Development of triazene prodrugs for ADEPT strategy: New insights into drug delivery system based on carboxypeptidase G2 activation

Article abstract of DOI:10.1016/j.bmcl.2012.09.029

Six novel urea triazene prodrugs have been synthesized to apply in antibody-directed enzyme prodrug therapy (ADEPT). The chemical and plasmatic stability of l-glutamate triazene prodrugs were evaluated and the chemical reactivity was mainly attributed to

Full text of DOI:10.1016/j.bmcl.2012.09.029

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6903–6908
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of triazene prodrugs for ADEPT strategy: New insights
into drug delivery system based on carboxypeptidase G2 activation
⇑
Verónica Capucha, Eduarda Mendes , Ana Paula Francisco, M. Jesus Perry
Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Av. Professor Gama Pinto, 1649-003 Lisbon, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 July 2012
Revised 6 September 2012
Accepted 10 September 2012
Available online 18 September 2012
Six novel urea triazene prodrugs have been synthesized to apply in antibody-directed enzyme prodrug
therapy (ADEPT). The chemical and plasmatic stability of L-glutamate triazene prodrugs were evaluated
and the chemical reactivity was mainly attributed to an intramolecular catalysis promoted by the neigh-
bouring carboxylate group of the glutamic moiety. These prodrugs showed an elevated binding to plasma
proteins. The L-glutamate triazenes were evaluated as prodrugs of the alkylating agent’s monomethyl-
triazenes, by activation of the bacterial enzyme carboxypeptidase G2 (CPG2). The synthesized prodrugs
have been shown to be good substrates for CPG2, and therefore new candidates for ADEPT strategy.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Triazenes
ADEPT
Carboxypeptidase G2
Intramolecular catalysis
Systemic cancer therapy is limited by a lack of tumor selectivity,
which leads to harmful side effects in normal tissues, and by drug
resistance. Antibody-directed enzyme prodrug therapy (ADEPT) is
designed to overcome both problems.1–4 ADEPT is a two step ap-
proach for the treatment of cancer which seeks to generate a po-
tent cytotoxic agent selectively at the tumor site or its
metastases. In the first step, a tumor selective antibody chemically
linked to an enzyme (antibody–enzyme conjugate) is administered
and allowed to fix itself on the tumor site. In the second step after a
suitable time, to allow the clearance of the antibody–enzyme con-
jugate from other tissues, the process is followed by the adminis-
tration of a relatively nontoxic prodrug. This prodrug will be
enzymatically converted into the active drug only at the tumor site
and the usually nonspecific cytotoxicity will be minimized. The ac-
tive drug is generated extracellularly with a higher concentration
and diffuses itself throughout the tumor mass, killing not only cells
expressing tumor antigen but also neighboring antigen-negative
tumor cells. Thus selectivity is achieved by the tumor specificity
of the antibody and by delaying prodrug administration until there
is a large differential between tumor and normal tissue enzyme
levels. Drug resistance can be overcome by generating high levels
of an alkylating agent in the tumor, and this is achieved through
the capacity of each enzyme to convert many molecules of prodrug
into drug. Such a complex system has already been applied in clin-
ical trials and in the last few years, a number of new monoclonal
antibodies-based drugs have been clinically approved (Rituxan,
Herceptin, Erbitux, Avastin and Mylotarg), and numerous other
mAb-based agents have also progressed to phase II/phase III clini-
cal trials in cancer.5,6
CPG2, a metalloenzyme derived from Pseudomonas sp., was the
elected enzyme for the first pilot-scale clinical trial of ADEPT. This
enzyme has no mammalian homologue and activates glutamic acid
prodrug derivatives of several nitrogen mustards alkylating
agents.7–12 A bond cleavable by CPG2 is essential and was con-
structed between the active drug and the glutamic acid moiety in
the prodrug. Prodrugs with linkages –OCO– (oxycarbonyl) for the
phenol nitrogen mustards and –NHCO– (carbamoyl) for the aniline
nitrogen mustards have been shown to be substrates for CPG2.7
Triazene compounds of clinical interest, dacarbazine 1 and tem-
ozolomide 2, are a group of alkylating agents with similar proper-
ties. Their mechanism of action is mainly related to methylation of
DNA, mediated by methyldiazonium ion, a highly reactive metab-
olite of the two compounds.13
H2N
O
N
H
N
Me
N
N
N
N
N
N
Me
N
N
H2N
1
Me
O
O
2
Of all the chemotherapeutic agents tested against this malignancy,
dacarbazine (DTIC), 1, remains the reference drug in the treatment
⇑
Corresponding author. Tel.: +351 21 7946400; fax: +351 21 7946470.
E-mail address: ermendes@ff.ul.pt (E. Mendes).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.029

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V. Capucha et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6903–6908
Ar
Me
Me
N
Me
N
Ar
P450
- HCHO
Ar
Ar
Me
N
N
N
N
N
N
N
H
N
Me
CH OH
2
3
DNA
+
Me
Ar
DNA
Me
N
N
H2N
N2
+
+
N
N
H
MMT (4)
Scheme 1. Metabolic pathway for dimethyltriazenes: activation and mechanism of action.
of metastatic melanoma.14,15 More recently a new compound has
been approved by FDA, the vemurafenib which is an orally inhibitor
of mutated BRAF, for the treatment of latest stages of melanoma.16
Related 1-aryl-3,3-dimethyltriazenes, 3 (Scheme 1), are also
documented as anticancer drugs, but with reduced selectivity for
tumour cells. The biological action of DTIC and the dimethyltriaz-
enes 3, is a consequence of their capacity to alkylate DNA and
RNA via the corresponding monomethyltriazene 4 following meta-
bolic oxidation by cytochrome P450 enzymes (Scheme 1).17–20
Thus, the treatment of melanoma can be improved by developing
of a prodrug strategy that can specifically deliver the ultimate
methylating agent to malignant cells. Since monomethyltriazenes
are good leaving groups as alcohols, we considered that coupling
them to carboxypeptidase substrates would identify suitable pro-
drugs 5 capable of regenerating the alkylating metabolite 4 via
the pathway depicted in Scheme 2.21,22 Additionally, triazenes as
small molecules with adequate lipophilicity will be free to diffuse
throughout the tumor.13 We report here the synthesis, the chemi-
cal stability studies and the evaluation of triazene prodrugs, de-
rived from glutamic acid 5, as potential molecules to be
converted into the cytotoxic drug MMT by CPG2 activation.
Attempts to synthesize the desired compounds 5a–f proceeds
via the carbamate derivatives 6a–f, these having been previously
COOH
O
X
N
HOOC
N
H
N
N
CH3
5
CPG2
COOH
X
+
H
N
CO2
+
N
N
HOOC
NH
H
CH3
MMT
Scheme 2. CPG2 mediated conversion of triazene prodrugs into the alkylating
agent MMT and
L
-glutamic acid.
H
N
N
N
O
N
N
N
(i)
O
X
NO2
X
4
6
(ii)
O
O
H
N
N
N
H
N
(iii)
N
N
N
N
O
OH
O
O
X
O
O
X
O
OH
5
7
5a X = CN
5b X = Br
5c X = CH3
5d X = COCH3
5e X = CO2Et
5f X = CONH2
Scheme 3. Synthesis of triazene prodrugs 5. (i) 4-Nitrophenyl chloroformate, pyridine, CH2Cl2; (ii) L-glutamic acid di-tert-butyl ester hydrochloride, triethylamine, THF; (iii)
TFA, anisole, CH2Cl2.

V. Capucha et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6903–6908
6905
Figure 1. Time course for the hydrolysis of compound 5c in PBS, pH 7.4, 37 °C,
278 nm: N, 5c; j, MMT 4; ꢀ, corresponding aniline.
Figure 2. Hammet plot of the logkobs for the decomposition of glutamic acid-based
triazenes 5 against rp values for the triazene substituents.
pharmacophore. A comparison between the half-lives for several
mustard urea derivatives (Fig. 3) of glutamic acid (9), self-immolative
derivatives (10), N-mustard–quinoline conjugates (11), N-mustard–
tiramine conjugates (12) and urea derivatives of triazenes
conjugated with tiramine and dopamine (13), shows that some of
these derivatives revealed an abnormally high chemical instability
(9a–c with t1/2 <17 min and 10a,b,d,e with t1/2 <4 min), in PBS, a
common feature with our 5d and 5f derivatives (with t1/2 2.6 and
6.4 min, respectively).8–10,22,24,25
This fact raised the hypothesis of a mechanistic change in the
chemical hydrolysis in physiological conditions, namely the occur-
rence of an intramolecular chemical catalysis (Scheme 4).8,9 During
this process there occurs a privileged initial cyclization, due to the
close proximity of the carboxylate group. The observed rate is the
result of two partitioning constants: the hydrolysis rate constant
associated with nucleophilic attack by the solvent (Scheme 4b),
and the neighboring-group promoted component (Scheme 4a).26
To recognize the occurrence of an intramolecular process, we syn-
thesized a model structure that is structurally related to the 5
derivatives, though incapable of reacting by the assisted route.
When masking the carboxylate functions of these derivatives by
esterification, chemically stable compounds are obtained (8) (Sup-
plementary data). The dimethylester derivative 8 presents a half-
life value in PBS at 37 °C of 101.9 h, 34 times more stable than
5e. This is evidence of the participation of the free COOH groups
of prodrugs 5e in the carbonyl displacement during the hydrolysis
reaction that liberates the MMT. Intramolecular reactions are faster
than the corresponding bimolecular processes, because of the close
proximity of the reacting groups. So, this compound having a glu-
tamic acid moiety show an extraordinary reactivity under condi-
tions where ordinary ureas are very stable. Again, a comparison
efficiently synthesized by reaction of the corresponding monom-
ethyltriazenes (4) with 4-nitrophenyl chloroformate, according to
the published procedure (Scheme 3).22 The reaction of pure carba-
mates with L-glutamic acid di-tert-butyl ester in the presence of
triethylamine produced very good yields of 7a–f compounds
(77–100%). Finally, the deprotection of 7 to the potential prodrugs
5 was achieved with trifluoracetic acid (TFA) and anisole at À10 °C.
The presence of anisole appears to be important for the latter reac-
tion, since it acts like a scavenger for cations. Moreover, the pres-
ence of anisole prevents side reactions.23 Derivatives 5–7 were
prepared in good yields and with correct spectral data described
in Supplementary data.
The chemical hydrolysis of triazene derivatives 5a–f, to the cor-
responding 1-aryl-3-methyltriazenes, 4 was performed in isotonic
phosphate buffer at 37 °C. The reactions of the 5a–c and 5e com-
pounds were followed by the monitoring of both the loss of the
starting materials and the formation of products (MMT and amine)
by HPLC (Fig. 1) as detailed in Supplementary data. Prodrugs 5d
and 5f proved to be too unstable to be studied by HPLC so their
hydrolysis reactions were followed by UV. The kinetics of the
hydrolysis of each prodrug followed pseudo-first-order kinetics.
Half-lives of the hydrolysis in PBS are shown in Table 1. The
analysis of half-lives for prodrugs 5 reveals that their reactivity
is nearly not influenced by the nature of the substituent
ÀX (
q
= 0.65, r2 = 0.99) being the faster hydrolyzed compounds
those containing electron-withdrawing substituents (Fig. 2).
Compounds 5d and 5f were outliers for this equation being the
two most reactive compounds. The presence of outliers implies
that other factors or specific mechanisms are involved in their
chemical hydrolysis.
Generally urea derivatives prove to be highly stable in aqueous
solutions in vitro. Additionally, several works highlight the poten-
tial of increasing the stability and half-lives of N-mustards pro-
drugs by introducing urea rather than carbamate linkages. A urea
linker is able to lower the reactivity of the reactive N-mustard
of the t value for the compound 11 (16.5 h) with that of the com-
½
pound 9a (0.28 h), or of the compound 13d (364 h) with 5c (7.63 h)
reveals that urea derivatives with no carboxylic free groups are
respectively 59 and 48 times more stable.7,21,22
Me
Table 1
O
O
COOEt
Triazene prodrugs 5 half-lives, t1/2, in pH 7.4 isotonic phosphate buffer at 37 °C and
O
their ability to act as CPG2 substrates (0.01 U/mL)
O
N
kobs Â10À3 (minÀ1
)
t (min)
½
N
H
N
N
Compound
X
pH 7.4 Buffer
pH 7.4 Buffer
CPG2a
O
CH3
Me
5a
5b
5c
5d
5e
5f
CN
Br
CH3
COCH3
CO2Et
CONH2
5.34
3.16
1.52
272
0.45
108
130.2
219.6
457.8
2.6
173.4
6.4
3.5
n.d.
n.d.
n.d
5.7
n.d.
8
Another feature to consider is the presence of an abundant ion mass
fragment m/z 84, for all the prodrugs 5a–f in mass spectra (Fig. 4).
a
n.d. = non-determined.

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V. Capucha et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6903–6908
R
X
Cl
Cl
O
N
Cl
Cl
N
N
N
O
R
X
H
N
H
N
H
N
HN
Me
HN
CO2H
CO2H
HN
CO2H
CO2H
O
N
O
O
11
10
9
X
a orto, para: R=H, X=I
b orto, para: R=H, X=Br
c orto: R=H, X=Cl
a: R=H;
b: R=3-F;
c: R=3-Cl;
d: R=3-CN
N
N
d para: R=Me, X=I
e para: R=Me, X=Br
fpara: R=Me, X=Cl
Me
HO
N
R
N
O
Cl
H
13
N
a: R=H, X=Ac
Cl
b: R=H, X=CO2Et
c: R=H, X=CN
d: R=H, X=Me
e: R=OH, X=Ac
H
O
H
N
N
H
O
f: R=OH, X=CO2Et
g: R=OH, X=Me
Cl
12
N
Cl
H
N
HOOC
HOOC
O
14
Figure 3. Chemical structures of N-mustard derivatives (9–12, 14) and of urea triazene derivatives (13).
COOH
O
O
(a)
H
N+
X
O
N
N
H
N
N
m/z 84
–
O
CH3
Figure 4. Chemical structure of ion mass fragment m/z 84 resulting from the
cyclization of the amino acid carrier.
Blood serum and plasma contain a range of enzymes that could
potentially catalyze the hydrolysis of urea derivatives. Therefore it
is possible for the prodrugs to be prematurely hydrolyzed by the
plasma enzymes leading to the release of triazenes before reaching
the pre-localized CPG2. Consequently, we were interested in exam-
ining the stability of prodrugs 5 in human plasma. Experimental
procedure is detailed in Supplementary data.
(b)
O
COOH
X
O
N
N
H
N
N
–
O
CH3
During the assays we realize that these derivatives strongly
bind to plasma proteins, preventing the plasma stability studies.
As the reaction proceeds, the formation of both monomethyltriaz-
enes and aniline is observed, fact that we justify based on the equi-
librium character of the binding. However, due to the binding of
prodrugs to plasma proteins its quantitative measurement in ser-
um proved difficult and complete mass balance was not possible.
We tested the ability of increasing concentrations of plasma (or
albumin) to deplete the triazene prodrug from the PBS medium.
During sample centrifugation the prodrug precipitates with the
denatured plasma proteins. When analyzed by HPLC the protein-
free supernatant was depleted of prodrug. Above 20% plasma
O
H
H
Scheme 4. Intramolecular chemical catalysis for activation of glutamic acid-based
triazene prodrugs (a) and hydrolysis in physiologic conditions (b).
This mass fragment results from the cyclization of the amino acid
carrier, so it points out some easiness for intramolecular reactions
involving the carboxylate functions of the glutamic acid moiety.
These cyclic fragments are absent from the mass spectra of the com-
pounds 7 and 8 which have the COOH group protected with an ester
group.

V. Capucha et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6903–6908
6907
Table 2
Enzyme kinetic properties of the prodrugs against CPG2
Compound
Km
(lM)
Vmax
(
lM/min)
kcat (s-1
)
kcat/Km (sÀ1 MÀ1
l )
5a
5e
17.70 0.23
2.73 0.79
4.94 0.35
1.18 0.10
4.64
1.11
0.26
0.41
the reaction very rapidly. These results show that our proposed
compounds 5 are potential good prodrugs for ADEPT strategy
based on CPG2 activation.
In conclusion, the structure and the type of linkage present be-
tween the drug and the glutamic acid seems to have important
implications for the design of tumor-targeted drugs to be activated
by CPG2. Prodrugs should be substrates of CPG2, but simulta-
neously should not be prematurely hydrolyzed by intramolecular
catalysis, which results in highly unstable compounds, more than
their free drug counterpart. It is clear that catalytic efficiency de-
pends crucially on the structure of the compounds involved. The
intramolecular catalysis should be an additional parameter to be
considered in the rationalization of prodrugs containing glutamic
acid, to be activated by CPG2. The bioavailability of these prodrugs
is conditioned by their high plasma protein binding. Nevertheless,
not only this can play a protective role regarding the premature
chemical hydrolysis shown by some of these prodrugs as also
can potentially afford a albumin based drug delivery system for tu-
Figure 5. The albumin binding ability was evaluated by comparison of the
remaining triazene prodrug 5b amounts (supernatant) front to increased concen-
trations of albumin.
concentrations, or above 10À4 M human albumin concentration in
PBS (Fig. 5), no triazene prodrug remained in the supernatant.
Similar results were obtained by Antoniw et al. who studied the
disposition of 4-bis (2-chloroethyl)amino)benzoyl-L-glutamic acid
(14) and its active parent drug in mice. The protein binding was
measured in both mouse and human plasma. The prodrug and
the active drug were significantly bound to plasma proteins, with
prodrug 92% bound in human plasma.27
mor targeting. L-Glutamate triazene prodrugs show a good struc-
Present in abundant amounts in blood (0.63 mM), human ser-
um albumin (HSA) is the major transport protein and has the abil-
ity to bind reversibly to many endogenous and exogenous
compounds including drugs and xenobiotics. Weakly acidic drugs,
negatively charged, tend to bind to albumin. Therefore, the
remarkable binding property of HSA has a significant effect on a
number of pharmacokinetic parameters. Often more than 90% of
the drugs are bound to the protein, which significantly influences
the drug efficacy. The studies on protein drug interactions may
provide information on the structural features that determine the
therapeutic effectiveness of drugs.28 Additionally, HSA can serve
as a natural and therefore biocompatible and biodegradable car-
rier, as it is described for cytotoxic conjugates with HSA that spe-
cifically target angiogenic endothelial cells.29–31
Following these studies we evaluated the triazene derivatives as
a substrate for CPG2, the elected enzyme for this strategy. The
kinetics of activation of prodrugs 5a and 5e by hydrolysis of the
glutamic acid moiety after addition of CPG2 was measured by UV
analysis, determining the half-lives for each compound in the pres-
ence of the enzyme (Table 1). The UV analysis of reaction mixtures
showed that both the compounds are rapidly hydrolysed with half-
lives of 4 and 6 min, thus indicating that they are good substrates
for CPG2. The absorbencies of the prodrug and corresponding drug
were scanned from 200 to 400 nm and the wavelength on the max-
imal difference of absorbance (due to the cleavage of the urea link-
age) between prodrug and drug was determined and used in the
kinetic studies. Experimental protocol is described in Supplemen-
tary data. The Km and Vmax with CPG2 were determined by measur-
ing the initial rate of conversion of prodrug into drug at
the wavelengths previously selected, using a range of prodrug
tural fit for the CPG2 active site and the data compare favorably
to related systems that have been reported and enable testing of
these prodrugs against cancer cell lines.
Acknowledgments
The authors gratefully acknowledge the Fundação para a Ciên-
cia e Tecnologia (Portugal) for financial support through funding
to research unit iMed.UL.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.09.
029.
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