flow rate of 80
mL/min. Homogeneous fractions were pooled, concentrated to
a small volume in vacuo, and lyophilized to yield 25 mg (65%
yield) of white powder: anal.HPLC (system A) 27.0 min, 99.3%;
ES/FT-MS m/z 1550.0 (calcd [M + H]+ 1550.2); amino acid
compositional analysis (theory/found), Ser 3.0/2.60 (corr.), Glu
1.0/0.97 (Gln f Glu), Chg 1.0/1.02, Hyp 1.0/1.08, Pro 1.0/0.94.
P r ep a r a tion a n d Com p a r ison of Sta bilities of 4-O-
(Ser -Leu )-d es-a cetyl Vin bla stin e 4a a n d 4-O-(Ser -P r o)-
d es-a cetyl Vin bla stin e 4c. Samples of 6.3 mg each of 4-O-
(Leu)-dAc-VIN 2a and 4-O-(Pro)-dAc-VIN 2c, respectively, were
dissolved in 3 mL of DMF, along with 2.8 mg of Boc-Ser-OH,
followed by 2.7 mg of HOAt and 1 drop of 2,4,6-collidine, then
2.6 mg of EDC. After 22 h analytical HPLC (system B) showed
the reaction to be complete, and 1 mL of H2O was added. In 1
h the solvent was removed in vacuo, and the intermediate 4-O-
(Boc-Ser) derivative was isolated by partitioning with EtOAc/
H2O and washing in turn with dil. KHSO4, H2O, NaHCO3, and
NaCl (2X), followed by drying over Na2SO4, solvent removal
and drying in vacuo. Treatment with 3 mL of TFA/CH2Cl2 (2:1
v/v), then evacuation under reduced pressure and dissolution
in 1.0 mL of H2O, afforded the title compounds: 4a HPLC
(system B) 28.1 min (95.4% pure); 4c HPLC (system B) 23.3
min (96.7% pure).
The pH of each solution was adjusted to 7.5 by the addition
of saturated NaHCO3. After 1 h 4c had been converted to dAc-
VIN 1b (RT 24.3 min) to the extent of 8.6%, at 4 h 31.8%, and
at 24 h nearly completely, corresponding to a T1/2 of ap-
proximately 6 h. Within the same 24 h period, 4a had
generated e0.5% of 1b.
Ack n ow led gm en t. We thank Dr. J oel R. Huff for
his support and encouragement, Trina Bolyar, Kathleen
Haskell, David M. Kiefer, Karen Leander, and Elizabeth
McAvoy for their work in support of the cell culture and
mouse tumor xenograft studies, Drs. Harri G. Ramjit
and Charles W. Ross III for mass spectral analysis, Mei-
J y Tang for amino acid compositional analysis, and J ean
Kaysen for preparing the manuscript.
4-O -(Ac -H y p -S e r -S e r -C h g -G ln -S e r -S e r -P r o )-d es -
a cetyl Vin bla stin e 5. Samples of 522 mg (0.66 mmol) of
peptide carboxylic acid 7b and 555 mg (0.46 mmol) of 4-O-
(Pro)-dAc-VIN 2c, prepared as above, were dissolved in 17 mL
of DMF under N2. Then 163 mg (1.13 mmol) of HOAt was
added, and the pH was adjusted to 6.5-7 (moistened 5-10
range pH paper) with NMM, followed by cooling to 0 °C and
addition of 155 mg (0.81 mmol) of EDC. Stirring was continued
at 0-5 °C until completion of the coupling as monitored by
analytical HPLC (system A), approximately 8 h, maintaining
the pH at 6.5-7 by periodic addition of NMM. Analysis showed
the major component at 26.3 min preceded by a significant
minor component (ca. 20%) at 26.1 min, suggestive of possible
racemization at the C-terminal serine during the coupling.
After 12 h the reaction was worked up by addition of ∼4 mL
of H2O and, after stirring 1 h, concentrated to a small volume
in vacuo and dissolution in 150 mL of 5% HOAc, followed by
preparative HPLC in two portions on a Waters C18 Delta-
Pak column 15 µM 300A (A ) 0.1% TFA/H2O; B ) 0.1% TFA/
CH3CN), gradient elution 95% f 65% A/70 min. Homogeneous
fractions from both runs were pooled and concentrated to a
volume of ∼50 mL and passed through approximately 40 mL
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