Int. J. Med. Sci. 2010, 7
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s2010; 7(6):326-339
© Ivyspring International Publisher. All rights reserved


1. German Cancer Research Center, Dept. of Imaging and Radiooncology, INF 280, D-69120 Heidelberg, Germany
2. German Cancer Research Center, Central Peptide Synthesis Unit, INF 580, D-69120 Heidelberg, Germany
3. University of Heidelberg, Institute of Pathology, INF 220, D-69120 Heidelberg, Germany
4. German Cancer Research Center, Division of Biophysics of Macromolecules, INF 580, D-69120 Heidelberg, Germany
* The authors contributed equally to this work
 Corresponding author: Klaus Braun, Ph.D., German Cancer Research Center (DKFZ), Dept. of Imaging and Radiooncol-
ogy, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Phone: +49 6221-42 2495; Fax: +49 6221-42 3326; e-mail:

Received: 2010.07.02; Accepted: 2010.09.07; Published: 2010.09.21
Abstract
Clinical experiences often document, that a successful tumor control requires high doses of
drug applications. It is widely believed that unavoidable adverse reactions could be minimized
by using gene-therapeutic strategies protecting the tumor-surrounding healthy tissue as well
as the bone-marrow. One new approach in this direction is the use of “Targeted Therapies”
realizing a selective drug targeting to gain effectual amounts at the target site, even with
drastically reduced application doses. MCF-7 breast cancer cells expressing the α
v
β
3
[al-
p h a ( v ) b e t a ( 3 ) ] i n t e g r i n r e c e p t o r a r e c o n s i d e r e d a s a p p r o p r i a t e c a n d i d a t e s f o r s u c h a t a r g e t e d
therapy. The modularly composed BioShuttle carrier consisting of different units designed to
facilitate the passage across the cell membranes and for subcellular addressing of diagnostic
and/or therapeutic molecules could be considered as an eligible delivery platform. Here we
used the cyclic RGD-BioShuttle as a carrier for temozolomide (TMZ) at the α
v
β
3
integrin

therapy options should be used, such as immunothe-
rapy, surgery or chemotherapy sensibly using cytos-
tatic active agents with acceptable adverse reactions.
It is remarkable how dated medical treatment me-
thods are persistently continued [reported during the
“International Brain Tumor Research Conference 2010
(
Toxic side effects are documented for TMZ as
adverse reactions in the bone-marrow. Moreover, it is
known from clinical experience, that even higher ap-
plication doses are necessary for successful tumor
control. This approach seems obsolete now, because
’Targeted Therapy’ has reached the focus of scientific
interest in order to minimize such unavoidable drastic
side effects. Strategies were discussed during the
aforementioned meeting to protect the bone-marrow,
e.g. with gene-therapeutic methods. Another inter-
esting field is the regional chemotherapy in which
cytostatic drugs are being locally applied to certain
body regions. The topical application increases the
amount of active substances in the tumor and im -
proves efficiency, while lowering the side effect rate at
the same time.
However, many cell immanent obstacles inhibit
chemical therapy, such as the multidrug resistance
(MDR) mediated against cytotoxic agents like TMZ,
and apoptosis resistance with disruption of the com-
plex programmed cell death pathway network. The
Janicke group documented apoptosis resistant MCF-7
breast cancer cells treated with ionizing radiation,

tion. [4] Integrins, the corresponding ligands, are
evolutionarily old and have critical roles during de-
velopmental and pathological processes. The antibo-
dies to α
v
β
3
integrin and its antagonists like
arg-gly-a s p (R G D)-containing peptides, including
osteopontin, bone sialoprotein, vitronectin and fibri-
nogen are considered as efficient inhibitors which can
control the tumor progression.[5]
This α
v
β
3
integrin receptor is documented as an
outstanding target in the field of tumor imaging [6-8]
and is equally important as a chemotherapeutic target
in the field of targeted therapy.[9]
Endocytosis-mediated intracellular trafficking of
ligands via the α
v
β
3
receptor of MCF-7 cells and the
α
v
β
5

and α
v
β
5
integrin antagonist
cRGD.
Cell culture
The estrogen sensitive MCF-7 adenocarcinoma
breast cancer and HeLa cervix cancer cells (dkfz, tu-
morbank) were maintained at 37°C in a 5% CO
2
at -
mosphere in RPMI cell medium (Gibco, Germany)
supplemented with 5% fetal calf serum (Biochrome,
Germany). The cells were split twice a week.
Chemical Procedures
Synthesis of the RGD-BioShuttle
Derivatization of temozolomide
N-(2-Aminopropyl)-4-(6-(pyrimidine-2-yl)-1,2,4,5-tetrazine-3-yl)be
nzamide 4
4-(6-(Pyrimidine-2-yl)-1,4-dihydro-1,2,4,5-tetrazi
ne-3-yl)benzoic acid (3) was prepared from
2-cyanopyrimidine 1 a nd 4 -cyano-ben zoic ac id 2 by
reaction with hydrazine and then oxidized with so-
dium nitrite to the tetrazine derivative 4 according to
the following procedure [11]. The tetrazine derivative
was converted with thionyl chloride under standard
conditions to the chloride 5. To this suspension of the
acid chloride (2 mmol) in 20 ml CH
2

ture and isolated by evaporation to a solid residue (6)
(ESI: m/z 337.2 [M]
+
(as shown in Figure A).
Figure A shows the mass of the
N-(2-Aminopropyl)-4-(6-( py ri mi di ne-2-yl)-1,2,4,5-tetrazine
-3-yl )b en za m i de ( 6 i n s c heme 1 /Figure S1), as discussed by
Wiessler [12]. 3-Methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carb
oxylic acid chloride 7
3-Methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,
5]tetrazine-8-carboxylic acid was converted to the
corresponding chloride 7 as documented by Ar-
rowsmith [13]. The acid (2 mmol) was refluxed with
thionyl chloride (10 ml) until the acid was completely
dissolved. The excess of thionyl chloride was evapo-
rated under vacuum and the resulting solid was
stored over NaOH.

3-Methyl-4-oxo-N-(3-(4-(6-(pyrimidine-2-yl)-1,2,4,5-tetrazine-3-y l )
benzamido)propyl)-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-
8-carboxamide (TMZ-tetrazine diene) 9
Compound 8 (0.5 mmol) and the chloride 7 (0.5
mmol) were dissolved in 5 ml chloroform and 5 ml
TEA at 0-5 °C. After 4 h at room temperature, the so -

.0
2,6
.0
8,11
]3,5-diox o-4-aza-
9,12-t
ridecadiene (Reppe-anhydride) 12 was prepared
f r o m 4 2 m g o f ( 1 Z , 3 Z , 5 Z , 7 Z ) -cycloocta-1,3,5,7-tetraene
10 and 44 mg maleic anhydride 11 in chloroform as
documented by Reppe [14].

Int. J. Med. Sci. 2010, 7 329

Figure S1 (Scheme 1) the nitriles 1 and 2 react with hydrazine to the 4-(6-(pyrimidine-2-yl)-1,4-dihydro-
1,2,4,5-tetrazine-3-yl)benzoic acid 3. Oxidation to 4 and reaction with thionyl chloride result in the corresponding acide
c h l o r i d e 5 w h i c h r e a c t s w i t h N-Boc-1,3-diaminopropane to the product 6. Boc-deprotection and subsequent reaction with
3-Methyl-4-ox o-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carboxylic acid chloride 7 result in the product
3-Methyl-4-ox o-N-(3-(4-(6-(pyrimidine-2-y l) -1,2,4,5-tetrazin-3-y l) b en za mi do ) pr op yl ) -3,4-dihydroimidazo[5,1-d][1,2,3,5]tet
razine-8-carboxamide (TMZ-tetrazine diene) 9.

Figure S2 (Scheme 2) illustrates the chemical reaction of (1Z,3Z,5Z,7Z)-cycloocta-1,3,5,7-tetraene 10 and 44 mg maleic
anhydride 11 which produces the t
etrac
yclo-[5.4.2
1,7
.0
2,6

The structure
describes the shift calculation for protons of the compound
with ChemDraw Ultra 2004. (Numbers indicate the pre-
dicted shift of the signals in ppm, as discussed by Wiessler
[12].
Synthesis of the dienophile cRGD-Lys(Tct) (14)
3 0 µ m o l c R G D p e p t i d e ( 1 8 m g ) 13 and 40 µm o l ( 8
mg) t
etrac
yclo-[5.4.2
1,7
.0
2,6
.0
8,11
]3,5-d ioxo-4-aza-9,12-
t
ridecadiene 12 were dissolved in pyridine over 5
hours at 70° - 80°C. Yield: 6 mg 14. Empirical formula
C39H49N9O9; exact Mass: 787.37 Mol. Wt.: 787.86
m/e: 787,37 (100,0%), 788,37 (43,1%), 789,37
(12,3%), 788,36 (3,3%), 790,38 (1,2%), 790,37 (1,1%) C,
59.45; H, 6.27; N, 16.00; O, 18.28 m/e peak at 788.5 for
the product.
L i g a t i o n o f t h e c R G D -L y s ( T c t ) w i t h t h e T M Z -tetrazine 9
Equimolar amounts of the TMZ-tetrazine con-
jugate 9 (1.03 mg; 2 µmol) and cRGD-Lys(Tct) 14 (7.3
mg, 2 µmol) were dissolved in aqueous solution and
stored at room temperature for 24 h. The DAR
inv