New chemical adaptor unit designed to release a drug from a tumor targeting device by enzymatic triggering

Article abstract of DOI:10.1016/j.bmc.2004.01.041

A new controlled drug delivery system for selective chemotherapy was developed. It is based on a chemical adaptor unit, that releases a drug by a spontaneous cyclization mechanism after cleavage of an enzymatic substrate. It also provides a generic linkag

Full text of DOI:10.1016/j.bmc.2004.01.041

Bioorganic & Medicinal Chemistry12 (2004) 1853–1858
New chemical adaptor unit designed to release a drug from
a tumor targeting device by enzymatic triggering
Anna Gopin,a Christoph Raderb and Doron Shabata,*
aSchool of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel-Aviv University, Tel Aviv 69978, Israel
bExperimental Transplantation and Immunology Branch, Center for Cancer Research, National Cancer Institute,
National Institutes of Health, Bethesda, MD, USA
Received 15 October 2003; revised 21 January2004; accepted 27 January2004
Abstract—A new controlled drug deliverysystem for selective chemotherapywas developed. It is based on a chemical adaptor unit,
that releases a drug by a spontaneous cyclization mechanism after cleavage of an enzymatic substrate. It also provides a generic
linkage of a drug with a targeting device in a manner set to be triggered by defined enzymatic activity. The system is generic and
allows using a varietyof drugs, targeting devices, and enzymes byintroducing the corresponding substrate as a trigger for drug
release in the chemical adaptor.
# 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Lack of selectivityof chemotherapeutic agents is a
major problem in cancer therapyand typicallyis asso-
ciated with severe side effects. It is obvious that more
research is needed to develop more selective and effec-
tive anticancer agents. A promising approach to over-
come side effects and to achieve a more tumor selective
cancer treatment is prodrug therapy. In this concept, a
cytotoxic drug is synthetically converted into a non-
toxic derivative, teremed prodrug. Upon administra-
tion, the prodrug must be selectivelyactivated to regen-
erate the toxic parent drug at the tumor site.1 This
tumor selective activation should result from properties
distinguishing neoplastic from normal cells.2 We have
recentlyreported on a new concept that combines a
tumor targeting device, a prodrug, and a prodrug acti-
vation trigger in a single entity.3 For this, we designed a
generic module or chemical adaptor that is based on
three chemical functionalities. The first functionalityis
attached to an active drug and thereby, masks it to yield
Figure 1. 2-amino-3-methylamino-propionic-acid, the central core of
the new chemical adaptor unit.
a prodrug. The second is linked to a targeting moiety,
which is responsible for guiding the prodrug to the
tumor site, and the third is attached to an enzyme sub-
strate. When the corresponding enzyme cleaves the
substrate, it triggers a spontaneous elimination reaction
that releases the active drug from the targeting moiety.
As a result, prodrug activation will preferentiallyoccur
at the tumor site. Here we report on the design, synth-
esis and proof of concept of a new adaptor unit that
utilizes spontaneous cyclization, rather than elimin-
ation, to release the active drug.
Abbreviations: Boc-t-buthyl carbonate, DIPEA; Diisopropyl ethyl
amine; DMAP, Dimethyl aminopyridine; DMF, Dimethyl formamide;
EDC, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide; EtOAc, Ethyl
acetate; Et3N, Triethyl amine; Fmoc, 9-fluorenylmethoxycarbonyl;
He, n-Hexane; MeOH, Methanol; NHS, N-hydroxy succinimide; PNP,
4-nitrophenyl; THF, Tetrahydrofuran.
Keywords: Prodrug; Self-immolative; Enzyme; Cancer.
* Corresponding author. Tel.: +1-972-3640-8340; fax: +1-972-3640-
9293; e-mail: chdoron@post.tau.ac.il
The central core of our chemical adaptor (Fig. 1) is
based on N-methyl-diamino-propionic-acid, which has
three functional groups suitable for linkage. Group I is
a carboxylic acid that is conjugated to a targeting moi-
etyvia an amide bond. The drug is linked through the 2-
amino group II and the enzyme substrate is attached via
0968-0896/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.01.041

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A. Gopin et al. / Bioorg. Med. Chem. 12 (2004) 1853–1858
the 3-amino group III bya carbamate bond. Figure 2
illustrates the release mechanism of the drug. Cleaving
of the enzyme substrate from complex 1 generates a free
amine group (intermediate 2) that spontaneouslycyclizes
to form a urea derivative 3 and the active drug.
protection group was removed from compound 10
to afford free amine, which was further reacted with
p-nitrophenylcarbonate of camptothecin to give com-
pound 11. The Fmoc group of 11 was removed with
iso-propylamine to generate a free amine, which was
mixed with a commerciallyavailable form of activated
ester of HPMA copolymer to finally afford conjugate
12 (Fig. 3C).
The design of our generic module allows to potentially
link anytargeting device to a varietyof drugs and to
release them with anyenzyme byusing the correspond-
ing substrate as a trigger. As proof of concept, we
designed a pilot system for which we chose Escherichia
coli Penicillin-G-Amidase (PGA) as the triggering
enzyme.4 As a targeting device we chose the. water-
soluble synthetic copolymer; N-(2-hydroxypropyl)-
methacrylamide (HPMA) which is biocompatible, non-
immunogenic and non-toxic.5 Its in vivo distribution is
well characterized6 and it is known to accumulate
The drug–polymer conjugate was purified by dialysis
against water. Bymeasuring its UV spectrum, the num-
ber of drug molecules attached to the HPMA-copoly-
mer was determined. Based on the CPT chromophore,
which has a lmax at 365 nm, we found that on average
three molecules of the drug were linked to one molecule
of the HPMA-copolymer. Next we tested whether the
CPT drug can be released from conjugate 12 by
the catalytic activity of PGA. According to our design, the
drug should be spontaneouslyreleased after the genera-
tion of amine 5 as illustrated in Figure 4. We incubated
conjugate 12 with PGA in PBS (pH 7.4) at 37 ꢀC and
monitored the appearance of free CPT using an HPLC
assay. As Figure 5 shows, CPT was released bythe cat-
alytic activity of PGA to form compound 14 and the
free drug. No spontaneous CPT release was observed in
the absence of the enzyme.
selectivelyat tumor sites due to the enhanced perme-
7
abilityand retention (EPR) effect.
Camptothecin
(CPT) was selected as the drug in our pilot system.8
CPT exerts its antitumor activitymainlythrough inhi-
bition of topoisomerase I. This enzyme, which is found
in mammals, binds preferentiallyto double-stranded
DNA, cleaving one strand and forming an enzyme–
DNA covalent bond between a tyrosine residue and the
30 phosphate of the cleaved DNA. Drug-induced accu-
mulation of topoisomerase I-DNA complexes was
identified as an essential step, ultimatelyleading to cell
3
While our previouslyreported chemical adaptor system
9,10
death byapoptosis.
utilizes 4-hydroxymandelic-acid as the central core, in
this example we have used 2-amino-3-methylamino-
propionic-acid. Consequently, the release mechanism of
the active drug has changed from elimination to cycli-
zation. Both of these reactions are often used in prodrug
chemistryto generate a self-immolative spacer between
the drug and the enzymatic substrate. The chemical lin-
kages, which are used to attach the targeting moiety, the
drug and the trigger, are relativelystable in phsyio-
logical conditions, and are constructed from carba-
mates and amides bonds. Interestingly, another group
has alreadyused similar chemical adaptor systems for
organic synthesis.11 An enzyme-labile linker group was
attached to a solid support and a third functionality
was used for solid-phase synthesis of a target molecule.
The synthesis of the adaptor system was performed
straightforward as described in Figure 3. In brief, Na-
Boc-l-2,3-diaminopropionic acid (DAP) was reacted
with benzaldehyde and then with sodium borohydride
to give secondaryamine 4, which was further reacted
with formaldehyde and sodium borohydride to afford
tertiaryamine 5. The latter was deprotected bycatalytic
hydrogenation to generate mono-methylated amine 6
(Fig. 3A). Activated carbonate 7 was then reacted with
amine 6 to give compound 8. The carboxylate of 8 was
activated with EDC and NHS and reacted with com-
pound 9 (after removal of the Boc protection group
with TFA) to generate amide 10 (Fig. 3B). The Boc
Figure 2. General design of the chemical adaptor system. Cleavage of the enzyme substrate generates an intermediate that spontaneously rearranges
to release the drug from the targeting device.

A. Gopin et al. / Bioorg. Med. Chem. 12 (2004) 1853–1858
1855
The small molecule was detached from the polymeric
support, starting byenzmy atic cleavage, leading to
generation of an intermediate that spontaneously
released the product. The potential of such chemical
adaptor systems was very recently highlighted in the lit-
erature.12 The relative large concentration of the
enzyme used in this study, could be reduced after opti-
mization of the interaction between the phenyl-
acetamide substrate and the enzyme’s binding site. This
concept was solved with the use of self-immolative
linkers in a studythat described in vivo activityin a
catalytic antibody-prodrug system.13
In conclusion, we have developed a drug deliverysystem
based on a new chemical adaptor unit with the cen-
tral core 2-amino-3-methylamino-propionic-acid which
Figure 3. (A–C): Synthesis of a cyclization based chemical adaptor system, using PGA as the activating enzyme, an HPMA copolymer as a targeting
device and camptothecin as the drug.

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A. Gopin et al. / Bioorg. Med. Chem. 12 (2004) 1853–1858
Figure 4. Mechanism of CPT drug release from the HPMA-copolymer, using Penicillin-G-Amidase as the triggering enzyme.
provides a generic linkage of a drug with a targeting
device in a manner set to be triggered bydefined enzy-
matic activity. The system is generic and accommodates a
varietyof drugs, targeting devices, and enzymes byintro-
ducing the corresponding substrate as a trigger for drug
release in the chemical adaptor. Proof of concept was
demonstrated using CPT as the drug, an HPMA-copoly-
mer as the targeting device and PGA as the triggering
enzyme. Currently we are synthesizing a variety of chemi-
cal adaptor systems using different tumor targeting
devices, prodrugs and enzymatic triggers. In vivo studies
will be initiated after further optimization of this system.
temperature unless stated otherwise. All general
reagents, including salts and solvents, were purchased
from Aldrich (Milwaukee, MN). HPMA copolymer-
Gly-Gly-ONp (4.4 mole%), (ONp is p-nitrophenyl),
M.W 30,000D was obtained from Polymer Laboratories
(Church Stratton, UK).
Compounds 4–6 were prepared according to procedures
described before with some modifications.14
2.1.1. Compound 4. To a suspension of the commercially
available Na-Boc-L-2,3-diaminopropionic acid (DAP),
(1 g, 4.9 mmol ) in MeOH, were added Et3N (1.48g, 14.7
mmol) and benzaldehyde (1.04 gr, 9.8 mmol) while stir-
ring. After 30 min, the mixture was cooled to 0 ꢀC and
NaBH4 (931 mg, 14.7 mmol) was added. After 40 min
the solvent was removed under reduced pressure, the
mixture was dissolved in NaOH (0.1M) and extracted
with ethyl ether. The aqueous phase was acidified with
HCl (10% solution) to pH=6 and extracted with
CHCl3.The organic layer was washed with brine, dried
2. Experimental
2.1. General methods
Thin layer chromatography (TLC): silica gel plates
Merck 60 F254: compounds were visualized byirradia-
tion with UV light and/or bytreatment with a solution
.
of 25 g phosphomolybdic acid, 10 g Ce(SO4)2 H2O, 60
over MgSO4 and evaporated. Compound 4 was
obtained as a white powder (866 mg, 60%) and was
used for the next step without further purification.
mL concd H2SO4 and 940 mL H2O followed byheating
and/or bystaining with a solution of 12 g 2,4-dini-
trophenylhydrazine in 60 mL concd H2SO4, 80 mL H2O
and 200 mL 95% EtOH followed byheating and/or
immersing into an iodine bath (30 g I2, 2 g KI, in 400
mL EtOH/H2O (1:1) and warming. Flash chromato-
graphy(FC): silica gel Merck 60 (particle size 0.040–
1H NMR (200MHz, CDCl3): d=7.3 ppm (m, 5H); 4.24-
4.13 (brm,1H); 3.75 (1H, bm); 3.5 (1H, m); 3.15 (1H,
m); 1.46 (9H, s)
1
0.063 mm), eluent given in parentheses. H NMR spec-
2.1.2. Compound 5. Compound 4 (866 mg, 2.9 mmol)
was dissolved in a mixture of Et3N (1.2 mL, 8.84 mmol)
and 10 mL of MeOH. Formaldehyde (35% aqueous
solution) (0.76 mL, 8.84 mmol) was added, and the
mixture was stirred for 15 min. Then the reaction was
tra were measured in CDCl3 using Bruker AMX
200 MHz. The chemical shifts are expressed in d relative
to TMS (d=ppm) and the coupling constants J in Hz.
The spectra were recorded in CDCl3 as solvent at room

A. Gopin et al. / Bioorg. Med. Chem. 12 (2004) 1853–1858
1857
Figure 5. Determination of CPT release from the HPMA-copolymer by PGA versus background reaction in absence of PGA. -^-?Complex 12 (330
mM) with PGA (4.4 mM in PBS-7.4). -&- Complex 12 (330 mM) in PBS (pH 7.4). Reactions were incubated at 37 ꢀC for the indicated time. The drug
release concentration was monitored byHPLC analysis.
cooled to 0 ꢀC, NaBH4 (336 mg, 8.84 mmol) was added
and the mixture was stirred for additional 15 min.
Similarly, addition of formaldehyde and NaBH4 was
repeated twice more. Then the solvent was evaporated,
the residue was dissolved in water, acidified to pH=6
with hydrochloric acid (1M solution) and extracted with
CHCl3. The organic layer was washed with brine, dried
2.1.5. Compound 8. Compound 6 (100 mg, 0.46 mmol)
was dissolved in 2 mL DMF and triethylamine (0.193
mL, 1.38 mmol). Compound 73 (149 mg, 0.35 mmol)
was added and the mixture was stirred in room temper-
ature for 30 min. The reaction was monitored byTLC
(EtOAc:MeOH:AcOH=9:1:0.1). After completion the
DMF was removed under reduced pressure, and the crude
product was purified byflash chromatography(ethly
acetate:methanol:acetic acid=90:9:1) to give pure com-
pound 8 (150 mg, 88%), MS (FAB): C25H31N3O7
over MgSO4 and evaporated. Compound
5 was
obtained as a white powder (904 mg, 100%) and was
used for the next step without further purification.
1
[M+Na]+ 508.1; H NMR (200MHz, CDCl3): d=7.4–
1H NMR (400MHz,CDCl3): d=7.4 ppm (5H, s); 5.75
(1H, s); 4.3 (1H, m); 4.15 (1H, bs); 4.0 (1H, m); 3.4 (1H,
m); 3.0 (1H, m); 2.6 (3H, s); 1.5 (9H, s).
7.2 ppm (9H, m); 5.0 (2H, bs); 3.74 (2H, s); 3.7 (2H,d);
2.98 (3H,s); 1.41 (9H, s).
2.1.6. Compound 10. Compound 8 (150 mg, 0.31 mmol)
was dissolved in 2 mL DMF, EDC (72 mg, 0.37 mmol)
and NHS (43 mg, 0.37 mmol) were added and the mixture
was stirred at room temperature for 1 h and monitored
byTLC (EtOAc:MeOH=9:1) for completion. N-Fmoc-
N0-boc-dimethyl ethylene-diamine 9 (139 mg, 0.34
mmol) was stirred for 5 min in 1 mL TFA, monitored
byTLC (EtOAc: He=1:1) for completion, then TFA
was evaporated under reduced pressure. The NHS-ester
of 8 was poured into the N-Fmoc-dimethyl ethylene-
diamine salt, DIPEA (0.2 mL) was added to the mixture
and the reaction was stirred for 1 h. After completion of
reaction the solvent was evaporated under reduced
pressure, the crude product was purified bycolumn
chromatography(EtOAc 100%) to give compound 10
(119 mg, 50%) MS (FAB): C44H51N5O8 [M+Na]+ 800;
1H NMR (200 MHz, CDCl3): d=7.8 ppm (2H,m); 7.6
(2H, bs); 7.4–7.3 (9H, m); 5.0 (2H, bs); 4.4 (1H, m); 4.3
(1H, m); 3.74 (2H, s); 3.1–2.87 (10H, m); 1.38 (9H, s).
2.1.3. Compound 6. A solution of compound 5 (904 mg,
2.9 mmol) in MeOH with catalytic amount of Pd/C was
stirred vigorouslyunder H
at atmospheric pressure
2
overnight. After filtration and evaporation of the sol-
vent, the residue was precipitated from DCM/He to
afford compound 6 (628 mg, 96%). The product was
used for the next step without purification.
1H NMR (200 MHz,CDCl3): d=4.2 ppm (1H, m); 3.4
(1H, m); 3.2 (1H, m); 2.9 (3H, s); 1.44 (9H, s)
2.1.4. N-Fmoc-N0-Boc-dimethyl ethylenediamine 9. The
mono-Boc-N,N0-dimethyl ethylene-diamine3 (500 mg,
2.65 mmol) was dissolved in THF. DIPEA (1.8 mL,
10.6 mmol) was added. The reaction was cooled to 0 ꢀC
and 9-fluorenylmethoxy-chloroformate (688 mg, 2.65
mmol) dissolved in THF was added dropwise. The
reaction was stirred at room temperature for 15 min and
monitored byTLC (EtOAc:He=1:3). After completion
the reaction mixture was diluted with ethylacetate and
washed with NH4Cl. Then the organic layer was washed
with brine, dried over MgSO4 and the solvent was
removed under reduced pressure. The product was pur-
ified bycolumn chromatographyon silica gel (EtOAc:
He=2:3) (360 mg, 55%).
2.1.7. Compound 11. Compound 10 (55 mg, 0.07 mmol)
was cooled to 0 ꢀC and deprotected with 1 mL TFA to
remove the Boc group. The excess of the acid was
removed under reduced pressure and the residue was
dissolved in 2 mL DMF. Camptothecin-PNP-carbon-
ate3 (39 mg, 0.089 mmol) and 0.030 mL DIPEA were
added and the solution was stirred for 1 h. The reaction
was monitored byTLC (EtOAc:MeOH=9:1). After
completion the DMF was removed under reduced pres-
sure and the crude product was purified bycolumn
chromatography(EtOAc 100%) to give pure compound
11 in the form of white powder (40 mg, 54%).
MS (FAB):C24H30N2O4 [M+Na]+ 433; 1H NMR
(200MHz, CDCl3): d=7.77 ppm (2H, d, J=7.3); 7.6
(2H, d, J=7.07); 7.4-7.3 (4H, m); 4.59 (1H, d, J=5.3);
4.3 (1H, bs); 4.13 (1H, m); 3.4 (2H, bs); 3.2-2.9 (4H, m);
2.5 (2H, bd); 1.43 (9H, s)

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A. Gopin et al. / Bioorg. Med. Chem. 12 (2004) 1853–1858
Figure 6. UV spectrum chromatograms of CPT and HPMA conjugate with CPT-prodrug.
HRMS (MALDI): calcd for C54H68N4O21 1074.4013
[M+Na]+, found 1074.3990.
itored byan HPLC assayusing C-18 column, Wave-
length; 360 nm, eluent; acetonitrile: water (25:75), flow
rate; 1 mL/min.
2.1.8. Conjugate preparation (compound 12). Compound
11 (20 mg, 0.019mmol) was dissolved in DCM (1 mL)
iso-propylamine (0.160 mL, 1.9 mmol) was added. The
mixture of reaction was stirred for 2 h and monitored by
TLC (EtOAc:MeOH=9:1). After completion of the reac-
tion, the solvent was evaporated, the crude was dissolved
in DMF and HPMA co-polymer (25 mg, 0.83 mmol)
was added. The mixture of reaction was stirred for 12 h.
Then the solvent was removed under reduced pressure.
The crude was dissolved in water, centrifuged and dia-
lyzed through MWCO-10,000D membrane against
water. The conjugate (21 mg) was obtained after lipho-
lization of the water in the form of pinkish powder.
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Bymeasuring its UV spectrum, the number of drug
molecules attached to the HPMA-copolymer was deter-
mined. Based on the camptothecin chromophore, which
has a lmax at 360 nm, we concluded that on average
three molecules of the drug were linked to one molecule
of the HPMA-copolymer. There are approximately ten
linkage sites on the HPMA-copolymer. The mild cou-
pling’s yield can be probably improved by optimizing
the coupling reaction conditions (Fig. 6).
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2.3. Drug release analysis
HPMA-drug-conjugate (1 mg, 0.033 mmol) was dis-
solved in 100 mL solution of PGA (0.35 mg/mL, 4.4 mM)
and incubated at 37 ꢀC. The background sample was
prepared similarlyin PBS-7.4. Drug release was mon-
14. Andruszkiewicz, R. Pol. J. Chem. 1988, 62, 257.