Synthesis and in vitro antitumor effect of vinblastine derivative- oligoarginine conjugates

Article abstract of DOI:10.1021/bc100028z

Vinblastine is a widely used anticancer drug with undesired side effects. Its conjugation with carrier molecules could be an efficient strategy to reduce these side effects. Besides this, the conjugate could exhibit increased efficiency against resistant cells, e.g., due to the altered internalization pathway. Oligoarginines, as cell-penetrating peptides, can transport covalently attached compounds into different kinds of cells and enhance the efficiency of those compounds. We report here the coupling of vinblastine through its carboxyl group at position 16 with the N-terminal amino function of l-Trp methyl ester. After hydrolysis of the ester group, 17-desacetylvinblastineTrp was conjugated to the N-terminal amino group of oligoarginine via the C-terminal carboxyl group of the Trp moiety in solution. The antitumor effect of conjugates was studied on sensitive and resistant human leukemia (HL-60) cells in vitro. Our data suggest that all conjugates investigated possess an antiproliferative effect against the studied cells. However, the effect was dependent on the number of Arg residues in the conjugates: Arg₈ > Arg₆ Arg ₄. The conjugate with Arg₈ exhibited similar efficicacy as compared with free 17-desacetylvinblastineTrp. The in vitro studies also showed that the tubulin binding ability of vinblastine was essentially preserved even in the octaarginine conjugate. We also observed that two isomers were formed during conjugation. These isomers showed different levels of activity against tubulin polymerization in vitro and in vivo. The 17-desacetylvinblastineTrp- Arg₈-1 isomer conjugate possessed high selectivity against the mitotic spindles. HRMS and NMR data suggest that 17-desacetylvinblastineTrp- Arg₈-1 and 17-desacetylvinblastineTrp-Arg₈-2 are epimers at the tryptophan α carbon atom.

Full text of DOI:10.1021/bc100028z

1948
Bioconjugate Chem. 2010, 21, 1948–1955
Synthesis and in Vitro Antitumor Effect of Vinblastine
Derivative-Oligoarginine Conjugates
´
Zolta´n Ba´no´czi, Almos Gorka-Kereske´nyi, Judit Reme´nyi, Erika Orba´n, La´szlo´ Hazai, Nata´lia To˝ke´si, Judit Ola´h,
Judit Ova´di, Zolta´n Be´ni, Viktor Ha´da, Csaba Sza´ntay, Jr., Ferenc Hudecz,* Gyo¨rgy Kalaus, and Csaba Sza´ntay
Research Group of Peptide Chemistry, Eo¨tvo¨s Lora´nd University (ELTE), Hungarian Academy of Sciences, Budapest 112,
P.O. Box 32, H-1518, Hungary, Department of Organic Chemistry and Technology, Budapest University of Technology and
Economics, Budapest, Gelle´rt te´r 4, H-1521 Hungary, Research Group for Alkaloid Chemistry, Hungarian Academy of Sciences,
University of Technology and Economics, Budapest, Gelle´rt te´r 4, H-1521 Hungary, Institute of Enzymology, Biological
Research Center, Hungarian Academy of Sciences, Budapest, Karolina u 29, H-1113 Hungary, Spectroscopic Research, Gedeon
Richter Plc., Budapest 10, P.O. Box 27, H-1475 Hungary, and Department of Organic Chemistry, Eo¨tvo¨s Lora´nd University
(ELTE), Budapest, Pa´zma´ny P. s. 1./a, H-1117 Hungary. Received January 15, 2010; Revised Manuscript Received July 12, 2010
Vinblastine is a widely used anticancer drug with undesired side effects. Its conjugation with carrier molecules
could be an efficient strategy to reduce these side effects. Besides this, the conjugate could exhibit increased
efficiency against resistant cells, e.g., due to the altered internalization pathway. Oligoarginines, as cell-penetrating
peptides, can transport covalently attached compounds into different kinds of cells and enhance the efficiency of
those compounds. We report here the coupling of vinblastine through its carboxyl group at position 16 with the
N-terminal amino function of L-Trp methyl ester. After hydrolysis of the ester group, 17-desacetylvinblastineTrp
was conjugated to the N-terminal amino group of oligoarginine via the C-terminal carboxyl group of the Trp
moiety in solution. The antitumor effect of conjugates was studied on sensitive and resistant human leukemia
(HL-60) cells in vitro. Our data suggest that all conjugates investigated possess an antiproliferative effect against
the studied cells. However, the effect was dependent on the number of Arg residues in the conjugates: Arg8 >
Arg6 . Arg4. The conjugate with Arg8 exhibited similar efficicacy as compared with free 17-desacetylvinblastineTrp.
The in vitro studies also showed that the tubulin binding ability of vinblastine was essentially preserved even in
the octaarginine conjugate. We also observed that two isomers were formed during conjugation. These isomers
showed different levels of activity against tubulin polymerization in vitro and in vivo. The 17-desacetylvin-
blastineTrp-Arg8-1 isomer conjugate possessed high selectivity against the mitotic spindles. HRMS and NMR
data suggest that 17-desacetylvinblastineTrp-Arg8-1 and 17-desacetylvinblastineTrp-Arg8-2 are epimers at the
tryptophan R carbon atom.
breast cancer. However, vincristine is more effective against
Hodgkin’s disease and pediatric solid tumors. Vinblastine
treatment can result in leucopenia, while vincristine might be
neurotoxic. These differences indicate that there is a marked
relationship between the chemical structure and the biological
effect of these alkaloids. To eliminate or reduce the side effects
of these potent vinca-alkaloids, much effort has been directed
toward the synthesis of new derivatives.
In the first experiments, Barnett et al. synthesized 17-
desacetylvinblastine-amide (vindesine) by modifying the methyl
ester group at position C-16 of the vindoline part. Vindesine
and vincristine exhibited very similar biological activity spectra
in animal experiments, but the neurotoxicity of the former was
lower (5). Further compounds were prepared by modification
of the amide group by incorporating different alkyl groups (6)
or amino acids (7). Among the amino acid derivatives, the
hydrophobic amino acid (Leu, Ile, Val, and Trp) containing
derivatives showed pronounced activity. The Trp-OEt derivative
had an outstanding effect on P388 and L1210 leukemias
implanted intravenously in DBA2 mice (7).
INTRODUCTION
Vinca-alkaloids (e.g., vincristine, vinblastine) are widely used
antitumor drugs isolated first from the leaves of Catharanthus
roseus (L.) G. Don. They cause a cytotoxic effect by disturbing
the dynamics of the microtubular system (1, 2). In vivo, these
compounds interact with tubulin at the ends of microtubules in
mitotic spindles and promote microtubule instability (3). They
are bisindole alkaloids containing a rearranged catharanthine
and a vindoline moiety. Vindoline, a major alkaloid of Cantha-
ranthus roseus, constitutes the most complex half of vinblastine,
and it is both a biosynthetic and synthetic precursor of the natural
product. Upon studying the tubulin binding ability of the two
parts separately, it turned out that only the catharanthine part
could bind to tubulin, but this interaction is weaker than that
for the bisindole alkaloids (4). It was concluded that the indole
part of catharanthine could be responsible for the binding of
vinblastine/vincristine to tubulin and the vindoline moiety could
function as an anchoring residue.
The structure of vinblastine and vincristine differs only in
the nature of the substituent, a methyl or a formyl group,
respectively, on the vindoline N-atom, but this “minor” differ-
ence leads to different antitumor efficacies, potencies, and
toxicities. Vinblastine is in clinical use with success in the
treatment of testicular cancer, non-Hodgkin’s lymphoma, and
Various bioconjugates of vinblastine derivatives were also
prepared for targeting. Conjugates with tumor-specific antibodies
(8), for example, KS1/4 (9), carcinoembryonic antigen (CEA)
(10), epidermal growth factor receptor (11), or PF1/D, a
squamous carcinoma-reactive monoclonal antibody (12), were
also produced and tested extensively. In other experiments, 17-
desacetylvinblastine monohydrazide was coupled with folic acid
* Address correspondence to Ferenc Hudecz, Tel.: +36-1-2090-555/
6428, Fax: +36-1-372-2620; E-mail: fhudecz@caesar.elte.hu.
10.1021/bc100028z  2010 American Chemical Society
Published on Web 10/25/2010

Antitumor Effect of Vinblastine Conjugates
Bioconjugate Chem., Vol. 21, No. 11, 2010 1949
as a targeting unit via a peptide spacer (13, 14). The conjugate
could bind to the folate receptor positive cells and produced
specific, dose-responsive activity in vitro. In vivo tests confirmed
the activity of the conjugate in both syngeneic and xenograft
models with minimal to moderate toxicity.
gas flow rate, 10 arb; aux gas flow rate, 2 arb; sweep gas flow
rate, 2 arb; solvent, MeOH/H2O 1:1 + 1 v/v % cc. AcOH)
ionization technique.
All NMR measurements were performed on a Varian 800
MHz NMR spectrometer equipped with a 13C enhanced HCN
1
1
1
cold probe. H-1H, direct H-13C, long-range H-13C scalar,
and dipolar spin-spin connectivities were established from 1H,
2D-GHSQCAD, GCOSY, 2D-GHMBCAD, and 2D-ROESY
NMR experiments, respectively. All pulse sequences were
applied by using the standard spectrometer software package.
Oligoarginines are members of the cell-penetrating oligopep-
tide family. These synthetic or natural peptides permeate the
cell membrane and transport the covalently attached cargoes
into the cytosole. Among oligoarginines, hexa-, hepta-, and
octaarginine showed the most effective internalization (15, 16).
Different oligoarginines were used efficiently to transport
genetically or chemically attached protein, peptide, and non-
covalently bound plasmid DNA into cells (17). Oligoarginines
can also be used to transport covalently coupled small molecules
into cells, like cyclosporin A (18), ferrocene (19), and dauno-
mycin (20) derivatives or bisubstrate-analogue inhibitors of
basophilic protein kinases (adenosine, 5′-adenosine-carboxic
acid) (21).
In this work, we report on the synthesis of oligoarginine (Argn,
where n ) 4, 6, or 8) conjugates with the Trp-derivative of
vinblastine via amide bond formation in solution. We observed
and isolated two isomers after conjugation. The effect of these
compounds on sensitive and resistant HL-60 (human leukemia)
cells was studied. Furthermore, we described our findings on
the influence of conjugates on the in vitro and in vivo tubulin
polymerization. Data suggest that the cytotoxicity of the
conjugates was dependent on the chain length of the peptides.
Moreover, only the 17-desacetylvinblastineTrp-Arg8-1 conjugate
exhibited the capability to destroy only the mitotic spindle and
cause the apoptosis of the cells that are in mitosis. On the basis
of HRMS and NMR data, we also report on the structure
identification of the isomers proposing that 17-desacetylvin-
blastineTrp-Arg8-1 and 17-desacetylvinblastineTrp-Arg8-2 are
epimers at the R-carbon atom of the Trp residue.
Synthesis of Oligoarginines. Oligoarginines (tetra-, hexa-,
and octaarginine) were synthesized manually by solid-phase
methodology on Rink-amide resin (0.3 g, 0.69 mmol/g) using
Fmoc/tBu strategy. The side chain of Arg was protected with
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group. NR-
Fmoc group was removed with 2% piperidine plus 2% DBU in
DMF (2 + 2+5 + 10 min) followed by washing with DMF (8
× 0.5 min). For coupling, the Arg derivative and DIC and HOBt
dissolved in DMF were used in 3-fold molar excess for the resin
capacity. The reaction proceeded at RT for 60 min. Then, the
resin was washed (DMF 2 × 0.5 min, DCM 3 × 0.5 min). The
efficiency of coupling was checked by ninhydrin assay (22).
The amount of resin was divided into three parts during the
synthesis. After assembly, the peptide was cleaved from the
resin by 10 mL of TFA using 0.75 g of phenol, 0.5 mL of
distilled water, 0.5 mL of thioanisole, and 0.25 mL of 1,2-
ethandithiol as scavengers. The crude product was precipitated
by dry diethyl ether, dissolved in 10% acetic acid, and
freeze-dried. Purification was done by RP-HPLC as described
above. The following gradients were applied for purification: 0
min 0% B; 5 min 0% B; 50 min 50% B.
Synthesis of Conjugates. First, 17-desacetylvinblastineTrp
was prepared by coupling the unprotected 17-desacetylvinblas-
tine to Trp as described earlier (7) with some modifications.
Briefly, vinblastine was refluxed in an ethanol-chloroform
mixture with 98% hydrazine monohydrate instead of anhydrous
hydrazine used in the published paper mentioned above. The
corresponding acid azide was prepared with sodium nitrite in
the presence of hydrochloric acid. The latter compound, without
isolation, was allowed to react with L-Trp methyl ester in
dichloromethane at 4 °C to give the Trp derivative of 17-
desacetylvinblastine (23). After hydrolysis of the carboxylic
ester, the corresponding 17-desacetylvinblastineTrp carboxylic
acid (3 mg, 3.15 µmol) was coupled to oligoarginines (Argn,
where n ) 4, 6, or 8) (6.3 µmol) by DCC (1.3 mg, 6.3 µmol);
HOBt (1 mg, 6.3 µmol) in the presence of DIEA (1.1 µL, 6.3
µmol) in 0.5 mL DMF. The reaction proceeded at RT for 4 h.
The DMF was evaporated and conjugates were purified by RP-
HPLC using the following gradient: 0 min 5% B; 5 min 5% B;
50 min 80% B as described above.
Cell Cultures. HeLa (ATCC, CCL-2) cells were grown in
DME/F-12 medium supplemented with 10% FCS, 1 mM sodium
pyruvate, 100 U/mL streptomycin, and 100 µg/mL penicillin
(all from Sigma) (complete medium) in a humidified 37 °C
incubator with 5% CO2. The sensitive and resistant HL-60
human leukemia cells (ATCC: CCL-240) were cultured in
RPMI-1640 medium supplemented with 10% FCS, 2 mM
L-glutamine, and 160 µg/mL gentamycin. The cell culture was
maintained at 37 °C in a humidified atmosphere with 5% CO2.
MTT Assay. For cytostatic studies, sensitive and resistant
HL-60 cells were placed in a 96-well plate with each well
containing 5 × 103 cells. After incubation at 37 °C for 24 h,
the cultured cells were treated with the solution of conjugates
in serum-free RPMI-1640 medium for 3 h. Compounds were
used at the c ) 2 × 10-3 to 8 × 102 µM range. In control
experiments, cells were treated with serum-free medium at 37
°C for 3 h. After incubation, cells were washed with serum-
EXPERIMENTAL PROCEDURES
Fmoc-Arg(Pbf)-OH was purchased from Novabiochem
(La¨ufelfingen, Switzerland), while thioanisole, 1,2-ethanedithiol
(EDT), N-diisopropyl-ethylamine (DIEA), 1-hidroxybenztriazole
(HOBt), N,N′-diisopropylcarbodiimide (DIC), trifluoracetic acid
(TFA),phenol,ninhydrine,piperidine,1,8-diazabicyclo[5.4.0]undec-
7-ene (DBU) were Fluka (Buchs, Switzerland) products. The
Rink-amid MBHA resin was from Novabiochem (La¨ufelfingen,
Switzerland). Reagents and solvents used in the biological assays
were purchased from Sigma-Aldrich Ltc (Budapest, Hungary).
Solvents (dimethyl formaide (DMF), dichloromethane (DCM),
diethyl ether, acetonitrile (AcN)) for syntheses and purification
were obtained from Reanal (Budapest, Hungary).
General Procedures. Analytical RP-HPLC was performed
on a Knauer (Herbert Knauer GmbH, Berlin, Germany) system
using a Phenomenex SYNERGI MAX-RP column (250 × 4.6
mm I.D., 4 µm silica, 80 Å pore size) (Torrance, CA, USA) as
a stationary phase. Linear gradient elution (0 min 0% B; 5 min
0% B; 50 min 90% B) was generated using 0.1% TFA in water
as eluent A and 0.1% TFA in acetonitrile-water (80:20, V/V)
as eluent B. Flow rate of 1 mL/min was applied at ambient
temperature. The samples were dissolved in eluent B. The crude
products were purified on a semipreparative Phenomenex Jupiter
C18 column (250 × 10 mm I.D., 10 µm silica, 300 Å pore
size) (Torrance, CA, USA). The flow rate was 4 mL/min. Peaks
were detected at λ)220 nm. Positive ion electrospray ionization
mass spectrometric analysis was performed on a Bruker Esquire
3000 plus (Germany). The samples were dissolved in acetoni-
trile-water (50:50, V/V), containing 0.1% acetic acid. High-
resolution MS measurements were carried out on a Thermo LTQ
FT Ultra mass spectrometer using ESI (source voltage, 4.2 kV;
capillary voltage, 43 V; capillary temperature, 200 °C; sheath

1950 Bioconjugate Chem., Vol. 21, No. 11, 2010
Ba´no´czi et al.
Scheme 1. Synthesis of 17-DesacetylvinblastineTrp
free medium three times, and serum-containing medium was
added to the cells. After 3 days at 37 °C, the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay was carried out. The yellow solution of MTT at a
concentration of 0.2 mg/mL was added to each well. The
obtained purple crystal was dissolved in DMSO, and the optical
density (OD) of the samples was measured at λ ) 540 nm using
ELISA Reader (Labsystems MS Reader, Finland). The percent-
age of the antitumor effect was calculated using the following
equation:
antitumor effect % ) (1 - ODtreated/ODcontrol) × 100
where ODtreated and ODcontrol correspond to the optical densities
of the treated cells and the control cells, respectively, at λ )
540 nm. Results obtained from in vitro cytotoxicity measure-
ments were analyzed using sigmoidal curve fitting.
Tubulin Preparation. Microtubule-associated protein (MAP)-
depleted tubulin was purified from bovine brain by the method
of Na and Timasheff (24) and stored in 10 mM phosphate buffer,
pH 7.0, containing 1 M of sucrose, 0.5 mM of MgCl2, and 1
mM of GTP at -80 °C. Purified tubulin showed no contamina-
tion with MAPs on overloaded SDS-PAGE. Before use, stored
tubulin was dialyzed against 50 mM of 4-morpholine ethane-
sulfonic acid (MES) buffer, pH 6.8, at 4 °C for 3 h and then
centrifuged at 100 000 g at 4 °C for 20 min.
Polymerization Assay. Ten micromoles of tubulin was as-
sembled to microtubules at 37 °C in polymerization buffer (50 mM
of MES buffer, pH 6.6, containing 1 mM of dithioerythritol, 1 mM
of MgCl2, 1 mM of EGTA (ethylene glycol tetraacetic acid), and
100 mM of KCl. The polymerization was initiated with 20 µM of
paclitaxel at 37 °C. Vinblastine, 17-desacetylvinblastineTrp, and
17-desacetylvinblastineTrp-octaarginine conjugates in the
0.05-2 µM concentration range were incubated with tubulin at
37 °C for 5 min before starting the polymerization. For turbidity
measurements, the absorbance was monitored at λ ) 350 nm with
a Cary 100 spectrophotometer (Varian, Mulgrave, Victoria, Aus-
tralia). The error margin of the turbidity measurements was (5%
within a series of experiments.
Immunocytochemistry. For microscopic studies, HeLa cells
were grown on 12 mm diameter coverslips. The vinblastine, 17-
desacetylvinblastineTrp, and 17-desacetylvinblastineTrp-octa-
arginine conjugates were added to the complete medium of the
cells in the 0.25-5 µM concentration range for 24 h and then were
fixed with ice-cold methanol for 15 min and immunostained for
R-tubulin as described by Lehotzky et al. (25). Nuclei were
counterstained with 4′,6-diamidino-2-phenylindole (DAPI) after
coverslips were mounted on an Aqua Poly/Mount medium (Sigma).
Images were taken on a Leica DMLS microscope (Leica Micro-
systems, Wetzlar, Germany) equipped with a cooled CCD camera
(Spot, Digital Instruments, Sterling Heights, MI, USA). Statistical
analysis was performed manually by investigating random micro-
scopic fields. Data were analyzed with Microsoft Excel. Images
were processed with ImageJ software.
Table 1. Characteristics of 17-DesacetylvinblastineTrp-
Oligoarginine Conjugates
[M+H]+,b
cal. [meas.]
conjugate
Rta (min)
17-desacetylvinblastineTrp-Arg4-1
17-desacetylvinblastineTrp-Arg4-2
17-desacetylvinblastineTrp-Arg6-1
17-desacetylvinblastineTrp-Arg6-2
17-desacetylvinblastineTrp-Arg8-1
17-dezacetylvinblasztinTrp-Arg8-2
27.9
26.3
26.0
26.3
26.3
26.6
1564.9 (1564.3)
1564.9 (1564.3)
1877.3 (1876.7)
1877.3 (1876.7)
2189.6 (2189.1)
2189.6 (2189.1)
a RP-HPLC retention time, column: Phenomenex SYNERGI MAX-
RP (250 × 4.6 mm I.D., 4 µm silica, 80 Å pore size). Linear gradient
elution 0 min 0% B; 5 min 0% B; 50 min 90% B using 0.1% TFA in
water as eluent A and 0.1% TFA in acetonitrile-water (80:20, V/V) as
eluent B. b ESI-MS.
studies 17-desacetylvinblastineTrp (7, 23, 26) was prepared with
a modified procedure using 17-desacetylvinblastine (26), as the
major metabolite of vinblastine in humans considering its
pharmacological equivalency with vinblastine (27) (Scheme 1).
17-DesacetylvinblastineTrp was conjugated with tetra-, hexa-,
or octaarginine in solution. First, the oligoarginines were built
up on Rink-amide resin by Fmoc/tBu strategy, and after cleavage
from the resin by TFA, the unprotected and purified C-terminal
peptide amides were used for the conjugation. 17-Desacetylvin-
blastineTrp was coupled to the peptides by the active ester
method using DCC/HOBt coupling reagents in DMF in the
presence of DIEA as base. The conjugates were characterized
by analytical RP-HPLC and ESI-MS (Table 1 and Figure 1 in
Supporting Information).
The conjugation reaction resulted in two isomers with
different retention times, but with the same molecular mass in
all three cases. The isomers were separated, isolated, and
characterized, and their biological effects were examined. To
identify the structure of the two isomer conjugates, these
compounds were studied by high-resolution MS and NMR.
Identification of the Structure of the Two Isomers of
the Octaarginine Conjugates. The two compounds (17-
desacetylvinblastineTrp-Arg8-1 and 17-desacetylvinblastineTrp-
Arg8-2) obtained during the preparation of the 17-desacetylvin-
blastineTrp octaarginine conjugate were investigated after HPLC
separation.
RESULTS AND DISCUSSION
To avoid the undesired effect of antitumor drugs and to
increase their efficiency against resistant cells is a difficult task
to achieve. The conjugation of small drug molecules to carriers
could be considered as a potential strategy for utilization. In
this paper, we describe the synthesis and in vitro antitumor effect
of oligoarginine conjugates of the Trp derivative of 17-
desacetylvinblastine. The influence of these compounds on the
tubulin polymerization was also studied in vitro and in vivo.
Synthesis of Conjugates. Among the amino acid ester
derivatives of viblastine, the tryptophan derivative showed the
highest antitumor effect in mice (7). Accordingly, for the present
High-Resolution Mass Spectrometry. In the direct ESI mass
spectra of the samples 17-desacetylvinblastineTrp-Arg8-1 and
17-desacetylvinblastineTrp-Arg8-2 multiply charged ions of
[M+4H]4+, [M+5H]5+ and [M+6H]6+ can be detected. The
monoisotopic mass values were obtained by the deconvolution
of the mass spectra (using Xtract in Xcalibur 2.0.7): 17-
DesacetylvinblastineTrp-Arg8-1: m/z 2188.29693, calculated
value for C102H161O16N39: 2188.29780 (δ -0.4 ppm). 17-

Antitumor Effect of Vinblastine Conjugates
Bioconjugate Chem., Vol. 21, No. 11, 2010 1951
Figure 1. Structures proposed for the two isomeric Arg8 conjugates of 17-desacetylvinblastineTrp: 17-desacetylvinblastineTrp-Arg8-1 (left) and
17-desacetylvinblastineTrp-Arg8-2 (right).
DesacetylvinblastineTrp-Arg8-2: m/z 2188.29728, calculated
value for C102H161O16N39: 2188.29780 (δ -0.2 ppm).
(mainly those belonging to the vindoline half of the two
molecules) were still significantly broadened in this solvent
mixture, structurally informative spectral information could
nevertheless be obtained. In accordance with the HRMS results,
these data suggest that 17-desacetylvinblastineTrp-Arg8-1 and
17-desacetylvinblastineTrp-Arg8-2 are epimers at the tryptophan
R-carbon atom (Figure 1).
The most intense [M+5H]5+ ion peaks were fragmented by
CID (collision induced dissociation) at a normalized collision
energy of 25%. The monoisotopic mass values of the main
fragment ions in the deconvoluted MS-MS spectra are the
following: 17-DesacetylvinblastineTrp-Arg8-1: m/z 2129.25013,
2015.17064, 1876.09619, 1702.96880, 1660.94691, 1537.91516,
1501.89406, 1348.74426, 1234.66543, 1192.64356, 1174.63301,
1078.56404, 648.36752, 523.28346. 17-DesacetylvinblastineTrp-
Arg8-2: m/z 2129.25155, 2015.17057, 1876.09677, 1234.66544,
1192.64463, 1174.63280, 953.63348, 894.46857, 708.38901,
648.36785.
Partial H and 13C NMR assignments (determined from H,
2D-GHSQCAD, GCOSY, 2D-GHMBCAD, and 2D-ROESY
NMR spectra) that are indicative of the proposed epimeric
structures are listed in Table 1 in Supporting Information. These
structures are supported via the following argument. The
chemical shifts of the catharantine parts of 17-desacetylvin-
blastineTrp-Arg8-1 and 17-desacetylvinblastineTrp-Arg8-2 (note
that an almost complete assignment could be achieved for the
catharantine moieties) are practically the same, telling us that
the structural difference is located elsewhere in the two
molecules.
1
1
On the basis of the accurate mass values of the fragment ions
in the MS-MS spectra, elemental compositions were calculated
using the Xcalibur 2.0.7 software. In the range of 0.2-1.5 ppm
mass accuracy, plausible elemental compositions could be
obtained for all of the fragment ions. All of these elemental
compositions are in agreement with the expected 17-de-
sacetylvinblastineTrpArg8 chemical structure. However, when
comparing the MS-MS spectra of the two samples some
differences can be noticed regarding the obtained fragment ions
and their relative abundance values. Most of the fragment ions
were obtained by the cleavage of the Arg8 part of the molecule
for both samples. In the case of 17-desacetylvinblastineTrp-
Arg8-1, more fragment ions were produced by the cleavage of
the vindoline C ring (cleavage of the C2-C16 and C17-C20
bonds), while exclusively for 17-desacetylvinblastineTrp-Arg8-
2, a single minor fragment ion was observed in the deconvoluted
mass spectrum, which can originate from a C-C cleavage beside
the carbonyl group of the Tryptophan. According to these MS
data, compounds 17-desacetylvinblastineTrp-Arg8-1 and 17-
desacetylvinblastineTrp-Arg8-2 are two stereoisomers of 17-
desacetyvinblastineTrpArg8.
Due to the low sample concentrations and to the significant
broadening of some of the resonances belonging to the vindoline
half of the molecules, assignments for this part of the compounds
could not be given completely. Nevertheless, the similarity of
the signals due to the detectable and assignable functional groups
[the C(14)dC(15) double bond, the C(20)-Et, C(11)-OMe, and
N(1)-Me functions], as well as those belonging to the aromatic
resonances (8, 9) of the indole part, indicate that the vindoline
parts also remained intact during the formation of the conjugates.
The most significant differences were detected in signals
belonging to the tryptophan moieties, especially those belonging
to the Trp R and ꢀ protons of these residues. Not only do the
chemical shifts of the Trp R (5.09 and 4.84 ppm in 17-
desacetylvinblastineTrp-Arg8-2 and 17-desacetylvinblastineTrp-
Arg8-1, respectively) and ꢀ protons (3.73 ppm in 17-desacetylvin-
blastineTrp-Arg8-2 and 3.58 and 3.72 ppm in 17-desacetylvin-
blastineTrp-Arg8-1) differ significantly, but the coupling patterns
differ as well (Figure 3 in Supporting Information). In 17-
desacetylvinblastineTrp-Arg8-2, the Trp R proton resonance
appears as a triplet with a coupling constant of 5.9 Hz, while
that in 17-desacetylvinblastineTrp-Arg8-1 is a doublet of doublet
with coupling constants of 10.4 and 5.6 Hz. These findings
indicate that during the formation of the Arg conjugates
inversion of the Trp R carbon occurred. The Trp moieties are
expected to be in different conformational states in the two
epimers, which accounts for the observed difference in the
coupling patterns of the Trp R proton resonances.
NMR. Since both compounds 17-desacetylvinblastineTrp-
Arg8-1 and 17-desacetylvinblastineTrp-Arg8-2 were available
only in a limited amount for NMR measurements, only limited
spectroscopic information could be obtained on an affordable
time scale even on our ultrasensitive 800 MHz NMR spectrom-
eter. Furthermore, due to their conformational dynamics both
1
compounds gave broad H NMR resonances in all common
NMR solvents (such as DMSO-d6, CDCl3, ACN-d3, MeOD-d4,
or D2O), which prohibited easy access to the desired spectro-
scopic information (see Supporting Information Figure 2 as an
example) in this concentration. Finally, a solvent mixture
containing ACN-d3 and D2O in a one-to-one ratio was chosen
as the most suitable medium. Although most of the resonances
In order to identify the two epimers, we recorded the 1H NMR
spectra of two smaller model systems containing only the

1952 Bioconjugate Chem., Vol. 21, No. 11, 2010
Ba´no´czi et al.
Figure 3. Cytostatic activity of 17-desacetylvinblastineTrp-Arg8 con-
jugates on sensitive and resistant HL-60 cells. The cells were incubated
with the compound for 3 h, after grown in serum-containing medium
for 3 days. The IC50 values were determined by MTT assay as described
in the text: black -9-, 17-desacetylvinblastineTrp-Arg8-1/HL-60, IC50
) 1.61 µM; red -b-, 17-desacetylvinblastineTrp-Arg8-2/HL-60, IC50
) 1.59 µM; blue -2-, 17-desacetylvinblastineTrp-Arg8-1/HL-60-MDR1,
IC50 ) 2.62 µM; green -1-, 17-desacetylvinblastineTrp-Arg8-2/HL-
60-MDR1, IC50 ) 2.94 µM; cyan -[-, 17-desacetylvinblastineTrp-Arg8-
1/HL-60-MRP1, IC50 ) 2.94 µM; magenta -1-, 17-desacetylvinblastin-
eTrp-Arg8-2/HL-60-MRP1, IC50 ) 3.78 µM.
Figure 2. Cytostatic activity of vinblastine sulfate, 17-desacetylvinblastine,
and its conjugates on sensitive HL-60 cells. The cells were incubated with
the compound for 3 h, after growth in serum-containing medium for 3
days. The IC50 values were determined by MTT assay as described in the
text: black -9-, 17-desacetylvinblastineTrp, IC50 ) 0.49 µM; magenta -1-,
vinblastine sulfate, IC50 ) 1.26 µM; red -b-, 17-desacetylvinblastineTrp-
Arg4-1, IC50 ) 5.25 µM; green -2-, 17-desacetylvinblastineTrp-Arg4-2,
IC50 ) 4.97 µM; cyan -[-, 17-desacetylvinblastineTrp-Arg6-1, IC50 ) 0.97
µM; blue -1-, 17-desacetylvinblastineTrp-Arg6-2, IC50 ) 1.75 µM; purple
-9-, 17-desacetylvinblastineTrp-Arg8-1, IC50 ) 1.61 µM; teal -b-, 17-
desacetylvinblastineTrp-Arg8-2, IC50 ) 1.59 µM.
eTrp, and these values are comparable with those of vinblastine
sulfate (IC50 ) 0.97-1.75 µM for octa- and hexaarginineTrp
conjugates and IC50 ) 1.26 µM for vinblastine sulfate).
The activity of compounds was studied on HL-60/MDR1 and
HL-60/MRP1 resistant cells, too. The effect of vinblastine sulfate
on resistant HL-60/MRP1 cells (IC50 ) 1.31 µM) is similar to
that of sensitive HL-60 cells (IC50 ) 1.26 µM), but its efficacy
was somewhat lower in the case of resistant HL-60/MDR1 cells
(IC50 ) 2.96 µM).
The effect of 17-desacetylvinblastine decreased markedly
(IC50 ) 1.78 for MDR1 and 0.24 µM for MRP1), showing that
it is a good substrate of the MDR1 and MRP1 transporter
proteins, somewhat better than vinblastine sulfate. The efficiency
of the Trp derivative diminished dramatically in the presence
of the MDR1 protein (IC50 ) 59.6 µM), but the effect of the
MRP1 protein was less marked (IC50 ) 3.98 µM).
The antitumor effect of the conjugates increased with the
number of Arg residues present in the conjugates on both
resistant cell lines, but the IC50 values are somewhat higher than
in the case of the sensitive cells. The two isomer conjugates
(except the compounds with four Arg residues) show similar
effects on the resistant cells. The IC50 values of octaarginine
conjugates are shown in Figure 3.
Our results show that 17-desacetylvinblastine significantly
inhibits the division of sensitive human leukemia cells. This
effect, which is higher than that of vinblastine sulfate, decreases
if the molecule is modified by a Trp. The conjugation of 17-
desacetylvinblastineTrp to oligoarginine resulted in an antitumor
effect which is dependent on the number of Arg residues. The
presence of hexa- and octaarginine in the conjugates has almost
no effectsthese compounds exhibit essentially the same level
of antitumor activity. However, tetraarginine reduces the activity
significantly.
17-Desacetylvinblastine has significant cytotoxicity, higher
than that of vinblastine. While the attachment of Trp dramati-
cally decreases the effect of 17-desacetylvinblastine, this deriva-
tive still has a similar activity to vinblastine. The IC50 values
of octa- and hexaarginineTrp conjugates suggest that these
“vindoline” half bound to D- and L-tryptophan (unpublished
synthetic results), having the structures shown in Figure 4 in
Supporting Information. In the vindoline-L-tryptophan model,
the R proton appears as a triplet (5.8 Hz) similarly to epimer
17-desacetylvinblastineTrp-Arg8-2, while in the vindoline-D-
tryptophan model, it appears as a doublet of doublets (8.9 and
4.8 Hz) similarly to epimer 17-desacetylvinblastineTrp-Arg8-1
(Figure 5 in the Supporting Information). These similarities
indicate that 17-desacetylvinblastineTrp-Arg8-2 and 17-de-
sacetylvinblastineTrp-Arg8-1 contain the L- and D-tryptophan
moieties, respectively.
Additional synthetic and spectroscopic investigations are in
progress to further verify the proposed structures as well as to
explore the nature of and reasons behind the observed epimer-
ization of Trp octaarginine during its coupling to 17-de-
sacetylvinblastine.
Cytostatic Effect of Conjugates on HL-60 Human Leukemia
Cells. The cytostatic effect of conjugates as well as of the control
compounds was studied on sensitive and resistant HL-60 cells
using MTT assay. The cells were treated with vinblastine sulfate,
17-desacetylvinblastineTrp, and its conjugates at different
concentrations for 3 h, and then, the number of living cells was
determined after 72 h. Data are summarized in Figure 2. In
contrast to vinblastine sulfate (IC50 ) 1.26 µM), the 17-
desacetylvinblastine has more marked activity on sensitive HL-
60 cells (IC50 ) 0.0043 µM). The presence of Trp residue in
the amino acid conjugate decreased the activity by 2 orders of
magnitude (IC50 ) 0.49 µM), but it is still more effective than
vinblastine sulfate. On sensitive HL-60 cells, the increasing
number of Arg residues enhanced the cytostatic effect of the
conjugate. The octa- and hexaarginineTrp conjugate isomers
have slightly higher activity (IC50 ) 1.61 µM and 1.59 µM, for
octaarginineTrp conjugate and IC50 ) 0.97 µM and 1.75 µM
for hexaarginineTrp conjugates) as compared to the tetraargin-
ineTrp conjugates (IC50 ) 5.25 µM and 4.97 µM). It is
interesting to note that the octa- and hexaarginineTrp conjugate
isomers almost preserve the activity of 17-desacetylvinblastin-

Antitumor Effect of Vinblastine Conjugates
Bioconjugate Chem., Vol. 21, No. 11, 2010 1953
compounds exhibit in vitro an antitumor effect similar to that
of vinblastine.
In the case of resistant cells, the effect of 17-desacetylvin-
blastine is dramatically decreased, and the activity of both
the Trp derivative and its octaarginine conjugate is similar in
the case of HL-60/MRP1. The HL-60/MDR1 cells pump
out the Trp derivative so effectively that its activity decreases
to one tenth, while the octaarginineTrp conjugates show
comparable effect with 17-desacetylvinblastine. Interestingly,
the effect of vinblastine sulfate is reduced only by MDR1
proteins and only to a little extent. The biggest effect is measured
in the presence of the MDR1 protein when the cells are treated
by vinblastine derivatives. In the case of conjugates, the MRP1
proteins result in a bigger reduction in the activity. These results
show that 17-desacetylvinblastine is a very good substrate of
the transporter proteins. Considering the decreased effect of the
conjugates, it is attractive to speculate that the transport proteins
can also pump out the conjugated 17-desacetylvinblastine from
the cells. The extent of efflux increased in the case of conjugates
with a shorter oligoarginine, which could suggest that the
character of conjugates became similar to that of the drug. On
the other hand, the efficacy of the MRP1 protein to 17-
desacetylvinblastineTrp is increased by the conjugation. In the
resistant cells, the proteins pump out the compounds and the
resulting concentration reduction inhibits the effect of the drugs.
Because the cytotoxicity of 17-desacetylvinblastine and the
octaarginineTrp conjugate are almost similar on resistant cells,
it can be supposed that the reason for the significant difference
in the case of the sensitive cells is the difference between the
concentrations of active 17-desacetylvinblastine in the cells. In
the case of 17-desacetylvinblastine, the achieved effective
intracellular concentration is determined only by the diffusion
rate. However, with the conjugates at least two different
processes are involved, namely, the internalization of the
conjugates and the interaction of the released (free) or conju-
gated drug with the microtubular system at the site of action.
In order to analyze the influence of the presence of oligoarginine,
the effect of the conjugates was studied on tubulin polymeri-
zation in vitro and in vivo.
In Vitro and in Vivo Effect of Vinblastine Derivatives
on Tubulin Polymerization. In order to characterize the effect
of vinblastine and its conjugate derivatives on a microtubule
assembly, the polymerization of tubulin heterodimers was
investigated in vitro at different drug concentrations.
Figure 4. Effect of vinblastine and its conjugate derivatives on tubulin
polymerization. (A) Turbidimetric assay. The paclitaxel-induced tubulin
polymerization was followed at λ ) 350 nm. The concentration of
vinblastine and its derivatives was 1 µM. Tubulin control (solid line),
vinblastine (dashed line), 17-desacetylvinblastineTrp (dotted line), 17-
desacetylvinblastineTrp-Arg8-1 (short dashed line), 17-desacetylvin-
blastineTrp-Arg8-2 (dash-dot-dotted line). (B) The relative effect of
vinblastine and its derivatives on tubulin polymerization as a function
of drug concentration. (9, dashed line) vinblastine, (2, dotted line)
17-desacetylvinblastineTrp, (b, short dashed line) 17-desacetylvin-
blastineTrp-Arg8-1, (O, dash-dot-dotted line) 17-desacetylvinblastin-
eTrp-Arg8-2.
Figure 4 shows that vinblastine and its derivatives inhibit
paclitaxel-induced tubulin polymerization; however, this effect
was dependent upon the nature of the derivatives. The
dose-response curves obtained by plotting the polymerization
rate as a function of drug concentrations reveal that the inhibitory
effects of 17-desacetylvinblastineTrp and the 17-desacetylvin-
blastineTrp-Arg8-2 conjugates are comparable to those of
vinblastine in the polymerization assay. However, the inhibitory
potency of the 17-desacetylvinblastineTrp-Arg8-1 conjugate on
the microtubule assembly was less pronounced. It should be
noted that with the mixture of the two Trp-Arg8-containing
isomers (before their HPLC separation) we obtained a lower
inhibitory effect than with 17-desacetylvinblastineTrp-Arg8-2,
but a higher effect than with 17-desacetylvinblastineTrp-Arg8-
1. This result suggests that the 17-desacetylvinblastineTrp-
Arg8-1 conjugate is not a potential antimitotic agent. However,
the effect observed in an in vitro system with purified tubulin
cannot provide information on the specificity of the drugs.
Specificity, i.e., the antimicrotubular activity of the drug, was
studied at a cellular level using HeLa cells by investigating both
the interphase and spindle microtubules in parallel.
in cancer cells, which are characterized by rapid proliferation.
Therefore, if a drug specifically targets the mitotic microtubule
spindle, this effect is not necessarily reflected at a cellular level
by the cytotoxicity index.
In order to characterize the antimicrotubule activities of
vinblastine and its conjugate derivatives at a cellular level, we
performed comparative studies in human cervical carcinoma
(HeLa) cells. The drugs were added to the medium of the
growing cells at different concentrations. After 24 h, the
microtubular network of the cells was immunostained with
antitubulin antibody and visualized by immunofluorescence
microscopy.
Figure 5 shows that the interphase microtubular network can
be characterized by three well-defined stages: (i) Normal stage
(Figure 5a): the microtubular network is assembled into bundled
fibers of highly ordered fashion. The fibers originate from the
The majority of the eukaryote cells are at interphase, and
only the minority are in mitosissat a certain point in timeseven

1954 Bioconjugate Chem., Vol. 21, No. 11, 2010
Ba´no´czi et al.
Figure 5. Characteristic pictures for interphase (a-c) and mitotic (d-f) microtubule network formations in HeLa cells immunostained for R-tubulin
(green); normal, control (a and d), fragmentated (b) induced by 2.5 µM 17-desacetylvinblastineTrp, depolymerized (c) induced by 2.5 µM vinblastine,
asymmetric (e) induced by 0.25 µM, and multipolar (f) induced by 2.5 µM 17-desacetylvinblastineTrp treatment. Blue: DAPI. Bar: 5 µm.
Table 2. Effect of Vinblastine and 17-Desacetylvinblastine Derivatives on the Spindle and Interphase Microtubule Ultrastructuresa
vinblastine
17-desacetylvinblTrp
17-desacetylvinblTrpArg8-2
17-desacetylvinblTrpArg8-1
aberrant
interphase
aberrant
interphase
aberrant
interphase
aberrant
interphase
mitosis (%)
microtubules
mitosis (%)
microtubules
mitosis (%)
microtubules
mitosis (%)
microtubules
control
0.25 µM
1 µM
2.5 µM
5 µM
2
100
100
100
100
normal
2
47
100
100
100
normal
normal
fragmented
fragmented
depolymerized
2
22
75
98
100
normal
normal
normal
fragmented
fragmented
2
normal
n. d.
normal
normal
normal
depolymerized
depolymerized
depolymerized
depolymerized
n. d.b
45
75
100
a HeLa cells were treated with the drugs at different concentrations for 24 h. The percentage distribution of the aberrant mitotic spindle among all
cells in the phase of mitosis was quantified by investigating random microscopic fields. The structure of the interphase and mitotic microtubules was
visualized by immunofluorescence microscopy using monoclonal antitubulin antibody. b n.d: not defined.
microtubule organizing center and are concentrated around the
nucleus. Their free ends radiate outward toward the cell
membrane, forming a network. In drug-treated cells, microtu-
bules undergo significant reorganizations. (ii) Fragmented stage
(Figure 5b): long bundles of microtubules are fragmented into
short, disorganized fibers which radiate to the cell periphery in
a criss-cross fashion. In the vicinity of nuclei, the microtubules
acquire an amorphous appearance. The microtubule organizing
centers are no longer apparent. (iii) Depolymerized stage (Figure
5c): all microtubules are disassembled; the depolymerized
microtubules show homogeneous cytosolic distribution.
We also studied the changes of the spindle structures in
parallel with those of the interphase at various drug concentra-
tions. Treatment of the cells with vinblastine or its derivatives
caused significant alterations in the spindle structures. Typical
structures visualized in the drug-treated samples are shown in
Figure 5: normal, symmetric (Figure 5d), asymmetric (Figure
5e), and multipolar (Figure 5f).
For evaluation of the specificity of the vinblastine derivatives,
we quantified the relative amount (%) of the aberrant mitotic
spindles as compared to all mitotic cells. In addition, the
ultrastructures of the interphase microtubules were visualized
and ranked according to the categories presented in Figure 5.
According to the data summarized in Table 2, vinblastine,
even at the lowest concentration used in this study, depolymer-
ized the microtubular network completely; the tubulin homo-
geneously distributed within the cytosol corresponding to Figure
5c. 17-DesacetylvinblastineTrp causes much less damage to the
microtubular network than vinblastine, and 47% of the mitotic
cells are aberrant at 0.25 µM. The two Trp-Arg8 conjugates
displayed distinct effects, while the 17-desacetylvinblastineTrp-
Arg8-2 isomer conjugate caused significant fragmentation of the
interphase microtubule at 2.5 µM, the 17-desacetylvinblastin-
eTrp-Arg8-1 isomer conjugate did not affect them at all even at
a 5 µM concentration. These results clearly show that the 17-
desacetylvinblastineTrp-Arg8-1 isomer conjugate is highly selec-
tive against the mitotic spindles and destroys only these
structures in the mitotic cells. As among tumor cells there are
more cells in mitosis at a certain time point than among normal
cells, these data suggest that the 17-desacetylvinblastineTrp-
Arg8-1 isomer conjugate may have a selective cytotoxic effect
against tumor cells and may thus have lower side effects. As
the isomers are different in the configuration of Trp, further
studies are needed to rationalize this selectivity.
CONCLUSIONS
Vinblastine is an efficient antitumor agent, but its side effects
and resistance can restrict its use. A promising approach to avoid
the harmful effects is its conjugation with an appropriate partner
compound. The coupling of 17-desacetylvinblastineTrp with
cell-penetrating oligoarginines results in conjugates whose
biological effectiveness correlates with the chain length of
peptides. The octa- and hexaarginine conjugates preserve the
cytotoxicity of the original drug on both sensitive and resistant
HL-60 cells. Moreover, the 17-desacetylvinblastineTrp-Arg8-1
isomer conjugate preferentially destroys the mitotic spindle, at
least in the case of cancerous HeLa cells, resulting in cell cycle
inhibition. Further studies should be performed to demonstrate
that this conjugate can exclusively target the tumor cells and is
less toxic to normal cells in many other cases.
ACKNOWLEDGMENT
These studies were supported by grants from the Hungarian
Research Fund (OTKA, no. K68285 and K68734), Hungarian
Ministry of Education (NKFP Medichem 2), and from the
Hungarian Ministry of Health (ETT 209/2009). The authors are
grateful to Dr. Szilvia Bo˝sze for the helpful discussion and to

Antitumor Effect of Vinblastine Conjugates
Bioconjugate Chem., Vol. 21, No. 11, 2010 1955
regioselective synthesis of a new generation of targeted chemo-
therapeutics. Part 1: EC145, a folic acid conjugate of de-
sacetylvinblastine monohydrazide. Bioorg. Med. Chem. Lett. 16,
5093–5096.
Gedeon Richter Plc. (Budapest, Hungary) for sponsorship.
Moreover, one of the authors (A. G.-K.) is indebted to the Jo´zsef
Varga Foundation for financial support.
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