Optimization of the N-lost drugs melphalan and bendamustine: Synthesis and cytotoxicity of a new set of dendrimer-drug conjugates as tumor therapeutic agents

Article abstract of DOI:10.1021/bc900453f

Bendamustine and melphalan are very promising alkylating drugs with applicability in the treatment of various tumoral diseases, e.g., chronic lymphocytic leukemia (CLL) or breast cancer. However, numerous adverse effects limited their use. Therefore, 1,3,5-tris(3-aminopropyl)benzene (G0) and its G1 analogue 3,5-bis(3-aminopropyl)-N-(3-{3,5-bis[3-{3,5-bis(3-aminopropyl) benzoylamino}propyl]phenyl}propyl)benzamide were selected to design cytostatic drug-dendrimer conjugates to achieve tumor cell accumulation by endocytosis as already demonstrated for platinum complexes. The dendrimers act as carriers and an N-(2-hydroxyethyl)maleimide spacer between drug and carrier should guarantee a selective release of the cytostatics in the tumor cells. The resulting cytotoxicity was determined in vitro using the human MCF-7 and MDA-MB-231 breast cancer cell lines. It was demonstrated that melphalan caused cytotoxic effects depending on its free amino group (Boc protection strongly decreased the activity) but independent of a derivation of the carboxylic group (dendrimers and spacer binding). Esterification of bendamustine with the N-(2-hydroxyethyl)maleimide spacer strongly increased the hydrolytic stability of the N-lost moiety, so antiproliferative effects were yet observed in vitro.

Full text of DOI:10.1021/bc900453f

1728
Bioconjugate Chem. 2010, 21, 1728–1743
Optimization of the N-Lost Drugs Melphalan and Bendamustine: Synthesis
and Cytotoxicity of a New Set of Dendrimer-Drug Conjugates as Tumor
Therapeutic Agents
Ana Maria Scutaru,† Maxi Wenzel,† Heike Scheffler,† Gerhard Wolber,† and Ronald Gust*,†,‡
Institute fu¨r Pharmazie, Freie Universita¨t Berlin, Ko¨nigin Luise Str. 2 + 4, 14195 Berlin, Germany, and Institut fu¨r Pharmazie,
Universita¨t Innsbruck, Innrain 52, A-6020 Innsbruck, Austria. Received October 15, 2009; Revised Manuscript Received July 19, 2010
Bendamustine and melphalan are very promising alkylating drugs with applicability in the treatment of various
tumoral diseases, e.g., chronic lymphocytic leukemia (CLL) or breast cancer. However, numerous adverse effects
limited their use. Therefore, 1,3,5-tris(3-aminopropyl)benzene (G0) and its G1 analogue 3,5-bis(3-aminopropyl)-
N-(3-{3,5-bis[3-{3,5-bis(3-aminopropyl)benzoylamino}propyl]phenyl}propyl)benzamide were selected to design
cytostatic drug-dendrimer conjugates to achieve tumor cell accumulation by endocytosis as already demonstrated
for platinum complexes. The dendrimers act as carriers and an N-(2-hydroxyethyl)maleimide spacer between
drug and carrier should guarantee a selective release of the cytostatics in the tumor cells. The resulting cytotoxicity
was determined in vitro using the human MCF-7 and MDA-MB-231 breast cancer cell lines. It was demonstrated
that melphalan caused cytotoxic effects depending on its free amino group (Boc protection strongly decreased the
activity) but independent of a derivation of the carboxylic group (dendrimers and spacer binding). Esterification
of bendamustine with the N-(2-hydroxyethyl)maleimide spacer strongly increased the hydrolytic stability of the
N-lost moiety, so antiproliferative effects were yet observed in vitro.
Melphalan is a typical alkylating agent and contains two
functional groups: the amino acid phenylalanine (drug targeting
by using amino acid transporter of tumor cells) and the N-Lost
moiety (cross-link of DNA strands). It is used in the treatment
of cancer and other diseases, including ovarian cancer, malignant
melanoma, multiple myeloma (bone-marrow cancer), breast
cancer, and chronic myelogenous leukemia.
INTRODUCTION
Designing miscellaneous dendritic structures containing both
a drug and a targeting residue has played a very important role
in drug development during the past years (1-4). The main
objective has been to diminish the side effects induced through
drug therapy. Accumulation of these conjugates in the tumor
tissue is explained by the EPR (enhanced permeability and
retention) effect, the property by which macromolecules build
up in the tumor due to the special properties of the cells (e.g.,
poor lymphatic drainage) (5, 6). Current studies by Baker et al.
showed remarkable results and proved the selective uptake of
PAMAM dendrimer-based multifunctional conjugates in the
tumor tissues (7-9).
Bendamustine is a drug with high potency in the treatment
of several tumoral diseases. It was first synthesized by Oze-
gowski et al. and has been in clinical use since 1985 (10). The
molecule consists of three structural elements: a mechloretha-
namine group, a benzimidazole core, and a butyric acid chain.
It was proposed that the latter (together with the protonated
heterocycle) mediates water solubility and the benzimidazole
moiety should lead to tumor cell accumulation by purine base
transporters.
Bendamustine belongs to the class of nitrogen mustard agents
like chlorambucil, melphalan, and cyclophosphamide and should
induce cell death, possibly through apoptosis. Recent clinical
trials have illustrated remarkable results in the treatment of
chronic lymphocytic leukemia (CLL), and thus, in March 2008,
bendamustine was also approved in the USA for the treatment
of CLL (11). Additionally, bendamustine has a promising future
in the treatment of breast cancer (12). The mechanism of action
is thought to include both alkylating agent properties (compa-
rable to melphalan) and purine antimetabolite characteristics.
Both bendamustine and melphalan are very effective antitu-
mor drugs, without having the proposed tumor selectivity.
Therefore, we decided to use them in our approach to increase
the selectivity for malignant cells following the concept of Kratz
et al. They synthesized in 1998 a protein-conjugate with
chlorambucil capable of releasing the drug in breast cancer
(MCF-7) and leukemia (MOLT4) cells. Chlorambucil was
attached through an ester or amide bond to a maleimide spacer,
which is able to bind selectively to thiol groups of human serum
albumin (HSA) (13, 14). HSA is a handy protein in pure form,
is nontoxic and biodegradable, and is essentially assimilated
by the tumor cells (15, 16). The resulting drug-HSA conjugates
should accumulate in the tumors due to the EPR effect and then
release the drug in the cells (drug targeting). The in vitro cell
tests illustrated a relationship between the linker and the efficacy
of the conjugates, and in vivo studies demonstrated the
possibility of administering a conjugate with a lower IC50 value
than chlorambucil in a higher dose to mice.
These positive results induced us to bind melphalan and
bendamustine directly or through a cleavable maleimide spacer
to the dendrimers G0 and G1 (see Figure 1) synthesized in a
former SAR study (17). In vitro antitumor activity studies on
MCF-7 and MDA-MB-231 cell lines showed for G0 and G1
no cytotoxic effects (see Supporting Information), making them
suitable as carriers.
* Corresponding author. Phone: +43 512 507 5245; Telefax: +43
512 507 2940; E-mail: ronald.gust@uibk.ac.at.
† Freie Universita¨t Berlin.
Initially, three different spacers were considered for the
assembling of the conjugates (Figure 1). Due to the instability
of the hydrazone linkers under the given reaction conditions,
‡ Universita¨t Innsbruck.
10.1021/bc900453f  2010 American Chemical Society
Published on Web 09/17/2010

Optimization of N-Lost Drugs Melphalan and Bendamustine
Bioconjugate Chem., Vol. 21, No. 10, 2010 1729
Figure 1. Structures of the considered spacers and dendrimers.
the synthesis was first limited to the use of N-(2-hydroxyeth-
yl)maleimide 3.
(137 mg, 32%) as yellow solid: Rf ) 0.3 (hexane/ethyl acetate
2:1); mp 101 °C; 1H NMR (400 MHz, CDCl3) δ ) 1.29 (s, 9H,
CH3), 2.62 (m, 1H, CH2Ar), 2.74 (dd, 3J(H,H) ) 4.46 Hz, 9.44
Hz, 1H, CH2Ar), 3.62 (t, 3J(H,H) ) 5.28 Hz, 2H, CH2 maleimide),
3.66 (s, 8H, CH2CH2Cl), 4.09 (m, 2H, CH2O), 4.22 (m, 1H,
CH), 6.63 (d, 3J(H,H) ) 8.52 Hz, 2H, ArH), 7.02 (d, 2H+2H,
EXPERIMENTAL SECTION
1
General. H and 13C NMR spectra were taken on a Bruker
Avance/DPX 400 MHz with TMS as internal standard. Melting
points (uncorrected) were measured with a Bu¨chi Melting Point
B-545. All column chromatography purifications were done
using silica gel 60 (0.063-0.1 mm, Merck). TLC was performed
on silica gel 60 GF254 plates (Merck). Elemental analysis was
carried out on a Vario EL (Hanau). EI spectra were recorded
on a Thermo-Fisions VG Auto Spec (70 eV). The FAB spectra
were measured on a CH-5, Varian MAT, Bremen. The ESI-
TOF spectra were measured on an Agilent 6210 ESI-TOF,
Agilent Technologies, Santa Clara, CA, USA. Solvent flow rate
was adjusted to 4 µL/min, with spray voltage set to 4 kV. Drying
gas flow rate was set to 15 psi (1 bar). All other parameters
were adjusted for a maximum abundance of the relative
[M+H]+.
Synthesis. The following compounds were prepared accord-
ing to literature procedures: G0 (17), G1 (17), N-tert-butoxy-
carbonyl-4-[bis(2-chloroethyl)amino]-L-phenylalanine (2) (18).
Bendamustine hydrochloride was a gift from the company
Bendalis. Melphalan hydrochloride was synthesized as described
by Bergel (19). All other starting materials were purchased from
commercial sources and used without purification. Solvents were
dried under standard conditions.
3
ArH+Hmaleimide), 7.17 (d, J(H,H) ) 8.01 Hz, 1H, NH); 13C
NMR (CDCl3) δ ) 28.31 (CH3), 36.64 (CH2Ar), 36.98
(CH2Nmaleimide), 40.46 (CH2NAr+CH2Cl), 54.49 (CH), 62.11
(CH2O), 79.84 (C(CH3)3), 112.09 (Caromatic), 124.8 (C), 130.56
(Caromatic), 134.25 (CHmaleimide), 145.04 (Caromatic), 155.07 (COOB-
oc), 170.29 (COmaleimide), 171.75 (COO); MS (EI, 80 eV, 300
°C) m/z (%) 527.2 (5.93) [M+-1], 404.1 (0.36) [M-C6H6NO2]+,
230.3 (100) C15H14NCl2; Anal. (C24H31Cl2N3O6) C, H, N.
O-{4-[Bis(2-chloroethyl)amino]-L-phenylalanyl}-2-hydroxy-
ethylmaleimide Hydrochloride (5). MelBoc-spacer 4 (350 mg,
0.66 mmol) was dissolved in 6 mL of THF. To this solution,
250 µL of hydrochloric acid (w ) 25%) was added and the
mixture was stirred for 20 h at room temperature. The solvent
was removed under reduced pressure. The remaining oil was
dissolved in methanol, and the product was precipitated with
diethyl ether, filtered, and dried, to afford 245 mg (80%) as a
colorless solid: mp 130 °C; 1H NMR (400 MHz, CD3OD) δ )
3.00 (dd, 3J(H,H) ) 7.89 Hz, 14.59 Hz, 1H, CH2Ar), 3.14 (dd,
3J(H,H) ) 5.22 Hz, 14.60 Hz, 1H, CH2Ar), 3.69 (m, 4H,
CH2Cl), 3.78 (m, 4H+2H, CH2CH2Cl+CH2 maleimide), 4.18 (m,
3
1H, CH), 4.38 (m, 1H, CH), 6.77 (d, J(H,H) ) 8.81 Hz, 2H,
ArH), 6.86 (s, 2H, Hmaleimide), 7.11 (d, 3J(H,H) ) 8.75 Hz, 2H,
ArH); 13C NMR (CD3OD) δ ) 37.55 (CH2Nmaleimide), 39.67
(CH2Ar), 41.07 (CH2N), 42.6 (CH2Cl), 53.7 (CH), 60.14
(CH2O), 119.5 (Caromatic), 132.50 (Caromatic), 132.54 (Caromatic),
132.7 (Caromatic), 135.43 (CHmaleimide), 135.68 (Caromatic), 170.33
O-{N-tert-Butoxycarbonyl-4-[bis(2-chloroethyl)amino]-L-phe-
1
nylalanyl}-2-hydroxyethylmaleimide (4). MelBoc 2 (330 mg,
0.81 mmol), DMAP (tip of a spatula), and N-(2-hydroxyethyl)-
maleimide 3 (441 mg, 3.1 mmol) were dissolved in 30 mL of
dry dichloromethane at room temperature. DCC (176.5 mg, 0.85
mmol), dissolved in 25 mL of dry dichloromethane, was added
dropwise to this solution within 1 h, and the solution stirred
for additional 20 h. The solution was filtered and evaporated in
vacuo. The red residue was chromatographed on a silica gel
column (hexane/ethyl acetate 2:1) to afford the desired product
+
(COmaleimide), 172.3 (COO); MS (ESI) calcd. for C19H24N3O4Cl2
428.1138, found 428.1152; Anal. (C19H25Cl3N3O4 · HCl) C,
H, N.
O-{4-[6-Bis(2-chloroethyl)amino-3-methylbenzimidazoyl(2)]-
butanoyl}-2-hydroxyethylmaleimide (7). Bendamustine free base
6 (2 g, 5.6 mmol), DMAP (35 mg, 0.26 mmol), and N-(2-
hydroxyethyl)maleimide 3 (2.36 g, 16.7 mmol) were dissolved
in 250 mL of dry dichloromethane at room temperature. DCC
(1.27 g, 6.16 mmol), dissolved in 100 mL of dry dichlo-
romethane, was added dropwise to this solution within 1 h and
the solution stirred for additional 24 h. The solution was filtered
and evaporated in vacuo. The residue was chromatographed on
1Abbreviations: Mel, Melphalan; DMAP, 4-dimethylaminopyridine;
DCC, N,N-dicyclohexylcarbodiimide; DIPEA, N-ethyl-diisopropyl-
amine; Benda, Bendamustine; TFA, trifluoroacetic acid; TBTU, O-(1H-
benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate; HOBT,
hydroxybenzotriazole hydrate; EDC, N-(3-dimethylaminopropyl)-N′-
ethylcarbodiimide hydrochloride.

1730 Bioconjugate Chem., Vol. 21, No. 10, 2010
Scutaru et al.
Chart 2. Supporting Scheme for NMR Data of Compound 9
Chart 1. Supporting Scheme for NMR Data of Compound 7
a silica gel column (dichloromethane/methanol 20:1, dichlo-
romethane/acetone 3:1) to afford the desired product (1.89 g,
70.1%) as yellow solid: Rf ) 0.4 (dichloromethane/acetone 3:1);
CD3OD) δ ) 2.12 (m, 6H, 1 CH2), 2.76 (m, 6H, 2 CH2),
3.06-3.12 (m, 6H, 3 CH2), 3.18-3.26 (m, 6H+6H, 4+5 CH2),
3.68 (m, 12H+6H, 6+9 CH2), 3.82 (m, 12H, 8 CH2), 4.29 (m,
3H, 7 CH), 4.42 (m, 3H, 11 CH2), 4.52 (m, 3H, 11 CH2), 4.61
(m, 3H, 10 CH), 6.89 (d, 3J(H,H) ) 8.37 Hz, 6H, 12 CH), 7.07
(s, 3H, 13 CH), 7.23 (d, 3J(H,H) ) 8.37 Hz, 6H, 14 CH) (Chart
2); 13C NMR (CD3OD) δ ) 28.84 (1 CH2), 33.52 (3 CH2), 34.22
(2 CH2), 36.45 (9 CH2), 39.08 (5 CH2), 41.12 (8 CH2), 41.19
(6 CH2), 47.42 (4 CH2), 55.21 (10 CH), 59.29 (7 CH), 63.83
(11 CH2), 115.32 (12 CH), 127.97 (Caromatic), 131.95 (Caromatic+14
CH), 132.02 (Caromatic), 145.95 (Caromatic), 169.91 (COO), 171.34
(COmaleimide), 173.80 (COmaleimide); MS (ESI): calcd. for
C72H97N12O12Cl6 1535.5445, found 1535.5312.
1
mp 115 °C; H NMR (400 MHz, DMSO) δ ) 1.95 (quin,
3J(H,H) ) 7.31 Hz, 2H, CH2), 2.39 (t, 3J(H,H) ) 7.27 Hz, 2H,
3
CH2CO), 2.79 (t, J(H,H) ) 7.37 Hz, 2H, CH2CObenzimidazole),
3.64 (m, 4H+2H, CH2CH2Cl+CH2O), 3.70 (s, 4H+3H,
CH2CH2Cl+NCH3 benzimidazole), 4.14 (t, 3J(H,H) ) 5.32 Hz, 2H,
CH2 maleimide), 6.77 (dd, 3J(H,H) ) 2.21 Hz, 8.8 Hz, 1H, CH b),
6.91 (d, 3J(H,H) ) 2.08 Hz, 1H, CH c), 7.02 (s, 2H, Hmaleimide),
3
7.31 (d, J(H,H) ) 8.77 Hz, 1H, CH a) (Chart 1); 13C NMR
(CDCl3) δ ) 25.0 (CH2), 26.4 (CH2), 33.0 (NCH3), 33.9 (CH2),
36.9 (CH2), 40.8 (CH2CH2Cl), 61.4 (CH2N), 103.5 (Caromatic),
109.6 (Caromatic), 110.6 (Caromatic), 129.8 (Caromatic), 134.6
(Cmaleimide), 143.8 (Caromatic), 154.7 (Cbenzimidazole), 170.4 (CO-
maleimide), 172.9 (CO); MS (EI, 80 eV, 300 °C) m/z (%) 479.8
(32.78) [M+-1], 431.0 (95.45) [M-CH2Cl]+, 321.9 (100)
C16H20ClN3O2; Anal. (C22H26Cl2N4O4) C, H, N.
Chart 3. Supporting Scheme for NMR Data of Compound 10
1,3,5-Tris{N-[(2-(2,5-dioxopyrrolidin-1-yl)ethyl-3-(4-(bis(2-
chloroethyl)amino)phenyl)-2-(tert-butoxycarbonylamino)pro-
panoate)-1-yl]propylamino}benzene (8). MelBoc-spacer 4 (620
mg, 1.17 mmol) was dissolved in 20 mL of chloroform. To
this solution was added dropwise a mixture of G0·3HCl (125
mg, 0.35 mmol) and DIPEA (137 mg, 1.05 mmol, 186 µL)
dissolved in 2 mL of dry methanol. The reaction mixture was
refluxed for 72 h and was continuously monitored by TLC. The
solution was then washed once with sodium hydrogen carbonate
solution and once with brine. The organic layer was dried over
magnesium sulfate and the solvent evaporated in vacuo. The
residue was purified by column chromatography (silica gel,
dichloromethane/methanol 20:1) to afford the desired product
(192 mg, 30%) as a white solid: Rf ) 0.2 (dichloromethane/
methanol 20:1); mp 230 °C; 1H NMR (400 MHz, CDCl3) δ )
3
1.40 (s, 27H, 1 CH2), 1.79 (quin, J(H,H) ) 7.29 Hz, 6H, 2
1,3,5-Tris{N-[(2-(2,5-dioxopyrrolidin-1-yl)ethyl-4-(5-(bis(2-
chloroethyl)amino)-1-methyl-1H-benzo[d]imidazol-2-yl)butanoate)-
1-yl]propylamino}benzene (10). Benda-spacer 7 (470 mg, 0.97
mmol) was dissolved in 20 mL of chloroform. To this solution,
a mixture of G0·3HCl (77.8 mg, 0.216 mmol) and 116 µL of
DIPEA dissolved in 2 mL of dry methanol was added dropwise.
The reaction mixture was refluxed for 72 h, being continuously
monitored by TLC. The reaction mixture was then washed once
with sodium hydrogen carbonate solution and once with brine.
The organic layer was dried over magnesium sulfate and the
solvent was evaporated in vacuo. The residue was purified by
column chromatography (silica gel, chloroform/methanol 20:
1) to afford the desired product (290 mg, 79%) as an ochre
CH2), 2.35 (s, 3H, 1 NH), 2.49 (m, 3H, 3 CH2), 2.61 (m,
3H+6H, 3+4 CH2), 2.71 (m, 3H, 5 CH2), 2.88-3.03 (m,
3H+6H, 5+6 CH2), 3.59 (t, 3J(H,H) ) 6.30 Hz, 12H, 7 CH2),
3
3.68 (t, J(H,H) ) 6.57 Hz, 12H, 8 CH2), 3.75 (m, 3H+6H, 9
CH2), 4.33 (m, 3H, 10 CH2), 4.45 (m, 3H, 10 CH2), 4.94 (t,
3J(H,H) ) 7.56 Hz, 3H, 11 CH), 6.59 (d, J(H,H) ) 8.52 Hz,
3
12H, 12 CH), 6.82 (s, 3H, 13 CH), 7.00 (d, 3J(H,H) ) 7.08 Hz,
14 CH); 13C NMR (CD3OD) δ ) 26.79 (2 CH2), 28.79 (CH3),
32.31 (3 CH2), 34.80 (4+5 CH2), 38.64 (9 CH2), 41.73 (7+8
CH2), 48.2 (6 CH2), 54.49 (9+11 CH2), 62.42 (10 CH2), 80.6
(C), 113.41 (12 CH), 127.02 (Caromatic), 129.26 (14 CH), 129.97
(Caromatic), 131.59 (13 CH), 146.63 (Caromatic), 159.90 (CONH),
172.9 (COO), 173.76 (COmaleimide), 176.95 (COmaleimide); MS
(ESI) calcd. for C87H121N12O18Cl6 1835.7034, found 1835.6977;
Anal. (C87H121N12O18Cl6) C, H, N.
1
solid: Rf ) 0.44 (dichloromethane/methanol 10:1); H NMR
3
(400 MHz, CDCl3) δ ) 1.79 (quin, J(H,H) ) 7.22 Hz, 6H, 1
CH2), 2.17 (m, 6H, 2 CH2), 2.48 (m, 6H, 3 CH2), 2.60 (m,
6H+3H, 4+5CH2), 2.69-2.75 (m, 3H, 5 CH2), 2.93 (m, 6H, 6
CH2),3.64(m,12H+6H,7CH2),3.71-3.79(m,12H+3H+6H+9H,
1,3,5-Tris{N-[(2-(2,5-dioxopyrrolidin-1-yl)ethyl-3-(4-(bis(2-chlo-
roethyl)amino)phenyl)-2-aminopropanoate)-1-yl]propylamino}-
benzene trishydrochloride (9). The G0 dendrimer 8 (280 mg,
0.15 mmol)) was dissolved in 10 mL of THF. To this solution,
173 µL of hydrochloric acid (w ) 25%) was added and the
mixture was stirred at room temperature. After monitoring the
reaction by TLC, 400 µL of hydrochloric acid (25%) was added.
After 72 h, the solvent was removed under reduced pressure to
afford the deprotected product as a brown solid (240 mg, 95%)
after lyophilization from water: mp 255 °C; 1H NMR (400 MHz,
3
8 CH2), 4.23 (m, 6H, 9 CH2), 6.79 (dd, J(H,H) ) 2.17 Hz,
8.75 Hz, 3H, 10 CH), 6.83 (s, 3H, 13 CH), 7.08 (d, 3J(H,H) )
1.96 Hz, 3H, 11 CH), 7.19 (d, 3J(H,H) ) 8.76 Hz, 3H, 13 CH)
(Chart 3); 13C NMR (CDCl3) δ ) 22.2 (2 CH2), 26.3 (6 CH2),
29.4 (1 CH2), 31.9 (8 CH2), 32.9 (3 CH2), 33.1 (5 CH2), 33.3
(4 CH2), 37.8 (8 CH2), 40.86 (7+8 CH2), 47.1 (7 CH2), 54.6 (8
CH2), 60.8 (9 CH2), 102.6 (Caromatic), 110.0 (Caromatic), 110.9
(Caromatic), 126.1 (Caromatic), 129.1 (Caromatic), 141.8 (Caromatic), 142.9

Optimization of N-Lost Drugs Melphalan and Bendamustine
Bioconjugate Chem., Vol. 21, No. 10, 2010 1731
Chart 4. Supporting Scheme for NMR Data of Compound 11
Chart 5. Supporting Scheme for NMR Data of Compound 12
(Caromatic), 154.2 (Cbenzimidazole), 173.02 (CO), 175.06 (CO), 177.6
(CO); MS (ESI): calcd. for C81H106N15O12Cl6 1692.6258, found
1692.6193; C81H106N15O12Cl6 846.8165, found 846.8170.
(8 CH2), 55.52 (11 CH), 63.85 (7+12 CH2), 113.85 (13 CH),
118.45 (Caromatic), 121.22 (Caromatic), 126.47 (Caromatic), 128.85
(Caromatic), 131.7 (16 CH), 142.54 (Caromatic), 143.29 (Caromatic), 147.55
(Caromatic), 160.34 (CO), 160.70 (CO), 170.11 (CO), 174.64 (CO);
MS (ESI) calcd. for C168H219N27O27Cl12Na22+ 1760.1307, found
1760.1456.
2+
1,3,5-Tris{3,5-bis-[N-[(2-(2,5-dioxopyrrolidin-1-yl)ethyl-3-(4-
(bis(2-chloroethyl)amino)phenyl)-2-(tert-butoxycarbonylamino)-
propanoate)-1-yl]propylamino]-N-propylbenzamide}benzene
(11). To a solution of MelBoc-spacer 4 (700 mg, 1.32 mmol)
in 50 mL of chloroform was added dropwise a solution of
G1·CF3COOH (320 mg, 0.2 mmol) and DIPEA (156 mg, 0.12
mmol, 115 µL) in dry methanol. The reaction mixture was
refluxed for 70 h. After complete reaction (TLC), the solution
was washed once with sodium hydrogen carbonate solution and
once with brine. The organic layer was dried over magnesium
sulfate; the solvent was evaporated in vacuo and the residue
purified through column chromatography (silica gel, dichlo-
romethane/methanol 20:1 and chloroform/methanol 10:1) to
afford the compound as a yellow oil (200 mg, 24.5%): Rf )
0.26 (chloroform/methanol 10:1); 1H NMR (400 MHz, CDCl3)
δ ) 1.32 (s, 54H, 1 CH2), 1.71 (m, 12H, 2 CH2), 1.86 (m, 6H,
3 CH2), 2.40 (m, 6H, 4 CH2), 2.56 (m, 24H, 5 CH2), 2.80-2.95
(m, 12H+12H, 6 CH2), 3.35 (m, 6H, 7 CH2), 3.52 (m, 24H, 8
CH2), 3.61 (m, 24H+6H, 9 CH2), 3.68 (m, 12H, 10 CH2),
4.09-4.18 (m, 6H, 11 CH2), 4.27 (m, 6H, 11 CH2), 4.36 (m,
1,3,5-Tris[3-(4-(bis(2-chloroethyl)amino)phenyl)-2-(tert-bu-
toxycarbonylamino)-N-propylpropionamide]benzene (13). A
solution of 2 (910 mg, 2.5 mmol) in a mixture of dry
dichloromethane/DMF (14:1/v:v) was cooled to -20 °C. A
solution of TBTU (740 mg, 2.35 mmol) in dry DMF was added
slowly and the reaction mixture was allowed to warm to room
temperature. The reaction mixture was stirred at room temper-
ature for additional 30 min, cooled to -20 °C again, followed
by the dropwise addition of DIPEA (1040 µL, 6 mmol) and a
solution of G0·3HCl (179.39 mg, 0.5 mmol) in dry methanol.
The mixture was allowed to warm to room temperature and
was stirred for additional 20 h. After complete reaction (TLC),
the mixture was washed twice with sodium hydrogen carbonate
solution, then once with brine, and then dried over magnesium
sulfate. The solvent was removed under reduced pressure and
column chromatography (silica gel, dichloromethane/methanol
20:1) afforded the desired product (400 mg, 0.28 mmol, 57%)
as a brown oil: Rf ) 0.36 (dichloromethane/methanol 24:1); 1H
NMR (400 MHz, CDCl3) δ ) 1.36 (s, 27H, CH3), 1.7 (quin,
3J(H,H) ) 6.892 Hz, 6H, CH2), 2.55 (t, 3J(H,H) ) 6.9 Hz, 6H,
3
6H, 12 CH), 4.93 (m, 6H, 1 NH), 6.52 (d, J(H,H) ) 8.03
Hz, 12H, 13 CH), 6.74 (s, 3H, 2 NH), 6.82 (s, 3H, 14 CH),
3
6.92 (d, J(H,H) ) 7.38 Hz, 12H, 15 CH), 7.04 (s, 6H, 3
2
NH), 7.06 (s, 3H, 16 CH), 7.33 (s, 6H, 17 CH) (Chart 4);
13C NMR (CD3OD) δ ) 29.22 (CH3+2+3 CH2), 31.16
(CH2 maleimide+CH2Ar+CH2Ar), 39.9 (10 CH2+ArCH2 melphalan),
42.17 (CH2NHCO+CH2CH2Cl), 44.23 (CH2NHmaleimide), 54.86 (12
CH), 56.28 (CHmaleimide+11 CH2), 81.07 (C), 113.82 (13 CH),
126.69 (Caromatic), 131.92 (Caromatic), 132.28 (Caromatic), 140.13
(Caromatic), 143.95 (Caromatic), 150.3 (Caromatic), 158.15 (NH-
COO), 174.21 (CO), 175.25 (COmaleimide); MS (ESI) calcd.
CH2Ar), 2.68 (dd, J(H,H) ) 13.46 Hz, 3H, CH2Armelphalan),
2
2.83 (dd, J(H,H) ) 13.46 Hz, 3H, CH2Armelphalan), 3.1 (m,
6H, CH2N), 3.71 (s, 24H, CH2CH2Cl), 4.08 (m, 3H,
3
CHCH2Armelphalan), 6.68 (d, J(H,H) ) 8.168 Hz, 6H, ArHmelphalan),
3
6.82 (d, J(H,H) ) 8.45 Hz, 3H, NH-Boc), 6.86 (s, 3H, G0-
ArH), 7.12 (d, 3J(H,H) ) 8.19 Hz, 6H, ArHmelphalan), 7.9 (s, br,
3H, NH); 13C NMR (CDCl3) δ ) 28.3 (CH2), 30.5 (ArCH2),
37.6 (NHCH2), 40.4 (CH2CH2Cl), 54.5 (CHNH2), 79.8 (C-
(CH3)3), 112.1 (Caromatic), 124.9 (Caromatic), 126.4 (Caromatic), 130.6
(Caromatic), 144.9 (Caromatic), 155.7 (Caromatic), 171.6 (CO); MS (ESI)
calcd. for C69H99N9O9Cl6Na+ 1432.5572, found 1432.5566.
1,3,5-Tris[3-(4-(bis(2-chloroethyl)amino)phenyl)-2-amino-N-
propylpropionamide]benzene Tristrifluoroacetate (14). A solu-
tion of 13 (590 mg, 0.42 mmol) in 10 mL of TFA (w ) 95%)
was stirred for 4 h at room temperature. After complete reaction
(TLC), the solvent was repeatedly removed in vacuo to afford
the deprotected dendrimer (580 mg, 0.39 mmol, 95%) as brown
solid: mp 150 °C; 1H NMR (400 MHz, CD3OD) δ ) 1.63 (quin,
3J(H,H) ) 7.09 Hz, 6H, CH2), 2.44 (t, 3J(H,H) ) 7.28 Hz, 6H,
CH2Ar), 2.84 (dd, 2J(H,H) ) 12.85 Hz, 3H, CH2Armelphalan), 2.91
2+
for C198H269N27O39Cl12 2037.307, found 2037.3243.
1,3,5-Tris{3,5-bis-[N-[(2-(2,5-dioxopyrrolidin-1-yl)ethyl-3-
(4-(bis(2-chloroethyl)amino)phenyl)-2-aminopropanoate)-1-yl]-
propylamino]-N-propylbenzamide}benzene Hexatrifluoroacetate
(12). The G1 dendrimer 11 (180 mg, 0.044 mmol) was dissolved
in 10 mL of dichloromethane and treated with 2.8 mL of TFA
(w ) 95%). The mixture was stirred for 24 h at room
temperature and continuously monitored by TLC. The solvent
was then evaporated in vacuo to afford the deprotected den-
1
drimer as brown oil (170 mg, 93%): H NMR (CD3OD) δ )
1.92 (m, 12H, 1 CH2), 2.09 (s, br, 12H, 2 CH2), 2.65 (m, 12H,
3 CH2), 2.77 (m, 18H, 4 CH2), 2.99-3.05 (m, 6H+12H, 5 CH2),
3.21 (m, 6H+6H, 5 CH2+6 CH2), 3.40 (m, 6H, 7 CH2), 3.67
(m, 24H, 8 CH2), 3.74 (m, 24H, 9 CH2), 3.84 (m, 12H, 10 CH2),
4.19 (m, 6H, 11 CH), 4.37 (m, 12H, 12 CH2), 6.76 (d, 3J(H,H)
) 8.13 Hz, 12H, 13 CH2), 6.93 (s, 3H, 14 CH2), 7.14 (d, 3J(H,H)
) 8.19 Hz, 12H, 15 CH2), 7.32 (s, 3H, 16 CH2), 7.55 (s, 6H,
17 CH2) (Chart 5); 13C NMR (CD3OD) δ ) 27.77 (1+2 CH2),
28.84 (4 CH2), 31.17 (4 CH2), 33.31 (5 CH2), 36.29 (5 CH2),
36.44 (10 CH2), 39.01 (6 CH2), 41.67 (9 CH2), 47.6 (3 CH2), 54.32
2
(dd, J(H,H) ) 12.84 Hz, 3H, CH2Armelphalan), 3.02 (m, 3H,
CH2N), 3.15 (m, 3H, CH2N), 3.65 (s, 24H, CH2CH2Cl), 3.83
3
(m, 1H, CHCH2Armelphalan), 6.69 (d, J(H,H) ) 8.58 Hz, 6H,
ArHmelphalan), 6.8 (s, 3H, G0-ArH), 7.05 (d, 3J(H,H) ) 8.43 Hz,
3
6H, ArHmelphalan), 8.1 (s, br, 6H, NH2), 8.4 (t, J(H,H) ) 5.24
Hz, 3H, NH); 13C NMR (CD3OD) δ ) 31.9 (CH2), 34.0
(ArCH2), 40.2 (NHCH2), 41.6 (CH2CH2Cl), 56.0 (CHNH2),
113.6 (Caromatic), 123.8 (Caromatic), 127.3 (Caromatic), 131.7 (Caromatic),

1732 Bioconjugate Chem., Vol. 21, No. 10, 2010
Scutaru et al.
Chart 6. Supporting Scheme for NMR Data of Compound 15
Chart 7. Supporting Scheme for NMR Data of Compound 16
143.0 (Caromatic), 147.2 (Caromatic), 169.6 (CO); MS (ESI); Anal.
(C60H78Cl6N9O9F9) C, H, N.
1,3,5-Tris[4-(5-(bis(2-chloroethyl)amino)-1-methyl-1H-ben-
zo[d]imidazol-2-yl)-N-propylbutyramide]benzene (15). A solu-
tion of bendamustine free base 6 (430 mg, 1.2 mmol) in a mixture
of dry dichloromethane/DMF (7 mL/2 mL) was cooled to -20
°C. A solution of TBTU (352 mg, 1.128 mmol) in dry DMF was
added slowly, and the reaction mixture was allowed to warm to
room temperature. The reaction mixture was stirred at room
temperature for additional 30 min and cooled to -20 °C again,
followed by the dropwise addition of DIPEA (502 µL, 2.88 mmol)
and a solution of G0·3HCl (85.4 mg, 0.24 mmol) in dry methanol.
The mixture stirred was allowed to warm to room temperature and
was stirred for additional 20 h. After complete reaction (TLC),
the mixture was washed twice with sodium hydrogen carbonate
solution, then once with brine, and dried over magnesium sulfate.
The solvent was removed under reduced pressure, and column
chromatography (silica gel, chloroform/methanol 20:1) afforded
the desired product (130 mg, 1.02 mmol, 42%) as a brown oil: Rf
) 0.5 (chloroform/methanol 10:1); 1H NMR (400 MHz, CDCl3)
δ ) 1.71 (quin, 3J(H,H) ) 6.91 Hz, 6H, CH2), 2.05 (quin, 3J(H,H)
) 6.85 Hz, 6H, CH2 bendamustine), 2.23 (t, 3J(H,H) ) 6.66 Hz, 6H,
CH2CObendamustine), 2.48 (t, 3J(H,H) ) 7.01 Hz, 6H, CH2ArG0), 2.85
(t, 3J(H,H) ) 7.08 Hz, 6H, CH2 benzimidazole), 3.08 (m, 6H, CH2N),
3.50-3.67 (m, 24H, CH2CH2Cl), 3.50-3.67 (m, 9H,
CH2), 3.41 (m, 6H, 7 CH2), 3.53 (m, 24H, 9 CH2), 3.64 (m,
3
24H, 10 CH2), 4.32 (m, br, 6H, 11 CH), 6.55 (d, J(H,H) )
8.14 Hz, 12H, 12 CH), 6.92 (s, 3H, 13 CH), 6.98 (s, 3H, 14
3
CH), 7.06 (d, J(H,H) ) 8.28 Hz, 12H, 15 CH), 7.32 (s, 6H,
16 CH) (Chart 7); 13C NMR (CD3OD) δ ) 27.33 (CH3), 30.34
(1 CH2), 30.85 (2 CH2), 32.38 (3 CH2), 32.98 (4 CH2), 36.63
(8 CH2), 38.48 (5+6 CH2), 38.67 (7 CH2), 40.16 (9+10 CH2),
54.12 (11 CH), 79.3 (C), 113.69 (12 CH), 123.38 (Caromatic),
124.77 (Caromatic), 128.65 (Caromatic), 130.09 (Caromatic), 131.62
(Caromatic), 134.54 (Caromatic), 135.82 (Caromatic), 141.83 (Caromatic),
156.2 (NHCOO), 167.75 (NHCO), 167.92 (NHCOCH); MS
+2
(ESI) calcd. for C162H225N21O21Cl12
1615.1794.
1615.1781, found
3
NCH3 bendamustine), 6.71 (dd, J(H,H) ) 2.30 Hz, 8.86 Hz, 3H,
1,3,5-Tris{3,5-bis-[3-(4-(bis(2-chloroethyl)amino)phenyl)-2-
amino-N-propylpropionamide]-N-propylbenzamide}benzeneHexahy-
drochloride (17). The G1 dendrimer 16 (100 mg, 0.031 mmol)
was dissolved in 5 mL of THF. To this solution, hydrochloric
acid (w ) 25%) was added dropwise and the mixture stirred
for 3 days at room temperature. The reaction was continuously
monitored by TLC. The solvent was then removed under
reduced pressure to afford the deprotected product as a brown
oil (85 mg, 96%) after lyophilization from water: 1H NMR (400
MHz, CD3OD) δ ) 1.75 (m, 18H, 1 CH2), 2.49 (m, 12H, 2
CH2), 2.65 (m, 6H, 3 CH2), 3.02 (m, 12H+18H, 4+5 CH2),
3.54 (s, br, 24H, 6 CH2), 3.65 (s, br, 24H, 7 CH2), 3.8 (s, 6H,
8 CH), 6.75 (m, 12H, 9 CH), 6.89 (s, 3H, 10 CH), 7.15 (m,
12H+3H, 11 CH), 7.44 (s, 6H, 12 CH) (Chart 8); 13C NMR
(CD3OD) δ ) 27.78 (1 CH2), 35.12 (2+3 CH2), 40.21 (4+5+7
CH2), 42.32 (6 CH2), 53.00 (8 CH), 112.50 (9 CH), 127.70
(10+12 CH), 128.66 (11 CH), 129.16 (Caromatic), 130.45 (11 CH),
131.45 (Caromatic), 134.53 (Caromatic), 140.4 (Caromatic), 146.2
(Caromatic), 165.87 (CO), 170.2 (CO); MS (ESI) calcd. for
3
CH b), 6.73 (s, 3H, ArHG0), 6.93 (d, J(H,H) ) 2.09 Hz,
3
3H, CH c), 7.1 (d, J(H,H) ) 8.8 Hz, 3H, CH a) (Chart 6);
13C NMR (CD3OD) δ ) 26.0 (CH2 benzimidazole), 27.29 (CH2CH2CO),
29.33 (CH2), 31.91 (CH3), 32.76 (CH2Ar), 38.69 (CH2CO), 40.02
(CH2NHCO+CH2N), 42.1 (CH2Cl), 104.1 (CH c), 112.92 (CH a+b),
125.85 (CaromaticNCH3+Caromatic), 131.2 (CaromaticNC+Caromatic), 146.2
(Caromatic+CNCH3), 163.91 (CO); MS (ESI) calcd. for
C63H84N12O3Cl6Na+ 1291.4800, found 1291.4634.
1,3,5-Tris{3,5-bis-[3-(4-(bis(2-chloroethyl)amino)phenyl)-2-
(tert-butoxycarbonylamino)-N-propylpropionamide]-N-
propylbenzamide}benzene (16). MelBoc 2 (472 mg, 1.16 mmol)
was dissolved in 15 mL of dry dichloromethane, HOBT (165
mg, 1.22 mmol) was added, and the solution was stirred for
30 min at room temperature. Afterward, the orange mixture
was cooled to -20 °C, EDC (245.3 mg, 1.28 mmol) was
added, and the mixture was subsequently stirred for additional
2 h. During that time, the reaction mixture was allowed to
warm up to room temperature. After complete reaction (TLC),
the mixture was cooled to -30 °C. G1 · CF3COOH (440 mg,
0.3 mmol) dissolved in 10 mL of dry methanol and DIPEA
(1.22 mL, 7 mmol) were added dropwise slowly. The solution
was stirred for additional 16 h and during that time allowed
to warm up to room temperature. After complete reaction,
the solution was washed twice with sodium hydrogen
carbonate solution and once with brine. The organic layer
was dried over magnesium sulfate, the solvent was removed
in vacuo, and the crude product was purified by column
chromatography (silica gel, dichloromethane/methanol 20:
1) to afford the G1 dendrimer (30 mg, 3.1%) as a brown oil:
+2
C132H177N21O9Cl12Na2 1333.005, found 1333.0546.
HPLC Analytic Procedure. The stability of the compounds
was determined in aqueous solution at a concentration of 1 mg/
mL. Degradation was followed by HPLC analytic using a
Kontron HPLC system: HPLC-pump 420; HPLC-autosampler
460; UV-Detector 430; Kontron Data System Kroma 2000. An
RP 18 column (250 × 6 × 4 mm Nucleosil 100-5 C18
(Macherey-Nagel)) was used. The solutions were held at 37 °C,
and every hour, 20 µL was injected onto the column and
isocratically analyzed using a methanol/H2SO4 (0.001 N, 20
mmol Na2SO4) mixture (50:50). A wavelength of λ ) 254 nm
was selected for UV-detection.
1
Rf ) 0.33 (dichloromethane/methanol 10:1); H NMR (400
MHz, CDCl3) δ ) 1.37 (s, 54H, CH3), 1.66 (m, 18H, 1 CH2),
1.97 (m, 6H, 2 CH2), 2.43 (m, 12H, 3 CH2), 2.66 (m, 6H, 4
CH2), 2.93 (m, 6H+6H, 5+8 CH2), 3.08 (m, 6H+6H, 6+8
The stability in PBS (pH 5 and 7.4) was measured at a
concentration of 0.1 mg/mL. For protein binding, 40 mg/mL
HSA and 0.1 mg/mL compound were incubated at 37 °C. The

Optimization of N-Lost Drugs Melphalan and Bendamustine
Bioconjugate Chem., Vol. 21, No. 10, 2010 1733
Chart 8. Supporting Scheme for NMR Data of Compound 17
ing equations:
cytostatic effect: %T/Ccorr ) [(T - C0)/(C - C0)] × 100
(1)
cytocidal effect: %τ ) [(T - C0)/C0] × 100
(2)
in which T (test) and C (control) are the optical density values
at 578 nm of the crystal violet extract of the cells in the wells
(that is, the chromatin-bound crystal violet extracted with 70%
ethanol), and C0 is the density of the cell extract immediately
before treatment. A microplate reader at 590 nm (Flashscan
Analytik Jena AG) was used for the automatic estimation of
the optical density of the crystal violet extract in the wells. The
calculated %T/C values can be interpreted as follows:
T/C > 80% no antiproliferative effect
80% > T/C > 20% antiproliferative effect
20% > T/C > 0% cytostatic effect
τ ) T/C < 0% cytocidal effect
RESULTS
protein was separated by ethanolic precipitation. After centrifu-
gation, the supernatant was evaluated for drug content.
Conformational Analysis for Molecular Extent Predic-
tion. Conformational analysis was performed using the program
OMEGA (20) from OpenEye Scientific Software (www.
eyesopen.com). The maximum possible number of conforma-
tions was set to 10 000, and the MMFF94 force field was used
for conformational search and subsequent energy minimization
with a dielectric constant of 80 simulating a water environment.
For all other parameters, default values were kept. This setup
resulted in 918 conformations for 12 and 6005 for 10.
Subsequently, all conformations were converted and analyzed
using a custom routine implemented in the LigandScout
framework (21, 22) for determining the maximum extent of each
conformation.
Biological Methods. Cell Culture. The human MCF-7 and
MDA-MB-231 breast cancer cell lines were obtained from the
American Type Culture Collection (ATCC). The MCF-7 breast
cancer cell line originated from a 69-year-old Caucasian woman
and is a well-characterized estrogen receptor (ER) positive
control cell line (cells are positive for cytoplasmic estrogen
receptors). The human cell line MDA-MB-231 is a prototype
for the study of hormone-independent breast cancer. Cell line
banking and quality control were performed according to the
seed stock concept reviewed by Hay (23). Both cell lines were
maintained in L-glutamine and sodium pyruvate containing
DMEM High Glucose (4.5 g/L) supplemented with 5% fetal
calf serum (FCS, Gibco) using 25 cm2 culture flasks in a
humidified atmosphere (5% CO2) at 37 °C. The cell lines were
passaged weekly after treatment with trypsin (0.05%)/ethylene-
diaminetetraacetic acid (EDTA, 0.02%, Boehringer). Myco-
plasma contamination was regularly monitored, and only
mycoplasma-free cultures were used.
In Vitro ChemosensitiVity Assays. Briefly, 100 µL of a cell
suspension of 7500 cells mL-1 culture medium were plated into
each well of a 96-well microtiter plate and incubated at 37 °C
for 3 days in a humidified atmosphere (5% CO2). By adding an
adequate volume of a stock solution of the appropriate com-
pound (solvent: H2O, DMF, DMSO, MeOH) to the medium,
the desired test concentration was obtained. After the proper
incubation time, the medium was removed and the cells were
fixed with a glutardialdehyde solution and stored under phos-
phate buffered saline (PBS) at 4 °C. Cell biomass was
determined by a crystal violet staining technique described
previously (24, 25). The efficiency of the compounds is
expressed as corrected %T/Ccorr values according to the follow-
Synthesis. The synthesis first implicated the building of the
drug-spacer derivatives which were then attached to the
dendrimers.
Melphalan hydrochloride was Boc-protected in a first step
and then reacted in a second step with N-(2-hydroxyethyl)ma-
leimide using a Steglich reaction to give the ester 4 which was
purified by column chromatography. Treatment of 4 with
aqueous HCl yielded 5 as a colorless solid (Scheme 1).
Bendamustine hydrochloride was initially transformed to the
free base 6 (26) and then converted to 7 through a similar
approach as described for 4.
The binding of the drug-spacer to the dendrimers was
achieved in the last step of the synthetic route. Due to their
nucleophilic properties, the terminal amine groups of the
dendrimers were able to bind the maleimide residue. For this
purpose, the reactants were dissolved in chloroform and heated
to reflux. Cleavage of the Boc groups in melphalan derivatives
was performed with 25% HCl. The analytically pure products
were obtained after column chromatography. Schemes 2 and 3
depict the synthesis of the G0 generation dendrimers conjugates
9 and 10.
The melphalan-spacer 4 was bound to the G1 dendrimer via
the same synthetic route (see Scheme 4).
In order to evaluate the significance of the spacer 3 for the
mode of action, the cytostatics were also directly bound to the
dendrimer by formation of an amide bond using the standard
peptide coupling reactions (27-29) (Schemes 5 and 6).
For this reaction, it is necessary to activate the carboxyl group
of melphalan and bendamustine with peptide coupling agents,
which led to the formation of reactive anhydrides or ester
intermediates (for the mechanism of the reaction and the
methods used, see ref 17). The syntheses of G0 dendrimer-drug
conjugates were then carried out as depicted in Scheme 5 using
TBTU as coupling agent.
Structural Characterization. All products and those com-
pounds used in biological tests were characterized by their fully
1
assigned H and 13C NMR spectra and molecular ion peaks in
the mass spectra. Their purity was proven by high-resolution
ESI-TOF mass spectrometry. All deprotected dendrimers were
fully soluble in water and in buffered cell culture media as
judged by visual examination.
Computational Molecule Size Estimation for Assessing
Enhanced Permeation and Retention. In order to estimate
the molecule size in solution, the maximum extent of the

1734 Bioconjugate Chem., Vol. 21, No. 10, 2010
Scheme 1. Synthesis of the Drug-Spacera
Scutaru et al.
a Reagents and conditions: (a) Boc2O, TEA, methanol, RT, 1 h; (b) DCC, DMAP, CH2Cl2, RT, 24 h; (c) aqueous HCl, THF, RT, 24 h; (d) DCC,
DMAP, CH2Cl2, RT, 20 h.
Scheme 2. Synthesis of the Mel-spacer-G0 Conjugatesa
a Reagents and conditions: (a) chloroform, DIPEA, reflux, 72 h; (b) aqueous HCl, THF, RT.
compounds was calculated by performing an exhaustive
conformational analysis using the software OMEGA and
the cheminformatics framework of the software package
LigandScout (21, 22). Figure 2 shows representative confor-
mations of 10 and 12, respectively, while Figure S1 (see
Supporting Information) shows a histogram of the maximum
extent distributions over the calculated three-dimensional
conformational space for the two compounds. The relative
extent frequencies reach their maxima near the average values
of about 31 Å for compound 12 and at about 38 Å for 10
(Table S1, see Supporting Information).
Antitumor Activity. The antiproliferative activity of the
synthesized drug-dendrimer conjugates was determined in time-
dependent cytotoxicity tests on the human MCF-7 and MDA-

Optimization of N-Lost Drugs Melphalan and Bendamustine
Bioconjugate Chem., Vol. 21, No. 10, 2010 1735
Scheme 3. Synthesis of the Benda-spacer-G0 Conjugatesa
a Reagents and conditions: (a) DIPEA, chloroform, reflux, 72 h.
Scheme 4. Synthesis of the Mel-spacer-G1 Conjugatesa
a Reagents and conditions: (a) chloroform, DIPEA, reflux, 70 h; (b) aqueous HCl, THF, RT.

1736 Bioconjugate Chem., Vol. 21, No. 10, 2010
Scheme 5. Synthesis of the Drug-G0 Conjugatesa
Scutaru et al.
Scheme 6. Synthesis of the Mel-G1 Conjugatesa
a Reagents and conditions: (a) EDC, HOBt, CH2Cl2, -20°C/1 h,
RT/2 h, G1, DIPEA, -30°C/1 h, RT/16 h; (b) aqueous HCl, THF,
RT, 3 h.
a Reagents and conditions: (a) CH2Cl2, DMF, -20°C, TBTU/30 min,
G0, DIPEA, methanol, RT, 20 h; (b) TFA (95%), RT, 4 h; (c) CH2Cl2,
DMF, -20°C, TBTU/30 min, G0, DIPEA, methanol, RT, 20 h.
MB-231 breast cancer cell lines. From the graphs, antiprolif-
erative and cytostatic effects (80% > T/Ccorr > 0%), as well as
cytocidal effects (τ ) T/Ccorr < 0%), can be deduced.
Cisplatin (DDP) was used as positive control. It influenced
the cell growth in a concentration-dependent manner and
caused almost 100% growth inhibition at both cell lines at a
concentration of 5 µM. The two basic dendrimers G0 and
G1 were previously tested at concentrations of 1, 5, and 10
µM (17, 30). G0 was completely inactive, while the G1
dendrimer marginally reduced cell proliferation at 10 µM
(T/Ccorr ) 60%).
It should be mentioned that the cytotoxic properties of the
tested compounds were similar at the MCF-7 and MDA-MB-
231 cell lines (except where noted; MDA-MB-231 graphs, see
Supporting Information).
Melphalan hydrochloride 1 exhibited cytotoxic properties at
10 µM (Figure 3A). Boc protection led to the inactive compound
2 (data not shown) whose activity increased by esterification
with the maleimide 3. The resulting compound 4 showed
cytocidal characteristics at concentrations of 5 and 10 µM after
an incubation time of 48 h (Figure 3B).
Cleavage of the Boc group increased the cytotoxicity further.
The maleimide ester 5 was cytostatic at the lowest concentration
used (T/Ccorr ≈ 0%; conc. 1 µM), however, not before the end
of the test (incubation time 144 h, see Figure 3C). The
comparison of the time response curves in Figure 3A and C
clearly documented an ∼10-fold higher activity of 5 compared
to that of Melphalan 1.
Binding of 4 to G0 (f 8) decreased the activity at the MCF-7
cell line (compare Figures 3B and 4A) but marginally enhanced
the antiproliferative effects at the MDA-MB-231 compared to
the MCF-7 cell line. The activity of the G0-bound compound
Figure 2. Representative examples of minimized 3D conformations
for compounds 12 (left, extent: 38.3 Å) and 10 (right, extent: 30.6 Å).
Both molecules are shown as their Van-der-Waals surfaces colored by
hydrophobicity (blue: hydrophilic, gray: hydrophobic).
5 (f 9) remained nearly unchanged (compare Figures 3C and
4B). Only at 1 µM was a somewhat lower growth inhibition
observed.
The increased generation of the dendrimer reduced the
cytotoxicity of the Boc-protected compound further. Compound
11 showed only weak activity (T/Ccorr ≈ 40%) even at the
highest concentration used (10 µM). In contrast, the activity of
5, 9, and 12 is nearly independent of the presence of a dendrimer
and its generation (compare Figures 3C, 4B, and 5B).
The same trend was determined for bendamustine (6), its
maleimide derivative (7), and its G0 conjugate (10). Benda-
mustine was inactive at the MCF-7 and the MDA-MB-231 cell
lines (Figure 6A and Supporting Information). Esterification with
the maleimide 3 (f 7, Figure 6B) increased the antiproliferative
effects (cytostatic effects (T/Ccorr ) 0%) at 5 µM and cytocidal
effects (T/Ccorr < 0%) at 10 µM). Binding to G0 (f 10) reduced
the activity slightly (compare Figures 6B and 7A).

Optimization of N-Lost Drugs Melphalan and Bendamustine
Bioconjugate Chem., Vol. 21, No. 10, 2010 1737
Figure 3. In vitro cytotoxicity (τ ) T/Ccorr < 0%): (A) Mel, (B) MelBoc-spacer 4, and (C) Mel-spacer 5. For detailed information on the investigation
of the T/Ccorr [%] and τ[%] values, see Experimental Section.
Figure 4. In vitro cytotoxicity (τ ) T/Ccorr < 0%): (A) MelBoc-spacer-G0 8, (B) Mel-spacer-G0 9.
In order to estimate the significance of the maleimide spacer
for the cytotoxicity of bendamustine, the drugs were directly
bound to the dendrimers via an amide bond. This binding led
to a complete inactive compound (f 15, see Figure 7B). This
means that in vitro cytotoxicity of bendamustine needs binding
to a maleimide spacer.
In contrast to this, the amide binding of melphalan to G0
only slightly reduced the activity. The MelBoc-spacer-G0
derivative 8 reached the T/Ccorr ) 50% at 5 µM, its congener
without spacer 13 (MelBoc-G0) at 10 µM. The cleavage of the
Boc groups resulted in the compounds 9 and 14, which showed
similar antiproliferative effects at the low concentration of 1
µM. At 5 and 10 µM, 14 distinctly reached cytocidal effects
(see Figure 8B).
If dendrimers with higher generation were used, the effects
were independent of the presence of a maleimide spacer
(compare Figure 5 with Figure 9).
Investigations into the Hydrolysis of Bendamustine and
Melphalan. Bendamustine belongs to the class of alkylating
agents of the N-Lost type. Binding to the DNA occurs by
attachment to guanine bases. In aqueous solution, the drug
underwent hydrolysis into the hydroxyl (HP1, Figure 10) or
dihydroxyl species (HP2, Figure 10). This aquation is ac-
companied by an inactivation of the drug.
In order to investigate the stability of bendamustine in
aqueous solution, it was incubated in water for 24 h at 37 °C
and the degradation was followed by HPLC analysis (RP 18;
methanol/H2SO4 (0.001 N, Na2SO4 (20 mM)) and UV-detection

1738 Bioconjugate Chem., Vol. 21, No. 10, 2010
Scutaru et al.
Figure 5. In vitro cytotoxicity (τ ) T/Ccorr < 0%): (A) MelBoc-spacer-G1 11, (B) Mel-spacer-G1 12.
Figure 6. In vitro cytotoxicity (τ ) T/Ccorr < 0%): (A) Benda 6; (B) Benda-spacer 7.
Figure 7. In vitro cytotoxicity (τ ) T/Ccorr < 0%): (A) Benda-spacer-G0 10; (B) Benda-G0 15.
(254 nm). During the first 2 h, 55.6% of bendamustine
hydrolyzed to HP1 (36.8%) and HP2 (18.8%). After 8 h,
amounts of 81.6% of HP2, 14.7% of HP1, and only 3.7% of
bendamustine were detected. Incubation of 24 h led to a
complete hydrolysis into HP2.
The maleimide residue mediated 7 high hydrophobicity
resulting in an increased retention time of RT ) 57.3 min
(bendamustine RT ) 25.7 min). It is visible that two reactions
took place: (1) cleavage of the ester bond in 7 resulting in
bendamustine 6 and the maleimide 3; (2) hydrolysis of 7 to the
hydroxyl and dihydroxyl compounds 7-OH and 7-diOH.
Bendamustine itself hydrolyzed to HP1 and HP2 (see Figure
10).
Melphalan was distinctly less stable. An amount of 88.1%
hydrolyzed after 2 h to the monohydroxyl (24.7%) and dihy-
droxyl (63.5%) species, while after 8 h, only the dihydroxyl
species was present.
After 8 h, 14% (sum of 6, HP1, and HP2) of bendamustine
was set free. Aquation products of 7 were detected at RT ) 14.6
min (7-OH; 38%) and RT ) 5.28 min (7-diOH; 7.5%). Unchanged
7 amounted to 39.5%. It is obvious that the esterification of
The bendamustine-maleimide derivative 7 was tested under
the same conditions. A representative of a chromatogram is
depicted in Figure 11.

Optimization of N-Lost Drugs Melphalan and Bendamustine
Bioconjugate Chem., Vol. 21, No. 10, 2010 1739
Figure 8. In vitro cytotoxicity (τ ) T/Ccorr < 0%): (A) MelBoc-G0 13, (B) Mel-G0 14.
Figure 9. In vitro cytotoxicity (τ ) T/Ccorr < 0%): (A) MelBoc-G1 16, (B) Mel-G1 17.
Figure 10. Hydrolysis of 7 at 37 °C in water.
bendamustine increased the stability in aqueous solution, but
bendamustine was released over 36 h and held a level up to 7%.
HP1 and HP2 are not only the hydrolysis products of bendamus-
tine, but also the degradation products of 7-OH and 7-diOH.

1740 Bioconjugate Chem., Vol. 21, No. 10, 2010
Scutaru et al.
Figure 11. Chromatogram of 7 after incubation in water at 37 °C for 8 h.
The same modification done with melphalan did not increase
the stability. The maleimide derivative 5 degraded (t1/2 ) 37
min) comparable to melphalan (t1/2 ) 30 min). It should be
mentioned that 5 showed the same degradation profile as 7.
In order to model blood circulation (buffer pH 7.4) and
intracellular/endosomal environment (buffer pH 5), the same
hydrolysis experiments were performed with bendamustine and
7 in PBS at relevant pH.
Bendamustine showed pH-sensitive degradation. It was
completely hydrolyzed to HP2 during 2 h at pH 7.4, while at
pH 5, after 4 h only 50% and after 24 h 92% of bendamustine
degraded. Compound 7 showed distinctly increased stability.
The half-life in PBS was 8.5 h at pH 5 and 10 h at pH 7.4.
The hydrolysis rates at pH 5 and pH 7.4 were not as different
as observed for bendamustine. The half-lives was 120 and 98
min, respectively. Interestingly, after derivation to 5, a complete
degradation (100%) occurred at pH 7.4 and a slightly increased
stability at pH 5 (t1/2 ) 154 min).
HSA solution (40 mg/mL) was used to study protein binding.
Ethanolic precipitation indicated protein binding of bendamus-
tine. After incubation for 2 h, 50% were protein-bound and 6%
hydrolyzed to HP1 and HP2, respectively. In contrast, during
the same incubation time only 25% of 7 were HSA-bound and
18% hydrolyzed to 7-OH.
Melphalan was strongly HSA-bound. After 2 h, 14.3% of
hydroxyl derivatives and 18.9% of melphalan could be detected.
The amount of 66.8% was bound to the protein. A still faster
interaction with HSA was found for 5. No drug was identified
in the chromatograms after 30 min.
derivative 2 was inactive. Interestingly, the esterification with
N-(2-hydroxyethyl)maleimide 3 strongly increased the activity.
The melphalan-Boc-protected spacer analogue 4 was proven to
exhibit cytocidal effects on both cell lines. As expected, the
deprotection leading to compound 5 increased the influence on
the cells. Already, at a concentration of 1 µM cytostatic effects
were achieved.
A possible explanation could be the binding of the spacer
derivative to human serum albumin (HSA) present in the cell
culture media. In some previous work, Kratz et al. synthesized
such conjugates of the nitrogen mustard chlorambucil (14). They
also demonstrated an HSA binding of the spacer derivative in
situ after iv administration. Due to the considerable uptake of
the macromolecule-bound drug in tumor cells, this concept has
a promising future as a drug carrier system (31).
This mode of action can principally be realized for maleimide
derivatives 4, 5, and 7. HSA binding was confirmed on the
examples of 5 and 7. Fast binding also occurred with benda-
mustine and melphalan. After 2 h of incubation, more than 50%
were bound.
Besides HSA or transferrin as carrier molecules (32-34),
some synthetic polymers were designed to bind drugs for
selective accumulation in tumors and tumor cells (35, 36).
In previous studies (17), we used the 1,3,5-tris(3-aminopro-
pyl)benzene (G0) and its G1 analogue 3,5-bis(3-aminopro-
pyl)-N-(3-{3,5-bis[3-{3,5-bis(3-aminopropyl)benzoylamino}-
propyl]phenyl}propyl)benzamide for the transport of drugs
into MCF-7 and MDA-MB-231 cells. The concept was very
successful in the case of platinum(II) complexes (30).
Already, in the 1990s covalent conjugation of melphalan to
macromolecular carriers was described (37). It was, e.g., linked
to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers via
enzymatically cleavable peptide side chains. In 2008, Shukla
et al. published poly(ethylene glycol) conjugates of melphalan
demonstrating enhanced dose-dependent in vitro cytotoxicity
(38).
DISCUSSION
The preliminary in vitro tests on MCF-7 and MDA-MB-231
breast cancer cells confirmed the existing literature data about
the antitumor activity of melphalan hydrochloride (1). It reduced
the concentration-dependent growth of MCF-7 and MDA-MB-
231 breast cancer cells. At 10 µM, cytostatic effects (T/Ccorr
0%) were determined (Figure 3), which depended on the
presence of a free amino group. The Boc-protected melphalan
≈
Therefore, we decided to bind melphalan to G0 or G1
dendrimers via the maleimide spacer (8, 9 and 11, 12) or directly

Optimization of N-Lost Drugs Melphalan and Bendamustine
Bioconjugate Chem., Vol. 21, No. 10, 2010 1741
via an amide bond (13, 14 and 16, 17) to evaluate their cytotoxic
potency in vitro. The BOC-protected conjugates 8, 11, 13, and
16 were only marginally active at MCF-7 and MDA-MB-231
cells. Only the drug-spacer-G0 conjugate 8 showed low cyto-
static properties at the highest concentration. The G1-conjugates
with free NH2 groups (12 and 17) only slightly differ in their
activity. They showed cytostatic properties at 5 and 10 µM.
These results indicated a relatively high stability of the
conjugates. We assume that melphalan interacted after transport
into the tumor cells in free form and also dendrimer-bound with
the DNA via the N-lost group. The resulting cross-links led to
cell death. Bendamustine belongs to the same class of antitumor
agents. However, its cytotoxic behavior in free form, maleimide
bound, or as dendrimer conjugate differs from that of melphalan.
Melphalan and bendamustine are sensitive to hydrolysis in
water and were degraded at 37 °C to the bis(hydroxyethyl)amino
compounds over 24 h. The results of Chang et al. demonstrated
for melphalan a retarded hydrolysis in the presence of bovine
serum albumin or human plasma proteins (39). In a Roswell
Park Memorial Institute Medium containing 10% fetal bovine
serum, the half-life of melphalan at 37 °C was 1.13 ( 0.10 h
and was only slightly higher compared to that in pure water
(40). In our experiments, besides strong HSA binding a reduced
amount of hydrolysis was detected, too.
Bendamustine showed a half-life of about 100 min in pure
water and increased stability in physiological NaCl solution (41).
This correlates very well with the hydrolysis of melphalan (39).
However, the attempt failed to stabilize the aqueous solution
of bendamustine by HSA. A drug amount of 50% was HSA-
bound during an incubation time of 2 h and an amount of 12%
was hydrolyzed. In PBS buffer at pH ) 7.4, fast hydrolysis
took place. After 2 h, only HP2 was present. Therefore, we
assume that under cell culture conditions not enough active drug
was present to reduce the growth of MCF-7 and MDA-MB-
231 cells.
The mode of action of bendamustine is discussed in very
contradictory terms. However, it is still accepted that DNA-
alkylation is the main cause of cell death in vivo. Preclinical
data demonstrate that bendamustine induces rapid, sustained
single- and double-strand DNA damage, which results in
apoptosis, or programmed cell death, in the tumor. It also
induces mitotic checkpoint inhibition, which results in nonapo-
ptotic cell death (42-44).
Such a mode of action requires an intact N-lost moiety. The
dihydroxybendamustine derivative HP2 is inactive in cell culture
experiments. The participation of the partially hydrolyzed HP1
to the antiproliferative effects is still unknown. This assumption
is confirmed by the fact that in in vitro assays bendamustine is
inactive (45) (see also Figure 6A). If it were possible to prevent
hydrolysis in aqueous solution, antiproliferative properties
should be observed. The stability is enhanced in alcoholic
solution (unpublished results) which is not usable in in vitro
conditions. However, in this SAR study it was at first demon-
strated that esterification of the carboxylic group increased the
water stability.
The binding of 7 to G0 through the maleimide spacer slightly
reduced the activity from cytocidal to cytostatic. In contrast,
the direct binding of bendamustine to G0 via an amide bond
terminated the cytotoxic effects. The higher stability of the amide
bond prevents a release of bendamustine from the molecule.
Therefore, it is very likely that it was not 10 itself causing cell
death but the hydrolytically cleaved bendamustine 6. This
contrasts with the results obtained with melphalan, which was
active in free as well as dendrimer bound via the spacer and
the amide bond.
Our concept to increase the activity of melphalan and
bendamustine by an adequate carrier (dendrimer) was successful.
Through the binding to the dendrimer via spacer, not only can
it be assumed that the new bendamustine derivatives ac-
cumulated in the tumor cells probably through endocytosis, but
it can be also presumed that the hydrolysis of bendamustine is
suppressed.
The investigations pointed, on one hand, to a participation
of the butyric acid in the hydrolytic processes; on the other hand,
Pencheva et al. achieved for bendamustine immobilized onto
polyphosphoester a higher stability by the formation of molec-
ular associates (46). If this were true, the stability of benda-
mustine should increase using polymers such as poly(ethylene
glycol)s. In our lab, however, we cannot find increased stability
with PEG 400/water mixtures.
Hopwood and Stock demonstrated that macromolecules with
lipophilic moieties are capable of diminishing the hydrolysis
of drugs (47). Albumin proved to be the most efficient at
protecting the anticancer agents. This may also be true for the
bendamustine maleimide derivative 7. However, the binding
mode at HSA is yet unknown.
CONCLUSIONS
A new set of dendrimer and dendrimer-spacer conjugates
of melphalan and bendamustine have been successfully syn-
thesized. The antiproliferative activity of these compounds and
their maleimide precursors has been determined against MCF-7
and MDA-MB-231 cell lines. No selectivity for one of the cell
lines was detected. The cytotoxicity of melphalan depended on
a free amino group and was independent of the kind of bond to
the spacer or the dendrimer.
The antiproliferative activity of bendamustine was strongly
increased due to reduced hydrolytic processes after esterification
with the N-(2-hydroxyethyl)maleimide. An amide bond to 1,3,5-
tris(3-aminopropyl)benzene terminated the activity. Therefore,
it is very likely that the cell death promoting effects were caused
after transport into the tumor cells and release of the intact drug
from the core molecule G0.
In a following study, we will investigate the hydrolysis
reactions and the release of bendamustine from dendrimers of
higher generation. Furthermore, we will evaluate the binding
mode at the carboxylic acid necessary for increased activity.
ACKNOWLEDGMENT
The maleimide moiety of compound 7 is attached to benda-
mustine by a hydroxyethyl chain. The resulting ester was
incubated at 37 °C in water and the degradation was followed
by HPLC.
The presented study was supported by grants Gu285/
4-1, Gu285/5-1, and Gu285/5-2 from the Deutsche
Forschungsgemeinschaft.
After 8 h of incubation, degradation to bendamustine followed
by hydrolysis to HP1 was observed. Furthermore, besides intact
7, its monohydroxyl derivative 7-OH was determined, too.
Interestingly, the stability of 7 in PBS was higher than that of
bendamustine and independent of the pH. The half-life of 7 at
pH 7.4 was 10 h, and guaranteed higher amounts of active
compounds resulting in increased antiproliferative effects in cell
culture experiments.
Supporting Information Available: Elemental analyses of
the new compounds, their results (cytotoxicity tests) on the
MDA-MB-231 cell line, the cytotoxicity of G0 and G1 on the
MCF-7 cell line, a histogram of the maximum extent distribu-
tions over the calculated three-dimensional conformational space
for 10 and 12 and median, average and maximum values for
molecule sizes of 10 and 12. This material is available free of
charge via the Internet at http://pubs.acs.org.

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