Synthesis and evaluation of N-acylamino acids derivatives of triazenes. Activation by tyrosinase in human melanoma cell lines

Article abstract of DOI:10.1016/j.ejmech.2013.09.040

In this research work we report the synthesis of a new series of triazene prodrugs designed for Melanocyte-Directed Enzyme Prodrug Therapy (MDEPT). These compounds are derived from the N-acyltyrosine amino acid - a good enzyme substrate for the tyrosinase enzyme, which is significantly overexpressed in melanoma cells. We analysed their chemical stability and plasma enzymatic hydrolysis, and we also evaluated the release of the antitumoral drug in the presence of the tyrosinase. Subsequently, we performed the evaluation of the prodrug cytotoxicity in melanoma cell lines with different levels of tyrosinase activity. Prodrug 5c showed the highest cytotoxicity against melanoma cell lines, and this effect correlated well with the tyrosinase activity suggesting that prodrug cytotoxicity is tyrosinase-dependent.

Full text of DOI:10.1016/j.ejmech.2013.09.040

European Journal of Medicinal Chemistry 70 (2013) 1e9
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of N-acylamino acids derivatives of triazenes.
Activation by tyrosinase in human melanoma cell lines
,
Ana Sofia Monteiro a, Joana Almeida a, Guadalupe Cabral b c, Paulo Severino b,
Paula A. Videira b, Ana Sousa a, Rafael Nunes a, João D. Pereira a, Ana Paula Francisco a,
,
M. Jesus Perry a, Eduarda Mendes a
*
a Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculdade de Farmácia, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003
Lisboa, Portugal
b CEDOC, Immunology Department, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Campo Mártires da Pátria 130, 1169-056 Lisboa, Portugal
c Universidade Lusófona de Humanidades e Tecnologias, ULHT Lisboa, Campo Grande 376, 1749-024 Lisboa, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
In this research work we report the synthesis of a new series of triazene prodrugs designed for
Melanocyte-Directed Enzyme Prodrug Therapy (MDEPT). These compounds are derived from the N-
acyltyrosine amino acid e a good enzyme substrate for the tyrosinase enzyme, which is significantly
overexpressed in melanoma cells. We analysed their chemical stability and plasma enzymatic hydrolysis,
and we also evaluated the release of the antitumoral drug in the presence of the tyrosinase. Subse-
quently, we performed the evaluation of the prodrug cytotoxicity in melanoma cell lines with different
levels of tyrosinase activity. Prodrug 5c showed the highest cytotoxicity against melanoma cell lines, and
this effect correlated well with the tyrosinase activity suggesting that prodrug cytotoxicity is tyrosinase-
dependent.
Received 17 April 2013
Received in revised form
17 September 2013
Accepted 20 September 2013
Available online 2 October 2013
Keywords:
Prodrugs
Triazenes
Tyrosinase
Melanoma cells
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
Disseminated melanoma is a highly metastatic malignancy
which is generally lethal. Systemic chemotherapy is often the only
recourse, but to date the results have been very disappointing and
the lack of selective cytotoxicity often leads to intolerable side ef-
fects. Triazenes are a well-known class of anticancer drugs used in
the treatment of melanoma, which have a N]NeN structure type
generally close to an aromatic ring. Dacarbazine (DTIC) (1) and
Temozolomide (2) are the only triazenes in current chemotherapy
[1,2]. The mechanism of their antitumour activity works via the
alkylation of the DNA, through in vivo generation of methyl-
diazonium cations. Both temozolomide and DTIC are prodrugs of
the active alkylating agent 5-(3-methyltriazen-1-yl)imidazole-4-
carboximide e MTIC (3). Unlike DTIC, which requires metabolic
activation by cytochrome P450, temozolomide spontaneously
converts to 3 under physiologic conditions [3e7].
2
1
3
* Corresponding author. Tel.: þ351 217946413; fax: þ351 217946470.
E-mail address: ermendes@ff.ul.pt (E. Mendes).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.09.040

2
A.S. Monteiro et al. / European Journal of Medicinal Chemistry 70 (2013) 1e9
Scheme 1. Synthesis of triazene prodrugs 5. (i) Tetrahydrofuran (THF), 1 h, rt; (ii) Triethylamine (TEA), NaH, THF, 48 h, rt.
Tyrosinase (EC 1.14.18.1), is an enzyme which abounds in mela-
noma cells and is considered an ideal molecular target for the
developmentof anti-melanoma drugs [8]. This enzyme catalyzes the
mushroom tyrosinase and cytotoxicity studies in human mela-
noma cell lines.
oxidation of
L-tyrosine into the corresponding o-quinone, dop-
2. Results and discussion
aquinone, which is the first step in melanin pigment biosynthesis.
The degree of malignancy of pigment cells was shown to correlate
with tyrosinase activity [9]. Melanocyte-Directed Enzyme Prodrug
Therapy (MDEPT) is an acronym coined fora prodrugstrategy whose
target is the treatment of melanoma: since tyrosinase is naturally
overexpressed in the tumour, and virtually absent from the other
cells, it provides an in-built drug delivery mechanism that will be
selective of melanoma tumours [10]. The efficacy of the MDEPT
strategy relies on the fact that tyrosinase activity is aberrantly high
in melanoma tumour cells. In this tyrosinase mediated drug delivery
system the release of the cytotoxic agent from prodrugs is a conse-
quence of the recognition and oxidation of the promoiety of pro-
drugs. Our group had previously investigated the structureeactivity
relationship of a series of tyramine and dopamine derivatives of
triazenes. We synthesized and evaluated a series of prodrugs to be
activated by tyrosinase [11]. The release of the monomethyltriazene
(MMT) was based on an intramolecular Michael addition reaction.
Unfortunately, we had to conclude that this process required a much
better nucleophile than the urea nitrogen atom [12].
2.1. Synthesis
The synthesis of aminoacylderivatives of triazenes 5aef was
based on the coupling of the N-acyltyrosine with the triazene. The
synthetic methodology implies the use of a coupling activator such
as Dicyclohexylcarbodiimide (DCC), due to the mild conditions
under which it can be used, and which are important for the tri-
azene stability. The first step is the addition of the carboxy group of
N-acyl or N-Boc-L-tyrosine to the carbodiimide functionality, to
produce rapidly an O-acylisourea which is a potent acylating agent.
In the subsequent step, the addition of the nucleophile triazene
leads to the final prodrugs N-acyltyrosine triazene derivatives 5aef,
and N-Boc- -tyrosine triazene derivatives 6a,b with the concomi-
L
tant formation of dicyclohexylurea (Scheme 1). This urea is only
partially soluble in most solvents but in our case it gave rise to
purification problems. Another drawback of this methodology is
that the reactions are not complete and sometimes, depending on
the triazene, the yields are low.
Following the same strategy, with the purpose of developing
more potent and better targeted compounds for melanoma, we
developed new triazene derivatives 5aef with an amide linker
between the cytotoxic unit, the triazene, and the carrier, N-acyl-
tyrosine. The use of amino acids as promoieties offers several
advantages: (a) a number of structurally diverse amino acids
(aliphatic, aromatic, acidic, basic, neutral, etc.) are commercially
available, (b) there are fewer safety concerns regarding the use of
amino acids as promoieties, (c) amino acid and peptide chemis-
tries are well established, and (d) amino acid prodrugs can
potentially target carrier-mediated transporters for their transport
across cell membranes [13]. The aim of our analysis is to find out
whether or not the triazene prodrugs synthesized 5aef show the
hoped for tyrosinase dependent cytotoxicity and specificity. To
achieve this goal we performed assays of activation of prodrugs by
To improve the coupling rates and yields we also applied micro-
waveassistedsynthesis(MAS), whichfunctionsasanalternativetothe
traditional amide coupling described in Scheme 1. The syntheses were
found to be achievable, since we were concerned with the stability of
the triazenes obtained by MAS. The results reveal a much more rapid
and cleaner reaction, and e to our knowledge e these results provide
the first demonstration of MAS applied to MMT reactivity (Scheme 2).
However, the final yields obtained were found to be of the same order
of magnitude as the ones obtained by the DCC methodology.
2.2. Kinetic study
2.2.1. Stability in phosphate buffer
The
a-(acyltyrosine)acyltriazenes 5aef hydrolysis in an isotonic
phosphate buffer generates the corresponding 3-methyltriazenes
Scheme 2. Synthesis of prodrugs 5 by MAS.

A.S. Monteiro et al. / European Journal of Medicinal Chemistry 70 (2013) 1e9
3
Table 1
Log P and half-lives of prodrugs, t , in PBS buffer, 80% human plasma and in the presence of tyrosinase (300 U/mL) at 37 ꢁC.
½
Compound
X
R
Log Pexp
t1/2
pH 7.4 buffer (h)
80% Human plasma (h)
Tyrosinase (min)
5a
5b
5c
5d
5e
5f
COMe
CO2Et
CN
Me
2.33 ꢂ 0.02
2.96 ꢂ 0.02
2.14 ꢂ 0.09
3.36 ꢂ 0.07
1.27 ꢂ 0.02
2.12 ꢂ 0.07
3.85 ꢂ 0.05
3.90 ꢂ 0.09
56.6 ꢂ 1.0
70.3 ꢂ 0.8
42.7 ꢂ 0.5
a)
19.8 ꢂ 1.2
18.0 ꢂ 1.2
15.6 ꢂ 1.8
8.9 ꢂ 0.10
17.4 ꢂ 0.42
18.3 ꢂ 0.56
b)
1.41 ꢂ 0.13
5.32 ꢂ 0.08
1.23 ꢂ 0.17
6.80 ꢂ 0.01
5.19 ꢂ 0.08
2.80 ꢂ 0,26
9.54 ꢂ 0.86
8.10 ꢂ 0.62
Me
Me
Br
Me
CO NH2
CO2Me
COMe
CN
Me
40.6 ꢂ 0.6
75.1 ꢂ 1.7
72.2 ꢂ 0.8
48.5 ꢂ 0.7
Me
6a
6b
OC(Me)3
OC(Me)3
b)
a) stable for 15 days.
b) unchanged over 1 h.
[Concentration of the substrate] ¼ 10ꢀ5 M.
All the experiments were performed in triplicate and the data given are the corresponding average values.
which get spontaneously decomposed into the corresponding an-
iline. These reactions were easily followed by HPLC, monitoring
both the loss of starting material and the formation of products.
Table 1 show that with a physiological pH these compounds get
decomposed with half-lives over 41 h emphasizing their high level of
chemical stability. A comparison between the half-lives of prodrugs
2.2.2. Stability in human plasma
Blood serum and plasma contain a range of enzymes that ca-
talyse the hydrolysis of amides [15]. Consequently, we were inter-
ested in examining the stability of our triazene derivatives since
they contain an amide link.
The results in Table 1 show that in human plasma all the com-
pounds are substrates for the plasma enzymes with half-lives
ranging from 1.23 to 6.8 h. N-acyltyrosine derivatives hydrolyse in
80% human plasma containing 20% of isotonic phosphate buffer to
the corresponding MMT, which further break down into the cor-
responding anilines (Fig. 2). The hydrolysis rates are 8e40 times
faster than with phosphate buffer but still quite stable in human
5aef and the half-lives of several a-(acylamino)acyltriazenes 8aeh
(Fig. 1) shows that these derivatives revealed a higher chemical sta-
bility [14]. In fact, in PBS, N-acyltyrosine derivatives are four times
more stable than their analogues derived from N-acylalanine 8aee or
the analogues derivedfromN-acylglycine8fandN-acylphenylalanine
8g. However, it seems that these derivatives are less stable than the
prodrugs containing tyramine 9a or dopamine 9b (Fig. 1) promoiety
coupled withthe cytotoxic triazene via a ureafunctionalgroup[11,14].
plasma. Analysis of t for compounds 5aef reveals that the rate of
½
reaction is influenced by the nature of the substituent eX in the
triazene aryl group, the more reactive compounds being those
containing electron-withdrawing substituents. There is a correla-
Another feature affecting the relative magnitude of the t values for
½
thecompounds5istheinfluenceof thesubstituent, ꢀX,presentinthe
aryl group. Inspection of the table enables us to assert that electron-
withdrawing groups increase the rate of hydrolysis (these com-
tion between log t values and the Hammett rp values yielding a
r
½
value of 1.9 (r2 ¼ 0.80; n ¼ 6). When we compared the half-life
pounds present a Hammett correlation with a
r
value of þ0.92;
r2 ¼ 0.95; n ¼ 4). The same was also previously observed in triazene
prodrugs with peptide and urea linkage [14].
Fig. 1. Chemical structures of
azene derivatives 9a,b [11].
a
-(acylamino)acyltriazenes 8aeh [14] and of urea tri-
Fig. 2. Time course for the hydrolysis of 5c in 80% in human plasma at 37 ꢁC:
corresponding aniline; , corresponding MMT.
, 5c;
,

4
A.S. Monteiro et al. / European Journal of Medicinal Chemistry 70 (2013) 1e9
values of our N-acyltyrosine derivatives 5aef in human plasma
with the correspondent values obtained for derivatives 8aeh we
observed that for the recognition and hydrolysis promoted by
plasma enzymes, the compounds behaved the same way, that is,
presented the same easiness of hydrolysis, so the type of N-acyla-
minoacid seems to have little effect in this medium.
Comparison with prodrugs 9 (tyramine and dopamine triazene
derivatives) show that compounds 5 are more susceptible to
plasma enzymes although prodrug present an adequate plasma
stability for MDEPT strategy.
The partition coefficients between octanol and pH 7.4 phosphate
buffer at 25 ꢁC for compounds 5 (Table 1) give values of log P
ranging from 1.27 to 3.36. Our prodrugs 5 proved to be more
lipophilic than their analogues 8. There is a correlation between
prodrug stability in human plasma (log t1/2) and lipophilicity (log P)
(Fig. 3) with the more lipophilic prodrugs being the more stable.
Substrate lipophilicity is one of the main features that influence
esterase activity e decreasing enzyme activity being associated
with increasing lipophilicity as we observed in our study [16].
Fig. 4. Time course for the hydrolysis of 5c in the presence of tyrosinase at 37 ꢁC:
5c; , corresponding aniline; , corresponding MMT.
,
phenolic group as an essential functional group in the chemical
structure of the prodrugs, we synthesized the phenylalanine de-
rivative 8g. This compound is not a substrate for tyrosinase since in
the presence of the enzyme the half-life remains the same as in PBS.
ꢀ
ꢁ
logt1=2 ¼ 4:19logP ꢀ 7:33 n ¼ 5; r2 ¼ 0:88
(1)
Presented in Table 2 are the kinetic parameters (Km, Vmax, kcat
,
2.2.3. Triazene derivatives as substrates for tyrosinase
and kcat/Vmax) as well as the van der Waals volume calculated for
compounds 5c, 5f and 6a. The evaluation of comparable kinetic
parameters is crucial to obtain a more thorough understanding of
the reactivity of different tyrosinase substrates. The Km values for
compounds 5c and 5f compare favourably with other phenolic
Triazene prodrugs 5aef were studied as mushroom tyrosinase
substrates by HPLC. Since purified human tyrosinase is not
commercially available, in this study we used a mushroom tyrosi-
nase enzyme in vitro assays. For each compound, the half-lives in
the presence of the enzyme, were determined (Table 1) and the
products of activation identified and analysed. Prodrugs are
oxidized by tyrosinase into the quinone intermediate. However,
after a 15 min reaction it is possible to quantify the cytotoxic
monomethyltriazene. Actually it is present along the total reaction
time but in a very small proportion (below five per cent) (Fig. 4). A
complete mass balance for the reaction is not possible since the
aromatic amines (the final product of the reaction) are as also
tyrosinase substrates and are concomitantly oxidized by the
enzyme [17].
The results show that the N-acyltyrosine triazene derivatives
5aef are excellent tyrosinase substrates with half-lives ranging
from 9 to 20 min (Table 1). However, other molecule functionalities
seem to play a role in the recognition by the enzyme. In fact,
compounds like 6a and 6b that contains the N-Boc group are not a
substrate for the enzyme. The catalysis of the reaction by tyrosinase
depends on the capacity of the substrate to bind to a tyrosinase
active site. If the substrate binds tightly to the active site, tyrosinase
will oxidize the substrate efficaciously. To investigate the role of a
substrates including
L-tyrosine, tyramine and dopamine
(Km ¼ 0.25 ꢂ 0.03 mM, 0.54 ꢂ 0.05 and 0.28 ꢂ 0.01 mM, respec-
tively) indicating that they have structures that can bind to the
active site in a very effective way, and consequently, should oxidize
very easily [18,19]. On the contrary, tyrosinase could not effica-
ciously oxidize compound 6a and 6b, probably due to unexpected
steric interactions between the N-Boc group and the tyrosinase
binding motif, which results in an unfavourable alignment and
position of 6a and 6b on the active site. From what is known about
the substrate specificity of tyrosinase, the access to the active site is
quite limited, and the presence of the bulky tert-butyl group in the
lateral chain of substituted phenols or in the lateral chain of steri-
cally hindered amino acids phenylhydrazides is limitative of any
oxidation reaction [20e22].
The Kcat/Km ratio e the catalytic efficiency e that determines the
rate of reaction at low substrate concentrations, is significantly
greater than those of tyramine or dopamine triazene prodrugs (1.9
and 8.5 respectively) suggesting a better affinity for these new
substrates [11]. Comparing with our previous triazene prodrugs,
derived from tyramine and dopamine, these new derivatives pre-
sent the great improvement of liberating the cytotoxic mono-
methyltriazene through the action of the enzyme. These
experimental results led us to propose a mechanism for the release
of MMT based on the intramolecular cyclization of the quinone
Table 2
Kinetic parameters for the oxidation of acyltyrosine derivatives by mushroom
tyrosinase.
Prodrug Km
(mM)
Vmax
mM/min
kcat
Kcat/Km van der
(M-1sꢀ1
Waals
(sꢀ1
)
)
3
ꢀ
volume (A )
5c
5f
6a
0.20 ꢂ 0.09 5.4 ꢃ 10ꢀ6 ꢂ 5.3 ꢃ 10ꢀ7 0.03 151.2
0.22 ꢂ 0.04 9.7 ꢃ 10ꢀ6 ꢂ 3.9 ꢃ 10ꢀ7 0.05 246.5
329
374
388
e
e
e
e
Values reported are the average ꢂ one standard deviation for the three independent
determinations. The van der Waals volumes were calculated according to the
literature [23].
Fig. 3. Plot of the plasma half-lives (t ) versus lipophilicity for compounds 5.
½

A.S. Monteiro et al. / European Journal of Medicinal Chemistry 70 (2013) 1e9
5
Scheme 3. Proposed mechanism for MMT release after tyrosinase oxidation.
intermediate and the formation of a hemiaminal/iminium ion,
which subsequently suffers a chemical hydrolysis producing the
MMT triazene and a cyclic hemiacetal (Scheme 3).
Cyclization by nucleophilic attack of the amide nitrogen of N-
acyltyrosine to the quinone intermediate is less probable as pre-
vious works have demonstrated. N-acyltyrosine is considered a
non-cyclizing phenolic substrate of mushroom tyrosinase since the
presence of the acetyl substituent in nitrogen inhibited cyclization
(Scheme 4) [24].
All the prodrugs tested led to a dose-dependence increase in the
number of apoptotic/necrotic MNT-1 cells, confirming their cyto-
toxic effect (Fig. 6). Compared to non treated cells, the percentage of
apoptotic/necrotic cells increased by 7.88 and 11.23% respectively in
the presence of 50 and 100
respectively in the presence of 50 and 100
finally 10.67 and 29.36% respectively in the presence of 50 and
100 M of prodrug 5c (Fig. 6A). The cytotoxicity of prodrug 5c was
next analysed in another tyrosinase rich cell line (SKMEL-30), and a
tyrosinase-absent (M8) cell line. Exposure to 50 and 100 M of
m
M of prodrug 5a, 17.46 and 21.23%
mM of prodrug 5b and
m
m
2.3. Determination of prodrug cytotoxicity in melanoma cell lines:
tyrosinase activity dependence
prodrug 5c led to a significant increase of 8.50 and 17.01% respec-
tively, in the percentage of MNT-1 apoptotic/necrotic cells and
10.41 and 12.47% respectively in the percentage of SKMEL-30
apoptotic/necrotic cells (Fig. 6B). On the contrary, M8 cells
To investigate the cytotoxic effect of the prodrugs, three
different human melanoma cell lines, MNT-1, SKMEL-30 and M8,
were used as in vitro models.
Both MNT-1 and SKMEL-30 cell lines displayed a relevant level of
tyrosinase activity, namely 5.30 and 4.13 mU/mg respectively, con-
trary to M8 cell line whose tyrosinase activity was undetected (Fig. 5)
and was therefore used to represent a tyrosinase-absent cell line.
The triazene prodrugs 5a, 5b and 5c were selected because of
their partition coefficients (Table 1), which provide an estimate of
their permeability across the membrane.
The working concentration range of the prodrugs was first
determined by both the colorimetric (3-(4,5-dimethylthiazol-2-yl)-
5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)
(MTS) assay, which detects metabolic active but not inactive cells,
and by microscopic observations with trypan blue dye exclusion
exposed to 50 and 100 mM of prodrug 5c showed no significant
alteration in the percentage of apoptotic/necrotic cells. Interest-
ingly the percentage of apoptotic/necrotic cells obtained after
prodrug 5c exposure showed a direct correlation (r ¼ 0.9045,
p ¼ 0.0132) with the tyrosinase activity presented by the cell lines.
Moreover, we compared the cytotoxicity/specificity of prodrug 5c
and Temozolomide,
a commercially available prodrug with
tyrosinase-independent toxicity used for the treatment of mela-
noma. Interestingly, M8 cell line appeared to be more sensitive to
Temozolomide than MNT-1 and SKMEL-30 cell lines e as illustrated
e with an increased susceptibility at the highest prodrug concen-
tration (Fig. 6C).
3. Conclusions
assay (data not shown). The concentration of 50 and 100 mM was
then selected to analyse further the percentage of apoptotic/necrotic
cells derived after 48 h exposure of each cell line to the prodrugs.
In conclusion, most of the derivatives tested in this study un-
derwent metabolic oxidation by tyrosinase with the
Scheme 4. Oxidation of N-acetyltyrosine by tyrosinase.

6
A.S. Monteiro et al. / European Journal of Medicinal Chemistry 70 (2013) 1e9
tyrosinase dependent. When compared with Temozolomide pro-
**
10
8
**
drug 5c showed an increased specificity for tyrosinase expressing
cells, suggesting its strong therapeutic potential for melanoma
treatment.
6
4. Experimental
4
4.1. General information
2
All the triazenes used in this study should be considered as
mutagenic and/or carcinogenic and appropriate care should be
taken in order to handle them safely.
Microwave heating was carried out with a single-mode cavity
Discover Microwave Synthesizer (CEM Corporation, NC) producing
continuous irradiation at 2455 MHz.
Melting points were determined using a Kofler camera Bock-
Monoscop “M” and were not corrected. IR spectra were recorded
as KBr discs using a Perkin Elmer 1310 spectrophotometer. 1H and
13CNMR spectra were recorded in CDCl3, MeOD or DMSO-d6 solu-
0
1
0
8
-
3
M
-
T
L
N
E
M
M
K
S
Fig. 5. Tyrosinase activity in melanoma cell lines. Tyrosinase activity was determined
in the MNT-1, SKMEL-30 and M8 cell lines by measuring the formation of dopachrome
as referred in the Experimental section. Data represent the averages ꢂ SD of three
different experiments each carried out in duplicate. The statistical significance
(*p < 0.05, **p < 0.01) refers to the differences compared with the M8 cell line in which
no relevant tyrosinase activity was detected (0.00 mU/mg).
tions using a Brucker AM 400 WB spectrometer. Chemical shifts, d,
are reported as p.p.m. from Me4Si, and coupling constants, J, in Hz.
Mass spectra were recorded in a Micromass Quattro Micro API
benchtop, Waters mass spectrometer. All chemicals were reagent
grade except those for kinetic studies and HPLC, which were of
analytical or LiChrosolvÒ (Merck) grade. 1-Aryl-3-methyltriazenes
were synthesized by previously published methods [25]. The (S)-
N-acetyltyrosine and (S)-N-Boc-tyrosine were purchased from
Sigma and Mushroom tyrosinase (5350 U/mg) was purchased from
SigmaeAldrich.
correspondent liberation of triazene, the antitumoral agent. These
compounds prove to be extremely stable in a pH 7.4 buffer and in
human plasma and simultaneously are good substrates for tyros-
inase. The cytotoxicity assays also show that the all-newly-syn-
thesized prodrugs have a cytotoxic effect being the prodrug 5c the
highest cytotoxic. The correlation between this cytotoxicity and
the tyrosinase activity suggests that the prodrug effect is
A
B
*
*
*
***
**
*
**
*
*
*
***
*
40
30
20
10
0
60
0
0
50
40
100
50
100
20
0
5c
5a
5b
30
EL-
M8
NT-1
M
C
SKM
30
20
10
0
0
50
100
0
-1
MNT
3
M8
-
EL
SKM
Fig. 6. Drug cytotoxicity on melanoma cell lines. The percentage of apoptotic/necrotic cells was estimated by flow cytometry after 48 h exposure to 0, 50 or 100
m
M of the prodrugs.
Cells were double-stained with APC-labelled annexin V and 7-AAD as mentioned in the Experimental section. A: Effect of prodrugs 5a, 5b, 5c on MNT-1 cell line. B: Effect of prodrug
5c on MNT-1, SKMEL-30 and M8 melanoma cell lines. C: Effect of Temozolomide on MNT-1, SKMEL-30 and M8 melanoma cell lines. The values represent the averages of at least 3
independent assays. The degree of significant difference is represented by *p < 0.05; **p < 0.01 and ***p < 0.005.

A.S. Monteiro et al. / European Journal of Medicinal Chemistry 70 (2013) 1e9
7
4.2. Synthesis
1.84 (s, 3H, COCH3), 2.84 (HA, dd, J ¼ 8.4 and J ¼ 13.6 Hz, CH2), 2.92
(HB, dd, J ¼ 6.4 and J ¼ 13.6 Hz, CH2), 3.40 (s, 3H, NCH3), 4.35 (q,
J ¼ 8.0 Hz, 2H, CH3CH2), 5.56 (dd, J ¼ 7.2 and J ¼ 7.6 Hz, 1H,
ArCH2CH), 6.56e6.97 (dd, J ¼ 8.4 Hz, 4H, HOAr), 7.64e8.09 (dd,
J ¼ 8.4 Hz, 4H, Ar), 8.53 (d, J ¼ 7.6 Hz, 1H, NH), 9.18 (s, 1H, OH).
4.2.1. General procedures for the synthesis of prodrugs 5aef and 6a,
b
N,N0-Dicyclohexylcarbodiimide(2.4mmol)wasaddedtoasolution
of the N-acyltyrosine (1.9 mmol)intetrahydrofuran (THF,10 mL). After
60minatroom temperatureunderstirring, triethylamine (0.19 mmol)
and the appropriate 1-aryl-3-methyltriazene (1.9 mmol) previously
activated with NaH (1.9 mmol) inTHF (5 mL) were added. After 48 h at
room temperature, the N,N0-dicyclohexylurea by-product was
removed by filtration and the solvent removed under reduced pres-
sure. The crude residue was subjected to chromatography (silica gel
60, 70e230 mesh ASTM, Merck) using one of the following systems:
diethyl ether: methanol (for 5a), dichloromethane: methanol (for 5b,
c, 6a, b), ethyl acetate: methanol (for 5d, 5e), ethyl acetate: dichloro-
methane (for 5f) (see Supporting information, Table S1). The organic
layerscorrespondentstothepurecompoundswerefiltered afterbeing
dried on MgSO4 before evaporation under vacuum. The isolated N-
(acyltyrosine)acyltriazenes were subsequently recrystallized from
ethyl acetate/n-hexane (for compounds 5a, 5b, 5e, 6a, 6b, 17:3) and
dichloromethane/n-hexane (5c, 5d, 5f, 17:3). For the synthesis of
13CNMR (100 MHz, DMSO-d6):
d ppm 14.6 (CH3CH2), 22.70 (COCH3),
28.3 (NCH3), 37.0 (CH2Ar), 52.7 (CHCH2Ar), 61.4 (CH3CH2O), 115.4,
122.5, 127.5, 130.3, 130.3, 130.9, 151.9, 156.4 (CAr), 165.6
(CH3CH2OCO), 169.8 (COCH3), 175.0 (N]NeNeCO). EI: m/z 413.4
(M þ H)þ, 177.3 (CH3CH2OCOC6H4N2þ), 149.1 (CH3CH2OCOC6Hþ4 ).
4.2.5. 3-[2-(Acetylamino)-3-(4-hydroxyphenyl)propanoyl]-1-(4-
cyanophenyl)-3-methyltriazene (5c)
Yield 38%, m.p.196e198 ꢁC. IR (cmꢀ1): 3327, 2226,1702,1634. 1H
NMR (400 MHz, CDCl3):
d ppm 1.83 (s, 3H, COCH3), 2.84 (HA, dd,
J ¼ 8.0 and J ¼ 13.6 Hz, CH2), 2.91 (HB, dd, J ¼ 7.2 and J ¼ 13.6 Hz,
CH2), 3.30 (s, 3H, NCH3), 5.56 (dd, J ¼ 7.2 and J ¼ 7.6 Hz, 1H,
ArCH2CH), 6.54e6.95 (dd, J ¼ 8.0 Hz, 4H, HOAr), 7.67e8.00 (dd,
J ¼ 8.0 Hz, 4H, Ar), 8.53 (d, J ¼ 7.2 Hz, 1H, NH), 9.16 (s, 1H, OH).
13CNMR (100 MHz, CDCl3):
d ppm 22.6 (CH3CO), 28.5 (NCH3), 37.0
(CH2Ar), 52.6 (CHCH2Ar), 11.6 (CN), 115.4, 119.0, 123.5, 127.4, 130.3,
134.2, 151.6, 156.4 (CAr), 169.8 (CO), 175.1 (CO). EI: m/z 365.0 (Mþ),
206 (AcNHCH(CH2Ph)COþ), 178 (AcNHCHþ(CH2Ph)), 130
(CNC6H4Nþ2 ), 107 (C6H5CHþ2 ), 102 (CNC6Hþ4 ).
compounds 6 we used N-Boc-L-tyrosine as the starting material.
4.2.2. General procedure for the synthesis of
acyltriazenes by microwave synthesis
a-(acyltyrosine)
A 10 mL reaction vessel containing a small magnetic stir bar was
load in N,N,N0,N0-Tetramethyl-O-(benzotriazol-1-yl)uronium tet-
rafluoroborate (TBTU, 1 mmol), N-acyltyrosine (1 mmol), Dime-
thylaminopyridine (DMAP, 1 mmol) and the appropriate 1-aryl-3-
methyltriazene (1 mmol). The reagent mixture was dissolved with
2 mL of dimethylformamide (DMF) and the reaction was carried in
the presence of triethylamine (0.1 mmol). The tube containing the
reaction mixture was sealed with an ActiVent cap and then it was
exposed to microwave irradiation (200 W) for 30 min at a temper-
ature of 55 ꢁC. The buildup of pressure in the closed reaction vessel
was carefully monitored. After the irradiation, the reaction tube was
cooled with high pressure air through an inbuilt system inside in the
instrument until the temperature had fallen below 25 ꢁC (ca. 2 min).
The reaction mixture was diluted with ethylacetate and the mixture
was washed by 5% citric acid solution (10 mL), 5% NaHCO3 (10 mL)
and saturated NaCl solution (10 mL). The organic layer was dried
over anhydrous magnesium sulphate and the solvent removed un-
der reduced pressure. The residue was purified by column chro-
matography (silica gel 60, 70e230 mesh ASTM, Merck). The isolated
N-(acyltyrosine)acyltriazenes were subsequently recrystallized
from ethyl acetate/hexane or dichloromethane/hexane.
4.2.6. 3-[2-(Acetylamino)-3-(4-hydroxyphenyl)propanoyl]-1-(4-
bromophenyl)-3-methyltriazene (5d)
Yield 15%, m.p. 218e221 ꢁC. IR (cmꢀ1): 3327, 3215, 1709, 1651. 1H
NMR (400 MHz, DMSO-d6):
d ppm 1.87 (s, 3H, COCH3), 2.82e2.90
(2dd, J ¼ 8.0 Hz, 2H, CH2Ar), 3.27 (s, 3H, NCH3), 5.67 (dd, J ¼ 6.8 Hz,
1H, ArCH2CH), 6.53e6.87 (dd, J ¼ 8.0 Hz, 4H, HOAr), 7.39e7.51 (dd,
J ¼ 8.0 Hz, 4H, Ar), 8.02 (d, J ¼ 8 Hz, 1H, NHCOCH3), 8.81 (s, 1H,
ArOH); 13CNMR (100 MHz, DMSO-d6):
d ppm 22.8 (COCH3), 27.9
(NCH3), 37.8 (CH2Ar), 52.2 (CHCH2Ar), 115.3, 123.9, 130.0, 132.2
(CAr), 147.4 (BreCAr), 156.2 (ArCeOH), 170.0 (NCOCH3), 174.5 (N]
NeNeCO); EI: m/z 419.3/421.3 (Mþ); 185.0/183.0 (BrC6H4N2þ);
157.0/155.0 (BrC6Hþ4 ).
4.2.7. 3-[2-(Acetylamino)-3-(4-hydroxyphenyl)propanoyl]-1-(4-
aminocarbonylphenyl)-3-methyltriazene (5e)
Yield 38%, m.p.216e217 ꢁC. IR (cmꢀ1): 3334, 3301, 3251, 1738,
1698, 1657. 1H NMR (400 MHz, DMSO-d6):
d ppm 1.83 (s, 3H,
COCH3), 2.82 (HA, dd, J ¼ 8.4 and J ¼ 13.6 Hz, CH2), 2.93 (HB, dd,
J ¼ 8.4 and J ¼ 13.6 Hz, CH2), 3.30 (s, 3H, NCH3), 5.57 (dd, J ¼ 7.6 and
J ¼ 8.0 Hz, 1H, ArCH2CH), 6.57e6.98 (dd, J ¼ 8.4 Hz, 4H, HOAr), 7.50
(s, 1H, NH2), 7.60e8.01 (dd, J ¼ 8.4 Hz, 4H, Ar), 8.10 (s, 1H, NH2), 8.52
(d, J ¼ 7.6 Hz, 1H, NH), 9.20 (s, 1H, OH). 13CNMR (100 MHz, DMSO-
4.2.3. 3-[2-(Acetylamino)-3-(4-hydroxyphenyl)propanoyl]-1-(4-
acetylphenyl)-3-methyltriazene (5a)
d6): d ppm 22.1 (COCH3), 27.6 (NCH3), 36.3 (CH2Ar), 52.1 (CHCH2Ar),
114.8, 121.4, 1276.7, 128.6, 129.6, 134.3, 149.9, 156.1 (CAr), 167.0
(NH2CO), 169.2 (COCH3), 174.4 (N]NeNeCO). EI: m/z 384.3
(M þ H)þ, 148.1 (NH2COC6H4N2þ), 120.1 (NH2COC6Hþ4 ).
Yield 38%, m.p. 199e201 ꢁC. IR (cmꢀ1): 3318, 1709, 1683, 1641. 1H
NMR (400 MHz, CDCl3):
d ppm 1.83 (s, 3H, NHCOCH3), 2.64 (s, 3H,
COCH3), 2.84 (HA, dd, J ¼ 8.4 and J ¼ 13.6 Hz, CH2), 2.93 (HB, dd, J ¼ 6.4
and J ¼ 13.6 Hz, CH2), 3.30 (s, 3H, NCH3), 5.58 (dd, J ¼ 7.6 Hz, 1H,
ArCH2CH), 6.57e6.98 (dd, J ¼ 8.4 Hz, 4H, HOAr), 7.65e8.10 (dd,
J ¼ 8.4 Hz, 4H, Ar), 8.52 (d, J ¼ 7.6 Hz,1H, NH), 9.18 (s,1H, OH).13CNMR
4.2.8. 3-[2-(Acetylamino)-3-(4-hydroxyphenyl)propanoyl]-1-(4-
methoxycarbonylphenyl)-3-methyltriazene (5f)
Yield 24%, m.p. 195e197 ꢁC. IR (cmꢀ1): 3381, 3296, 1728, 1692,
(100 MHz, CDCl3):
d
ppm 22.7 (COCH3), 27.3 (COCH3), 28.3 (NCH3),
1655. 1H NMR (400 MHz, DMSO-d6):
d ppm 2.05 (s, 3H, COCH3), 3.01
37.0 (CH2Ar), 52.6 (CHCH2Ar), 115.4, 122.5, 127.5, 130.0, 130.3, 137.1,
151.7, 156.4 (CAr), 169.8 (COCH3), 175.0 (N]NeNeCO), 197.7
(COCH3). EI: m/z 382.0 (Mþ), 133.0 (CH3COC6H4Nþ2 ), 119.1
(CH3COC6Hþ4 ) (see Supporting Information).
(HA, dd, J ¼ 6.8 and J ¼ 13.8 Hz, CH2), 3.12 (HB, dd, J ¼ 6.4 and
J ¼ 13.6 Hz, CH2), 3.44 (s, 3H, NCH3), 5.98 (q, J ¼ 6.8 Hz, 1H,
ArCH2CH), 6.36 (d, J ¼ 8.4 Hz, 1H, NH), 6.63e6.95 (dd, J ¼ 8.4 Hz, 4H,
HOAr), 7.64e8.13 (dd, J ¼ 8.4 Hz, 4H, Ar), 13CNMR (100 MHz, DMSO-
d6): d ppm 23.7 (COCH3), 28.4 (NCH3), 38.7 (CH2Ar), 52.5 (CHCH2Ar),
4.2.4. 3-[2-(Acetylamino)-3-(4-hydroxyphenyl)propanoyl]-1-(4-
ethoxycarbonylphenyl)-3-methyltriazene (5b)
52.6 (OCH3), 115.8, 122.6, 126.7, 130.4, 130.7, 131.0 (CAr), 151.9 (Ne
CAr) 156.5 (ArCeOH), 166.8 (COCH3), 170.2170.0 (NCOCH3), 174.8
(N]NeNeCO). EI: m/z 399.3 (M þ H)þ, 163.1 (CH3OCOC6H4N2þ),
135.1 (CH3OCOC6Hþ4 ).
Yield 66%, m.p. 212e215 ꢁC. IR (cmꢀ1): 3318, 1724,1703,1649. 1H
NMR (400 MHz, DMSO-d6):
d
ppm 1.35 (t, J ¼ 8.0 Hz, 3H, CH3CH2),

8
A.S. Monteiro et al. / European Journal of Medicinal Chemistry 70 (2013) 1e9
4.2.9. 3-[2-(N-BOC)-3-(4-hydroxyphenyl)propanoyl]-1-(4-
acetylphenyl)-3-methyltriazene (6a)
4.6. Hydrolysis in human plasma
Yield 20%, m.p. 195e197 ꢁC. IR (cmꢀ1): 3358, 3330, 1713, 1674,
Human plasma was obtained from the pooled, heparinised
blood of healthy donors, and e prior to use e was frozen and stored
at ꢀ70 ꢁC. For the hydrolysis experiments, the substrates were
incubated at 37 ꢁC in human plasma that had been diluted to 80%
(v/v) with a pH 7.4 isotonic phosphate buffer. At appropriate in-
1626. 1H NMR (400 MHz, CD3OD):
d ppm 1.38 (s, 9H, COC(CH3)3),
2.63 (s, 3H, ArCOCH3), 2.84 (dd, J ¼ 16.0 and J ¼ 8.0 Hz, 2H, CH2Ar),
3.32 (s, 3H, NCH3), 5.52 (t, J ¼ 8 Hz, 1H, CHCH2Ar), 6.50e6.92 (dd,
J ¼ 8.0 Hz, 4H, HOAr); 7.65e8.08 (dd, J ¼ 8.0 Hz, 2H, Ar). 13CNMR
(100 MHz, DMSO-d6):
d
ppm 27.0 (CH3COAr), 28.4 (C(CH3)3), 33.7
tervals, aliquots of 200 mL were added to 400 mL of acetonitrile in
(NCH3), 36.6 (ArCH2), 54.2 (ArCH2C), 78.7 (C(CH3)3), 115.2, 122.7,
127.8, 129.9, 130.3, 137.1, 151.7, 155.8 (CAr), 156.2 (NHCO), 175.3
(CONCH3), 197.6 (ArCOCH3). EI: m/z 463.4 (M þ Na)þ, 147.2
(CH3COC6H4Nþ2 ), 118.9 (CH3COC6Hþ4 ).
order both to quench the reaction and precipitate plasma proteins.
These samples were centrifuged and the supernatant analysed by
HPLC for the presence of substrate and products.
4.7. Tyrosinase degradation studies
4.2.10. 3-[2-(N-BOC)-3-(4-hydroxyphenyl)propanoyl]-1-(4-
cyanophenyl)-3-methyltriazene (6b)
Yield 10%, m.p. 95e98 ꢁC. IR (cmꢀ1): 3350, 2228, 1703, 1680. 1H
Mushroom tyrosinase was used at a concentration of 300 U/mL
in a solution of phosphate buffer (pH 7.4, 3.0 mL) at 37 ꢁC. A solution
NMR (400 MHz, CD3OD):
d ppm 1.42 (s, 9H, COC(CH3)3), 2.98 (t,
of the prodrug under investigation (15 m
L of 10ꢀ2 M solution) was
J ¼ 8.0 Hz, 2H, CH2eAr), 3.39 (s, 3H, NCH3), 5.30 (d, J ¼ 8.0 Hz, 1H,
NH), 5.62 (q, J ¼ 8.0 Hz, 1H, CHCH2Ar), 6.61e6.91 (dd, J ¼ 8.0 Hz, 4H,
HOAr), 7.62e7.71 (dd, J ¼ 8.0 Hz, 4H, Ar). 13CNMR (100 MHz, CDOD):
added and the mixture incubated and gently stirred at 37 ꢁC. Ali-
quots were collected at selected time intervals and quenched by
addition of an organic solvent (acetonitrile). The disappearance of
the prodrug, with the concomitant appearance of metabolites, was
followed by HPLC for evaluation of the hydrolysis kinetics.
d
ppm 28.4e28.3 (C(CH3)3), 39.1 (HNCHCH2), 53.2 (OCCeHNH),
115.3, 122.9, 130.3, 133.2 (CAr).
4.3. HPLC analysis
4.8. Determination of Km and Vmax
The analytical high-performance liquid chromatography (HPLC)
system included a Hitachi UV Detector L-2400 Elite LaChrom,
Hitachi Pump L-2130 Elite LaChrom Intel Core2 Duo, a Samsung
Scala Monitor, a manual sample injector module equipped with
Km and Vmax values were determined by HPLC method as
described in 4.3 and 4.7. The tyrosinase concentration was calcu-
lated taking the value of Mr as 120,000 Da. Protein content was
determined by Bradford’s method using bovine serum albumin as
standard [26]. Each substrate was incubated with 15.0 units of
mushroom tyrosinase in a water bath at 37 ꢁC at six different
prodrug concentrations (0e10 mM), following simple saturation
kinetics. Initial rates were determined by the slope of the straight
line of the initial reaction for decrease of the prodrugs concentra-
tion 5c and 5f versus time. Values are the mean of triplicates. Data
were fit using a simple MichaeliseMenten equation to yield Km and
Vmax values. Linear regression analysis was performed using
GraphPad Prism 5, Graphpad Software Inc.
20
(5
m
L loop, a Merck LichrospherÒ 100 RP18 125 mm ꢃ 4.6 mm
mm) column equipped with a Merck Lichrocart pre-column
(Merck, Germany). For compounds 5a, 6a and 6b an acetonitrile/
water (60:40 to 40:60) an isocratic solvent system was used. For
compounds 5bef we used a gradient elution beginning with a
mobile phase of 95% water/5% acetonitrile ramping to 40% aceto-
nitrile/60% water over 15 min. The column effluent was monitored
at 300 nm for all compounds and a flow rate of 1.0 mL/min was
used.
4.4. Apparent partition coefficients
4.9. Melanoma cell lines
The apparent partition coefficients (log Pexp) were determined
at room temperature using 1-octanol-pH 7.4 phosphate buffer. Each
phase was mutually saturated before the experiment. The volumes
of each phase were chosen so that the solute concentrations after
distribution in the aqueous phase were readily measurable. The
compounds were dissolved in 1-octanol and the 1-octanol-phos-
phate buffer mixtures were shaken for 30 min to reach the distri-
bution equilibrium. Each phase was analysed separately by HPLC,
but the organic phase was diluted in acetonitrile (1:10) before HPLC
analysis. Partition coefficients, log Pexp, were calculated from the
ratio of the peak area in octanol to the peak area in the buffer.
The human melanotic melanoma cell line SKMEL-30 was pur-
chased from the Leibniz-Institut DSMZ (Germany), the MNT-1 cell
line was kindly offered by Dr. José Ramalho (Faculdade de Ciências
Médicas, Lisbon, Portugal) and the amelanotic melanoma cell line e
the M8 e was kindly offered by Dr. Edgardo D. Carrosela (Institut
Universitaire d’Hématologie, Paris, France). Cells were grown in
Roswell Park Memorial Institute medium (RPMI), supplemented
with 10% foetal bovine serum, 2 mM
penicillin/streptomycin.
L-glutamine and 100 mg/mL
4.10. Analysis of cell proliferation and viability
4.5. Hydrolysis in buffer solution
Cancer cell lines were seeded in 96-well microtitre plates at
3 ꢃ 105 cells/mL, and different concentrations of prodrugs were
added. After 24 h and 48 h incubations, the effect on proliferation/
viability were determined by using the 3-(4,5-dimethylthiazol-2-
yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazo-
lium (MTS) assay and by using the hemocytometer-based trypan
blue dye exclusion cell quantitation and viability assay. Control
assays were conducted in the absence of prodrugs and in the
presence of similar amounts of organic solvent only.
Reaction mixtures were analysed using HPLC. Usually, a 10 mL
aliquot of a 10ꢀ2 M stock solution of prodrug 5 in acetonitrile was
added to 10 mL of the appropriate thermostated buffer solution at
37 ꢁC. At regular intervals, samples of the reaction mixture were
analysed by HPLC along the following conditions: mobile phase,
acetonitrile/water (the composition of which depended on the
compound). The substrate reaction progress curve was analysed to
obtain the reaction rate constants.

A.S. Monteiro et al. / European Journal of Medicinal Chemistry 70 (2013) 1e9
9
4.11. Analysis of cell apoptosis
Appendix A. Supplementary data
Cell lines were cultured for 48 h at 1.5 ꢃ 105 cells/mL in serum
free medium, to synchronize cells at G0/G1 phase. After 24 h in
complete medium, the cells were incubated for another 48 h with
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.09.040.
50 or 100
medium alone (0
m
M of the prodrugs. Temozolomide (Sigma) and the
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cells was analysed by flow cytometry and data elaborated with
Paint-a-Gate (BD Bioscience) software.
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Acknowledgements
The authors gratefully acknowledge the Fundação para a Ciência
e Tecnologia (Portugal) for financial support through funding to
research unit iMed.UL.