The selective cytotoxicity of new triazene compounds to human melanoma cells

Article abstract of DOI:10.1016/j.bmc.2017.04.049

Metastatic melanoma still remains one the most difficult cancers to overcome. The aim of our research was the design of anti-tumour triazene compounds 3 for application to a melanoma-specific therapy. The strategy exploits the unique enzyme pathway of melanin biosynthesis for conversion of non-toxic prodrugs into toxic drugs in the melanoma cell. The compounds 3 were designed by coupling two active moieties, the alkylating triazenes and different tyrosinase substrates. All compounds 3 revealed to be chemically stable in isotonic phosphate buffer (PBS) at physiologic pH (t_??≥?48?h), and most of them showed to be slowly hydrolysed in human plasma (1.5?≤?t_? (h)?≤?161). Compounds 3c–n revealed to be excellent tyrosinase substrates (0.74?≤?t_? (min)?≤?6) with the best tyrosinase substrate 3l releasing MMT 45?s after tyrosinase activation. Structure-activity relationship studies allowed the identification of the better structural features for enzyme affinity. Furthermore, the derivatives 3l and 3m showed cell selectivity with significant cytotoxic effects (IC₅₀ values of 46–65?μM) against melanoma cell lines with tyrosinase overexpression MNT-1 and B16F10.

Full text of DOI:10.1016/j.bmc.2017.04.049

Accepted Manuscript
The Selective Cytotoxicity of New Triazene Compounds to Human Melanoma
Cells
Ana Sousa, Fábio Santos, Maria Manuela Gaspar, Susana Calado, João D.
Pereira, Eduarda Mendes, Ana Paula Francisco, Maria Jesus Perry
PII:
S0968-0896(17)30468-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.04.049
BMC 13772
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
10 March 2017
12 April 2017
27 April 2017
Please cite this article as: Sousa, A., Santos, F., Gaspar, M.M., Calado, S., Pereira, J.D., Mendes, E., Francisco,
A.P., Perry, M.J., The Selective Cytotoxicity of New Triazene Compounds to Human Melanoma Cells, Bioorganic
&
Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.04.049
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THE SELECTIVE CYTOTOXICITY of NEW TRIAZENE COMPOUNDS TO HUMAN
MELANOMA CELLS
a,1
a,1
a
a
Ana Sousa , Fábio Santos , Maria Manuela Gaspar , Susana Calado , João D.
a
a*
a*
a
Pereira , Eduarda Mendes , Ana Paula Francisco , Maria Jesus Perry
a
Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de
Lisboa. Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal.
1
AS and FS contributed equally to this work
*Corresponding authors
Abstract
Metastatic melanoma still remains one the most difficult cancers to overcome. The aim of our
research was the design of anti-tumour triazene compounds 3 for application to a melanoma-
specific therapy. The strategy exploits the unique enzyme pathway of melanin biosynthesis for
conversion of non-toxic prodrugs into toxic drugs in the melanoma cell. The compounds 3 were
designed by coupling two active moieties, the alkylating triazenes and different tyrosinase
substrates. All compounds 3 revealed to be chemically stable in isotonic phosphate buffer (PBS) at
physiologic pH (t
½
≥ 48h), and most of them showed to be slowly hydrolysed in human plasma (1.5
(min) ≤ 6)
≤
t
½
(h) ≤ 161). Compounds 3c-n revealed to be excellent tyrosinase substrates (0.74 ≤ t
½
with the best tyrosinase substrate 3l releasing MMT 45 seconds after tyrosinase activation.
Structure-activity relationship studies allowed the identification of the better structural features for
enzyme affinity. Furthermore, the derivatives 3l and 3m showed cell selectivity with significant
cytotoxic effects (IC50 values of 46-65 µM) against melanoma cell lines with tyrosinase
overexpression MNT-1 and B16F10.
Key words: Triazenes, Melanoma cell lines, tyrosinase, target approach, enzyme
activation
1

1.
Introduction
Melanoma is a significantly growing public health problem not only in the United States but
worldwide. In the United States, the incidence rate of melanoma has increased faster than
any other cancer. Over the last two decades, the melanoma incidence rates have overall,
escalated by 3.1% per annum. Indeed, in the 1930s, the lifetime risk of an American devel-
1
oping invasive malignant melanoma was 1 in 1,500. Currently, that risk is 1 in 59. The
American Cancer Society’s estimates for 2017 that 87,110 new melanomas will be diag-
nosed in the United States about and about 9,730 people are expected to die of melano-
2, 3
ma.
The observed improvement in the 5-year survival rate for invasive melanoma, is a
consequence of earlier detection and ablation by surgical excision, the primary treatment
modality of cutaneous melanoma. However advanced and metastatic cancers often require
other treatments, namely radiation therapy, chemotherapy, immunotherapy and targeted
therapy. Sometimes treatment is accomplished by a multiple approach. Studies suggest
that combining chemo drugs with immunotherapy such as interferon-alpha and/or interleu-
kin-2 may be more effective than a single chemo drug alone. This form of treatment is also
4
called biochemotherapy or chemoimmunotherapy. However combination
biochemotherapeutic regimens have not been shown to have an overall survival benefit,
5, 6
and have added toxicity.
Vemurafenib (Zelboraf®, Genentech, South San Francisco, CA) is a BRAF inhibitor that has
demonstrated its activity on patients with metastatic melanoma who harbour the V600E
BRAF mutation. Vemurafenib proves rapidly responsive and has improved by 6-month the
overall survival and progression- free survival, but lacks durability. Ipilimumab is a
monoclonal antibody that blocks cytotoxic T lymphocyte antigen-4, an inhibitor of T cell
activation, thereby potentiating an immune response. Its unique mechanism of action
differentiates it from chemotherapies in that it targets the immune system rather than
directly targeting the tumour itself. However, it is important to note that all these agents
have been used singly or occasionally in combination with chemotherapy. Nowadays a
2

combination of therapies remains the most promising approach since it prevents the
6-10
development of resistance.
Dacarbazine (DTIC) and Temozolomide are alkylating chemotherapeutic agents used to
treat advanced or metastatic melanoma. They are capable of methylating DNA and promote
cell death. Dacarbazine demonstrated response rates of approximately 15%. The major
problem associated with chemotherapy is the low selectivity of the cytotoxic effect on
tumour cells, consequently the side-effects may constrain the effectiveness of the
chemotherapeutic drugs. One of the ways to reduce the side effects is to design prodrugs
that can be selectively activated in the tumour site. In recent years, our research group has
designed and developed triazene derivatives as anti-melanoma agents. Such drugs will
selectively release the alkylating moiety in the melanoma cells through the action of a
11, 12
specific enzyme: tyrosinase.
The biological functions of this enzyme, the up-regulation of melanogenesis with the
overexpression of tyrosinase in malignant melanocytes and its potential applications
renders tyrosinase an extensively explored target. With regard to melanomas, it has been
exploited in multifaceted ways, either as a melanocytic-associated immunohistochemical
marker (in the diagnosis of melanomas, approximately 80%-90% of the melanomas
express this marker), or as a way of treating specifically melanoma, being this the area we
13-16
are concerned with.
Tyrosinase is the key enzyme involved in the human pigmentation
process. It catalyzes the hydroxylation of L-tyrosine, and dopaquinone is produced as the
immediate product. Dopaquinone undergoes the intramolecular addition of the amino group
giving cyclodopa (Scheme 1-i). It is precisely this step of melanogenic pathway that
underpins the development of the first prodrugs. These prodrugs are specifically made for
activation by tyrosinase by incorporating a phenol group capable of being oxidized by this
enzyme. The release of the cytotoxic agent takes advantage of the same framework that
leads to the formation of dopaquinone which takes place by an intramolecular Michael
addition reaction involving the nitrogen lone pair of electrons on the side-chain. By attaching
the cytotoxic agent through a hydrolysable linkage to the nitrogen, the process of cyclization
13-17
has the effect of rendering the link vulnerable to a hydrolytic attack (Scheme 1-ii).
3

Scheme 1. Activation pathway for tyrosinase substrates-based prodrugs.
1
1
Our previous work with tyramine and dopamine derivatives of triazenes demonstrated that
the reduced nucleophilicity of the modified amine nitrogen hindered the release of the
alkylating triazene after enzymatic activation. The more recent results obtained with N-
1
2
acyltyrosine prodrugs demonstrated the advantage of an amide linker , since these
compounds presented a better affinity and the yield of effective delivery of the cytotoxic
triazene increased. Based on these results, we developed new series of triazene
derivatives to be activated in a more efficient way by tyrosinase, exploring the SAR
evaluation of the new derivatives 3. In this article we describe their synthesis, the stability
in physiologic conditions, their activation by tyrosinase and the cytotoxic activity in
melanoma cell lines.
2
. Results and discussion
.1. Chemistry
2
The synthetic approach used for the preparation of triazene prodrugs derivatives (3a-n,
Table 1) is shown in Scheme 2.
4

Scheme 2. Synthetic pathway of triazene derivatives 3
Amide coupling was enhanced by the use of coupling reagents along with the activation of
the amino group. In our experiments the secondary amine MMT (1) was activated with NaH.
This deprotonation process generates a negative charge in the nitrogen atom, increasing its
nucleophilicity and enhancing the amide coupling between MMT (1) and the activated
18
carboxylic acid 2. When the amide coupling was not efficient at room temperature,
12
conventional heating or microwave irradiation was used.
Table 1
Triazene derivatives synthesized (3) and respective coupling agents.
Compound Yield
%)
R
1
R
2
n
X
Coupling Reaction
(
3a
3b
3c
3d
3e
3f
15
20
17
15
24
23
H
H
OH
OH
H
2
2
2
2
2
2
COOMe
CN
DCC/DMAP
TBTU (MW irradiation)
TBTU (MW irradiation)
DCC/DMAP
OH
OH
OH
OH
COOMe
CN
H
H
COMe
CONH2
TBTU (MW irradiation)
DMT/MM
H
5

3
3
3
3
3
3
3
3
g
h
i
25
30
18
28
30
26
20
15
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
H
4
4
4
4
4
4
4
1
CN
DCC/DMAP
DCC/DMAP
DCC/DMAP
DCC/DMAP
DMT/MM
COOMe
CONH2
COOEt
COMe
Me
j
k
l
DMT/MM
m
n
Br
DMT/MM
CN
DMT/MM
DCC/DMAP-N,N’-Dicyclohexylcarbodiimide/
Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate ; DMT/MM- 2-Chloro-
,6-dimethoxy-1,3,5-triazine/ 4-Methylmorpholine
4-(Dimethylamino)pyridine ;TBTU-
O-
(
4
The yields obtained from the different processes were low and did not exceed 26 %.
Microwave irradiation provided the best yields along with the reaction time and purification
process which are shorter. The poorer yields can be explained by the intrinsic reactivity of
the main reactants (hydroxyphenylalkyl acids 2 and MMT derivatives 1), which can easily
suffer decomposition in the reaction mediums.
During the synthesis of 3c and 3e, under microwave irradiation, dimers 4a and 4b were
formed as by-products (Figure 1 and Supplementary material S2).
Figure 1. Dimers obtained from the synthesis when using TBTU as an activating agent and
MW irradiation.
The dimerization process occurs between two activated molecules of 3-(4-hydroxyphenyl)
propionic acid probably before the amide coupling and the formation of the final dimers. A
19
similar process was observed by Bejugam and co-workers.
6

2.2. Biological Evaluation
2.2.1. Stability in PBS and Human Plasma
The stability of triazene derivatives 3 was studied both in isotonic phosphate buffer (PBS)
o
and in 80% human plasma at 37 C. These reactions were followed by HPLC by monitoring
both the loss of compounds 3 and the formation of products (Supplementary material S3).
Half-lives for the hydrolysis in PBS and in human plasma are presented in Table 2.
These chemical reactions followed pseudo first-order kinetics and were monitored during at
least 3 half-lives. Pseudo first-order rate constants (kobs) were calculated from the slopes of
plots of ln (Area) vs time (equation 1) and half-lives (t1/2) were calculated using equation 2.
ln (area) = -kobs x time + b
(Equation 1)
(Equation 2)
ꢀꢁ(ꢂ)
t =
½
ꢃꢄꢅꢆ
Triazene derivatives 3 decompose in PBS leading to the formation of 1-aryl-3-
methyltriazenes 1 and hydroxyphenylpropionic or pentanoic acid 2. Under the reaction
conditions the MMTs 1 are also unstable and hydrolyze further to the corresponding anilines
11
(
Scheme 3).
Scheme 3. Decomposition pathway of triazene derivatives 3 in physiological conditions
Table 2 shows that triazene derivatives 3a-f, h-i, decompose in PBS with half-lives ranging
from 60 to 183 hours. Derivatives 3g, j-m, are even more stable and remain intact after 8
7

days. The analysis of these data allows us to state that all the derivatives are chemically
o
stable in physiological conditions (37 C and pH 7.4).
Table 2
Half-lives (t ) for triazene derivatives 3, both in PBS buffer and 80% human plasma at 37
½
o
C, their experimental logP values, and their ability to act as tyrosinase substrates (100
U/mL)
t
½
Compound
X
logPexp
pH 7.4 buffer (h)
80% human
plasma (h)
Tyrosinase
(min)
a
3
3
3
3
3
3
3
3
3
3
3
3
3
a
b
c
d
e
f
COOMe
CN
3.42 ± 0.6*
3.24 ± 0.6*
3.23 ± 0.23
3.40 ± 0.17
3.05 ± 0.25
2.80 ± 0.07
4.3 ± 0.05
3.9 ± 0.23
3.6 ± 0.05
4.5 ± 0.22
4.0 ± 0.05
4.0 ± 0.30
4.32 ± 0.27
2.99 ± 0.24
94.7 ± 10.4
60.1 ± 3.9
101.5 ± 9.5
76.0 ± 9.0
122.7 ± 1.9
118.2 ± 3.4
82.5 ± 5.5
123.8 ± 6.4
7.8 ± 0.1
1270 ± 120
a
2.7 ± 0.1
1167 ± 108
2.46 ± 0.24
1.5 ± 0.12
3.66 ± 0.3
4.62 ± 0.3
4.2 ± 0.89
3.6 ± 0.47
5.8 ± 1.15
4.8 ± 0.85
1.31 ± 0.19
0.74 ± 0.19
6.46 ± 1.92
28.1 ± 4.3
COOMe
CN
7.3 ± 0.2
5.8 ± 0.3
COMe
14.6 ± 0.4
48.5 ± 2.9
11.84 ± 0.87
17.45 ± 0.34
55.42 ± 8.24
26.30 ± 2.85
32.74 ± 6.78
79.67 ± 0.05
161.5 ± 10.6
1.48 ± 0.18
CONH
2
g
h
i
CN
COOMe
CONH
2
182.4 ± 7.8
b
j
COOEt
COMe
b
b
b
k
l
CH
3
m
n
Br
3
CN
48 ± 0.53
a
Tyrosinase (300 U/mL)
b
100% of the compound remaining after 8 days
*
Calculated values
8

A further analysis of these results enables us to say that the position of the OH group in the
phenolic moiety does not have a significant influence on the chemical hydrolysis of
compounds 3, since the meta-phenolic derivatives (3a,b) have half-lives similar to the
corresponding para-phenolic counterparts (3c,d). The compounds with higher logP values
(
3g-m) also seem to be more stable in PBS.
A complete mass balance was observed for all compounds which hydrolyse in a convenient
time in this assay (Figure 2). The percentages of these compounds in each assay were
calculated based on concentrations obtained using calibration curves.
1
00
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
Triazene derivative 3b
Aniline
0
50
100
Time (h)
150
200
Figure 2. Time-course curve for the decay of compound 3b and the formation of aniline in
PBS.
Derivatives with an amide linkage have in general a suitable stability in vivo, due to reduced
20
chemical and enzymatic hydrolysis. A comparison of our results with others previously
published in the literature, for compounds with significant structural resemblance (Figure 3),
reveals that the decomposition rate depends on the carrier unit. Carvalho and co-workers
analyzed the chemical stability of a range of aminoacyltriazenes 5a and showed that these
21, 22
compounds are more unstable than compounds 3.
Perry and co-workers also
9

evaluated the chemical stability of a range of N-acylamino acid derivatives of triazenes 5b
in PBS. Although these derivatives 5b gain some stability upon aminoacyl derivatives 5a,
23, 24
they are less stable than our derivatives 3.
Since the only difference between MMT-
based derivatives 3 and 5a-b is the trigger/carrier unit, it is possible to state that the
hydroxyphenylpropionic or pentanoic acid trigger 2 is more efficient in the stabilization of
the amide linker than the amino acid unit. Wanner and co-workers also synthesized
prodrugs 6a-d with an amide linkage having half-lives between 22.1 and 58.3 hours and
25
prodrugs 6e-k with a carbamate linkage having half-lives ranging from 0.4 to 58.3 hours.
Figure 3. N-Acyl amino acid derivatives of triazenes
Perry and co-workers have also evaluated the stability of compounds 7, which have a urea
linker. Compounds 7a-d are stable in PBS for 15 days and prodrugs 7e-g have half-lives
longer than 15 hours. Triazene derivatives 3a-f revealed themselves to be more stable than
prodrugs 7e-g, and compounds 3g-m revealed an ideal chemical stability like prodrugs 7a-
11
d.
In conclusion, derivatives 3 proved to be stable enough to reach the tumor cells
undecomposed.
Hydrolysis in human plasma
Blood serum and plasma contain a variety of enzymes that catalyse the hydrolysis of ester
and amide functions. Since triazene derivatives 3 have in their structure an amide function,
tests in human plasma were performed in order to evaluate if compounds 3 are stable in
2
3, 26
this medium.
These assays followed the experimental procedure described in the
10

experimental section. The calculated pseudo first-order rate constants (kobs) and half-lives
) for the hydrolysis of derivatives 3 in human plasma are given in Table 2.
(t
½
In this assay it was possible to observe the hydrolysis of triazene prodrugs 3b,d in the
corresponding MMT-CN. Over time MMTs hydrolysed in the corresponding aniline
(
(
Supplementary material S4). Complete mass balance was observed for derivatives 3b, d-f
Figure 4).
Figure 4. Time-course for the formation and decay of intermediates in the plasma
hydrolysis of compound 3b
The half-lives calculated in these assays range from 3 to 162 hours. These results clearly
show that all derivatives are substrates for plasma enzymes but pentanoic acid derivatives
are more stable. A comparison of the hydrolytic rates between derivatives 3 and
compounds 6 and 7, shows that they have an intermediate stability between carbamate and
2
1-23
urea linkers.
plasma is favored by the presence of electron-withdrawing groups in the triazene moiety,
presenting a Hammett correlation with a value of + 1.08 (Supplementary material S3).
It was also possible to quantify for 3g-m that enzymatic hydrolysis in
ρ

Triazene derivatives 3 (with the exception of 3b) seem to have an adequate stability in
plasma to reach the tumour site undecomposed.
2.2.2. Evaluation of the compounds as Tyrosinase substrates
The evaluation of compounds 3 as tyrosinase substrates was followed by HPLC by
determining the half-lives of derivatives in the presence of the enzyme (present in high level
in tumour melanocyte cells). The assays were performed in order to evaluate the ability of
triazene derivatives 3 to release the cytotoxic agent MMT 1 after tyrosinase activation.
These assays are fundamental to demonstrate the concept and validate the design of
triazene derivatives 3.
The half-lives (t1/2) for the activation of compounds 3 by mushroom tyrosinase are given in
Table 2. The % of MMT formation and derivative consumption for compounds 3a-f is given
in Table 3.
Table 3
Results from HPLC analysis of the assays performed in the presence of mushroom
o
tyrosinase at 37 C for compounds 3 and dimers 4a-b.
-
1
Mushroom
tyrosinase
(units/mL)
%Compound
consumption
%Aniline
formation
%MMT
formation
kobs (s )
t
½
Triazene
prodrugs
(
min)
-
6
-6
-6
-3
-3
-3
-3
3
3
3
3
3
a
b
c
d
e
300
300
100
100
100
100
100
100
97.5 ± 3.4
91.1 ± 3.6
100
54.9 ± 11.2
68.9 ± 11.4
36
2.0 ± 0.2
9.1x10 ± 0.9x10
1270
1167
2,5
-6
4.6 ± 0.7
9.9x10 ± 0.9x10
-3
5
4.7x10 ± 0.4x10
-3
100
46
4
7.9x10 ± 0.5x10
1,5
-3
100
24
5
3.2x10 ± 0.3x10
3,6
-3
3
f
100
20
4
2.5x10 ± 0.2x10
4,6
-
3
4
a
b
------------
------------
------------
------------
-----------
-----------
1.8x10
2.1x10
6,4
-
3
4
5,5
12

Different processes were observed, depending on the meta/para-hydroxyphenylalkyl acid
derivative 2. Compounds 3a and 3b, having the hydroxyl group in meta position relative to
the linker did not seem to be substrates for tyrosinase. Two observations substantiate this
conclusion: i) when activated by tyrosinase derivatives 3a, b release the corresponding
MMTs, which in the course of the reaction are hydrolysed to the corresponding aniline
(
Supplementary material S4).
No formation of intermediate species was observed during the potential tyrosinase
oxidation; ii) derivatives 3c and 3d, with the OH group in the para position, have half-lives
approximately one thousand shorter in the presence of tyrosinase, relatively to their
analogs 3a and 3b respectively. They are undoubtedly tyrosinase substrates. Previous
studies suggested that tyrosinase hydroxylates substrates substituted in para position more
readily than those substituted in meta or ortho positions because of the lower steric
27
hindrance.
The triazene derivatives 3c-m, when exposed to mushroom tyrosinase were oxidized into
intermediate specie 8 before the MMT release (Supplementary material S5).This
intermediate could be a quinone specie that is stable enough to be detected. In the
literature the formation of a similar quinone specie is also referred when 3-(4-hydroxyphenyl)
28
propionic acid is oxidized by tyrosinase. Perry and co-workers using LC-MS had already
observed the formation of a similar intermediate in the oxidation of compounds 7b,c and 7e
11
by mushroom tyrosinase.
With the data collected from the assays of derivatives 3c-f in the presence of mushroom
tyrosinase, we hypothesized a tyrosinase-dependent mechanism of MMT release (Scheme
4).
13

Scheme 4. Proposed mechanism for MMT release from derivatives 3c-f after tyrosinase
activation
In this drug release pathway, triazene derivatives 3c-f are oxidized by tyrosinase into the
corresponding orthoquinone 8. Then this specie 8 can initiate an intramolecular cyclization
pathway and MMT is released from a reactive intermediate instable in aqueous media.
The release of MMT 1, was detected after 250 seconds of exposure of triazene derivatives
3c-f to mushroom tyrosinase. The maximum percentage of MMT 1 formation ranged from 4
to 5%. Because aniline is always derived from the precursor MMT and in its mass balance
sometimes reaches 100%, it is our understanding that the 5% value obtained for MMT is
not necessarily a low value. In fact, the MMT release from derivatives 3c-f is much faster in
comparison with other drug release pathways described in the literature for tyrosinase
29
activation.
In the assays in which we did not observe a complete mass balance for the aniline, the
observation could be explained by the fact that aromatic amines (e.g.: anilines) are also
tyrosinase substrates. In a previous work Toussaint and co-workers have found that
several p-anilines are oxidized by tyrosinase in a two-step mechanism Firstly, p-anilines
30
suffer an ortho hydroxylation and then they are converted into o-quinone imines. The
interaction mechanism between p-anilines and the active site of tyrosinase was proposed
27
by Munoz-Munoz and co-workers. The formation of this o-quinone imine specie can be
the main reason why we have not detected the complete formation of aniline in some
mushroom tyrosinase assays.
14

Comparing the half-life values for compounds 3a,b and 3c-m the importance of the position
of the hydroxyl group in acid 2 is obvious in terms both of early recognition and higher
affinity for tyrosinase when the acid is p-hydroxylated. A similar difference has already been
observed in a previous work by Fenoll and co-workers, in which they found that the catalytic
3
1
efficiency of tyrosinase is much higher for 4-hydroxyanisole than for 3-hydroxyanisole.
The length of the linker-arm seems to be particularly important in the activation of the drug
release by tyrosinase since compound 3n (n=1) has a peculiar t of 28 min, 6.7x and 18.7x
½
greater than their respective homologues 3g (n=4) and 3d (n=2). Therefore it seems there
is an ideal length for the linker arm.
Triazene derivatives 3c-m reveal themselves to be excellent tyrosinase substrates with half-
lives that range from 0.7 to 7 minutes. These derivatives 3c-m have a better affinity for
tyrosinase in comparison with other potential derivatives.
Tyrosinase activation over dimeric compounds 4a,b was also evaluated and surprisingly it
was found that these compounds are excellent tyrosinase substrates despite their length
(
large molecules). Compounds 4a,b (Table 3) have a half-life of approximately 6 minutes. In
this assay it was possible to observe the formation of two intermediate species, probably
the same type of quinone specie already observed during the hydrolysis of prodrugs 3c-f.
The analysis of enzymatic kinetics for compounds 3g and 3h proved to satisfy Michaelis-
Menten kinetics equation and the values of K
compounds are in accordance with the values for natural tyrosinase substrates like tyrosine
= 0.25±0.03 mM) and dopamine (K = 0.28±0.01 mM), or other monophenols such as
p-hydroxyphenylacetic acid (K = 1.91±0.08 mM) and p-hydroxyphenylpropionic acid (K
.44±0.01 mM), and confirm that the compounds are good substrates for mushroom
m
and Vmax obtained (Table 4) for these
(
K
m
m
m
m
=
0
32
tyrosinase.
Table 4
RT (min)
Kinetic parameters for the oxidation of triazene derivatives 3 by mushroom tyrosinase
-
1
Compound K
m
(mM)
Vmax
µM/min)
K
cat (s )
K
cat/K
m
Van
der
-
1 -1
(
(M s )
Waals
volume
3
(
Å )
15

3
g
h
0.86 ± 0.22 0.012 ± 0.001 0.009
1.08 ± 0.25 0.018 ± 0.001 0.013
1.39
325.92
352.44
3
13.88
The values reported are the average ± SD (standard deviation) for the three independent
33
determinations. The van der Waals volumes were calculated according to the literature.
2.2.3 Anti-proliferative assay in melanoma and non-melanoma cell lines
The cytostatic activity of all compounds under study were determined both in murine and
human melanoma cell lines, the B16F10 and the MNT-1, respectively which are described
to have an overexpression of tyrosinase.12, 34 Moreover, in order to evaluate the possible
toxicity of the compounds to healthy cells, the human keratinocyte cell line - the HaCat -
was also included in this study. The IC50 values obtained are shown in Table 5. Taking into
account the results obtained the most promising compounds were the 3l and 3m which
displayed the lowest IC50 against MNT-1 and B16F10 cell lines. In particular the compound
3
l presented IC50 values of 64.6 and 57.6 µM respectively and concomitant higher values of
IC50 against HaCaT cell line. In addition this compound presented a high affinity to
tyrosinase, with a t of 0.74 min (Table 2). An important remark was the fact that our
½
derivatives are more cytotoxic to those melanoma cell lines than TMZ a triazene prodrug in
clinical use and tested in the assays as a reference drug.
Table 5
Inhibitory effect of tested compounds against the proliferation of MNT-1, B16F10 and
HaCaT cells
Compound
IC50 ± SD (µM)
B16F10
MNT-1
HaCaT
3a
3b
3c
>75
>75
>75
>75
>75
>75
75-150
75-150
75-150
16

3
d
e
>75
>75
>75
>75
75-150
75-150
75-150
75-150
75-150
75-150
>150
3
3
f
>75
>75
3
g
h
>75
>75
3
>75
>75
3
i
>75
>75
3
j
>75
>75
3k
>75
>75
>100
3
l
64.6 ± 1.0
45.8 ± 1.8
>75
57.6 ± 5.2
52.2 ± 5.7
>75
>150
3m
>75
Temozolomide
-
3.
Conclusions
In summary, we have successfully synthesized a new series of triazene derivatives 3
selective for melanoma cells overexpressing tyrosinase. The results show that the
compounds are chemically stable in physiologic conditions and in the presence of tyrosinase
they oxidize with the liberation of the alkylating monomethyltriazene. The synthesized
compounds (3c-m) differ in the extension of the linker unit intercalated between the triazene
and the p-hydroxyphenyl group. The extension of the linker arm influences the affinity of the
enzyme tyrosinase to these derivatives with better affinities when n=2.
The kinetics of the tyrosinase catalytic activity on these derivatives has been characterized
and compared with analogs previously developed: Km values for p-hydroxypentanoic
derivatives indicated a good affinity for the enzyme. The ratios between the catalytic
m
efficiencies (Kcat/K ) are lower than the correspondent values obtained with N-acetyltyrosine
derivatives, probably reflecting a higher stability of the oxidation intermediates.
Regarding the cytotoxic activity of all the compounds in murine (B16F10) and human (MNT-1)
melanoma cell lines with tyrosinase overexpression, the most promising compounds were
the 3l and 3m. These compounds displayed the lowest IC50 against MNT-1 and B16F10 cell
lines, with a concomitant lesser toxicity against healthy human keratinocytes cells.
In conclusion, triazene compounds 3c-n had great stability, excellent tyrosinase affinity, and
3
l and 3m are more cytotoxic than the standard temozolomide, a triazene prodrug in clinical
17

use. These results are a proof of concept of the designed strategy providing a strong
potential for melanoma treatment.
4. Experimental
4.1 General information
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 2455MHz.
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
1
13
spectrophotometer. H and C-NMR spectra were recorded in CDCl
DMSO-d using a Brucker AM 400 WB spectrometer. Chemical shifts, δ, are reported as
p.p.m. from Me Si, and coupling constants, J, in Hz. Mass spectra were recorded in a
3 3
, CD CN, MeOD or
6
4
Waters Micromass Quattro micro API Benchtop Mass Spectrometer. All chemicals were
reagent grade except those for kinetic studies and HPLC, which were of analytical or
®
LiChrosolv (Merck) grade. 1-Aryl-3-methyltriazenes were synthesized by previously
35, 36
published methods.
The 3-(4-hydroxyphenyl) propionic acid, 3-(3-hydroxyphenyl)
propionic acid, 5-(4-hydroxyphenyl) pentanoic acid, 2-(4-hydroxyphenyl) etanoic acid and
-
Mushroom tyrosinase (5350 U mg ) were purchased from Sigma-Aldrich.
1
4.2 General procedures for the synthesis of derivatives 3a,d and 3 g-j (DCC/DMAP)
Hydroxyphenyl alkyl carboxylic acid (1.12 mmol) was dissolved in dried THF (3 mL) and
DCC (0.29 g, 1.4 mmol) was added to the solution at room temperature. The reaction
mixture was stirred for one hour. MMT (1.12 mmol) was dissolved apart in dried THF (2 mL)
and NaH (0.027 g, 1.12 mmol) was added to the solution. MMT solution, TEA (0.156 mL,
18

1.12 mmol) and DMAP (0.014 g, 0.112 mmol) were all added into the reaction mixture. The
reaction was stirred at room temperature for 72 hours. The solution was concentrated under
reduced pressure and the crude product was purified by column chromatography (diethyl
ether:petroleum ether
or dichloromethane:methanol). The isolated products were
subsequently recrystallized from dichloromethane / n-hexane.
4.3 General procedures for the synthesis of derivatives 3b,c,e (TBTU)
Hydroxyphenylalkyl carboxylic acid (0.3 mmol), MMT (0.33 mmol) and TBTU (0.101 g,
0.315 mmol) were dissolved in dried DMF (3 mL) in a microwave tube and TEA (0.088 mL,
0.63 mmol) was added. The resulting mixture was irradiated in a first cycle with 100 W, 50
0 0
C, 15 min and in a second cycle with 100 W, 55 C, 15 min. Reaction progress was
followed by TLC. Then, the reaction mixture was extracted with a solution of citric acid 5%
(
3
2x5mL), a solution of NaHCO 5% (2x5mL) and a saturated NaCl solution (2x5mL). The
organic phase was dried with anhydrous sodium sulphate and concentrated under reduced
pressure. The final products were recrystallized from dichloromethane/ n-hexane.
4.4 General procedures for the synthesis of derivatives 3f and 3k-n (DMT/MM)
N-methylmorpholine (0.33 mL, 3 mmol) was added to a solution of 2-chloro-4,6-dimethoxy-
1,3,5-triazine (0.176 g, 1 mmol) in 4 mL of dried DCM. The reaction mixture was stirred for
o
0-40 min at 0-5 C. Then, 3-(4-hydroxyphenyl)alkylcarboxylic acid (0.166 g, 1 mmol) in 10
3
mL of dried DCM was added to the reaction mixture and the resulted mixture stirred at room
temperature for one hour. Afterwards the appropriate MMT (0.178 g, 1 mmol) was added to
the reaction mixture and stirred for 48 hours. The reaction mixture was treated with 2x5 mL
3 2
of NaHCO (10%) and 3x5 mL of H O. The organic phase was dried with anhydrous sodium
sulphate and concentrated under reduced pressure. The final products were recrystallized
from dichloromethane / n-hexane.
4.5.1. 3-[3-(3-hydroxyphenyl)propanoyl]-1-(4-methoxycarbonylphenyl)-3-methyltriazene (3a)
19

o
-1
1
Yield 15%; yellow crystals; mp 136-138 C; νmax/cm 1686, 1713, 3410; H NMR (400 MHz,
CDCl ) δ 3.04 (2H, t, J = 7.7 Hz, CH ), 3.26 (2H, t, J = 7.7 Hz, NCOCH ), 3.44 (3H, s,
NCH ), 3.94 (3H, s, COOCH ), 5.31 (1H, s, ArOH), 6.69 (1H, dd, J = 7.8, 2.0 Hz, Ar), 6.75
1H, br s, Ar), 6.81 (1H, d, J = 7.8 Hz, Ar), 7.16 (1H, t, J = 7.8 Hz, Ar), 7.60 (2H, d, J = 8.4
3
2
2
3
3
(
1
3
Hz, Ar), 8.11 (2H, d, J = 8.4 Hz, Ar); C NMR (100 MHz, CDCl
CH ), 35.99 (CH ), 52.48 (COOCH ), 113.42 (Ar), 115.57 (Ar), 120.90 (Ar), 122.09 (Ar),
29.89 (Ar), 130.28 (Ar), 130.90 (Ar), 142.76 (Ar), 152.10 (Ar), 155.96 (Ar), 166.74 (C=O),
3
) δ 28.14 (NCH
3
), 30.97
(
2
2
3
1
1
+
+
-
-
75.20 (C=O); ESI -MS: m/z 364.14 ([M+Na] ); ESI -MS: m/z 340.53 ([M-H] ).
4.5.2. 3-[3-(3-hydroxyphenyl)propanoyl]-1-(4-cyanophenyl)-3-methyltriazene (3b)
o
-1
1
Yield 20%; yellow crystals; mp 119-121 C; νmax/cm 1684, 2228, 3410; H NMR (400 MHz,
CDCl ) δ 3.03 (2H, t, J = 7.8 Hz, CH ), 3.25 (2H, t, J = 7.8 Hz, NCOCH ), 3.45 (3H, s,
NCH ), 5.39 (1H, s, ArOH), 6.69 (1H, dd, J = 7.8, 2.2 Hz, Ar), 6.75 (1H, br s, Ar), 6.80 (1H, d,
J = 7.8 Hz, Ar), 7.15 (1H, t, J = 7.8 Hz, Ar), 7.63 (2H, d, J = 8.4 Hz, Ar), 7.73 (2H, d, J = 8.4
3
2
2
3
1
3
Hz, Ar); C NMR (100 MHz, CDCl
3
) δ 28.34 (NCH
3
), 30.88 (CH
2
), 35.95 (CH
2
), 112.18 (Ar),
113.46 (Ar), 115.57 (Ar), 118.64 (C≡N), 120.84 (Ar), 122.85 (Ar), 129.89 (Ar), 133.46 (Ar),
-
42.62 (Ar), 151.76 (Ar), 156.01 (Ar), 175.12 (C=O); ESI -MS: m/z 307.21 ([M-H] ).
-
1
4.5.3.
3-[3-(4-hydroxyphenyl)propanoyl]-1-(4-methoxycarbonylphenyl)-3-methyltriazene
(
3c)
o
-1
1
Yield 17%; yellow crystals; mp 101-103 C; νmax/cm 1697, 1728, 3368; H NMR (400 MHz,
CDCl ) δ 3.01 (2H, t, J = 7.6 Hz, CH ), 3.23 (2H, t, J = 7.6 Hz, NCOCH ), 3.44 (3H, s,
NCH ), 3.94 (3H, s, COOCH ), 4.97 (1H, s, ArOH), 6.76 (2H, d, J = 7.8 Hz, Ar), 7.11 (2H, d,
J = 7.8 Hz, Ar), 7.60 (2H, d, J = 8.2 Hz, Ar), 8.11 (2H, d, J = 8.2 Hz, Ar); C NMR (100 MHz,
CDCl ) δ 28.10 (NCH ), 30.36 (CH ), 36.48 (CH ), 52.49 (COOCH ), 115.50 (Ar), 122.07
Ar), 122.71 (Ar), 129.71 (Ar), 130.87 (Ar), 132.87 (Ar), 152.14 (Ar), 154.30 (Ar), 166.74
3
2
2
3
3
13
3
3
2
2
3
(
(
-
C=O), 175.31 (C=O); ESI -MS: m/z 340.23 ([M-H] ).
-
20

4.5.4. 3-[3-(4-hydroxyphenyl)propanoyl]-1-(4-cyanophenyl)-3-methyltriazene (3d)
o
-1
1
Yield 15%; yellow crystals; mp 140-141 C; νmax/cm 1711, 2234, 3389; H NMR (400 MHz,
CDCl ) δ 3.01 (2H, t, J = 7.6 Hz, CH ), 3.22 (2H, t, J = 7.6 Hz, NCOCH ), 3.44 (3H, s,
NCH ), 4.74 (1H, s, ArOH), 6.76 (2H, d, J = 7.6 Hz, Ar), 7.11 (2H, d, J = 7.6 Hz, Ar), 7.63
3
2
2
3
1
3
(
(
3
2H, d, J = 7.8 Hz, Ar), 7.73 (2H, d, J = 7.8 Hz, Ar); C NMR (100 MHz, CDCl ) δ 28.30
NCH
3
), 30.24 (CH
2
), 36.42 (CH
2
), 112.16 (Ar), 115.53 (Ar), 118.65 (C≡N), 122.83 (Ar),
-
29.70 (Ar), 132.72 (Ar), 133.45 (Ar), 151.82 (Ar), 154.37 (Ar), 175.18 (C=O); ESI -MS: m/z
1
-
07.15 ([M-H] ).
3
4.5.5. 3-[3-(4-hydroxyphenyl)propanoyl]-1-(4-acetylphenyl)-3-methyltriazene (3e)
o
-1
1
Yield 24%; yellow crystals; mp 134-136 C; νmax/cm 1661, 1695, 3244; H NMR (400 MHz,
CDCl ) δ 2.64 (3H, s, COCH ), 3.01 (2H, t, J = 7.8 Hz, CH ), 3.23 (2H, t, J = 7.8 Hz,
NCOCH ), 3.45 (3H, s, NCH ), 5.15 (1H, s, ArOH), 6.77 (2H, d, J = 8.6 Hz, Ar), 7.11 (2H, d,
J = 8.6 Hz, Ar), 7.63 (2H, d, J = 8.8 Hz, Ar), 8.03 (2H, d, J = 8.8 Hz, Ar); C NMR (100 MHz,
CDCl ) δ 26.89 (COCH ), 28.14 (NCH ), 30.34 (CH ), 36.48 (CH ), 115.51 (Ar), 122.28 (Ar),
29.71 (Ar), 132.90 (Ar), 137.01 (Ar), 152.18 (Ar), 154.27 (Ar), 175.27 (C=O), 197.63 (C=O);
3
3
2
2
3
13
3
3
3
2
2
1
-
ESI -MS: m/z 324.21 ([M-H] ).
-
4.5.6. 3-[3-(4-hydroxyphenyl)propanoyl]-1-(4-aminocarbonylphenyl)-3-methyltriazene (3f)
o
-1
1
Yield 23%; yellow crystals; mp 172-174 C; νmax/cm 1670-1686, 3306, 3348-3410; H
NMR (400 MHz, CDCl ) δ 2.90 (2H, t, J = 7.6 Hz, CH ), 3.14 (2H, t, J = 7.6 Hz, NCOCH ),
.35 (3H, s, NCH ), 6.65 (2H, d, J = 8 Hz, Ar), 6.98 (2H, d, J = 8 Hz, Ar), 7.52 (2H, d, J = 8
3 2
Hz, Ar), 7.83 (2H, d, J = 8 Hz, Ar); C NMR (100 MHz, CDCl ), 30.41 (CH ),
3
2
2
3
3
13
3
) δ 27.79 (NCH
3
6.44 (CH
51.30 (Ar), 155.13 (Ar), 175.66 (C=O); ESI -MS: m/z 349.09 ([M+Na] ).
2
), 115.29 (Ar), 122.10 (Ar), 128.61 (Ar), 129.37 (Ar), 131.64 (Ar), 133.26 (Ar),
+
+
1
4.5.7. 3-[5-(4-hydroxyphenyl)pentanoyl]-1-(4-cyanophenyl)-3-methyltriazene (3g)
o
-1
1
Yield 25%: mp 156-159 C; νmax/cm 1639, 2228, 3418; H NMR (400 MHz, CDCl
3
) δ 1.70
(
2H, m, NCOCH CH ), 1.76 (2H, m, CH CH Ar), 2.60 (2H, t, J = 8 Hz, NCOCH ), 2.94 (2H,
2
2
2
2
2
21

t, J = 8 Hz, CH
2
Ar), 3.44 (3H, s, NCH
3
), 6.74-7.04 (4H, dd, J = 8 Hz, Ar), 7.63-7.74 (4H,dd,
J = 12 Hz, Ar); C NMR (100 MHz, CDCl ) δ 24.49 (NCOCH CH ), 28.15 (NCH ), 31.29
Ar), 112.00 (Ar), 115.16 (Ar), 118.57 (C≡N),
22.66 (Ar), 129.44 (Ar), 133.36 (Ar), 134.13 (Ar), 151.79 (Ar), 153.75 (Ar), 175.72 (C=O).
1
3
3
2
2
3
(
2 2 2 2
CH CH Ar), 34.16 (NCOCH ), 34.75 (CH
1
+
ESI -MS: m/z 359.12 ([M+Na] ).
+
4.5.8. 3-[5-(4-hydroxyphenyl)pentanoyl]-1-(4-methoxycarbonylphenyl)-3-methyltriazene (3h)
o
-1
1
Yield 30%: mp 105-107 C; νmax/cm 1611, 1713, 2851, 2928, 3327; H NMR (400 MHz,
CDCl ) δ 1.68 (4H, m, CH CH ), 2.54 (2H, t, J = 8 Hz, NCOCH ), 2.94 (2H, t, J = 8 Hz,
CH Ar), 3.39 (3H, s, NCH ), 3.90 (3H, s, COOCH ), 6.64-6.96 (4H, dd, J = 8 Hz, Ar), 7.63-
.01 (4H,dd, J = 8 Hz, Ar); C NMR (100 MHz, MeOD) δ 24.31-22.82 (CH CH ), 26.62
OCH ), 29.29 (NCH ), 33.36-31.03 (COCH ), 34.34-33.69 (CH Ar), 114.63 (Ar), 121.64
Ar), 128.88 (Ar), 129.88 (Ar), 130.38 (Ar), 132.83 (Ar), 152.30 (Ar), 154.96 (Ar), 166.48
3
2
2
2
2
3
3
13
8
2
2
(
(
(
3
3
2
2
-
-
3
COOCH ), 176.12 (C=O). ESI -MS:m/z 368.30 ([M-H] ).
4.5.9. 3-[5-(4-hydroxyphenyl)pentanoyl]-1-(4-aminocarbonylphenyl)-3-methyltriazene (3i)
Yield 18%: mp (NCH ), 31.06 (CH
3
2 2 2 2
CH ), 33.73 (COCH ), 34.32 (CH Ar), 114.65 (Ar),
1
1
21.59 (Ar), 128.54 (Ar), 128.89 (Ar), 132.84 (Ar), 133.68 (Ar), 151.33 (Ar), 154.94 (Ar),
-1
1
21-124 ˚C; νmax/cm 1645, 1659, 3372; H NMR (400 MHz, CDCl
3
) δ 1.68 (4H, m,
CH
2
CH ), 2.53 (2H, t, J = 8 Hz, NCOCH ), 2.93 (2H, t, J = 8 Hz, CH Ar), 3.38 (3H, s, NCH
2
2
2
3
),
1
3
6
.94-6.96 (4H, dd, J = 8 Hz, Ar), 7.62-7.96 (4H,dd, J = 8 Hz, Ar); C NMR (100 MHz,
+
NCO), 176.14 (C=O). ESI -MS: m/z
MeOD) δ 24.36-22.82 (NCOCH
2
CH
2
), 29.30 170.09 (H
2
+
77.21 ([M+Na] ).
3
4.5.10. 3-[5-(4-hydroxyphenyl)pentanoyl]-1-(4-ethoxycarbonylphenyl)-3-methyltriazene (3j)
-
1
1
Yield 28%: mp 113-117 ˚C; νmax/cm 1641, 1738, 3404; H NMR (400 MHz, CDCl
3
) δ 1.41
), 2.59 (2H, t, J = 8 Hz, NCOCH ), 2.97
), 4.39 (2H, q, J = 8 Hz, CO CH CH ), 6.62-7.05
4H,dd, J = 8 Hz, Ar), 7.69-8.13 (4H, dd, J = 8 Hz, Ar), C NMR (100 MHz, ACN) δ 14.47
(
(
(
3H, t, J = 8 Hz, CO
2H, t, J = 8 Hz, CH
2
CH
2
CH
3
), 1.71 (4H, m, CH
2
CH
2
2
2
Ar), 3.40 (3H, s, NCH
3
2
2
3
13
22

(
CO
2
CH
5.16 (CH
31.27-130.21 (Ar), 131.41 (Ar), 134.34 (Ar), 153.20 (Ar), 155.76 (Ar), 166.63
2
CH
3
), 25.21 (NCOCH
2
CH
2
), 30.80 (CH
2 2 3 2
CH Ar), 32.00 (NCH ), 34.61 (NCOCH ),
3
1
2
Ar), 61.96 (CO
2
CH
2
CH
3
), 115.74 (Ar), 115.83 (Ar), 118.26 (Ar), 122.76 (Ar),
+
+
(
2 2 3
CO CH CH ), 176.54 (C=O). ESI -MS: m/z 384.10 ([M+H] ).
4.5.11. 3-[5-(4-hydroxyphenyl)pentanoyl]-1-(4-acetylphenyl)-3-methyltriazene (3k)
-
1
1
Yield 30%: mp 115-118 ˚C; νmax/cm 1697, 1748, 3300; H NMR (400 MHz, CDCl
2H, m, NCOCH CH ), 1.78 (2H, m, CH CH Ar), 2.64 (3H, s, COCH ), 2.59 (2H, t, J = 8 Hz,
NCOCH ), 2.95 (2H, t, J = 8 Hz, CH Ar), 3.45 (3H, s, NCH ), 6.76-7.03 (4H, dd, J = 8 Hz,
Ar), 7.61-8.03 (4H,dd, J = 8 Hz, Ar); C NMR (100 MHz, CDCl ) δ 21.43 (NCOCH CH ),
6.87 (COCH ), 28.06 (NCH ), 31.43 (CH CH Ar), 34.39 (NCOCH ), 34.82 (CH Ar), 115.30
Ar), 122.21 (Ar), 129.53 (Ar), 129.73 (Ar), 134.02 (Ar), 136.88 (Ar), 152.25 (Ar), 154.10 (Ar),
3
) δ 1.70
(
2
2
2
2
3
2
2
3
1
3
3
2
2
2
3
3
2
2
2
2
(
+
+
1
3
76.11 (C=O), 197.88 (COCH ). ESI -MS: m/z 376.20 ([M+Na] ).
4.5.12. 3-[5-(4-hydroxyphenyl)pentanoyl]-1-(4-toluyl)-3-methyltriazene (3l)
-
1
1
Yield 26%: mp 120-124 ˚C; νmax/cm 1675, 3390; H NMR (400 MHz, CDCl
NCOCH CH ), 1.77 (2H, m, CH CH Ar), 2.40 (3H, s, CH 2.59 (2H, t, J = 8 Hz, NCOCH
.94 (2H, t, J = 8 Hz, CH Ar), 3.41 (3H, s, NCH ), 6.73-7.03 (4H, dd, J = 8 Hz, Ar), 7.23-
.47 (4H, dd, J = 8 Hz, CH Ar); C NMR (100 MHz, CDCl ) δ 21.43 (H CAr), 27.73
CH ), 31.10 (NCH ), 31.52 (CH CH Ar), 34.29 (NCOCH ), 34.91 (CH Ar), 115.26
Ar); 122.01 (Ar), 129.54 (Ar), 129.93 (Ar), 134.33 (Ar), 139.35 (Ar), 146.79 (Ar), 153.91 (Ar),
3
) δ 1.69 (2H, m,
2
2
2
2
3
)
,
2
),
2
7
2
3
1
3
3
3
3
(
NCOCH
2
2
3
2
2
2
2
(
+
76.08 (C=O). ESI -MS: m/z 348.51 ([M+Na] ).
+
1
4.5.13. 3-[5-(4-hydroxyphenyl)pentanoyl]-1-(4-bromophenyl)-3-methyltriazene (3m)
-
1
1
Yield 20%: mp 117-119 °C; νmax/cm 1680, 1692, 3404; H NMR ((400 MHz, CDCl
3
) δ 1.71
), 2.62 (2H, t, J = 7.6 Hz, NCOCH ), 2.95
3
), 4.83 (1H, s, ArOH), 6.77-7.57 (4H, dd, J =
(
(
2H, m, CH
2H, t, J = 7.2 Hz, CH
.4 and 8.8 Hz, Ar), 7.05-7.47 (4H,dd, J = 8.4 Hz, Ar). C NMR (100 MHz, CDCl ) δ 24.59
2
CH CH Ar), 27.92 (NCOCH CH ), 31.46 (NCH ), 34.20 (CH Ar), 34.88 (NCOCH ), 115.30
CH
2 2
Ar), 1.80 (2H, m, NCOCH
CH
2 2
2
2
Ar), 3.43 (3H, s, NCH
1
3
8
3
(
2
2
2
2
3
2
23

(
Ar), 122.93 (Ar), 123.66 (Ar), 129.40 (Ar), 132.48 (Ar), 134.21 (Ar), 147.88 (Ar), 153.84 (Ar),
-
75.88 (C=O). ESI -MS:m/z 388.11 and 390.11 ([M-H] ).
-
1
4.5.14. 3-[2-(4-hydroxyphenyl)etanoyl]-1-(4-cyanophenyl)-3-methyltriazene (3n)
o
-1
1
Yield 15%; yellow crystals; mp 162-165 C; νmax/cm 1696, 2232, 3376; H NMR (400 MHz,
CDCl ) δ 3.48 (3H, s, NCH ), 4.22 (2H, s, NCOCH ), 5.57 (1H, s, ArOH), 6.79 (2H, J = 8.4
Hz, Ar), 7.16 (2H, d, J = 8.4 Hz, Ar), 7.68 (2H, d, J = 8.8 Hz, Ar), 7.77 (2H, d, J = 8.4 Hz, Ar);
3
3
2
13
3
C NMR (100 MHz, CDCl ) δ 28.40 (NCH
3 2
), 40.13 (NCOCH ), 112.16 (Ar), 115.66 (Ar),
118.50 (C≡N), 121.73 (Ar), 121.77 (Ar), 122.77 (Ar), 125.93 (Ar), 130.37 (Ar), 130.48 (Ar),
-
30.54 (Ar), 133.40 (Ar), 151.69 (Ar), 154.98 (Ar), 174.15 (C=O). ESI -MS: m/z 293.10 ([M-
1
-
H] ).
4.6 HPLC analysis
The analytical high-performance liquid chromatography (HPLC) system comprised a Merck
Hitachi L-7110 pump with an L-7400 UV detector, a manual sample injection module
equipped with a 20 µL loop, a Merck Lichrospher 100 RP18 125 mm 4.6 mm (5 µm) column
equipped with a Merck Lichrocart pre-column (Merck, Germany). In all studies, the HPLC
conditions were: detector wavelength 300 nm; mobile phase, acetonitrile/water (the
composition of which depended on the compound-supplementary material S2); flow rate 1.0
mL/min.
4.7 Apparent partition coefficients
The apparent partition coefficients (log Pexp) were determined at room temperature using 1-
octanol–phosphate buffer (pH 7.4). Each phase was mutually saturated before the
experiment. The volumes of each phase were chosen so that solute concentrations in the
aqueous phase after distribution were readily measurable. The compounds were dissolved
in 1-octanol, and the 1-octanol–phosphate buffer mixtures were shaken for 30 min to reach
an equilibrium distribution. Each phase was analyzed separately by HPLC, but the organic
24

phase was diluted in acetonitrile (1:10) before HPLC analysis. Partition coefficients, Pexp
,
were calculated from the ratio of the peak area in octanol to the peak area in the buffer.
4.8 Stability studies
4.8.1 Phosphate Buffer
-2
A 30 µL aliquot of a 10 M stock solution of compounds 3a-n in ACN was added to 10 mL
o
of PBS (0.01 M, pH=7.4) thermostated at 37 C. At different times, several aliquots of the
reaction mixture were taken and analysed by HPLC at λ = 300 nm.
4.8.2 Human plasma (80% v/v)
The human plasma was collected from several healthy donors in sodium heparin and
o
stored at -70 C until required.
A mixture of 2 mL of human plasma and 0.5 mL of PBS (0.01 M, pH=7.4) was thermostated
o
-2
at 37 C. To this mixture were added 10 µL of a 10 M stock solution of compound 3a-n in
ACN. Several aliquots (200 µL) of the reaction mixture were taken at different times and
added to eppendorfs already containing 400 µL of cold ACN. The eppendorfs were
centrifuged at 14000 rpm for 5 min and the supernatant was analysed by HPLC at λ = 300
nm.
25

4.8.3 Mushroom tyrosinase
Tyrosinase (Sigma, UK) was received as a lyophilised powder. The enzyme was dissolved
in PBS (pH 7.4) to produce a stock solution of 10 000 U/mL. Aliquots of the stock solution
o
89.4 µL, 900 units / 29.8 µL, 300 units) were stored at -20 C until used. Depending on the
(
enzyme units needed for each assay, the aliquots were added to a solution of 2.4 mL of
o
PBS (0.01 M, pH=7.4) and 0.6 mL of DMSO at 37 C. To this mixture were added 10 µL of a
-
2
1
0 M stock solution of compounds 3a-n in ACN. Several aliquots (200 µL) of the reaction
mixture were collected at selected times and added to eppendorfs with 400 µL of cold ACN.
The Eppendorf’s were centrifuged at 14000 rpm for 5 min and the supernatant was
analysed by HPLC at λ = 300 nm.
4
.9 General procedure for the determination of K and Vmax
m
The K
conversion of prodrug into drug at the wavelength previously selected, using a range of
prodrug concentrations (0–10 µM) in the presence of the enzyme (5 U/mL). K
m
and Vmax with mushroom tyrosinase were determined by measuring the initial rate of
m
and Vmax
were calculated through a non-linear Michaelis–Menten regression with the Graphpad
Prism program. The kcat was calculated from the Vmax by dividing it by the amount of enzyme
(
M) in the reaction mixture.
4
.10 General procedure for the citotoxicity screening assays
.10.1 Cell lines culture conditions
4
The human melanoma cell line MNT-1 (kindly offered by Dr. José Ramalho, Faculdade de
Ciências Médicas, Lisbon, Portugal), the murine melanoma B16F10 cell line (ATCC#CRL-
6
475) and the non-melanoma cell line (the human keratinocyte cell line) HaCaT, (CLS,
00493), were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with high-glucose
3
(
4500 mg/L), supplemented with 10% fetal bovine serum (FBS) and 50 U/mL penicillin, 50
o
µg/mL streptomycin (hereafter complete medium), at 37 C in a 5% CO
2
humidified
atmosphere.
26

4.10.2 Cell proliferation and viability assays
The viability of melanoma and non-melanoma cells in the presence of the synthesized
compounds was performed by the MTS assay according to the manufacturer’s instructions.
3
Cells were seeded in 96-well plates (200 µL; 1.5 x 10 cells/mL) for 24h. The culture
medium was removed and the compounds at concentrations ranging from to 37.5 to 150
µM prepared in complete medium were added. Cells were allowed to proliferate for 72h at
o
7 C in a humidified CO
3
2
controlled atmosphere. After that, the media was removed and
replaced with incomplete fresh media. Subsequently, 20 µL of MTS were added to each
well, and incubated for 2 h under the same culture conditions as above mentioned. The
absorbance was measured at 490nm in a BioRad microplate reader Model 680 (Bio-Rad,
Hercules). Control assays were conducted in the absence of compounds and in the
presence of similar amounts of organic solvent used in the highest concentration tested. All
the tests were performed with six samples for each concentration under study.
The IC50 (50% inhibitory concentration) was defined as the concentration of the compound
that inhibited cell proliferation by 50% calculated by linear regression analysis.
Acknowledgements
The authors gratefully acknowledge the Fundação para a Ciência e Tecnologia (Portugal)
for financial support through funding of the research unit iMed.ULisboa PEst-
OE/SAU/UI4013/2014.
The authors also gratefully acknowledge
counseling and proofreading.
Mme Françoise Girard for her valuable
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